THE 16924 - UQ eSpace - University of Queensland [PDF]

example of this is the patched gene, which is abbreviated as ptc and Ptc in Drosophila and .... Drosophila melanogaster.

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Idea Transcript


THE 16924

H . B U^viVE2^.-rry OF QoE£?

anlerior posterior

proximal <

dorsal

> distal

posterior

B ectopic hh

removal of hh > •

Figure 1.4

^

The hedgehog pathway and insect wing development. (A) Normal wing development in Drosophila. Left panel shows the third instar stage imaginal disc, which is divided into anterior-posterior and dorsal-ventral (indicated by dotted line) compartments. Posterior compartment cells express hedgehog (hh), as indicated by dark shading. The wing blade of the adult that is formed from this structure is shown on the right. The five longitudinal veins of the wing are numbered. Bristles and sensory areas (dots on vein three) are in specific positions. (B) Ectopic expression of hh in the anterior compartment leads to duplication of wing tissue. The highly abnormal wing in this example is caused when hh is ectopically expressed at the dorsalventral compartment boundary in the anterior region. (C) When the normal action of hedgehog is removed small and poorly patterned wings are formed. Reproduced with permission from Capdevila and Johnson, (2000).

In summary. Hedgehog is involved in a diverse range of developmental processes and at multiple stages in the formation of the fly. Furthermore, the hedgehog pathway is intertwined with additional signalling pathways, through which many of its actions are exerted.

32

Chapter 1: General Introduction and Literature Review

1.4 The hedgehog pathway in mammalian development In vertebrates the hedgehog pathway controls a large number of diverse patterning events during embryogenesis. The following sections provide an overview of the roles of the three mammalian hedgehogs in mammals. Functionally, Sonic hedgehog appears to play the major role in mammalian development, whilst Indian and Desert Hedgehog appear to play important roles in skeletal development and male gonad function, respectively.

1.4.1 Role of Sonic hedgehog Shh is expressed in the developing embryo at many sites of epithelial-mesenchymal interaction (Bitgood and McMahon, 1995). A large body of work has now been pubUshed on the specific role of Shh in a varied range of tissues and a complete review of all primary literature is beyond the scope of this introduction. Extensive reviews on Shh patterning of vertebrate tissues have been published by Hammerschmidt et al, (1997) and Ingham and McMahon, (2001). This section outlines several well studied systems that highlight the diverse nature of patterning, and illustrate how the Shh signal can have quite different effects on different tissues. Early in embryogenesis Shh plays a key role in the determination of cell fate along the midline. Shh protein is expressed from the notochord, and later the floor plate, structures that extend along the centre of the embryo and are involved in patterning the developing neural tube. In this system Shh acts as a classical morphogen with concentration differences at various distances from the source altering cellular responses (reviewed by Briscoe and Ericson, 1999). Earlier however, when the notochord causes differentiation of the floor plate itself, short-range Shh signalling is used and physical contact is necessary (Placzek et al, 1993; Yamada et al, 1993). Shh induces a number of neural cell types, and in addition plays a role in promoting cell survival and proliferation (Echelard et al, 1993; Krauss et al, 1993; Roelink et al, 1994; Marti et al, 1995; Roelink et al, 1995). Other areas of the cenfral nervous system (CNS) are also extensively patterned by Shh, including the developing oligodendrocytes (myelin forming cells), various regions of the developing brain and associated sensory structures (reviewed by Marti and Bovolenta, 2002). Hedgehog also patterns the differentiation of the somites, blocks of mesodermal tissue that form adjacent to the neural tube. Under the guidance of Shh (and to some extent Ihh) these regions give rise to

1.4 The hedgehog pathway in mammalian development

33

a number mesodermal derived structures including the vertebrae, ribs and associated cartilage, the body wall, muscles and the dermis of the skin. Another area in which the role of Shh has been investigated extensively is in the developing vertebrate limbs (Figure 1.5), with data coming from avian as well as mammalian model systems. Correct patterning in limbs relies on signalling from a specific set of posterior mesenchymal cells in a region known as the zone of polarising activity (ZPA). Shh secreted from the ZPA acts as a concentration dependent morphogen and is the primary determinant of limb anterior-posterior polarity. Evidence for this came from studies showing that ectopically supplied Shh leads to the formation of mirror image duplication of digits, in a similar fashion to those induced by grafting secondary ZPA regions to the developing limb (Riddle et al, 1993; Lopez-Martinez et al, 1995). Further support for Shh as the ZPA signal comes from studies showing the converse; that loss of Shh in the limb bud leads to limb truncation (Chiang e? a/., 1996). Shh expression is critical for normal hair follicle morphogenesis in mammals (St-Jacques et al, 1998; Chiang et al, 1999; Karlsson et al, 1999) and may have a role in post-natal hair cycling (Sato et al, 1999; Wang, L. C. et al, 2000). Regulation of follicle development by Shh is of particular interest since basal cell carcinoma, a key feature of hedgehog pathway disruption in humans (refer Section 1.6), is thought to arise from cells residing in or near follicles. Recent studies have identified a role for Shh in regulating aspects of the developing circulatory system, including both the formation of new blood vessels through angiogenesis (Pola et al, 2001), and confrol over haematopoietic stem cell proliferation and differentiation of both red and white blood cells (Detmer et al, 2000; Outram et al, 2000; Bhardwaj et al, 2001). Other diverse roles include regulating left-right asymmetry during vertebrate development (reviewed by Levin, 1997). Shh also plays a key role in patterning the gut, heart, lungs, bladder, prostate, pancreas and a range of other internal structures.

Chapter 1: General Introduction and Literature Review

34

humerus AER

radius normal

development Shh

antenor

anlenor

proximal •*——• distal

proximal

removal of Shh >•

Figure 1.5

The hedgehog pathway and vertebrate limb development. The diagrams shown illustrate the importance of hedgehog action on vertebrate limb development using the chicken wing as an example. (A) The left hand diagram shows the limb bud after 3 days of development, at which stage it is composed of mesenchyme tissue surrounded by an ectodermal sheath. The apical ectodermal ridge (AER), a thickened epithelial structure, is labelled. Sonic hedgehog (Shh; dark shading) is secreted from posterior mesenchyme in a region known as the zone of polarising activity (ZPA). At 10 days the normal aduh structures are apparent, as shown on the right (digits numbered). (B) If Shh is ectopically expressed in anterior regions mirror image duplications occur. (C) Removal of the ZPA region that normally expresses Shh results in severe truncation of the limb. Reproduced with permission from Capdevila and Johnson, (2000).

1.4.2 Role of Indian hedgehog Indian hedgehog appears primarily involved in regulating growth and proliferation of developing bone and cartilage (Vortkamp et al, 1996; Vortkamp et al, 1998; St-Jacques et al, 1999). Ihh induces its affects by stimulating parathyroid hormone-related peptide (PTHrP)

1.5 Downstream targets of the hedgehog pathway

35

and bone morphogenetic proteins (BMPs), that in turn head further signalling pathways that regulate chondrocyte activity in bone formation (Lanske et al, 1996; Vortkamp et al, 1996; Zou et al, 1997; Pathi et al, 1999). PTHrP and Ihh control regulation of each other through a negative-feedback loop that that prevents chondrocytes fi-om exiting the mitotic cycle (reviewed by Kronenberg et al, 1998). There is also an apparent role for Ihh in the early differentiation of the visceral endoderm (Maye et al, 2000), and later in patterning of the gut (Ramalho-Santos et al, 2000). The gut displays a distinct overlap of Ihh and Shh expression during development (Bitgood and McMahon, 1995) and there may be some redundancy between the functions of the two proteins. Patterning is influenced by both genes during early bone development, heart morphogenesis, lateral asymmetry, vasculogenesis and haematopoesis (reviewed by Ingham and McMahon, 2001). Thus it appears that Shh and Ihh work in concert during development in some regions.

1.4.3 Role of Desert hedgehog Of the three mammalian hedgehogs, Dhh displays the most restricted expression pattern. In mouse development it is expressed principally in the Sertoli cells of the testes and Schwann cells of peripheral nerves (Bitgood and McMahon, 1995). In the male germ line, Dhh appears to regulate various stages of spermatogenesis and is crucial for the formation of Leydig cells and other testicular features (Clark et al, 2000; Yao et al, 2002). In the developing CNS Dhh is secreted from Schwann cells and signals to fibroblasts surrounding the peripheral nerves to produce the perineurium, a protective sheath that surrounds nerve fibre bundles (Mirsky et al, 1999; Parmantiere^a/., 1999).

1.5

Downstream targets of the hedgehog pathway

Much of what is known about Hedgehog signalling components was initially gleaned from studies in Drosophila, and the knowledge of downstream targets is more advanced in the fly than in any vertebrate system. This is largely due to the applicability of this organism to large scale mutation screens, and the success of a number of techniques using either mosaic animals or complex combinatorial mutants to reveal hierarchies of genes. There are currently many molecules implicated downstream of the hedgehog family in mammals, but few bona fide

36

Chapter 1: General Introduction and Literature Review

targets. In particular, the specific molecules that signal neoplastic cell proliferation upon disruption of hedgehog signalling remain largely elusive.

1.5.1 The "Universal" hedgehog target genes Hedgehog pathway target genes differ in different tissues, and this is one mechanism by which hedgehog is able to cause such diverse effects on target cells. Upon reviewing the literature from many organisms where the hedgehog pathway has been ectopically activated, two genes appear universally expressed in all responsive vertebrate tissues that have been studied. These are Patched and Glil, both of which are induced at the level of transcription in response to pathway activation (Goodrich et al, 1996; Marigo et al, 1996b; Marigo et al, 1996c; Marigo and Tabin, 1996; Lee, J. et al, 1997; Murone et al, 1999). These molecules are also up-regulated in a number of tumours arising from hedgehog pathway disruption (Gailani et al, 1996; Dahmane et al, 1997; Unden et al, 1997; Vorechovsky et al, 1997b; Ghali et al, 1999; Bonifas et al, 2001). Furthermore, the up-regulation of Ptc and Glil transcription also holds tme when cells grown in vitro are hedgehog stimulated (Nakamura et al, 1997; Pepinsky et al, 1998; Taipale et al, 2000). A third gene. Hip (refer Section 1.2.4) has been studied less extensively but may also be a "universal" pathway target (Chuang and McMahon, 1999; Bonifas et al, 2001). These three genes were used extensively in this project, as markers of successful pathway stimulation. In Drosophila, which lacks a known homologue for Hip and regulates its Gli homologue ci posttranscriptionally, ptc is the only target gene that has been observed to have mRNA upregulated by Hh in all responsive cell tjqpes (Hidalgo and Ingham, 1990; Tabata and Komberg, 1994).

1.5.2 Additional downstream target genes As mentioned in Section 1.3, the key transcriptional targets of hedgehog signalling in a range of tissues in Drosophila are ptc, wingless and dpp, though the latter two are not necessarily activated in all responsive cell types. In vertebrate development the situation is thought to be analogous, whereby genes related to wg and dpp would play a major hedgehog mediated developmental role. The single wg gene of the fly is represented by a large family of vertebrate homologues known as the Wnt family. Likewise, the vertebrate genome contains a considerable number of genes related to dpp, which are known collectively as the

1.5 Downstream targets of the hedgehog pathway

37

transforming growth factor p (TGPP) superfamily. In close analogy with processes in Drosophila, embryonic development in vertebrates depends upon critical interactions between Hedgehog, Wnt and TGFp family members (reviewed by Roelink, 1996). The sub-group of the TGpp superfamily most closely resembling dpp are the BMPs, and as such are considered prime candidates for regulation by the hedgehog pathway in vertebrates. Within the BMP family the members most closely resembling dpp stmcturally are Bmp2 and Bmp4, both important developmental molecules (Hogan, 1996). A strong line of evidence supporting BMPs as Hedgehog regulated molecules came from expression studies showing that BMPs and Hedgehogs are frequently expressed in adjacent regions during mouse development (Bitgood and McMahon, 1995). Analysis of BMP-hedgehog interrelationships is confounded by the fact that not only do hedgehogs appear to regulate BMPs, but BMPs themselves are also implicated in regulating Hedgehog expression (Arkell and Beddington, 1997; Zhang, Y. et al, 2000; Grimsmd et al, 2001; Ohkubo et al, 2002). Direct evidence for Hedgehog mediated regulation of BMP genes has come from studies in a limited number of tissue systems (Fan, H. et al, 1997; Bhardwaj et al, 2001; Kawai and Sugiura, 2001). Like the BMP family, the Wnt family contains a large number of vertebrate family members. The mechanisms by which Wnt proteins signal show many parallels to the wingless signalling system in Drosophila, and this has been the subject of extensive review (Cadigan and Nusse, 1997; Dale, 1998; Wodarz and Nusse, 1998). A recent study in the frog has shown that members of the Gli family, which are themselves controlled by Hedgehogs, can regulate the expression of Wnt5A, 7B, 7C, 8 and 8B in ectodermal explants (Mullor et al, 2001). An additional twist to the Hh-Wnt story is the recent finding that some molecules that act as antagonists or modulators of Wnt signalling are also regulated by the hedgehog pathway. Sfrpl and Sfrp2, members of the secreted frizzled related protein family, have been found to be up-regulated by ectopic Shh in developing mouse mesoderm tissue (Lee, C. S. et al, 2000). Members of this family have a stmcture similar to the Frizzled proteins that act as membrane tethered Wnt receptors (refer Section 1.3.1). SFRPs lack transmembrane domains, can bind to specific Wnt ligands in the extracellular space, thereby modulating Wnt signalling (reviewed by Polakis, 2000). In the developing CNS an important target induced by Shh is Hepatocyte nuclear factor-3P {HNF-3P), a winged-helix transcription factor (Roelink et al, 1995). This is one of the few target genes for which regulation by Hedgehogs has been studied extensively, and is directly

38

Chapter 1: General Introduction and Literature Review

mediated by Gli proteins (Sasaki et al, 1997). Ectopic expression of Shh up-regulates expression of Shh itself, and this is mediated, at least in neural tissues, by HNF-3p (Roelink et al, 1994; Ruiz i Altaba et al, 1995; Epstein et al, 1999). There is some evidence that Patched2, like Patched, may be a target of hedgehog regulation (Taipale et al, 2000; Pathi et al, 2001), but there is some conflict in reports of human PATCHED2 {PTCH2) up-regulation in basal cell carcinoma (Zaphiropoulos et al, 1999; Bonifas e? a/., 2001). Other known hedgehog responsive genes include COUP-TFII (refer Section 1.2.8) and Isletl (Chiang et al, 1996; Nakagawa et al, 1996; Dutton et al, 1999) in the developing neural tube, SWiP-1 in somitic mesoderm and limb buds (Vasiliauskas et al, 1999), Myf5 (Borycki et al, 1999; Gustafsson et al, 2002) and MyoD (Munsterberg et al, 1995) in muscle formation and Angiopoietin2 (Pola et al, 2001) in developing vasculature. Other tissuespecific targets include members of the PAX (Ericson et al, 1996; Ericson et al, 1997; Borycki et al, 1998), SOX (Hargrave et al, 2000), TBX (Gibson-Brown et al, 1998; Garg et al, 2001), NKX (Nakagawa et al, 1996; Briscoe et al, 1999; Cai et al, 2000; Pabst et al, 2000; Murtaugh et al, 2001) and HOX (Riddle et al, 1993) families. Recently, links between hedgehog signalling and regulation of the cell cycle have been uncovered with the discovery that several cyclins are transcriptionally regulated by Hedgehog (Kenney and Rowitch, 2000; Duman-Scheel et al, 2002; Yoon et al, 2002). Several genes have also been implicated in the pathway due to changes in expression observed in animal models. IgfZ, for example, is elevated in tumours of the patched knockout mouse (Hahn et al, 1998).

1.6 The hedgehog pathway in human disease Perturbation of hedgehog signalling is present in a range of human diseases. Such disorders generally involve either developmental abnormalities (when genetic lesions or environmental factors dismpt signalling during embryogenesis) or tumour formation (when the pathway becomes abnormally activated in an individual cell by acquisition of mutations). This section provides an outline of diseases of interest that provide further clues to the normal function of the hedgehog pathway. Figure 1.6 outlines the points in the hedgehog signalling cascade where disease causing mutations have been identified.

39

1.6 The hedgehog pathway in human disease

Sonic Hedgehog

Indian Hedgehog

>

Figure 2.7

Histochemical alkaline phosphatase assay in C3H/10T1/2 cells shows transfected HaCaT cells express Shh protein with potent biological activity. (A) Media collected from HaCaT cells that have been transfected with pShh-N-PMT21 induces alkaline phosphatase activity in C3H/10T1/2 cells, as indicated by blue staining. (B) Media collected in parallel from a negative control transfection has no detectable effect on alkaline phosphatase activity in C3H/10T1/2 cells. The principle of the C3H/10T1/2 alkaline phosphatase assay is described in detail in Chapter 3. The media used was collected from the cells for which RNA was obtained for the northem blot shown in Figure 2.6. Media was collected 24 hours post-transfection. The above result shows that the lack of a convincing response to Shh in HaCaT cells was not due to any problems with the construct or transfection procedure, as it confirms that the cells were exposed to high levels of active Shh protein during the experiments.



Chapter 2: Strategies for Hedgehog Pathway Activation and Initial Studies in Keratinocytes

2.4 Discussion Basal skin keratinocytes were considered a cell type of interest for studies of the hedgehog pathway due to their probable involvement in the formation of basal cell carcinoma. Both of the keratinoc)^e systems investigated, primary murine cells and the immortal human line HaCaT, expressed Patched and Smoothened at readily detectable levels, suggesting they could potentially receive the Shh signal. However, neither cell type was successfully stimulated with rShh N-terminal protein. Subsequently the Cos7-derived rShh-N used for this work was found to have very low biological activity, and thus the results could not be considered conclusive. Preliminary transfection studies in HaCaT cells did not yield convincing data to indicate they were responsive to Shh when Patched was investigated as a marker of pathway activation, even when the cells were exposed to Shh-N shown to have high biological activity. Transfection of a constitutively active SMOH mutant also failed to elicit an obvious response. There was an apparent induction in the level of PTCH in response to stimulation with GLIl, but this was so slight that extensive replicated studies to see if the response was significant were not performed. A subtle change in a key indicator would have made for a poor model system in terms of hedgehog target gene discovery. At this stage a different tissue system involving embryonic mesodermal cells was found to be extremely amenable to hedgehog pathway activation, and it was decided that resources should be devoted to this alternative cell type (as described in the following chapter). A recent publication has reported up-regulation of pathway markers in HaCaT cells in response to stimulation with GLIl, with maximal induction of PTCH mRNA observed 48 hours after treatment (Regl et al, 2002). Thus, it appears that HaCaT cells can indeed display a response when the pathway is activated at a level of the signalling cascade more distal than hedgehog itself The greater magnitude of PrC//induction in this study compared with GLIl experiments such as that shown in Figure 2.6 may reflect differences in GLIl stimulation methods. Regl and colleagues used a stably transfected line such that GUI expression could be tightly controlled using a tetracycline sensitive construct. It is possible that keratinocytes do indeed respond directly to Shh in vivo, but that this response is retarded when keratinocytes are grown in monoculture. In an animal, basal epidermal keratinocytes sit near the interface with the dermal tissue layer. Here they would be exposed to any cofactors potentially secreted by the dermal cells. Interestingly, BCCs are

2.4 Discussion

71

recalcitrant to being grown in a tissue culture environment and this may be for similar reasons. It is also possible that the human HaCaT cells did not show a strong up-regulation of PTCH in response to Shh because the transfected construct encoded the mouse protein, though this is considered unlikely given the high homology between the mouse and human proteins at the amino acid level. HaCaT cells exhibit trisomy of the long (q) arm of chromosome 9 (Boukamp et al, 1988), and as such presumably have three copies of PTCH. If all three are transcribed then elevated Patched protein levels could potentially lead to either increased inhibition of Smo, or increased sequestering of Shh, thereby contributing to the lack of a convincing response upon stimulation with Shh. One study has shown that the hedgehog pathway can be activated in human keratinocytes, though in this case a full length human Shh construct was retrovirally expressed in cultured cells before they were differentiated into a skin layer and grafted onto immune deficient mice (Fan, H. et al, 1997). Due to the difficulties in eliciting a clear cut hedgehog pathway activation response in the keratinocytes used in this work further studies were conducted in an alternative system, a embryonic mouse mesodermal line called C3H/10T1/2. This line proved highly amenable to pathway activation and is discussed in detail in the following chapter.

Chapter 3: C3H/10T1/2 as a Model System for Hedgehog Target Gene Discovery 3.1

Introduction

The experiments outlined in the previous chapter indicated that basal keratinocj^es present some difficulties in terms of their use as a model for hedgehog pathway activation. In addition, production of biologically active purified recombinant Shh protein proved difficult. At this stage in the project it was decided that a change in both activation methodology and cell type was essential to maximise the chance of discovering novel hedgehog target genes. When this project was initiated most known hedgehog responsive systems involved organ explant culture. In 1997 two cell lines, C3H/10T1/2 clone 8 (hereafter referred to as lOTl/2) and MC3T3-E1, were reported to be responsive to Shh (Kinto et al, 1997; Nakamura et al, 1997). Both cell lines were investigated and their responsiveness to Shh confirmed. Unlike lOTl/2 cells, which are pluripotent, MC3T3-E1 cells are aheady committed to the osteoblastic lineage. For this reason lOTl/2 cells were chosen as the system of interest, as they would potentially express a more diverse suite of target genes in response to Sonic hedgehog. The lOTl/2 cell line is examined extensively in this chapter and was found to be particularly suitable as a model to meet the aims outlined in Chapter 1. Studies in lOTl/2 cells have led to a greater understanding of the fimctions of Shh (Pepinsky et al, 1998; Katsuura et al, 1999; WilHams, K. P. et al, 1999; Pepinsky et al, 2000; Saeki et al, 2000), Smo (Murone et al, 1999) and Gli family members (Ruiz i Altaba, 1999), as well as providing a means to investigate comparative effects of vertebrate Hh proteins (Pathi et al, 2001). In the work described in this thesis, the use of lOTl/2 cells has been extended to provide a useftil model system for the discovery of novel downstream target genes regulated by the Hh pathway.

74

Chapter 3: C3H/10T1/2 as a Model System for Hedgehog Target Gene Discovery

3.1.1 Origin and characteristics of the 10T1/2 cell line The lOTl/2 cell line was initially estabhshed and characterised by Reznikoff e? al, (1973). The line was isolated from a pool of 14 to 17 day old C3H mouse embryos using a methodology that selects for cells showing inhibition of cell division upon confluence (Aaronson and Todaro, 1968), and was named following the convention of Todaro and Green, (1963). The lOTl/2 line is clonal. Cells are adherent and motile and when sub-confluent display a fibroblast-like morphology. lOTl/2 cells are pluripotent, having the ability to differentiate into a number of mesodermal lineages, including myoblasts, adipocytes, chondrocytes and osteoblasts, upon stimulation with various growth factors or chemicals (Taylor, S. M. and Jones, 1979; Katagiri et al, 1990; Asahina et al, 1996). The plasticity of lOTl/2 cells makes them of particular interest for hedgehog target gene discovery, since they have the potential to express a number of genes of critical importance to cell fate determination in embryogenesis. The cell line undergoes a low level of spontaneous differentiation and displays occasional transformation to a non-contact inhibited phenotype. The latter leads to "foci", small piles of proliferating cells within the otherwise single cell thickness monolayer observed in confluent populations. If this cell type reaches high cell density the morphological appearance of the cells becomes changed, and this change remains even after re-seeding and further passaging. To maintain the features of the cell line and to minimise the build up of imdesirable transformed and differentiated cells, lOTl/2 stocks were never allowed to reach confluence and were only used at early passage.

3.1.2 Known responses of 10T1/2 cells to hedgehog stimulation Initial studies exposing lOTl/2 cells to Shh protein showed that this causes a large percentage of cells to enter the osteoblastic lineage, as measured by the induction of the osteoblastic marker alkaline phosphatase (AP), and that the resulting cells able to induce ectopic bone formation in mice (Kinto et al, 1997). Other studies have taken advantage of the induction of AP in response to Shh to provide a measure of hedgehog pathway activation that can be readily assayed (Nakamura et al, 1997; Pepinsky et al, 1998; Spinella-Jaegle et al, 2001). Nakamura et al, (1997) demonstrated induction of Patched by RT-PCR after Shh stimulation, and this induction has been confirmed by other studies (Williams, K. P. et al, 1999).

3.2 Basal expression of key pathway genes in lOTl/2 cells

75

Transcriptional responses to Shh in lOTl/2 cells have also been reported for Ptc2 and Hip using RT-PCR (Pathi et al, 2001). Glil has also been established as a target of Shh signalling in lOTl/2 cells through several approaches (Murone et al, 1999; Pathi et al, 2001). As with Shh, exposure to Bmp2 causes lOTl/2 cells to become osteoblastic (Katagiri et al, 1990). BMPs are implicated as candidate targets regulated by the hedgehog pathway, but surprisingly Bmp-2, 4, 5, 6 and 7 have been found to show no significant expression changes in lOTl/2 cells upon stimulation with Shh (Nakamura et al, 1997). This suggests that genes other than BMPs are critical in inducing the osteoblastic phenotype in this cell type. When Bmp2 and Shh added in combination to lOTl/2 cultures they produce a synergistic effect on osteoblastic differentiation, much greater than for either protein alone. Furthermore, expression of the BMP inhibitor Noggin abolished osteoblast differentiation in Bmp2 treated cells, while Noggin does not inhibit Shh induced differentiation (Spinella-Jaegle et al, 2001). Though a large percentage of lOTl/2 cells stimulated with Shh have been shown to enter the osteoblastic lineage, little is known about the differentiation of cells to other mesodermal cell types under Shh control. It is clear however that treatment with Shh inhibits the ability of the pluripotent cells to enter the adipocytic lineage (Spinella-Jaegle et al, 2001).

3.2 Basal expression of key pathway genes in 10T1/2 cells Before initiating pathway activation studies in the lOTl/2 cell line investigations were made into the basal level of key pathway genes. Though this line is already known to be responsive to Shh, it was thought prudent begin with such work. The basal level of Shh expression had not previously been addressed in the literature, nor had several other genes of interest. Ptc expression in lOTl/2 has previously been vaguely described as "low" (Nakamura et al, 1997) and "not highly expressed" (Pepinsky et al, 1998), with the later work going so far as to suggest that other Hedgehog receptors may contribute to the response of the cell line. Initial northem blotting studies used the mouse Ptc probe "mPtcl263", a 1263 base pah Pvull restriction fragment fi-om the mouse Ptc cDNA beginning midway through the first extracellular loop and extending through transmembrane domains 2 to 6. On untreated poly(A)^ RNA, this probe detected a band of the size expected for the fiill length Ptc transcript. In addition, several smaller bands were observed, all of which remained bound at high stringency. These additional bands are of particular interest as some appear, along with

Chapter 3: C3H/10T1/2 as a Model System for Hedgehog Target Gene Discovery

76

the full length transcript, to be Hedgehog regulated. For northem blot data showing basal Patched expression in lOTl/2 cells the reader is referred to Figure 3.11 and Figure 3.13. Further studies investigating the level of Patched protein in lOTl/2 cells by immunofluorescence showed a diffiise pattem of cell staining, indicating that the produced mRNA is indeed translated under basal conditions (data not shown). Basal Smoothened expression was also assessed by northem blotting (Figure 3.1), revealing a single band of a size comparable to the 3.7 kb transcript previously reported in rat tissues (Traiffort et al, 1998). Immuno-fluorescence indicated that Smoothened protein, like Patched protein, is produced at detectable levels in untreated lOTl/2 cells (data not shown).

28s RNA (4.7 kb)

Smo

18s RNA (1.9 kb)

Figure 3.1

lOTl/2 cells express Smo mRNA, as shown by northern blotting of total RNA harvested from untreated cells.

Glil is not detected by northem blot in untreated lOTl/2 RNA, even after long exposure (data not shown); however, it can be readily detected by RT-PCR (Figure 3.2). Shh expression was investigated by northem blotting, using a probe known to detect the gene in spiked samples. Expression was not detected in lOTl/2 cells (data not shown). Taken together the finding of significant Patched and Smoothened expression, along with extremely low basal levels of Glil and Shh in lOTl/2 cells make the lOTl/2 line suitable for pathway activation studies.

77

Gli1 (251 bp product)

Figure 3.2

^

Ladder Untreated 10T1/2

U

+ Control

3.3 Comparison of potential pathway activation strategies

-

2

CD

-^

CD —I

O 1

RT-PCR showing Glil is expressed in untreated lOTl/2 cells. Mouse Glil primers were modified from those published by Walterhouse et al., (1993), in that the superfluous overhangs were removed and a miss-match between GenBank sequences and the published reverse primer was corrected. Template for lOTl/2 reaction template was cDNA transcribed from 70 ng of total RNA. Negative control represents the same volume of a "no RNA" control cDNA synthesis reaction. Positive control PCR template was 1 ng of a plasmid containing the mouse Glil cDNA. PCR had 35 cycles. Ladder is "1 kb ladder" from Gibco BRL.

3.3 Comparison of potential pathway activation strategies As outlined in Section 2.1.2, there are a number of levels at which the hedgehog pathway could potentially be activated in mammalian cells. Although addition of "home-made" recombinant Shh protein was chosen as the ideal method of stimulation, the results outlined in Chapter 2 highlighted a nimiber of difficulties with achieving this. At the stage where the work moved its primary focus from keratinocytes to lOTl/2 cells, various strategies were compared to determine the best altemative activation strategy.

3.3.1 Alkaline phosphatase and Patched as key indicators of pathway activation The success of different treatments in activating the hedgehog pathway was compared by investigating the induction of AP activity and the up-regulation of Patched transcription in

78

Chapter 3: C3H/10T1/2 as a Model System for Hedgehog Target Gene Discovery

lOTl/2 cells. The increase in AP activity provided a robust measure of hedgehog induced osteoblastic differentiation, which was readily amenable to both histochemical and quantitative spectrophotomeric assays. Changes in the level of Patched transcription were assessed in parallel. Initial studies with semi-quantitative RT-PCR indicated this method was not robust, displaying a high level of variability in amplification between replicates and frequent lack of reproducibility between experiments. At the time this work was conducted real-time quantitative PCR systems were not accessible to the author, and it was felt that the more laborious task of northem blotting was worthwhile for all experiments due to its ability to yield high quality quantitative data when coupled with densitometry. Patched was chosen as the primary gene to analyse in initial studies comparing activation methodologies due to its status as a universal marker of hedgehog stimulation (refer Section 1.5.1).

3.3.2 Alternate recombinant protein based strategies for pathway activation Although the techniques outlined in the previous chapter failed to produce rShh protein with high levels of biological activity, the addition of pure Shh protein was still seen as the ideal method of pathway stimulation. For this reason, other sources of suitable protein were sought both commercially and through collaborators. Once established the lOTl/2 system allowed straightforward testing of Shh protein from various sources. A small amount of commercially available mouse rShh protein (461-SH) was purchased fi-om R&D Systems (Minneapolis, USA) and used as a positive control in a limited number of small scale experiments. This protein proved active, and provided a benchmark by which to judge the quality of recombinant Shh from other sources (Figure 3.3). The commercial protein was not viable for use in any fiill scale experiments involving RNA harvests, nor the project as a whole, as its cost was prohibitive. Recombinant Shh potentially available for fiill scale experiments was sourced fi-om two collaborating groups. A small amount of baculovims produced Shh protein, made by the method of Ericson et al, (1996) was obtained (a kind gift of the late Dr. T. Yamada). When assayed in the lOTl/2 system this protein was shown to have potent biological activity (data not shown), however due to unforeseen circumstances fiirther supplies were unable to be obtained for the remaining experiments. A fiirther source of recombinant protein, HIS-tagged mouse N-terminal Shh produced in bacteria (a kind gift of Dr. P. Bartlett), was obtained and

3.3 Comparison of potential pathway activation strategies

79

assayed. This protein had no detectable activity in subsequent lOTl/2 assays (data not shown), and at this stage perseverance with a purified protein based approach was abandoned. Time constraints meant that the project had to move immediately to assessing altemative activation strategies. One available option was the production of cmde Shh-N conditioned media. The possibility of producing this successfully was based on the assumption that biological activity of this molecule was difficult to maintain during purification procedures, but that if it was secreted into growth media that was then used directly, then this obstacle could be overcome. This indeed proved to be the case. Conditioned media was produced in lOTl/2 cells to ensure that any secondary secreted products induced by Hedgehog stimulation were limited to the suite of molecules that the target cell type would normally produce. Initial studies involved collecting post-transfection growth media from lOTl/2 cells transfected with the constmct pShh-N-PMT21 (this constmct encodes the N-terminal active region of mouse Shh and is discussed in detail in Section 3.4), or an empty vector control constmct. Media was collected after various time periods of conditioning, and found to have potent activity when assayed for AP induction (Figure 3.3).

3.3.3 Shh transfection strategy In parallel to work on conditioned media (outlined above), various fransfection based strategies were investigated to determine which were suitable for studying expression changes caused by hedgehog pathway activation. The strategies investigated were those infroduced in Section 2.1.2. Direct fransfection of lOTl/2 cells with the constmct pShh-N-PMT21 ehcited a strong pathway response, with dramatic induction of both AP activity (Figure 3.4) and Patched transcription. These responses are fiirther investigated in Section 3.5, where figures detailing the profile of induction are presented, along with the responses of other known target genes. Shh transfection proved to be a robust sfrategy to achieve pathway activation.

80

Chapter 3: C3H/10T1/2 as a Model System for Hedgehog Target Gene Discovery

Figure 3.3

Shh conditioned media has potent biological activity when compared to commercially available Shh protein in the lOTl/2 alkaline phosphatase (AP) induction assay. (A) Commercially obtained rShh protein (461-SH, R&D Systems) causes a substantial increase in AP enzyme activity in lOTl/2 cells. Assay was performed 7 days after addition to growth media. The effect was most dramatic when the growth media was changed and fresh Shh added half way through the growth period. At low concentrations the effect increased in magnitude with increasing Shh concentrations, before starting to plateau at 1 |j,g/ml. (B) Shh conditioned growth media (Shh CM) collected for various time periods after transient transfection of lOTl/2 cells with pShhN-PMT21, is highly active. The three batches of conditioned media tested in this experiment showed higher activity, in terms of AP induction, than the commercially available protein at 5 \iglm\. Error bars are not included on these graphs as the author only had access to a very limited amount of rShh from R&D systems and this did not allow replication. The results are intended simply as an indication that the conditioned media produced did have significant biological activity. The actual conditioned media used for stimulation in large scale experiments (outlined in the following chapters) was collected over an even wider time period that that used in the above investigative assays, and fully replicated experiments showed it also had dramatic AP inducing activity (data not shovra).

82

Chapter 3: C3H/10T1/2 as a Model System for Hedgehog Target Gene Discovery

GLI1

%r'

M

o

/

< # ••.'•- i W V

m

m

(S^

A

%

Neg Control

•.5.

4^

J r.

T

. - , ^ ' ^

•*

•M' .

Figure 3.4

.

^



'

'

..",.

Histochemical assay shows lOTl/2 cells transfected with Shh or GUI show marked induction of AP enzyme activity. Cells transfected with an empty vector have very low AP activity, whereas activity in cells transfected with mouse Shh (pShh-N-PMT21) or human GLIl (pRK7-N-MychGli), indicated by blue staining, is greatly increased. Assay was performed four days post-transfection.

3.3 Comparison of potential pathway activation strategies

3.3.4

i3

Oncogenic Smoothened mutant transfection strategy

A full length human SMOH expression construct containing the base pair 1604 G-to-T transversion (SMOH^^^^; as discussed Section 1.6.4) was kindly provided by Dr M. Narang, who had introduced the mutation into a wild-type construct, originally the gift of Dr. F. de Sauvage. The mutated insert was cloned into the expression vectors pcDNA3 (Invitrogen, Carlsbad, USA) and pEFBOS (Mizushima and Nagata, 1990) and the resulting constructs, pcDNA3-hSmo-mutl604 and pEFBOS-hSmo-mutl604, were transiently transfected into lOTl/2 cells. RNA was harvested from these cells (and appropriate controls) at a number of timepoints after transfection and AP enzyme assays were also performed (Figure 3.5).

Negative Control

SMOH

1604

GLM

-if

t

«

if lh

Figure 3.5

lOTl/2 cells transiently transfected with an oncogenic Smoothened mutant show no evidence of AP induction. Assay shown was performed 5 days post-transfection on cells treated with pEFBOShSmo-mutl604 (SMOH^^^"^). An empty vector (negative control) and GLIl transfections provide negative and positive controls respectively. A range of timepoints gave similar results. A negative result was also obtained when the alternate construct pcDNA3hSmo-mut 1604 was used (data not shown).

Surprisingly, expression of SMOH^^^"^ did not result in detectable changes in either Patched or Hip expression (data not shown) or in AP enzyme activity. This implied that pathway activation was not occurring through expression of this allele, or that if a response was occurring it was partial and did not cause either the up-regulation of Patched expression nor

Chapter 3: C3H/10T1/2 as a Model System for Hedgehog Target Gene Discovery

84

osteoblastic differentiation. Immuno-fluorescence studies indicated that the construct was indeed expressed at high levels, and the lack of response was not simply a consequence of poor transfection or a defect in vector fimction (Figure 3.6).

DAPI

a-Smoothened • M^^^H

H^^^^^^^^^^^^^^^^^H is^^^l o ^^^^^^^^^^^^^^^^^^^^^^1 c

S'S

^^^^^^^^^^^^^^^1 ^^^^^^^^^^^^^^^1 ^^^^H

t i CD ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^H ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^H cO **^ ^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^H

o § 1_

H

c

^^^^^1 ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^H

^H^^^^^H ^1 •^^^^H^^^^^^^^^^^^l

s .9 ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^H ^^^^^^^^H^^^^^^^^^H ^ ^ ^

MOH nsfec

CO

CO 2

1-

Figure 3.6

^^^^^^^^^H'^^^^^^^^^^^Hil^l

^^^^^^l^^^^^^^n^l

^^^^Hl

Immuno-fluorescence studies show transfection with mutant Smoothened construct was successful. Transfection of the pcDNA3-hSmo-mutl604 construct (Panels C and D) into lOTl/2 cells leads to an increase in the level of Smoothened protein present compared to cells treated with a negative control construct (Panels A and B). Cells shown 1 day posttransfection. Panels A and C show labelling of Smoothened (using a-Smoothened rabbit-"Edith" antibody, a gift of the late Dr. M. Gailani), visualised via a Cy3 conjugated secondary antibody. Panels B and D show signal from DAPI, which stains cell nuclei, for the fields shovra in A and C respectively.

3.3 Comparison of potential pathway activation strategies

85

3.3.5 Gli1 transfection strategy Transfection of GUI into lOTl/2 cells proved to be a potent method of inducing markers of hedgehog pathway activation. Two fiill length GUI clones were obtained. One of these, pGli-K12, was a kind gift of Dr, K. Kinzler and contained the GUI coding region originally cloned from a human glioma cell line harbouring GUI amplification (Kinzler et al, 1988). The insert from this clone was moved to the expression vector pcDNA3, creating construct pcDNA3-hGlil ready for use in transfection studies. A second clone, pRK7-N-Myc-hGli, contained Myc-tagged human GUI and was kindly provided by Dr. F. de Sauvage. Initial transfection studies confirmed the findings of Murone et al, (1999), who had reported induction of AP activity in lOTl/2 cells upon transfection of pRK7-N-Myc-hGli. The increase in AP activity, as indicated by both histochemical and quantitative spectrophotomeric assays, was strong and reproducible. This was in contrast to the results of transfection with pcDNA3hGlil, for which a significant change in quantitative AP activity was not detected. In corresponding histochemical AP assays, positively stained cells were observed that were not seen in control transfections, but they were at a much lower frequency and with considerably fainter signal than those observed with pRK7-N-Myc-hGli transfection (data not shown). For all fijrther investigations the more potent of the two available constructs, pRK7-N-Myc-hGli, was used. Transfection with pRK7-N-Myc-hGli gave a pattem of AP induction distinct to that given by Shh transfection when investigated by histochemical assay (Figure 3.4). In the case of GUI transfection the cells tended to have very intense staining when a response was observed. In contrast, the AP staining pattem after Shh transfection was distinct, with both darkly stained cells and a larger number of more lightly stained cells. The former presumably represent the transfected cells themselves, whilst the additional cells are thought to represent cells that have secondarily responded to Shh protein secretedfi-omthe primary transfectants. GUI transfection into lOTl/2 cells leads to an increase in Pa/c/zeJ transcription, indicating hedgehog pathway activation (Figure 3.7). The increase in Patched expression in response to GUI expression occurs within six hours, much more rapidly than that observed after transfection with Shh (compare Figure 3.7 below, with Figure 3.11).

Chapter 3: C3H/10T1/2 as a Model System for Hedgehog Target Gene Discovery

9.5 kb 7.5 kb -

+•



O

CD

O

-1

^»'.

48 hours

m

Patched





Figure 3.7

GLI1

c

24 hours

Cont

12 hours

GLI1

GLI1

Contr

6 hours

Cont

SI



GAPDH

Up-regulation of Patched expression in lOTl/2 cells in response to Glil. Northem blots contain RNA obtained from cells transfected with pRK7-N-Myc-hGli (GLIl), or from control cells. GAPDHprohQ provides loading control.

Sequence data from the pcDNA3-hGlil and pRK7-N-Myc-hGli constmcts was analysed to see if any obvious cause for the dramatic difference in potency between the plasmids could be found. During these investigations a nucleotide sequence difference between the two cDNA clones was identified. However, fiirther investigations (refer to Appendix C) showed that this probably represents a common human GUI polymorphism. As such, it is unlikely that this change is responsible for any difference in fiinction between the two constmcts. A number of other factors could altematively account for the poor function of pcDNA3-hGlil, such as differing cloning positions with reference to the promoter regions of the vectors, differences in promoter activity, or differing transfection efficiencies between the two plasmids. The pRK7-N-Myc-hGli plasmid, which has the stronger effect on pathway activation markers, was used for all subsequent experiments involving Glil.

3.4

Mutant Shh control construct

When investigating differential expression patterns it is of utmost importance that expression differences between the cell population of interest and the control population are only due to the treatment of interest. Any other factors influencing one population but not the other may lead to false positive results. With transfection studies great care must be taken, as differences can arise at a number of levels. It is important that untreated cells are not used as the reference population for transient transfection studies, since these cells would not have experienced the

3.4 Mutant Shh control construct

87

events and stresses of the transfection procedure. The use of untreated cells would therefore represent a scenario where there would be a high risk of false positive results. Transfection of pShh-N-PMT21 (encoding the N-terminal active region; amino acids 1 to 198 of the mouse Shh protein) was chosen as a key strategy for hedgehog target gene discovery experiments, complemented by studies using Shh conditioned media. For this reason, constmcting a negative control to complement pShh-N-PMT21 transfection was of particular importance. For this work a null-mutant control constmct was designed so the cell would produce a near fiill length Shh-N mRNA and translate this into a tmncated protein. This was used, rather than an empty vector control, to ensure the general transcription and translation mechanisms of the cells were stimulated in both cell populations to be compared. The negative control constmct to complement pShh-N-PMT21 was created by deleting the 64 base pair EcoBI to Smal region from the original constmct, followed by blunting of the EcoBJ site and re-closure of the vector (Figure 3.8). This removed the start ATG codon, and the resulting constmct was named pA64-Shh-N-PMT21. This constmct was expected to be a fiinctional null mutant as the next in frame ATG is not present until approximately half way through the Shh N-terminal active region coding sequence. In addition, the deleted region contained the signal peptide, so even if protein were produced it would not be targeted properly for processing and subsequent secretion. Test experiments confirmed that the new constmct was indeed fiinctionally null. The pA64-Shh-N-PMT21 constmct gave no detectable increase in AP activity when transfected into lOTl/2 cells (Figure 3.9 and Figure 3.10), and subsequent investigations of a range of markers of pathway activation yielded negative results for this constmct by northem blotting (as outlined in experiments forming the remainder of this Chapter).

Chapter 3: C3H/10T1/2 as a Model System for Hedgehog Target Gene Discovery

pShh-N-PMT21 Codons 1-198 of mouse Sonic Hedgehog (encoding entire N-terminal active region)

EcoR1 cDNA insert:

Hind\\\ Xho\

Smal

I

4f

STOP

ATG

V Produced protein:

t

t

Ends at auto-cleavage point

Signal sequence

pA64-Shh-N-PMT21 64 bp deletion EcoRI to Smal

Next in-frame

(Remainder re-IJgated after EcoRI blunting)

start COdon

Hind\\\ Xho\ cDNA insert:

I

ATG

STOP

V Produced protein:

t Ends at auto-cleavage point

Figure 3.8

Regions of mouse Shh encoded by the expression construct pShh-N-PMT21 and the negative control mutant pA64-Shh-N-PMT21. Both constmcts are in the vector PMT21, with expression in mammalian cells driven by the adenovirus major late promoter. The functionally null mutant (bottom) was created from pShh-N-PMT21 (top; originally constructed by the late Dr. T. Yamada) by deleting a 64 bp region from the initial plasmid such that the first 63 base pairs of the Shh-N coding region were removed. Control cells transfected with pA64-Shh-N-PMT21 express a near fiill length mRNA, however the protein these cells could hypothetically produce (based on the position of the next in-fame ATG start codon) is a truncated protein lacking the first 97 amino acids. Further studies showed the mutant control construct lacked any detectable Shh activity.

3.5 Profile of the Hedgehog Response in lOTl/2 cells

.>

u < (A

3 (0

a.

A-t

c (0

Z •

S

CO

•ti .c ^ sz

4.4 kb

-

2.4 kb

-

c CO

•4-»

3

:^

c CO

/' JZ

^3-» ^

.c CO

3 days 4 days +-*

Z x: .c CO

c

(0 +-• 3

^

y sz x: CO

CO CD

c (0

4-»

:3

^

CO

Gli1 Transcripts

GAPDH

Mutant

B

9.5 kb 7.5 kb

-

4.4 kb

-

2.4 kb

-

1.4 kb

-

Shh-N

4 days

1

m m

Gli1 Transcripts

GAPDH

Figure 3.12 Up-regulation of Glil in lOTl/2 cells in response to Shh. Northem blots of RNA obtained from cells transfected with pShh-N-PMT21 (Shh-N) or pA64-Shh-N-PMT21 (mutant) plasmids, or treated with conditioned media collected from cells expressing the same constmcts. GAPDH probe provides loading control. (A) Up-regulation of Glil transcripts in response to Shh stimulation by transfection and conditioned media treatment shown on total RNA. (B). Four day post-transfection poly(A)"^ RNA from a second independent experiment. When run in the absence of ribosomal RNA, the Glil transcripts have apparent sizes of 4.2 kb (major transcript) and 3.5 kb (minor transcript).

H

Chapter 3: C3H/10T1/2 as a Model System for Hedgehog Target Gene Discovery

GUI Up-regulation in response to hedgehog signalling was initially observed two days posttransfection by northem hybridisation (Figure 3.12). However, as basal expression of this gene is below the threshold for detection by northem blot, it is possible that some degree of up-regulation had occurred before this time. A doublet of induced Glil transcripts were observed with sizes estimated at 4.2 kb (major transcript) and 3.5 kb (minor transcript). Previous studies have also reported two Glil transcripts in some mouse tissues, with previous size estimates of 4.0 and 4.7 kb (Walterhouse et al, 1993) and 4.0 and 4.4 (Hui et al, 1994) for the major and minor transcripts respectively. To further investigate the origin of the smaller bands revealed by the Patched probe, poly(A)^ RNA was harvested from untreated cells and used to make a set of high quality northem blots with the aim of giving strong signals even with very small probes. Four new sub-probes were designed that together covered the region in mPtcl263. These probes were made by RT-PCR from lOTl/2 RNA, and used individually on the lOTl/2 poly(A)^ blots. The probe ends were placed at exon boundaries where possible. Figure 3.13 shows the pattem of bands hybridised by the sub-probes. As expected, all four probes hybridise to the full length Patched transcript, but there were some differences in detection of the other bands. In particular the approximately 950 base pair transcript appears to have homology to the regions in sub-probes C and D (the large intracellular loop, and to a lesser degree to the preceding transmembrane regions). Probe B recognises a number of medium sized transcripts while probe A, representing part of the first extracellular loop, only detects the fiill length transcript. Overall, at least six different sized transcripts can be clearly distinguished. Probing of untreated lOTl/2 RNA with a probe to Patched2 exon 12, a region with minimal homology to Patched, gave a major band at approximately 4.7 kb, along with several smaller transcripts (Figure 3.14). Since the Patched probes bind more transcripts that can be accoimted for by crosshybridisation to Patched2, this suggests the existence of highly homologous related genes or complex altemate slicing of Patched transcripts in vivo. BLAST algorithm searches of the NCBI nucleotide database do not reveal other genes with strong homology to the mPtcl263 probe region, suggesting the majority of the bands represent altemately spliced Patched transcripts.

3.5 Profile of the Hedgehog Response in lOTl/2 cells

A

95

B

m 28s RNA 1 (4.7 kb) •

^^

C

D

1

i

i

18s RNA (1.9 kb) •

^4—

^ •

:.. E

-

Pvull 1 Probe A region Probe B region Probe C region Probe D region

Figure 3.13 Probes to different regions of mouse Patched highlight different transcript patterns in untreated lOTl/2 RNA when investigated by northern blot hybridisation. Panels A to D show northern blots containing 2 jiig of untreated lOTl/2 poly(A)^ RNA, each hybridised with a small cDNA probe containing sequence corresponding to regions of the Patched protein, as indicated in the schematic (Panel E). Numbers indicate transmembrane domains (as proposed by Goodrich et al., 1996). Probe mPtcl263 (as used in Figure 3#1) corresponds to the entire 1263 bp region between the two Pvull sites indicated. PCR primers were designed to split this region into four sub-probes for investigation (probe D extends slightly beyond the end Pvull site). All probes bind the full length 8.5 kb transcript, but differ in their specificity for detecting smaller transcripts.

Chapter 3: C3H/10T1/2 as a Model System for Hedgehog Target Gene Discovery

m

3.5.3 Induction of Patched2 in 10T1/2 cells Up-regulation of Ptc2 after Shh stimulation was observed in lOTl/2 cells, but was more difficult to detect by northem blotting than the response observed for Ptc (Figure 3.11). Ptc was elevated to such high levels by Shh that the induced transcripts for a range of timepoints gave strong signals when total RNA was used for blot constmction. This was the case even for exposures of a few hours. In contrast, the signal with Ptc2 on the same blots was very faint, even after a two week exposure, and could not be reproduced as a figure. It appeared from these investigations that Ptc2 was induced with Shh conditioned media four days posttreatment, however signals were too weak to allow meaningfiil quantitative analysis. In order to obtain conclusive data on the status of Ptc2 as a Shh target in lOTl/2 cells (particularly for directly transfected samples which had extremely faint total RNA northem bands) poly(A)+ RNA was prepared from the four day transfection timepoint. Subsequent poly(A)'^ northem blotting showed that these samples had undergone considerable mRNA enrichment, and that Ptc2 transcripts were now readily detected (Figure 3.14).

7.5 kb

-

4.4 kb

-

2.4 kb

-

1.4 kb

-

Shh-N

Mutant

4 days

Patched2 ^*— Transcript 1



A^-'j,

-

i

m

-

-1.4

-1.6

m

4

-

(for panel C)

-2.4

Amh

*

1



N/A

N/A

-1.6

-1.8



-2.3

«l

GAPDH (for panel D)

176

Chapter 6: Further Investigation into Regulation of Identified Hedgehog Target Genes and Related Family Members

6.4 Analysis of upstream and intronic gene sequences obtained from the Celera database DNA binding studies have indicated that human GLIl binds the nine base pair consensus sequence GACCACCCA (Kinzler and Vogelstein, 1990). This motif and a number of closely related sequences have been identified in the upstream regions of several hedgehog target genes in vertebrates (Sasaki et al, 1997; Gustafsson et al, 2002; Yoon et al, 2002). Motifs in mammals that have been shown to be functional, in terms of either binding Gli proteins in vitro or allowing activation of reporter constructs in biological systems, are summarised in Table 6.1 (bases differing from the initially reported Gli consensus motif are shown in white). The consensus binding motif is well conserved between species, and is also found in regulatory regions of Ci target genes in Drosophila (Alexandre et al, 1996). The Gli binding motif acts as an enhancer element, and as such does not need to be in the immediate vicinity of the promoter region of target genes in order to influence transcription. As an example, a single copy of the Gli consensus related sequence GACCACCAA in the enhancer region of Myf5 is able to direct expression of the gene in a hedgehog dependent manner during muscle development, even though it is positioned 6.6 kb upstream of the transcriptional start site (Gustafsson et al, 2002).

Table 6.1

Gli binding consensus motif and related mammalian sequences which have been shown to be functional in Gli protein binding or reporter activation studies.

Motif

Gene or origin

Species

Reference

G A C

c

A C

c c

A

CONSENSUS

human

Kinzler and Vogelstein, 1990

G A A

c

A C

c c

A

HNF-3P

mouse

Sasaki e? a/., 1997

G A C

c

A

c c

Myf5 Plakoglobin

mouse human

Gustafsson et al, 2002 Yoon ef a/., 2002

C A C

c

A

c c c

A

Cyclin D2

human

Yoon effl/., 2002

c c

T

c c c

A

Osteopontin

rat

Yoon etal, 2002

G A

A A

6.4 Analysis of upstream and intronic gene sequences obtained from the Celera database

177

6.4.1 Identification of known and putative Gli protein binding motifs Genomic DNA sequence from regions in and adjacent to each of the eleven newly identified Shh target genes was obtained from the Celera mouse genome database. Sequence information for Patched, Patched2, Hip and COUP-TFII was also obtained. Analysis was performed using the sub-sequence searching procedures in the DNA analysis software package MacVector (Accelrys). Table 6.2 summarises the findings for the Shh induced genes when sequence extending from the first exon to 30 000 base pairs upstream was searched for either the Gli consensus binding site or regions differing from it by a maximum of one base. Intronic gene regions were also investigated. Table 6.3 shows similar Gli motif location data obtained for the Shh repressed genes. Sequences known to be functional (as summarised in Table 6.1) are indicated using the name of the gene they were first investigated in (column two of Table 6.1) as an identifier. Putative and known Gli binding motifs are present in each of the four Shh target genes shown to be regulated by Glil in the previous section, and are also observed to varying degrees for the other Shh responsive genes. The 30 000 base pair cut off point for upstream sequence was set at an arbitrary distance, and it is worth mention that enhancer region binding sites for certain transcription regulating proteins have been reported to function at even greater distances for some genes (reviewed by Blackwood and Kadonaga, 1998).

178

Chapter 6: Further Investigation into Regulation of Identified Hedgehog Target Genes and Related Family Members Table 6.2

Glil Induced by Northem

STATUS

Putative Gli binding motifs in murine genomic sequence surrounding genes induced by Shh in lOTl/2 cells. "-" indicates the region is upstream of the first exon of the gene under investigation.

GENE Patched

Glil Induced by Northem

Hip

Bf2

Glil Induced by Northem

Glil Induced by Northem

N/A

Patchedl

Igft

MOTIFS OF NOTE

REGION Intronic Sites

1X 1x 16 x

Functional "HNF-Sp type" motif Functional "Osteopontin type" motif 1 bp variant from Consensus motif

0 to -2 000 bp

1X 1X

CONSENSUS Functional "Osteopontin type" motif

-2 000 to-10 000 bp

2X 2X

CONSENSUS 1 bp variant from Consensus motif

-10 000 to-30 000 bp

1X 4X

CONSENSUS 1 bp variant from Consensus motif

Intronic Sites

1X 1X 1X

CONSENSUS Functional "Osteopontin type" motif 1 bp variant from Consensus motif

0 to -2 000 bp

1X 1X

CONSENSUS 1 bp variant from Consensus motif

-2 000 to-10 000 bp

1X

CONSENSUS

-10 000 to-30 000 bp

1X 1X

Functional "HNF-3p type" motif Functional "MyfS/Plakoglobin type" motif

Intronic Sites

1X 1X 2X 2X 9X

CONSENSUS Functional "HNF-3p type" motif Functional "MyfS/Plakoglobin type" motif Functional "Cyclin D2 type" motif 1 bp variant from Consensus motif

0 to -2 000 bp

1X

1 bp variant from Consensus motif

-2 000 to-10 000 bp





-10 000 to-30 000 bp

1X

Functional "MyfS/Plakoglobin type" motif

Intronic Sites





0 to -2 000 bp





-2 000 to-10 000 bp

1X 1X

Functional "Cyclin D2 type" motif 1 bp variant from Consensus motif

-10 000 to-30 000 bp

1X

1 bp variant from Consensus motif

Intronic Sites

1X 4X

CONSENSUS 1 bp variant fi:om Consensus motif

0 to -2 000 bp





-2 000 to-10 000 bp





-10 000 to-30 000 bp

1X

1 bp variantfiromConsensus motif

6.4 Analysis of upstream and intronic gene sequences obtained from the Celera database

Table 6.2 continued... REGION

Gilz

Intronic Sites



— (3' exon position unclear)

0 to -2 000 bp

1X

CONSENSUS

-2 000 to-10 000 bp





-10 000 to-30 000 bp

1X 1X 4X

Functional "Osteopontin type" motif Functional "HNF-3P type" motif 1 bp variantfiromConsensus motif

Intronic Sites





0 to -2 000 bp





-2 000 to-10 000 bp

2X

1 bp variant from Consensus motif

-10 000 to-30 000 bp

2X

1 bp variantfi-omConsensus motif

Intronic Sites

3X

1 bp variant from Consensus motif

0 to -2 000 bp





-2 000 to-10 000 bp

1X 2X 1X

Functional "Osteopontin type" motif Functional "MyfS/Plakoglobin type" motif 1 bp variant from Consensus motif

-10 000 to-30 000 bp

2X 4X

CONSENSUS 1 bp variant from Consensus motif

Intronic Sites

1X 1X 1X 1X

Functional "Osteopontin type" motif Functional "MyfS/Plakoglobin type" motif Functional "HNF-3p type" motif 1 bp variant from Consensus motif

0 to -2 000 bp





-2 000 to-10 000 bp





-10 000 to-30 000 bp

2X 2X

Functional "Cyclin D2 type" motif 1 bp variant from Consensus motif

Intronic Sites

1X 1X 3X 8X

CONSENSUS Functional "Osteopontin type" motif Functional "Cyclin D2 type" motif 1 bp variant from Consensus motif

0 to -2 000 bp





-2 000 to-10 000 bp

2X

1 bp variant from Consensus motif

-10 000 to-30 000 bp

2X

1 bp variantfi-omConsensus motif

No evidence of regulation by GUI

Thbd

N/A

Nr4al

N/A

Pmp22

No evidence of regulation by GUI

MOTIFS OF NOTE

GENE

N/A

STATUS

Laspl

179

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Chapter 6: Further Investigation into Regulation of Identified Hedgehog Target Genes and Related Family Members Table 6.3

Glil Repressed by Northem

Glil Repressed by Northem

STATUS

Putative Gli binding motifs in murine genomic sequence surrounding genes repressed by Shh in lOTl/2 cells

REGION

Amh

Intronic Sites

1X

1 bp variantfi-omConsensus motif

0 to -2 000 bp

2X

1 bp variant from Consensus motif

-2 000 to-10 000 bp

1X

1 bp variantfiromConsensus motif

-10 000 to-30 000 bp

1X 2X

CONSENSUS 1 bp variantfiromConsensus motif

Intronic Sites

1X

1 bp variantfiromConsensus motif

0 to -2 000 bp

1X 1X

Functional "MyfS/Plakoglobin type" motif 1 bp variantfi-omConsensus motif

-2 000 to-10 000 bp





-10 000 to-30 000 bp

1X

1 bp variant from Consensus motif

Intronic Sites

1X 3X

Functional "HNF-3p type" motif 1 bp variant from Consensus motif

0 to -2 000 bp

1X

1 bp variant from Consensus motif

-2 000 to-10 000 bp

1X

CONSENSUS

-10 000 to-30 000 bp

1X 1X 4X

Functional "Osteopontin type" motif Functional "Cyclin D2 type" motif 1 bp variant from Consensus motif

Intronic Sites

1X

1 bp variant from Consensus motif

0 to -2 000 bp





-2 000 to-10 000 bp

1X

1 bp variant from Consensus motif

-10 000 to-30 000 bp

2X

1 bp variant from Consensus motif

Sfrp2

N/A

Sfrpl

No evidence of regulation by GUI

MOTIFS OF NOTE

GENE

Mip-ly

6.4.2 Absence of the Sonic hedgehog response element (ShhRE) Available genomic sequence for each of the newly identified Shh target genes was investigated to see if any of them contained the eighteen base pair Shh response element (ShhRE, refer Section 1.2.8). The ShhRE (GTTCTACATAATGCGCCG) is reported to direct transcription of the Gli-independent Shh target gene COUP-TFII (Krishnan et al, 1997b). Though published work on the ShhRE was conducted in chicken, analysis of Celera upstream sequence of the corresponding mouse gene showed the motif is conserved between the two species. The motif was not identified in any of the eleven Shh target genes identified in the previous chapter, nor were any related motifs found when searches allowing for up to three base pair changes from the published sequence were performed.

6.5 Discussion

6.5

181

Discussion

6.5.1 TSC-22 family members display tissue dependent responses to Shh stimulation Gilz is a strongly up-regulated target of the hedgehog pathway in the embryonic mesodermal cell line lOTl/2. The two other characterised members of this family, TSC-22 and Thgl do not appear to be transcriptional targets of Shh in this cell type. The structure and biological functions of Gilz, along with its relevance to the hedgehog pathway, is discussed in the following chapter. Interestingly, one of the Gilz clones present in the UniGene set on the microarray chips used in Chapter 5 was annotated in the supplied grid position information file as "5' similar to SW;TSC2_M0USE Q00992 putative regulatory protein TSC-22". When the clone corresponding to this location was sequenced it was found to correspond to the expected accession number and was indeed similar to TSC-22. However, BLAST searching revealed it was actually far more similar (100 percent) to Gilz. This highlights the fact that it is important in microarray studies to check not only that a clone represents the EST it should, but that current databases are checked for any clones that do not already show a complete match to a known gene. In this way it can be ensured that the correct gene, and not a near relative, is attributed with a particular response. If sequence validation and BLAST searching of the current GenBank database had not been performed on all microarray derived lead gene clones TSC-22, rather that Gilz, may have been attributed with the Shh response in lOTl/2 cells reported earlier in this thesis. The absence of a response of TSC-22 to Shh in lOTl/2 cells, even though TSC-22 is a known target of Glil, highlights the fact that different hedgehog response profiles are present in different tissue systems. Variations in the genes responding to Hedgehog in different cell types, as well as in other spatial and temporal situations, may help explain how a single molecule such as hedgehog can cause such a diverse range of responses in patterning of the mammalian embryo.

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6.5.2 Gli1 regulation in the control of Shh target genes The finding that at least four of the eleven newly identified targets of Shh signalling are also regulated by Glil further supports the conclusions of the previous chapter. It adds weight to the evidence that the microarray approach, and subsequent validation procedures, did indeed yield genuine targets of the hedgehog signalling pathway. In addition the finding of a response to Glil is of interest as it provides evidence that Igf2, Bf2, Amh and Sfrp2 are regulated by what is considered the "classical" hedgehog response, with Shh acting (presumably through Patched and Smoothened) to control members of the Gli gene family, which then orchestrate further responses. An altemative mechanism, by which the hedgehog pathway bifurcates at some point (as discussed in Chapter 1, Section 1.2.8) may explain why some of the other newly identified hedgehog target genes do not show evidence for any response to Glil. They may be regulated instead by other transcription factors. However, this is not the only possibility to explain the absence of a detectable response to Glil. It is possible that the other Shh targets are regulated by Glil at timepoints outside of those investigated in this study, or are only regulated via Gli2 or Gli3 but not Glil. Furthermore, the level of Glil stimulation may have failed to reach some critical threshold for some genes which did not show an apparent response. It is relatively straightforward to show that a factor does cause a transcriptional effect, but very difficult to prove the converse.

6.5.3 Gli1 can act as both an activator and a repressor The finding that GLIl expression in lOTl/2 cells can cause an increase in transcript abundance, as it does in the case of Bf2 and Igf2, or that it can decrease mRNA levels, as is the case with Sfrp2 and Amh, provides evidence that Glil has both activator and repressor actions. This supports similar observations first made by Yoon et al,

(2002). It is not yet

known if Glil itself directly causes the repression. It remains a possibility that Glil may induce transcription of a secondary inhibitory factor that then mediates negative effects on the transcription of Shh repressed genes.

6.5 Discussion

183

6.5.4 Insights from genomic sequence analysis Each of the genes shown conclusively in this chapter to be regulated by Glil {Patched, Hip, Bf2, Igf2, Sfrp2 and Amh) display a higher number of Glil consensus related sites in surrounding genomic regions than would be expected by chance. The presence of putative and known Gli interacting sequences in the upstream regions of Igf2, Bf2, Amh and Sfrp2 suggests that positive and negative regulation of these newly identified Shh target genes via Glil may occur by a direct mechanism. It is also possible that Gli2 or Gli3 may interact with the identified sequences. It is interesting that a number of the putative and known Gli binding sites occur in the intronic regions of the investigated genes, a phenomenon also observed for other enhancers (Blackwood and Kadonaga, 1998). The finding of a number of known and putative Gli binding motifs in the newly identified Shh target genes other than the four shown conclusively to be regulated by Glil suggests the actual number of Gli regulated genes may be higher. If so a number of factors may have prevented their identification in transient transfection studies with Glil. The level of Glil stimulation may not have reached high enough levels to initiate a response, the expression of Gli family members other than Glil be necessary, or the timepoints investigated may not have been appropriate to detect the response. In the case of Sfrpl there were problems in obtaining northem blot data for inclusion in this work, however the presence of a number of Gli binding sites (induing a consensus motif and sequences known to be functional in the HNF-3fi, Osteopontin and Cyclin D2 genes) suggests that Sfrpl, like Sfrp2, may be Glil regulated. The number of consensus and known functional Gli binding motifs identified in the introns and upstream regions of Patched2 were striking, suggesting that Patched2, like Patchedl, will prove to be a Glil regulated gene. Of the four newly identified Shh target genes that were demonstrated to be regulated through Glil, only one. Brain factor 2, had no Gli consensus related sequences of note, either within the gene, or within 2000 base pairs upstream of the first exon. Of particular note however was the existence of a tight cluster of five perfect Gli consensus sites (plus several closely related sequences) approximately 360 kilobase pairs upstream of the Bf2 gene. This putative enhancer region is located within the intron sequence of another gene, Rho-guanine nucleotide exchange factor {Rgnef). Whether or not this cluster acts as an extremely long distance enhancer for Bf2, or whether it actually regulates Rgnef will be an interesting question to address in future investigations.

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It is important to stress that the finding of Gli consensus related motifs that putatively control the regulation of genes in a hedgehog directed manner is currently speculative and awaits detailed functional studies such as gel shift analysis or reporter activation studies. Though a number of the identified motifs match those previously found to be functional in other studies, it is important to note that function may be considerably effected by surrounding DNA sequences. Enhancer action in general is not yet well understood, particularly with regard to the actual mechanisms transcriptional control and how such elements can act at considerable distancesfiromtarget gene promoters. The most important findings of this chapter are that Bf2, Igf2, Sfrp2 and Amh have been shown to be able to be regulated under the control of the Glil transcription factor, firmly establishing their role as downstream targets of the hedgehog pathway and providing clues to the mechanism of their transcriptional regulation. The fact that Glil expression in lOTl/2 cells was able to elicit either activation or repression, depending on the particular Sonic hedgehog target gene under investigation, is a significant finding.

Chapter 7: Discussion - Discovered Hedgehog Target Genes and Overview The major aim of this thesis, the identification of novel downstream targets of Sonic hedgehog, has been met with the identification and validation of eight genes that have not previously been described in the literature as being involved in the hedgehog pathway. In addition, microarray generation of lead genes and their subsequent verification led to the corroboration of three other genes already implicated in hedgehog pathway biology firom other studies. The eleven genes confirmed as genuine targets of Shh in this work encode a wide range of proteins with diverse putative functions. All are "known genes" in that they have previously had their full coding sequence recorded in the NCBI GenBank database. Whilst some of them have been intensively studied with regard to their function, others have only recently been named and published with far less known about their biological roles. In this chapter each of the hedgehog responsive genes is discussed with particular emphasis on the eight genes not previously described as having links to the hedgehog pathway. The biological implications of the findings are discussed, along with known links to aspects of human disease. The remainder of this chapter provides an overview of the conclusions drawn in this thesis and their implications, a critical review of the strategies employed and a discussion of possible future studies leading on from the findings of this work.

7.1 Possible roles of identified pathway targets and links to human disease 7.1.1 Brain factor 2 (Bf2) Bf2 has not previously been linked to the hedgehog pathway. Bf2 is a member of the wingedhelix (W-H)/forkhead box (FOX) family of transcription factors and is expressed in small number of embryonic tissues including developing CNS and kidney (Hatini et al, 1994), Winged-helix proteins, named fi^om the conserved protein stmcture of their DNA binding domains, have been shown to have important roles in the control of cell growth, proliferation and differentiation. Some family members are capable of inducing a transformed phenotj^e in

Iffi

Chapter 7: Discussion - Discovered Hedgehog Target Genes and Overview

cells and have a role in neoplasia (reviewed by Vogt et al, 1997). Homozygous Bf2 knockout mice die within a day of birth, having severely malformed kidneys and anomalies of the forebrain, retina and adrenal gland (Hatini et al, 1996). Several other members of the W-H/FOX family have been implicated downstream of Shh, the best studied being HNF-3p (refer Section 1.5), which is a well known target of Shh signalling in neural tissues and a gene also shown to be a transcriptional target in lOTl/2 cells fi-om the work presented in Chapter 3. HNF-3J3 transcription is controlled via Gli binding sites in its promoter (Sasaki et al, 1997). Recently another member of the family, FoxMl, has been shown to be up-regulated in basal cell carcinoma and regulated through a Gli dependent mechanism (Teh et al, 2002). The data presented in Chapter 6 shows Bf2 is also GHl regulated, suggesting a common mechanism of regulation for winged-helix genes under the transcriptional control of Shh. When lOTl/2 cells respond to Shh, many cells differentiate down the osteoblastic lineage. Interestingly, several other winged-helix members have been implicated in regulating osteogenesis, and one of these, Mfh-l, is thought to directly stimulate a forkhead response element in the AP promoter (Yang, X. L. et al, 2000; Hatta et al, 2002). It will be of particular interest in future studies to obtain a full length clone of Bf2 to investigate whether its expression in lOTl/2 cells is sufficient for their induction to the osteoblastic lineage, or whether Bf2 represents a more general component of the hedgehog response. In Xenopus, Bf2 expression can convert cells from an epidermal to neural cell fate, and it may play a similar role in patteming the mammalian CNS (Mariani and Harland, 1998). Since Bf2 is a transcription factor it presumably in tum controls the transcription of its own target genes, a mechanism that may be either mled or fine tuned by the actions of Shh.

7.1.2 Glucocorticoid induced leucine zipper (Gilz) Gilz was found to be induced in lOTl/2 cells in response to Shh stimulation. It has already been discussed in Chapter 6 with relevance to homology to related genes, some of which are also implicated as downstream targets of hedgehog signalling in other systems. As discussed previously members of the TSC-22 family, which includes Gilz, contain a leucine zipper region, a motif commonly found in transcription factors. However, unlike most leucine zipper proteins, members of the TSC-22 family lack an obvious DNA binding domain

7.1 Possible roles of identified pathway targets and links to human disease

187

and it is thought that they regulate transcription indirectly through the formation of heterodimers with various other transcription control proteins, Gilz was independently discovered by groups using two different biological approaches, and its nomenclature can be confusing. Initially its existence was determined by researchers who isolated it as an unexpected protein bound by an antibody to the nine amino acid Delta sleep inducing peptide (DSIP) in porcine brain tissue (Sillard et al, 1993). Though Gilz and DSIP cross react immuno-chemically they were found to have no similarity in protein sequence and at that time the function of Gilz, then called DSIP-immunoreactive peptide (referred to as DSIPI or DIP), remained elusive (Sillard et al, 1993). Later the gene corresponding to this new protein was cloned and recognised as a member of the TSC-22 family (Vogel et al, 1996). Meanwhile, the gene was isolated independently by researchers investigating modulation of apoptosis in T-lymphocytes under the control of glucocorticoids, a group of steroid hormones produced by the adrenal cortex. From this work it gained its currently accepted name of Glucocorticoid induced leucine zipper (D'Adamio et al, 1997). Recent work on Gilz has highlighted further aspects of its involvement in apoptotic control in T-cells, including links to the NF-KP and Fas/FasL systems (Ayroldi et al, 2001; Riccardi et al, 2001 and earlier studies reviewed by Riccardi et al, 1999). In contrast to the above studies little work has been published on the role of Gilz outside of the immune system, though it is known to be expressed in a wide range of human tissues (Vogel et al, 1996). Northem blot studies on a small number of mouse tissues conducted in this work have shown that Gilz is expressed in skin keratinocytes and brain, and at a lower level in kidney and spleen (data not shown). As yet transgenic and knockout animal studies on Gilz have not been published, making this a particularly interesting area to initiate future work. Given that mutation of the single Drosophila TSC-22 family homologue, bunchedlshortsighted (refer references in previous Chapter), causes defects in embryonic development, it will be interesting to see if mutation of Gilz in a mammalian system results in a similar phenotype. In this way it will be possible to deduce more about the fiinction of Gilz in a developmental setting. Given the importance of Gilz in apoptotic control, it may also be informative to monitor and compare apoptotic markers in lOTl/2 cells after Shh or Gilz expression, to see if up-regulation of Gilz in response to Shh has an effect on cell survival in embryonic mesodermal cells.

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Chapter 7: Discussion - Discovered Hedgehog Target Genes and Overview

7.1.3 Thrombomodulin (Thbd) One of the most intriguing responses identified in this work is the induction of Thbd transcription upon Shh stimulation. Thbd encodes an endothelial cell membrane receptor that forms a 1:1 complex with thrombin, changing its conformation and converting it from a procoagulant to an anticoagulant molecule. Thrombin then catalyses activation of Thrombinactivatable fibrinolysis inhibitor (TAFI) and Protein C, which in tum cleaves activated cofactors mediating coagulation. In this respect Thbd acts as an anticoagulant, slowing blood clotting (reviewed by Wu and Matijevic-Aleksic, 2000). It is initially unclear what role such a gene might play in embryonic pluripotent mesodermal cells, however clues come fi-om Thbd knockout mice which display embryonic lethality before the cardiovascular system develops, suggesting a fiinction in growth and development independent of its anticoagulant activities (Healy et al, 1995). Follow up studies using a reporter gene linked to the Thbd promoter revealed expression in the murine trophoblast, as well as in mesodermal precursors of the endothelial cell lineage, suggesting Thbd may play a developmental role during vascular differentiation (Weiler-Guettler et al, 1996). Other studies have revealed that in addition to the well known expression of Thbd in endothelial cells, Thbd is also transcribed in osteoblasts (Maillard et al, 1993) as well as several other cell types (reviewed by Zhang, Y. et al, 1998). In keratinocytes and some other cells Thbd is proposed to play a role in cellular differentiation (Mizutani et al, 1994; Raife et al, 1994). Recently studies have proposed mechanisms by which both Thbd and Thrombin may be involved in signalling to control cell proliferation (reviewed by Freedman, 2001). Although a direct link to the Hh pathway has not previously been described there is evidence that TGFp family members (which have some homology to the key Drosophila Hedgehog target dpp) can down-regulate Thbd expression in neural tissue (Tran et al, 1999). In terms of neoplasia Thrombomodulin is a hedgehog target gene of interest, given that it shows expression changes in a variety of human cancers where it has been hypothesised to play a role in tumour invasion and metastasis (Wilhelm et al, 1998; Zhang, Y. et al, 1998). It also appears to regulate the growth of tumour cells using mechanisms other than its anticoagulant fiinctions (Zhang, Y. et al, 1998). This gene will be of interest for fiiture studies, in particular if embryos from homozygous Shh knockout mice can be obtained for in situ hybridisation analysis. Possible changes in Thbd

7.1 Possible roles of identified pathway targets and links to human disease

189

expression could be informative to understanding the biological link between Hedgehog and Thrombomodulin during mammalian development, whilst its investigation in tumours with and without associated hedgehog pathway dismption would be informative.

7.1.4 Nuclear receptor subfamily 4, group A, member 1 (Nr4a1) Another newly identified Shh target, Nr4al, is a growth factor inducible orphan nuclear receptor, that functions as a transcriptional regulator mediating mitogenic effects, and also regulates apoptosis by a secondary independent mechanism (Li, H. et al, 2000). A name for this gene has yet to become standardised and it is referred to in the literature by a multitude of different aliases including NGFl-B, Nur77, NAK-1, NIO and TR3. Nr4al is an inducible orphan nuclear receptor, a member of the steroid receptor family with an unknown ligand. It consists of an N-terminal AFl trans-activation domain, a double zinc finger DNA binding motif and a C-terminal ligand binding region. It is known to bind an eight base pair consensus DNA sequence directly, and it can also regulate transcription by modulating the activity of other orphan steroid receptors, (reviewed by Winoto and Liftman, 2002). Nr4al is induced by a variety of factors, suggestive of a role in a range of biological fiinctions. Several target genes of Nr4al have been identified that play a role in steroid metabolism (reviewed by Sladek and Giguere, 2000), further hinting at the "steroid theme" that comes up often in aspects of hedgehog pathway biology. It is thought that orphan steroid receptors probably have some degree of redundancy in function, since Nr4al deficient mice have no observable phenotype (Lee, S. L. et al, 1995). Nr4al has not previously been described as a hedgehog target gene, though it is generally upregulated by a range of cellular growth factors. Another orphan steroid hormone receptor, COUP-TFII (refer Section 1.2,8), is a known target of hedgehog regulated independently of the Gli transcription factors, and it will be interesting to see if Nr4al is also regulated by a COUP-TFII-like mechanism. Also of note, Nr4al shares several functional properties with Gilz, another new Shh target described above. Both are involved in the response to steroid hormones, both are regulators of transcription, and both are important regulators of cellular apoptosis. It is possible that both of these genes are induced to address a similar biological goal.

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Chapter 7: Discussion - Discovered Hedgehog Target Genes and Overview

7.1.5 Insulin-like growth factor 2 (Igf2) Igf2 was identified as an up-regulated target of Shh. By chance this gene was represented on the microarrays by four independent clones, and when the data from the experiments was followed up by northem blots the induction in lOTl/2 cells in response to hedgehog stimulation was found to be dramatic. Igf2 provides an example of a gene already implicated in the hedgehog response that has been corroborated by this work. Previous studies suggested Shh mediates Igf2 expression due to the observation of increased Igf2 mRNA levels in both normal tissues and tumours arising on hetrozygous patched mutant mice (Hahn et al, 1998), with further studies suggesting that Igf2 is indispensable in the formation of both meduUoblastoma and rhabdomyosarcoma in mice (Hahn et al, 2000). The work presented in Chapters strongly supports these findings, and additionally shows that Igf2 is up-regulated by Glil, firmly establishing its role as a transcriptional target of the hedgehog pathway. Igf2 is a member of the insulin-like growth factor (IGF) family, whose other members include Igfl and Insulin itself Like Insulin, Igfl and IgfZ have effects on metabolism, but they also play a role in the control of cellular proliferation. A number of IGF-binding proteins have been identified and these modulate the IGF response, which is mediated by binding to the IGF receptor/g/^7i?. Binding activates the receptor's tyrosine kinase activity, triggering changes in downstream molecules (reviewed by Le Roith and Butler, 1999). A long history of studies exist showing increased expression of Igf2 in a diverse range of human tumour types (reviewed by Toretsky and Helman, 1996). Increased Igf2 in cancer often arises from mechanisms involved with loss of imprinting or from transcriptional activation, the latter of which is likely to be associated with hedgehog pathway activation. It is thought that increased expression of Igfl in some cancers sets up an autocrine feedback loop such that cell proliferation is continuously stimulated, and Igf2 may act similarly (Chen, J. C. et al, 1994). Igf2 can also act synergistically with other growth factors and steroids to antagonise the effects of anti-proliferative molecules in cancer cells (Yu and Rohan, 2000). The importance of this gene in human neoplasia makes it a prime suspect as a gene directly involved in tumour initiation when the hedgehog pathway is activated.

7.1 Possible roles of identified pathway targets and links to human disease

191

7.1.6 Peripheral myelin protein 22 (Pmp22) Studies presented in this thesis have shown Pmp22, also known as Growth arrest-specific 3 {Gas3), is up-regulated in lOTl/2 cells in response to hedgehog. Pmp22 was first cloned during a screen to identify genes induced during the semm starved growth arrest of NIH 3T3 fibroblasts (Manfioletti et al, 1990), and is a member of a larger protein family, members of which are hydrophobic molecules featuring four conserved transmembrane domains. It is widely expressed during embryonic development as well as in adult tissues, with the highest expression in the Schwann cells of the peripheral nervous system (reviewed by Jetten and Suter, 2000), Pmp22 protein forms a component of the myelin sheath of peripheral nerves where it regulates various aspects of Schwann cell biology. Several lines of evidence suggest that Pmp22 is a dual function protein that also plays a more general role in growth control and apoptosis regulation in non-neuronal tissues (Jetten and Suter, 2000), A number of natural mouse mutants are known for the Pmp22 locus, and its dismpted function is involved in several human diseases. The Trembler {Tr) mouse was identified more than half a century ago, followed later by another mouse, Trembler-J {Tr-J) with an independent mutation (Falconer, 1951; Henry et al, 1983; Henry and Sidman, 1983), Both mutants represent missense mutations in Pmp22 that dismpt normal protein trafficking, and phenotypically they exhibit shaking and muscle control abnormalities. In humans mutation of PMP22 is associated with several neuropathies including Charcot-Marie-Tooth neuropathy type 1 (a chronic demyelination disorder), Dejerine-Sottas disease (featuring severe infantileonset demyelination) and Hereditary neuropathy with liability to pressure palsies, an autosomal dominant disorder that results in episodic demyelinating neuropathy (reviewed by ^e\is etal, 1999). Pmp22 has not previously been implicated in Hh signalling and it will be interesting to see if its expression can be induced by hedgehog molecules other than Shh. In particular future studies will aim to see if Pmp2 2 might also be a downstream target of Desert hedgehog. Dhh has a greatly restricted expression pattem compared to the other mammalian hedgehog genes, but one specific area it is involved in is patteming of the peripheral nerves where it is involved in formation of the peripheral nerve sheath (Parmantier et al, 1999). The possibility exists that Pmp22 might be regulated by Dhh during this developmental process.

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It is of note that another gene. Growth arrest specific 1 {Gasl), was identified whilst this work was in progress as a protein that can bind to Shh and act as an antagonist of the hedgehog pathway (Lee, C. S. et al, 2001). Although the Gas genes are part of a similar fiinctional grouping it is important to note that Gasl and Gas3 are quite distinct in sequence and protein stmcture and are not directly related.

7.1.7 Lim and SH3 Protein 1 (Laspl) Laspl, also called Metastatic lymph node 50 {MLN50), is a unique member of the LIM protein family featuring an N-terminal LIM domain and a C-terminal Src homology region 3 (SH3) motif (Tomasetto et al, 1995a). The LIM domain (named from the Lin-11, Isl-1 and Mec-3 genes) is a cysteine-rich double zinc finger consensus sequence implicated in mediating protein-protein interactions (reviewed by Dawid et al, 1998). The SH3 domain is thought to serve a similar function and is found in cytoskeleton-associated molecules and signal transduction t5n-osine kinases (reviewed by (Mayer, 2001). Given that both ends of the Laspl protein feature regions using in protein-protein interactions it is possible that Laspl may function as an adaptor molecule with a role in cellular signal transduction pathways or in regulating the architecture of the cytoskeleton (Schreiber et al, 1998). Recent work suggests that phosphorylation of Laspl could potentially regulate actin-associated ion transport activities (Chew et al, 2000). In this study Laspl was found to be an up-regulated target of Shh in lOTl/2 cells. Although the response occurred from a relatively high basal expression level it was subtle but significant (refer Chapter 5 and Appendix). This gene showed a lower magnitude fold change to that observed with the other newly identified target genes discussed in this chapter, but nonetheless it may play an important biological role. Laspl was originally isolated from a differential screen of clones from a human metastatic lymph node cDNA library to identify genes involved in breast cancer progression (Tomasetto et al, 1995b). It was found to be located on the long arm of human chromosome 17, between qll and q21.3, a region known to contain the oncogene c-erbB-2 oncogene and the BRCAl breast cancer susceptibility gene, and overexpression of all three is dependent on gene amplification in breast cancer cell lines. Further work showed that Laspl is frequently over expressed in breast carcinomas, where it is hypothesised to cause cytoskeletal changes during neoplastic transformation (Bieche et al, 1996).

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193

Given that Laspl is a relatively late target of Shh in lOTl/2 cells there is a possibility that changes in its expression relate to architectural changes in cell stmcture during Shh induced differentiation, however its apparent importance in human cancer formation warrants further investigation.

7.1.8 Secreted frizzled related proteins 1 and 2 (Sfrpl and Sfrp2) Of all the Shh target genes identified in this work the most dramatic responses (in terms of fold change) were for Secreted frizzled related protein-1 {Sfrpl; also known as Sarp-2) and Secretedfrizzled related protein-2 {Sfrp2; also known as Sarp-1 or SdfS), on which Shh had a strong inhibitory effect on transcription. In addition one of these genes, Sfrp2, has been conclusively shown as a downstream target mediated through the actions of the transcription factor Glil, providing further information of the mediation of the signal between Shh and Sfrp2. The author also had access to a Sfrp3 clone; however this gene did not appear to be regulated by hedgehog in lOTl/2 cells when tested by northem blotting even though microarray data was promising (refer Appendix and data not shown). As discussed in Chapter 5, Sfrpl and Sfrp2 have previously been implicated as Shh targets from a study using presomitic mesoderm (Lee, C. S. et al, 2000). In this tissue both genes are induced by Shh, the opposite response to that seen in the lOTl/2 system. The transcriptional response of the two Shh target genes is intriguing since they already show extreme changes in expression in untreated cells across the timepoints investigated in this study. The action of Shh is superimposed over the natural expression pattem, which is a dramatic increase in Sfrpl and Sfrp2 expression as the cells reach high density and age in the cellular monolayer. The expression of SFRPs in quiescent but not in exponentially growing lOTl/2 cells that was encountered in this work had previously been observed by other researchers (Melkonyan et al, 1997). When exposed to Shh this natural increase in Sfrpl and Sfrp2 expression was greatly diminished. As with most of the observed responses this was most apparent with conditioned media stimulation. It is important to note that SFRP gene responses would have been missed if the microarray studies had simply used untreated "time zero" cells (which were not fully confluent) as the reference RNA on each chip. In this case the Shh response for these genes would mimic the expression pattem seen at lower cell density, masking the sfrong inhibitory effect. Put more

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simply it is not possible to detect a decrease in the expression of a gene, no matter how strong the inhibitor, if the sample compared to does not express the gene in the first place. It was a strength of the experimental design, which involved meticulous pair-matching of confrols over each timepoint in the timecourse, that allowed this response to be readily detected. The SFRP family, also known as SARPs (secreted apoptosis-related proteins), feature a cysteine rich domain homologous to that found in the transmembrane Wnt receptors known as Frizzled proteins, however unlike the hydrophobic Frizzled receptors, SFRPs are secreted molecules. The family was first discovered with the identification of four members {Sfrpl to 4) on the basis of this homology (Rattner et al, 1997). SFRPs are expressed in mesenchymal condensates and in a range of subsequent epithelial stmctures during murine embryogenesis (Leimeister et al, 1998). Members of the family bind Wnt proteins outside of cells and are thought to antagonise, or in some cases enhance, Wnt fiinction. Vertebrate SFRPs, like the frizzled receptors themselves, exhibit fiinctional specificity with respect to various Wnt proteins, (reviewed by Polakis, 2000; Jones, S. E. and Jomary, 2002). The Wnt pathway, and the genes controlled by it, are strongly implicated in cancer formation, particularly in colorectal cancer, where mutations in the tumour suppressor APC activate a Wnt response through the stabilisation of P-catenin (reviewed by Taipale and Beachy, 2001). A direct link between the hedgehog and Wnt pathways has been known for some time in Drosophila, whereby the orthologous Wnt gene wingless is a key target of Hedgehog signalling. More recently Hedgehog mediated expression of Wnt genes has been established in higher organisms (refer Chapter 1). It is not unexpected therefore, that SFRPs would be involved in cancer given there close functional relationship to Wnt signalling pathway, and indeed it appears that this is the case. In lOTl/2 cells, Sfrpl and Sfrp2 are both involved in modulating cellular sensitivity to apoptotic stimuli, dismption of which may also influence tumour formation (Melkonyan et al, 1997). SFRPs are hypothesised to act as tumour suppressors, and abnormally low expression of various SFRPs has been reported in tumour samples from kidney, ovary and breast (Jones, S. E. and Jomary, 2002). Although there are not yet any studies directly associating SFRP mutation with human disease, a recent study of non-medullary invasive breast carcinomas found loss of SFRP-1 expression in more than 80% of cases (Ugolini et al, 2001). The hedgehog pathway may provide a mechanism to fine tune Wnt signalling in vertebrates through the regulation of Sfrpl and Sfrp2, and predispose cells to neoplasia when this control is dismpted.

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7.1.9 Macrophage inflammatory protein-1 gamma (Mip-ly) Mip-ly was found to be down-regulated by Shh in lOTl/2 pluripotent mesodermal cells. As with all Shh target genes reported in this work, the response was observed both by Shh transfection and by stimulation with Shh conditioned media. Since its identification in the mid 1990s by several groups (Hara, T. et al, 1995; Poltorak et al, 1995; Youn et al, 1995) Mip-ly has become known by a myriad of names in human and mouse including CCF18, Small inducible cytokine A9 {Scya9), Small inducible cytokine AlO {ScyalO), Chemokine C-C motif ligand 9 {Ccl9), and Macrophage inflammatory proteinrelated protein-2 {MRP-2). The role of Mip-ly is not yet fiilly understood. Mip-ly is a member of the superfamily of chemokines, a group of secreted molecules that act as chemo-attractants for various cells involved in haematopoiesis and inflammation by binding to target cell surface receptors of the G protein-coupled family. Specifically Mip-ly belongs to the C-C (cys-cys) group of chemokines, named due to the stmcture of their consensus protein sequence. Mip-ly is closely related to the chemokines Mip-la, Mip-lfi, and CIO and it displays inflammatory, pyrogenic and chemokinetic properties. Unlike other C-C cytokines Mip-ly is constitutively expressed by a wide range of tissues in adult mice and circulates in normal blood (Poltorak et al, 1995). The inhibition of Mip-ly mediated by Shh is intriguing. Some cytokines are produced by osteoblasts, which is what many lOTl/2 cells become after Shh exposure, however in this case the opposite is observed. Whether of not the down-regulation of Mip-ly is correlated with mesodermal differentiation or a more general response of the hedgehog pathway awaits further investigation.

7.1.10 Anti-Mullerian hormone (Amh) In this work Amh, also known as Mullerian inhibiting substance {MS), was found to be a down-regulated target gene of Shh in lOTl/2 cells that is regulated imder the control of Glil. Amh encodes a glycoprotein with a critical role in mammalian sex determination. In male embryos Amh is produced by the Sertoli cells of the foetal testes, causing regression of the Mullerian duct that would otherwise form into female stmctures (reviewed by Josso et al, 2001). Male mice mutant for Amh develop into intemal pseudohermaphrodites, having both

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male and female reproductive organs (Mishina et al, 1996), while mutations in human AMH have been linked to persistent Mullerian duct syndrome in a number of patients (Knebelmann et fl/., 1991; Imbeaud et al, 1994). Amh is expressed in the aduh gonads of normal males and females, though at much lower level than that observed in foetal testes (Teixeira et al, 1999). In contrast to its role in the development of sex organs the role of Amh in adults is less clear. There is some evidence that Amh may be involved in oocyte maturation, blocking granulosa cell proliferation and reducing steroidogenesis in the ovary (Kim et al, 1992; Seifer et al, 1993). The phenotypes of transgenic and knockout mouse models for Amh, and an Amh receptor, provide support for Amh playing a role after birth in maintaining steroid hormone balance in both male and female gonads (Behringer et al, 1990; Behringer et al, 1994; Mishina et al, 1996). Recent work has shown a direct effect of Amh at the transcriptional level in regulating an enzyme involved in androgen biosynthesis (Teixeira et al, 1999). A possible link to hedgehog biology is suggested by the fact that Amh is a member of the TGFp superfamily distantly related to Drosophila dpp; however, to the author's knowledge this work provides the first evidence for Amh as a downstream transcriptional target of the hedgehog pathway. Shh acts to inhibit the expression of Amh observed in quiescent lOTl/2 cells, however the roles that Amh might play in embryonic mesodermal cells are unclear. At this stage it is not known what phenotypic changes the reduction in Amh transcript levels under the control of Shh and Glil might have in a developmental context.

7.1.11 Summary of Hedgehog target genes: common themes The eleven Shh target genes identified in this work as downstream targets in pluripotent embryonic mesoderm represent molecules with a diverse range of protein stmctures. Many of the genes represent important developmentally regulated molecules and there are a number of common themes apparent in terms of their putative functions. Three genes, Bf2, Gilz and Nr4al are transcription factors or are involved in regulating transcriptional responses. These genes, under the influence of Shh, may confrol fiirther cascades of secondary target molecules through which Shh exerts its action. The majority of the genes have putative roles in the regulation of cell growth and survival. Thrombomodulin, Nr4al, Igf2 and Pmp22 are thought to have important roles in the regulation of cellular proliferation, while Gilz, Nr4al, Amh and the two Sfrp genes have known functions in the regulation of apoptotic cell death. Aberrant expression of any of these downstream targets when the Hh pathway is perturbed could be a

7.2 Importance of a well characterised response profile to the success of comparative gene expression studies

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factor in the uncontrolled cell growth leading to tumour formation. In addition, a number of the downstream targets of Shh identified in this work (including Thrombomodulin, Igf2, Laspl, and the Sfrps), show altered expression in human cancer tissue, or are known to be involved in tumourigenesis.

7.2 Importance of a well characterised response profile to the success of comparative gene expression studies With the rapid advances in microarray technology, and the reaching of a stage where laboratories without specialised robotic equipment can now access microarray slides by commercial means, many groups are now embarking on microarray based projects. One factor that must not be overlooked is that the final data obtained will only be as relevant to the specific biological question being asked as the cell populations themselves are at the time of harvest. This applies to the use of any technique used for the analysis of differential gene expression. The two most important factors in addressing this in terms of investigating the response to a factor are: 1. Selection of suitable model tissue or cell system. 2. Selection of the optimal reference (negative control) cell population. 3, Selection of suitable treatment sfrategy that avoids introducing irrelevant transcriptional effects, 4, Selection of relevant timepoints for investigation.

Unless all of the above decisions are made carefiilly there is a risk that any analysis of gene expression will not actually provide answers to the question of interest, no matter how meticulously the data is collected. On one hand a particular response may be missed completely and on the other hand poor choices involving points two and three above can lead to a high level of false positives. In order to make these decisions correctly the researcher needs to be armed with an adequate body of knowledge about the biological system of interest so that decisions on experimental design can be made in an informed way. For this reason much of the research conducted in

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this thesis aimed to establish a framework of knowledge so that comparative gene expression studies would yield as much useful information as possible on the transcriptional response to hedgehog stimulation. The above points have been discussed in detail in Chapters 2 and 3, In particular the knowledge gained in Chapter 3 regarding the kinetics of the response based on a range of known hedgehog target genes has been critical to the success of this project. Without this knowledge it is likely that timepoints within a matter of hours from treatment would have been selected, as is usually the case in fransfection studies. If it had not been shown that lOTl/2 cells have a cell density dependent component to their competence to respond to hedgehog that dramatically delays the response of any known pathway marker tested, then it is possible that new target genes may have been missed completely. In hindsight the work involving the subtracted library approach for detection of differentially expressed transcripts was conducted at too early a timepoint, as the libraries were constmcted before the discovery of density dependence was made. The choice of timepoint for this approach was made on the basis of Bmp2 expression data at an early stage that was later found to be artefactual (refer Chapter 4). By the time microarray work was initiated a much larger body of knowledge had been accumulated, as summarised in Chapter 3, and this ultimately made the discoveries of Chapter 5 possible.

7.3 Power of the 10T1/2 system as a tool for hedgehog target gene discovery 7.3.1 Plasticity of the 10T1/2 line and diverse nature of hedgehog target genes The corroboration of genes previously implicated in hedgehog signalling, along with the finding of novel targets, demonstrates both the validity and power of using the lOTl/2 system for Shh target gene discovery. The wide range of target genes found to be regulated by Shh in this cell type is probably a reflection of its pluripotent nature. A number of the targets, discovered either by microarray or via the candidate gene approach in Chapter 3, are known to be important in developing neural tissues (for example HNF-3fi). It will be interesting in fiiture studies to see if lOTl/2 cells have a role as progenitors of a tissue involved in neural development, or altematively if such genes play non-neural roles in embryonic mesoderm.

7.3 Power of the lOTl/2 system as a tool for hedgehog target gene discovery

199

The use of lOTl/2 cells has allowed the identification of Shh target genes potentially involved in decisions of cell fate during embryogenesis. Few mammalian cell lines are known to be Hedgehog responsive, and those that are known tend to be differentiated cell types. These would be expected to have a more limited response profile in terms of the types of target genes able to be regulated. lOTl/2 cells, on the other hand, are known to have the ability to form a number of cell types including chondrocytes, osteoblasts, myoblasts and adipocytes. In particular, the large percentage of Shh stimulated lOTl/2 cells entering the osteoblastic lineage raises the possibility that some of the genes identified in this work may be involved in regulating embryonic bone development. It will be of interest in fiiture work to express flilllength cDNA constmcts of each of the newly identified Shh target genes in lOTl/2 cells to see if any are sufficient themselves to induce an osteoblastic phenotype. Overall lOTl/2 cells have provided an ideal model system for investigation of the hedgehog response and fiiture studies with microarrays containing a large percentage of the mouse genome should allow even more detailed analysis of role of the transcriptional response to hedgehog signalling.

7.3.2 Implications of the density dependence of 10T1/2 cells to the hedgehog response The discovery that lOTl/2 cells must reach a critical cell density before showing any detectable hedgehog response has interesting implications in terms of hedgehog pathway biology. It suggests that some critical event occurs in this cell type that acts as a switch to allow them to mount a fiill response to the Shh stimulus. It will be particularly interesting in future studies to attempt to understand this molecular mechanism. A number of mechanisms can be envisaged that could account for this phenomena. It may be that certain pathway components or essential cofactors are not expressed until a certain level of cell contact is reached. On the other hand the change might involve a post-transcriptional mechanism, such as the movement of essential factors spatially within the cell. Another hypothesis is that this competence to Shh may involve the direct interaction of a contact induced cofactor with the Smoothened protein. As discussed in Chapter 1, Smoothened has a stmcture highly reminiscent of a receptor molecule, though a ligand has never been identified. If this hypothesis were correct then lOTl/2 cells harvested at low and high density would provide negative and positive confrol populations for further studies. Methods that detect

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protein interactions, such as yeast two hybrid screening, could then be employed to see if such a ligand could be isolated. The mechanism controlling the density dependence of the Shh response in lOTl/2 cells must act upstream of Glil, since expression of Glil can bypass the effect. Transient transfection of Glil results in an up-regulation of Pa/c/zet/transcription within six hours, a time at which the cells are still well below confluence. It is important to note that while the work in Chapter 3 has established that high cell density is essential for a full response to Shh in lOTl/2 cells, this does not preclude some as yet undiscovered aspects of the response still being present at earlier times. For this reason an open minded approach was taken in the microarray studies, with one timepoint investigated that was earlier than any responses had previously been detected. However, the early timepoint (24 hours) did not lead to the identification of any Hedgehog responsive genes.

7.4 Comparison to recently published studies with related aims Two recent sttidies have also used microarrays to investigate aspects of the Hedgehog signalling pathway. Kato et al, (2001) transfected a neuroepithelial cell line, MNS-70, with a Shh expression constmct and screened chips containing 2304 mouse clones from day 14 embryos and adult brain. From this work two Shh responsive genes were isolated: the metalbinding protein Ceruloplasmin {Cp) and the serine protease inhibitor Inter-alpha-trypsin inhibitor heavy chain H3 {ITIH3). Both were confirmed using RT-PCR. The RT-PCR approach was extended to explore the response of these genes to Glil, though the resuhs of this procedure may not be conclusive given in that the results of Kato and colleagues appear to show that that Glil fransfection did not up-regulate Glil itself Neither of the genes reported in this paper had previously been reported as Shh targets. One criticism of the above study is that inadequate negative control populations were used to provide reference RNA for the microarray experiments, and that this deficiency was carried through to the "validation" procedure. In this fransfection based study, untreated cells were used as a reference population rather than cells that had also undergone the transfection procedure in parallel with a negative confrol plasmid. Microarrays are very powerful and do not discriminate between the origins of expression changes, they simply report them. While

7.5 Future directions

201

the response of ITIH3 was shown to be regulated by Glil, Cp was not and while it may be a Gli independent target of Shh the authors have made no attempt exclude the possibility that Cp may be regulated by the stress of the transfection procedure itself, or as a general response to the introduction of foreign DNA. This study was quite preliminary, investigating a single timepoint without the support of any data as to why this particular timepoint was chosen. The second and more comprehensive study, Yoon et al, (2002), aimed to identify target genes regulated through Glil, and used transformed foci generated by stable Glil expression in a rat epithelial kidney cell line (RK3E) as a source of experimental material. In this case 4608 rat UniGene clones were screened, revealing 30 differentially expressed targets (15 upregulated and 15 down-regulated). Seven of the genes (one of them being Patched) were independently verified, two were shown to display up-regulation in tumours with Gli amplification, and the rest were presented solely on the basis of microarray data. One substantial difference between the experimental design of Yoon et al, (2002) and the current study is that this work, while powerful in finding permanent gene expression changes in transformed foci cells, lacked the power to detect direct but transient expression changes which may not occur after long term continued stimulation. The genes identified in both of these previous studies are distinct from those reported in this work. This may reflect tissue specific pathway responses, differences in potency and timing of stimulation methods, or a lack of overlap between cDNA sets used in studies to date. A further study, Zhao et al, (2002), also involved microarray analysis of cells freated with Shh, though in this case the specific goal was to identify genes expressed in immature granule cell neuron precursors that were maintained by Shh expression. This study used mouse tissues and employed oligonucleotide rather than cDNA microarrays. From 13179 genes, 76 were reported as up-regulated. None of the eight new target genes discovered in this work were amongst these, and given the number of genes investigated in this work this may reflect differences between the genes regulated in neonatal cerebellum compared with pluripotent embryonic mesoderm.

7.5

Future directions

The findings of this work will provide a basis for a range of investigations to further understand the biological roles of the newly discovered hedgehog responsive genes. Such

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Studies would initially involve a thorough investigation of the normal expression of the genes in developing mouse tissues, using techniques such as RT-PCR and in situ hybridisation, especially for genes such as Gilz where little expression data has previously been reported. Genes which show developmentally regulated expression in tissues known to be pattemed by hedgehog will be of particular interest for more detailed experiments. Expression studies in existing animal models of hedgehog pathway dismption will be a key aspect of future research. Such studies would provide useful information as to whether the new target genes are Shh responsive in tissues other than embryonic mesoderm. Systems of interest would include mice over- or under-expressing Shh in specific tissues, for example the spontaneous extra-toes mutant, which shows ectopic Shh expression in the developing mouse limb (Buscher et al, 1997), or man-made models such as that provided by Oro et al, (1997). Heterozygous and homozygous Patched knockout mice (Goodrich et al, 1997; Hahn et al, 1998) and various Gli gene under- and over-expression models already reported in the literature would also be informative in assessing the status of the newly identified targets in a range of tissues. Long term studies could involve production of transgenic mice overexpressing Shh target genes of interest. Specific promoters could be used, for example to target ectopic expression to skin in cases where expression studies suggest the gene may be of importance in BCC formation. In other cases knockout mice for Shh target genes that prove to be of particular research interest will be more relevant, and where not already in existence these could be produced. One limitation with complete knockout animal models is that these sometimes show dramatic phenotypes, and when gene function is critical to development this may be embryonically lethal. This is the case for mouse models of genes such as Shh and Ptc (refer Section 1.7) and some of the newly identified pathway targets may behave similarly. In such cases it is not possible to infer functions that the particular gene may have in older embryonic or adult tissues. To address this problem methods such as Cre-lox technology can be used to create "conditional knockouts", which ablate expression of the target gene only at a specific time and in a specific tissue of interest. Emerging methods, such as double-stranded RNA and morpholino antisense technologies, may potentially be employed for future spatial and temporal "transient knock down" studies of gene expression in vivo. For the newly identified target genes, and known components of the hedgehog pathway signalling mechanism, these methods may provide a more elegant and powerfiil means to examine gene function than is provided by a complete knockout model.

7.5 Future directions

203

Studies in animal models would be complimented by an investigation of relative expression differences between normal and tumour tissues from human patients for selected target genes. In situ hybridisation of human homologues of the target genes will allow identification of those which show altered expression in BCCs and other NBCCS related tumours. Genes with known roles in cellular proliferation and apoptosis control, especially those which have historically received little research attention in studies of tumours, would be priorities for such analysis (for example Gilz, Sfrpl and Sfrp2). Consistent finding of altered expression for any of the targets would warrant mutation searching of the corresponding gene, if it occurred in tumours known not to harbour detectable mutations in other pathway genes such as Patched and Smoothened. This would help determine if any of the target genes can act as tumour suppressors or oncogenes in their own right. Mutations could be identified using single stranded conformational polymorphism analysis, chromatography methods or direct sequencing. Detailed functional studies would provide fiirther clues to the biological roles of each of the new hedgehog target genes. Further experiments in lOTl/2 cells, particularly with relevance to osteoblastic differentiation, are of interest. Over-expression of the target genes will show if any are sufficient to induce an osteoblastic phenotype in the absence of hedgehog stimulation. Bf2 would be of particular interest for such experiments, since it has homology with other proteins known to be involved in bone development. Osteoblast inducing molecules would provide key information about the mechanism of Shh mediated bone development, which is currently not well understood. Investigation of the response of each target gene after Shh stimulation in responsive tissue explants, or in cell lines other than lOTl/2, will be useflil in determining if the genes are also targets in other cell types. This could be assessed by northem blot or RNase protection assay analysis, or by employing a "mini" microarray chip to assess multiple targets at a time, to see if they warrant further investigation in the particular cell system. Other anticipated functional studies could involve identification of proteins physically interacting with the products encoded by the new target genes, using techniques such as yeasttwo-hybrid analysis or fluorescence resonance energy transfer. This would be of particular interest for molecules known to have protein-protein interaction motifs for which the exact partners are not known, such as Gilz and Laspl.

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Studies investigating regulation of the Shh induced genes in this work could be extended to the protein level, for example using immuno-fluorescence analysis, in order to see if the changes at the mRNA level are indeed reflected in a detectable variation in the amount of corresponding protein produced by cells. Stimulation of lOTl/2 cells with Shh protein after treatment with cycloheximide would be useful to determine if any of the discovered Shh regulated genes are direct transcriptional targets. Similar studies in P19 (mouse embryonic carcinoma) cells have shown that induction of COUP-TFII by Shh does not require new protein synthesis, and it will be interesting to see if any of the eleven new targets are regulated similarly. For the genes found to be regulated by Glil it would be of interest to perform gelshift binding experiments or reporter activation assays with the discovered upstream motifs related to the Gli binding consensus sequence, to determine if they are indeed fimctional. There are a number of other findings from this work that warrant further investigation. The presence of a number of transcripts hybridising to the mouse Patched probe, at least three of which are induced by Shh stimulation, is intriguing, particularly since several are substantially shorter than the frill length mRNA. Isolation of cDNA clones corresponding to these transcripts and their subsequent sequence analysis would indicate whether these represent new genes with homology to Patched, or whether they represent altemate splice variants of Patched itself. If the latter is tme, then evaluating which motifs are missing from the resulting protein sequences, if they are indeed translated, will give clues to their putative function. Preliminary work has indicated that similar sized transcripts are present in other mouse tissues (data not shown). Isolation of a clone corresponding to the large Shh induced transcript hybridising with mouse Hip probes will also be of interest. Such issues would be able to be addressed after the constmction of a cDNA library from Shh stimulated lOTl/2 cells. Such a library would also provide a key resource for the isolation of fiill length clones corresponding to the newly discovered Shh regulated genes, in cases where such clones are otherwise unavailable. The establishment of a robust lOTl/2 based system for discovery of hedgehog target genes provides the opportunity for more detailed gene discovery studies. In particular, future microarray experiments, with a chips containing a much larger proportion of the mouse genome, should yield more Shh responsive genes. The experimental system will also provide a usefiil platform for studies comparing the set of target genes regulated by Shh with those controlled by Indian hedgehog and Dessert hedgehog.

7.6 Overall conclusions

7.6

205

Overall conclusions

The reporting in this thesis of eleven Sonic hedgehog target genes, eight of them novel, in lOTl/2 cells provides a significant contribution to the current body of knowledge of the fiinction of the hedgehog pathway. In every case these genes were validated by multiple independent studies both in terms of the method of analysis and stimulation technique. In addition, the response of a number of downstream targets of Sonic hedgehog signalling implicated either by analogy to the pathway in the fruit fly, or known from other tissue systems, has been investigated. The finding that a number of the discovered hedgehog targets are regulated through Glil provides fiirther weight to there importance as Shh regulated genes. The discovery that Glil can not only cause up-regulation of Shh induced targets, but that it can also cause inhibition of the expression of Shh repressed targets provides evidence supporting initial observations by Yoon et al, (2002), suggesting that Glil has both activator and repressor actions. It is not known whether these actions are direct for the genes identified in this study, but the presence of sequences with close homology to the consensus Gli binding motif in upstream regions of these genes, some of them known from previous studies to bind Glil, suggests this may be the case. However, until fiinctional binding studies are conducted with the motifs identified in this work in the context of surrounding sequences, the direct nature of the regulation mechanism remains speculative. The approach taken in this work, of using cDNA microarrays in conjunction with a vigorous verification strategy to identify hedgehog target genes has proved pmdent. Microarray technology was used to investigate a number of pairwise comparisons at different time points, and with independent activation sfrategies. This provided a robust screening sfrategy whereby false positive genes unrelated to the biology of interest where minimised, and when they did occur they were readily identified and discounted. Microarrays used in this work contained a relatively small percentage of genes from the mouse genome, and future studies will be possible with substantially larger cDNA sets. Such studies will provide even more Hedgehog responsive genes, relieving a bottleneck that has hindered previous studies trying to imderstand the role of the hedgehog pathway. Overall the findings of this work have provided a substantial number of markers for use in fiirther studies of hedgehog pathway biology. In the past the lack of knowledge of such targets has limited our ability to understand both the mechanism and consequences of hedgehog

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signalling in mammalian cells. In particular, the identification of novel Sonic hedgehog responsive genes provides clues as to how hedgehog pathway dismption causes developmental defects and tumour formation in humans. As well as providing fundamental knowledge on events in embryonic development, continuation of this work may ultimately pinpoint key potential targets for therapeutic intervention in cancer treatment.

Chapter 8: Materials and Methods 8.1 Source of materials 8.1.1 General reagents and chemicals All chemicals were of Analar grade or equivalent, and in most cases were purchased from Sigma Chemical Co. (St Louis, USA). Restriction enzymes were sourced from New England BioLabs (Beverly, USA), while the sources of other specific enzymes were as stated in the following sections. Custom oligonucleotides were synthesised by Pacific Oligos (later renamed Genset; Brisbane, Australia) and Geneworks (Adelaide, Ausfralia).

8.1.2 Radioisotope Radioisotope labelled nucleotide [a- PJdCTP was obtained from Geneworks (Adelaide, Australia).

8.1.3 Microarray chips Glass microarray slides were a kind gift of Dr. L. Fowles, and were manufactured by Dr. L. Fowles and A. Forrest under the guidance of Dr. S. Grimmond (then at the Queensland Institute of Medical Research). Slide manufacture used a four pin arraying robot and involved printing onto poly-L-lysine coated glass slides using standard protocols (Grimmond et al, 2000), except that PCR amplification was performed using a small volume of bacterial culture as the template for each reaction.

8.1.4 Tissue culture reagents and cell lines C3H/10T1/2 clone 8 cells were obtained from the American Type Culture Collection (ATCC; Manassas, USA) at passage 10. Cos7 cells were also from ATCC. HaCaT cells were from Dr. N. Fusenig and MC3T3-E1 cells were a gift of Dr. T. Martin. Reagents used in mammalian tissue culture are outlined in Section 8.3.11, and were all of cell

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culture grade. Cells were grown on gamma irradiated disposable plastic ware (TPP brand. Life technologies. Paisley, UK).

8.1.5 Bacterial strains The two following E. coli stains (obtained from Invitrogen, Carlsbad, USA) were used as standard hosts for storage and amplification of plasmid DNA (genotype indicated on right): ToplOF'

F'[lact^ TnlO (Tet^)] mcrA A{mrr-hsdRMS-mcrBC) mOlacZAMlS MacXlA deoRrecAl araDl39 A{ara-leu)7697 galU gallL rpsL end Al nupG

DH5a

F- O80/acZAM 15 A{lacZYA-arg¥)U 169 deoR recA I end A1 hsdRll{r\i^', m^)phoA supEAA thi-l gyrA96 relAl X

The following E. coli strain (obtained from Novagen, Madison, USA) was used for production of recombinant Shh protein: BL21 (DE3)pLysS

¥' ompT hsdSn (rs" ms") gal dcm (DE3) pLysS (Cm^)

8.1.6 cDNA clones and gene expression constructs The source of plasmid vectors containing clones of key importance has been acknowledged as each constmct has been introduced in the main text. Further clones (used only as probes) are acknowledged in Section 8.5.

8.2

Buffers, solutions and media Solutions were generally sterilised by autoclaving, except in cases where the components were not heat stable. In these instances sterilisation was achieved by passing solutions through 0.22 |j,m gamma irradiated filter units (from Millipore, Watford, UK).

8.2 Buffers, solutions and media

209

8.2.1 Buffers TAE (DNA electrophoresis buffer). 50x stock 2 M Tris-acetate, 50 mM EDTA TE (DNA storage buffer) 10 mM Tris-HCl (pH 8.0), 1 mM EDTA (pH 8.0) Phosphate Buffered Saline (PBS) Tablets were obtained from Amresco (Solon, USA). Final solution (pH 7.4) contained 137 mM NaCl, 2 mM KCl with 10 mM phosphate buffer. Tris Buffered Saline (TBS) 140 mM NaCl, 3 mM KCl, 25 mM Tris MOPS buffer (RNA electrophoresis buffer) 0.2 M MOPS, 0.05 M Sodium acetate, 0.01 M EDTA, adjusted to pH 7.0 with Glacial acetic acid Tris-Glvcine mnning buffer (protein electrophoresis buffer) 25 mM Tris, 192 mM Glycine, 0.1% SDS Saline Sodium Phosphate EDTA buffer (SSPE). 20x stock 3.0 M NaCl, 0.2 M NaH2P04, 0.02 M EDTA, adjusted to Ph 7.4 with NaOH Church hybridisation buffer 3.5% SDS, 325 mM Sodium dihydrogen orthophosphate, 0.5 mM EDTA, pH adjusted to 7.2 with NaOH Orange loading buffer (for DNA/RNA electrophoresis), 6x 0.25% orange-G dye in 30% (v/v) glycerol Blue loading buffer (for DNA/RNA electrophoresis), lOx 0,25% bromophenol blue and 0.25% xylene cyanol dyes in 50% (v/v) glycerol

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Laemmli loading buffer (for protein elecfrophoresis), 5x 67 mM Tris-HCl (pH 6.8), 2.2% SDS, 11% (v/v) glycerol, 0.1% bromophenol blue, 5% (v/v) P-mercaptoethanol (added immediately before use). SDS gel-loading buffer (for cell Ivsis and protein electrophoresis), 2x 100 mM Tris-HCl (pH 6.8), 4% SDS, 20% (v/v) glycerol, 0.2% bromophenol blue, 200 mM DTT (added immediately before use). Mammalian cell lysis buffer 100 mM Tris-HCl pH 8.8, 5 mM EDTA, 0.2% SDS, 200 mM NaCl, 100 ^ig/ml Proteinase K (added immediately before use).

8.2.2 General solutions and reagents Denhardt's solution (50x stock) 1% (w/v) Bovine semm albumin (BSA), 1% (w/v) Ficoll 400, 1% (w/v) Polyvinylpyrrolidone in sterile water. Solution was not sterilised, but was kept in aliquots at -20°C and thawed immediately prior to use. Sheared salmon sperm DNA Lyophilised salmon sperm DNA (Sigma Chemical Co., St Louis, USA) was resuspended in sterile water at 10 mg/ml (by heating to 65°C for several hours), then sonicated (approximately 10 minutes in 30 second bursts) until in the 100 to 3000 base pair range. Solution aliquots were stored at -20°C and denatured by heating to 100°C for 5 minutes immediately prior to use. 20x SSC 3 M NaCl, 0.3 M tri-Sodium citrate, adjusted to pH 7.0 with HCl. Water saturated phenol Crystalline phenol (Wako Pure Chemical Industries, Osaka, Japan) was melted at 65°C, followed by addition of an approximately equal volume of RNA grade water. After mixing well the phases were allowed to separate for several hours, the aqueous phase was removed, and the procedure repeated two fiirther times. After the final separation

8.2 Buffers, solutions and media

211

much of the aqueous phase was removed, leaving about 1/5 the volume of water. Aliquots were frozen in polypropylene tubes at -20°C. 4% Paraformeldehvde (PFA) PFA used for fixation of mammalian cells was made as an 8% (w/v) stock in water, and heated at 65°C until dissolved. Aliquots were stored at -20°C and thawed on the day of use. An equal volume of 2x PBS was added to give a 4% PFA/lx PBS working solution. Coomassie blue protein staining and destaining solutions 0.25% Coomassie brilliant blue in 45% (v/v) Methanol, 10% (v/v) Glacial acetic acid [Destain is same solution without Coomassie brilliant blue.] Turbo Juice 2x SSC, 5% SDS Solution D 4M guanidine thiocyanate, 25 mM sodium citrate (pH 7.0), 0.5% sarcosyl with 0.1 M P-mercaptoethanol added immediately prior to use. This solution was not autoclaved (forms toxic fiimes if heated). It was stored at 4°C, protected from light.

8.2.3 Media General bacterial growth media Liquid Luria Bertani (LB) broth was made from powder (USB Corporation, Cleveland, USA), as per the manufacturers instmctions, and sterilised by autoclaving. For plates agar was added at 15 g/1. When required for selection, ampicillin was added at 100 |j,g/ml. For "blue-white" (beta-galactosidase a-complementation) screening of colonies 1 mg of IPTG (in aqueous solution) and 1 mg of X-gal (in dimethylformamide) were top spread on solidified 10 cm diameter LB plates.

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Hanahan SOC Media (for incubation of .g. coli cells post transformation) 2% Bactotiyptone, 0.5% Yeast exfract, 8.5 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, sterilised by autoclaving. 10 mM MgS04 and 20 mM Glucose added just before use from sterile solutions. Growth media for mammalian cell culture All reagents were cell culture grade and Gibco BRL brand (Life technologies. Paisley, UK) unless otherwise stated. Details of specific media are given in Section 8.3.11.

8.3

Procedures and Techniques

8.3.1 DNA extraction and purification Small scale plasmid DNA preparation ("miniprep") Bacteria harbouring plasmids of interest were sfreaked to ensure single colonies, which were used to inoculate 5 ml LB/Amp broth and grown ovemight (37°C, shaking 225 rpm). Plasmid DNA was isolated by alkaline lysis followed by binding to silica, using the Ulfraclean mini plasmid preparation kit (Mo Bio Laboratories Inc., Carlsbad, USA), and re-suspended in 10 mM Tris (pH 8.0). Large scale plasmid DNA preparation ("maxiprep") Bacteria harbouring plasmids of interest were sfreaked to ensure single colonies, which were used to inoculate 5 ml LB/Amp broth, and incubated approximately 10 hours (37°C, shaking 225 rpm). Starter was used to inoculate 250 ml of LB/Amp and grown (with shaking) ovemight. Cells were spun down and media removed before bulk plasmid purification using "Jetstar maxi" columns (Genomed, Bad Oeynhausen, Germany) as per the manufacturers instmctions, prior to re-suspension in TE and storage at -20°C. "Heat and Spin" PCR grade plasmid DNA preparations from bacterial colonies This method was used to obtain DNA to rapidly screen large numbers of transformed colonies for a sequence of interest by PCR, for which cmdely prepared plasmid was sufficient. Half the bacterial mater from each colony of interest (grown over night on LB/Amp plates) was transferred to a tube containing 100 |j,l sterile water. This was

8.3 Procedures and Techniques

213

mixed, heated at 95 °C for 5 minutes, vortexed vigorously, chilled on ice, then spun at 12 000 g for 5 minutes. 1 /il of supematant was subsequently used in each 20 jiil PCR screening reaction. Phenol-chloroform DNA purification DNA to be purified was extracted once with tris equilibrated phenol (pH 7.9), once with a mixture of tris equilibrated phenol/chloroform/isoamyl alcohol (25:24:1 v/v), and once with chloroform/isoamyl alcohol (24:1 v/v). Each exfraction involved mixing the DNA solution with the exfraction mixture by vortexing, followed by separation of phases by centrifiigation (12 000 g for 15 minutes) and recovery of the aqueous phase. DNA precipitation DNA precipitation was generally performed by adding 1/10 volume of 3M sodium acetate (pH 5.2) and 2 volumes ethanol, followed by precipitation at -20°C. DNA was recovered by centrifiigation (12 000 g for 20 minutes), then washed with 70% ethanol. DNA was dried prior to re-suspension as required. General PCR product and enzvmatic reaction purification Purification of PCR amplified DNA was carried out with the Ulfraclean PCR clean-up kit (Mo Bio Laboratories Inc., Carlsbad, USA), as per the manufacturers instmctions. Procedure was based on DNA absorption to silica column filters. DNA from enzymatic reactions was frequently purified using the same procedure. Post-agarose gel purification DNA from bands excised from agarose gels was purified by binding to glass milk after dissolving agarose in Nal solution, using the GeneClean procedure (Biol 101, La Jolla, USA), as per the manufacturers instmctions. Buccal cell "PCR quality" human DNA preps Genotj^jing of human DNA was performed on PCR products amplified using buccal cell DNA as a template. DNA was obtained from inner cheek scrapes using disposable plastic fransfer pipettes. Recovered material was mixed with 1 ml of PBS and spun for 5 minutes at 800 g, resuspended again in 1 ml of PBS and spun again. The cell pellet was resuspended in approximately 40 /il of PBS, followed by 300 /il of mammalian cell lysis

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buffer, and incubated at 37°C ovemight prior to purification by ethanol precipitation (without additional salt). After washing, the pellet was resuspended in 30 /xl of water and 3 /il was used per PCR reaction.

8.3.2 Quantification of nucleic acid concentration Nucleic acids were quantified by spectrophomeric absorbance measurement at 260 nm against an reference blank of the appropriate resuspension solution. The 260 nm/280 nm absorbance ratio was used for assessment of purity. The following conversions were used for concentration determination (for spectrophotometer path length of 1 cm): Concentration of DNA = A260 x 50 ng//il Concentration of RNA = A260 x 40 ng//il Altematively, DNA concentration was estimated by comparison to standards of known mass (Gibco BRL "low DNA mass ladder". Life Technologies, Paisley, UK) mn in parallel on ethidium bromide stained agarose gels.

8.3.3 General molecular techniques for cloning and manipulating DNA sequences Unless otherwise stated, all standard molecular manipulations were performed as in Sambrooke?a/.,(1989). Agarose gel electrophoresis Nucleic acids were separated in agarose gels submersed in Ix TAE buffer and nm at 50 to 125 V. Agarose was sourced from Amresco (Solon, USA) or Progen Indusfries (Brisbane, Australia). Agarose concenfration depended on the expected size range of DNA under analysis (0.8 to 1.5%). Nucleic acid was stained with ethidium bromide (0.5 /ig/ml) prior to viewing under UV light. For separation of DNA prior to ligation reactions 0.65% low melt agarose (Progen Industries, Brisbane, Ausfralia) was used. Restriction digests Restriction enzyme digests were performed as per the enzyme manufacturers recommended conditions (New England BioLabs, Beverly, USA).

8.3 Procedures and Techniques

215

End filling of protmding single stranded DNA termini DNA (in any of the four standard New England BioLabs restriction enzyme buffers supplemented with 33 |xM each dNTP) was incubated for 15 minutes at 25 °C with DNA polymerase I large Klenow fragment (1 U/|j,g DNA, New England BioLabs, Beverly, USA) in order to fill in bases complementary to 5' overhangs. This created blunt ends where required for subsequent ligation reactions. Ligation of DNA fragments General ligations were carried out using T4 DNA Ligase (New England BioLabs, Beverly, USA) using the manufacturers recommended protocol. Gel derived fragments were ligated directly in low melt agarose, which was melted and mixed with buffer and enzyme. Blunt ligations were incubated at room temperature, while reactions involving complementary sticky overhangs were incubated at between 10 and 16°C (depending on overhang base composition). Where necessary, digested plasmid vectors were dephosphorylated

using

Shrimp

alkaline phosphatase

(Boehringer

Mannheim,

Mannheim, Germany). PCR derived products were ligated by means of generated 3'T overhangs into the "pGEM-T Easy" vector (Promega corporation, Madison, USA) using the supplied buffer and enzyme at 4°C.

8.3.4 Establishment of RNase free conditions Preparations to avoid RNase degradation of RNA All equipment that came into direct contact with RNA during its manipulation was treated to remove any RNase activity. Reusable plastic ware and elecfrophoresis tanks were decontaminated by 10 minutes contact with 3% H2O2. Glassware was baked for 8 hours at 180°C. Sterile disposable plastic ware was assumed RNase free. DEPC treatment of water and solutions RNase free water was prepared by addition of 0.02% DEPC, residual traces of which were removed by autoclaving after ovemight reaction. Solutions for RNA work were made in sterile RNase free bottles from sterile RNase free components and were not autoclaved.

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8.3.5 RNA extraction and purification RNA was prepared by several altemative techniques depending on the intended use. "Solution D" method for total RNA preparation RNA was prepared using the method of Chomczynski and Sacchi, (1987). Briefly, tissue or cultured cells were homogenised with Solution D prior to addition of 1/10 volume 2 M sodium acetate, 1 volume of water saturated phenol and 1/5 initial volume of chloroform/isoamyl alcohol (24:1 v/v). After chilling and subsequent centrifiigation at 4°C (15 minutes at 12 000 g) the aqueous phase was recovered. RNA was precipitated with an equal volume of isopropanol, washed in 70% ethanol, then resuspended in solution D for a second isopropanol precipitation. The final pellet was resuspended in an appropriate volume of RNase free water. RNeasy total RNA preparation For microarray and northem blot analysis, cells were washed with PBS prior to lysis directly on culture plates with buffer "RLT" (Qiagen, Hilden, Germany). Lysates were stored at -80°C prior to purification using the RNeasy procedure (Qiagen, Hilden, Germany), as per the manufacturers instmctions. RNA was eluted in RNase free water ready for use in subsequent procedures. Residual DNA contamination was avoided by including a DNase digestion step during purification (using RNase free DNase I, Qiagen, Hilden, Germany). Poly(A)"^ RNA purification Poly(A)'^ RNA was used in the constmction of subtracted libraries and for northem blot analysis of lowly expressed franscripts. Total RNA was prepared using one of the above techniques, and after quantification was purified by oligo(dT) cellulose chromatography using the Message Maker reagent assembly (Gibco BRL, Life Technologies, Paisley, UK). Two rounds of selection were performed. Efficiency of purification was assessed after quantification by separating equal amounts of total and poly(A)"^ purified RNA by gel elecfrophoresis and observing the reduction in ribosomal RNA present.

8.3 Procedures and Techniques

8.3.6

217

PCRandRT-PCR

Polymerase chain reaction (PCR) PCR was performed using 1 U Taq DNA polymerase (Boehringer Mannheim, Mannheim, Germany) and 2.5 ng/|j,l of each oligonucleotide primer per 20 |xl reaction. Reactions were performed under mineral oil in thin walled tubes. Standard buffer was 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCb, 0.2 mM each dNTP. Peltier thermal cycling was performed in the PTC-200 "DNA Engine" (MJ Research, Watertown, USA). cDNA svnthesis Total RNA was reverse transcribed to cDNA using Moloney murine leukaemia vims reverse transcriptase (M-MLV; Invitrogen, Carlsbad, USA). Reactions with 1 |xg total RNA input were performed in a 30 p-l volume with 100 U of enzyme. Reaction buffer contained 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 6.5 mM MgCb, 0.83 mM each dNTP and 90 U/|xl random hexamers (Amersham Pharmacia, Little Chalfont, UK). Synthesis was performed at 42°C for 1 hour. Produced cDNA was stored at -20°C. Reverse Transcription - Polvmerase chain reaction (RT-PCR) Reactions were performed as for standard PCR (above), except that buffer with reduced Mg^ was used so that the high level of carryover Mg"^ from the cDNA synthesis step did not take the Mg"^ level out of its normal working range. Between 0.5 and 6 ^il of cDNA was used per 20 ^il PCR reaction, as optimised for the primer set of interest.

8.3.7 Transformation of plasmid DNA into E. Coli Plasmid constmcts were infroduced into chemically competent E. coli cells prepared using the method of Inoue et al, (1990). DNA of interest was added to cells, chilled on ice for 20 minutes, then heat shocked at 42°C for 1 min. Cells were immediately retumed to ice for 2 minutes prior to the addition of 900 p,l of SOC media and outgrowth without antibiotic for 1 hour (37°C, shaking 225 rpm). Cells were then plated on LB/Agar plates containing the appropriate antibiotic (usually ampicillin), and where appropriate reagents for blue-white screening (refer Section 8.2.3). After incubation of plates (37° ovemight) colonies were picked and streaked to ensure pure isolates were obtained.

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For long term storage of plasmids in E. coli, glycerol (final concentration 20% v/v) was added to a small volume of ovemight culture to prior to storage at -80°C. Ahematively, cells were grown directly in LB media containing 10% glycerol prior to freezing.

8.3.8 "Turbo" hybridisation screening of transformed bacterial colonies This rapid colony screening technique (adapted from Buluwela et al, 1989) was used to identify ligated constmcts of interest after bacterial transformation, and also as a method to fiher out undesirable colonies containing hedgehog sequences in the analysis of subtracted libraries. Colonies of interest were inoculated onto LB agar plates and grown at 37°C ovemight. Plates where either made in duplicate, with one being used for lifts and the other as an archive, or altematively a single plate was inoculated. For single replicate plates a short (~3 mm) streak was made with the agar surface broken at one end, so that some bacteria would remain after lifting. Dry uncharged nylon filters (Osmonics, Minnetonka, USA) were laid onto the warm (at least room temperature) agar plates and allowed to suck down onto the surface. A needle stab pattem was made to allow later alignment. After two minutes filters were carefully peeled from the plates, and placed colony side upon gel blotting paper (GB002; Schleicher & Schuell, Dassel, Germany) dampened with Turbo Juice (2x SSC, 5% SDS). Filters were microwaved for two to four minutes, until all liquid had evaporated and membranes were extremely dry and curled. Denatured DNA was now bound to the membrane and no further treatment was required. Purified plasmids were able to be investigated directly using a modification of the procedure whereby 1 ^1 of miniprep DNA was spotted directly onto dry membrane then tteated as above. Membranes were pre-hybridised for at least one hour in Church hybridisation buffer at 65°C. The liquid was changed (to remove bacterial debris and salts) prior to addition of radioactive probe (labelled as for northem blotting), and hybridised for at least four hours (or ovemight). Membranes were washed five times for approximately 15 minutes with large volumes of O.lx SSC/0.1% SDS. Probe preparation and autoradiography was as for northem blots (see below).

8.3 Procedures and Techniques

219

8.3.9 DNA sequencing DNA sequencing was performed using ABI Prism dye terminator cycle sequencing (Perkin Elmer, Wellesley, USA) as per the manufacturers instmctions (based on the principle of Sanger et al, 1977). Completed reactions were electrophoresed and scanned by the Australian Genome Research Facility.

8.3.10 Northern blotting and hybridisation Preparation and radioactive labelling of cDNA probes DNA sequences to use as probes were prepared either from purified PCR products or DNA isolated from agarose gel bands. Each labelling reaction used 25 ng of DNA as a template, which was radioactively labelled using Rediprime II (Amersham Pharmacia, Little Chalfont, UK), as per kit instmctions. 50 |j,Ci of [a-''^P]dCTP was used for each labelling reaction. After incubation, unincorporated nucleotides were removed using Sephadex-G50 columns. These were formed just before use by adding 1 ml of Sephadex-G50/3x SSC slurry to a 1 ml disposable syringe, then spinning at 450 g for 5 minutes. 150 p.1 of 3x SSC was added to each labelling reaction to give a total volume of 200 |xl. This was added to the top of the compacted column and collected into a fresh tube by spinning again as above. Probe incorporation was checked by holding tube containing the purified probe exactly 20 cm from a Geiger counter and comparing cps to that of the reaction prior to purification. (This method provides only a cmde estimate of incorporation, but is sufficient to detect occasional poorly labelling probes that have to be remade.) Elecfrophoretic separation and capillary transfer to membrane Total RNA was prepared using the RNeasy RNA isolation kit (Qiagen, Hilden, Germany), with poly(A)"^ isolated using "Message Maker" (GIBCO BRL, Life Technologies, Paisley, UK) when required. 10 jig total, or 2 pg poly(A)"^ RNA, was used per lane, and loaded in 50% formamide/6% formaldehyde/Ix MOPS buffer/lx "blue loading buffer" prior to elecfrophoresis on 1% agarose/3% formaldehyde/Ix MOPS gels.

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Chapter 8: Materials and Methods

Size estimates were made by including 0.24 to 9.5 kb RNA ladder (Gibco BRL, Life Technologies, Paisley, UK) in one lane to provide molecular weight standards (cut off and ethidium bromide stained prior to transfer step). RNA was moved to uncharged Magna nylon membrane (Osmonics, Minnetonka, USA) by capillary transfer in 20x SSC (ovemight). After transfer the membrane was washed in 2x SCC, then baked at 80°C for 2 hours before being cross-linked with 120 mJ of light in a UV multilinker (Ultra-Lum Inc., Claremont, USA). Northem hvbridisation procedure Pre-hybridisation (4 hours) and hybridisation (ovemight) were performed in 5x SSPE, 5x Denhardt's solution, 0.5% SDS in 50% formamide at 42°C, with 125 pg/ml heat denatured salmon sperm DNA added immediately prior to use. Extensive washing was performed at hybridisation temperature, using solutions in the range of 3x SSC/0.1% SDS (low stringency) to O.lx SSC/0.1% SDS (high stringency), with final washing concentration dependent on the probe of interest. Band detection, normalisation and densitometrv Bands were detected by autoradiography using Fuji "SuperRX" medical x-ray film and Kyokko High Plus intensifying screens (both from Fuji Photo Film Co., Tokyo, Japan). Autoradiographs were quantified using a GS-700 imaging densitometer (Bio-Rad, Hercules, USA) and Molecular Analyst 1.0.2 software (also from Bio-Rad). A 600 bp fragment of mouse GAPDH cDNA was used as a "loading control" probe for normalisation of differences in RNA amounts attached to the membrane between different samples on each blot. Such differences can occur because unequal amounts of RNA are initially elecfrophoresed (this was minimised in by spectrophotomeric quantification of all samples) or because capillary ttansfer efficiency is not uniform across the gel. The GAPDH conttol also addresses variations in the level of covalent bond formation to the membrane occurring post-transfer (such as those caused by shadows or gradients during UV cross-linking). GAPDH was chosen for northem blot normalisation as preliminary experiments (data not shown) indicated that GAPDH mRNA levels in the cell lines used were not significantly altered by Shh stimulation.

8.3 Procedures and Techniques

221

Probe stripping and blot storage After detection, radioactive blots were striped by placing them in near-boiling 0.1% SDS with gentle shaking, after which they were checked ovemight against x-ray film to test for residual radioactivity. Blots were always stored damp (in 0.1% SDS) at -20°C, prior to reuse.

8.3.11 Culture and manipulation of mammalian cell lines General culture conditions lOTl/2, HaCaT, MC3T3-E1 and Cos7 cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco BRL, Life Technologies, Paisley, UK) supplemented with sfreptomycin (50 |ig/ml, Gibco BRL), penicillin (50 U/ml, Gibco BRL, Life Technologies, Paisley, UK), additional glutamine (2 mM, Gibco BRL, Life Technologies,

Paisley, UK)

and heat inactivated

"Semm

Supreme"

(9.1%,

BioWhittaker, Walkersville, USA). Cells were passaged after dissociation with 0.05% Trypsin/0.53 mM EDTA/lx PBS (Gibco BRL, Life Technologies, Paisley, UK) using standard techniques. In the case of HaCaT cells pre-treatment with 5 mM EDTA in PBS was required prior to trypsinisation. Cells were generally split at between 1 in 10 and 1 in 20, with the exception of lOTl/2 cells for which stock cells were plated at 2000 cells/cm^. To avoid the build up of differentiated cells and spontaneous non-contact inhibited transformants, lOTl/2 cells were never allowed to reach confluence and discarded at passage 20. All manipulations were performed in a class II biological safety cabinet. Cells were maintained at 37°C in a 5% CO2 atmosphere. For long term storage, cell lines were maintained above liquid nitrogen in culture medium containing 10% v/v DMSO (5% for lOTl/2), with additional serum added where necessary. Transfection of expression constmcts into mammalian cell lines For transient transfection, expression constmcts were introduced into cells by liposome mediated delivery. Liposomes were formed using Plus reagent and Lipofectamine (Gibco BRL, Life Technologies, Paisley, UK), as per the manufacturers protocol. Conditions for optimal transfection efficiency were determined by freating cell lines of interest with a constmct encoding a protein for which a plentiful antibody was available, using a range of DNA and Lipofectamine concenttations. Immuno-fluorescence detection and DAPI counterstaining were performed to determine the percentage of

222

Chapter 8: Materials and Methods

successfully ttansfected cells in each case, in order to determine optimal transfection conditions. For lOTl/2 cells the maximum ttansfection efficiency under optimised conditions in preliminary experiments was around 30 percent, though this did show considerable variation from experiment to experiment. Cells were ttansfected in the absence of semm. The time of liposome addition was considered "time zero" in all experiments. After liposome treatment (3.5 hours) cells were washed with PBS and retumed to normal growth media (without antibiotics). For experiments involving RNA harvest cells were grown in 55 cm^ dishes, which were randomly assigned to treatment or conttol groups prior to ttansfection. For microarray and northem blot RNA harvests lOTl/2 cells were transfected with 4.6 fig of DNA per 55 cm^ dish, using 16.5 pi of Plus reagent and 27 pi of Lipofectamine. Transfection for stable cell line generation was performed as for ttansient ttansfection, except that 1/10 the amount of a G418 resistance plasmid (a gift of T. Evans) was transfected along with the constmct of interest. G418 (Gibco BRL, Life Technologies, Paisley, UK) selection was begun one day after ttansfection, using an appropriate concenttation determined previously from "kill-curve" investigations on sensitive cells. Direct stimulation of cells with Sonic hedgehog Recombinant protein experiments were performed using 100 nM Shh, an amount above Patched binding saturation levels from kinetic studies (Marigo et al, 1996a). Cells were kept in growth media appropriate to the cell line under investigation. When using Shh conditioned media this was diluted 1:1 with fresh growth media and incubated with cells. Negative conttols were always employed, which consisted of the appropriate protein resuspension/storage buffer in the case of recombinant protein, or pair-matched media collected after expression of a mutant control constmct in the case of Shh conditioned media. Isolation of primary murine neonatal keratinocvtes Primary cultures of mouse epidermal keratinocytes were established and maintained using a protocol based on the Rheinwald and Green technique (Rheinwald and Green, 1975; Rheinwald, 1989; Leigh and Watt, 1994), with modifications as described in Jones, S. J. et al, (1997). Skin was dissected from euthanased 1 to 2 day old Quackenbush-Swiss mice, divided into 6 pieces, and incubated ovemight in Dispase solution containing 2.5% Bacillus polymyxa Dispase (equivalent to 22.5 U/ml final

8.3 Procedures and Techniques

223

concentration; Gibco BRL, Life Technologies, Paisley, UK), 0.02 mg/ml Gentamicin (Sigma, St Louis, USA), 100 U/ml Penicillin, 100 pg/ml Streptomycin, 8 pg/ml Fungizone (all from Gibco BRL, Life Technologies, Paisley, UK) and Ix PBS. This allowed separation of epidermis from dermis. Epidermal cell sheets were then passed repeatedly though a large bore plastic pipette for 5 minutes, during which time the cells were dissociated with 0.05% Trypsin/0.53 mM EDTA/1 X PBS at room temperature. After neutralisation, cells were collected by centrifiigation (200 g for 8 minutes), and washed with PBS (a procedure that was repeated three times). Cells were resuspended in "3:1 keratinocyte selection media" (see below), counted, and plated at 4x10^ cells per 55 cm^ dish. Culture dishes were coated in rat tail collagen (Boehringer Mannheim, Mannheim, Germany) before use. Cells were cultured without a feeder layer and used for experiments without further passaging. Cultures were incubated at 37°C in 5% CO2, with the media changed after 24 hours to Keratinocyte semm free media without calcium, with the supplied epidermal growth factor and bovine pituitary extract supplements added as per the manufacturers instmctions (k-SFM; Gibco BRL, Life Technologies, Paisley, UK). Media was subsequently changed every two days and experiments initiated when the cells approached confluence. 3:1 keratinocyte selection media: Major media components were DMEM and Ham's F12 media at a ratio of 3:1, with foetal calf semm added to a final concentration of 9.1% (all major components supplied by Gibco BRL, Life Technologies, Paisley, UK). The following supplements (all from Sigma, St Louis, USA) were added to the base media: 4.5 pg/ml Transferrin, 4.5 pg/ml Insulin, 7.6 ng/ml Cholera toxin, 0.2 pg/ml Hydrocortisone, 30 pg/ml adenine and 20 pg/ml Gentamicin,

8.3.12 Production of recombinant Sonic hedgehog protein Production of rShh protein in Cos7 Constmct pShh-N-PMT21 (codons 1-198 of the mouse Shh cDNA encoding the N-terminal active region) was transfected into Cos7 (SV40 transformed African green monkey kidney cells) as described in Section 8.3.11. Both the pShh-N-PMT21 constmct

224

Chapter 8: Materials and Methods

and protocol for rShh production in Cos7 cells were provided by the late Dr. T. Yamada. Briefly, media was changed 24 hours after transfection to semm free Optimem media (Gibco BRL, Life Technologies, Paisley, UK), without additives. Other altemate media were tried, such as kSFM or DMEM, but westem blotting indicated this was ineffective compared with the high yields obtained using Optimem. Cells were allowed to grow for a fiirther 48 hours prior to harvest of the collection media. Cell debris was removed by centtifugation (250 g for 5 minutes) and the media concenttated 50 fold using Centriprep columns (10 000 molecular weight cut-off "YM-10" from Millipore, Watford, UK) as per the manufacturers instmctions. Produced rShh was stored in small aliquots at -80°C until required. Quantification was performed by comparison to a series of BSA mass standards mn in parallel by acrylamide gel electrophoresis. Production of rShh protein in E. coli cells An expression vector for production of Shh in bacteria, pETmShhl98[6HisT-pETlld] (encoding amino acids 25-198 of the mouse protein and an attached hexa-histidine tag), was kindly provided by Dr. A. McMahon, and was derived from the parent plasmid pETlld from Novagen (Madison, USA). Recombinant Shh protein production and purification was performed as described in the manufactures instmctions for the pET system (Novagen, Madison, USA) with specific conditions as in Marti et al, (1995). Briefly, this involved transforming the above constmct into E. coli strain BL21(DE3)pLysS, and inducing Shh expression in 50 ml LB cultures using IPTG. Harvested cells were sonicated, filtered, and mn on "His-Bind" resin columns (Novagen, Madison, USA) where the hexa-histidine tag bound to divalent nickel cations in the matrix, allowing protein purification and concentration. After extensive washing, recombinant protein was eluted by addition of imidazole. Collected fractions containing rShh protein were pooled and dialysed using "Slide-A-Lyzer" cassettes (Pierce, Rockford, USA), prior to storage of small aliquots at -80°C. Quantification was performed as above. Production of conditioned media containing Shh in lOTl/2 cells For production of rShh conditioned media from lOTl/2 cells, transfections were performed as outlined in Section 8.3.11 (using the Shh expression and null-mutant plasmids described in detail in Section 3.4). Protein was secreted into standard growth media without added antibiotics (DMEM/9.1% heat inactivated semm supreme/2 mM

8.3 Procedures and Techniques

225

additional glutamine), that was added after liposome removal. Media was collected 3 to 4 days post-transfection and centrifuged at 3000 g for 5 minutes to remove cell debris, prior to storage at 4°C. Media from a number of production plates was pooled to give a large volume stock to ensure stimulation with an equal concentration of Shh in all experiments. This was tested in lOTl/2 cells and shown to be potent in inducing AP activity. The activity of conditioned media produced by this method was found to be quite stable when stored at 4°C, and achieved consistently higher levels of AP induction than 1 pg/ml of commercial recombinant Shh protein (#461-SH, R&D systems Inc., Minneapolis, USA) when small scale 7-day quantitative assays were performed in parallel.

8.3.13 Immuno-fluorescence studies in adherent cell lines Cells were grown on glass coverslips that were removed from culture dishes for analysis. Cells were fixed for 1.5 hours with 4% PFA, washed with Ix PBS/0.5 BSA, treated with PBS containing 0.1% Triton-X-100 for 5 minutes. After washing again in Ix PBS/0.5 BSA, cells were incubated in the same solution with the primary antibody (ovemight at 4°C). After washing, cells were incubated with an appropriate secondary antibody (1 hour at room temperature), before staining nuclei with DAPI, washing again, then mounting in 0.1% N-propyl gallate/50% glycerol/lx PBS. Immuno-fluorescence was detected using an Olympus AX70 microscope and photographed on Kodak fihn. The antibody to Patched (a-Ptc chicken-23) was kindly provided by Dr. C. Wicking, while that to Smoothened (a-Smoothened rabbit-"Edith") was a gift of the late Dr. M. Gailani.

8.3.14 Protein harvesting, electrophoresis and western blotting Total protein was obtained from cultured mammalian cells using standard methods (Sambrook et al, 1989). Briefly, cells were lysed in SDS gel-loading buffer, with sonication and boiling performed when necessary prior to electrophoresis. Purified or concentrated rShh protein was analysed by directly adding solutions containing it to Laemmli loading buffer. Size was estimated by comparison to molecular weight standards (Kaleidoscope pre-stained standards (Bio-Rad, Hercules, USA), or Benchmark pre-stained protein ladder (Gibco BRL, Life Technologies, Paisley, UK)).

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Protein separation was performed on poly-acrylamide gels using standard dual layer vertical electtophoresis (refer Sambrook et al, 1989). Separating gels contained 375 mM Tris-HCl (pH 8.8), 0.1% SDS and typically used 12% bis-acrylamide that was pol)mierised in the presence of ammonium persulphate (APS) and N,N,N',N'tettamethylethylenediamine (TEMED). Stacking gel was polymerised similarly, except that 500 mM Tris-HCl (pH 6.8) was used and bis-acrylamide concentration was 3.5%. Gels were mn in Tris-Glycine mnning buffer (at 40 mA), prior to either Coomassie staining or westem blotting. Westem blotting was performed by ttansfering separated proteins onto "Hybond-C extta" supported nitrocellulose membrane (Amersham Pharmacia, Little Chalfont, UK) using a Sartoblot apparatus (Sartorius, Goettingen, Germany), as per the manufacturers instmctions. Membrane was blocked in PBS containing 5% skim milk powder and 0.1% Tween-20 (4°C ovemight), prior to primary antibody incubation in the same solution (1 hour at room temperature). Membrane was then washed with PBS containing 1% heat inactivated normal goat semm and 0.1% Triton-X-100, then incubated with an appropriate peroxidase labelled secondary antibody. After washing, blots were incubated for 15 minutes in TBS containing 0.5% diaminobenzidine, prior to colour development by addition of 0.006% H2O2.

8.3.15 Alkaline phosphatase enzyme activity assays Histochemical alkaline phosphatase assav AP activity was detected histochemically using a procedure modified from Katagiri et al, (1994), and was usually performed on cells grown on glass coverslips. Cells were fixed with 4% PFA for 10 minutes, washed with PBS, then incubated in darkness for 1 hour with AP staining reagent (0.09 mg/ml Fast Blue BB sah, 0.5%) dimethylformamide, 0.1 mg/ml Naphthol AS-MX phosphate, 2 mM MgCl2, 0.1 M Tris-HCl pH 8.5). After washing with Ix PBS, coverslips were mounted on glass slides in 0.1% N-propyl gallate/50% glycerol/lx PBS. Cells were photographed using Kodak film on an Olympus IMT-2 microscope using Nomarski interference conttast optics.

8.3 Procedures and Techniques

227

Quantitative alkaline phosphatase assav Quantitative AP measurement was performed using "ALP Procedure 104" (Sigma Diagnostics, St Louis, USA), which was scaled down to a microtiter plate format. Cells were harvested by dissociating with 0.05% Trypsin/0.53 mM EDTA, which was neutralised with growth media prior to cenfrifiigation (12 000 g for 30 seconds). Cells were washed in TBS prior to lysis in 0.9% NaCl with 0.2% Triton X-100, after which the manufacturers protocol was followed. Sample inputs were normalised to total protein concentration measured with Bradford reagent. Each reaction was read in triplicate at 415 nm against p-Nitrophenol standards (Sigma Diagnostics, St Louis, USA), with a secondary read after acid addition to correct for background lysate absorbance. Since assays were performed at a different scale to the Sigma Diagnostics 104 procedure, and under modified conditions (the Sigma procedure is designed for blood testing, whereas lysed cultured cells in buffer are used here instead), the assay is no longer under Sigma "standard conditions". For this reason the amounts of p-Nitrophenol produced have not been expressed as units of enzyme activity. Instead the amounts of p-Nitrophenol produced provide a relative measure (with arbitrary units) of the level of AP activity between samples in any given experiment.

8.3.16 Construction and analysis of normalised subtracted libraries All steps were carried out exactly as stated in the PCR-Select cDNA Subtraction Kit User Manual, using supplied enzymes, buffers and oligonucleotides (K1804-1, Clontech, Palo Alto, USA). PCR amplification was performed using "Advantage cDNA polymerase mix" (also from Clontech) and resulting normalised subtracted products were cloned into the plasmid vector pGEM-T-Easy (Promega corporation, Madison, USA). Libraries were fransformed into Epicurian Coli XLIO-Gold ulfra-competent cells (Stratagene, La Jolla, USA). Differential screening and assessment of subtraction success was carried out using the manufacturers recommended procedures.

8.3.17 Microarray hybridisation and subsequent analysis Great care was taken to minimise the risk of introducing gene expression differences unrelated to the treatment of interest during cell culture experiments for microarray analysis. This included Shh stimulated and control cells being treated (either by

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transfection or conditioned media addition) for a particular experiment coming from the same batch of seeded cells on the same day at the same time, with plates allocated randomly to the Shh or the confrol groups prior to treattnent. At the desired time after treatment cells were harvested in parallel using the same batch of lysis buffer. Every RNA preparation for microarray analysis was checked for quality by gel electtophoresis after processing, and again on the day of reverse transcription after concentration and aliquoting. All subsequent steps were performed in parallel for matched pairs of samples to be hybridised. Total RNA samples (40-65 pg) from treatment and pair-matched control samples were labelled with Cy-5 or Cy-3 dUTP, using oligo d(T) primer and Superscript II reverse transcriptase (Invitrogen, Carisbad, USA). RNA was removed by alkaline hydrolysis. Labelled cDNA was purified using YM-30 microcons (Millipore, Watford, UK). Hybridisation (in 0.25 mg/ml CotI DNA, 0.5 mg/ml Poly d(A), 4x SSC, 0.5% SDS, 50% formamide) was performed ovemight at 45°C, under coverslips in humidified chambers ("Arraylt" brand from TeleChem Intemational, Sunnyvale, USA). Slides were washed for 3 minutes in 0.2x SSC/0.05x SDS, and 2x 3 minutes in 0.2x SSC, then spun dry prior to obtaining 16-bit greyscale fluorescence images for each channel with a GMS 418 array scanner (Genetic MicroSystems, Wobum, USA). Images were imported into the image analysis software ImaGene (BioDiscovery; Marina del Rey, USA), which was used for the conversion of pixelated image data into an overall signal strength for each spot on each channel. ImaGene was able to fit a precise grid to each image, such that the perimeter of the spot at each position was accurately located. Every image was manually checked for poor quality spots, which were flagged accordingly. This procedure involved making sure spots that would have given artifactual results, such as those obscured by fluorescent fluff or dust, did not lead to erroneous results. The package was then used to ttansform the image files into an overall signal strength for each spot on each channel, and to provide an estimate of local area background signal for each data point. Normalisation and statistical analysis of local background adjusted signals was performed using Excel (Microsoft, Redmond, USA) and the GeneSpring package (Silicon Genetics, Redwood City, USA). In the former normalisation was performed by

8.4 Primers for PCR and DNA sequencing

229

applying a constant to all values such that the average intensity of spots on both channels was equivalent, in accordance with Hegde et al, (2000). GeneSpring analysis was performed with the data normalised using the median of signals on each channel as a synthetic positive confrol. Procedures for identification and prioritisation of putative differentially expressed clones were as outlined in Chapter 5. Clones corresponding to spots of interest were obtained from the 1MB microarray facility. Bacterial cultures were stteaked to ensure isolation of a single colony, and then re-grown in LB broth, after which aliquots were frozen as glycerols for storage and the remaining culture harvested for purification of plasmid DNA. Each of the plasmids (which corresponded to a spot of interest on the microarrays) was sequenced with Ml3 forward and reverse primers as described by Hegde et al, (2000). Resulting sequences were used to search the GenBank (NCBI) database using the BLAST search algorithm. Genes were then assigned an identity. In any cases of ambiguity or low quality sequence the reactions were repeated. In most cases the clone sequence had a 100% match with a known mouse gene. In some cases the clones did not match known genes, but did match ESTs that represented the 3' UTR of known genes identified from tentative consensus sequences generated by The Institute for Genomic Research (TIGR) gene indices database. A small number of clones could not be matched to known genes, and may represent so called "unknown genes". Altematively such sequences may be from the UTRs of known genes for which sequence is not yet available on public databases. Plasmids were digested to liberate insert without poly(A)^ tail, separated by agarose gel electrophoresis, and purified with Geneclean (Biol 101) ready for use as northem hybridisation probes.

8.4

Primers for PCR and DNA sequencing In general, primers have a nucleotide position from an appropriate GenBank mRNA reference sequence included in their name. This represents the nucleotide corresponding to the 5' most base of the oligo, such that the difference between the numbers can be used to calculate the length of the product amplified from cDNA.

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General plasmid insert amplification and sequencing For routine screening and sequencing of clones the following primers were used: M13FNEB-20 (forward)

5' gta aaa cga egg cca g t 3 '

M13R-24 region (reverse) T7 Terminator (reverse) T7 Promoter (forward) Sp6 Promoter (forward)

5' 5' 5' 5'

gga get taa att

aac agt tae tag

age tat gac gtg

tat tgc tea aca

gac tea eta eta

cat geg tag tag

g 3' g 3' gg 3 ' 3'

PCR primers used for general amplification of cDNA microarray inserts, and in array chip manufacture, were the "Quackenbush" Ml3 primers (Hegde et al, 2000). These were also used for sequence verification of microarray clone inserts: Q-M13 (forward)

5' gtt tte eea gte acg aeg ttg 3'

Q-M13 (reverse)

5' tga geg gat aae aat tte aea eag 3'

General pEFBOS vector primers for insert amplification/sequencing: Efbos 3780FHdIII (for)

5' a t t aag e t t geg egg a t t c t t t a t eac 3 '

Efbos 4833R (reverse)

5' t t g t a a aae gae gge eag 3 '

Primers for RT-PCR and/or amplification of cDNA for northem blot probes Mouse Glil Mouse Glil primers were modified from those published by Walterhouse et al, (1993), in that the superfluous overhangs were removed and a miss-match between GenBank sequences and the published reverse primer was corrected. The resulting primers were: mGliF2 (forward) mGli R2 (reverse)

5'eag gga aga gag eag aet ga 3' 5'age tga tge age tga tee age eta 3'

Human GLIl General amplification and genotyping: G2506F (forward) G3440R (reverse)

5' gga eaa gtg eaa gte aag eea g 3' 5' ett agg aaa tge gat etg tga tgg 3'

Additional sequencing primers: G616F (forward) G1090F (forward) G1647F (forward) G221 OF (forward) G821R(reverse)

5' 5' 5' 5' 5'

aag tge tea aag gag

tet agt aae age tea

gag aaa tge eag aat

etg gee eea aag tee

gae tte get ttg tgg

atg age tgt gga etg

etg g 3' 3' ee 3' e 3' c 3'

231

8.4 Primers for PCR and DNA sequencing

G1223R G1979R G2409R G3100R

(reverse) (reverse) (reverse) (reverse)

5' 5' 5' 5'

tgt eet ttg ttg

ttt gea ggt tge

ege ttg ggg ete

age ggg gte eea

gag ttg agg ett

eta tat ata tga

gg 3' e 3' tg 3' gag 3'

Mouse Patched General: Ptc6 73IF (forward) Ptc8 1146R (reverse)

5' aet ttg aee eet tgg aat te 3' 5' eag gat gge gge tge eet g 3'

Sub-probe "A": mP 791F (forward) mP 1175R (reverse)

5' ggg agg aaa tge tga ata aag 3' 5' aee tee aeg taa gte ete tg 3'

Sub-probe "B": mP1175F (forward) mP 1465R (reverse)

5' tgg tte ate aaa gtg teg 3' 5' aaa eet gag ttg teg eag 3'

Sub-probe "C": mP 1462F (forward) mP 1794R (reverse)

5' gtt ttg ceg ttt ett get e 3' 5' gea gaa aat ate eaa tet tet gte e 3'

Sub-probe "D": mP 1797F (forward) mP2161R (reverse)

5' ttt eac aag ecc etg tgt eag eag 3' 5' aeg aag aga gtg tec aet tgg tge 3'

Human Patched PTCH 3074F (forward) PTCH 3550R (reverse)

5' tgc tgt tea geg tgg tg 3' 5' tgg etg gag aea eet eag gat atg 3'

Mouse Shh (sequencing primer) mShh 150 (reverse)

5' ggg aat aaa etg ett gta gge 3'

Human Shh Setl: SHH IF (forward) SHH3R (reverse)

5' geg a t t t a a gga a e t eac ecc c 3 ' 5' egg t t g a t g aga a t g g t g ec 3 '

Set 2 (as in Fan, H. et al, (1997)): SHH 139F (forward) 5' gte ate agt tec atg gge gag 3' 5' etg agt ggt gge eat ett egt 3' SHH 557R (reverse) Mouse Hip 5' end of coding region: mHIP 759F (forward) mHIP 1453R (reverse)

5' t t e t g c eac eaa eaa e t c 3 ' 5' a t a e t g t g t a t t cca eaa ecc 3 '

3' end of coding and 3'UTR region: mHIP 1922F (forward) 5' t e a age e a t t e a g t a aeg 3 '

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Chapter 8: Materials and Methods

mHIP266IR(reverse)

5' age aga agg aea gte tet

e 3'

Mouse Bmp2 m B M P 2 e2F (forward) m B M P 2 e3R (reverse)

5' gtt tgg eet gaa gea gag ac 3' 5' atg gtt ggt gtg tee etg tg 3'

Human Bmp2 hBMP2 556F (forward) hBMP2 925R (reverse)

5' aea t g c tag aee tgt ate gea gg 3' 5' gea t t e tga tte aee aae etg g 3'

Mouse Bmp4 mBMP4 418F (forward) 5' ega gee atg eta gtt tga tae etg 3' mBMP4 800R (reverse) 5' gat get get gag gtt gaa gag g 3' Human Bmp4 (as in Fan, H. et al, 1997) BMP-2BF (forward) BMP-2BR (reverse)

5' eac cat gat tee tgg taa ec 3' 5' tet cca gat gtt ctt egt gg 3'

Mouse Thgl (as in Fiorenza et al, 2001) mThg-1 1263F (forward) 5' gag age ett ate gte gag gt 3' mThg-1 1565R(reverse) 5' t e t cca eet tag eet tge c 3' Mouse TSC-22 mTSC22 10IF (forward) 5' ttt gaa eea gge tge tgg ag 3' mTSC22 163OR (reverse) 5' geg eag aae gae tat aca ggt gag 3'

8.5

Northern hybridisation probes DNA Probes were prepared either as isolates from digests of parent plasmids, or by RT-PCR amplification. Probes corresponding to microarray clones were prepared as described in Section 8.3.17. Additional probes were sourced as described in previous chapters.

Shh Shh probe for both northem hybridisation and subtracted library screening was the 0.6 kb N-terminal coding region from pShh-N-PMT21. Patched The probe for mouse Patched (mPtcl263) was the 1.3 kb intemal

PVMII

fragment

isolated from a cDNA constmct kindly provided by Dr. M. Scott (plasmid 617-1). A

8.5 Northem hybridisation probes

233

human probe was made from the corresponding Pvull fragment from the human cDNA (plasmid IB PTCH, provided by Dr. C. Wicking). Mouse Patched "sub-probes" were PCR amplified from plasmid 617-1 using primer sets A to D (described above). Patched2 Patched2 expression was detected using a 0.3 kb probe from exon 13 of the mouse cDNA, corresponding to the intracellular loop region, as obtained from a constmct provided by Dr. C. Wicking. Glil Human and murine Glil transcripts were detected by hybridisation to the 1.6 kb mouse Glil coding region (plasmid provided by Dr. A. Joiner). Smoothened Mouse Smo mRNA was detected with a homologous 1.2 kb intemal Smal fragment from the human SMOH coding region (constmct provided by Dr. F. de Sauvage). Hip Murine probes (695 bp and 740 bp) were amplified by RT-PCR from mouse brain, using the primer sets described in the previous section. Bmp2 Murine Bmp2 was detected with a 1.2 kb cDNA fragment containing 5' UTR and the majority of the coding region (ending at the E'coRI site at position 1406 of GenBank entry NM_007553). Source plasmid was kindly provided by Dr. P. Koopman. A 370 bp human Bmp2 probe was RT-PCR amplified from human keratinocyte RNA using the human Bmp2 primer set described in the previous section. Bmp4 A 0.4 kb human Bmp4 probe was generated by RT-PCR from human keratinocytes using the primer set described in the previous section. Mouse Bmp4 mRNA was detected using a 1.0 kb cDNA fragment containing 5' UTR and coding sequence outside of the conserved Bmp2IBmp4 region (Jones, C. M. et al, 1991; constmct (pSP72-BMP4) kindly provided by Dr. A. McMahon). HNF-3P The murine HNF-3p probe (657 bp) contained coding sequence located between intemal Sacll restiction sites (positions 254 bp and 911 bp on GenBank entry X74937), and was digested from a parent plasmid obtained from Dr. S. Grimmond.

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Chapter 8: Materials and Methods

Angiopoietin2 Angiopoietin2 probe consisted of the complete mouse cDNA (-2.5 kb), amplified by PCR from plasmid B(1575d2), which was provided by Dr. S. Grimmond. GAPDH A 600 bp sequence from the central coding region of the mouse GAPDH cDNA was used as a loading conttol probe on both mouse and human northem blots, detecting a single transcript of-1.3 kb in both cases (plasmid kindly provided by Dr. M. Little).

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Appendix A: Use of the Geometric IVIean in IVIicroarray Analysis While biologists are familiar with measures of central tendency such as the arithmetic mean, median and mode another related statistic, the geometric mean, is less well known. This appendix is intended as a brief outline as to why it is appropriate in this thesis for the averaging of replicate microarray data points. Where R„ is a ratio and N is the number of replicates:

Geometric Mean = i(l{R^x R^x R^x.. .R^)

or alternatively it can be represented as:

/^ 1

Geomettic Mean = anti log

Af

^ZM^J

K^ti

The geometric mean is used instead of an arithmetic mean as an estimator of central tendency when working with numbers that represent relative quantities. Although an arithmetic mean is often used in the literature for a range of work that generates fold change (e.g. microarrays, assays involving reporter constmct responses and relative densitometry) it is inappropriate in such cases. Attempts to calculate arithmetic means on data in the form of relative numbers leads to the formation of "harmonic" estimates of central tendency such that two different estimators are obtained depending on which number was the denominator when the original ratios were calculated. This means that different conclusions may be drawn depending on the arbitrary decision of which data is considered the "baseline" response. Clearly an appropriate estimator should allow unbiased comparison, such that the same net conclusion is reached no mater whether a hypothesis is constructed to consider a population A verses B or visa versa. The geomettic mean avoids this problem in that its reciprocal is the number that is obtained if the ratios themselves are expressed as fractions the opposite way up prior to calculation.

278

Appendix A: Use of the Geometric Mean in Microarray Analysis

In Summary (where R„ represents a data point as a ratio):

Arithmetic Mean(/?i, i?2,..., i?;^) ?^ Arithmetic Mean

but the following does hold true:

Geometric Mean(i?] ,R2,...,Rff)

= Geometric Mean

f 1

1

V^l

^2

^N J

and therefore in terms of microarray analysis: •-7

C

1

Geometric Mean of -^— data points = y Geomettic Mean of - ^ data points Cy5 The geomettic mean is frequently used in other fields, particularly economics and finance where it is used to analyse another form of "relative" data, that of the rates of returns on investments over time. Many other statistically valid methods also exist for dealing with replicates in microarray studies. Most microarray papers deal with this issue appropriately, but historically some authors have not.

Appendix B: Supplementary Microarray Data B1. Raw lists of putatively regulated clones with associated microarray data The data presented in the following tables provides normalised microarray ratios for individual gridded clones at each timepoint, along with the results of validation tests where conducted. Extreme values caused by very low signal on one channel but a large signal on the other have not been removed, since the data in this form was used largely as a screening tool. All data has been filtered to remove spots with low signal strength on both channels, using a threshold calculated two standard deviations above the average background signal. In the case of the 48 hour transfection timepoint ratios were calculated from two replicates and therefore this data is of lower confidence as an estimate of the true ratio than other timepoints where four replicate data points were obtained. In all cases the chips represent Sonic hedgehog stimulation (either by transient transfection or conditioned media) compared with a pair-matched Shh null mutant treatment.

Criteria for inclusion in "putative clone of interest" lists Clones showing putative differential expression have been categorised into the following categories for the purposes of display in the following tables: Set 1:

Ratios of highest tmst. Normalised geometric mean ratio greater than 1.5 or less than 1/1.5. Signal at least 2 standard deviations above average slide background signal on both channels for all replicates.

Set 2:

High tmst in direction but not in magnitude of ratio. Normalised geometric mean ratio greater than 1.5 or less than 1/1.5. Gene lowly expressed on one channel for one or more replicates, but other channel

280

Appendix B: Supplementary Microarray Data

has significant expression for all replicates (possibility of a number near zero on one chaimel which can lead to non-representative ratios). Set 3:

Data of lower confidence. Normalised geometric mean ratio greater than 2 or less than 1/2. Average calculated from reduced mmiber of replicates as some data points rejected (those which fall within 2 standard deviations of average slide backgrotmd or have been manually flagged as "bad" spots and been removed from the analysis).*

* Clones in this category tend either to represent lowly expressed genes or printed spots of lesser quality that do not hybridise well. Lowly expressed genes can fall below the noise threshold easily, especially if one of the replicate hybridisations has yielded lower quality signal or high noise (as occurred for several slides).

Key to clone set abbreviations and formatting U =

mouse UniGene 2k clone set

N =

NMEBA (normalized mouse embryonic branchial arch library)

I =

1MB "in-house" clone set Ratio greater than 2 or less than 0.5, for spots meeting Set 1 or Set 2 screening criteria (key clones of interest).

Validated Sonic hedgehog responsive genes are indicated in the tables with coloured text: red indicates confirmed up-regulated genes, while confirmed down-regulated genes are shown in blue.

CAl'TION: Clone "Gene ID's" and corresponding descriptions should be interpreted with care and only assumed correct if specifically indicated as sequence verified by the author in the right hand columns of the table. Approximately 16% of UniGene set clones DO NOT match their expected identity when sequenced or are contaminated. Miss-identifications have also been found with a small number of "in house" (1MB) clones.

281

Summary: clones of interest 4 day conditioned media timepoint

Clone Set

Sequence matches accession #

5' similar to SW;GALE RAT P18645 UDP-GLUC0SE4EPIMERASE

U

Yes

No

XM_136069 Mus musculus glucocorticoidinduced leucine zipper (Gilz)

Geometric mean ratio

Description from manufacturers supplied chip file

Obtained and Resequenced

4dCM Putative Up-regulation Set 1 ID from supplied chip file

3.1

AA050732

2.5

W42107

5' similar to gb;X14432 Mouse mRNA for thrombomodulin(MOUSE)

U

Yes

Yes

[as on left]

1.8

AA060795

5' similar to WP:F42H10.4 CE00166 CRIP

U

Yes

Yes

NM 010688 Mus musculus LIM and SH3 protein 1(Laspi)

1.6

AI327133

3" similar to SW;FBN1 BOVIN P98133 FIBRILLIN 1 PRECURSOR

U

3.7

AA050733

5'similar to SW;TSC2 MOUSE Q00992 PUTATIVEREGULATORY PROTEIN TSC-22. [1]

u

Yes

Yes

XM_136069 Mus musculus glucocorticoidinduced leucine zipper (Gilz)

1.5

AA050002

u

Yes

Yes

[as on left]

1.5

AA116287

5' similar to gb;M38337 Mouse milk fat globulemembrane protein E8 mRNA, complete (MOUSE) 5'similarto gb;Z19054 BETA-CATENIN (HUMAN); gb;M90364 Mouse(MOUSE)

u

1.6

AA116711

5'similar to gb;D37874 Mouse FcRn gene. (MOUSE)

u

AA145018

5' similar to gb;U09874 Mus musculus SKD3 mRNA.complete cds (MOUSE)

u

Yes

Yes

[as on left]

1.6

Updated True applicable)

ID from GenBank

BLAST Of

Status post Verification (if applicable)

Validated Shh Target Gene (Induced) Validated Shh Target Gene (Induced) Validated Shh Target Gene (Induced)

Validated Shh Target Gene (Induced) False Positive (highly expressed gene)

no evidence of differential expression

Sequence matches accession # ?

U

Yes

Yes

XM_124281.1| Mus musculus tenascin C (Tnc),

Passed some but not all validation criteria

1

Yes

Yes

HUMBDNFB Homo sapiens brain-derived neurotrophic factor precursor (BDNF)

Passed some but not all validation criteria

NM_008242 Mus musculus forkhead box Di (Foxdi)/Brain factor2

ID from supplied chip file

Description from manufacturers supplied chip file

Clone Set

No

Geometric mean ratio

Obtained and Resequenced

4dCM Putative Up-regulation Set 2

1.6

IMAGE; 3168457

-

N

1.6

BE334139

2.0

BE333652

Similar to TR:Q13485 Q13485 DPC4. ;, mRNA

N

2.6

AA003942

5'similartogb;X56160 ma1 TENASCIN PRECURSOR(HUMAN); gb;X56304 Mouse mRNA fortenascin (MOUSE)

3.5

W48569

human BDNF

BE334139

N

5' similar to gb;D13738 Mouse mRNA for receptortyrosine kinase, complete cds (MOUSE) 5' similar to gb;M80360 Mouse Rep-3 protein mRNA, complete cds(MOUSE) 5" similar to SW;YJAD ECOLI P32664 HYPOTHETICAL 29.8KD PROTEIN IN THIC-HEME INTERGENIC REGION.

U

5' similar to gb;L38607 Mus musculus (MOUSE)

u

Yes

Yes

2.0

AA017847

1.5

AA122755

1.8

W34412

2.3

W54549

im

AA117096

5'similar to gb;X76850 M. musculus mRNA for MAP kinaseactivatedprotein kinase (MOUSE)

u

Yes

Yes

1.6

BE333945

BE333945

N

No

?

1.6

BE333957

similar to TR:Q15131 015131 PISSLRE MRNA.; BE333957

N

Yes

Yes

0.00

N

U U

[as on left]

Validated Shh Target Gene (Induced) False Positive (highly expressed gene)

NM 008445 Mus musculus kinesin family member 3c (Kif3c)

1.9 1.5

BF147370

BF147370

N

No

?

[no GenBank gene hits - "Unknown"]

1.8

BE333748

BE333748

N

No

?

XM_134453 Mus musculus similar to EGLN1 protein (Eglnl)

2.2

BFi47369

similar to SW:BF2_MOUSE Q61345 TRANSCRIPTtON

N

No

?

NM_008242 Mus musculus forkhead box D1 (Foxdi) / Brain factor2

1.7

BF147372

similar to SW:AFAR_RAT P38918 AFLATOXIN B1

1.6

BE456175

N

No

?

NM 025475 Mus musculus RIKEN cDNA 2410007P03 gene (2410007P03Rik)

Description from manufacturers supplied chip file

Clone Set

Sequence matches accession #

[as on left]

IMAGE; 3168545

Updated True ID from GenBank BLAST (if applicable)

BF147397

N

No

?

BE456175

Status post Verification (if applicable)

BC025475 Mus musculus. Similar to hypothetical gene supported by U92995

Obtained and Resequenced

I

Updated True ID from GenBank BLAST (if applicable)

no evidence of differential expression

Validated Shh Target Gene (Induced)

N

Geometric mean ratio

4dCM Putative Up-regulation Set 3 ID from supplied chip file

2.4

BF147397

Status post Verification (if applicable)

AF020308 Mus musculus HRS gene

CAUTION: Clone "Gene ID's" and corresponding descriptions should be interpreted w^ith care and only assumed correct if specifically indicated as sequence verified by the author in the right hand columns of the table. Approximately 16% of UniGene set clones DO NOT match their expected identity when sequenced or are contaminated. Miss-identifications have also been found with a small number of "in house" (1MB) clones.

282

Appendix B: Supplementary Microarray Data

2.8

8E333469

similar to gb:M36332 Moule insulin-like growth

N

Ves

Ves

(as on left. NM_01051.& Mus musculus like growth factor 2 Ilgf2))

2.1

BE333773

BE333773

N

Ves

Ves

{no GenBank gene hits - "Unknownj

U

8E332067

8E332467

N

No

?

2.2

BF011713

BF011713

N

No

?

BF011922

N

No

?

No

?

2.1

BF011922

2.3

BE333715

similar to gb:L08115 Mus musculus antigen (MOUSE

N

2.1

BF147798

BF147798 similar to TR:09VHG4 Q9VHG4 CG8444 PROTEIN. :.

N

2.3

W44060

2.2

AA139715

2.1

AA003328

2.0

AA038306

2.1

AI385846

5' similar to gb;L14677 Mus musculus Epoc-1 mRNA,compiete cds (MOUSE) 5'similar to gb;X62753 FOLATE RECEPTOR, ADULT PRECURSOR (HUMAN);gb;M64782 Mouse folate-binding orotein 1 (MOUSEl 5' similar to gb;U22396 Mus musculuspolyomavirus late initiator promoter binding (MOUSE) 5' similar to gb;X93037 M.musculus mRNA forWDNM1protein (MOUSE) 5'similar to TR;082887 062687 RENAL OSMOTIC STRESSINDUCED NA-CLORGANIC SOLUTE COTRANSPORTER.

XM_136069 Mus musculus glucocorticoidinduced leucine zipper (Gilz)

Validated Shh Target Gene (Induced) no evidence of differential expression

Validated Shh Target Gene (Induced)

XM 124686 Mus musculus pleckstrin homology. Sea and coiled/coil domains 3 (Pscd3) BC025496 Mus musculus. Similar to veside amine transport protein 1

BC014706 Mus musculus, Similar to ATPase, H+ transporting, lysosomal (vacuolar proton pump) membrane sector associated protein MB-9

U U U U U

2.1

A1385848

5'slmilar to TR;041172 041172 ENV.

U

2.2

AA023770

~~~~~~~~ ~:~:::;:t A48184 transcriptioninitiation factor lID

U

2.1

AA060403

~~~~i~~~~,~~~lf~6~s~tmusculusosteocalcin-rerated protein

U

2.1

AA125379

5'similar to gb;D26532 Mouse gene for PEBP2aB2 (MOUSE)

U

2.1

AAI66187

2.2

AA013594

2.1

AA023595

5' similar to gb;L06465 Musmusculus cytochrome oxidase subunit Vla (MOUSE) 5' similarto SW;KC1A_BOVIN P35506 CASEIN KINASEI, ALPHA ISOFORM 5' similar to PIR;A45054 A45054 probableintercellular signal transducer or transmitter Fz-1

insulin~

U U U

4dCM Putative Down-regulation Set 1 ~

" E

.gg Gif! E

" ~"'~ " ~ ai~·2 "c" " ~ &~ ~ ~~ " E 0::

IDfrom supplied chip file

Description from manufadurers supplied chip file

~ .§" u

2

Cl

],l~

If)

Updated True 10 from GenBank BLAST {if applicable)

Status post Verification (If applicable)

False Positive (highly expressed gene)

"

0

0.53

AA162879

5' similar to gb;L12029 Mus musculus cytokine (MOUSE)

0.58

AA139961

5' similar to gb;D26089 Mouse mRNA for c:dc21 homolog(MOUSE)

U

0.57

AA145048

~~:,a~~~~~~~~~U~~)2Mouse testis-specifice-abl protein

U

0.66

AA114591

0.49

AA124045

0.65

C (1343111)

0.62

BE335884

5' similar to gb;L21993 ADENVLATE CVCLASE. TYPEII (HUMAN) 5'similar to gb;D10523 2-0XOGLUTARATE DEHVDROGENASE El COMPONENT(HUMAN) (C (1343111» Sean Grimmond 1~IE335884 similar to TR:060493 060493 SORTING NEXIN 3.

similar to TR:088584 088584 VERSICAN V3 ISOFORM PRECURSOR.; BF011818 5'similar to gb;M64429 Mouse B-raf oncogene mRNA, complete cds(MOUSE) 5' simirarto \\IP;F09E5.3 CE02610 DEOXYRIBOSEPHOSPHATEALDOLASE 5' similar to PIR;S44774 844774 C30A5.1 proteinCaenorhabditis elegans

U

Ves

No

NM 016752 Mus musculus UDP-galactose transloeator 2 (Uga~2)

Got

?

[clone obtained but poor growth, poor plasmid yield, PCR has numerous bands, problems making orobOt

U U I U

0.56

BF011818

0.64

AA119599

0.62

W09388

0.61

AA028564

0.65

1IV62914

5' similar to SW;ACCC_ANASP 008862 BIOTINCARBOXYlASE

U

0.49

W91173

5' similar to SW;NIDO_HUMAN P14543 NIDOGENPRECURSOR

U

0.63

AI892413

5' similar to gb;X70398 M.musculusP311 mRNA (MOUSE)

U

0.21

Sftpl

(Sftp1) se.n Grtmmond

I

V••

(ty po)

Sfrp.1 (typing error in manufacturers entry]

0.61

L (Sfrp-2)

(L (Sfrp-211 se.n Grtmmond

I

V••

V.s

Sfrp-2

U

Ves

Ves

[as on left)

U

Ves

Ves

[as on left]

no evidence of differential expression

no evidence of differential expression

N U U U

? {uncooperative clone]

V.Iid8tod Shh

Target Gene (R.p....sod) Validated Shh Target Gene (R.p....sod)

5' similar to gb;X61399 Mouse F52mRNA for a novel protein (MOUSE) 5' similar to PIR;A41735 A41735hyaluronate-binding protein TSG-6 precursor - human 5' similar to gb;U00674 Musmusculus NMRI fibroglycan (MOUSE) 5' similar to gb;D32040 Mouse mRNA forproteoglycan, PG·M{V3) (MOUSE)

0.56

AA154597

0.62

AA051341

0.64

AA106952

0.47

AA049816

0.41

Rpx3

(Rpx3) Sean Grimmond

I

Ves

Ves

las on left)

0.51

HTK

(HTK) sean Grimmond

I

Ves

No

HTK Ugand

U U

CAPTION: Clone "Gene !D's" and corresponding descriptions should be interpreted with care and only assumed correct if specifically indicated as sequence verified by the author in the right hand columns of the table. Approximately 16% of UniGene settlones DO NOT match their expected identity when sequenced or are contaminated. Miss-identifications have also been found with a small number of "in house" (1MB) clones.

283

ID from supplied chip file

Obtained and Resequenced

Sequence matches accession #

0.61

AA153570

5* similarto TR;G468012 G468012 PKR INHIBITOR P58.

U

0.64

AA000892

5' similarto rii-(,654676 B54676 antiquitin - rat

U

0.33

Nkx2.5

(Nkx 2.5) Sean Grimmond

1

Yes

Yes

(Fgf8) Sean Grimmond

1

Los t

?

BE335886

similarto gb:X83536 M.musculus mRNA for

N

No

?

BE456918

similarto gb:X55885 ER LUMEN PROTEIN RETAINING RECEPTOR 1 (HUMAN); BE456918 similarto gb:X55885 ER LUMEN PROTEIN RETAINING RECEPTOR 1 (HUMAN); BE332646

N

0.28

BF012253

similarto SW:PTN_MOUSE P20935 PLEIOTROPHIN

0.44

BF147023

No

?

mm 0.66

0.62

Fgf8

0.53

AA061454

0.67

AA153700

0.16

AA154035

0.47

W84068

0.65

AA004018

1^

W91144

0.56

AA060051

0.64

AA066354

Description from manufacturers supplied chip file

Clone Set

Geometric mean ratio

4dCM Putative Down-regulation Set 2

BF147023 5' similarto SW;S61A CANFA P38377 PROTEIN TRANSPORTPROTEIN SEC61 ALPHA SUBUNIT. 5'similar to gb;L08266 Mouse Face mRNA, complete cds (MOUSE) 5' similarto gb;X58957 TYROSINE-PROTEIN KINASE ATK (HUMAN);gb;L08967 Mus musculus B cell cytoplasmic tyrosine kinase (MOUSE) 5' similarto gb;X00734_cds1 TUBULIN BETA-5CHAIN (HUMAN); gb;X04663 Mouse mRNA for beta-tubulin (MOUSE) 5' similarto gb;X08058_rna1 GLUTATHIONES-TRANSFERASE P (HUMAN); gb;U15654_ma1 Mus musculus glutathionestransferase pi class (MOUSE) 5' similarto SW;ANGT MOUSE P11859ANGIOTENSINOGEN PRECURSOR. [1] 5' similarto gb;S62138 GROWTH ARREST ANDDNA-DAMAGEINDUCIBLE PROTEIN GADD153 (HUMAN) 5' similarto gb;M64174 TYROSINE-PROTEIN KINASE JAK1 (HUMAN)

U

3'similartoTR;P97329 P97329 KINESIN-LIKE PROTEIN 174.

U

(LhxS) Sean Grimmond

1

Yes

(typ 0)

(Sfrp-2) Sean Grimmond

1

Yes

Yes

(B (1575d2)) Sean Grimmond

1

Yes

?

human syndecan 1

1

Yes

Yes

No

?

U

U U U U

AA137337 LhxS

e.11

Sfrp-2

0.47

B(1575d2)

0.58

r01486

0.53

BE456989

similar to gb:X51703 Mouse mRNA for ubiquitin (MOUSE); BE456989 similarto gb:X51703 Mouse mRNA for ubiquitin (MOUSE); BE333409

N

0.57

BE334020

similarto TR:O76058 076058 DJ1409.2 ;, mRNA

N

0.58

BF147350

0.61

BE333462

0.63

BE333686

0.59

BF011644

0.04 \

Sfrp-4

0.66

HNF35

0.55

BE332524

0.51

L22473

^Bi

Sox9 mouse

0.56

IMAGE; 3169081

0.64

BF147690

; 0.37

BF147749

NM_019766 Mus musculus telomerase binding protein, p23 (Tebp-pending)

U

0.49

BE333686

no evidence of differential expression ? [uncooperative clone]

U

0.27

BF147350 similarto gb:M60474 Mouse myristoylated

[as on left] [could not obtain clone and altemative from submitter failed] 3' similarto gb:X83536 M.musculus mRNA for membrane-type matrix (MOUSE);, mRNA sequence

Status post Verification (if applicable)

N N

similar to gb:D12907 Mouse gene for47-kDa heat

Updated True ID from GenBank BLAST (if applicable)

Lhx5 (typing error in manufacturers entry) [as on left] Angiopoietin2 [as on left]

no evidence of differential expression Validated Shh Target Gene (Repressed) Validated Shh Target Gene (Induced) no evidence of differential expression

N N N

similarto SW:RL6_MOUSE P47911 60S RIBOSOMAL

N

(Sfrp-4) Sean Grimmond

1

(HNF35) Sean Grimmond

1

similarto TR:O65204 065204 ACTIN.;, mRNA

N

HUMAN BAX ALPHA

U

-

1

NM_010336 Mus musculus endothelial differentiation, lysophosphatidic acid G-proteincoupled receptor, 2 (Edg2)

Yes

No

MUSALGL Mus musculus alpha-globin

Yes

(typ 0)

HNF3beta [typing error in manufacturers entry]

Yes

Yes

[as on left]

No

?

BC021403 Mus m., ADP-ribosylation factor 1

no evidence of differential expression Validated Shh Target Gene (Induced)

False Positive (highly expressed gene)

N BF147690 similarto TR:O70341 070341 TAIPOXINASSOCIATED CALCIUM BINDING PROTEIN 49. ; BF012221 BF147749

N N

0.57

BF147799

similarto TR:088665 088665 BROMODOMAIN-CONTAINING PROTEIN BP75.; BF147799 similarto TR:088665 088665 BROMODOMAIN-CONTAINING PROTEIN BP75. ; BF011673

0.11

BF147800

BF147800 similarto TR:035297 035297 SECRETED APOPTOSIS

0.55

BF011889

0.54

W65601

0.64

AA168789

0.29 0.53

BF011889

N

N

No

?

NM_009144 Mus musculus secreted frizzledrelated sequence protein 2 (Sfrp2)

N

No

?

XM 124528 Mus musculus WD repeat domain 1 (Wdr1)

5' similarto gb;M76231 SEPIAPTERIN REDUCTASE(HUMAN)

U

5' similarto gb;J03210 72 KD TYPE IV COLLAGENASEPRECURSOR (HUMAN); gb;M84324 Mus musculus type IV collagenase mRNA.complete cds (MOUSE)

U

Amh

(Amh) Sean Grimmond

1

Yes

Yes

[as on left]

Sfrp-3

(Sfrp-3) Sean Grimmond

1

Yes

Yes

[as on left]

'*^'

Validated Shh Target Gene (Repressed)

^

Validated Shh Target Gene (Repressed) no evidence of differenfial expression

CAUTION: Clone "Gene ID's" and corresponding descriptions should be interpreted with care and only assumed correct if specifically indicated as sequence verified by the author in the right hand columns of the table. Approximately 16% of UniGene set clones DO NOT match dieir expected identity when sequenced or are contaminated. Miss-identifications have also been found with a small number of "in house" (1MB) clones.

284 0.55

0.62

Appendix B: Supplementary Microarray Data P(Ms~

BE332452

(P (Msf) Sean Glimmond

similar to gb:XOS021 Murine mRNA with homology to yeast l29 ribosomal protein (MOUSE); BE332452 similar to gb:XOS021

Murine mRNA with homology to yeast L29 ribosomal protein

I

N

(MOUSE); BE332607 0.33

hl3471

human HSPG

I

0.60

BF011570

BF011570

N

0.65

BF147807

similar to SW:TDXN_MOUSE 008807 THIOREOOXIN

N

0.55

BF147061

similar to TR:Q07065 007065 P63 PROTEIN. ;, mRNA

N

Yes

Yes

HUMHSPGC Human heparan suttate proteoglycan (HSPG) core protein

no evidence of differential expression

Updated True ID from GenBank BLAST ~f applicable)

Status post Verification (If applicable)

4dCM Putative Down-regulation Set 3

..~ c

~~ E g

"

;;

10 from

supplied chip file

&¥ " E m

n"

~~

Updated True 10 from GenBank BLAST (If applicable)

"

0

U

Yes

No

XM_123629 Mus musculus Kruppel·like factor 7 (ubiquitous) (Klf7)

U

Yes

Yes

las on left]

U

Yes

Yes

las on leftJ

;;

"g8 '" " c "ua5~.!2 me

Status post Verification (If applicable)

5'similar to gb;X17500 Mouse mRNA for putative transcription fadoraf the insulin (MOUSE) 5' similar to gb;L25444 60S RIBOSOMAL PROTEINL35A (HUMAN) 5' similar to SW;RL39_RAT P02404 60S RIBOSOMALPROTEIN L39. (2J PIR;R6RT39 5' similarto SW;GBG5_BOVIN P30670 GUANINENUCLEOTIOEBINDING PROTEIN G(IYG(S)IG(O) GAMMA-5 SUBUNIT. 5' similar to gb;Z26676 60S RIBOSOMAL PROTEIN L36(HUMAN) 5' similar to SW;COPZ_BOVIN P35604 COATOMER ZETASUBUNIT

1.9

M002476

1.5

BE456172

similar to SW:RS9...;.HUMAN P46781 40$ RIBOSOMAL

1.6

M166606

5' similar to gb;K02928 Mouseribosomal protein L30 gene, complele cds (MOUSE)

1dT Putative Up-regulation Set 2 c

m

~

u 0

~~

~

IDfrom supplied chip file

U>

Description from manufacturers supplied chip file

M033401

1.5

M163512

~~

g~ ~

Updated True 10 from GenBank BLAST (if applicable)

0

5' similar to gb;M19643 Mouse Krox-24 protein mRNA,

U

Yes

Yes

(also known as EGR-1]

I

Yes

Yes

[as on left]

Status post Verification (if applicable)

m

U>

5' similarto SW;SUI1_HUMAN P41567 PROTEINTRANSLAnON FACTOR SUl1 HOMOLOG 5' simiiartogb;X12517 U1 SMAU NUCLEARRIBONUCLEOPROTEIN C (HUMAN) 5' similar to PIR;S40989 540989 hypotheticalprotein F55H2.6 Caenorhabditis elegans

?,9

W56963

2,9

M162264

3.6

AJ243425

human EGR-1

BF147661

similar to SWCOXO MOUSE P17665 CYTOCHROME C OXIDASE POLYPEPTIDE VIIC PRECURSOR; BF147661 similar to SW:COXO MOUSE P17665 CYTOCHROME C

1.7

al~

"

CI

1.6

§"

~

False Positive (DNA prepeffed) False Positive (DNA prep effect)

1dT Putative Up-regulation Set 3 c

~

.g~

a;r!! E

g

,l!

10 from supplied

'gB " " " al~ ~ ~.~ U ~:: £E ~ ;jj

Description from manufacturers supplied chip file

me

u '" c

c 0

chip file

CI

Updated True 10 from GenBank BLAST (if applicable)

Status post Verification (if applicable)

0

2.2

M123346

2.6

W09926

2.1

M026564

2.1

M145793

2.2

BF012276

2.5

M163695

2.0

M163916

2.2

W34432

2.4

BE332452

2.0

IMAGE; 3169306

2.1

BF011912

5' similar to SW;P120_MOUSE P30999 P120 PROTEIN. 5' similar to gb;J03544 GLYCOGEN PHOSPHORYLASE, BRAINFORM (HUMAN) 5' similarto PIR;S44774 544774 C30A5.1 protein Caenorhabditis elegans 5'similar 10 gb;X57351 INTERFERON-INDUCIBLE PROTEIN 16D(HUMAN) similar to TR:092530 092530 PROTEASOME INHIBITOR

5' similar to gb;U20156 Mus musculus macrophagemigration inhibitory factor (MOUSE) 5' similar to gb;U11027 Mus musculus C57BU6J Sec61 protein complexgamma subunit (MOUSE) 5' similar to PIR;A45174 A45174 eyes absent

U

Fail

?

similar to gb:X05021 Murine mRNA with homology to yeast L29 ribosomal protein (MOUSE); BE332452 similar to gb:X05021 Murine mRNA with homology to yeast l29 ribosomal protein (MOUSEl; BE332607

BF011912

CAlITlON: Clone "Gene !D's" and corresponding descriptions should be interpreted with care and only assumed correct if specifically indicated as sequence verified by the author in the right hand columns of the table. Approximately 16% of UniGene sef clones DO NOT match their expected identity when sequenced or are contaminated. Miss-identifications have also been found with a small number of "in house" (1MB) clones,

295

1dT Putative Down-regulation Set 1 m

~

m

E

.g~ a;:~

~

0::

10 from supplied chip file

m

rn

Description from manufacturers supplied chip file

m ~ .2 0

~

~8 m ~

m m '

"C

~~

8(1)~

~ ~.~

!e ~

Updated True applicable)

ID

from

GenBank BLAST

(~

Status post Verification (if applicable)

0

0.65

AAOO2979

5' similar to SW;ATNe_CHICK P33879 sodium/potassium-

transporting atpase beta-2 chain mouse bAC:;"

0.48

X03672

0.64

AA116745

5' similar to gb;M36829 Mouse heat-shock proteinhsp84 mRNA (MOUSE) 5'similarto SW;PAD1_SCHPO P41876 PADl PROTEIN. 111

0.64

AA139447

0.39

BF147395

0,48

BF147652

0.62

BF012160

0.61

AA036274

~~3~:)SOR(HUMAN);gb;M66620 Mouse thrombospondin 3

0.65

AA123346

5' similar to gb;X12597 HIGH MOBILITY GROUPPROTEIN HMG1 (HUMAN); gb;UD0431 Mus musculus HMG-1 mRNA, com leteCds (MOUSE)

BF147395 similar to gb:X56468_ma114-3-3 PROTEIN THETA (HUMAN); BE334300 BF141652 similar to SW:143T_HUMAN P2734814-3-3 PROTEIN TAU

similar to $W:CNBP_MOUSE P53996 CELLULAR NUCLEIC

5' similar to gb;Z19585 THROMBOSPONDIN 4

0.65

AA162371

5'similar to TR:G55535 G55535100 KDA PROTEIN.

0.59

BE334329

similar to TR:095406 095406 CORNICHON. ; BE334329

0.67

BF147721

0.56

AA044542

0.39

AA116762

0,33

BE332606

0.58

IMAGE; 3166549

0.58

BF147439

0.66

AAOO6793

0.63

AA066708

0.61

Al692436

0.61

AA066481

0.60

AI692633

0.49

BF147497

similar to SW:ANX2_MOUSE P07356 ANNEXIN II ;, mRNA 5' similar to gb;J04046 CALMODULIN (HUMAN);gb;M19380 Mouse calmodulin (MOUSE) 5'similar to gb;S65738 OESTRIN (HUMAN); gb;D00472 Mouse mRNA forcofilin, complete cds and flanks (MOUSE) similar to gb:M33212 Mouse nucleolar protein N038 mRNA, complete cds (MOUSE); BE332606

similar to SW:ROF_HUMAN P52597 HETEROGENEOUS 5' similar to gb;l25080 TRANSFORMING PROTEINRHOA (HUMAN) 5' similar to gb;D90151 Mouse mRNAforCArG-binding fador-A. complete cds (MOUSE) 5' similar to gb;J04181 Mouse A-X adin mRNA, completecds (MOUSE) 5' similar to gb;X65488_cds1 HETEROGENOUSNUClEAR RIBONUCLEOPROTEIN U (HUMAN) 5'similarto gb;X63526 ELONGATION FACTOR 1-GAMMA (HUMAN) similar to sw:nu4rn_mouse p03911 nadh-ubiquinone ~:dOreductase chain 4; bf147497 similar to sw:nu4m_mouse 3911 nadh-ubiauinone

1dT Putative Down-regulation Set 2 ~

~

0

~~ E g

10 from supplied chip file

'"

Description from manufadurers supplied chip file

3lm

§ 0

~

BU)~

"g~ m

ai2·9

m' .5 g

mE

~

"co>

5-~ ~

:5U) rn

Updated True 10 from GenBank BLAST (if applicable)

m

Status post Verification (if applicable)

0

0.64

AA123466

5'similar to gb;M21019 Mouse R-ras mRNA, complete cds (MOUSE)

0.4>

aa083969

human ODC

0.52

IMAGE; 3166664

0.48

BF147705

0.48

IMAGE; 3169515

0.61

AA014475

0.63

AA003942

0.66

AAOO2277

0.67

AA139665

0.53

Sfrp-2

similar to TR:089113 0891131ER5.;, mRNA

5' similar to gb;X58965 NUCLEOSIDE DIPHOSPHATEKINASE B (HUMAN) 5' similarto gb;X56160_ma1 TENASCIN PRECURSOR(HUMAN); gb;X56304 Mouse mRNA fartenascin (MOUSEl 5' similar to PIR;S49172 549172 translationinitiatianfador elF4gamma - human 5' similar to SW;YB48_YEAST P38129 HYPOTHETICALTRPASP REPEATS CONTAINING PROTEIN IN PGll-KTR4 INTERGENIC REGION.

U

Yes

Yes

XM_124281.11 Mus musculus tenascin C (Tne),

I

Ye.

Yes

(as on teft)

Passed same but not all validation crtleria

Validated Shh (Sfrp-2) Sean Grimmond

Target GeM (Re....sed)

0:38

BF147535

similar to SW:MAPR_MOUSE 055022 MEMBRANE ASSOCIATED PROGESTERONE RECEPTOR COMPONENT. ; BF147535 similar to SW:MAPR MOUSE 055022 MEMBRANE

0.62

BF147025

0.21

AA041626

5' similar to SW;IPP2_RABIT P11845 PROTEINPHOSPHATASE INHIBITOR 2

BF147025

0.3'4

aa129991

human Elongation factor 1.a1

-

CAUTION: Clone "Gene !D's" and corresponding descriptions should be interpreted with care and only assumed correct if specifically indicated as sequence verified by the author in the right hand columns of the table. Approximately 16% of UniGehe set clones DO NOT match their expected identity when sequenced or are contaminated. Miss-identifications have also been found with a small number of "in house" (1MB) clones,

296

Appendix B: Supplementary Microarray Data

0.61

BE332524

0.63

BE456962

0.48

l

BF011932

0.57

BF147798

0.47

BF147804

0.19

AA067001

0.66

AA034560

similar to TR:065204 065204 ACTIN. ;, mRNA BE456962 similar to SW;N358_HUMAN P49792 NUCLEAR PORE similar to SW:RB48_HUMAN a09028 CHROMATIN ASSEMBLY FACTOR 1 P48 SUBUNIT; BF011932

BF14n98 similar to TR:Q9VHG4 Q9VHG4 CG8444 PROTEIN.;,

N

No

?

BC014706 Mus musculus. Similar to ATPase, H+ transporting, lysosomal (vacuofar proton pump) membrane sector associated protein M8-9

similar to SW:ARP3_HUMAN P32391 ACTIN·L1KE PROTEIN

5' similar to gb;M15990 PROTD-ONCOGENETYROSINEPROTEIN KINASE YES (HUMAN); gb;X67677 M.musculus c;.. vesmRNA (MOUSEl 5' similar to WP;ZK945.3 CEOl734 PUMILIO-REPEATLIKE DOMAIN

0.54

BF147388

BF147386 similarto SW:SE15_HUMAN 06061315 KD

0.65

BE331853

similar to SW:VBP1 HUMAN Q15765 VON HIPPEL-LiNDAU BINDING PROTEIN-I; BE331853

0.30

IMAGE; 3168999

0.60

BF147561

similar 10 SW,NPL I_MOUSE P28856 NUCLEOSOME

1dT Putative Down-regulation Set 3 c

~

E

.g .g Q)~

E

g

10 from supplied chip file

;; (f)

Description from manufacturers supplied chip file

m

c

.Q

u

Cl

"

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