Chapter 1: Literature review

1 A REVIEW OF THE EPH/EPHRIN RECEPTOR

TYROSINE KINASES

1.1 Overview of the Eph/ephrin receptor-ligand family

The Eph receptor tyrosine kinases and their ephrin ligands are cell surface molecules with a wide range of biological functions which influence cell behaviour during both embryogenesis and adult life (Poliakov et al., 2004). These functions include roles in:- (1) the direction of cell positioning and migration (Klein, 2004); (2) axon guidance during development (Flanagan, 2006); (3) control of tissue morphogenesis and patterning (Coulthard et al., 2002; Hinck, 2004; Tepass et al., 2002); (4) defining tissue boundary formation during somitogenesis (Sela-Donenfeld and Wilkinson, 2005); (5) the development of the vascular system (Zhang and Hughes, 2006); (6) neural plasticity (Murai and Pasquale, 2004; Yamaguchi and Pasquale, 2004); (7) tumour invasion and metastasis (Wimmer-Kleikamp and Lackmann, 2005); (8) immune function (Wu and Luo, 2005), haematopoiesis (Okubo et al., 2006) and blood clotting (Prevost et al., 2005); (9) an emerging role in the biology of stem cells (Sancho et al., 2003) and (10) tissue repair and maintenance (Hafner et al., 2004).

The Eph/ephrin receptor-ligand family, (which will be subsequently referred to jointly as the Eph/ephrin proteins), principally modifies cytoskeletal organisation and cell adhesion (Wilkinson, 2000). Cytoskeletal modification regulates the dynamics of cellular protrusions, affects cell–cell repulsion and adhesion and attachment to the extracellular matrix, and influences cell migration (Boyd and Lackmann, 2001). The signalling cascades that Eph-ephrin interactions initiate ultimately converge on targets such as integrins and small Rho-family GTPases (Noren and Pasquale, 2004). This is in contrast to many receptor tyrosine kinases (RTKs), which were first identified as oncogenes, because they activate signalling pathways that target gene transcription, and regulate cell proliferation and/or differentiation (Schlessinger and Ullrich, 1992; Ullrich and Schlessinger, 1990).

The Eph/ephrin proteins, which constitute the largest class of RTKs in the human genome, are conserved amongst vertebrates, insects, nematodes and sponges (Drescher, 2002; Gu and Gu, 2003; Tuzi and Gullick, 1994; Vidovic et al., 2006). The Eph/ephrin family of receptors are so designated because they are related to Eph (pronounced

Queensland Institute of Medical Research 1 Chapter 1: Literature Review

“eff”), the first receptor described, which was named for its expression in an erythropoietin producing human hepatocellular carcinoma cell line (Hirai et al., 1987).

The ligands of the Eph receptors are known as ephrins (pronounced “effrins”), an abbreviation derived from Eph family receptor interacting proteins. There are ten EphA receptors, EphA1–EphA10 (pronounced “eff-A”), and six EphB receptors, EphB1– EphB4 and EphB6 in vertebrates (Eph Nomenclature Committee, 1997; Lemke, 1997), and an additional EphB receptor, EphB5, which exists in chickens (Wilkinson, 2001). The nematode Caenorhabiditis elegans possesses a single Eph receptor, Vab-1, and four ephrins, Vab-2/EFN1, EFN2–4 (George et al., 1998; Wang et al., 1999). The fruitfly Drosophila melanogaster has a single Eph receptor, Dek, and a single transmembrane ephrin, dephrin (Bossing and Brand, 2002; Scully et al., 1999).

Figure 1.1 Eph receptor and ephrin ligand binding preferences. (Modified from Wilkinson 2000).

The initial distinction between EphA and EphB receptors was based on sequence differences within the extracellular ligand binding domain, but also corresponds to the binding preferences for the six glycosylphosphatidylinositol (GPI)-linked ephrin-A ligands and the three transmembrane ephrin-B ligands respectively (Eph Nomenclature Committee, 1997; Gale and Yancopoulos, 1997; Himanen and Nikolov, 2003a; Lemke, 1997) (Figure 1.1).

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The Eph/ephrin receptor-ligand interactions are promiscuous within each A or B class, with variations in binding affinities (Flanagan and Vanderhaeghen, 1998), although EphB4 only binds ephrin-B2 (Blits-Huizinga et al., 2004). There are also exceptions in the binding preferences between A and B class, as EphA4 binds to ephrin-B ligands (ephrin-B2–ephrin-B3) (Blits-Huizinga et al., 2004), and EphB2 binds to ephrin-A5 (Himanen et al., 2004).

1.2 The structural basis for Eph/ephrin receptor-ligand interaction

The Eph RTKs are type-I transmembrane proteins (Hubbard, 1999; Ullrich and Schlessinger, 1990). Their extracellular portion includes an 180-amino acid N-terminal “globular” ligand (ephrin) binding domain (Labrador et al., 1997), a cysteine rich region containing an epidermal growth factor (EGF)-like motif and two fibronectin type III repeats (Himanen and Nikolov, 2003b). This is separated by a transmembrane segment from the cytoplasmic portion of the receptor, which includes a juxtamembrane segment, tyrosine kinase domain, a sterile alpha motif (SAM) domain (Schultz et al., 1997) and a PDZ (post-synaptic density protein-95, Drosophila disc large tumour suppressor (Dlg), zona occludens-1) (Fanning and Anderson, 1999; Himanen and Nikolov, 2003a) (Figure 1.2).

The ephrin receptor-binding domain also contains a globular N-terminal binding, or receptor-binding domain, which is separated from the membrane by a linker of approximately 40 amino acids (Himanen and Nikolov, 2003a; Himanen and Nikolov, 2003b). The ephrin-A ligands are attached via a GPI linkage to the plasma membrane (Himanen and Nikolov, 2003a; Himanen and Nikolov, 2003b). The ephrin-B ligands have a short highly conserved cytoplasmic domain consisting of 80 amino acids, which includes a C-terminal PDZ-binding motif (Gauthier and Robbins, 2003; Himanen and Nikolov, 2003a; Himanen and Nikolov, 2003b; Lin et al., 1999).

The extracellular domain of the EphB2 receptor has a compact globular structure with a β-sandwich “jellyroll” folding topology, which comprises two anti-parallel β-sheets, a concave sheet composed of six strands and a convex sheet composed of seven strands, which together form a compact β-sandwich that has an extensive hydrophobic core (Himanen et al., 1998). The β-strands are connected by loops of varying length; in particular a long well-ordered loop (H–I) and two partially disordered loops which play a central role in ligand recognition and binding (Himanen et al., 1998). This β-jellyroll

Queensland Institute of Medical Research 3 Chapter 1: Literature Review folding topology shares no precise sequence or structural homology to other known proteins, although a similar folding topology is found in some carbohydrate binding proteins (lectins).

Figure 1.2 The structure of the Eph receptors and their ephrin ligands. The EphA and EphB receptors have a conserved domain structure. The ephrin-A ligands are attached to the cell membrane by a GPI anchor. The ephrin-B ligands are transmembrane proteins. (Modified from Murai and Pasquale, 2003)

The only sequence feature that is conserved within but differs between the EphA and EphB receptor classes is the length of the H–I loop, and consequently this loop is referred to as the class-specificity loop. The H–I loop consists of 17 amino acid residues in EphA receptors and 13 amino acid residues in EphB receptors (Himanen et al., 1998).

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In mutagenesis experiments, a chimaeric EphB2 receptor constructed with an EphA3 H–I loop recognised both ephrin-A and ephrin-B ligands (Himanen et al., 1998).

SAM is a protein interaction domain of approximately 70 amino acids, which was identified originally in yeast sexual differentiation and Drosophila proteins. Subsequently, SAM domains have been identified in over 100 proteins of fungi, protozoa and animals, in which they play a role in developmental regulation (Schultz et al., 1997). All the Eph receptors contain a highly conserved C-terminus SAM domain with approximately 50% identity (Stapleton et al., 1999). The X-ray crystal structure of the SAM domain of EphA4 has been resolved and consists of four short α-helices and a longer C-terminal α-helix in a “lobster claw” configuration, which form homo-dimers within both a crystal environment and in solution (Stapleton et al., 1999).

The ephrins also contain a conserved extracellular receptor binding domain, which has novel topology for a signalling protein, shared only with plant phytocyanins and nodulins. X-ray crystallographic studies of ephrin-B2 indicate that the ephrin receptor binding domain has a globular β-barrel structure with a Greek key folding topology, and crystals of uncomplexed ephrin-B2 form homo-dimers by burying the hydrophobic regions around the G–H loop (Toth et al., 2001). The G–H loop is involved in receptor binding, so it is likely that there is significant rearrangement of the ephrin homo-dimers upon Eph receptor interaction (Himanen et al., 2001). In contrast, ephrin-B1 is a monomer and its ectodomain has a distinct G–H loop conformation, which is not involved in homo-dimerisation as has been reported for ephrin-B2. Ephrin-B1 is glycosylated at Asn 139 in contrast to ephrin-B2 which is glycosylated at Asn 39, which may account for the lower binding affinity of ephrin-B1 to EphB2 (Nikolov et al., 2005). The entire cytoplasmic domain of ephrin-B2 has been studied using nuclear magnetic resonance (NMR) spectroscopy, revealing that the 48 N-terminal residues of the cytoplasmic domain are unstructured and prone to aggregation, whereas the highly conserved 33 C-terminal residues form a well-packed hairpin structure followed by a flexible PDZ-binding tail (Song, 2003; Song et al., 2002).

The structural basis of Eph and ephrin interaction, in particular the interaction of EphB2 with ephrin-B2, has been elucidated by X-ray crystallographic studies (Himanen et al., 2001). A comparison of the bound and free EphB2 and ephrin-B2 suggests that the long G–H loop of ephrin-B2 inserts into a channel on the surface of EphB2 during binding, which proceeds via an induced-fit or ligand-induced receptor folding mechanism. The

Queensland Institute of Medical Research 5 Chapter 1: Literature Review loops which form the sides of this binding channel on EphB2 are unstructured in the unbound receptor, but ligand binding induces folding of the EphB receptor, generating an extensive interaction surface that is complementary to the G–H loop of ephrin-B2 (Himanen et al., 2001). Two EphB2–ephrin-B2 dimers then join to form a hetero- tetramer, in which each ligand interacts with two receptors and each receptor interacts with two ligands (Figure 1.3).

Figure 1.3 Eph-ephrin interaction. Dimerisation and tetramerisation interfaces of a receptor-ligand tetrameric complex. (Modified from Murai and Pasquale, 2003)

One interface is extensive and is responsible for the 1:1 high affinity receptor-ligand dimerisation, whereas the second interface is smaller and responsible for the assembly of Eph-ephrin dimers into the circular tetramer. The high affinity dimerisation interface does not provide a clear reason for Eph class discrimination and suggests the possibility of cross-subclass Eph-ephrin interaction. The lower affinity Eph-ephrin tetramerisation shows a clear structural basis for subclass specificity due to interaction with the Eph “class-specificity” loop (Himanen and Nikolov, 2003b).

The Eph/ephrin interaction model elucidated by the crystal structure has also been analysed using a novel random mutagenesis approach. The interaction of EphA3 with ephrin-A5 has been studied by the generation of an expression library of EphA3 extracellular domain point mutants with reduced ephrin-A5 binding (Smith et al., 2004a). These studies confirm that there are high and low affinity binding interfaces within the EphA3–ephrin-A5 receptor-ligand interaction which are necessary for signalling, and suggest that there also exists a third Eph/ephrin interface within the cysteine-rich linker (Day et al., 2005; Smith et al., 2004a).

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1.3 The molecular mechanisms of Eph/ephrin protein signalling

1.3.1 Introduction

The Eph/ephrin proteins regulate cell migration, and cell-cell repulsion and attachment to the extracellular matrix (Davy and Soriano, 2005). Consequently, the signalling pathways that are activated upon receptor-ligand interaction ultimately converge on the cytoskeleton and cell adhesion molecules (Noren and Pasquale, 2004; Vearing and Lackmann, 2005). The mechanisms of Eph receptor kinase activation are similar to those of other RTKs (Eswarakumar et al., 2005; Schlessinger and Ullrich, 1992). The Eph receptor tyrosine kinases catalyse the transfer of the γ-phosphate of adenosine triphosphate (ATP) to the side-chain hydroxyl groups of tyrosine residues in protein substrates (Hubbard, 1999). Eph/ephrin receptor-ligand binding results in the formation of an Eph-ephrin tetramer (Himanen and Nikolov, 2003b), which brings the cytoplasmic domains of the two catalytically auto-inhibited Eph receptor monomers into close proximity in an orientation favourable for phosphorylation of the receptor monomers by one another (= trans-autophosphorylation) (Hubbard, 1999) (Figure 1.4).

The complete activation of Eph receptor function requires both the presence of receptor catalytic activity and the creation of docking sites for downstream signalling proteins (Hubbard, 1999). The phosphorylation of a specific tyrosine within the activation-loop (A-loop) of the kinase domain is necessary for the catalytic activity of the receptor (Schlessinger, 2000). In the non-phosphorylated form, the A-loop folds into the catalytic pocket of the kinase domain and inhibits kinase activity; however, phosphorylation changes the steric forces within the molecule and liberates the phosphorylated A-loop outside the pocket and results in kinase activation (Binns et al., 2000). In addition, the non-phosphorylated juxtamembrane region forms a well-ordered helical structure, which contributes to auto-inhibition of catalytic activity through interaction with the N-terminal region of the kinase domain which distorts a key α-helix. This distortion prevents kinase activation by displacing one of the catalytic site glutamate residues away from the active site (Hubbard, 2001). The p+1 loop is a small conserved motif (WTAPE) immediately downstream of the A-loop which plays a role in activation loop phosphorylation (Yuan et al., 2004b). Subsequent phosphorylation of two specific conserved juxtamembrane tyrosine residues (tyrosines 596 and 602 in EphA4, and tyrosines 604 and 610 in EphB2) results in steric and electrostatic forces pushing the

Queensland Institute of Medical Research 7 Chapter 1: Literature Review phosphorylated juxtamembrane region away from the kinase site (Binns et al., 2000; Ellis et al., 1996; Kalo and Pasquale, 1999a; Zisch et al., 2000). This relieves the structural constraints which distorted the active site and allows binding of ATP in the catalytic cleft and therefore kinase activation (Wybenga-Groot et al., 2001).

Figure 1.4 Receptor-ligand engagement induces bi-directional signalling. Both the Eph receptors and the ephrin-ligands become tyrosine phosphorylated either through trans-autophosphosphorylation (receptors) or recruitment of other tyrosine kinases. (Modified from Murai and Pasquale, 2003)

Furthermore, the phosphorylated juxtamembrane region becomes available for interaction with downstream signalling proteins, thus contributing to the full function of the Eph receptor (Holland et al., 1997; Zisch et al., 1998). The protein kinase domain contains the consensus sequence GXGXXGX (15–20) K, which allows ATP binding at the catalytic site, and replacement of the invariant lysine residue (Lys 662) completely abolishes kinase activity (Hanks et al., 1988; Ullrich and Schlessinger, 1990). In summary, the formation of the Eph-ephrin hetero-tetramer results in the trans- autophosphorylation of conserved tyrosine residues between the Eph receptor

8 Queensland Institute of Medical Research Chapter 1: Literature review monomers, which in turn leads to activation of the catalytic domain, phosphorylation of associated downstream signalling molecules and the production of docking sites for adaptor proteins (Himanen and Nikolov, 2003b).

Several recent papers have presented findings that suggest that Eph receptor kinase activation is not completely explained by the model described above, and indicated that ligand binding may not be required for receptor phosphorylation. The phosphotyrosine content of EphA2 did not correspond to enzymatic activity in mammary epithelial cells (Zantek et al., 1999). All the Eph receptors have a threonine (Thr) in the p+1 loop in contrast to all other RTKs which have a methionine (Met) at this position. A Met→Thr point mutation, in the p+1 loop of the receptor tyrosine kinase, RET, resulted in medullary thyroid tumours with higher metastatic potential, suggesting that this mutation (Met→Thr) resulted in gain of function. Therefore, by extrapolation, the Eph receptors, with a wild type p+1 loop Thr, could be constitutively active. In vitro studies confirmed that Eph receptors activate the signalling protein, STAT3, in the absence of ligand, suggesting that receptor dimerisation was not required for Eph receptor autophosphorylation and downstream signalling (Yuan et al., 2004b). In contrast to these findings, the kinase domains from EphA10 (Aasheim et al., 2005a; Aasheim et al., 2005b) and EphB6 (Freywald et al., 2002) are non-functional; therefore, these two receptors cannot function by phosphorylating cytoplasmic proteins, but presumably interact with other signalling molecules and may be trans-phosphorylated by other EphB receptors (Freywald et al., 2003).

1.3.2 Adaptor proteins

The phosphorylation of conserved tyrosine residues activates Eph receptors and the consequent structural rearrangements in the cytoplasmic domain allow binding and recruitment of adaptor proteins and downstream signalling molecules required for the interpretation and propagation of the Eph/ephrin receptor-ligand binding signal (Kullander and Klein, 2002). The adaptor proteins contain functional protein-protein interaction domains such as the Src-homology 2 (SH2) and Src-homology-3 (SH3) domains, which bind specific sequence motifs (e.g. pYXN), and phospho-tyrosine binding (PTB) domains, which recognise the sequence NPXpY (Schlessinger and Lemmon, 2003). The SH2 domain-containing proteins include:- (1) various proteins with enzymatic function (Src, Btk, Zap70, Shp1, PLC, GAP, Vav); (2) adaptor proteins with no intrinsic enzymatic activity (Grb2, Grb10, Crk, Nck); (3) regulatory subunits of

Queensland Institute of Medical Research 9 Chapter 1: Literature Review signalling proteins (SOCS and SAP); (4) transcription factors (STAT) and (5) scaffold proteins (Shc, SLP76, Grb7) (Schlessinger and Lemmon, 2003).

The following SH2 domain-containing adaptor proteins and enzymes have been reported to bind activated Eph receptors:- PI3 kinase (Pandey et al., 1994); SLAP (Src- like adaptor protein) (Pandey et al., 1995a); Fyn (Davy et al., 1999; Ellis et al., 1996) ; Grb2, Grb10 (Stein et al., 1996) ; Ras GTPase activating protein (Ras GAP) (Holland et al., 1997) ; Yes (Zisch et al., 1998) ; γ isoform phospholipase C (PLCγ) (Zisch et al., 1998); Nck (Stein et al., 1998a) ; SHEP1 (SH2 domain-containing Eph receptor-binding protein 1) (Dodelet et al., 1999) and Src-family non-receptor tyrosine kinases (Hock et al., 1998a). The conserved tyrosine in the carboxy-terminal tail of EphB1 (Tyr 929) has been identified by mutational analysis to interact with Grb2 (Stein et al., 1996). The SH2 domain of the adaptor, Grb4, bound to the SH2 binding domain of ephrin-B1 and through the interaction of the SH3 domain of Grb4 with various downstream effectors, mediated cytoskeletal changes by reverse signalling (Cowan and Henkemeyer, 2001). A yeast two-hybrid screen identified the adaptor protein Grb7 bound to EphB1 and confirmed the interaction between the Grb7 SH2 domain and EphB1 (Han et al., 2002). The binding of these downstream signalling molecules occurs at specific phosphorylated tyrosines; for example, PLCγ binds at Tyr 611 of EphB2 (Zisch et al., 1998), and plays an explicit role in mediating the specificity of the final biological response (Kullander and Klein, 2002). The Tyr 604/610 residues of EphB2 both have a proline residue at the +3 position, and pTyr-X-X-Pro is the preferentially recognised SH2 domain-binding motif for Ras-GAP (Tong et al., 2003).

The adaptor protein Crk (CT10 regulator of kinase, pronounced “crack”) links phospho- tyrosine containing molecules, and alternative splicing of human Crk generates several proteins, CrkI, CrkII, CrkIII and CrkL, each with one SH2 domain, a regulatory tyrosine residue (except CrkI), and either one or two SH3 domains (Chodniewicz and Klemke, 2004). The SH2 binding domain of Crk binds CAS and paxillin, and the SH3 binding domain binds C3G (Crk SH3 binding GEP) and DOCK180 (180-kDa protein downstream of Crk), both of which activate the low molecular weight G proteins, Rap1 and Rac1, therefore providing a link between the Eph tyrosine kinases and the small G proteins (Feller, 2001). The family of docking proteins, CAS (previously referred to as p130CAS, Crk-associated substrate), has an SH3 domain, a proline rich domain, multiple SH2 binding motifs (YXXP) and a serine rich region (Chodniewicz and

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Klemke, 2004). In ephrin-B1 induced endothelial migration, membrane ruffling was mediated by the CrkI-Rac1 signalling pathway (Nagashima et al., 2002). CrkII bound ephrin-A5 activated EphA3 at tyrosine 602 and caused de-adhesion of melanoma and human epithelial kidney (HEK) 293T cells, which was effected through a Rho-mediated signalling pathway (Lawrenson et al., 2002).

SHEP1 (SH2 domain-containing Eph receptor binding protein 1) was identified in mouse brain by the yeast two-hybrid screening method to be associated with EphB2. SHEP1 contains an SH2 domain and a guanine exchange factor (GEF) domain, suggesting that SHEP1 links the Eph receptors to the small Ras family GTPases (R-Ras and Rap1A) (Dodelet et al., 1999). The Abelson (Abl) and Abelson related gene (Arg) are non-receptor tyrosine kinases with SH2 domains which regulate the actin cytoskeleton. Yeast-two hybrid screening determined that both Abl and Arg associated with the phosphorylated juxtamembrane tyrosines of EphA4 and EphB2. Thus, activated EphB2 causes tyrosine phosphorylation of Abl/Arg and vice versa. Abl and EphB2 are co-expressed in the developing brain and retina; suggesting that Eph receptors, via Abl/Arg signalling, influence axon guidance through changes in cell adhesion and actin cytoskeleton organisation (Yu et al., 2001). The Src-family kinases (SFKs) are involved in other receptor-induced signal transduction pathways including the T-cell and B-cell receptor pathways, adhesion to the extracellular matrix through the integrins, cadherins, selectins and cell adhesion molecules, receptor protein tyrosine kinases, cytokine receptors and GPI-linked receptors (Thomas and Brugge, 1997). The tyrosine residues of the Eph receptor cytoplasmic domain were phosphorylated both by trans-phosphorylation and by SFKs associated with the receptors. The SFKs contributed to axon guidance in a stripe assay through phosphorylation of the EphA associated proteins, cortactin and the RhoGEF, ephexin (discussed later in more detail) (Knoll and Drescher, 2004; Zisch et al., 1998).

1.3.3 The small GTP-binding proteins

The small GTP-binding proteins are monomeric G proteins (MW 20–40kDa) and were initially discovered as the oncogenes of sarcoma viruses. Their homologues in humans were subsequently identified, and it was determined that mutated forms of these viral oncogenes stimulated proliferation and transformation of cultured mammalian cells, hence the term cellular oncogenes. The Ras and Rho proteins represent two of the five families of the small GTP-binding protein (small G protein) superfamily. The Ras

Queensland Institute of Medical Research 11 Chapter 1: Literature Review family proteins mainly regulate gene expression. The Rho/Rac/Cdc42 proteins regulate both cytoskeletal organisation and gene expression. The small G proteins have both GDP/GTP-binding and GTPase activity, which is required for the termination of small G protein function (Takai et al., 2001) (Figure 1.5).

.

Figure 1.5 Regulation of small G protein activity. (Modified from Takai et al., 2001)

The small G proteins act as molecular switches that cycle between an inactive GDP- bound state and an active GTP-bound state. The guanine nucleotide exchange factors (GEFs or GEPs = guanine exchange proteins) promote the exchange of GDP for GTP and facilitate activation, whereas the GTPase activating proteins (GAPs) promote GTP hydrolysis and inhibit activation. The guanine nucleotide dissociation inhibitors (GDIs) also negatively regulate Rho-GTPase family proteins (Noren and Pasquale, 2004; Takai et al., 2001). The Rho family proteins (Rho-family GTPases) link membrane receptors with the organisation of the actin cytoskeleton. RhoA promotes actin-myosin contractility and the formation of actin stress fibres and focal adhesions, thereby regulating cell shape, attachment and mobility. Rac1 promotes actin polymerisation and the formation of lamellipodia (curtain-like cellular extensions consisting of actin sheets at the leading edge of migrating cells) and membrane ruffles. Cdc42 causes filopodia formation (thin finger-like cytoplasmic extensions that contain tight actin bundles which

12 Queensland Institute of Medical Research Chapter 1: Literature review are involved in recognition of the extracellular environment) (Burridge and Wennerberg, 2004; Wennerberg and Der, 2004) (Figure 1.6).

Figure 1.6 Rho, Rac and Cdc42 control actin polymerisation. (Modified from Takai et al., 2001)

RhoA activation results in neuronal growth cone repulsion by enhancing actin cytoskeleton contractility, and Rac1 and Cdc-42 induce the formation of actin-based lamellipodia and filopodia which results in extension. In other words, actin polymerisation within the growth cone drives extension of growing axons. In neurons, RhoA is under the control of chemo-repellents, which could result in either localised protrusion or retraction of the growth cone, and Rac and Cdc42 are under the control of chemo-attractants, resulting in axon extension (Hall, 1998; Wennerberg and Der, 2004).

Both RhoA and the Rho associated kinase (ROCK), a downstream effector of RhoA GTPase, are expressed in the lamellipodia and filopodia of retinal ganglion cells. In ephrin-A5 treated retinal ganglion cells, RhoA was activated, Rac was downregulated and inhibition of Rho-GTPase activity reduced retinal growth cone collapse. This suggests that in retinal ganglion cells the effects of Rac and RhoA are antagonistic (Wahl et al., 2000). Rac1 activity is required for endocytosis of the retinal growth cone

Queensland Institute of Medical Research 13 Chapter 1: Literature Review plasma membrane and reorganisation of F-actin, but not for the depolymerisation of F-actin during growth cone collapse in response to ephrin-A2 and semaphorin 3A (Jurney et al., 2002).

When murine fibroblast NIH3T3 cells are plated on ephrin-A1 coated surfaces, the cells both adhere and spread in a specific phenotype due to actin cytoskeletal reorganisation, which is mediated by the RhoA pathway and is both focal adhesion kinase (FAK) and p130cas dependent (Carter et al., 2002). In a Xenopus laevis model system using a chimaeric EGFR extracellular domain fused to the EphA4 catalytic domain, EphA4 activity was associated with loss of cell-cell adhesion and cell polarity (Winning et al., 2001). A follow-up study elucidated that EphA4 activity inhibited RhoA GTPase, which was responsible for the loss of cell adhesion and polarity (Winning et al., 2002).

1.3.4 The MAPK pathway

The activity of Ras proteins is induced by a large variety of extracellular signals and regulated by GEFs and GAPs. The Ras proteins mediate their effects by activation of the Ras-MAPK pathway (Figure 1.7) (Takai et al., 2001). The mitogen activated protein kinase pathway (MAPK) is an important signalling pathway with roles in differentiation, transformation, stress response, apoptosis and cell migration. MAPK stimulation occurs by activation of MAPK kinase kinase (MAPKKK), which phosphorylates MAPK kinase (MAPKK), which in turn phosphorylates MAPK (Huang et al., 2004). The MAPK signalling pathways consist of three related protein kinase cascades named after the final enzyme in each pathway:- (1) extracellular signal- regulated kinases (ERKs); (2) c-Jun N-terminal kinases (JNKs) and (3) p38s (Johnson and Lapadat, 2002). The Ras-MAPK pathway is activated when RTKs engage the Grb2/SOS1 complex, which induces the exchange of GDP for GTP on Ras proteins, and the association of Ras with Raf. Raf phosphorylates MEK1 and MEK2, which then activate the MAPKs, ERK1 and ERK2 (Takai et al., 2001).

Activation of EphA2 by ephrin-A1-Fc inhibits the Ras-MAPK pathway in non-neuronal cell types (Miao et al., 2001) and inhibits integrin mediated cell adhesion, spreading and migration by dephosphorylation and inactivation of FAK by phosphatases (Miao et al., 2000). In endothelial cells, TNF-α binding to either the TNF receptor 1 or TNF receptor 2 upregulates ephrin-A1 expression through the p38 MAPK and stress- activated protein kinase (SAPK)/JNK MAPK pathways, which are independent of the

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NF-κB signalling pathway (Cheng and Chen, 2001). The homeobox transcription factor, HoxB3, which is induced by TNF-α, induces ephrin-A1 expression in endothelial cells and may be the link between TNF-α and ephrin-A1 (Myers et al., 2000).

Figure 1.7 The MAPK pathway. (Modified from Takai et al., 2001).

In stably transfected NIH 3T3 cells, activated EphB2 phosphorylates R-Ras at Tyr 66, which results in decreased integrin mediated activity and loss of cell-extracellular matrix adhesion (Zou et al., 2002; Zou et al., 1999). In NG108 neuronal cells, EphB2 activation by clustered ephrin-B1 induced growth cone collapse, and neurite retraction was associated with downregulation of the Ras-MAPK pathway which is dependent on p120-Ras-GAP activity (Elowe et al., 2001). The Nck interacting kinase (NIK)

Queensland Institute of Medical Research 15 Chapter 1: Literature Review increased in cells stimulated by EphB1 and EphB2 and co-immunoprecipitation formed a complex with p62dok, Ras-GAP and a 145 kDa tyrosine phosphorylated protein. NIK couples EphB1 to the JNK-MAPK pathway (Becker et al., 2000). In HEK 293T cells, the JNK-MAPK pathway is involved in ephrin-B1 reverse signalling which is independent of ephrin-B1 phosphorylation (Xu et al., 2003b). In breast cancer cell lines, Ras-mediated upregulation of MAPK increases EphA2 expression in mesenchymal cells, which attenuates ephrin-A1 expression in adjacent epithelial cells (Macrae et al., 2005). EphA and EphB receptor activation inhibits the activity of GTP-bound R-Ras through phosphorylation and decreases the levels of GTP bound R-RasGTP through the activity of the GTPase-activating protein, p120RasGAP (Dail et al., 2006).

1.3.5 Ephexins

The ephexin family, (Eph interacting exchange protein), consists of guanine nucleotide exchange factors (GEFs) composed of five mammalian genes with a Dbl homology (DH) domain followed by a pleckstrin homology (PH) domain and a C terminal SH3 domain. Ephexin1 was identified through demonstration of binding to the EphA4 kinase domain in a yeast two-hybrid screen. All ephexins bind constitutively to Ephs and interact efficiently with EphA receptors, and poorly with EphB receptors (Shamah et al., 2001). Ephexin1 facilitates guanine nucleotide exchange with RhoA, Rac1 and Cdc42, leading to a state of GTPase activation. However, ephrin-A mediated EphA activation results in Src-dependent phosphorylation of ephexin1 (Tyr 87), which potentiates ephexin1 activity towards RhoA and inhibits activity towards Rac1 and Cdc42. These different responses of RhoA, Rac1 and Cdc42 to ephexin1 mediated by EphA signalling may be important regulators of the attractive and repulsive guidance of the growth cone through manipulation of the actin cytoskeleton (Shamah et al., 2001). In ephexin1-/- neurons there was impaired growth cone collapse following treatment with clustered ephrins (Sahin et al., 2005). As ephexin1 is constitutively bound to Ephs, then the absence of ephrin stimulation may result in localised extension of the growth cone; whereas in portions of the growth cone that have made contact with ephrin, the ephexin1 will promote growth cone collapse. This would result in directed movement of the growth cone away from the ephrin-expressing zone and towards the ultimate target (Murai and Pasquale, 2005). The Rho family GEF, Vav2, binds ephrin activated EphA4 and triggers Vav-dependent endocytosis of the receptor-ligand complex, thus converting an initially adhesive interaction into a repulsive event. Analysis of Vav-/- knockout mice

16 Queensland Institute of Medical Research Chapter 1: Literature review revealed abnormal retinal axon projections (Cowan et al., 2005). In vascular smooth muscle, Vsm-RhoGEF (ephexin5) functions as a GEF for RhoA, but not Rac1 or Cdc42. Following ephrin induced EphA4 activation, Vsm-RhoGEF becomes phosphorylated and contributes to vascular contraction by regulating RhoA (Ogita et al., 2003). Ephrin-A1 induced EphA2 activity inhibits spreading of vascular smooth muscle cells on laminin and fibronectin, which is mediated through the Rac1/PAK pathway (Deroanne et al., 2003).

EphB receptor stimulation by ephrin-B2-Fc results in activation of Cdc42 but not Rac1. Intersectin-1 is a GEF for Cdc42 expressed in the brain. The neuronal Wiskott-Aldrich syndrome protein (N-WASP), which binds intersectin-1 and upregulates GEF activity, is also a regulator of actin polymerisation (Irie and Yamaguchi, 2002). The downstream signalling of ephrin-B1–activated EphB2 was mediated by phosphorylation of the Rho- GEF kalirin which regulates hippocampal neuron dendritic spine morphogenesis via its downstream effector, p21-activated kinase (PAK) (Penzes et al., 2003), which is responsible for actin cytoskeletal reorganisation including the formation of actin stress fibres and focal adhesions (Zhao and Manser, 2005).

1.3.6 Ephrin-B reverse signalling

The final 33 residues of the cytoplasmic tail of the three ephrin-Bs are highly conserved, (in fact the last 33 residues of ephrin-B1 and ephrin-B2 are identical), and include five tyrosine residues which are potential phosphorylation sites (Bergemann et al., 1998). The stimulation of cells expressing ephrin-Bs with soluble EphB-Fc or EphB expressing cells results in subsequent tyrosine phosphorylation at sites -23, -18 and -4 (Kalo et al., 2001). The PDZ binding motif in ephrin-Bs, YYKV, contains a potential tyrosine phosphorylation site (Lin et al., 1999). Ephrin-B phosphorylation has also been observed by activation of cis-expressed platelet derived growth factor (PDGF) or fibroblast growth factor (FGF) receptors, suggesting that ephrin-Bs are components of these signalling pathways (Bruckner and Klein, 1998; Bruckner et al., 1997; Chong et al., 2000).

Ephrin-Bs bind constitutively to PDZ-RGS3, a cytoplasmic protein containing one PDZ and one regulator of G-protein signalling (RGS) domain. The RGS proteins serve as downregulators of G-protein signalling through binding to the α subunit of heterotrimeric G proteins. Ephrin-B reverse signalling via PDZ-RGS interferes with

Queensland Institute of Medical Research 17 Chapter 1: Literature Review signalling of the stromal derived factor (SDF)-1 via its G-protein coupled receptor, the chemokine receptor-4 (CXCR4). SDF-1 is an attractant for migrating cerebellar granule cells and the inhibitory action of ephrin-B reverse signalling has been implicated in the correct layering of granule cells during cerebellar development (Lu et al., 2001).

A modified yeast two-hybrid screen determined that the SH2/SH3 domain binding adaptor protein, Grb4 (Nckβ), binds ephrin-B1. In ephrin-B expressing cells activated with EphB-Fc, there was phosphorylation of focal adhesion kinase (FAK), reduction in F-actin stress fibres, disassembly of focal adhesions and detachment of the cells from the substratum, which was not observed when a dominant negative form of Grb4 (i.e. where the mutated receptor acts as a competitive inhibitor of the wild type receptor) was overexpressed. Grb4 also binds to Abl interacting protein (Abi), axin (a scaffold protein in the Wnt signalling pathway) and the c-Cbl associated protein (CAP) (Cowan and Henkemeyer, 2001). Ephrin-Bs also interact with several PDZ domain proteins including GRIP1, GRIP2 (glutamate receptor interacting protein) (Meyer et al., 2004) and syntenin (which contains only PDZ domains) (Grembecka et al., 2006), protein interacting with C-kinase (PICK-1) and protein tyrosine phosphatase-basophil like (PTP-BL) (Kalo and Pasquale, 1999b; Lu et al., 2001; Palmer et al., 2002). Ephrin-B signalling also promotes integrin mediated adhesion of endothelial cells through a pathway which involves Jun kinase (JNK) and is independent of PDZ binding to ephrin- B1 (Huynh-Do et al., 2002).

1.3.7 The PI3 kinase pathway

Phosphatidylinositol 3-kinase (PI3K) is a heterodimer composed of a p85 regulatory subunit and a p110 catalytic subunit, which phosphorylates phosphatidylinositol lipids in the plasma membrane at the D-3 position of the inositol. PI3-kinase converts phosphatidylinositol-4-5-bisphosphate to phosphatidyl-inositol-3-4-5-trisphosphate. Stimulation of the PI3K pathway sets off a complex chain of events, which influences cell survival, gene regulation, cell metabolism and cytoskeletal rearrangements. Phosphatidylinositol (3,4,5)-trisphosphate (PIP3) acts as a lipid anchor in the plasma membrane for assembling signalling proteins at specific membrane locations. PIP3 recruits signalling proteins with pleckstrin-homology domains, including the guanine exchange factors for Rac, which provides a link between the PI3K pathway and the actin cytoskeleton (Cantley, 2002). In a yeast two-hybrid screen, the p85 subunit of PI3K binds to EphA2. The p85 subunit contains two SH2 domains and one SH3

18 Queensland Institute of Medical Research Chapter 1: Literature review domain, and activation of EphA2 with ephrin-A1 in vascular smooth muscle cells increases PI3K activity (Pandey et al., 1994). In NIH3T3 and HEK293 cells, EphA8 activation recruits the p110γ subunit of phosphatidylinositol 3-kinase (P13Kγ isotype), promoting integrin mediated adhesion and cell migration (Gu and Park, 2001; Gu and Park, 2003). In human microvascular endothelial cells, stimulation of EphB4 with ephrin-B2-Fc increases proliferation mediated by the PI3K pathway (Steinle et al., 2002). In human retinal endothelial cells, EphB4-Fc activation of ephrin-B2 results in migration and proliferation, which can be blocked by inhibitors of the PI3K pathway (Steinle et al., 2003). Ephrin-B2-Fc induces migration of human umbilical vein endothelial cells via the PI3K pathway and induced angiogenesis in vivo in a corneal neovascularisation assay (Maekawa et al., 2003). In EphA2-/- endothelial cells, ephrin- A1 stimulation failed to induce endothelial cell migration and vascular assembly in vitro, and EphA2 deficient mice displayed a reduced response to ephrin-A1 in an angiogenic assay. Expression of dominant negative forms of PI3K and Rac1 in endothelial cells suggests that EphA2-mediated endothelial cell migration proceeds by PI3K dependent activation of Rac1 (Brantley-Sieders et al., 2004b).

1.3.8 Chemokine signalling

In a Xenopus embryo de-adhesion assay, the cytoplasmic protein, PDZ-RGS3 (PDZ– regulator of heterotrimeric G-protein signalling), binds the PDZ domain of ephrin-Bs and binds via a RGS domain to heterotrimeric G-proteins, and mediates the signalling between the cytoplasmic tail of ephrin-B with the G-protein coupled receptors (Lu et al., 2001). The chemoattractant effect of stromal derived factor 1 (SDF-1) on cultured cerebellar granule cells, which is mediated via the G-protein coupled receptors, can be inhibited by EphB-Fc, suggesting that ephrin-B1 reverse signalling through PDZ-RGS3 counteracts the chemoattractant signal of SDF-1 (Lu et al., 2001).

1.3.9 Integrin signalling

Eph receptors regulate integrin-mediated adhesion to substrate and can either enhance or inhibit adhesion. The integrins are a large family of cell surface receptors for a number of cell surface (e.g. ICAM-1, ICAM-2, ICAM-3, VCAM-1) and extracellular matrix (ECM) proteins (e.g. collagen, laminin, fibronectin, vitronectin). The cell-ECM adhesion sites are termed focal adhesions (Hynes, 1992). The integrins are heterodimeric proteins consisting of α- and β- subunits, including at least 18 distinct α-

Queensland Institute of Medical Research 19 Chapter 1: Literature Review subunits and eight β-subunits. The binding of inactive integrins to ECM proteins, the ligands for integrins, induces conformational changes in the αβ heterodimer, which are conveyed to the cytoplasmic domains (Schwartz, 1992). The ligand-bound, activated integrins have no intrinsic enzymatic activity; however, the cytoplasmic domains of activated integrins interact with a variety of membrane bound proteins and adaptor proteins or cytoplasmic binding partners. These binding partners include:- (1) membrane proteins (e.g. integrin associated proteins, tetraspanin proteins); (2) caveolin (αv, α5, α1 subset of integrins); (3) talin, α-actinin, filamin and paxillin and (4) tyrosine kinases (e.g. FAK, syk) (Schwartz, 2001).

The integrin signalling is both “outside-in” and “inside-out,” such that not only does ligand-binding induce cytoplasmic changes and downstream signalling, but changes within the cytoplasm also modify the interaction between the integrin cytoplasmic tails, thus regulating the affinity of the extracellular domain of the integrin molecule for the extracellular ligands (“inside-out”) (Hynes, 1992). Integrin forward (“outside-in”) signalling induces assembly of actin filaments, higher-order stress fibres and focal adhesions, which is mediated by pathways involving the Rho family GTPases and actin- binding proteins (vinculin, talin, α-actinin, actopaxin), therefore physically linking the integrins to the actin cytoskeleton (Juliano, 2002; Schwartz, 2001) (Figure 1.8).

NIH3T3 cells overexpressing ephrin-A5 activated with EphA5-Fc demonstrated increased adhesion to fibronectin (and to a lesser extent laminin), which was mediated by the Src-family kinase, Fyn (Davy et al., 1999). The ephrin-A5 overexpressing cells were subjected to competitive-binding adhesion assays with the soluble RGD peptide which confirmed that ephrin-A5 activation regulated integrin function via an “inside- out” signalling pathway (Davy and Robbins, 2000). Following ephrin-A5 activation in ephrin-A5 overexpressing fibroblasts, isolated murine astrocytes and a neuroblastoma cell line (NG108-5), vinculin was redistributed to the focal adhesion complexes. Ephrin- A5 and β-integrin1 were localised in close proximity at the cell surface. These effects were mediated by both a MAPK (ERK-1 & ERK-2) pathway and a MAPK independent pathway, and were associated with the formation of filopodia (Davy and Robbins, 2000). Activation of ephrin-A2 or ephrin-A5 by EphA3 increased adhesion of cells to laminin in a β1-integrin dependent manner mediated by tyrosine phosphorylation of a p120 raft membrane protein (Huai and Drescher, 2001).

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Figure 1.8 Integrin-mediated signalling. (Modified from Gauthier and Robbins, 2003)

In a mouse corneal micro-pocket assay, EphB1-Fc induced endothelial ephrin-B1 phosphorylation, migration and integrin-mediated attachment and neovascularisation. In vitro EphB-Fc stimulation of ephrin-B1 expressing CHO cells resulted in phosphorylation of p46 JNK but not ERK1/2 or p38 MAP kinases, which was dependent on four C-terminal amino acids which include the PDZ domain (Huynh-Do et al., 2002). In human kidney cells, EphB1 promotes integrin adhesion via the SH2/SH3 Nck adaptor (Huynh-Do et al., 2002). The docking protein p130CAS (Crk- associated substrate) and its binding to Crk plays a vital role in integrin dependent processes through the organisation of the actin cytoskeleton (Chodniewicz and Klemke, 2004). Eph receptor activation decreases integrin mediated activity and loss of adhesion to the extracellular matrix by either of two methods:- (1) inhibiting R-Ras through phosphorylation which impairs binding to downstream effectors or (2) enhancing the GTPase-activating protein, p120RasGAP, which reduces R-Ras GTP levels.

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1.3.10 Interaction with the cadherins

Epithelial-cadherin (E-cadherin), which is located at the adherens junctions and mediates calcium dependent cell-cell adhesion between epithelial cells through homophilic interactions with E-cadherin on the opposing cell, is the principal adhesion molecule of early embryonic and adult epithelial cells (Juliano, 2002). E-cadherin co- localises with EphA2 which is tyrosine-phosphorylated upon E-cadherin mediated adhesion, suggesting that E-cadherin regulates EphA2 function (Zantek et al., 1999). In epithelial cells, the absence of E-cadherin results in the localisation of EphA2 to the perinuclear region, suggesting that E-cadherin may be required for the correct membrane localisation of EphA2 at sites of cell-cell contact. In cancer cells E-cadherin function is often lost, and transfection or pharmacological therapy to restore E-cadherin function will block cancer cell growth and induce differentiation (Zantek et al., 1999). In E-cadherin null mutant ES cells rescued with E-cadherin cDNA, the expression of EphA2 was restored and several other Eph/ephrin proteins including EphB3, EphB4, ephrin-B1 and ephrin-B2 were downregulated (Orsulic and Kemler, 2000). In cultured hippocampal neurons, EphB2 activation resulted in syndecan-2 (a cell adhesion molecule), clustering and the formation of new dendritic spines (Ethell et al., 2001; Ethell and Yamaguchi, 1999)

1.3.11 The SAM domain

The X-ray crystal structure of the SAM domain of EphA4 and EphB2 was resolved, confirming the overall structure and suggesting a mechanism by which SAM domains facilitate the formation of receptor homo-dimers and could also mediate the formation of oligomeric receptor complexes. A conserved residue (Tyr 8 in EphB-SAM and Phe 910 in EphA4-SAM) on the N-terminal protruding arm anchors into a hydrophobic cleft in a neighbouring SAM domain (Stapleton et al., 1999; Thanos et al., 1999b). Subsequently, a new crystal structure of SAM domain monomers of EphA2, EphB1 and EphB2 was solved (Thanos et al., 1999a). In contrast to the previously determined crystal structure of the EphB2 SAM domain, there were no substantial inter-molecular contacts and the N-terminal arm protruded from the core of the molecule and left the hydrophobic cleft exposed, suggesting there must be a different mechanism for receptor oligomerisation. In fact, mutation of the EphB-SAM Tyr 8 to another hydrophobic residue (Ala8) did not significantly alter its stability or structure (Thanos et al., 1999a).

22 Queensland Institute of Medical Research Chapter 1: Literature review

The solution structure of EphB2 was determined by NMR and suggested that the SAM domains were involved in receptor clustering. A second interface was presumed to allow oligomerisation; however, the weak tendency for self-association would be due to the heterotetrameric forces imposed by the ligand binding regions of the Eph-ephrin oligomer (Smalla et al., 1999). The oligomeric structure currently proposed has exposed binding sites for the low molecular weight phosphotyrosine phosphatase (LMW-PTP), AF6, with Grb10 providing a platform for the formation of larger protein complexes (Thanos et al., 1999b).

1.3.12 Interactions with PDZ domain-containing proteins

The PDZ binding domains are stretches of 85–90 amino acid sequences identified in over 75 proteins which act as central organisers of protein complexes at the plasma membrane and mediate:- (1) the adhesive properties of cells; (2) the formation of the paracellular “tight” junctions; (3) ion transport and (4) transmission of growth and differentiation signals between adjacent cells. The PDZ domains comprise six β strands forming two opposing anti-parallel sheets flanked by two α-helices such that the carboxy-terminus of the interacting protein which has a recognition consensus C- terminal peptide sequence, originally (T/S)XV, inserts into the groove of the PDZ domain. The interactions between the PDZ domain recognition sequence (e.g. YKV in ephrin-Bs) and the PDZ binding domain-containing proteins (PDZ proteins for short) binds together macromolecular protein complexes and recruits these complexes to additional membrane structures including neurotransmitter and ion channels. For example, the PDZ proteins, AF-6 and Zona Occludens-1, bind directly to actin filaments, and the Na/H exchange regulatory factor co-localises with the cystic fibrosis transmembrane regulator chloride channel (Fanning and Anderson, 1999).

The yeast two-hybrid system combined with site-directed mutagenesis and glutathione- S-transferase (GST)-fusion protein “pull-down” assays confirms that PDZ domain- containing proteins interact with Eph/ephrin proteins. Many EphA and EphB receptors end in the carboxy terminal sequence, VXV, or a variation where another hydrophobic amino acid replaces the -3 valine. The PDZ proteins, PICK1 (protein kinase C interacting protein), and GRIP were confirmed to bind to EphB2, and in addition, ephrin-B1 and EphA7 also bind syntenin. In vitro co-localisation experiments revealed that PICK1 induced clustering of EphB2 and ephrin-Bs (but not ephrin-As) through the PDZ domain (dependent on the C-terminal VEV residues) (Torres et al., 1998). The

Queensland Institute of Medical Research 23 Chapter 1: Literature Review three ephrin-Bs have an extreme carboxy-terminal motif, YYKV, which was confirmed by expression cloning to interact with PDZ domain-containing proteins including FAP- 1 (Fas associated peptide-1, also called PTP-BL), GRIP, syntenin, PHIP (ephrin interacting protein) and PDZ-RGS (Lin et al., 1999; Lu et al., 2001; Palmer et al., 2002; Songyang et al., 1997). The GRIP family of PDZ proteins (GRIP1 and GRIP2) provide a scaffold for the recruitment and assembly of macromolecular multi-protein signalling complexes downstream of the ephrin-Bs in membrane lipid microdomains termed rafts (Bruckner et al., 1999). The PDZ domain of the Ras-binding protein, AF6, binds the SAM domain of various Eph receptors (Hock et al., 1998b). Maturation of neuronal dendritic spines is dependent on syntenin, a PDZ-domain protein, which interacts with syndecan-2 and EphB2 (Ethell et al., 2001). A pathway involving Rac1 and PAK links the EphB receptor to actin dynamics in dendritic spines. EphB2 interacts with intersectin, an activator of Cdc42 (Irie and Yamaguchi, 2004).

1.3.13 Clustering, lipid rafts and signal transduction

Signal transduction is mediated by complex protein-protein interactions. The ephrin-B ligands were observed not to activate EphB1 receptors unless the ligands were presented in a membrane-bound form or in an antibody-mediated clustered form (Davis et al., 1994). During signalling, lipid microdomains or lipid rafts within the cell surface membrane, which contains specific proteins, change their size and composition to facilitate specific protein-protein interactions and the subsequent activation of downstream signalling cascades (Simons and Toomre, 2000). Protein-protein interaction domains which may contribute to oligomerisation of Eph-ephrin tetramers include:- (1) the juxtamembrane part of the ephrin-B cytoplasmic tail (Song et al., 2002); (2) the PDZ domain proteins for EphB and ephrin-B proteins (Bruckner et al., 1999; Lin et al., 1999); (3) the SAM domain (Thanos et al., 1999b); (4) the EGF-like domain (Lackmann et al., 1998; Smith et al., 2004a) and (5) the GPI anchor in ephrin-A proteins (Davy et al., 1999).

When expressed in NIH3T3 cells, ephrin-A5 is localised to “caveolae-like” lipid microdomains (Davy et al., 1999). EphA and ephrin-A receptor-ligand pairs activate the Src-family kinase, Fyn, which is also associated with lipid rafts via its myristoyl moiety (Davy et al., 1999). Ephrin-A5 reverse signalling regulates integrin mediated adhesion through phosphorylation of “raft” proteins (Davy and Robbins, 2000; Huai and Drescher, 2001).

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In both human renal microvascular endothelial cells and teratocarcinoma-derived pluripotent murine P19 cells, varying concentrations of either dimeric ephrin-B1 (ephrin-B1-Fc) or multimeric ephrin-B1 (ephrin-B1-Fc + anti-Fc) resulted in tyrosine phosphorylation of EphB1. However, in endothelial cells, only ephrin-B1 multimers (tetramers) promoted capillary-like assembly, cell attachment and the recruitment of LMW-PTP. In P19 cells, only multimeric ephrin-B1 promoted attachment to fibronectin. The downstream signalling proteins, Grb2, Grb10 and Nck were recruited with equal efficiency; however, the LMW-PTP depended on both phosphorylation of Tyr 929 and ephrin-B1 multimers (Stein et al., 1998b). A biphasic response to cell attachment and migration following ephrin-B1 activation of EphB1 suggests that the integrin-mediated signalling response in endothelial cells and human embryonic kidney cells “senses” the surface density of ephrin-B1 (Huynh-Do et al., 1999).

Further functional studies using fluorescence resonance energy transfer (FRET) and fluorescence lifetime imaging (FLIM) to analyse the interaction of pre-clustered fluorescent ephrin-A5-Fc with cells expressing EphA3-green fluorescent protein (GFP) using confocal time lapse photography have allowed real time visualisation of Eph- ephrin signalling clusters (Wimmer-Kleikamp et al., 2004). The binding of EphA3-GFP mutants with oligomeric ephrin-A5-Fc triggers the formation of EphA3 clusters, which exceed the size of the ligand and receptor populations in direct contact. This suggests that once initiated by clustered ephrins, the propagation of Eph signalling clusters does not depend on ephrin contact, but on interaction between the Eph receptors themselves (Wimmer-Kleikamp et al., 2004).

1.3.14 Signal attenuation and repulsion versus adhesion

The predominant cellular response that results from the interaction of Eph and ephrin receptor bearing cells is repulsion (Gale and Yancopoulos, 1997; Holmberg and Frisen, 2002). The Eph receptors and ephrin ligands are membrane bound; hence Eph/ephrin receptor-ligand interaction requires cell to cell contact (Xu and Wilkinson, 1997). Therefore, although the final cellular response may be the separation or repulsion of the two interacting cells, there must be initial cell to cell adhesion (Gale and Yancopoulos, 1997). However, in addition to repulsion, adhesive and attractive responses to Eph/ephrin receptor-ligand interaction can occur, and curiously these opposing responses can be mediated by the same Eph/ephrin molecules within the same cell types (Poliakov et al., 2004). The adhesive/attractive role of Eph protein signalling was

Queensland Institute of Medical Research 25 Chapter 1: Literature Review initially suggested by gene knockout studies in which the phenotypic features were midline defects (Dravis et al., 2004; Holmberg et al., 2000).

The first described mechanism that allowed the separation of two interacting cell surfaces in Eph/ephrin mediated contact was cell detachment following proteolytic cleavage of the ephrin (Hattori et al., 2000). The Kuzbanian/ADAM 10 (Kuz) metalloprotease identified in Drosophila is required for normal axon extension (Primakoff and Myles, 2000). The interaction of EphA3 and ephrin-A2 activates ADAM 10 (a disintegrin and metalloprotease 10), which results in cleavage of the extracellular domain of the ephrin. In ephrin-A2 transfected cell lines treated with clustered EphA3-Fc, the ephrin-A2 disappeared from the cell membrane and appeared in the supernatant (Hattori et al., 2000). All vertebrate ephrins contain a conserved motif of 10 amino acids which is the cleavage site recognised by ADAM 10. Indeed, an ephrin-A2 mutant which could not be cleaved by ADAM 10 was examined in vitro, and although growth cone collapse proceeded at the same rate as the wild type ephrin-A2, axon withdrawal was greatly delayed (Hattori et al., 2000). The ADAM 10 protease is associated on the cell surface with EphA3, hence the substrates ephrin-A2 or ephrin-A5 are in trans on the opposing cell surface, which ensures that only Eph bound ephrins are proteolytically cleaved (Janes et al., 2005). The ephrin-B ligands can also be released from the cell membrane through cleavage by serine proteases of the rhomboid family, which cleave substrate proteins within their membrane domains (Pascall and Brown, 2004).

Another mechanism for allowing cell separation following Eph/ephrin mediated contact is endocytosis of the Eph/ephrin receptor-ligand complex. Using cell cultures of NIH3T3, HeLa or cultured neurons transfected with full-length or truncated EphB2 or ephrin-B1, a form of bi-directional endocytosis was observed which removed the adhesive Eph/ephrin complexes from the cell surfaces and thereby allowed the cells to disengage and detach (Zimmer et al., 2003). The full-length cytoplasmic domain of both Eph and ephrin, and hence bi-directional signalling, was required for endocytosis. In fact, cells expressing truncated Eph/ephrins did not detach and strongly adhered to each other, suggesting a possible mechanism for Eph/ephrin mediated adhesive responses (Zimmer et al., 2003). The expression of EphB4 and ephrin-B2 in adjacent Swiss 3T3 fibroblasts led to localised activation of Eph/ephrin proteins, and observation by phase contrast microscopy concluded that following endocytosis of the phosphorylated EphB

26 Queensland Institute of Medical Research Chapter 1: Literature review receptor and trans-endocytosis of the full-length ephrin-B ligand, the receptor and ligand bearing cells retract from one another. The EphB4/ephrin-B2 internalisation and cell retraction were dependent on actin polymerisation, which was mediated by Rac signalling within the receptor-expressing cells (Marston et al., 2003). EphB1 and ephrin-B1-GFP expressing CHO cells were placed in contact with each other, which resulted in ephrin-B1 clustering followed by bi-directional receptor-ligand endocytosis. The ephrin-B1 internalisation was inhibited by a dominant-negative dynamin mutant and potassium depletion, which suggested that ephrin-B1 reverse endocytosis is mediated by a clathrin-dependent pathway (Parker et al., 2004). The phosphatidylinositol-phosphatase protein, synaptojanin 1, which is required for clathrin- mediated endocytosis, was demonstrated to be a downstream target for ephrin-B1 mediated phosphorylation, which provides further evidence for the role of EphB/ephrin-B signalling in endocytosis (Irie et al., 2005). The protein tyrosine phosphatase basophil-like (PTP-BL) is recruited to activated ephrin-B ligands via PDZ domain interactions, followed by dephosphorylation of the ephrin-B ligand and switching off the phosphotyrosine dependent signalling and replacing it with PDZ- dependent signalling. It is postulated that phosphatases may promote adhesion if they terminate signalling before the cells have separated (Palmer et al., 2002).

The ubiquitination of proteins is involved in the regulation of a number of cellular processes including the regulation of the duration and intensity of signalling by tyrosine kinases (Sanjay et al., 2001; Thien and Langdon, 2005). EphA2 activation by antibody or ligand binding is followed by receptor internalisation and degradation, which is mediated by interaction with the c-Cbl adaptor protein, which can function as a ubiquitin ligase (Walker-Daniels et al., 2002). EphA2, but not EphB1 or EphB2, was negatively regulated by Cbl (Wang et al., 2002a). In the Jurkat T-cell line, EphA, but not EphB, activation induced tyrosine phosphorylation of c-Cbl (Sharfe et al., 2003).

The Eph/ephrin receptor-ligand interaction which typically results in a repulsive response also mediates attraction and adhesion. A sub-population of ephrin-A5 null mice (17%) displayed severe craniofacial abnormalities and the majority of affected mice lacked a brain and died shortly after birth. During the process of neurulation, ephrin-A5 and its cognate receptor EphA7 are co-expressed at the edges of the developing dorsal neural folds. This suggests that closure of the neural folds requires ephrin-A5 which, in this situation, participates in cell adhesion (Holmberg et al., 2000).

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There are three alternative splice variants of EphA7 (one full-length and two truncated kinase deficient forms), and all three variants are co-expressed with ephrin-A5 at the edges of the developing neural folds. In vitro co-expression of a truncated kinase deficient EphA7 suppresses tyrosine phosphorylation of the full-length EphA7, and shifts the cellular response measured by cell aggregation assays from repulsion to adhesion. This suggests that alternative splice variants of Eph receptors may mediate the switch between cellular repulsion and adhesion during embryological development (Holmberg et al., 2000). A splice variant of ephrin-A1 which lacked Exon 3 and bound weakly to EphA2-Fc, but did induce EphA2 phosphorylation, resulted in a downstream protein phosphorylation signalling pattern which was different to the full-length ephrin-A1 pattern and inhibited the cell surface shedding mediated by ADAM 10. The alternatively spliced variant of ephrin-A1 may regulate EphA receptor activation (Finne et al., 2004). In the C. elegans VAB-1 Eph receptor mutants (vab = variably abnormal), there is defective ventral closure, a process which resembles neurulation. Analysis of various VAB-1 mutants confirms that ventral closure in C. elegans requires the adhesive function of VAB-1, independent of kinase activity. This finding suggests that the adhesive functions of Eph receptors are evolutionarily conserved (George et al., 1998).

In the migration of neural crest cells (NCCs) in the chick embryo, an early wave of NCCs migrate via the ventral pathway through the anterior sclerotome to form the adrenal medulla, sympathetic ganglia, or dorsal root ganglia. However, a migrating wave of NCCs (melanoblasts) 12–18 hours later invades the dorsolateral pathway between the dermomyotome and the overlying ectoderm to colonise the skin and differentiate into melanocytes (Perris and Perissinotto, 2000; Robinson et al., 1997). In EphB expressing NCCs, ephrin-B1 can both repel or attract different cell populations of NCCs, such that the early migratory NCCs are prevented from invading the dorsolateral pathway, and hence migrate into the ventral pathway (Santiago and Erickson, 2002; Wang and Anderson, 1997). However, the later migration of melanoblasts into the dorsolateral pathway is stimulated by ephrin-B ligands, possibly by increasing their adhesion to fibronectin (Santiago and Erickson, 2002).

A similar phenomenon was observed in the dorsoventral mapping of retinal axons and retroviral mediated overexpression of ephrin-B1, which resulted in ectopic domains of ephrin-B1 expression. The branching response of axons from retinal ganglion cells

28 Queensland Institute of Medical Research Chapter 1: Literature review changed from attraction to repulsion dependent on the level of expressed ephrin-B1, as interstitial branches of EphB expressing RGC (retinal ganglion cell) axons moved either up (attracted) or down (repulsed) the ephrin-B1 gradient to find their correct position within the termination zone (McLaughlin et al., 2003b). Using in ovo electroporation mediated overexpression experiments of EphA4 or ephrin-A5, the trajectory of motor axons from the medial motor column (MMC) and lateral motor column (LMC) towards chick embryo hind limbs was studied. It was observed that EphA4 positive LMC axons were repelled by ephrin-A5 in hindlimb mesoderm; however, EphA4 positive MMC axons were attracted by ephrin-A5 expressing axial mesoderm (Eberhart et al., 2004). In summary, the experimental findings in an number of situations including:- (1) neural crest cell migration (Santiago and Erickson, 2002); (2) the role of ephrin-B1 in retinotectal mapping (McLaughlin et al., 2003b) and (3) the migration of motor neuron axons into the chick hindlimb (Eberhart et al., 2004) confirmed that the interaction between Ephs and ephrins can be bi-functional, either repulsive or attractive, and that the specific cellular response is determined by the cellular context of the interaction.

It is possible that components of the downstream signalling pathways may act as the “molecular switches” due to activation of distinct downstream signalling pathways. The guanine nucleotide exchange factors, which control the activity of the Rho-family small GTPases, Rho, Rac and Cdc42, and hence control the downstream effects of Eph/ephrin signalling on the actin cytoskeleton, could be differentially expressed in different cell populations and could explain the opposite responses of repulsion and attraction (Burridge and Wennerberg, 2004). Another possible switching mechanism involves the SH2 binding domain of Grb4, which binds the phosphorylated tyrosines of ephrin-B1 (Song, 2003). It is possible that ephrin-B reverse signalling is controlled by a switch between two pathways:- (1) phosphorylation dependent binding of the SH2 domain protein Grb4 and (2) PDZ-dependent G-protein receptor coupled signalling, which is responsible for the change from attraction to repulsion through modulation of cytoskeletal dynamics (Palmer et al., 2002).

The EphB6 receptor is catalytically inactive due to several changes in the kinase domain. However, the EphB6 receptor undergoes transphosphorylation upon stimulation with ephrin-B1 through association with co-expressed EphB1. It is possible that the kinase-inactive EphB6 receptor modifies the signalling outcome of the catalytically competent EphB1 receptor (Freywald et al., 2002).

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In vitro observations of Xenopus retinal axon turning behaviour in stripe assays suggest that EphA expressing RGCs are repulsed by higher concentrations of ephrin-A5 when the substratum is fibronectin; however, when the substratum is laminin, the RGCs are “attracted” and turn towards the higher concentration of ephrin-A5 (Weinl et al., 2003). Another guidance molecule, netrin-1, which mediates growth cone attraction towards the optic nerve head, is repulsive in the presence of the ECM molecule, laminin-1 (Hopker et al., 1999). There is evidence of crosstalk between Eph receptors and adhesion molecules such as L1 and cadherins (Orsulic and Kemler, 2000; Weinl et al., 2003; Zantek et al., 1999; Zisch et al., 1997).

In a cell culture system of endothelial capillary-like assembly and the P19 teratocarcinoma cell line, observations suggest that differences in oligomerisation of EphB/ephrin-B receptor-ligand complexes regulate the recruitment of LMW-PTP. Multimeric ephrin-B1-Fc was found to promote endothelial capillary-like assembly, whereas dimeric ephrin-Fc did not, although EphB1 phosphorylation was similar. In the P19 cell line, preclustered ephrin-B1-Fc multimers promoted P19 attachment to fibronectin, whereas ephrin-B1-Fc dimers modestly decreased cell attachment. The EphB1 receptors recruited LMW-PTP following multimeric but not dimeric stimulation, suggesting that the distinct ephrin-B signalling complexes could mediate their response via the response to a specific receptor switch mechanism (Stein et al., 1998b). If Eph/ephrin receptor-ligand interactions are the principal cues directing migrating axons to their specific “addresses”, then the receptor-ligand interaction which results from the relative density of the ligand expressed on the migrating cell may be coupled to the guidance cues within the extracellular matrix and underlying receptor signalling mechanism. When human renal microvascular endothelial cells, EphB1 transfected HEK293 cells, or P19 teratocarcinoma cells were coated on surfaces displaying defined densities of ephrin-B1, a biphasic response in cell attachment was observed depending upon the ephrin-B1 density and the presence of defined extracellular matrix components (e.g. fibrinogen, vitronectin) that interact with cellular integrins, suggesting that EphB1 discriminates ephrin-B1 surface density to direct integrin-mediated cell attachment. These findings suggest that low levels of Eph forward signalling can mediate attractive responses which change to repulsion when signalling levels increase (Huynh-Do et al., 1999).

30 Queensland Institute of Medical Research Chapter 1: Literature review

The EphBs and ephrin-Bs are present in countergradients along the dorsoventral axis of the retinal ganglion cell layer and the lateral-medial (LM) axis of the superior colliculus. Mice which lack both EphB2 and EphB3, and kinase-deficient EphB2 mice, establish aberrant projections along the LM axis of the superior colliculus due to the lack of EphB forward signalling which mediates branch attraction rather than repulsion (Hindges et al., 2002). This suggests that ephrin-B1 is bi-functional, acting both as a repellent and attractant (McLaughlin et al., 2003b). A similar finding has been observed for ephrin-A2 which promotes axon growth at low concentrations and inhibits growth at higher concentrations (Hansen et al., 2004). The precise mechanism by which this bi- functional action is mediated is not completely understood; however, the oligomeric state of ephrin-B1 can result in different EphB1 and EphB2 signalling and cell attachment (Stein et al., 1998b), and the concentration of ephrin-B1 is critical for EphB1-induced integrin-mediated cell attachment (Huynh-Do et al., 1999).

1.3.15 Summary of Eph/ephrin protein signalling

The interaction of an ephrin ligand with its cognate Eph receptor involves both forward and reverse signalling (Murai and Pasquale, 2003), which can result in both cell-cell adhesion or repulsion (de-adhesion) (Davy and Soriano, 2005). These effects are ultimately mediated through changes in cytoskeletal proteins (responsible for cell shape and motility) and cell surface receptors for extracellular matrix proteins (responsible for cell adhesion) (Cowan and Henkemeyer, 2002). The specificity of the cellular response, both forward and reverse signalling events, is mediated by:- (1) the particular receptor or ligand involved (Eph Nomenclature Committee, 1997; Pasquale, 1997); (2) receptor- ligand surface density (Hansen et al., 2004; Huynh-Do et al., 1999); (3) the receptor- ligand oligomerisation state (Stein et al., 1998b); (4) the specific cell type and (5) the subsequent downstream signalling pathway which is activated (Eberhart et al., 2004; McLaughlin et al., 2003b; Santiago and Erickson, 2002).

The changes in Eph/ephrin protein cytoplasmic conformation following receptor-ligand interaction (Hubbard, 2001; Wybenga-Groot et al., 2001) allow the specific binding of SH2 domain-, SH3 domain-, PTB domain- (Schlessinger and Lemmon, 2003) or PDZ domain-containing downstream signalling molecules (Lin et al., 1999). This facilitates the construction of macromolecular signalling complexes anchored to the plasma membrane (Pawson and Scott, 1997) at precise locations through protein-lipid interactions into lipid raft microdomains, which results in the association of downstream

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Figure 1.9 Eph-ephrin signalling pathways. (Modified from Vearing and Lackmann, 2005)

32 Queensland Institute of Medical Research Chapter 1: Literature review signalling proteins in exact positions for efficient interactions (Gauthier and Robbins, 2003; Simons and Toomre, 2000) (Figure 1.9).

Eph/ephrin protein signalling results in the activation of several cytoplasmic downstream signalling pathways which include:- (1) Src-family kinases (Thomas and Brugge, 1997); (2) MAPK (Huang et al., 2004); (3) PAK (Zhao and Manser, 2005); (4) PDZ-dependent (Garner et al., 2000); (5) chemokine (Laurence, 2006); (6) heterotrimeric G-protein (Lu et al., 2001) and (7) integrin-mediated pathways (Chodniewicz and Klemke, 2004; Nakamoto et al., 2004; Schwartz, 2001).

The activity of each of these pathways is dependent on the activity of the Rho family GTPases (Takai et al., 2001) and the availability of the guanine nucleotide exchange factors, each of which is specific for the cell type (Burridge and Wennerberg, 2004). This would suggest that Eph signalling is also regulated by the Rho-GTPases, RhoA, Rac1 and Cdc42, and a variety of guanine nucleotide exchange factors, including ephexins (Cowan et al., 2005; Sahin et al., 2005). This further implies that the downstream Eph signalling mechanism could vary with cell type, and this in part accounts for the specific cell responses, either cell adhesion or cell-cell repulsion (de- adhesion), following Eph receptor activation (Schmitz et al., 2000). The attenuation and termination of Eph/ephrin protein signalling involves proteolytic cleavage (Hattori et al., 2000; Pascall and Brown, 2004), receptor-mediated endocytosis (Zimmer et al., 2003) and tyrosine phosphatase activity (Palmer et al., 2002).

The cytoplasmic signalling proteins are viewed increasingly as forming networks of interaction rather than simple linear pathways (Pawson and Saxton, 1999), and the mechanistic model is being replaced with a spatio-temporal dynamic model of signal transduction (Kholodenko, 2006). The application of this approach to Eph/ephrin protein signal transduction may help understand the complexities of the system and explain some of the paradoxes of signalling which have been outlined; in particular, how the intensity of signalling can mediate opposite effects and why there is redundancy in the Eph/ephrin signalling system. 1.4 Eph/ephrin proteins in axon guidance

The neuron growth cone is a specialised motile structure at the tip of developing axons (Lander, 1990). The chemoaffinity hypothesis proposed by Sperry 40 years ago suggested that the pattern of synaptic connections in the brain was regulated by specific

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“cytochemical affinities” which arose systematically during the growth of a neuron towards its terminal contact, such that a single neuron connected via its axon establishes a synaptic connection only with certain other neurons (Sperry, 1963). The chemoaffinity theory explained nerve specificity in a more precise way than previous theories and also suggested that the ordering of brain neuronal networks could account for inherited components of behaviour (Sperry, 1963). Neuron growth cones are guided by at least four different mechanisms-: (1) contact-mediated attraction; (2) chemoattraction; (3) contact-mediated repulsion and (4) chemorepulsion (Goodman, 1996). An increasing number of molecules contribute to axon guidance functions in addition to the Eph/ephrin proteins, including neural cell adhesion molecules, netrins, slits, semaphorins, fasciclins and members of the classic three morphogen families:- (1) wingless/Wnt; (2) hedgehog and (3) decapentaplegic/bone morphogenic protein/transforming growth factor-β families (Charron and Tessier-Lavigne, 2005; Garbe and Bashaw, 2004). The individual molecules or families, including the Eph/ephrin proteins, do not always fit into the four categories and none are exclusively attractive or repulsive, and all play different roles in different neuron growth cones (Dickson, 2002; Goodman, 1996).

The activity of the neuron growth cone is mainly determined by axon guidance and elongation, and as the axon extends through the extracellular environment, the growth cone samples and responds to a variety of molecular guidance cues (Kalil and Dent, 2005). The growth cones sample their environment by extending slender fingerlike projections called filopodia and veil-like structures termed lamellipodia. In filopodia, F-actin is organised into bundles, and in lamellipodia, F-actin forms a meshwork of filaments (Dickson, 2002). The movement of filopodia and lamellipodia depends on the organisation and polymerisation of F-actin filaments, which are the major intracellular targets for extracellular guidance cues that alter growth cone behaviour (Zhou and Cohan, 2004). The Rho-family GTPases, including RhoA, Rac1 and Cdc-42, which are the downstream effectors of Eph/ephrin receptor-ligand signalling, and myosin II are the proteins identified as regulators of F-actin polymerisation in growth cones (Gallo and Letourneau, 2004; Huot, 2004).

A topographic map is a projection from one set of neurons to another such that the spatial relationships of the receiving set of neurons reflect the neighbouring spatial relationships of the projecting set (Tessier-Lavigne and Goodman, 1996). These maps

34 Queensland Institute of Medical Research Chapter 1: Literature review include the sensory and motor maps of the body, tonotopic maps for auditory stimuli, and maps for the visual fields. The retinotopic map consists of the primary visual projections formed by the axons of the RGCs to their midbrain targets in the optic tectum (OT) of zebrafish, frogs and chicks, and the superior colliculus (SC) of the mouse. These model systems have been the most utilised for investigating the role of guidance molecules (Goodman, 1996; McLaughlin et al., 2003a). The definitive retinotopic map depends on contributions from:- (1) molecular axon guidance cues; (2) axon-fibre to axon-fibre competition and (3) refinement of synaptic strength by patterned neural activity (Flanagan, 2006; Ruthazer and Cline, 2004). Although the retinotopic map is the most studied model system of axon guidance, the same molecular guidance cues are used in multiple maps and provide the basis for the organisation of other topographic projections within the human brain, including the olfactory (reviewed in (Key and St John, 2002; St John et al., 2002)), vomero-nasal, thalamo-cortical, somatosensory and auditory maps (reviewed in (Pickles, 2003) and (Cramer, 2005)). Similarly, the peripheral nervous system neurons arise from the neural crest cells which migrate away from the neural tube to the site where they form the dorsal root ganglia (reviewed in (Young et al., 2004).

The predominant model for studying the development of the topographic maps is the retinotectal system. The Eph/ephrin molecules fulfil the criteria for topographic guidance molecules, as they are expressed in a graded or restricted manner in the retina and optic tectum/superior colliculus (OT/SC), and they are required for the development of a proper topographic map (McLaughlin et al., 2003a). The RGCs bear a unique profile of guidance receptors along the temporal-nasal (TN) and dorsal-ventral (DV) axes. Similarly, each point of the OT/SC has a unique molecular “address”, also determined by the graded distribution of guidance molecules along its two orthogonal axes, the anterior-posterior (AP) and LM axes. The RGC axons along the TN axis of the retina project to the AP axis of the OT (fish, frogs, chicks) or SC (mice), and the DV axis of the retina projects to the LM axis of the OT/SC. (In fish, frogs and chicks, the AP axis of the OT is also referred to as the rostral-caudal axis, and the LM axis of the OT may be referred to as the DV axis). These species have differences in the absolute size of their OT/SC (the chick OT is 50 times larger that the fish OT) and other important differences in the development of their respective visual systems (discussed later) (McLaughlin et al., 2003a; Tessier-Lavigne, 1995). The diversity and complexity of Eph and ephrin spatial and temporal expression patterns and the features of their

Queensland Institute of Medical Research 35 Chapter 1: Literature Review function (attraction/repulsion) provide the signalling possibilities and guidance activities required for the development of the adult retinal topographic map. The elucidation of the signalling pathways that connect the Eph/ephrin proteins to the cytoskeleton provides the mechanism for the observed functional guidance activities (McLaughlin and O'Leary, 2005).

The first retino-topographic guidance molecules to be identified were ephrin-A2 (originally Eph ligand family-1, ELF-1) (Drescher et al., 1995) and ephrin-A5 (originally called repulsive axon guidance signal, RAGS) (Cheng et al., 1995), both of which are ligands for EphA3 (originally called Mek4). Initially, a number of functional in vitro experiments suggested that ephrin-As interacting with EphAs were responsible for RGC axon guidance and the formation of the retinotopic map. In the classic in vitro experiment, the stripe assay, RGCs derived from the temporal retina, when presented with a choice, preferred growing on membranes derived from the anterior tectum and avoided growing on posterior tectal membranes, whereas nasal RGCs showed no preference (Rosentreter et al., 1998; Walter et al., 1987a; Walter et al., 1987b). This finding led to the identification of ephrin-A5 (RAGS) which was shown to have repellent activity in the stripe assay (Drescher et al., 1995). Subsequently ephrin-A2, (ELF-1), a ligand for EphA3 (Mek4) and EphA4 (Sek1), was cloned by the receptor affinity probe method (Cheng et al., 1995), and in vitro stripe assay studies confirmed that ephrin-A2 was also a repellent retinal axon guidance molecule (Ciossek et al., 1998).

In chick and mouse, both Eph receptors and ephrin ligands are expressed on the growth cones of the RGCs and on the OT/SC. In chick retina, EphA3 and EphA6 are expressed in a high-to-low, temporal-to-nasal gradient, whereas EphA4 and EphA5 are almost uniformly expressed. In mouse retina, EphA5 and EphA6 are expressed in a high-to- low, temporal-to-nasal gradient, whereas EphA4 is expressed uniformly. In chick, the ephrin ligands ephrin-A2, ephrin-A5 and ephrin-A6, and in mouse, ephrin-A5, are also expressed on the RGCs in a low-to-high, temporal-to-nasal gradient. In chick OT, the levels of ephrin-A2 and ephrin-A5 are expressed in a low-to-high, anterior-to-posterior gradient and the ephrin-A5 gradient is steeper and more posterior, with a low uniform level of ephrin-A6. In mouse SC, ephrin-A2 levels are in a low-to-high, anterior-to- posterior gradient and the ephrin-A2 levels fall away at both ends of the SC and there is also a steeper, more posterior ephrin-A5 gradient. In chick, along the DV retinal axis,

36 Queensland Institute of Medical Research Chapter 1: Literature review

EphB1 is expressed uniformly and EphB2 and EphB3 are expressed in a low-to-high, dorsal-to-ventral gradient. In mouse retina, EphB1, EphB2, EphB3 and EphB4 are expressed in a low-to-high, dorsal-to-ventral gradient. In mouse and chick, ephrin-Bs are also expressed in the retina with high-to-low, dorsal-to-ventral gradients of ephrin- B1 and ephrin-B2, with additional uniform expression of ephrin-B3 in mouse. EphB2 and EphB3 are expressed in mouse SC in a high-to-low, lateral-to-medial gradient. In both mouse SC and chick OT, ephrin-B1 is expressed in a low-to-high, lateral-to-medial gradient (summarised in Figure 1.10).

In general, RGCs expressing high levels of EphA receptors project to regions expressing low levels of ephrin-As and vice versa, suggesting a receptor-ligand mediated cell-cell repulsive interaction. The EphA/ephrin-A interaction is best characterised by the fact that the repellent activity of ephrin-As, expressed in a low-to- high AP gradient along the OT/SC, controls the advance of the RGC axon expressing EphAs in high-to-low levels depending on its origin along the TN axis of the retina. Further, RGC axons expressing high levels of EphB receptors project to regions expressing high levels of ephrin-Bs and vice-versa, consistent with a receptor-ligand mediated cell-cell attractive interaction (Figure 1.11). These statements summarise the original model of Eph/ephrin mediated retinotopic mapping and were consistent in principle with Sperry’s original chemoaffinity theory.

However, several factors suggested that this model was an incomplete explanation, including:- (1) the observation that both Eph receptors and ephrin ligands were expressed on both the retina itself and co-expressed on the same axons of retinal ganglion cells; (2) the precise quantitative analysis of the Eph-ephrin model and (3) a review of previous theories of retinotopic mapping. This has led to much subsequent work to explain the formation of the precise retinotopic map which will be subsequently discussed in more detail (summarised in (Goodhill and Xu, 2005; McLaughlin and O'Leary, 2005).

Initial in vitro and mutant mouse studies followed and supported the general principles of the EphA/ephrin-A repulsive and EphB/ephrin-B attractive model. When the chick tectal ephrin-A2 gradient was modified by retroviral overexpression, temporal but not nasal axons avoided the ectopic ephrin-A2 expressing patches (Nakamoto et al., 1996). Studies in mutant mice confirmed that ephrin-As interacting with EphAs controlled the

Queensland Institute of Medical Research 37 Chapter 1: Literature Review projection of RGC axons of the TN axis along the AP axis of the SC (Dutting et al., 1999; Feldheim et al., 2000; Frisen et al., 1998).

Figure 1.10 Mouse and chick retinal and superior colliculus/optic tectum expression of Eph/ephrins. Modified from (McLaughlin and O'Leary, 2005)

In ephrin-A5 null mice, temporal retinal axons overshot the SC or targeted the posterior SC due to the lack of repulsion; however, some targeted normally (Frisen et al., 1998). In ephrin-A2/ephrin-A5 double knockout mice, many axons had normal and aberrant projections but there was no overshoot. These studies supported the theory that both ephrin-A2 and ephrin-A5 played a role in retinotopic mapping, which depended on both the amount of EphA present on the RGC growth cone and the shape of the ephrin-A gradient which it encountered. Indeed, reducing signalling through EphAs resulted in a decreased repellent response of temporal RGC axons to ephrin-As and increased

38 Queensland Institute of Medical Research Chapter 1: Literature review posterior overshoot (Feldheim et al., 2000; Frisen et al., 1998). Further in vitro studies with both linear and non-linear ephrin gradients confirmed these results (Rosentreter et al., 1998).

Figure 1.11 Retinotectal mapping. Modified from (Knoll and Drescher, 2002; Pasquale, 2005; Pittman and Chien, 2002).

Transgenic mice generated using a gene knock-in strategy resulted in two subpopulations of RGCs uniformly scattered across the retina, one with wildtype gradients of EphA receptors (EphA5 and EphA6), and the other with additional overexpression of EphA3. This strategy was used because although EphA3 is not normally expressed on retinal axons, it binds both ephrin-A2 and ephrin-A5 with similar Queensland Institute of Medical Research 39 Chapter 1: Literature Review efficacy to the native EphA5 and EphA6 (Brown et al., 2000). The projection of EphA3 overexpressing RGCs was compressed to the anterior half of the SC, and wildtype RGCs were compressed to the posterior half of the SC, indicating that relative rather than absolute levels of EphA signalling are important in mapping, supporting earlier in vitro work (Brown et al., 2000). Further genetic manipulation of the EphA levels expressed by RGCs in the retina was undertaken by crossing EphA3 knock-in and EphA4 knockout mice. These experiments confirmed that EphA signalling by RGCs depends on the relative rather than absolute differences in Eph signalling (Reber et al., 2004). In summary, these in vitro and in vivo methods provide evidence that the incremental or relative changes in ephrin ligand concentration were more important than the absolute ligand concentration, and that increased signalling through EphAs by overexpression or ectopic expression of either EphAs or ephrin-As resulted in an increased repellent response of EphA expressing axons to ephrin-As in vitro and a decreased extension of RGC axons along the AP axis in vivo (Reber et al., 2004).

The control of topographic mapping of the DV retinal axis along the LM OT/SC axis was suggested when the EphB and ephrin-B gradients were initially described in the developing mouse and chick visual systems (Braisted et al., 1997; Holash and Pasquale, 1995; Marcus et al., 1996; Sefton et al., 1997). The gradient orientations suggested that, in contrast to the EphA/ephrin-A interactions, the EphB/ephrin-B interactions are attractant and not repellent, because the areas of RGC axons with high EphB expression projected to areas of high ephrin-B expression in the OT/SC (Connor et al., 1998). The retinotopic mapping of the EphB2 (Henkemeyer et al., 1996) and EphB3 knockout mice (Orioli et al., 1996) and EphB2/EphB3 double knockout mice was analysed, and the EphB2/EphB3 null embryos displayed axon pathfinding errors with a more severe phenotype than either single mutant alone (Birgbauer et al., 2000). The generation of double knockout mice using a kinase dead EphB2-β-galactosidase construct determined that the EphB2/EphB3 guidance function was kinase-independent (Birgbauer et al., 2000). However, although the EphBs have an attractant rather than repellent role in retinal axon guidance, EphB1, EphB2 and EphB3 extracellular domains fused to Ig-Fc inhibited retinal growth cone navigation on a laminin substrate (Birgbauer et al., 2001).

The precise development of the retinotectal map involves significant “fine-tuning” following the arrival of the RGC growth cones in the OT/SC, which is partly controlled by Eph/ephrin protein interactions. In both chicks and rodents, the arrival of the RGC

40 Queensland Institute of Medical Research Chapter 1: Literature review axon in the OT/SC is characterised by posterior overshoot and interstitial branching before the RGC axon growth cone finally becomes located at its termination zone (TZ) (Nakamura and O'Leary, 1989; Yates et al., 2001). RGC axons from given retinal DV locations have broad distribution along the LM tectal/superior colliculus axis with a peak centred on the location of the future TZ. The branches arborise at the appropriate LM and AP location of their TZ, followed by refinement of the map which coincides with the death of substantial numbers of RGCs (Simon and O'Leary, 1992). In frogs and fish, initial DV mapping along the LM axis is more accurate, and the RGC axons extend along the AP axis to the correct location of their TZ. In chicks and mice, the degree of the RGC axon posterior overshoot is probably controlled by the single repellent EphA/ephrin-A interaction. In chicks and rodents with an extended posterior overshoot, the shape of the ephrin-A gradient in the OT/SC predicts the extent of the overshoot. In chicks the relatively shallow gradient of ephrin-A in the anterior OT compared with the posterior OT results in temporal RGC axons extending a greater distance past the TZ than do nasal RGC axons (Yates et al., 2001), summarised in (McLaughlin and O'Leary, 2005).

Eph/ephrin gradients and interactions are clearly important parts of the formation of the retinotopic map, and quantitative modelling has been used to describe and predict the results of genetic experiments (Reber et al., 2004). However, as mentioned above, some investigators have reviewed the data, interpreted the findings differently, and concluded that the interaction of Ephs with ephrins in retinotopic mapping is somewhat more complex than the initial in vitro and mutant mouse experiments have suggested (Flanagan, 2006; Mann et al., 2004). For example, the gene targeting of ephrin-A2 and ephrin-A5 predicted that removal of the repellent ephrin from the posterior SC would result in a shift of all RGC axons posteriorly; however, there was an approximately equal and opposite shift of temporal axons posteriorly and nasal axons anteriorly, which suggested a role for additional guidance cues (Flanagan, 2006). The EphA/ephrin-A repellent model has been analysed by simulation in computational modelling which has led to the conclusion that the precise retinotopic map with condensed termination zones requires additional guidance parameters. This may be the role played by competitive interactions between RGC axons and the presumed patterned neural activity (Yates et al., 2004). A recent study proposed a quantitative model which combined the effects of Eph/ephrin receptor-ligand interactions, axon-axon competition and correlated (patterned) neural activity to precisely predict the aberrations observed in the retinotopic

Queensland Institute of Medical Research 41 Chapter 1: Literature Review map in ephrin-A deficient mice (Tsigankov and Koulakov, 2006). Another recent study has re-analysed the data from the EphA3 knock-in and EphA4 knockout mice (Brown et al., 2000), using computational simulation to determine the relationship between EphA receptor density and retinal position. The modified retinal induction model of retinotopic mapping suggests that the level of EphA on the arriving RGC modifies the level of ephrin-As in the target SC (Willshaw, 2006).

In addition to axon-axon competition and patterned neural activity, there are several other experimental findings which support the basic Eph/ephrin attractant-repellent model and provide additional complexity to explain some of the puzzling results. These findings include:- (1) multiple Eph and ephrins are expressed in complex overlapping gradients including co-expression of both Eph and ephrins on the same cells (McLaughlin and O'Leary, 2005); (2) EphAs co-expressed with ephrin-As may interact in cis with downregulation of signalling function (Feldheim et al., 2000; Hornberger et al., 1999); (3) Eph and ephrins have the ability to signal bi-directionally (Mann et al., 2002); (4) there are concentration-dependent positive and negative effects which specify the termination point of an ephrin gradient (Flanagan, 2006; Hansen et al., 2004; Matsuoka et al., 2005) and (5) other guidance molecules are required. Other retinotopic guidance molecules include:- (a) RGM (repulsive guidance molecule), a 33 kDa GPI- anchored protein and its receptor neogenin (Monnier et al., 2002); (b) Wnt3 and its receptors, Ryk and Frizzled and (c) Semaphorin3a (Campbell et al., 2001).

Axons moving towards their target adhere to each other in bundles to form fascicles, and ephrin-As which promote the fasciculation in EphA-positive cortical axons (Caras, 1997; Winslow et al., 1995) have an inhibitory effect on the neurite outgrowth of chick motor neurons cultured on clustered ephrinA-Fc (Ohta et al., 1997). In EphB2/EphB3 knockout mice, EphB receptor forward signalling promotes de-fasciculation of hippocampal axons as they near their target regions to allow the individual axons to reach their respective target neurons (Chen et al., 2004). An in vitro approach in which both retinal position and ephrin concentration were varied continuously showed that ephrin-As can act both negatively in repelling axon growth at higher concentrations, and positively in promoting axon growth at lower concentrations (Hansen et al., 2004). In fact, ephrin-A5 can induce attraction on a laminin substrate and repulsion on a fibronectin substrate (Weinl et al., 2003). Similarly, studies in Xenopus suggesting possible concentration dependent changes of ephrin-Bs from repulsion to attraction

42 Queensland Institute of Medical Research Chapter 1: Literature review

(Mann et al., 2002) were confirmed by in vitro experiments (McLaughlin et al., 2003b). Cell migration assays also suggested a change in ephrin-B signalling from attraction at low concentrations to repulsion at high concentrations (Matsuoka et al., 2005).

Finally, it is intriguing to reflect on the underlying factors that determine retinal and tectal polarity which are responsible for the formation of the Eph/ephrin gradients. Retinal polarity is controlled by the morphogens, sonic hedgehog (Shh) and bone morphogenetic protein (BMP) and the complementary expression of the paired homeodomain proteins, Pax2 and Pax6. The distinction between the optic stalk and retina involves the homeodomain proteins, Vax1 and Vax2. Patterning of the temporal- nasal axis of the retina involves the expression of the winged transcription factors, BF1 and BF2. The regulatory genes, SOHo1 and GH6, are expressed in nasal retina, whereas dorsal-ventral retinal axis polarity is determined by the actions of Shh, BMP4, Tbx5 and ventropin. The polarity of the tectum is controlled by the engrailed homeodomain proteins (En-1 and En-2) (reviewed in (Flanagan, 2006; McLaughlin and O'Leary, 2005). 1.5 Adult nervous system and synaptic plasticity

The role of the Eph/ephrin proteins in the development of the central and peripheral nervous systems is well described (O'Leary and Wilkinson, 1999; Wilkinson, 2001); however, there is additional evidence that the Eph/ephrin proteins play an important role in the adult nervous system and have important functions in learning and memory (Gerlai, 2001). In adult rat brain, EphA4 was immunolocalised to the cerebral cortex, hippocampus, cerebellum and spinal cord (Martone et al., 1997). In a kainate induced excito-toxic model of brain injury in rats, reverse transcriptase (RT)-PCR on pooled hippocampal RNA showed increased expression of EphA4, EphA5 and EphB2 post- injury (Moreno-Flores and Wandosell, 1999). The intracellular distribution of EphA4 protein in the brain was observed from embryonic day 11 (E11) until post-natal day 6 (P6), which confirmed expression in the adult mouse brain (Greferath et al., 2002). In adult rat brain using in situ hybridisation and immunohistochemical analysis, EphA5 was detected in the hippocampal fields, cortex and amygdala (Gao et al., 1998b). Similar patterns of EphA5 expression results have been obtained in strains of inbred mice using in situ hybridisation (Gerlai et al., 1999), and a subsequent study detected complementary expression patterns of EphA5 and ephrin-A3 in the adult rat hippocampal mossy fibre system (Xu et al., 2003a).

Queensland Institute of Medical Research 43 Chapter 1: Literature Review

In rat cortex at E13.5 when neurogenesis is the predominant event, the mRNAs of EphA3, EphA4 and EphA5 were expressed at high levels, with lower EphA6 and EphA7 levels and no detectable EphA1, EphA2 or EphA8. All the ephrin-B mRNAs were present, and EphB3, EphB4, EphB5 and EphB6 were low (Zhou et al., 2001). As limited neurogenesis continues into adult life, EphB signalling may regulate this process. In vitro studies of rat cortical neurons determined that ephrin-A5 and ephrin-B1 stimulate neurite outgrowth (Zhou et al., 2001). An in situ hybridisation study comparing expression levels of EphB1, EphB2, EphB3, EphA4 and ephrins B1, B2 and B3 in the neonatal and adult mouse central nervous system (CNS) confirmed the presence of Eph and ephrins throughout the adult CNS. The complex expression pattern in neonatal and adult mice supported the role of Eph/ephrin proteins in the control of neuronal migration during development and suggested a role in the plasticity of the adult CNS (Liebl et al., 2003; Willson et al., 2006). In the adult human brain, expression of EphA4, EphA6, EphA7, EphB4 and EphB6 and the ephrin-A5 and ephrin-B2 ligands has been observed using RT-PCR (Hafner et al., 2004). The spinal cord is also a site for important Eph protein function, and the local neuronal circuits within the spinal cord, referred to as the central pattern generators, are responsible for locomotion. Mice lacking EphA4 or ephrin-B3 lost left-right limb alternation and instead exhibited synchrony and a hopping type gait (Dottori et al., 1998; Yokoyama et al., 2001). Initially it was hypothesised that loss of the normal decussation of the cortico-spinal tract (CST) was the cause of the abnormal gait (Kullander et al., 2001a); however, the abnormal gait may be due to the axons of excitatory EphA4 positive neurons, which have been identified in the central pattern generator crossing the midline (Kullander et al., 2003).

The subventricular zone (SVZ) of the lateral wall of the lateral ventricle remains a germinal zone in the adult mammalian brain. Electron microscopy has revealed that EphB1, EphB2, EphB3, EphA4 and ephrin-B2/ephrin-B3 ligands are expressed on SVZ astrocytes. Infusion of EphB2 or ephrin-B2 into the lateral ventricle disrupts migration and increases cell proliferation (Conover et al., 2000). In the lateral ventricle of the adult mouse brain, disruption of EphA7 induced ephrin-A2 signalling results in neural progenitor cell proliferation, suggesting that EphA7 and ephrin-A2 are negative regulators of neurogenesis (Holmberg et al., 2005). In vivo gain of EphA7 function by ectopic ephrin-A5 expression results in neural progenitor apoptosis and decreased cortical size (Depaepe et al., 2005).

44 Queensland Institute of Medical Research Chapter 1: Literature review

The neuronal synapse is a highly specialised site of cell-cell communication in the nervous system. The typical neuronal synapse consists of pre-synaptic (axonal) and post-synaptic (dendritic) compartments separated by a synaptic cleft. An electrical signal travelling down an axon arrives at a pre-synaptic terminal and is converted into a chemical signal through the release of neurotransmitters. The neurotransmitters diffuse across the synaptic cleft and bind to ligand-gated ion channels, resulting in the propagation of the electrical signal in the post-synaptic neuron (Murai and Pasquale, 2004). Neuroglial cells surround the pre-synaptic and post-synaptic terminals and influence neurotransmission by modifying the extracellular milieu. The complex organisation of the synapse allows nerve transmission to be regulated pre-synaptically, post-synaptically and through alterations to the glial cells. This allows fine tuning of synaptic function and imparts the plasticity required to encode and store information within the nervous system, and determines the molecular basis for learning and memory (Haydon, 2001).

During development, the post-synaptic axonal dendrites form extensive arborisations covered with motile filopodial protrusions. When the neuronal migration stage of nervous system development is completed and the dendritic filopodia have established synaptic connections, these filopodia are replaced by mature dendritic spines (Murai and Pasquale, 2004). The dendritic spines comprise numerous tiny protrusions on the dendrite surface and are the sites that receive the majority of the excitatory inputs. A mature dendritic spine has a narrow stalk connected to a spherical head that contains the post-synaptic density, where ion channels and post-synaptic signalling proteins are assembled in macromolecular complexes (Garner et al., 2000). The synaptic role of the EphB/ephrin-B receptor-ligands has been most completely studied with respect to dendritic spine morphogenesis. The Eph/ephrin proteins involved were determined in GST-pull down experiments and observed by immunohistochemistry to be co-localised with PDZ proteins at neuronal synapses, suggesting a role for Eph/ephrin proteins in synaptic function (Torres et al., 1998). The EphB/ephrin-B receptor-ligand interaction in cultured neurons induces the formation of lipid raft microdomains which contain the N-methyl-D-aspartate (NMDA) neurotransmitter receptors and also influences the number of pre- and post-synaptic specialisations (Dalva et al., 2000). The role of EphB2 signalling pathways was investigated by transfecting cultured hippocampal neurons with a kinase-inactive dominant negative EphB2. The observation that the dendritic

Queensland Institute of Medical Research 45 Chapter 1: Literature Review protrusions remained filopodial rather than maturing to the dendritic spine indicated that EphB signalling is required for dendritic spine morphogenesis (Ethell et al., 2001).

The rodent hippocampal slice preparation is a model experimental system for the investigation of long-term potentiation (LTP), defined as the long-lasting strengthening of the connection between two neurons (synaptic strength), which increases the ability of the pre-synaptic cell to activate the post-synaptic cell, and is considered to be the basis of learning and memory (Bliss and Lomo, 1973). The hippocampus contains three main synapses:- (1) the perforant pathway which connects the entorhinal cortex to dentate gyrus (DG) neurons; (2) the mossy fibres which connect DG neurons to CA3 (cornus ammonis) region neurons and (3) the Schaffer collaterals which connect CA3 to CA1 neurons (Bliss and Lomo, 1973; Lomo, 2003). In EphB2 mutant mice with an inactive kinase domain, CA1 hippocampal neuronal LTP was normal, whereas EphB2 null mice had reduced LTP (Henderson et al., 2001). In another study of EphB2 null mice, LTP was impaired despite normal hippocampal morphology. A truncated EphB2 (EphB2lacZ/lacZ) rescued the phenotype, confirming that LTP is a kinase-independent function of EphB2 (Grunwald et al., 2001). In vitro CA3 hippocampal neurons require interactions between post-synaptic EphB receptors and pre-synaptic ephrin-B receptors for induction of mossy fibre LTP (Contractor et al., 2002). EphB receptors cluster with NMDA receptors at excitatory synapse, and ephrin-B2 activation enhances NMDA receptor-dependent signalling pathways (Takasu et al., 2002). In EphB1, EphB2 and EphB3 triple knockout mice, the dendritic spines of hippocampal neurons displayed an immature filopodial phenotype in vivo and failed to form in vitro (Henkemeyer et al., 2003). Although different EphB knockout combinations indicated that EphB1 and EphB2 were the major determinants of normal dendritic spine morphology, EphB3 also contributed to normal spine shape in vitro. Mice expressing truncated EphB2 (EphB2lacZ/lacZ) also showed dendritic spine abnormalities, confirming that EphB forward signalling is required for normal spine formation. In the double and triple EphB knockout mice, the most pronounced reduction in spine size and post-synaptic densities was in the CA3 area of the hippocampus (Henkemeyer et al., 2003). The addition of ephrin-B1-Fc or ephrin-B2-Fc to cultured hippocampal and cortical neurons rapidly induced the formation of dendritic spines (Penzes et al., 2003). Ephrin-B3 null mice exhibited a marked reduction in CA3-CA1 LTP, increased levels of pre- and post- synaptic proteins and reduced levels of NMDA receptors, but these abnormalities were not observed in ephrin-B3 lacZ (ephrin-B lacZ/lacZ) mice. However, both ephrin-B3 null

46 Queensland Institute of Medical Research Chapter 1: Literature review and ephrin-B3 lacZ mutant mice showed an increased number of excitatory synapses, indicating that ephrin-B3 reverse signalling regulates synapse number (Rodenas-Ruano et al., 2006).

The EphB receptors regulate the cytoskeleton of dendritic spines, which are composed largely of F-actin filaments. The EphB receptors are constitutively associated with ephexins and regulate the activity of the Rho-GTPases via two separate pathways to contribute to the expansion of the dendritic head through actin cytoskeletal rearrangement (Irie and Yamaguchi, 2002; Penzes et al., 2003). In an animal injury model of chronic pain induced by tissue damage, EphB/ephrin-B receptor-ligand interactions modulated NMDA receptor-dependent spinal cord synaptic plasticity which may contribute to chronic pain (Battaglia et al., 2003). Ephrin-B2 has been localised to the post-synaptic density and in experiments with an ephrin-B2-CamK-Cre mouse, in which ephrin-B2 is knocked-out in the forebrain only, both ephrin-B2 and ephrin-B3 contributed to NMDA receptor-dependent LTP. In ephrin-B2-Nestin-Cre mice, in which ephrin-B2 is removed from the entire developing nervous system, the hippocampal structure was normal, but the mutant mice displayed a defect in long-term depression (Grunwald et al., 2004).

The EphA/ephrin-A receptor-ligands also participate in intercellular communication in the extra-synaptic regions of the dendritic spine where the spines are in close proximity to astrocytes (Murai et al., 2003). Ephrin-A3 is expressed on dendritic spines of hippocampal pyramidal neurons, and ephrin-A3-Fc stimulation causes dendritic spines to shorten and collapse (Murai et al., 2003). The application of EphA5-Fc to mouse hippocampal slices impairs activity-induced changes in synaptic responsiveness, such as LTP in Schaffer collateral-CA1 neuron synapses, (Schaffer collaterals connect hippocampal regions CA1 and CA3), probably through post-synaptic mechanisms; whereas ephrin-A5-Fc induces a LTP-like enhancement of synaptic transmission (Gao et al., 1998a). Infusion of EphA5-Fc directly into the mouse hippocampus impaired performance on behavioural testing (Gerlai et al., 1999), but infusion of ephrin-A5-Fc enhanced performance and ameliorated anaesthesia-induced memory loss (Gerlai and McNamara, 2000). In an experimental model of human temporal lobe epilepsy, the development of kindling-induced behavioural seizures was examined in conjunction with a continuous intraventricular infusion of EphA5-Fc or ephrin-A3-Fc. The intraventricular EphA5-Fc or ephrin-A3-Fc infusions altered the extent and pattern of

Queensland Institute of Medical Research 47 Chapter 1: Literature Review hippocampal mossy fibre sprouting, suggesting that EphA/ephrin-A receptor-ligand interactions modulate activity-dependent axonal growth in the adult CNS (Xu et al., 2003a). EphA4 null mice have defects in LTP; however, EphA4 mutant mice with the entire cytoplasmic domain replaced by GFP have normal LTP, suggesting that EphA4 plays a kinase independent role in synaptic plasticity (Grunwald et al., 2004). It is likely that these EphA mediated effects on synaptic plasticity and memory are due to EphA signalling induced changes in dendritic spine morphology (Yamaguchi and Pasquale, 2004).

The Eph/ephrin proteins play an important role in the structural and electrophysiological properties of synapses within the adult nervous system (Gerlai, 2001) and regulate neural progenitor proliferation and therefore control brain size (Depaepe et al., 2005; Holmberg et al., 2005). The EphB/ephrin-B receptor-ligands regulate synaptic structure through axon to dendritic signalling; whereas the EphA/ephrin-A receptor ligands are involved in glial cell (astrocyte) to dendritic communication (Murai and Pasquale, 2004). Eph protein dysfunction may play a role in nervous system disorders and mental illness (Yamaguchi and Pasquale, 2004). The role of Eph/ephrin proteins following injury to the adult brain will be discussed in a later section. 1.6 Tissue boundary formation and cell migration

The segmentation of the principal body axis is a basic feature of the development of complex multicellular organisms. The seven rhombomeres of the developing vertebrate hindbrain have been a model for the investigation of the process of segmentation (Tepass et al., 2002). The partition of the hindbrain into segmental blocks or rhombomeres implies the existence of a “boundary” between rhombomeres such that cells from “even” rhombomeres do not spread to the adjacent “odd” rhombomeres (Cooke and Moens, 2002; Lumsden, 1999). The cells at the boundaries have distinct molecular and cellular characteristics and, in fact, rhombomere boundaries are distinguished by enlarged intercellular spaces suggestive of de-adhesion (Heyman et al., 1993). In cell sorting experiments in the 1950s it was demonstrated that the dissociated cells of vertebrate embryonic organs could re-aggregate to resemble the original organ structure (Moscona and Moscona, 1952). This was initially attributed to a phenomenon of “directed cell movement” (Steinberg and Gilbert, 2004), however, in vitro experiments with cells expressing different levels of the homophilic cell adhesion molecule, P-cadherin, determined that cell sorting is mediated by differential cell

48 Queensland Institute of Medical Research Chapter 1: Literature review adhesion (Steinberg and Takeichi, 1994). This selective cell sorting was also observed when cells from different rhombomeres were dissociated, mixed and allowed to re- aggregate, which can be explained by the differential adhesive properties of the “odd” and “even” rhombomeres (Cooke and Moens, 2002).

Experiments using ectopic expression of mutated receptors (dominant negative) in the frog hindbrain and zebrafish suggest that a role in the compartmentalisation of cell populations can be attributed, at least in part, to the Eph/ephrin proteins (Winning and Sargent, 1994; Xu et al., 1995). In zebrafish, EphA4, EphB2 and EphB3 are expressed in the odd-numbered rhombomeres, r3/r5, whereas ephrin-B1, ephrin-B2 and ephrin-B3 are expressed in the even-numbered rhombomeres, r2/r4/r6. Therefore, interactions between EphA, EphB receptors and the ephrin-B ligands can occur at the interfaces of adjacent rhombomeres (Xu and Wilkinson, 1997). When ephrin-B2 is expressed in a mosaic fashion together with lacZ by injecting ephrin-B2 and lacZ mRNA into one cell of the eight-cell-stage zebrafish, the ephrin-B2/lacZ expressing cells in r3/r5 become restricted to the rhombomere boundaries; however, in r2/r4/r6, the distribution of ephrin-B2/lacZ expressing cells is scattered. Thus, if an ephrin-B2/lacZ expressing cell is within a rhombomere (r2/r4/r6) which corresponds to its normal expression pattern, then that cell will occupy any position within the rhombomere. However, if the ephrin- B2/lacZ expressing cell is in a rhombomere (r3/r5) where it is not normally expressed, then the ephrin-B2/lacZ expressing cell is “sorted” by the repulsive interaction with Ephs to the rhombomere boundary. A similar result is found when truncated ephrin-B2 is injected; however, injection of truncated ∆EphA4, (which will activate ephrin- B2/ephrin-B3 ligands and act as a “dominant negative” inhibitor of the wild type receptor), results in segregation of ∆EphA4 expressing cells at the boundaries of r2/r/4/r6. In summary, mosaic activation of Eph receptors results in cell sorting to odd- numbered rhombomere boundaries, whereas mosaic activation of ephrins results in cell sorting to even-numbered rhombomere boundaries (Xu et al., 1999). Further experiments in rhodamine or fluorescein-labelled juxtaposed zebrafish animal caps injected with truncated or full-length ephrin-B2, EphB2 or EphA4 confirmed that bi- directional Eph protein signalling is required to restrict cell intermingling of the adjacent cell populations (Mellitzer et al., 1999). In antisense morpholino (MO) knock- down experiments of EphA4 and ephrin-B2a in zebrafish, the rhombomere boundaries were disorganised, confirming that Eph-ephrin signalling is required for boundary formation. In mosaic zebrafish embryos in which EphA4MO cells were transplanted

Queensland Institute of Medical Research 49 Chapter 1: Literature Review into the hindbrain of wild-type embryos, the EphA4MO and EphA4 expressing cells sorted from one another, suggesting that EphA4-dependent adhesion also contributes to the cell sorting process which underlies rhombomere boundary formation. The ephrin ligand which contributes to this proposed adhesion has not been identified (Cooke et al., 2005).

In another model system in the sensory rays of C. elegans, the Eph/ephrin proteins may also play a role in cell segregation and boundary formation. Each sensory ray is composed of a row of cells connected by adherens junctions, but with no connection between the cells of the adjacent rays (Baird et al., 1991). In the Vab-26 mutant, in which ectopic adhesive contacts form between cells in adjacent rays, there is a mutation in ephrin-4, one of the four C. elegans ephrin genes (Chin-Sang et al., 2002). However, the role of ephrin-4 is somewhat more complicated because it does not actually bind Vab-1, the single C. elegans Eph receptor (Chin-Sang et al., 2002), and the signalling pathway for ephrin-4 may involve Sema2A (mab-20) (Hahn and Emmons, 2003).

More recent studies in ephrin-B1 knockout mice have confirmed the role of Eph protein signalling in direct cell sorting throughout the body. Because ephrin-B1 is located on the X-chromosome, random X-inactivation in heterozygous ephrin-B1 knockout female mice will create two populations, consisting of ephrin-B1 positive and ephrin-B1 negative cells. These two populations of cells have been observed to segregate from one another, such that ectopic patches of Eph-ephrin-B1 signalling caused defects in tissue morphogenesis (Compagni et al., 2003). Furthermore, the phenotype of heterozygous females is more severe than that of hemizygous (null) males, because there are two distinct populations of ephrin-B1 expressing cells in the heterozygous female compared with only one single homogenous cell population in null males (Compagni et al., 2003). In mice rendered ephrin-B1 deficient only in NCCs by using a Cre-loxP strategy, the NCC ephrin-B1 null mice exhibited cleft palate at a lower penetrance than the complete ephrin-B1 null animals, with defects in other NCC derived tissues, suggesting that ephrin-B1 is required in a cell autonomous fashion in NCCs. However, closer examination of the difference between palatal shelf development in complete ephrin-B1 null and ephrin-B1 NCC null mice suggests that ephrin-B1 is required both cell autonomously (reverse signalling) and cell non-autonomously (forward signalling) in craniofacial development. The migratory behaviour of NCCs can be tracked in detail by co-expression of β-galactosidase in these mutant mice, and the migration of NCCs from

50 Queensland Institute of Medical Research Chapter 1: Literature review the post-ototic rhombomeres to branchial arches 3 and 4 exhibits a wandering behaviour which has also been observed in cardiac NCCs but not trunk NCCs (Davy et al., 2004; Soriano, 1999).

The subdivision of the paraxial mesoderm into somites, in which mesenchymal cells differentiate into epithelial cells to delineate the boundaries between the developing somites, is the initial mesodermal segmentation event of vertebrate development. This process is the result of three distinct processes:- (1) pre-patterning of the mesoderm; (2) boundary formation and (3) patterning within the somite itself. Dominant negative experiments in zebrafish, in which RNA encoding full-length or truncated Eph/ephrin proteins are injected into zebrafish eggs, indicated that Eph protein signalling is required for segmentation and differentiation of the somites (Durbin et al., 1998). In the fused somites (fss) zebrafish mutant, which lacks intersomitic boundaries and epithelial somites (Nikaido et al., 2002), restoration of EphA4 signalling rescues most aspects of somite morphogenesis. EphA4-ephrin interactions cause the cells at the boundary interface to detach from one another, resulting in a morphological furrow separating adjacent somites. There are two changes involved in this process. Firstly, the cells on either side of the furrow acquire a columnar morphology. Secondly, the cells become polarised with:- (1) relocalisation of the β-catenin component of the adherens junctions to the apical junctions; (2) relocalisation of the centrosome to the apical pole and (3) relocalisation of the nucleus to the basal pole. These changes are mediated by Eph/ephrin protein bi-directional signalling; however, the precise signalling pathways remain to be elucidated (Barrios et al., 2003).

In mice which lack the Hoxa13 gene, the mesenchymal condensations that form the autopod skeletal elements were poorly developed, resulting in missing digits, carpal and tarsal elements, in addition to disorganised mesenchymal and endothelial cell layers of the umbilical arteries which resulted in embryonic death (Stadler et al., 2001). There was marked reduction in EphA7 expression in affected digits and decreased expression of both EphA4 and EphA7 in the umbilical arteries (Stadler et al., 2001). In the developing chick limb bud, retroviral mediated ectopic expression of ephrin-A2 resulted in malformations of the limb bud (Wada et al., 2003). Epithelial branching morphogenesis gives rise to many epithelial organs including the kidney, lung, prostate, salivary and mammary glands. Hepatocyte growth factor/scatter factor (HGF), which functions through the C-Met receptor tyrosine kinase, induces branching

Queensland Institute of Medical Research 51 Chapter 1: Literature Review morphogenesis. HGF-induced branching of Madin-Darby canine kidney (MDCK) cells in vitro was suppressed by ephrin-A1 activation of EphA2 (Miao et al., 2003).

Both tissue boundary formation and cell migration are important for the correct positioning of cells throughout the body during development, and many molecules in addition to the Eph/ephrin proteins are responsible for coordinating this process (Hinck, 2004). Repulsive and possibly adhesive Eph signals drive the cell sorting and prevent intermingling of sub-populations of cells that can be identified by the cell surface expression of different Eph and ephrins (Cooke et al., 2005). The interfaces created by differential Eph/ephrin expression, and mediated by Eph bi-directional signalling, contribute to the pattern of cellular organisation within tissues (Poliakov et al., 2004).

1.7 Cell proliferation and differentiation

The determination of cell fate is regulated not only by intrinsic factors such as the expression of transcription factor genes, but also by cell position. The classical developmental literature demonstrated that the spatial location of a cell within the embryo places that cell in a specific environment, which influences final outcome. More recent work has identified a multitude of signalling factors expressed in restricted domains, also referred to as micro-environments or niches, which influence the fate of surrounding cells. Furthermore, these signalling factors have to be experienced by the cell in the correct temporal sequence. Clearly the position of a cell within the embryo determines which signals are experienced in the local environment (Gilbert, 2003). The Eph/ephrin proteins control not only the migration and final position of neural crest cells and axon growth cone guidance, but also control the movement of developmentally competent cells into the appropriate environment to allow them to experience differentiation signals which determine cell fate (Moody, 2004).

The small intestinal epithelium consists of flask-shaped invaginations, known as the crypts of Lieberkühn, which are responsible for cellular renewal, and the intestinal villi, which comprise the mature absorptive surface of the gastrointestinal tract. The crypts contain an estimated one to six intestinal stem cells (stem cell compartment), which slowly duplicate and give rise to progenitor cells which rapidly proliferate (proliferation compartment) and migrate from the bottom of the crypt to the tip of the intestinal villus where they are finally shed into the intestinal lumen. The intestinal progenitor cells become committed towards one of four lineages:- (1) mucous-secreting; (2) absorptive; (3) entero-endocrine or (4) Paneth cells. The Paneth cell, which is an intestinal

52 Queensland Institute of Medical Research Chapter 1: Literature review macrophage, does not migrate towards the villus tip, but moves in the opposite direction and resides at the bottom of the crypts (Sancho et al., 2003).

The expression of EphB and ephrin-B in the intestinal epithelium is regulated by β-catenin and T-cell transcription factor (TCF), which is under the influence of the Wnt signalling pathway (Batlle et al., 2002; van de Wetering et al., 2002). Wnt signalling upregulates EphB2 and EphB3 expression in proliferating cells, whereas differentiated cells downregulate EphB receptors and upregulate ephrin-B1 (Sancho et al., 2003). In the intestinal epithelium there are opposing gradients of EphB2/EphB3 and ephrin- B1/ephrin-B2 along the intestinal crypt-villus axis. These gradients regulate stem cell renewal by controlling cell position, which in turn determines the exposure of the stem cells to differentiation factors that are released from the proliferative zone at the bottom of the crypts. Analysis of EphB2/EphB3 null mutant mice revealed that complementary EphB-ephrin-B expression serves to maintain the sorting of precursor and differentiating cell populations into distinct domains (Batlle et al., 2002). The EphB2/EphB3 null mutant mice also exhibited reduced proliferation within the intestinal stem cell niche (Holmberg et al., 2006). The Paneth cells express high levels of EphB3, which explains their migration away from high levels of ephrin-B1 expressed at the villus tip. Conversely, in EphB3 null mutants, the Paneth cells no longer move down towards the base of the crypts and are mis-positioned in the upper crypt region (Batlle et al., 2002).

The production of red blood cells from haematopoietic progenitor cells is regulated in part by erythropoietin and the micro-environment of the spleen and bone marrow (Bollerot et al., 2005). The spleen and bone marrow comprise resident stromal cells, extracellular matrix, secreted cytokines and growth factors and provides the micro- environment required for self-renewal and differentiation of haematopoietic stem cell and progenitor cells through complex cell-cell interactions (Heissig et al., 2002). Haematopoietic progenitor cells express various receptors during proliferation and maturation and respond to different cytokines and growth factors, which drives them down a particular maturational pathway (Lataillade et al., 2004).

The position of haematopoietic progenitors within specific “niches” in the spleen or bone marrow micro-environment determines to which particular maturational pathway progenitors become committed (Arai et al., 2005; Heissig et al., 2005). This positional information is determined by cell-cell interactions, and is mediated at least in part by

Queensland Institute of Medical Research 53 Chapter 1: Literature Review

Eph-ephrin interactions. EphB4 is predominantly expressed on human erythroid progenitors in bone marrow, and on nearly half of the CD34+/c-Kit+ human erythroid progenitors in human umbilical cord blood (Bennett et al., 1995; Inada et al., 1997). Ephrin-B2 is expressed on bone marrow stromal cells (Inada et al., 1997; Sakano et al., 1996). Under hypoxic conditions ephrin-B2 is upregulated, triggering the detachment of EphB4-positive haematopoietic progenitor cells and their differentiation into mature red blood cells (Suenobu et al., 2002). The mesodermal differentiation of EphB4 null embryonic stem cells was altered with impaired rate and magnitude in the production of haemangioblasts, mature blood cells, vascular endothelium and cardiomyoctes, suggesting that EphB4 plays a role in control of differentiation (Wang et al., 2004a). It was previously noted that retro-viral mediated overexpression of EphB4 in primary cord blood cells resulted in accelerated transition of primitive cells from a stem cell to a lineage-restricted adult phenotype (Wang et al., 2002b). Using co-culture of a stroma- dependent haematopoietic progenitor cell line, ephrin-B2 expression on haematopoietic cells was regulated by the interaction with stromal cells. Furthermore, an ephrin-B2 overexpressing pre-B cell line reduced transmigration into a stromal cell layer, presumably via ephrin-B2 interaction with cell surface expressed EphB4. These findings support a role for Eph protein interactions in providing positional information to haematopoietic progenitor cells, thus determining differentiation (Okubo et al., 2006). The thymus of EphA4-deficient mice was hypocellular with decreased proportions of CD4+/CD8+ cells, reduced numbers of cycling thymocytes and increased apoptosis. Reconstitution of severe combined immunodeficient (SCID) mice with EphA4 null bone marrow suggested that the altered T-cell development in EphA4 mutant mice was due to a defective thymic environment rather than the absence of EphA4 signalling in thymocytes (Munoz et al., 2006).

The Eph/ephrin proteins regulate the differentiation of stem cells and progenitor cells in organs other than the bone marrow. In neural precursor cells both in vivo and in vitro, EphA signalling alters the fate towards neuronal commitment (Aoki et al., 2004). Furthermore, neural cell progenitor proliferation in the SVZ is regulated by EphA7– ephrin-A2 signalling (Holmberg et al., 2005). In frogs, ephrin-B1 reverse signalling promotes the movement of retinal progenitor cells into the eye field, which is required for the progenitor cells to acquire a retinal cell fate (Moore et al., 2004). It is likely that Eph protein signalling is responsible for the differentiation of stem cells in other organs

54 Queensland Institute of Medical Research Chapter 1: Literature review and tissues by controlling the position, and therefore, the micro-environment of the immature cells and the various maturational factors which they encounter.

1.8 The role of Eph/ephrin proteins in cancer

The protein tyrosine kinases are the largest family of oncogenes because of their central role in signal transduction pathways which control cell differentiation and proliferation (Bishop, 1991; Blume-Jensen and Hunter, 2001; Hunter and Cooper, 1985). However, the Eph family of RTKs are not classical oncogenes, and Eph receptor stimulation does not result in a proliferative cellular response (Dodelet and Pasquale, 2000). The originally described Eph receptor, EphA1 (formerly eph) was found to be overexpressed in an erythropoietin producing hepatocarcinoma cell line (Hirai et al., 1987). It has been suggested that the upregulation of expression of the Eph/ephrin proteins in human cancers and tumour cell lines contributes to the mechanism of tumour invasion, tumour neo-angiogenesis and metastasis (Nakamoto and Bergemann, 2002). Indeed the re- expression of Eph/ephrin proteins and the uncontrolled re-emergence in their developmental role in controlling cell adhesion/repulsion and cell migration suggests that Eph/ephrin mediated tissue disorganisation and dysregulated neo-angiogenesis may be important in cancer biology (Nakamoto and Bergemann, 2002; Wimmer-Kleikamp and Lackmann, 2005).

The gene expression patterns of Eph and ephrins has been defined in a variety of benign and malignant human tumours, and a number of subsequent studies report upregulation or downregulation of Eph/ephrin proteins in tumour cell lines and human cancers (Hafner et al., 2004; Nakamoto and Bergemann, 2002). A genome-wide screen of mutations in tyrosine kinases in colorectal cancer cells revealed that EphA3, EphA7 and EphA8 are frequently mutated in the kinase domain (Bardelli et al., 2003; Wang et al., 2004b). The role of EphA2 in colorectal, lung, oesophageal, ovarian, renal and cervical cancers and melanoma has been recently reviewed, and the correlation of increased EphA2 expression with disease stage, increased tumour metastasis and poor prognosis suggests a critical role for EphA2 in cancer progression (Ireton and Chen, 2005; Walker-Daniels et al., 2003). In a study of implanted mammary adenocarcinoma growth, the tumours isolated from EphA2 null mice had decreased tumour volume, decreased microvascular density and fewer lung metastases and, in addition, the EphA2 deficient endothelial cells did not contribute to the tumour vasculature and did not support tumour growth (Brantley-Sieders et al., 2005). The EphA2 locus on

Queensland Institute of Medical Research 55 Chapter 1: Literature Review chromosome 1p36.1 has previously been identified as a genetic “hot spot” in cancer and is deleted or altered in neuroectodermal tumours (Sulman et al., 1997).

In normal adult melanocytes there is no Eph protein expression, but EphA3 was detected in 50–80% of melanoma samples by semi-quantitative PCR (Chiari et al., 2000; Lawrenson et al., 2002). In metastatic melanoma cell lines there are higher expression levels of EphA2, EphA3 and ephrin-A1 compared with primary melanoma (Easty and Bennett, 2000). Overexpression of ephrin-A1 and ephrin-B2 correlated with degree of malignancy, and tumour promoting drugs or repeated high dose UVB irradiation of human melanocytes and melanoma cell lines increased ephrin-B2 expression (Vogt et al., 1998). Aggressive metastatic melanoma was characterised by the ability of the melanoma cells to express endothelial-associated genes and to form tubular structures and three dimensional patterned networks in vitro known as “vasculogenic mimicry”. In an experimental approach designed to investigate vasculogenic mimicry, utilising three dimensional collagen matrices preconditioned by metastatic cells, poorly aggressive melanoma cells were induced to express angiogenic, vasculogenic, and matrix remodelling genes including EphA2 (Hendrix et al., 2003).

In breast cancer there is also a strong correlation between Eph protein expression levels and tumour aggressiveness and invasiveness (Walker-Daniels et al., 2003). Semi- quantitative analysis by RT-PCR and immunohistochemistry correlated high human tumour expression of EphB2 protein with the poorest long-term survival, whereas EphB4 protein expression was associated with a less malignant histological grade (Wu et al., 2004). In the Ha-ras transgenic mouse model of breast cancer, EphA2 and EphB4 were overexpressed in poorly differentiated and invasive mammary tumours (Andres et al., 1994; Andres and Ziemiecki, 2003). Overexpression of ephrin-B2 and EphB4 has been reported in human endometrial hyperplasia and carcinoma (Berclaz et al., 2003).

Analysis of the EphB2/EphB3 null mouse has revealed the importance of these Ephs in cell positioning in the normal intestinal epithelium (Batlle et al., 2002). In a colon tumour mouse model (multiple intestinal neoplasia = “Min mouse”), elevated levels of EphB2 and EphB3 expression were associated with abnormal epithelial cell migration at the crypt-villus junction (Batlle et al., 2002). Analysis by RT-PCR, Northern blot and immunohistochemical survey found co-expression of EphB2, EphB3, EphB4 and ephrin-B2 in colon carcinoma cell lines and tumour specimens (Liu et al., 2002; Stephenson et al., 2001). Most sporadic colorectal cancers are initiated by activating

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Wnt signalling pathway mutations and constitutive transcription of the β-catenin/T-cell factor 4 (Tcf-4) complex. The EphB receptors are target genes for Tcf-4 which ultimately controls the intestinal epithelial cell architecture (van de Wetering et al., 2002). It has been suggested that EphB2 may be a tumour suppressor gene, as it is located on chromosome 1p35-36, which is a frequent site of allelic loss in colorectal tumours (Zhou et al., 2004). A strong association was observed between histological tumour grade, EphB2 silencing and EphB2 expression loss in most tumours at the stage of adenoma to carcinoma transition (Batlle et al., 2005). In metastatic colon cancer, elevated EphA2 and decreased E-cadherin correlated with liver metastases and lymphatic vessel involvement (Saito et al., 2004).

In brain tumours, glioblastomas and derived tumour cell lines, elevated levels of EphA2, EphA3, EphB2 and EphB4 are expressed (Hafner et al., 2004; Weiner et al., 1996). In glioma cell lines and human brain tumour specimens, both EphB2 expression measured by real-time quantitative PCR (Q-PCR) and EphB2 phosphorylation were associated with a more invasive phenotype. In a glioma cell line, treatment with ephrin- B1-Fc promoted migration and invasion, which could be inhibited by a EphB2 blocking antibody (Nakada et al., 2004).

The pathogenesis of haematological and lymphoid malignancy also involves Eph/ephrin signalling. EphA3 was cloned from pre-B cell acute lymphoblastic leukaemia cells (Boyd et al., 1992), and other haematopoietic tumour cell lines show variable expression levels (Dottori et al., 1999). Ephrin-A1 modulates T-cell chemotaxis in response to chemokine CXCR4 receptor signalling (Sharfe et al., 2002). In the Jurkat T- cell tumour line, ephrin-A1 activation of EphA3 downregulates Cbl-mediated proteasomal degradation (Sharfe et al., 2003). In a cytokine/receptor cDNA array following CD28 stimulation of Jurkat cells, EphA3 was identified as a CD28 responsive gene. EphA3 expression was detected by immunohistochemistry and semi-quantitative RT-PCR in 38% of a panel of primary lymphomas (n = 42). Stimulation of EphA3 by clustered ephrin-A5 was associated with CrkII recruitment and decreased Jurkat T-cell adhesion to fibronectin surfaces (Smith et al., 2004b).

Malignant tumours require the development of a local circulation in order to support tumour survival and continued growth (Folberg et al., 2000; Hendrix et al., 2003). Using immunohistochemistry in human tumours grown in nude mice and surgical human tumour specimens, both EphA2 and ephrin-A1 were detected in endothelial

Queensland Institute of Medical Research 57 Chapter 1: Literature Review cells, co-expressed with CD31, an endothelial cell marker, and may play a role in tumour neovascularisation (Ogawa et al., 2000). Antagonising EphA2 function with soluble EphA2-Fc receptors inhibited tumour neovascularisation and growth in a mouse pancreatic islet cell tumour model and inhibited the migration of endothelial cells in response to tumour cells (Brantley et al., 2002). Both EphB4 and ephrin-B2 are essential for the establishment of the arterial-venous boundary during the development of the cardiovascular system (Adams, 2003), and ephrin-B2 expression is found in some tumour blood vessels (Gale et al., 2001).

The transition from normal cell through to the malignant phenotype involves the acquisition of new characteristics including cell survival, cell morphology, cell adhesion and motility (Bogenrieder and Herlyn, 2003). Many of the cellular and molecular processes which are involved in embryological development, tissue repair and regeneration following injury are similar to those observed during malignant transformation and include the re-expression of Ephs and ephrins (Friedl and Wolf, 2003). It appears that Eph/ephrin mediated control of cell-cell contact, cell motility and adhesion are involved in the increase in malignant cell motility, tumour cell spreading and final lodgement of metastatic tumour cells (Wimmer-Kleikamp and Lackmann, 2005). The re-emergence of Eph/ephrin protein expression in malignancies and their limited expression in adult tissues provides an opportunity for the development of novel anti-cancer therapeutic agents. In particular, the development of antibodies to Eph/ephrin proteins and Eph/ephrin-Fc fusion proteins to disrupt the intercellular signalling pathways is a potential method of treating metastatic tumours and tumour neovascularisation (Brantley et al., 2002; Dobrzanski et al., 2004; Ireton and Chen, 2005; Martiny-Baron et al., 2004). 1.9 Vasculogenesis and angiogenesis

The development and maintenance of the heart and blood vessels requires the processes of:- (1) vasculogenesis, which involves the de novo differentiation of endothelial precursors from mesoderm, followed by the assembly of blood vessels by migration, proliferation, cell-cell aggregation and morphogenesis, initially resulting in the formation of an unorganised meshwork of capillaries or primary vascular plexus and (2) angiogenesis, in which the mature blood vessels are formed by remodelling of the primary vascular plexus by branching, sprouting and fusion (Risau, 1997; Risau and Flamme, 1995). The development of the lymphatic system starts when a population of

58 Queensland Institute of Medical Research Chapter 1: Literature review venous endothelial cells become committed and sprout from the major central veins (Eichmann et al., 2005b). A number of other RTKs interact with the Eph/ephrin proteins and mediate the development of the blood vessels, including vascular endothelial growth factors (VEGFs), their corresponding receptors Flk-1 (VEGF-R2) and Flt-1 (VEGF-R1); angiopoietin-1 and its receptor Tie2; activin receptor-like kinase 1 and the Notch signalling pathway (Adams, 2003; Eichmann et al., 2005a; Eichmann et al., 2005b; Gale and Yancopoulos, 1999; Vogel, 2005).

The initial evidence for the role of Eph/ephrin proteins in vascular development was the identification of ephrin-A1 (previously B61) as a TNF-α responsive gene in endothelial cells (Dixit et al., 1990; Holzman et al., 1990). Ephrin-A1 is chemo-attractive for migrating endothelial cells in vitro, induces tubule formation in assays with human umbilical vein endothelial cells and has been shown to induce sprouting blood vessels in a corneal pocket assay (Pandey et al., 1995b). Ephrin-A1 mRNA was expressed in the mouse embryo in dorsal aorta, primary head veins, intersomitic vessels and limb bud vasculature (Flenniken et al., 1996; McBride and Ruiz, 1998). Antisense to HoxB3 blocks capillary endothelial morphogenesis which can be rescued with ephrin-A1-Fc (Myers et al., 2000). The cyclic growth of the endometrial lining of the uterus involves angiogenesis, and ephrin-A1 expression has been observed in normal human endometrial epithelial cells (Fujiwara et al., 2002). In biopsy material from uterus with endometriosis, ephrin-A1 expression is downregulated (Kao et al., 2003b).

The vertebrate cardiovascular system is composed of arteries and veins, and previously the anatomical differences between these vessels reflected their physiological roles, for example oxygen saturation, blood pressure and shear forces. Both EphB1 (ELK) and ephrin-B1 (LERK-2) are expressed on mesenchymal progenitors of vascular cells, on the glomerular capillary endothelium of adult kidney and human umbilical vein endothelial cells (Daniel et al., 1996). In human renal microvascular endothelial cells, ephrin-B1 promotes assembly of capillary-like structures (Daniel et al., 1996). Genetically modified mice lacking either ephrin-B2 or EphB4 die before E11 from cardiovascular defects including an enlarged heart and poor development of the yolk sac vasculature (Adams et al., 1999; Gerety et al., 1999; Wang et al., 1998). Analysis of an ephrin-B2/tau-β-galactosidase reporter mouse determined that ephrin-B2 is a marker of arterial endothelial cells during early development (Callahan and Thomas, 1994; Wang et al., 1998). The ephrin-B2 null mice (homozygous ephrin-B2/tau-β-galactosidase

Queensland Institute of Medical Research 59 Chapter 1: Literature Review mice) display absent angiogenesis in the yolk sacs and heads, with ablation of both the venous and arterial capillary beds, and die in utero at around E11. The expression of ephrin-B2 and its receptor EphB4 was in a complementary pattern on embryonic arteries and veins respectively, suggesting reciprocal interaction in the vascular remodelling process (Wang et al., 1998). Subsequent expression studies of ephrin-B1, ephrin-B2, EphB2, EphB3 and EphB4 were examined using fusion proteins of alkaline phosphatase and either EphB3 or ephrin-B2 and confirmed by RT-PCR (Adams et al., 1999). The complementary expression of EphB4 and ephrin-B2 was confirmed, and additional findings included ephrin-B1 and ephrin-B2 expression in the aortic arches, and co-expression of EphB3, EphB4 and ephrin-B1 on veins, suggesting a more complex interaction between the Ephs and ephrins in vascular morphogenesis (Adams et al., 1999).

In separate mutant mouse experiments, ephrin-B2 null mice displayed a phenotype similar to that described in the ephrin-B2/tau-β-galactosidase homozygous null mice, with embryonic lethality, growth retardation, a dilated pericardium and abnormal yolk sac vasculature (Adams et al., 1999). The expression pattern of ephrin-B2 was further analysed following generation of an ephrin-B2/β-galactosidase reporter mouse (Gale et al., 2001). In the adult mouse, ephrin-B2 expression extended from the arterial endothelium to the surrounding arterial smooth muscle and pericytes and in some capillary beds to the mid-point of the terminal arterioles and post-capillary venules. This suggests that during embryological development, ephrin-B2 expression marks the developing arterial vascular tree (Gale et al., 2001). In a carcinoma model in which Lewis lung carcinoma cells were transplanted into the muscle or subcutaneous tissue of ephrin-B2 heterozygous reporter mice, ephrin-B2 marked the arteries but not the veins in the pathological angiogenesis which accompanied tumour invasion (Gale et al., 2001). The function of ephrin-B2 has been studied in vitro in an endothelial cell system, in which OP9 stromal cells are co-cultured with para-aortic splanchno-pleura mesodermal cells. This study confirmed that ephrin-B2 promotes overexpressing OP9 stromal cells to form a vascular network with endothelial cell proliferation and recruitment of α-actin smooth muscle positive cells. Stromal cells transfected with EphB4 inhibited these ephrin-B2 effects, suggesting that ephrin-B2 and EphB4 mediate reciprocal interactions between arterial and venous endothelial cells and the surrounding cells in the formation of mature blood vessels (Nakano et al., 1994; Zhang et al., 2001).

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In 30% of the EphB2/EphB3 double knockout mice (n=32) the vascular phenotype displayed was similar but not as severe as the ephrin-B2 null mice (Orioli et al., 1996). The hearts of both the ephrin-B2 null and EphB2/EphB3 double knockout mice were smaller and had less intricately folded trabeculae within the ventricles. There were no vascular defects in either the single EphB2 or EphB3 knockout mice (Adams et al., 1999). A cDNA knock-in strategy in which the ephrin-B2 cytoplasmic domain was deleted and tagged with a haemagglutin (HA) epitope (ephrin-B2-∆C-HA) was used to study the function of the cytoplasmic domain in vivo. The homozygous ephrin-B2 cytoplasmic domain deleted mice (ephrin-B2-∆C-HA) died at around E11 with a phenotype similar to ephrin-B2 knockout mice, suggesting that the cytoplasmic domain of ephrin-B2 and reverse-signalling is required for vascular morphogenesis (Adams et al., 2001). However, other investigators prepared ephrin-B2 mutant mice such that the cytoplasmic domain was replaced by β-galactosidase (ephrin-B-2∆C-β-galactosidase). In these mice, vascular development was normal with the birth of live pups suggesting that ephrin-B2 reverse signalling was not required for vascular development. However, these mice displayed urogenital defects not seen in the ephrin-B2 null mice (Dravis et al., 2004). The explanation for these discordant results is that the ephrin-B2-∆C-HA protein became trapped in the Golgi apparatus and was not expressed on the cell surface, so this was essentially an ephrin-B2 protein null mouse. However, the ephrin- B2-∆C-β-galactosidase protein did pass through the Golgi apparatus and was successfully expressed on the cell membrane and participated in EphB4 forward signalling (Dravis et al., 2004). The generation of EphB4/tau-β-galactosidase reporter mice confirmed the in situ hybridisation evidence that EphB4 is expressed on veins and not arteries. The EphB4/tau-β-galactosidase null mice had a phenotype similar to the ephrin-B2 null mice with embryonic lethality and cardiovascular defects confirming the reciprocal functions for EphB4 and ephrin-B2 during angiogenesis and vascular development (Gerety et al., 1999).

In VEGF induced angiogenesis in the cornea and a wound healing model of neovascularisation using ephrin-B-2∆C-β-gal reporter mice, ephrin-B2 was expressed in new corneal vessels and small vessels around the wound region. In an implanted lung carcinoma or melanoma tumour model in these mice, ephrin-B2 was expressed within the tumour vasculature (Shin et al., 2001). The role of EphB/ephrin-B signalling was studied by in situ hybridisation in X. laevis embryos. This confirmed that EphB4 and its ligands ephrin-B1 and ephrin-B2 were expressed in a complementary fashion in the

Queensland Institute of Medical Research 61 Chapter 1: Literature Review developing vasculature. EphB4 is expressed on the intersomitic veins and ephrin-Bs are found in the adjacent tissues. Further gain of function (overexpression of ephrin-B) and loss of function (expression of truncated EphB4) experiments demonstrated that EphB4 and ephrin-Bs are required for angiogenic growth and guidance of the intersomitic veins (Helbling et al., 2000). A study of EphB4 null embryonic stem cells indicated that EphB4 is essential for haematopoietic, endothelial, haemangioblast, cardiomyocyte and primitive mesoderm development (Wang et al., 2004a).

An ephrin-B2 mutant mouse with the C-terminal valine of the PDZ interaction site deleted (ephrin-B2-∆PDZ) and another mutant mouse with the five conserved C- terminal tyrosines replaced (ephrin-B2-5F) with phenylalanine were generated. The ephrin-B2-∆PDZ homozygous mutant mice developed chylothorax and displayed major lymphatic vessel defects; however, the ephrin-B2-5F homozygous mutant mice had a milder phenotype (Makinen et al., 2005). Recently, a conditional ephrin-B2 knockout mouse was generated to selectively disrupt ephrin-B2 expression in pericytes and vascular smooth muscle cells. Observation of these mutant mice indicated that ephrin- B2 is a regulator of vascular smooth muscle cell migration, spreading and adhesion during blood vessel wall assembly (Foo et al., 2006).

The normal development of the cardiovascular and lymphatic system requires the coordinated function of a number of important transcription factors, receptor-ligand pairs, growth factors and guidance molecules (Hinck, 2004). These molecules include the Eph/ephrin proteins, VEGF and receptors (VEGF-R1 & VEGF-R2), angiopoietins (Ang-1 and Ang-2) and receptors (Tie1 and Tie2), netrins, slits and their receptors (Robo), semaphorins and plexins (the receptors for the semaphorins) and neuropilins. The genetic studies summarised above highlight the importance and complexity of the Eph/ephrin proteins in normal vascular development and adult vascular remodelling (Carmeliet and Tessier-Lavigne, 2005; Eichmann et al., 2005a; Eichmann et al., 2005b), and support their emerging role in tumour angiogenesis and the development of other human vascular diseases (Brantley-Sieders and Chen, 2004). For example, in retinopathy of prematurity, diabetic retinopathy, neovascular glaucoma and age-related macular degeneration, vision loss results from abnormal retinal angiogenesis. The VEGF-induced angiogenic responses of cultured retinal endothelial cells are inhibited by EphA2-Fc. In a rat model of retinopathy of prematurity, intra-ocular injection of a soluble EphA2-Fc receptor resulted in a significant reduction in abnormal retinal

62 Queensland Institute of Medical Research Chapter 1: Literature review neovascularisation without affecting normal retinal vessels (Chen et al., 2006). In a mouse model of proliferative retinopathy, intra-ocular injection of ephrin-A1-Fc suppressed ischaemic retinal neovascularisation in a dose dependent manner by inhibiting VEGF-induced angiogenesis and vasopermeability (Ojima et al., 2006).

1.10 The molecular biology of EphA1

EphA1 was isolated during a search for novel tyrosine kinases with oncogenic potential. A human genomic library was probed with the 0.6 kB HindIII/Sst fragment of the viral oncogene v-fps and a 12.5 kB EcoRI fragment with homology was isolated. Northern blot analysis of poly(A)+ RNA from normal human liver and an erythropoietin- producing-hepatocarcinoma cell line (ETL-1) determined that hEphA1 was overexpressed 10 to 20 fold in the ETL-1 cell line. The hEphA1 gene also hybridised on Southern blotting to DNA from chicken, mouse, rat and human cell types. A panel of 50 human tumours analysed by Northern blotting detected overexpression of the hEphA1 gene in several carcinomas including colon, lung, mammary and hepatocellular carcinoma (Hirai et al., 1987). Further human tumours analysed by Northern blot confirmed hEphA1 overexpression without gene amplification in hepatoma, lung, pancreatic, gastric, breast, thyroid and colon carcinomas (Maru et al., 1988). Overexpression of hEphA1 in NIH3T3 cells resulted in colony formation in soft agar and tumour formation when injected into nude mice, suggesting that hEphA1 is an oncogene (Maru et al., 1990).

The hEphA1 cDNA was isolated and sequenced from a library constructed from poly(A)+ RNA isolated from the ETL-1 cell line. The novel sequence suggested that EphA1 was a transmembrane glycoprotein which was either a receptor or a growth factor. The evolutionary relationship of the EphA1 gene with the other receptor tyrosine kinase family members was investigated by defining the splicing position of domain- encoding exons and determining exon-intron organisation (Patthy, 1987). This analysis indicated that EphA1 represented a fourth class of receptor tyrosine kinase. The presence of EphA1 was confirmed in chicken, mouse, rat and D. melanogaster by Southern blotting. EphA1 expression was also examined in rat by Northern blot analysis and a single 3.5 kB transcript was confined to tissues of epithelial cell origin including liver, lung, kidney and testis (Maru et al., 1988).

The murine homologue of hEphA1, originally termed Esk for embryonic stem cell kinase, now mEphA1, was cloned by RT-PCR using degenerate oligonucleotide primers

Queensland Institute of Medical Research 63 Chapter 1: Literature Review and RNA prepared from embryonic stem cells in culture. Northern blot analysis revealed expression of a 4.2 kB transcript in E12 mouse embryo, adult mouse thymus, liver, kidney, lung and placenta (Lickliter et al., 1996). A larger transcript of ~6.0 kB was observed in liver, kidney and lung samples, presumably due to alternatively spliced transcripts (unpublished observations, JD Lickliter and AW Boyd). There was no signal from lymph node, spleen, heart, brain or skeletal muscle. Northern blot analysis of embryonic stem cells, embryoid bodies and embryonic fibroblasts for mEphA1 (Esk), EphA2 (Eck) and EphA3 (Mek 4) determined that EphA1 and EphA2, but not EphA3, were expressed at significant levels in embryonic stem cells and embryoid bodies (Lickliter et al., 1996).

1.11 The potential role of the Eph/ephrin proteins in medicine

A substantial proportion of the burden of human disease results from the consequences of cardiovascular and cerebrovascular disease, cancer, infectious diseases and injury (Murray and Lopez, 1997; Thom et al., 2006; Van Der Weyden, 1999). While substantial improvements in the quality of the lives of the majority of the world’s people will be achieved through sociopolitical change and prevention, advances in science and medicine promise considerable hope to those already suffering (Lancet Editorial, 2003). The Eph/ephrin proteins play a major role during development, with roles in neural guidance, tissue boundary formation, determination of stem cell fate, angiogenesis and vasculogenesis (Pasquale, 2005). The Eph/ephrin proteins also have an emerging role in the adult in carcinogenesis (Brantley-Sieders et al., 2004a), neural plasticity (Gerlai, 2001) and inflammation (Romanovsky et al., 2006) and are consequently being increasingly recognised as important in human health and disease.

The Eph/ephrin proteins play a role in tissue repair and maintenance (Hafner et al., 2004). The pathological features that follow traumatic injury and tissue damage include the coagulation of extravasated blood, which initiates a complex signalling cascade to recruit inflammatory cells, stimulate fibroblast and epithelial cell proliferation, direct cell migration and induce angiogenesis in order to restore tissue integrity (Baum and Arpey, 2005). The Eph/ephrin proteins play an important role in development by determining cell movement and position, so it is a plausible hypothesis that the infiltration of specific cells to the injury site, removal of tissue debris, and growth of new cells in a highly ordered fashion includes a role for the Eph/ephrin proteins.

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Tissue injury can also result from vascular disease in which the blood and nutrient supply is interrupted with severe consequences to major organs, for example acute myocardial infarction and cerebrovascular stroke. The ischaemic damage is then followed by reperfusion injury when blood flow is restored, either as part of the natural history of the disease, or as a result of therapeutic measures (Kaminski et al., 2002). The ischaemia-reperfusion injury is characterised by:- (1) an inflammatory response regulated by the pro-inflammatory cytokines, TNF-α, IL-1 and IL-6; (2) upregulation of endothelial adhesion molecules and (3) recruitment of inflammatory cells to the damaged tissue (Berti et al., 2002; Kaminski et al., 2002). In a mouse renal ischaemia- reperfusion model, DNA micro-array and Q-PCR were used to study changes in gene expression; however, there were no changes in Eph protein gene expression in this study (Yoshida et al., 2002). However, in an in vitro mouse model of renal ischaemia- reperfusion, EphA2 was upregulated and caused a decrease in apoptosis (Chen and Lemay, 2005). A mouse skin flap model was used to determine the response of Eph/ephrin proteins to hypoxia. Partial cutaneous oxygen tension and tissue lactate/pyruvate measurements confirmed tissue hypoxia, and Q-PCR confirmed induction of hypoxia inducible factor-1α (HIF-1α) and VEGF. The expression of EphB4, ephrin-B2, EphA2 and ephrin-A1 were upregulated in hypoxic skin and the temporal expression pattern was determined, which supports the hypothesis that Eph/ephrin proteins are involved in neovascularisation following hypoxic injury (Vihanto et al., 2005).

The classic histological observation that many of the features of normal wound healing are similar to the tumour micro-environment suggested that the tumour stroma is “normal wound healing gone awry” (Dvorak, 1986). The emerging role of the Eph/ephrin proteins in cancer biology has been reviewed and, by extrapolation, their role in wound healing needs to be confirmed. The fibroblast response to serum, which stimulates fibroblasts to express many of the genes involved in wound healing, was investigated with a cDNA micro-array genome wide survey and confirmed a gene expression signature similar to metastatic cancer. However, this approach of measuring gene expression has thus far not revealed Eph/ephrin proteins as candidates at the time points considered (Chang et al., 2004). In chronic heart failure, the levels of circulating cytokines, TNF-α and IL-1β are elevated. Differential display identified EphA3 as a cytokine responsive gene in cultured rat cardiomyocytes (Li et al., 1998). The action of EphA2 (Eck) and ephrin-A1 (B61) has been described in maintenance of the intestinal Queensland Institute of Medical Research 65 Chapter 1: Literature Review barrier (Rosenberg et al., 1997). Real-time PCR and cDNA micro-array analysis determined that EphA2, ephrin-A1, EphB2 and ephrin-B1/B2 expression were upregulated in the intestinal epithelial cells of mucosal lesions of patients with inflammatory bowel disease (Hafner et al., 2005a). Stimulation of the ephrin-B2 reverse signalling pathway induced the expression of wound healing associated genes in an intestinal epithelial cell line (IEC-6) (Hafner et al., 2005b).

In fish and amphibians, damage to the optic nerve or spinal cord is followed by infiltration of microglial cells and macrophages and significant subsequent axon regrowth and functional recovery. In contrast, the injury site in mammals appears to actively inhibit axonal regeneration and there is poor functional recovery. In fish, the neurons which undergo successful axonal regeneration have a similar but not identical molecular profile to neurons in the embryonic state (Bernhardt, 1999). In adult goldfish which underwent optic nerve injury, immunohistochemical studies indicated that there was transient upregulation of EphA3/EphA5 in the retinal ganglion cells coincident with upregulated tectal ephrin-A2 expression, both of which were required for restoration of the normal retinotectal topographic map (King et al., 2004; King et al., 2003; Rodger et al., 2000; Rodger et al., 2004). A more complete understanding of the molecular basis of recovering axons in the fish and amphibian central nervous system could provide valuable insight into potential therapeutic advances following brain and spinal cord injury in humans.

Spinal cord injury in rats resulted in a marked increase in EphB3 mRNA at day 7 post- injury, and was confirmed by immuno-localisation of EphB3 expression in white matter astrocytes and gray matter neurons (Miranda et al., 1999). In a traumatic brain injury model in rats, Eph protein expression was analysed by semi-quantitative PCR, and EphB1 expression increased slightly at day 7 post-injury (Biervert et al., 2001). In mice that underwent optic nerve de-afferentation, the graded expression patterns of ephrin-A2 and ephrin-A5 in the superior colliculus were similar to those found during development (Knoll et al., 2001). In rat optic nerve injury there was upregulation of ephrin-A2 in the superior colliculus and EphA5 in the retina (Rodger et al., 2001). In another study using semi-quantitative PCR of the injured adult rat spinal cord, EphA3, EphA4 and EphA7 mRNAs were upregulated. Furthermore, EphA3, EphA4, EphA6 and EphA8 immunoreactivity was increased in the ventro-lateral white matter. The EphA receptor expression localised in the white matter to glial cells, both astrocytes and

66 Queensland Institute of Medical Research Chapter 1: Literature review oligodendrocytes, and localised to neurons in the gray matter (Willson et al., 2002). EphB3 expression detected by in situ hybridisation was upregulated in rats subjected to complete thoracic spinal cord transection and was confirmed by immunohistochemistry (Willson et al., 2003). Immunohistochemical data suggested that ephrin-B2 was expressed on reactive CNS astrocytes and EphB2 was present on fibroblasts invading the injury site from the adjacent meninges (Bundesen et al., 2003). The expression of EphA3 mRNA and protein following spinal cord injury was elevated from day 2 to day 28 and EphA3 immuno-reactivity was observed in reactive astrocytes (Irizarry- Ramirez et al., 2005).

Following spinal cord hemi-section, EphA4 was upregulated in wild type mice on astrocytes associated with the glial scar at the injury site, whereas EphA4 null mice showed markedly reduced astrocytic gliosis and scar formation. The EphA4 null mice exhibited axonal regeneration, characterised by axons growing across the injury site, associated with significant functional recovery one to three months following the injury (Goldshmit et al., 2004). EphA4 upregulation following spinal cord contusion injury in rats was blocked by infusing EphA4 antisense oligonucleotides. This did not result in enhanced locomotor recovery, but did improve chronic pain scores (Cruz-Orengo et al., 2006). The optic nerve is a site of substantial axonal plasticity and remodelling following injury and mounts a significant attempt to regrow. A strain of mutant mice, which expressed the yellow fluorescent protein in a small, fixed proportion of RGC axons, was bred with EphB3 null mice. Following optic nerve crush injury, macrophages expressing EphB3 accumulated at the injury site, and ephrin-B3 was expressed on RGC axons at the injury site. In mice with reduced EphB3 function there was decreased axon sprouting following optic nerve crush injury. This suggests a role for EphB3 expressing macrophages interacting with ephrin-B3 expressing RGC axons in the remodelling events which follow optic nerve injury (Liu et al., 2006).

The Eph/ephrin proteins are important in brain development and synapse function in the adult brain and have been implicated in brain disorders (Yamaguchi and Pasquale, 2004). Eph dysfunction might contribute to mental retardation and autism, which are both characterised by abnormal dendritic processes (Kaufmann and Moser, 2000), and schizophrenia, which is associated with decreased NMDA receptor function and abnormal dendritic spines (Glantz and Lewis, 2000). The Eph protein expression profiles in both active and inactive central nervous system lesions of multiple sclerosis

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(MS), normal adjacent white matter and control tissues have been characterised by immunohistochemistry. Inflammatory cells in active MS lesions expressed ephrin-A1– 4, and EphA1, A3, A4, A6, A7 and not EphA2, EphA5 and EphA8. In axons adjacent to active MS lesions, EphA3, EphA4, EphA7 and ephrin-A1 expression was increased (Sobel, 2005).

The Hendra (HeV) and Nipah (NiV) paramyxoviruses cause fatal infections in both animals and humans. The ephrin-B2 ligand is the cell surface receptor required for the cellular entry of these viruses. The HeV and NiV possess two principal glycoproteins in their membrane envelope required for virion attachment (Eaton, 2001). The observation that there are permissive (allowing virion attachment) and non-permissive HeLa cell lines in cell fusion assays, allowed the subsequent determination by micro-array analysis that ephrin-B2 is the cell surface attachment receptor for both HeV and NiV (Bonaparte et al., 2005). A similar observation that an NiV G-glycoprotein Fc fusion protein (NiV-G-Fc) bound to permissive 293T, HeLa and Vero cells, but not Chinese hamster ovary cells (CHO-pgsA745), confirmed by a co-immunoprecipitation method that ephrin-B2 is the receptor for NiV attachment. Transfection of ephrin-B2 into the non-permissive CHO-pgsA745 cell line rendered them permissive to NiV entry. Furthermore, a soluble ephrin-B2-Fc prevented viral-envelope mediated cell-cell fusion (Negrete et al., 2005).

Craniofrontonasal syndrome [CFNS, OMIM #304110] is an X-linked craniofacial disorder characterised by craniofacial asymmetry, hypertelorism, coronal synostosis, strabismus, bifid nasal tip, grooved nails, thoracic skeletal abnormalities, as well as wiry and curly hair. A genotypic analysis of three affected families determined that a mutation of ephrin-B1 was the cause of CFNS (Wieland et al., 2004; Wieland et al., 2005). Most affected patients are females, whereas male carriers show no, or only mild, manifestations, although in two unrelated families both mothers with classical features of CFNS gave birth to sons with congenital diaphragmatic hernia with a mutation in EFNB1 (Vasudevan et al., 2006). Another study of 59 affected females from unrelated families, including 20 previously published (Twigg et al., 2004), confirmed that the affected females had significant mutations, either frameshift, splicing or nonsense mutations, in the extracellular domain of ephrin-B1 (Twigg et al., 2006).

The role of Eph/ephrin proteins in human disease is an emerging field and much remains to be explored. This new information raises the prospect of novel therapies that

68 Queensland Institute of Medical Research Chapter 1: Literature review precisely modulate the molecular mechanisms of disease through the administration of specifically targeted molecules, such as Eph-Fc, ephrin-Fc, or tyrosine kinase inhibitors, which disrupt Eph-ephrin interactions (Krause and Van Etten, 2005). For example, it may be possible to encourage spinal cord axons to regenerate across an area of spinal cord injury by the administration of ephrin-A5-Fc to antagonise EphA4, reduce astrocytic glial scarring and promote functional recovery. Similarly, retardation of viral dissemination in a patient infected with Hendra or Nipah virus could be achieved by preventing viral-cell fusion by the intravenous infusion of soluble ephrin-B2-Fc. The identification of ephrin-B1 mutations as the underlying molecular cause of CFNS will allow antenatal diagnosis in affected families.

1.12 Aims and hypothesis of this thesis

On the basis of previous work performed in this laboratory, this thesis presents the hypothesis that mEphA1 is the murine homologue of hEphA1 and will be expressed in epithelial tissues, predominantly skin and kidney. Further, as mEphA1 is expressed in the developing embryo and several important adult tissues, it is further hypothesised that the EphA1 knockout mouse could potentially have alterations in these organs and display a characteristic physical phenotype which will help define the functional role of EphA1 during development and in adult life.

This led to the following specific aims:-

Aim 1: To characterise mEphA1 expression and function using standard molecular laboratory techniques.

Aim 2: To determine if data could be derived in another animal model by isolating the zebrafish homologue of mEphA1.

Aim 3: To generate the hPLAP EphA1 reporter knockout mouse and analyse EphA1 expression and characterise the (possible) phenotype.

Aim 4: To generate the EphA1 conditional knockout mouse and undertake a preliminary phenotypic analysis.

Queensland Institute of Medical Research 69

Chapter 2: General materials and methods

2 GENERAL MATERIALS AND METHODS

2.1 Microbiological methods

2.1.1 Bacterial cultures

Cultures of Escherichia coli were generated by plating out an inoculum retrieved from a glycerol stock stored at –80ºC on Luria Bertani (LB) agar plates and incubated overnight at 37°C. A single colony was then picked to inoculate a starter culture in 5– 10 ml of LB broth in a 50 ml conical tube which was shaken overnight at 37°C. Glycerol stocks were generated by combining 400 µl of overnight bacterial culture with 80% glycerol to a final concentration of glycerol 15%, mixed completely and immediately frozen on dry ice, and transferred for storage at –80°C. Ampicillin resistant cultures were selected with ampicillin at a concentration of 100 µg/ml.

2.1.2 Preparation of electrocompetent E. coli

For the preparation of recombinant DNA molecules, the following strains of E. coli were used: DH5α, DH10β, STBL2, XL-1 Blue and XL-10 Gold. A 500 µl aliquot of bacterial cell starter culture was used to inoculate 50 ml of LB broth and was grown at 37°C with shaking (220 rpm) until the OD600 [Thermospectronic Biomate 3 Spectrophotometer] reached 0.75 (approximately 3–6 x 108 cells/ml). The bacterial cell suspension was collected in chilled polypropylene tubes (capacity 250 ml) and centrifuged at 2600 x g for 15 minutes and the supernatant was carefully decanted. The bacterial pellet was resuspended in half the original culture volume of 10% glycerol in Milli-Q standard water. The centrifugation step was repeated and the bacterial cell pellet resuspended in several drops of excess liquid. The OD600 was then determined by diluting 2 µl of bacterial cell suspension to 500 µl and the bacterial cell pellet 600 resuspended to a OD of 200–250 OD units/ml. The bacterial cell suspension was then dispensed in convenient sized aliquots of 100–200 µl in chilled 1.5 mL microcentrifuge tubes [Eppendorf™], immediately frozen on dry ice and transferred for long-term storage at –80°C. The transformation efficiency was tested with 100 pg of pBluescript (pBst) [Stratagene] and calculated in cfu/µg of plasmid DNA.

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2.1.3 Preparation of heat shock competent E. coli

2.1.4a Method 1

A 50 ml culture of E. coli was commenced with the addition of 500 µl of an overnight starter culture to Medium A (see Appendix 5) and grown at 37°C with shaking at 220 rpm to mid-logarithmic phase (OD600 of 0.4–0.6). The culture was incubated on ice for 10 minutes, then pelleted by centrifugation at 1500 x g for 10 minutes at 4°C. The bacterial cells were resuspended in 500 µl of Medium A and incubated on ice, to which 2.5 ml of Medium B (see Appendix 5) was added and mixed well without vortexing. The competent cells were divided into 200 µl aliquots in chilled microcentrifuge tubes and stored at –80°C until required.

2.1.4b Method 2

The Inoue transformation buffer was prepared in advance. A stock solution of 0.5 M PIPES was prepared by dissolving 15.1 g of PIPES [Sigma-Aldrich] in 80 ml of Milli-Q water, adjusting the pH to 6.7 with 5 M KOH [BDH Merck], then adjusting the final volume to 100 ml. The 0.5 M PIPES, pH 6.7, was filter sterilised by passage through a pre-rinsed Nalgene™ 0.45 µm filter [Nalge Nunc International], divided into aliquots and stored at –20°C. The transformation buffer was prepared by dissolving all the solutes: 10.88 g MnCl2.4H2O, 2.20 g CaCl2.2H2O, 18.65 g KCl and 20 ml PIPES (0.5 M, pH 6.7) and adjusting the total volume to 1 litre with Milli-Q water.

A bacterial starter culture, which had been picked from a freshly streaked LB plate, was used to inoculate three flasks containing 250 ml of LB broth and grown overnight at room temperature. The following morning the OD600 of all three cultures were read and monitored at 45 minute intervals. When the OD600 of one culture reached 0.55, the culture flask was placed in an ice bath for 10 minutes and the other cultures were discarded. The bacterial cells were harvested by centrifugation at 2500 x g for 10 minutes at 4°C. The medium was carefully poured off and the centrifuge bottle inverted for two minutes before aspirating the remaining drops of medium. The bacterial cell pellet was resuspended by swirling in 80 ml of ice-cold Inoue transformation buffer. The cells were again harvested by centrifugation at 2500 x g for 10 minutes at 4°C. The excess medium was again carefully removed with inversion then aspiration. The bacterial cells were then gently resuspended in 20 ml of ice-cold Inoue

72 Queensland Institute of Medical Research Chapter 2: General materials and methods transformation buffer, to which 1.5 ml DMSO [Sigma-Aldrich] was added and mixed by swirling before incubating for a further 10 minutes on ice. The competent cells were dispensed in 200 µl aliquots into pre-chilled 1.5 mL microcentrifuge tubes and immediately snap frozen in liquid nitrogen and stored at –80°C until required.

As competent bacterial cells were needed, a tube of competent cells was removed from the freezer, thawed by holding in the palm of the hand, and just as the cells thawed, the tube was transferred to an ice bath. 2.2 Molecular biology methods

2.2.1 Plasmid purification procedures

2.2.1a Small scale plasmid purification (“EasyPreps”)

Colonies for screening were picked from the LB ampicillin agar plate with a sterile disposable pipette tip then inoculated into a 1 ml culture of LB ampicillin (100 µg/ml) in a 15 ml yellow cap tube [Sarstedt] and the culture was grown overnight at 37ºC with shaking at 220 rpm. This overnight 1 ml culture was transferred to a 1.5 ml microcentrifuge tube with the lid punctured with an 18 gauge needle and centrifuged at maximum speed in a microcentrifuge at room temperature. The supernatant was aspirated and the bacterial cell pellet resuspended in 30 µl of EasyPrep Lysis Buffer (see Appendix 5) and shaken for 5 minutes at room temperature. The suspension was boiled for 60 seconds, then quenched for 2 minutes on ice before centrifugation at maximum speed for 20 minutes to remove cellular debris. The supernatant was transferred to a fresh microcentrifuge tube prior to sequencing or restriction analysis.

2.2.1b Medium scale plasmid purification

Plasmid DNA was purified using the Qiagen Midi-Prep column system [Qiagen] as per the manufacturer’s instructions. In summary, the bacterial cells were pelleted by centrifugation and resuspended in Buffer P1. Lysis was achieved with Buffer P2 and gentle inversion. Cellular debris was removed by precipitating with Buffer P3 and pelleting by centrifugation. Plasmid DNA was bound to an equilibrated Qiagen-tip 100 by passing the cleared lysis solution over the column. The column was washed twice with Buffer QC and the plasmid DNA eluted in Buffer QF. Plasmid DNA was concentrated by precipitating with isopropanol, centrifuging, and resuspending in a

Queensland Institute of Medical Research 73 Chapter 2: General materials and methods smaller volume of TE buffer. The concentration was calculated from its absorbance at 260 nm and size verified by agarose gel electrophoresis (detailed in Section 2.2.7).

2.2.1c Large scale plasmid purification

Bacterial cultures were grown overnight in LB media with antibiotic selection at 37ºC and shaking at 200 rpm. The cells were pelleted by centrifugation at 3500 x g for 10 minutes at 4ºC. The pellet was resuspended in MaxiPrep Solution I (Appendix 5). The cells were lysed by addition of an equal volume of MaxiPrep Solution II (Appendix 5.1) and the solution mixed gently and incubated on ice for 20 minutes. The viscous solution was then centrifuged at 27,000 x g for 40 minutes at 4ºC to remove cellular debris. The supernatant was transferred to a 30 ml Corex™ tube and the DNA precipitated by adding 5 M NaCl and PEG 6000 to final concentrations of 0.5 M and 10% w/v respectively. After incubating at 4ºC for 1 hour, the DNA was pelleted by centrifuging at 2,500 g for 5 minutes at 4ºC. The DNA was resuspended in MaxiPrep Solution III and CsCl added to a final concentration of 1.55 g/ml. Ethidium bromide was added to 0.25 mg/mL to aid in visualising plasmid DNA. The insoluble material was removed by centrifugation at 10,000 x g for 10 minutes at room temperature. The supernatant was transferred to a QuickSeal™ polyallomer tube (Beckman 13x51 mm) and centrifuged at 250,000 x g (55,000 rpm) for 16 hours at room temperature. The plasmid DNA was recovered with a 23 gauge needle attached to a 5 ml syringe and diluted in two volumes of TE buffer. Ethidium bromide was removed by butanol extraction until the solution was clear. The plasmid DNA was precipitated with two volumes of 100% ethanol and pelleted by centrifugation at 4,000 x g for 15 minutes at 4ºC. The DNA pellet was air dried and resuspended in TE buffer. If the plasmid DNA was to be used for transfection into mammalian cells then the CsCl was removed by passage over a NAP-5 column [Amersham Biosciences] in accordance with the manufacturer’s instructions. The concentration of plasmid DNA was calculated from its absorbance at 260 nm and size verified by agarose gel electrophoresis (detailed in Section 2.2.7).

2.2.2 Screening of a lambda bacteriophage genomic library

Initially, library screening plates, dimensions 20 cm x 20 cm [Bio-Rad], were poured using 200 ml of “bottom LB agar” per plate (see Appendix 5). The plates hardened

74 Queensland Institute of Medical Research Chapter 2: General materials and methods overnight at 37˚C. The λ bacteriophage genomic library was diluted such that the estimated plaque density was 200,000 viral plaques per plate. The phage of known titre was diluted in SM buffer (see Appendix 5) in a volume of 1 ml in a 50 ml conical tube to which 1 ml of phage plating E. coli (OD600 = 0.9) were mixed by gentle agitation and incubated for 20 minutes at 37˚C. The phage incubated E. coli were added to 35 ml of molten “top LB agarose” and the mixture was poured over the “bottom LB agar”. The “top agarose” was allowed to harden at room temperature, and the plates were inverted and incubated overnight at 37°C.

Bacteriophage plaques from the near confluent lysis of the bacterial lawn on the plates were transferred in duplicate to a Hybond N nylon membrane [Bio-Rad]. The membrane was carefully placed on the surface of the bacterial lawn and its position and orientation was marked with patterned needle pricks and allowed to “soak” for 5 minutes. The filters were lifted and bathed in denaturation solution (Appendix 5) for 5 minutes and then bathed in neutralisation solution (Appendix 5) for 5 minutes. The denaturation and neutralisation step was repeated twice, then the filters were rinsed in 2X SSC (Appendix 5) and air-dried on Whatman™ paper [Whatman]. The filters were dried in a vacuum oven at 80°C for 30 minutes. The DNA was covalently bound to the membrane by exposure to 125 mJ of ultraviolet irradiation in a Bio-Rad GS Gene Linker.

The filters were pre-hybridised in 6X SSPE, 5X Denhardt’s solution (Appendix 5), 1% SDS, 10 mM sodium pyrophosphate, 100 µg/ml heat-denatured sheared salmon sperm DNA for at least 4 hours at 65˚C, then hybridised with a denatured [α-32P]-dCTP labelled DNA probe (see Section 2.2.15) in a Hybaid™ rotating oven [Thermo Electron Company] overnight at 65˚C. The next day the filter was washed in a shaking water bath at 60˚C in 0.1% SDS/1X SSPE (Appendix 5) in plastic containers. The filters were rinsed in 2X SSPE, air-dried on Whatman™ paper and sealed in Glad Wrap™. The filters were exposed to X-OMAT AR film [Kodak] in X-ray cassettes with intensifying screens at –80°C. The phage clones that were positive in duplicate were excised from the agar plates with cut-down 1 ml pipette tips and resuspended in 1 ml SM buffer and 10 µl chloroform [BDH Univar] at 4°C.

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2.2.3 In vivo excision from a λZAP library

The in vivo excision procedure was performed as per the manufacturer’s guidelines [Stratagene]. The basic principles of the in vivo excision are briefly outlined. The initial co-infection of XL-1 Blue E. coli with λZAP phage and filamentous ExAssist® helper phage results in “excision” of the pBst II (SK-) phagemid from within the λZAP phage by the filamentous phage gene II protein. The excised pBst II (SK-) is packaged into new filamentous phage particles and the subsequent heating step inactivates both the λZAP phage and the XL-1 Blue cells. The supernatant containing the pBluescript II (SK-) packaged in filamentous phage particles is used in the next step to infect the SOLR strain of E. coli. Any contaminating λZAP phages are unable to infect SOLR cells, and because they are suppressor negative, the ExAssist® helper phage is unable to replicate. Consequently, the SOLR cells will contain the excised pBst II (SK-) plasmid that can be selected on LB-ampicillin plates (Short et al., 1988).

An agarose plug of the duplicate positive plaque (identified above on library screening) was transferred to a microcentrifuge tube containing 500 µl of SM buffer and 20 µl of chloroform. The plug was vortexed and incubated at 4°C overnight. Separate overnight cultures of the XL-1 Blue MRF’ and SOLR strains of E. coli were commenced in LB broth supplemented with 10 mM MgSO4 and 0.2% maltose (w/v). The next day the XL- 1 Blue MRF’ and SOLR cells were pelleted by centrifugation at 1,000 x g at 4°C for 600 10 minutes and resuspended at an OD of 1.0 in 10 mM MgSO4. In a Falcon™ 2059 10 ml tube, 200 µl of XL-1 Blue MRF’, 250 µl λZAP phage stock (titre > 1 x 105 pfu/µl) and 1 µl of ExAssist® helper phage (titre > 1 x 106 pfu/µl) were incubated at 37°C for 15 minutes. Then 3 ml of LB broth was added to the Falcon™ 2059 tube and the incubation continued at 37°C for 3 hours with shaking. The Falcon™ tube was heated at 70°C for 20 minutes to inactivate λZAP phage and the XL-1 Blue cells, then centrifuged at 1,000 x g for 15 minutes. The supernatant contained the pBst II (SK-) phagemid packaged as filamentous particles. Then 100 µl of supernatant was incubated with 200 µl freshly grown SOLR E. coli (OD600 = 1.0) at 37°C for 15 minutes, then 200 µl was plated out on LB-ampicillin plates and incubated at 37°C overnight. The colonies were screened for inserts and sequenced.

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2.2.4 Extraction of genomic DNA from mouse tail tips

The distal few millimetres (< 5 mm) of the tails of mice between the ages of 10 days and two weeks were cut off with clean sharp scissors under general anaesthesia with inhaled Isoflurane™ (2-chloro-2-(difluoromethoxy)-1,1,1-trifluoro-ethane) [Laser Animal Health, Brisbane]. At this time the sex and identity of the mouse was recorded with a unique ear punch. The tail tips were digested overnight at 55ºC in 700 µl of Tail Tip Solution II (Appendix 5) The next day the samples were chilled on ice for 10 minutes, then 350 µl of saturated NaCl solution was added and mixed by inversion. The samples were incubated on ice for a further 10 minutes and centrifuged in a microcentrifuge at maximum speed for 10 minutes at 4ºC. The supernatant containing the genomic DNA was carefully aspirated and transferred to a sterile microcentrifuge tube, followed by the addition of 700 µl isopropanol [BDH Univar]. The sample was mixed by inversion and the precipitating strands of genomic DNA were visible on inspection. The genomic DNA was pelleted by centrifugation and the pellets were rinsed with 70% ethanol followed by a further centrifugation before a final rinse in 100% ethanol, after which they were allowed to air dry. The pellets were resuspended in 50 µl of TE pH 8.0 containing 1 µl of RNase A (10 mg/ml) [Sigma-Aldrich].

2.2.5 Extraction of genomic DNA from other organs

Genomic DNA was also isolated from other mouse organs, usually liver or spleen, following euthanasia of the mice and dissection of the required organ. The organ was minced in a 50 ml conical tube with a tissue blender [Dupont Sorvall Omni Mixer] in Tail Tip Solution II in a volume adjusted for the size of the organ fragment (usually 5 ml), and Proteinase K [Promega] was added to a final concentration of 0.5 mg/ml. The sample was allowed to digest at 55ºC overnight until the solution was clear, otherwise a further aliquot of Proteinase K was added and the digestion was allowed to continue until clear. The solution was transferred to a 30 ml Corex™ tube and the cellular debris was precipitated by adding 750 µl of 5 M KAc per 5 ml of solution followed by incubation on ice for 15 minutes. This resulted in a thick white precipitate to which 10 ml of chloroform was added, followed by centrifugation at 15,000 x g for 15 minutes at 4ºC. The upper aqueous phase containing the genomic DNA was transferred to a fresh Corex™ tube and the chloroform extraction was repeated. The upper aqueous

Queensland Institute of Medical Research 77 Chapter 2: General materials and methods phase was carefully removed with a pipette and the genomic DNA was precipitated by the addition of 1.5 volumes of 100% ethanol. The genomic DNA was spooled with a glass pipette and rinsed initially in 70% ethanol and then 100% ethanol before being transferred to a microcentrifuge tube, air dried for 10 minutes, then resuspended in 500 µl of 10mM Tris-Cl, pH 8.0, to which 10 µl of RNase A (10 mg/ml) was added. The genomic DNA was incubated at 55ºC to assist dissolution of the pellet.

2.2.6 Quantification of nucleic acid concentration

The concentration of solutions of nucleic acids was determined from the absorbance measured at 260 nm (A260) by a spectrophotometer in accordance with the principles of the Beer-Lambert law. The concentration of the final solution was calculated on the assumption that 1 A260 unit is equivalent to 50 µg/ml of DNA, 40 µg/ml of RNA and 33 µg/ml of single stranded DNA/oligonucleotides.

2.2.7 Agarose gel electrophoresis

DNA was electrophoresed through 0.8%–1% w/v agarose gels [Invitrogen] using the Bio-Rad submarine electrophoresis system at voltages between 50 V and 100 V. The agarose was melted in a microwave oven [LG Electronics] in 0.5X TBE buffer (45 mM Tris-borate, 1 mM EDTA) supplemented with ethidium bromide (final concentration 0.5 µg/ml)[Sigma-Aldrich] to allow visualisation of the DNA. Gel loading buffer (6X) (Appendix 5) was added to samples prior to loading. DNA size markers λHindIII and/or ΦX174/HaeIII [New England Biolabs] were run adjacent to samples to permit quantification and size estimation of unknown DNA fragments.

2.2.8 Phenol-chloroform extraction of DNA

In order to remove restriction enzymes, modifying enzymes or protein contamination from DNA solutions, such that the DNA could be used for additional purposes, the volume of the solution was adjusted to 50 µl in a microcentrifuge tube with TE buffer, pH 8.0, and an equal volume of Tris-buffered phenol, pH 8.0, was added and the mixture was vortexed. Subsequent centrifugation at 14,000 rpm for 2 minutes at room temperature resulted in the separation of the DNA into the upper aqueous phase, which was carefully transferred to a clean microcentrifuge tube. The DNA sample was then mixed with an equal volume of Tris-buffered phenol, pH 8.0, mixed with an equal

78 Queensland Institute of Medical Research Chapter 2: General materials and methods volume of chloroform (phenol:chloroform) before centrifugation and careful transfer of the upper aqueous phase. The aqueous phase containing the target DNA was then ethanol precipitated as described in detail below.

2.2.9 Ethanol precipitation of DNA

DNA was precipitated by the addition of one tenth of the volume of 3 M NaOAc and at least two volumes of 100% ethanol in a microcentrifuge tube. The sample was incubated at –20ºC for one hour, then centrifuged in a microcentrifuge at maximum speed for 30 minutes at 4ºC. The DNA pellet was washed carefully in cold 70% ethanol (v/v) to remove excess salt and centrifuged again for 10 minutes. The pellet was vacuum dried and resuspended in a small volume of 10 mM Tris-Cl, pH 8.0.

2.2.10 Isolation of DNA fragments from agarose gels

Following electrophoresis, DNA fragments of interest were excised from the agarose gel with a clean scalpel blade and transferred to a microcentrifuge tube. The DNA was extracted using the QiaQuick gel purification kit [Qiagen] as per the manufacturer’s protocol with particular care taken to centrifuge for an additional minute to remove all traces of the PE buffer prior to elution of the DNA fragment in 10 mM Tris-Cl, pH 8.0.

The bacteriophage λ DNA was extracted from agarose gels using the QiaExII kit [Qiagen] in accordance with the manufacturer’s instructions. This step ensured the removal of contaminating substances from the agar, which could inhibit subsequent restriction enzyme digestion of the λ DNA.

2.2.11 Recombinant DNA methods

2.2.11a Restriction enzyme digestion

Digestion of DNA with restriction endonucleases was undertaken with reagents purchased from New England Biolabs (NEB) in accordance with the manufacturer’s instructions. Briefly, between 1 and 5 µg of PCR product or plasmid DNA was added to a reaction mix containing the restriction enzyme at a final concentration of 1 U/ml, 1x recommended reaction buffer and, if required, 0.1 mg/ml BSA in a 1.5 ml microcentrifuge tube for at least one hour at 37ºC. If double digestion was required, the method suggested in the NEB catalogue was followed.

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2.2.11b Partial restriction digestion of plasmid DNA

Partial digestion of plasmid DNA was required when there were two restriction sites within the same plasmid, usually one restriction site within the multiple cloning sequence of the plasmid and the other site within the fragment of DNA for manipulation. Typically the site within the DNA fragment was required to remain intact for the subsequent cloning step. This was achieved by preparing a master mix containing 1–2 µg of the plasmid DNA to be partially digested, the appropriate NEB buffer and 0.1 mg/ml BSA without the restriction enzyme in a volume of 80 µl in a 1.5 ml microcentrifuge tube, which was then divided into four separate aliquots of volumes 30 µl, 20 µl, 20 µl and 10 µl. The restriction enzyme was added to the 30 µl aliquot and mixed thoroughly by pipetting, then 10 µl was transferred to the second tube, which was then mixed thoroughly, then 10 µl was transferred to the third tube and mixed thoroughly, and then finally 10 µl transferred to the fourth tube. Thus the final volume in all four tubes was 20 µl and the enzyme concentration in each successive tube was reduced by a factor of three. The digest was continued at 37°C for 15 minutes only. The digestion of all of the plasmid DNA was incomplete because the plasmid would be cut at either, but not both, of the restriction sites in tube number four, which contained the least amount of restriction enzyme. Consequently in each tube there were three species of DNA including intact plasmid, completely digested plasmid and DNA digested at one restriction site only. All four of the restriction digests were loaded in separate wells and subjected to agarose gel electrophoresis for separation of the DNA fragments and identification of the DNA cut at one site only. The appropriate DNA band was excised and gel purified, then subjected to complete restriction digestion with the second restriction enzyme, which would result in the generation of two DNA species, followed by agarose gel electrophoresis to allow identification of the DNA fragment required for the next cloning step.

2.2.11c Dephosphorylation of plasmid vectors

A plasmid vector, usually pBst, was digested with a restriction enzyme to generate the required restriction enzyme sites. The digested plasmid vector was then isolated from an agarose gel, purified and subjected to dephosphorylation with thermosensitive alkaline phosphatase (TsAP) [Gibco-BRL] to remove the 5’ phosphate group and prevent self- ligation. Following the restriction digest of the plasmid vector, an aliquot of the digest

80 Queensland Institute of Medical Research Chapter 2: General materials and methods was added directly to the phosphatase reaction such that the magnesium chloride concentration was between 2 mM and 10 mM. As per the manufacturer’s recommendations, the amount of TsAP required for removal of the phosphate on 5’ overhangs was 0.02 U per pmol of DNA ends and 0.2 U for removal of the phosphate on 3’ overhangs per pmol of DNA ends. The phosphatase reaction was performed in 1X TsAP reaction buffer (diluted from the supplied 10X stock) for 15 minutes at 65ºC in a water bath or heating block. The phosphatase reaction was halted by the addition of the supplied stop buffer and heated for a further 15 minutes at 65ºC. The digested and dephosphorylated plasmid vector DNA was then gel purified before subsequent ligation steps.

2.2.11e Phosphorylation of DNA adaptors and linkers

Because DNA ligase requires 5-phosphoryl termini, unphosphorylated adaptors or linkers must be modified by transferring the γ-phosphate from ATP to the 5-hydroxyl group of the linker. This reaction is catalysed by T4 polynucleotide kinase. For a total reaction volume of 50 µl, 5 µl of 10X polynucleotide kinase buffer, 5 µl of 1 mM ATP and the linker for phosphorylation (initial concentration 10 µM) were mixed in a clean microcentrifuge tube and the reaction volume made up to 50 µl with Milli-Q water, then heated at 70°C for 5 minutes to denature any double-stranded structures, followed by immediate quenching on ice prior to the addition of 2 µl of T4 polynucleotide kinase (2 Weiss units). The phosphorylation reaction mixture was incubated at 37°C for 30 minutes. The T4 polynucleotide kinase was heat inactivated at 65°C for 20 minutes. An aliquot (5–10 µl) of the reaction mixture was transferred directly to the ligation reaction (total volume 20 µl).

2.2.11f Ligation of DNA fragments into plasmid vectors

DNA fragments were cloned if possible by directional cloning of cohesive ends into plasmid vectors. The dephosphorylated plasmid vector (approximately 50–100 ng) and the insert to be ligated were mixed with a vector:insert ratio of 1:3 with 200 U of T4 DNA ligase in 1X ligase buffer containing ATP in a 16ºC water bath overnight. A dephosphorylated vector only negative control and a non-dephosphorylated vector positive control were always included. The ligation reactions were ethanol precipitated

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(see Section 2.2.8) with the addition of 1 µg of glycogen [Boehringer-Mannheim] and resuspended in 7 µl of ddH2O and transformed into competent E. coli (see below).

2.2.11g Ligation of DNA fragments generated by PCR into plasmid vectors

The DNA polymerase, Taq polymerase, preferentially adds a single 3’-A deoxynucleotide to double stranded DNA by a template independent extension reaction. Consequently, the ligation of PCR products can be facilitated by using a T-A cloning system in which the linearised plasmid vector is a “T-vector” with a complementary overhanging 3′-T. The pGEM-T vector system [Promega] was used to ligate PCR products into a plasmid vector for sequencing and further manipulation in accordance with the manufacturer’s instructions.

2.2.11h Electro-transformation of E. coli

Competent bacterial cells were thawed rapidly at room temperature and immediately placed on ice. In a cold microcentrifuge tube, 20 µl of competent cells was mixed with 5–10 ng of DNA (e.g. plasmid, ligation reaction) and incubated on ice for 60 seconds, then transferred to a chilled 0.1 cm gap electroporation cuvette [Bio-Rad] and electro- transformed (pulsed) in a Bio-Rad Gene Pulser (1.0 kV, 25 µF, 200 Ω). The cells were recovered in 1 ml LB broth on a shaker at 37°C for one hour before 100 µl of cells were plated on to LB ampicillin plates and incubated overnight at 37°C. A negative control of competent cells transformed in the absence of DNA and a positive control of competent cells with a known quantity of plasmid DNA (usually 100 pg of pBst) were also performed alongside the experimental ligation to ensure the quality of the transformation and selection procedure.

2.2.11i Heat shock transformation of E. coli

The competent E. coli were thawed as described above and placed on ice, then 100 µl of cells were transferred to chilled Falcon 2059 polypropylene tubes [BD Biosciences] and 2 µl of β-mercaptoethanol was added to a final concentration of 1.22 M. The cells were incubated on ice for 10 minutes and swirled gently every two minutes. The DNA (plasmid, ligation) was added in a volume of 1 µl and incubated on ice for 30 minutes. The NZCY+ broth was preheated to 42°C in a water bath. The cells were heat-pulsed for exactly 30 seconds in the water bath at 42°C, and immediately incubated for 2 minutes on ice. The cells were rescued with the addition of 900 µl of pre-heated

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NZCY+ broth and shaken at 220 rpm at 37°C for one hour before 100 µl of cells were plated out on LB ampicillin plates and incubated at 37°C overnight. Negative and positive controls were transformed as outlined above in electro-transformation to ensure the quality of the selection procedure.

2.2.11j Identification of recombinant DNA molecules following transformation

The LB ampicillin plates from the transformations were examined following 16 hours of incubation at 37ºC for bacterial colonies. The negative and positive controls were inspected to determine if the transformation had been successful. Subsequently 24 colonies were picked (if possible) with yellow disposable filter tips and transferred to a yellow conical tube containing 1 ml of LB ampicillin (100 µg/ml). The cultures were grown with shaking at 220 rpm overnight at 37°C. The cultures were decanted to a 1.5 ml microcentrifuge tube and the DNA was isolated using the EasyPrep method (see Section 2.2.1a). The mini-prep isolated DNA was subjected to restriction enzyme analysis to determine the presence and orientation of the cloned insert. Ideally a restriction enzyme that cut both within the vector multiple cloning site and asymmetrically within the insert was chosen. However, if there was no single restriction enzyme, then a pair of enzymes which required the same buffer and excised within the multiple cloning site on either side of the insert was chosen (for example, in pGEM-T, the plasmid DNA would be cut with NdeI and SacII to release the insert.) The digest was then separated by agarose gel electrophoresis and visualised by UV transillumination (Section 2.2.6).

2.2.12 DNA sequencing

All sequencing of plasmid, PCR product and bacteriophage DNA was performed by the QIMR in-house sequencing facility using the ABI Prism Dye Terminator Cycle Sequencing Ready Reaction kit with subsequent analysis on the ABI Prism DNA Sequencer Model 3100 (with 80 cm capillaries) in accordance with the manufacturer’s instructions [Applied Biosystems]. The template DNA which varied in amount between 100 ng (for PCR products) and 1 µg (for genomic DNA) was mixed with sequencing primer (1.6 pmoles for standard PCR product or plasmid DNA template or 10 pmoles for genomic DNA) and Big Dye Terminator Ready Reaction Mix (BD 3.1) in a total volume of 10 µl. The reaction was cycled for 25 cycles of denaturation at 96ºC for

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30 seconds, primer annealing at 50ºC for 15 seconds, extension at 60ºC for 4 minutes in a thermocycler. The unincorporated dye terminators were removed from the completed reaction mix by centrifugation through a Centrisep [Princeton Separations] or AutoSeq G-50 columns [Amersham Biosciences] before drying down the eluent containing the sequencing reaction in a Speed Vac [Savant].

2.2.13 Sequence analysis

The FastA (Pearson and Lipman, 1988) and SEQED functions of the GCG Version 7 (Genetics Computer Group) were used to align and manipulate DNA sequences. The BLASTN search program using the default parameters (http://www.ncbi.nlm.nih.gov/ National Center for Biotechnology Information) was used to determine sequence homologies.

2.2.14 Polymerase chain reaction (PCR) methods

In general all PCR reactions were undertaken in the purpose built room following “sterilisation” with overhead UV irradiation for 10 minutes. Usually “hot-start” AmpliTaq Gold™ [Applied Biosystems] was used to amplify DNA in accordance with the manufacturer’s instructions. In brief, a master mix was prepared of sufficient total volume to allow for the total number of samples including pipetting error for reaction volumes of 50 µl. The master mix included the supplied 10x PCRII buffer, MgCl2 to a final concentration of 2.5 mM, dNTPs to a final concentration of 200 µM, forward and reverse primers to 0.4 µM, and 1.0 U AmpliTaq Gold™. The template DNA was added at the laboratory bench in a volume of 1.0 µl. A negative water control and a positive control were always run simultaneously. The thermocycling reaction was performed on a thermocycler with an initial denaturing step of 94ºC for 10 minutes, followed by 30 cycles of denaturing at 94ºC for 30 secs, annealing at 50–60ºC for 30 secs, depending on the annealing temperature of the primer pair, and extension at 72ºC for 30 secs to 3 minutes depending on the amplicon size. The PCR products were visualised by agarose gel electrophoresis. Primers were designed either with the MacVector [Accelrys] (http://www.accelrys.com/products/macvector/) or the Primer 3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) to be 20–24 base pairs in length with an annealing temperature between 50ºC and 60ºC. Oligonucleotides were purchased from Sigma-Genosys and resuspended in 10 mM Tris-Cl, pH 8.0, at a

84 Queensland Institute of Medical Research Chapter 2: General materials and methods concentration of 200 µM, stored at –20ºC and diluted to 10 µM prior to use. Several variations of the PCR including degenerate PCR, “touch-up” and “touch-down” PCR were used in particular situations, and these will be discussed in detail in the relevant sections.

2.2.15 Radioactive labelling of double stranded DNA probes

All double stranded DNA probes were labelled with the Megaprime Labelling Kit [Amersham Biosciences] in accordance with the manufacturer’s instructions using [α-32P]-dCTP; 3,000 Ci/mmol [Amersham Biosciences]. In summary, 25–50 ng of the DNA probe to be labelled was transferred to a clean microcentrifuge tube with a needle hole puncture in the lid, and diluted to a volume of 5 µl, then mixed with 5 µl of random hexamers. This mixture was denatured in a heating block at 100ºC for 5 minutes, then immediately chilled on ice for several minutes followed by a quick centrifuge spin. The remaining reagents for the labelling reaction, including 10X reaction buffer (which includes dATP, dGTP and dTTP) and the Klenow fragment (DNA polymerase), were added and mixed thoroughly by a quick centrifuge spin before the addition of 5 µl of [α- 32P]-dCTP to a total reaction volume of 50 µl. The labelling reaction proceeded at 37ºC in a water bath for 30 minutes, when it was stopped with the addition of 5 µl 0.5 M EDTA and 45 µl 10 mM Tris-Cl, pH 8.0. The unincorporated radioactive [α-32P]-dCTP was removed by centrifugation of the stopped reaction mixture through a Sephadex G- 50 column [Pharmacia] equilibrated in TE buffer. The probe was denatured in a heating block at 100ºC for 5 minutes, then added to 5 ml of hybridisation buffer, which had been removed from the Hybaid™ bottle, before returning it to the remainder of the hybridisation solution. A 1 µl aliquot of the labelled probe was counted on a Scintillation Counter Bioscan QC 2000 [Bioscan Inc.] to check the efficiency of incorporation of the [α-32P]-dCTP. All radioactive labelling procedures were undertaken in the specifically designated isotope rooms at QIMR under the guidelines of the Radiation Safety Act, taking routine precautions to avoid radiation exposure whilst wearing a personal dosimeter.

2.2.16 Southern blot analysis

Southern blot analysis was used to genotype samples from mouse tail tips. A master mix was prepared containing the desired restriction enzyme, the appropriate reaction buffer

Queensland Institute of Medical Research 85 Chapter 2: General materials and methods and 0.1 mg/ml of BSA, then a 20 µl aliquot was pipetted into a microcentrifuge tube to which approximately 10 µg of the genomic DNA sample (in a volume of 10 µl) was subsequently added. The samples were centrifuged briefly to assist complete mixing, and incubated overnight in a humidified incubator at 37ºC. The digested samples were mixed with 8 µl of 6x Gel Loading Buffer and loaded on 0.8% agarose gel, then electrophoresed at 100 V for a period of between 4 and 8 hours to allow adequate separation of the digested genomic DNA. The gel was photographed in the gel tray with a ruler and the position of the λ HindIII molecular weight markers was indicated by aspirating a plug with a glass pipette.

The gel was carefully processed in the gel tray as follows before the transfer assembly was set up. The gel was initially depurinated in 0.25 M HCl for no longer than 20 minutes, then rinsed in copious deionised tap water. The gel was then denatured by incubating it in denaturing solution (Appendix 5) for 30 minutes on a gently rocking platform, following which the gel was again rinsed extensively in deionised tap water. The gel was then incubated in neutralisation solution (Appendix 5) for another 30 minutes, and again rinsed extensively in deionised tap water, then rinsed briefly in 2X SSPE (Appendix 5).

The denatured genomic DNA was transferred to a nylon membrane, either Zeta probe [Bio-Rad] or Hybond N [Amersham Biosciences], by upward capillary attraction overnight with 20X SSPE solution (Sambrook and Russell, 2001). The following morning the transfer apparatus was disassembled, the positions of the λHindIII molecular weight markers and the gel slots were marked on the filter with a soft 2B pencil to identify lane position. The filter was rinsed in 2X SSPE, briefly air dried on Whatman™ paper and the DNA was covalently linked to the nylon membrane by exposure to an ultraviolet light dose of 125 mJ in a Bio-Rad GS UV Cross Linker. The damp nylon membrane was stored between Glad Wrap™ until required.

The filter was pre-hybridised in 5–10 ml of Express-Hyb™ solution [Clontech] at 65ºC in a Hybaid™ oven for at least 4 hours. A few millilitres of pre-hybridisation liquid was removed from the Hybaid™ bottle and the labelled and denatured probe added to this before returning it to the Hybaid™ bottle. The filter was hybridised at 65ºC overnight. The filter was rinsed in 2X SSPE at room temperature initially then consecutively washed in 2X SSPE for 15 minutes at 60ºC, then 2X SSPE/0.1% SDS for 15–

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30 minutes, or until there were between 5 and 10 counts per second on the Geiger counter. The filter was rinsed in 2X SSPE, briefly air dried on Whatman™ paper, wrapped between Glad Wrap™, orientated with fluorescent markers and exposed to X- OMAT AR film [Kodak] with intensifying screens at –80ºC for 3 to 5 nights.

2.2.17 RNA isolation methods

2.2.17a Total RNA isolation

Total RNA was isolated from various mouse organs. All solutions were prepared with DEPC treated water, all pipetting was performed with sterile filter pipette tips, and gloves were worn at all times and changed frequently. The mice were euthanised and the organs dissected free and cut into small pieces with sharp scissors, then transferred into 3–5 ml of RNA tissue lysis buffer (Appendix 5) in a 50 ml conical tube and homogenised with the tissue homogeniser. One tenth of a volume of 20% N-lauryl sarcosine (= sarkosyl) [Sigma-Aldrich] was added to the lysate (therefore final sarkosyl concentration = 2%) and mixed prior to centrifugation at 3,800 rpm for 10 minutes at 12ºC. The supernatant was transferred to a fresh tube and stored at –20ºC. The supernatant containing the tissue lysate was layered onto a cushion of 5.7 M CsCl in polyallomer tubes (14 x 89 mm Cat No 331372) [Beckman], balanced precisely, then centrifuged in an NVT 100 rotor on a Beckman Optima XL-100 Ultracentrifuge at 30,000 rpm for 20 hours at 20ºC. Following centrifugation, the supernatant was removed by gentle suction to remove the genomic DNA, then the tube was quickly inverted to remove the CsCl. The RNA pellet remained attached to the bottom of the tube and all the liquid was carefully removed with a suction pipette without touching the RNA pellet. The pellet was then rinsed with 500 µl of 70% ethanol and air dried. The pellet was resuspended in tissue resuspension buffer (Appendix 5). Following resuspension of the pellet, the RNA was precipitated with 2 M NaOAc and ethanol. The final pellet was resuspended in DEPC treated Milli-Q water containing RNasin [Promega] then quantitated by spectrophotometry and agarose gel electrophoresis, then stored at –80ºC at a concentration of approximately 1 µg/ml until required.

2.2.17.b Preparation of poly A+ mRNA

Poly(A)+ mRNA was extracted from total RNA using the oligo (dT)25 Dynabeads® mRNA purification kit [Dynal] with the supplied buffers in accordance with the

Queensland Institute of Medical Research 87 Chapter 2: General materials and methods manufacturer’s instructions. As indicated in the technical manual, approximately 2 µg of poly(A)+ mRNA can be isolated with 200 µl of Dynabeads®. A 500 µl aliquot of Dynabeads® was conditioned and resuspended in 250 µl of 2X binding buffer. The total RNA was heated for 2 minutes at 65ºC in a heating block then added to the Dynabeads®. The beads and RNA were completely mixed on a rotating wheel for 5 minutes at room temperature, then placed in the magnet stand [Dynal MPC-E-1 Prod No. 120.07] and the buffer aspirated. The beads were washed twice with 500 µl of washing buffer. The poly(A)+ mRNA was eluted from the beads by adding 20 µl of elution buffer followed by heating at 65ºC for 2 minutes in the heating block. The samples were placed in the magnet and the eluant containing the poly(A)+ mRNA was transferred to a fresh microcentrifuge tube. The beads were resuspended in 400 µl of reconditioning solution (0.1 N NaOH) and incubated at 65ºC for 2 minutes in the heating block. The buffer was removed from the magnet stand and this step was repeated twice. The beads were washed in 250 µl storage buffer several times until the pH was below 8.0. The beads could be then re-used with another aliquot of total RNA until sufficient poly(A)+ mRNA, usually 5–10 µg, had been isolated.

2.2.18 Northern blot analysis

The electrophoresis equipment, including the gel comb and gel tray, was soaked overnight in detergent to remove ribonucleases. A 1.2% agarose gel containing deionised formaldehyde [Sigma-Aldrich] and 1X MOPS was poured in the fume hood. The RNA samples, either 10–20 µg of total RNA or 5 µg of poly(A)+ mRNA, were thawed on ice. A sample master mix was prepared with deionised formaldehyde (7 µl), deionised formamide (20 µl) and 10X MOPS (4 µl), then 31 µl aliquots of master mix were transferred to each sample tube to which the RNA samples were added in a volume of 5 µl. The samples were heated for 15 minutes at 65ºC, then quenched on ice for 15 minutes before the addition of 4 µl of 10X RNA gel loading buffer (Appendix 5) to make a total volume of 50 µl. The gel was electrophoresed at 65 V for 2.5 hours until the bromophenol blue dye front reached the end of the gel. The gel was processed by staining in ethidium bromide (5 µg/ml), destained in Milli-Q water and photographed beside a ruler. (The ribosomal bands, if visible, were marked by aspirating a plug with a glass pipette.) The gel was then washed twice in 0.05 N NaOH for 15 minutes, rinsed extensively in Milli-Q water, then washed twice in 10X SSPE for 15 minutes. The RNA

88 Queensland Institute of Medical Research Chapter 2: General materials and methods was transferred overnight on to Hybond N by upward capillary transfer with the transfer assembly in a wide-mini gel tank with 20X SSPE. The next morning the Hybond N filter was rinsed in 2X SSPE and the gel slots marked with a soft 2B pencil before UV cross-linking and storage between Glad Wrap™ at –20ºC. The filter was pre-hybridised, hybridised and washed as described previously for Southern blot analysis, and the filter was autoradiographed.

2.2.19 Quantitative PCR methods

First-strand cDNA synthesis was achieved with SuperScript™ III [Invitrogen] as per the manufacturer’s instructions. Briefly, 250 nmol of random hexamers were added to 200 ng of poly(A)+ mRNA or 2 µg of total RNA and 1 µl of 10 mM dNTPs and made up to 20 µl with Milli-Q water. The mixture was heated at 65ºC for 5 minutes, then quenched on ice for 1 minute before the addition of 5 µl of 5x First-Strand Buffer, 1 µl of 0.1 M DTT, 1 µl of RNasin® [Promega] and 1 µl of SuperScript III RT (200 U/ml). The reagents were mixed thoroughly by pipetting, followed by incubation at 25ºC for 10 minutes to allow the random hexamers to bind, then incubated for 60 minutes at 50ºC. The reaction was inactivated by heating for 15 minutes at 70ºC. The cDNA generated was used for subsequent PCR amplification.

The subsequent real-time PCR reaction used 1/20th volume of the first-strand cDNA reaction mixture diluted to 5 µl and was carried out in a Corbett Research Rotor-Gene 3000™ [Corbett Research, Australia] using Quantitect™ SYBR® Green PCR Master Mix [Qiagen] following the manufacturer’s instructions. Forward and reverse primers were added to a final concentration of 0.3 µM. All primers were designed using the Primer3 software http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi. The PCR cycling conditions included activation for 15 minutes at 95ºC, 35 cycles of 30 seconds at 95ºC, 30 seconds at 60ºC, and 30 seconds at 72ºC. Fluorescence data was recorded at the end of each 72ºC step. A DNA melt profile was run subsequently from 72ºC to 95ºC with a ramp of 1ºC/5 seconds during which fluorescence data was continuously recorded. All reactions were performed in duplicate for reproducibility. The relative expression of EphA1, EphA2 and ephrin A1 was calculated using the most appropriate housekeeping genes, including 18S rRNA, β-Actin, HMBS and GAPDH..

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2.3 Histological procedures

2.3.1 Tissue sections

Mice suitable for analysis were euthanised by asphyxiation in CO2 or overdose of Isoflurane™ [Laser Animal Health, Brisbane] by inhalation. The abdomen was washed in 70% ethanol and the organs were dissected free and fixed in 5–10 volumes of 0.2% glutaraldehyde in PBS overnight at 4ºC. The tissue samples were washed copiously in phosphate buffered saline (PBS) then cryoprotected in 15% sucrose in PBS for 2 hours, then in 30% sucrose in PBS overnight at 4ºC. The cryoprotected tissue samples were washed copiously in PBS to remove the sucrose and incubated in Tissue-Tek OCT [Sakura] for at least one hour, then embedded in OCT in an Alfoil™ cup over dry ice (Lobe et al., 1999). Alternatively tissues were dissected free and placed immediately in Alfoil™ cups containing Tissue-Tek® OCT™ [Sakura], frozen carefully in liquid nitrogen, then placed on dry ice before storage at –80°C prior to sectioning. The frozen sections were processed by the histology laboratory at QIMR, and sections were generally 5–10 µm thick. Additionally, tissues for paraffin section or immunohistochemistry were fixed overnight in 2% paraformaldehyde [Sigma-Aldrich] at 4ºC or in formalin in PBS at room temperature. Following fixation, tissues were processed by the histology laboratory at QIMR. Additionally, tissues were fixed in HOPE solution [DCS Innovative Diagnostik-Systeme, Hamburg] as per the manufacturer’s instructions (Olert et al., 2001) as described in Section 2.3.3.

2.3.2 Human placental alkaline phosphatase (hPLAP) staining

Those sections which had been fixed prior to sectioning and stored at –80ºC were thawed, refixed in 0.2% glutaraldehyde in PBS for 10 minutes, then rinsed in PBS. The remaining sections which had been frozen immediately in OCT™ [Sakura] on liquid nitrogen were thawed and fixed in ice-cold acetone (–20°C) for 5 minutes, air dried for 5–10 minutes, then rinsed in PBS (pH 7.4). The endogenous alkaline phosphatase was inactivated by incubating slides in PBS in a glass coplin jar in a water bath at 73ºC for 35 minutes. The slides, up to twenty at a time and always included a positive control, were transferred to a perspex humidified staining chamber and overlaid with alkaline phosphatase buffer (Appendix 5) and incubated for 10 minutes. The alkaline

90 Queensland Institute of Medical Research Chapter 2: General materials and methods phosphatase buffer was poured off and the slides were overlaid with NBT/BCIP stain (nitro-blue tetrazolium 337 µg/ml, 5-bromo-4-chloro-3-indolyl phosphate 175 µg/ml, toluidinium salt)[Roche] or BM Purple SP substrate [Roche] and placed at 4ºC overnight (Lobe et al., 1999). The slides were protected from light with Alfoil™ and the perspex humidified staining chamber was wrapped in a wet towel. The next day the slides were washed in PBS and counterstained with Nuclear Fast Red [Sigma-Aldrich] for 5 minutes. The slides were rinsed again in PBS, and either dehydrated through an ethanol series and mounted with a cover slide using Depex™ [Gurr BDH] mounting medium, or allowed to dry completely, even overnight, and mounted using VectaMount™ [Vector Laboratories] mounting medium and dried at room temperature or 37°C.

2.3.3 hPLAP staining of HOPE-fixed tissue sections

The HOPE technique was used to prepare paraffin embedded tissues for hPLAP staining (Olert et al., 2001). Tissues were dissected and immediately placed in a 5 ml tube containing HOPE I solution at 4°C for 1–3 nights (i.e. between 16–64 hours) [Innovative Diagnostic Services]. The HOPE I solution was poured off carefully and 5 ml of pre-mixed ice-cold acetone solution, containing 100 ml acetone and 100 µl of HOPE II solution, was added and the tube placed immediately back into the ice bath and incubated at 0–2°C for 2 hours. The tube was emptied after 2 hours and refilled with ice-cold pure acetone. This step was repeated twice with each incubation of 2 hours. (It is critical that the temperature of the acetone should not rise above 2°C during the total 8 hours of the incubation.) After the 8 hours of dehydration, 5ml of pre-warmed low- melting paraffin was added taking care to remove air bubbles, and incubated overnight at 54–55°C. The next morning the paraffinised tissues were sectioned by the histology laboratory. The tissue sections were stored at 4°C prior to hPLAP staining.

The slides were deparaffinised in isopropanol at 60°C by placing up to 20 slides back- to-back in a glass coplin jar in a water bath for 10 minutes, then transferred to a further coplin jar containing isopropanol for a further 10 minutes. The slides were drip-dried on filter paper then air dried. The tissue was rehydrated by incubation in 70% ice-cold acetone for at least 10 minutes in the refrigerator (4°C). The slides were removed from the 70% acetone, drip-dried briefly on filter paper and, whilst still moist, transferred to a

Queensland Institute of Medical Research 91 Chapter 2: General materials and methods coplin jar containing distilled water for 10 minutes, then transferred to a second batch of distilled water for a further 10 minutes, drip-dried on filter paper, then dried completely in a 45°C oven or hot plate (only takes 1–2 minutes on a hot plate). The tissue was then ready for heat-inactivation of endogenous alkaline phosphatases before staining for hPLAP with BM purple as described above (Section 2.3.2).

2.3.4 Immunohistochemistry

The paraffin sections (3–4 µm) were dewaxed using the Antigen Retrieval Dewaxing (ARD) solution [ImmunKontact] as recommended by the manufacturer, or in xylene, and rehydrated to water through descending graded alcohols. The 10X ARD solution was diluted prior to use, and the paraffin slides were immersed in 1X ARD solution in a glass coplin jar, then autoclaved for 15 minutes. Alternatively, heat antigen retrieval was performed by boiling the sections in target retrieval solution [Dako], pH 9.0, for 4 minutes at 125°C using a pressure cooker (Shi et al., 1991). Once antigen retrieval was complete, the slides were allowed to cool for 20 minutes and transferred to 1X PBS/1% BSA (pH 7.4) and washed thoroughly with three changes for 5 minutes each. All reagents from the Dako ARK kit were equilibrated at room temperature prior to use.

Alternatively after cooling, the sections were transferred to Tris buffered saline (TBS), pH 7.4. Endogenous peroxidase activity was quenched by incubating the sections in 1%

H2O2, 0.1% NaN3 in TBS (Appendix 5), pH 7.4, for 10 minutes. After a brief wash in TBS, the sections were transferred to a humidified chamber and incubated with a “Fab” fragment (goat anti-mouse Fab fragment, [Jackson Immunoresearch], diluted 1/200, for 30 minutes. This was followed by incubation with 10% non-immune goat serum for 20 minutes, before excess serum was decanted.

The sections were incubated with a monoclonal mouse anti-human placental alkaline phosphatase antibody [Dako Code No. M 7191, Clone 8A9, Lot No. 023] to detect the revealed hPLAP antigenic sites. The Dako ARK Peroxidase [DakoCytomation] was used to detect the primary antibodies in accordance with the manufacturer’s instructions. Briefly, the slides were rinsed in wash buffer and the biotinylated primary antibody was added to cover the specimen, which was then incubated for 15 minutes at room temperature. The slides were rinsed again in wash buffer, and streptavidin-

92 Queensland Institute of Medical Research Chapter 2: General materials and methods peroxidase was added and incubated for 15 minutes at room temperature. The slides were again rinsed in wash buffer and the Dako DAB+ substrate chromogen was added and the slides were incubated for only 10 minutes. Alternatively, the sections were washed in TBS and transferred to a humidified chamber and incubated with prediluted horseradish peroxidase conjugated goat anti-mouse immunoglobulins [EnVisionTM, Dako] for 45 minutes. The antigenic sites were revealed using 0.5% 3,3'- diaminobenzidine tetrahydrochloride [Sigma Aldrich] with H2O2 as substrate in TBS. Finally the slides were rinsed with distilled water to stop the reaction, and then lightly counterstained with Mayer’s haematoxylin [Sigma Aldrich]. The slides were dehydrated through an alcohol series and mounted with cover slips using Depex™ [BDH Gurr] mounting medium.

2.3.5 Photography

All photography of tissue sections was performed using a Leica DM IRB [Leica] inverted microscope and a M2F111 Leica DC digital camera [Leica] with the default software. In addition, fixed histology slides were scanned using the Aperio™ system and viewed in Imagescope™ to allow manipulation of the images. Images were manipulated in digital format with Adobe Photoshop Version 7 [Adobe] or ACD See™ Photo Editor [ACDSee]. 2.4 Animal husbandry

The inbred C57BL/6J and 129T2/SvEms strains of mice and outbred Animal Research Centre Swiss (ARC(S)) mice were housed in the Herston Medical Research Centre (HMRC) facility under the supervision of the Queensland Cancer Fund Transgenic Laboratory (The Queensland Institute of Medical Research, Herston Road, Herston Q4029, Brisbane, Australia). The Herston Medical Research Centre is a specific pathogen free facility with a 12 hour fixed light/dark cycle and controlled temperature (19–21ºC) and humidity (50%). Mice housed here were fed on standard mouse chow with free access to acidified and UV treated water. Once germ line transmission was confirmed, heterozygous mice were transferred to the Animal Facility at QIMR for convenience. Mice at QIMR had the same environmental conditions, but were fed with mouse pellets [Norco] and had free access to tap water.

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The mice numbers and matings were recorded and managed with entries in a standard laboratory notebook, and mouse data from PCR and Southern blotting were entered into a specifically designed Access Database [Microsoft]. All animal experiments were undertaken with approval from the QIMR Ethics Committee in accordance with the standards set by the Office of Gene Technology and Regulation. The QIMR E-form system was used to report the use of mice.

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Chapter 3: The characterisation and genomic organisation of mEphA1

3 THE CHARACTERISATION AND GENOMIC

ORGANISATION OF MEPHA1

3.1 Introduction

The discovery of a novel gene, even if it is probably the orthologue (homologue) of an already known gene, requires a systematic approach to characterise the gene, which includes determining the organisation of the gene and elucidating the structure and function of the encoded protein. The past 20 years have witnessed the development of molecular laboratory techniques that allow the scientist to explore gene structure and protein function. Our current understanding of the molecular biology of the cell follows from the study of viruses, bacteria, yeasts, fungi, flies, worms and various vertebrates including frogs, fish, chickens and mice. The evolutionary conservation of genes, proteins, organelles and cell types allows the application of lessons learned in less complicated experimental organisms to higher organisms including humans. This approach may allow us to achieve the ultimate goal of medical science, to understand the structure and function of humans in order to prevent and alleviate human disease. The laboratory mouse shares over 90% of its genome with humans, and is a well- established experimental model to study gene function. The application of gene knockout technology is currently only feasible in the mouse, and allows the inactivation or modification of individual genes, enabling us to study the function of a particular gene within the whole animal and consequently develop models of human disease for the investigation and development of new therapies. This project was commenced in late 1996 some years before the sequence of the mouse genome was published. Hence, the technical approach described in this chapter reflects the methods available at that particular time.

The cDNA for mEphA1 (esk), the supposed mouse homologue of human EphA1 (hEphA1), had been isolated prior to the commencement of this project (Lickliter et al., 1996). The next step was to prove that mEphA1 was indeed the mouse homologue of hEphA1 and to undertake some preliminary characterisation of the receptor function of mEphA1. In order to generate a knockout mouse, the genomic organisation of the candidate gene, in this case mEphA1, needed to be elucidated, to allow the construction

Queensland Institute of Medical Research 97 Chapter 3: The characterisation and genomic organisation of mEphA1 of a replacement-type gene targeting vector. The first step in generating the EphA1 knockout mouse was the isolation of mEphA1 genomic clones for sequencing, and the construction of the targeting vector. The ligand-binding domain of mEphA1 was determined by alignment of EphA1 with the more completely characterised EphA3 sequence (Lackmann et al., 1998). A 672 bp cDNA probe corresponding to the ligand binding domain and including both Exon 3a and Exon 3b was generated by PCR to allow screening of a bacteriophage λ library with the most specific and characteristic sequence of mEphA1. Subsequently, the λDASH mEphA1 genomic clones were subcloned into the pBst plasmid vector for sequencing and the manipulation required to generate a replacement type gene-targeting vector. 3.2 Aims

(1) To confirm that mEphA1 is the mouse orthologue (homologue) of hEphA1.

(2) To determine the chromosomal localisation of mEphA1.

(3) To characterise the binding properties of the mEphA1 receptor.

(4) To determine mEphA1 expression by in situ hybridisation studies.

(5) To isolate genomic mEphA1 clones from a bacteriophage λ library.

(6) To subclone suitably sized fragments of genomic mEphA1 DNA for manipulation and sequencing.

(7) To characterise the genomic structure of mEphA1. 3.3 Material and methods

3.3.1 Determining the chromosomal localisation of mEphA1

The methods used to prepare mouse chromosomes and perform in situ hybridisation have been previously described (Webb et al., 1989). Briefly, the 1.6 kB cDNA fragment of mEphA1 was labelled by nick-translation with tritiated dATP, dCTP, dTTP to a specific activity of 1.16 x 108 cpm/µg. The chromosomes of C57BL and BALB/c mice were hybridised with 200 ng/µl of probe and 200 µg/ml of salmon sperm DNA. Slides were then dipped in Ilford L4 emulsion and exposed for 25 days, then silver grains over chromosomes were scored onto idiograms of all mouse chromosomes (Nesbitt and Francke, 1973) and mouse chromosome 6 (Evans, 1989).

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3.3.2 Preparation of recombinant mEphA1 protein

An EphA1-Fc construct was prepared by cloning the extracellular domain of mEphA1 into the unique BamHI site of the pIgBOS vector (generous gift of Dr A van der Merwe, Sir William Dunn School of Pathology, Oxford University). The mEphA1 cDNA was used as the template to PCR amplify the mEphA1 extracellular domain for ligation into pIgBOS. A correctly orientated clone was sequenced to exclude mutations and transfected into CHO cells as previously described (Lackmann et al., 1997). The CHO cell clones were screened by Western blotting using a peroxidase-conjugated rabbit anti-human IgG antibody [Dako, Australia] and membranes developed with ECL chemiluminescence [Amersham, Australia]. The clones with strongest expression were selected and grown in serum-free medium, and the mEphA1-Fc protein was purified from conditioned medium on a protein-A Sepharose [Pharmacia, Australia] column.

The full-length mEphA1 coding sequence was cloned into the XbaI site of pEFBOS (Mizushima and Nagata, 1990), and the correct orientation and sequence was confirmed by DNA sequencing. The DNA was transfected into CHO cells, and neomycin resistant clones were tested for EphA1 protein staining on the cell surface (Lackmann et al., 1997). The cells were incubated with rabbit anti-EphA1-Fc polyclonal antibody [LFQ laboratory reagent] (25 µg/ml) for 30 minutes, washed and stained with FITC-labelled sheep anti-rabbit Ig antibody [Silenus, Australia], washed again twice and positive clones detected by flow cytometry on a FACS Calibur [Becton Dickinson].

3.3.3 Determining the binding characteristics of mEphA1

The binding characteristics of mEphA1 with various ephrin ligands were analysed by surface plasmon resonance binding and the recombinant proteins were immobilised on CM5 biosensor chips [BIACore™, Pharmacia] as previously described (Lackmann et al., 1996; Lackmann et al., 1997). In short, immobilisation was performed in the BIACore 2000 biosensor by passing 30 µl of protein at a concentration of 50 µg/ml over the surface of the activated chip at a flow rate of 5 µl/min. Parallel channels on the chip were used to immobilise up to four proteins to allow simultaneous comparison of binding to different receptors. The human ephrins used in these BIACore experiments bind equally well to mouse and human Eph proteins. In these experiments, the ephrin-

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Fc constructs were prepared at concentrations of 10 µg/ml and assayed on the biosensor as previously described (Lackmann et al., 1996).

3.3.4 In situ hybridisation studies

Antisense and sense control probes were synthesised from non-overlapping regions of the mEphA1 sequence and amplified by PCR using the mEphA1 cDNA as a template. The PCR products were subcloned into the pGEM7 vector [Promega] and the DIG RNA labelling kit [Boehringer Mannheim] was used to prepare digoxigenin labelled antisense and sense control riboprobes from the T7 and SP6 promoters of pGEM7. The riboprobe concentration was estimated by comparison with an RNA standard on a dot blot. Tissue sections were deparaffinised, rehydrated then refixed in 4% paraformaldehyde and 0.2% glutaraldehyde and the hybridisation procedure was performed as described (Rosen and Beddington, 1993), except that 2 mM levamisole was included to inactivate endogenous alkaline phosphatases.

3.3.5 Preparation of mEphA1 probe

A 672 bp probe which includes the entire length of Exon 3 (the ligand binding domain) of mEphA1 was prepared by polymerase chain amplification (Section 2.2.14) of the mEphA1 cDNA with Pfu polymerase [Stratagene] using the following primers:-

Forward: 5’– GGTGGAGTGAGGTGCAACAACTG – 3’ (NM_023580).

Reverse: 5’– CCACTGCTTTCTTCATAGCCAGG – 3’.

The thermocycler program was set with a time delay melting step at 94ºC for 2 minutes 30 seconds, an annealling temperature at 60ºC for 60 seconds and extension at 72ºC for 10 minutes. The size of the amplified product was confirmed by agarose gel electrophoresis, and the fragment was excised from the gel and purified with the QiaQuick gel purification spin columns [Qiagen] and resuspended to a concentration of approximately 50 ng/µl.

3.3.6 Estimation of the titre of the λDASH phage genomic library

The λDASH 129 s/v mouse genomic bacteriophage library (provided by Dr Frank Köntgen, Walter and Eliza Hall Institute, Melbourne) was titred. This bacteriophage library was a Sau3A partial digest cloned into λDASH [Stratagene]. For the phage

100 Queensland Institute of Medical Research Chapter 3: The characterisation and genomic organisation of mEphA1 library titration, 100 µl of serial 1:10 dilutions of phage diluted in SM buffer (100 mM

NaCl, 10 mM MgSO4, 50 mM Tris-Cl) were mixed in a 15 ml conical tube with 100 µl of LE392 cells (OD600 = 0.9) at 37°C for 20 minutes, to which 3 ml of molten “top LB agar” (0.7% LB agarose containing 10 mM MgSO4), was added and poured onto the “bottom agar” (LB agar 1.1%) plates. The plates were allowed to harden at room temperature and incubated overnight at 37°C. The viral plaques were counted next morning and the phage library titre was calculated using the formula:- phage titre = (number of plaques on the plate) x (plating volume converted to millilitres) x 1/(dilution factor).

3.3.7 Isolation of λDASH phage mEphA1 positive genomic clones

The λDASH 129 s/v mouse genomic library was plated out and screened using the LE392 E. coli phage plating cells as described in Section 2.2.2. The 672 bp Exon 3 probe was used to identify mEphA1 positive λ phage clones. The phage titre of the primary clones was estimated by preparing serial dilutions of the phage plug supernatant and incubating with LE392 E. coli. The secondary filters were generated in duplicate and positive phage clones were picked, and the process was repeated until tertiary filters had been screened. At the tertiary stage, the λ phage clones on each plate were considered identical.

Prior to expansion of the positive λ phage clones, an aliquot of each of the eight isolated clones was amplified by PCR (Section 2.2.14) using the Exon 3 primers used to prepare the probe.

3.3.8 Expansion of positive λDASH genomic clones

The positive λ phage clones were expanded using the plate-lysis method (Sambrook and Russell, 2001). A 100 µl aliquot of freshly prepared LE392 phage plating E. coli was incubated with 100 µl of supernatant from the tertiary filter plugs resuspended in SM buffer overnight. The incubated LE392 cells were added to 15 ml “top agarose” and plated out on 10 cm diameter bottom agar plates. The plates were inverted and incubated overnight at 37ºC. The plates were inspected the next morning, and if the plates were nearly confluent with viral plaques, then 10 ml of freshly prepared SM buffer was pipetted onto each inverted plate on the rotary shaker the following night at

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4ºC. The supernatant was aspirated carefully next morning (total volume approximately 7.5 ml) and transferred to a 50 ml conical tube. In addition, the top agarose was scraped off using a cell scraper [Costar] and transferred to the same 50 ml conical tube, and centrifuged in a benchtop centrifuge at 1,500 rpm for 10 minutes at 4°C.

Alternatively, a 500 µl aliquot of LE392 E. coli was incubated with bacteriophage for 20 minutes at 37ºC. The phage infected LE392 cells were added to 100 ml of LB (with

10 mM MgSO4) and grown on the rotary shaker at 37ºC overnight until bacterial lysis had occurred as suggested by the presence of bacterial debris.

3.3.9 Preparation of genomic DNA

The supernatant containing the phage suspended in SM buffer was collected, 100 µl of chloroform was added, and the tubes were shaken at 37ºC for 10 minutes while bacterial lysis occurred. The bacterial debris was pelleted by centrifugation in an Oakridge™ tube in a JA-17 rotor at 3,000 rpm for 15 minutes at 4ºC. The supernatant containing the phage was transferred to a 30 ml Corex™ tube and the phage was pelleted by centrifugation in a JA-17 rotor at 12,000 rpm for 90 minutes at 4ºC. There were visible pellets and the supernatant was poured off carefully. The phage pellet was air dried, and freshly prepared bacteriophage DNA lysis buffer (Appendix 5) was added including Proteinase K (10 mg/ml) at 25 µl per 5 ml of DNA lysis buffer. The phage pellets were resuspended in 500 µl of DNA lysis buffer at 50ºC in a heating block overnight. The bacteriophage DNA was subsequently extracted with phenol/chloroform (Section 2.2.8) and precipitated with isopropanol, rinsed in 70% ethanol, and the pellets air dried prior to resuspension in 50 µl SM buffer. The bacteriophage DNA was run through a 1% agarose gel and the band excised and purified with the QiaExII gel purification kit [Qiagen] according to the manufacturer’s instructions to remove impurities to facilitate the restriction enzyme digest of the recovered DNA. At least 50 µl of resin matrix was used for each sample. The bacteriophage DNA was resuspended in 10 mM Tris-Cl, pH 8.0. Alternatively, the lambda bacteriophage DNA was isolated using the Wizard™ Lambda DNA Purification Kit [Promega] according the manufacturer’s protocol.

3.3.10 Southern blot analysis of the bacteriophage clones

The phage DNA was digested with several restriction enzymes including NotI, SalI, EcoRI and XbaI and the fragments separated by agarose gel electrophoresis prior to

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Southern blotting. The Southern blot was probed with the P32-labelled and denatured 672 bp Exon 3 probe.

3.3.11 Sub-cloning of bacteriophage fragments into pBluescript™

The XbaI and EcoRI digested fragments of the bacteriophage DNA were sub-cloned into pBluescript [Stratagene] as described in Section 2.2.11. The resultant DNA fragments were excised from the gel and purified, then ligated into XbaI or EcoRI digested phosphatased pBst. The colonies were picked, and mini-prep DNA was prepared and screened by restriction enzyme digestion or by PCR amplification using the T3 and T7 primers. The sub-cloned fragments of bacteriophage genomic DNA were amplified by the MaxiPrep method (Section 2.2.1c) to produce adequate amounts of DNA for further manipulation.

3.3.12 Sequencing of genomic DNA

The bacteriophage DNA was directly sequenced (Section 2.2.12) initially using the T3 and T7 primers. The amount of template bacteriophage DNA was increased to 1 µg and the quantity of sequencing primers increased to 10 pmoles in order to improve the sequence signal. The genomic DNA fragments sub-cloned into pBst were also directly sequenced initially with the T3 and T7 primers. As the genomic DNA sequence was generated, fresh primers were designed using the MacVector program [Accellrys Inc] to sequence the contiguous DNA. In this manner the entire sequence of each sub-cloned genomic fragment was characterised in both directions.

The DNA sequence of these genomic clones was manipulated using the FastA and SEQED functions of GCG [Version 7, Genetics Computer Group]. Overlapping sequence was joined using the SEQED function to create a contiguous genomic sequence of mEphA1 (see Appendix 1: EphA1 genomic sequence contig).

3.3.13 PCR amplification of Exons 15, 16 and 17

The purported exon-intron boundaries for Exon 15, 16 and 17 were determined by aligning the mEphA1 cDNA with the mEphA3 cDNA and genomic sequence. PCR primers were designed from the cDNA sequence using the MacVector program which would cross the exon-intron boundaries. In this manner mouse genomic DNA was used as the template to amplify the intronic DNA between Exons 15 and 16 and between

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Exons 16 and 17 (see Table 3.1). The amplified genomic DNA products were subsequently ligated into the pGEM-T vector [Promega], sequenced, aligned and incorporated into one contiguous mEphA1 sequence using the GCG program as previously described (see Appendix 1).

Table 3.1 Primers used to amplify Exons 15, 16 and 17

Primer Name Sequence (5’– 3’)

Exon 15F ATGAAGAACTGCTGGGCTTA

Exon 16R AGCCGTCAGCTCCAGCACA

Exon 16F GGATCCCTTATCGAAGTGTC

Exon 17R CAGCACCAATAGCCTACATTT

Exon 15RA TGGAACCTTCAGAGTCATTC

Exon 16RA GCTCCCTTCTCCTAAGATGG

3.4 Results

3.4.1 Confirmation that mEphA1 and hEphA1 are interspecies homologues (orthologues)

A comparison of a corrected DNA sequence of hEphA1 with mEphA1 sequence (Figure 3.1) showed 86% identity at the nucleotide level and 88% at the amino acid level (Lackmann et al., 1998). Interestingly, neither human nor mouse EphA1 possess a C- terminal PDZ protein binding motif. The chromosomal location of mEphA1 on mouse chromosome region 6B (Figure 3.2) forms a syntenic group with genes from region 7q32-36, the location to which the hEphA1 gene was assigned (Tsui et al., 1989).

104 Queensland Institute of Medical Research Chapter 3: The characterisation and genomic organisation of mEphA1

1 M E R R W P L G L A L L L L L L C A P L P P G A R A E E V T L M D T S T A Q G E L G W L L D P P E T 51 G W S E V Q Q M L N G T P L Y M Y Q D C P I Q E G G D T D H W L R S N W I Y R G E E A S R I Y V E L 101 Q F T V R D C K S F P G G A G P L G C K E T F N L F Y M E S D Q D V G I Q L R R P L F Q K V T T V A 151 A D Q S F T I R D L A S D S V K L N V E R C S L G H L T R R G L Y L A F H N P G S C V A L V S V R V 201 F Y Q R C A E T V H G L A H F P D T L P G P G G L V E V A G T C L S H A Q I S L G S S G T P R M H C 251 S P D G E W L V P V G Q C Q C E P G Y E E S S G N V G C T A C P T G F Y R V D M N T L R C L K C P Q 301 H S I A E S E G S T I C T C E N G H Y R A P G E G P Q V A C T R P P S A P Q N L S F S T S G T Q L S 351 L R W E P P R D T G G R H D I R Y S V E C L Q C R G I A Q D G G P C Q P C G K G V H F S P A A S G L 401 T T S T V Q V Q G L E P Y A N Y T F T V K S Q N R V S G L D S S S P S S A S L S I N M G H A E S L S 451 G L S L K L V K K E P R Q L E L T W A G S R P R N P G G N L S Y E L H V L N Q D E E W H Q M V L E P 501 R V L L T K L Q P D T T Y I V R V R T L T P L G P G P F S P D H E F R T S P P V S R S L T G G E I V 551 A V I F G L L L G I A L L I G I Y V F R S R R G Q R Q R Q Q R Q R E R T T N V D R E D K L W L K P Y 601 V D L Q A Y E D P A Q G A L D F A Q E L D P A W L I V D T V I G E G E F G E V Y R G A L R L P S Q D 651 C K T V A I K T L K D T S P D G Y W W N F L R E A T I M G Q F N H P H I L R L E G V I T K R K P I M 701 I I T E F M E N G A L D A F L K E R E D Q L A P G Q L V A M L L G I A S G M N C L S G H N Y V H R D 751 L A A R N I L V N Q N L C C K V S D F G L T R L L D D F D G T Y E T Q G G K I P I R W T A P E A I A 801 H R I F T T A S D V W S F G I V T W E V L S F G D K P Y G E M S N Q E V M K S I E D G Y R L P P P V 851 D C P A P L Y E L M K N C W A Y D R A R R P H F L Q L Q A H L E Q L L T D P H S L R T I A N F D P R 901 V T L R L P S L S G S D G I P Y R S V S E W L E S I R M K R Y I L H F R S A G L D T M E C V L E L T 951 A E D L T Q M G I T L P G H Q K R I L C S I Q G F K D

Figure 3.1 The deduced amino acid sequence of mEphA1. The signal peptide is boxed and the transmembrane domain is double underlined. Within the extracellular domain, conserved cysteine residues are highlighted, the EGF domain is boxed and the two fibronectin type III repeats are shaded gray. In the cytoplasmic domain, the highly conserved GXGXXG motif and invariant lysine are highlighted. Three motifs associated with substrate specificity for tyrosine and the conserved phosphotyrosine docking site (YEDP) are shown in gray. The C terminal SAM domain is boxed (Lackmann et al., 1998).

3.4.2 Restriction analysis and Southern blotting of λ phage clones

The restriction digest analysis of genomic DNA isolated from the λ phage of the eight isolated tertiary bacteriophage clones revealed that there were five distinct λ genomic clones. The genomic clones were subjected to Southern blot analysis to confirm which fragments contained the Exon 3 sequence (data not shown).

A Southern blot of mouse genomic DNA digested with various restriction enzymes and probed with probes from corresponding regions of hEphA1 and mEphA1 revealed identical sized fragments with both probes for all the restriction enzymes tested (Figure 3.3).

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Figure 3.2 The chromosomal location of mEphA1. Plot of grains after scoring approximately 130 chromosomes 6, showing probable localisation of mEphA1 to bands B1-B2. Grains scored from C57BL and BALB/c mice are scored by solid and open dots, respectively. 3.4.3 Characterisation of the binding properties of mEphA1

The binding of mEphA1 and hEphA3 to human ephrin ligands determined on the BIACore sensor confirmed previous findings for EphA3. EphA1 bound strongly to h- ephrin-A1, weakly to h-ephrin-A3 and h-ephrin-A4, with no specific binding to h- ephrin-A5, h-ephrin-B1 or h-ephrin-B3 (Figure 3.4).

Figure 3.3 Analysis of mEphA1 and hEphA1 by Southern hybridisation. The filter was probed initially with an mEphA1 probe (A) then stripped and re-probed with a probe from the equivalent region of hEphA1 (B).

106 Queensland Institute of Medical Research Chapter 3: The characterisation and genomic organisation of mEphA1

3000

2500 EphA1 )

U EphA3

R 2000 ( s t i

n 1500 u e s 1000 n o p

s 500 e r 0 A1 A3 A4 A5 B1 B3 CD48Fc

Figure 3.4 Binding of ephrins to mEphA1 and hEphA3. The binding of a panel of ephrin-Fc fusion proteins to purified mEphA1 and hEphA3 immobilised to a BIAcore biosensor chip was analysed on a BIAcore 2000 biosensor (BIAcore, Sweden). Peak binding for a typical experiment is shown; these results were highly reproducible over several experimental runs. (Note: hEphA3 data previously published Lackmann et al., 1996; Lackmann et al., 1997).

The binding of soluble mEphA1 to a panel of human ephrins analysed by surface plasmon resonance binding [BIACore] indicated that recombinant mEphA1-Fc protein bound strongly to ephrin-A1 but only weakly to ephrin-A3 and ephrin-A5. FACS analysis revealed that recombinant full-length mEphA1 expressed on CHO cells bound strongly to ephrin-A1-Fc, weakly to ephrin-A3-Fc, but not to ephrin-A5-Fc (Figure 3.5).

A B

Fluorescence Intensity

C D

Fluorescence Intensity

Figure 3.5 Binding of ephrins to EphA1-transfected CHO cells. A: ephrin-A1-Fc; B: ephrin-A3-Fc; C: ephrin-A5-Fc; D: ephrinA1-Fc on control CHO cells.

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3.4.4 PCR of genomic clones for Exon 3

PCR amplification also confirmed that the isolated genomic clones contained Exon 3 of mEphA1; however, the amplified DNA in all five of the genomic clones was 1.4 kB larger than the band amplified from the mEphA1 cDNA control. This suggested the presence of an additional intron; however, restriction digestion and Southern blot analysis were not sufficiently sensitive to confirm the presence of the additional intron.

3.4.5 The genomic organisation of mEphA1

The genomic organisation of mEphA1 is summarised in Figure 3.6. There are a total of 18 exons distributed over 22 kB. The results of direct sequencing confirmed that Exon 3 is separated into two exons by 1.4 kB of intervening sequence. The important ligand binding domain is encoded by Exon 2, Exon 3a, Exon 3b and Exon 4. The entire EphA1 genomic contig, which was constructed from sequence of the subcloned genomic DNA, is displayed in Appendix 1: EphA1 genomic sequence contig.

.

Figure 3.6 Genomic organisation of mEphA1. (mEphA1 aligned with the corresponding cDNA)

108 Queensland Institute of Medical Research Chapter 3: The characterisation and genomic organisation of mEphA1

3.4.6 In situ hybridisation analysis of mEphA1 expression

In situ hybridisation confirmed the results of Northern analysis (unpublished observations, AW Boyd and JD Lickliter). mEphA1 expression was observed in the basal layer of the epidermis and developing hair follicles. In adult thymus, mEphA1 expression was observed in thymic epithelial cells. In adult kidney, mEphA1 expression was localised in the proximal and distal convoluted tubules; however, the glomeruli and interstitium did not stain (Figure 3.7).

Figure 3.7 mEphA1 expression in embryoid tissues and adult mouse tissues. A: embryoid bodies differentiated in vitro; B: thymus; C: renal cortex; D: day 18 embryonic skin. Scale bars: A 160 µm sense, antisense/LP, 39 µm antisense/HP; B: 390 µm sense antisense/LP, 39 µm antisense/HP; C: 160 µm sense, antisense/LP, 16 µm antisense/HP; D: 39 µm sense, antisense/LP, antisense/HP. Abbreviations: LP, low power; HP, high power.

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3.5 Discussion

The murine homologue of EphA1 (originally Esk) was identified through a degenerate PCR strategy which cloned a 1.6 kB cDNA fragment subsequently used to screen a mouse liver cDNA library constructed in λZAP [Stratagene] and to identify the full- length mouse EphA1 (Lickliter et al., 1996). The characterisation described in this chapter confirms that mEphA1 is the mouse homologue of the human gene hEphA1.

A comparison of the genomic organisation of the Eph genes reveals a highly conserved exon-intron structure between Ephs of different classes and species. Now that both the human and mouse genome databases have been sequenced and released, the number and localisation of the Eph and ephrin genes can be examined. There are three clusters of Eph genes, on human chromosome 1 (EphA2, EphA8, EphB2), chromosome 3 (EphA3, EphA6, EphB1 and EphB3) and on chromosome 7 (EphA1, EphB4, EphB6) (Pasquale, 2005). Similar clusters are found on the corresponding mouse chromosomes. The mEphA1 gene is localised on chromosome 6B which forms a syntenic group with human genes from region 7q32–36, which is the location assigned to the hEphA1 gene.

The genomic organisation of mEphA1 matches that of hEphA1 and supports the conservation of genomic structure of all the Eph genes with the exception that in both mouse and human EphA1, Exon 3 is split by an additional intron into what we have denoted Exon 3a and Exon 3b (Owlshalimpur and Kelley, 1999). The additional intron divides the region coding for the N-terminal complex β-jellyroll structure, which is critically important in ephrin-binding (Coulthard et al., 2001). The phase at the boundaries of this additional intron differs from that of the 5′ and 3′ boundaries of the “ancestral” Exon 3, which suggests that this intron was inserted later in the evolution of the EphA1 gene (Patthy, 1987). There are 18 coding exons in EphA1 — two more than EphB2. The genomic organisation of the kinase domain of hEphA1 has been previously described and is encoded by five exons (Maru et al., 1988), in comparison with EphB2 which has only four exons and lacks the intron between domain 1 (D1) and domain 2 (D2) of the catalytic domain (Connor and Pasquale, 1995).

Previous Northern blot analysis of E12 mouse embryo, adult thymus, liver, kidney, lung and placenta revealed a primary 4.2 kB EphA1 transcript with faint bands at 6.0 kB consistent with the presence of alternatively spliced transcripts (unpublished observations, JD Lickliter and AW Boyd). The function of the additional intron within

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Exon 3 of mEphA1 is currently not known, but might be consistent with the presence of alternatively spliced EphA1 variants. There have been splice variants described of several ephrins (Eph variants), including ephrin-B2 (Bergemann et al., 1995), ephrin-A3 (Lai et al., 1999), ephrin-A4 (Aasheim et al., 2000) and ephrin-A5 (Flenniken et al., 1996), which result in different isoforms. The isoforms of ephrin-A3 and ephrin-A5 both inhibit neurite outgrowth, indicating that the deleted region is not required for functional activity (Lai et al., 1999). However, an isoform of ephrin-A1 did have altered receptor binding properties and was resistant to cleavage from the cell membrane (Finne et al., 2004).

The presence of alternatively spliced variants of Ephs has also been previously described (Ciossek et al., 1995; Connor and Pasquale, 1995; Valenzuela et al., 1995). In vitro co-expression of a truncated form of EphA7 (lacking the tyrosine kinase domain) with the full-length EphA7 receptor suppressed the phosphorylation of the full-length receptor and shifted the cellular response from repulsion to adhesion (Holmberg et al., 2000). In fact a recent genome-wide in silico review of mouse tyrosine kinases and phosphatases reported the existence of likely dominant negative forms for many receptor tyrosine kinases including EphA1 (Forrest et al., 2006). In fact the role of alternatively spliced variants may be to shift the cellular response of the Eph/ephrin receptor-ligand interaction from repulsion to adhesion (Holmberg et al., 2000).

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Chapter 4: Library screening for the zebrafish EphA1 homologue

4 LIBRARY SCREENING FOR THE ZEBRAFISH

ORTHOLOGUE OF MEPHA1

4.1 Introduction

The zebrafish has become a useful model for studying gene function and, in terms of relevance to humans, has an obvious advantage over Drosophila which is not a vertebrate (Grunwald and Eisen, 2002). There are a number of characteristics of the zebrafish, Danio rerio, which have made it a model system for the study of vertebrate function:- (1) the short generation time; (2) external fertilisation allows eggs to be harvested and (3) the fertilised eggs are large and transparent so all embryonic development is visible, which allows relatively easy manipulation and the identification of mutations (Westerfield, 2000). In particular, the ability for genetic manipulation and the short generation time facilitates large-scale genetic study [http://zfin.org/zf_info/zfbook/zfbk.html]. The initial chemical mutagenesis screens with ethyl-nitrosurea, which caused single gene mutations, were followed up by careful visual analysis of zebrafish embryos, and this approach implicated at least 500 genes of developmental importance (Driever and Fishman, 1996). A number of the zebrafish mutant phenotypes identified in the genetic screens were reminiscent of human diseases, which allows further insight into the pathophysiological mechanisms of these disease states (Dooley and Zon, 2000). Further techniques, including positional cloning, the candidate gene approach, insertional mutagenesis and reverse genetic approaches have allowed the development of several databases of zebrafish genes [http://wwwmap.tuebingen.mpg.de/ ; http://zfin.org/] and have revealed important information about vertebrate biology (Talbot and Hopkins, 2000). The zebrafish model system has been used to identify the specific genes responsible for cardiac development (Stainier, 2001) and angiogenesis (Chan et al., 2002), and it is promising as a cancer model system (Amatruda et al., 2002). The first step towards utilising the zebrafish model for understanding the function of a particular gene is to isolate the zebrafish orthologue of the gene of interest, in this case EphA1. At the time this PhD project commenced the laboratory had set up a zebrafish facility. Therefore, a search for the zebrafish orthologue of EphA1 was logical and would have provided useful insight into the function of EphA1.

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4.2 Aims

(1) To isolate the zebrafish orthologue of EphA1 from a bacteriophage library.

(2) To isolate the zebrafish orthologue of other Eph-like receptor tyrosine kinases from a bacteriophage library using degenerate primers in the polymerase chain reaction.

4.3 Materials and methods

4.3.1 Screening the λZAP cDNA library

A number of zebrafish cDNA libraries were kindly received from Andrew Oates (Ludwig Institute for Cancer Research, Melbourne), including λZAP cDNA libraries prepared from zebrafish at 6–9 hours post fertilisation (hpf), 9–16 hpf, 20–28 hpf and adult. The λZAP libraries had been prepared in 1989 in the Department of Human Genetics, University of Utah, Salt Lake City, using the Stratagene ZAP cDNA synthesis library kits (personal communication from David Jonah Grunwald) in accordance with the manufacturer’s instructions. Briefly the cDNA was derived from zebrafish poly(A)+ RNA with a XhoI linker added to the 5′ end and an EcoR1 linker added to the 3′ end. The modified cDNAs were cloned into λZAP II from which pBst plasmids could be excised (Short et al., 1988). Additionally, the laboratory acquired a zebrafish genomic library and zebrafish random primed library for screening. The libraries were titred on RY1090 E. coli grown in NZCYM+ broth as described in Section 3.3.2. An aliquot of the library was diluted in SM buffer so that the plaque density would be approximately 100,000 pfu per plate. Library screening plates were prepared with NZCYM bottom agar (1.1%), and the diluted zebrafish library was incubated with RY1090 E. coli and plated out as described in Section 2.2.2. A set of filters on which a plasmid artificial chromosome (PAC) library had been blotted also became available for screening.

4.2.2 In vivo excision from the λZAP library

In vivo excision was carried out on positive clones as described in Section 2.2.3. The excised pBst (SK-) phagemid was screened for insert by restriction digestion with EcoRI and XhoI and clones containing the insert were sequenced.

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4.3.2 Screening for zebrafish EphA1 by PCR

The zebrafish libraries were screened by PCR using degenerate oligonucleotides with the primers listed in Table 4.1. The zebrafish 20–28 hpf library was also screened with the mEphA1 Exon 3 primers with an annealing temperature of 50°C. The thermocycler program used hot start AmpliTAQ® Gold with an annealing temperature of 37°C for 60 seconds and extension at 63°C for 60 seconds for 30 cycles. (Section 2.2.12)

Table 4.1 Primers used in degenerate PCR

Primer Name Sequence (5′– 3′)

MIITEY/S GCG ATG ATC AT(CG) AC(AGT) GAG (AG) TA (CT) ATG G

ASDVWS/AS GGA ATT CCA (CGT)AC ATC (AG)CT (AG) GC

ASDVWS/AS CC(AG) (AT)A(AG) CTC CA(CT) AC(AG) TCG CTG GC

ASDVWS/AS CC(AG) TAG CTC CA(CG) ACA TC(AG) CT(GC) GC

Exon 3F GGTGGAGTGAGGTGCAACAACT C

Exon 3R CCACTGCTTTCTTCATAGCCAGG

The PCR products were run on an agarose gel, and sub-cloned into pGEM-T (Section 2.2.11g) for sequencing. The specificity of the PCR was improved by using the “touch up” technique with an initial annealing temperature of 46°C, which was increased over three cycles to 52°C (Don et al., 1991).

4.3.3 Probing a ZF genomic Southern blot

A Southern blot of zebrafish genomic DNA digested with several different restriction enzymes had been prepared in the laboratory and this was probed with the mouse Exon 3 probe as described (Section 2.2.16) with a hybridisation temperature of 50°C. The filter was washed to a stringency of 2X SSPE/0.1% SDS at 50°C then exposed at –80°C for seven nights.

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4.4 Results

4.4.1 Results of library screening

The screening of the λZAP 20–28 hpf zebrafish cDNA library did not reveal any strongly positive clones on the primary filter. However, there were two weakly positive clones, which were isolated and re-screened. At the secondary screening stage it was clear that none of the clones was truly positive.

4.4.2 Result of probing the ZF genomic Southern blot

The ZF genomic Southern blot filter was washed at low stringency and was exposed for seven nights. The filter was completely clear of bands indicating that the mouse EphA1 Exon 3 probe did not hybridise significantly to ZF genomic DNA. A control filter spotted with pBst_mEphA1 was hybridised simultaneously, and this was strongly positive.

4.4.3 Results of PAC library screening

The results of the PAC library screening were negative. There were no positive clones.

4.4.4 Results of degenerate PCR screening

The results of degenerate PCR yielded products of the expected size, suggesting the presence of Eph-like receptor tyrosine kinases in all the libraries tested. The PCR products were sub-cloned into pGEM-T for sequencing.

4.4.5 Sequencing degenerate PCR products

The sequences of degenerate PCR products were checked against the GenBank database using the BLASTn program. There were over 50 clones sequenced, and although several Eph-like sequences were identified based on high homology with mammalian sequences, including previously reported putative zEphA2, zEphA5, zEphA6, zEphA7, zEphB2 and zEphB3 (see Figures 4.1 and 4.2). However, none of the sequences had significant homology to mouse or human EphA1.

116 Queensland Institute of Medical Research Chapter 4: Library screening for the zebrafish EphA1 homologue

Figure 4.1 Zebrafish sequence line-up. Deduced amino acid sequence lineup of sequences derived from degenerate PCR of various ZF libraries as described in the text (BioManager software). An example of PCR derived sequence (ZFB) is highlighted in comparison with other zebrafish sequence from the database.

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Figure 4.2 Phylogenetic tree of obtained zebrafish sequences. The ZF sequences derived from degenerate PCR are highlighted.

4.5 Discussion

At the time that the ZF library screening project was commenced the zebrafish genome had not been completely sequenced, so based on the presence of zebrafish orthologues of other Eph (and ephrin) genes, it seemed likely that there would be a ZF orthologue of EphA1. However, the ZF genome has now been sequenced completely [http://wwwmap.tuebingen.mpg.de/ ; http://zfin.org/] and this confirms the conclusion from the results presented, that zebrafish lack an orthologue of EphA1. A recent search of the genomes of the two pufferfish, Tetraodon nigroviridis (http://www.genoscope.cns.fr) and Fugu rubripes (http://fugu.biology.qmul.ac.uk/;

118 Queensland Institute of Medical Research Chapter 4: Library screening for the zebrafish EphA1 homologue http://www.fugu-sg.org/) confirmed the absence of EphA1-related sequence. Over 50 clones were isolated by degenerate PCR of zebrafish cDNA and genomic libraries, and although some of the sequences had homology to known Ephs, none matched EphA1.

The zebrafish orthologue of EphA3 was identified and cloned in the laboratory using similar experimental methods (Oates et al., 1999). However, a concurrent search using the same experimental techniques for the chicken orthologue of EphA1 was also unsuccessful. The recently described chicken EphA9 shares 63% homology with hEphA1 and 52% with hEphA2 at the amino acid level, and the expression pattern is distinct from both EphA1 and EphA2. The amino acid sequence of the chicken orthologues of EphA3, EphA4, EphA5 and EphA7 are more than 92% identical, which suggests that although EphA9 is the closest orthologue to hEphA1, it is a distinct, single gene (Sasaki et al., 2003). This failure to find the chicken orthologue suggested that EphA1 does not occur in all vertebrates and may be a product of later evolution only expressed in mammals. It is likely that the 14 Eph receptors are not identical in all vertebrates, and to further support this idea, EphB5 has been found in chickens but not in mammals, despite experimental approaches and in silico searching (Soans et al., 1996). There is one chicken ephrin, ephrin-A6, which does not have a mammalian orthologue and probably arose separately through gene duplication during the evolution of the avian lineage (Menzel et al., 2001). It was disappointing to search persistently in the hope a zebrafish orthologue of EphA1 existed. However, in hindsight, its absence does seem to fit with the notion that EphA1 may be present only in mammals. Consequently, this made success in the following gene knockout approach to elucidating the expression and function of EphA1 more important.

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Chapter 5: Generation and analysis of the EphA1 reporter mice

5 THE GENERATION AND EXPRESSION

ANALYSIS OF THE EPHA1 REPORTER MICE

5.1 Introduction

5.1.1 General comments on analysing gene function

There are basically two methods of genetic analysis utilised to answer questions about the function of a particular gene and its encoded protein. Firstly, there is the classical genetic approach in which experimental animals or individuals with the mutant phenotype of scientific interest are screened in order to identify the abnormal gene and hence imply the normal function of that gene and its role in producing the phenotype. This approach has been successful in the zebrafish model system where random mutagenesis screens produce many mutant phenotypes by introducing single gene mutations into developmentally important genes (Talbot and Hopkins, 2000). The next step in this approach is to identify the gene through systematic library screening and mapping processes. An example in the zebrafish is the gridlock mutation, in which there was impeded blood flow to the tail similar to the human disease of coarctation of the aorta (Weinstein et al., 1995; Zhong et al., 2001). Subsequent gene mapping work has identified the gridlock gene encoding a basic helix-loop-helix protein expressed in lateral plate mesoderm before vessel formation (Zhong et al., 2000).

Secondly, a newly discovered gene and its encoded protein can be investigated by “reverse genetics”. In this method, the gene of interest can be mutated; for example, removing the kinase activity of the tyrosine kinase, modifying specific residues at critical binding sites, or silencing the gene function by “knocking-out” the gene completely. The resultant change in phenotype of the animal allows the investigator to imply the function of the molecule in the whole animal.

5.1.2 General comments on gene targeting

The mouse is very similar to the human species, particularly in developmental processes, and all or nearly all human genes are present in the mouse. Furthermore, the arrangement of mouse and human genes on the chromosomes is comparable. The mouse

Queensland Institute of Medical Research 121 Chapter 5: Generation and analysis of the EphA1 reporter mice model is highly equivalent to the human with respect to organ systems, in contrast with the model systems of the lower organisms such as worms (C. elegans) and flies (D. melanogaster). The ability to express transgenes or modify gene expression has allowed the mouse to become one of the most useful animal models in biomedical research. There have been a number of technical developments, including the generation of transgenic mice, pluripotent embryonic stem cell cultures, gene knockout mice, tissue- specific knockout mice and gene knock-in mice, which have made the mouse model system even more appealing for scientists to study gene function. The small size and relatively rapid breeding time of mice results in some advantages over other mammalian models such as rat, rabbit and pig. There are now hundreds of genetically homogenous inbred strains of mice and a wide availability of pluripotent embryonic stem cells to allow genetic manipulation of the germ line.

In order to determine the function of a particular gene of interest, in this case EphA1, a gene knock-in strategy allows the replacement of the gene of interest with a reporter gene and a selectable marker (Joyner, 2000; Mansour et al., 1988). The design of a replacement-type targeting vector requires consideration of the introduced mutation, the selectable marker and the method of screening for homologous recombination. These principles have been thoroughly investigated and have become standard practice in transgenic laboratories involved in producing “knockout” mice (Hasty, 2000). The specific use of gene targeting to replace the endogenous gene of study with a marker or reporter for gene expression has been termed a “knock-in” (Cui et al., 1994). In a “knock-in” experiment, when an endogenous gene is replaced by a reporter gene, such as beta galactosidase (LacZ), or human placental alkaline phosphatase (hPLAP), under the transcriptional control of the endogenous gene, then the spatial and temporal expression pattern of the endogenous gene can be monitored both during development and in the adult mouse (DePrimo et al., 1996). The positive selection marker can be removed using the Cre-loxP system (discussed more fully below), which assures that the strong promoter driving the expression of the selectable marker does not interfere with the normal regulation of the knocked-in allele. The additional benefit of the knocking-in of a reporter gene is that the vector design can include features which result in the loss of function of the endogenous gene, leading to complete loss of the targeted gene in homozygous mice. This project was commenced in late 1996 some years before the

122 Queensland Institute of Medical Research Chapter 5: Generation and analysis of the EphA1 reporter mice sequence of the mouse genome was published. Hence, the technical approach described in this chapter reflects the methods available at that particular time.

5.1.3 The IRES element

The translation of eukaryotic mRNA is dependent upon 5′-cap-mediated ribosome binding. The internal ribosomal entry site (IRES), which is derived from the Picornaviruses, provides an alternative to the 5′-cap-mediated ribosome binding site which allows efficient initiation of translation. The use of an IRES element in the targeting construct enables a single transcription unit to provide efficient production of both the gene of interest (in our case a reporter gene) and a selectable marker (in our case neomycin resistance). This obviates the need to generate fusion proteins between the selectable marker and linker reporter gene, and also avoids the possibility of splicing around the inserted transgene. In fact the combination of a splice acceptor sequence– IRES–transgene allows the knock-in vector to be cloned in anywhere without regard for in-frame fusion after splicing. In summary, the use of the IRES element in gene targeting allows the use of a promoterless gene targeting construct which allows regulation of the transgene by the endogenous promoter of the targeted gene. This allows the reporter gene to provide an expression profile of the targeted gene (Mountford and Smith, 1995).

5.1.4 hPLAP as a reporter enzyme

The alkaline phosphatases are widely expressed in mice during embryonic development and adult life; however, in embryonic tissues and their derivatives these enzymes are heat-sensitive and can be inactivated by treatment of the tissues at 73°C for 35 minutes. In contrast, the human placental alkaline phosphatase (hPLAP) enzyme is largely restricted to the placenta and is a thermostable enzyme (Knoll et al., 1988). hPLAP enzyme activity can be identified by a simple colour reaction in heat-treated sectioned tissues using alkaline phosphatase substrates such as nitroblue tetrazolium/5-bromo-4- chloro-3-indolyl-phosphate (NBT/BCIP) [Boehringer-Mannheim] or BM purple [Roche] (see Section 2.3.2). This allows hPLAP to be used as a very effective reporter gene in the mouse (Nagy, 2000).

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5.1.5 The Cre-loxP system

The utilisation of the site-specific Cre-recombinase systems allows removal of regions of DNA inserted into the genome through transgenesis or homologous recombination and in particular selection genes (neomycin resistance) driven by strong promoters such as the phosphoglycerokinase (PGK) promoter. The presence of the strong promoter can affect reporter gene expression, thus changing the pattern of expression conferred by the endogenous gene promoter through the function of the IRES element.

The Cre-recombinase (cyclisation recombination protein) of bacteriophage P1 Cre is a 343 amino acid recombinase which mediates recombination events between two loxP recognition sites. The 34 bp loxP site (locus of crossing over [X] in P1) consists of two 13 bp inverted repeats separated by an 8 bp spacer (Figure 5.1). The asymmetry of the loxP site gives the sequence polarity. A Cre-mediated recombination event between loxP sites with the same polarity results in excision of the DNA flanked by those sites (hence the term “floxed”) with preservation of one loxP site. However, recombination between loxP sites in the opposite orientation results in inversion of the intervening DNA. It should be noted that Cre+ strains of E. coli excise and circularise segments of DNA that are flanked by loxP sites (Sambrook and Russell, 2001).

5' A T A A C T T C G T A T A G C A T A C A T T A T A C G A A G T T A T 3'

3' T A T T G A A G C A T A T C G T A T G T A A T A T G C T T C A A T A 5'

Figure 5.1 Structure of the 34 bp loxP site. There are two perfect inverted repeats 13 bp in length separated by an 8 bp spacer.

5.2 Aims

(1) Generate a replacement-type targeting vector with hPLAP as the reporter gene.

(2) Generate mice that express hPLAP in the same spatial and temporal distribution as EphA1.

(3) Analyse EphA1 expression.

(4) Generate a complete EphA1 knockout mouse.

(5) Characterise the phenotype.

124 Queensland Institute of Medical Research Chapter 5: Generation and analysis of the EphA1 reporter mice

5.3 Materials and methods

5.3.1 General comments

The overall homologous recombination strategy was to produce a mutant allele with the IRES-hPLAP-bpA reporter cassette knocked-in towards the 5' end of mEphA1. This would allow the IRES to be controlled by the endogenous EphA1 promoter and enhancer elements, therefore, hPLAP enzyme activity would reflect EphA1 gene expression. The presence of a BamHI site within Exon 2 facilitated this strategy. Staff of the QIMR transgenic facility, under the supervision of Dr Graham Kay, performed the ES cell culture, electroporation of ES cells with linearised vector, screening of homologous recombinant ES cell clones, injection of blastocysts and production of chimaeric mice. Therefore, in mice homozygous for the knocked-in or “reporter allele”, a normal EphA1 transcript is not produced as Exon 2 is disrupted by the reporter cassette. However, transcription of hPLAP is directed by the IRES element under the control of the upstream EphA1 enhancer and promoter elements.

5.3.2 Generation of the reporter cassette

The reporter cassette, IRES_hPLAP_bpA_PGK_neo, was constructed as summarised below (Figure 5.2). Initially the bovine growth hormone polyadenylation signal (bpA) was XbaI – XhoI double digested from pBst_PGK_βgeo_bpA (Adra et al., 1987; Pfarr et al., 1986) [generous gift from Dr Ian Tonks, QIMR].

The IRES_hPLAP plasmid received from Dr Phil Leighton [Howard Hughes Medical Institute, University of California, San Francisco, CA, USA] was partially digested with XbaI followed by complete digestion with XhoI so that the polyadenylation signal, bpA, could be inserted at the 3’ end of the hPLAP. The PGK_neo cassette was directionally cloned into pBst_IRES_hPLAP_bpA as follows. A three-way ligation of (1) SalI and NotI digested pBst_floxed PGK_neo, (2) SpeI and XhoI digested IRES_PLAP_bpA and (3) SpeI and NotI digested pBst (SK-) was performed to complete preparation of the IRES_hPLAP_bpA_PGK_neo reporter cassette, as shown in Figure 5.2.

5.3.3 Generation of the EphA1 reporter targeting construct

The construction of the targeting vector used a multiple cloning step strategy, which is briefly described below and outlined in Figure 5.3. In summary, the pBst_λ4.2 EphA1

Queensland Institute of Medical Research 125 Chapter 5: Generation and analysis of the EphA1 reporter mice genomic clone was used as the backbone of the targeting construct to provide sufficient homology on either side of the IRES_hPLAP_bpA_PGK_neo reporter cassette for homologous recombination events to occur. The IRES-hPLAP-bpA_ floxed PGK_neo reporter cassette was knocked-in to the BamHI site of Exon 2 of mEphA1. This resulted in loxP sites flanking the PGK_neo cassette, which could be later removed by excision with Cre-recombinase.

I I t l o a PGK-neo N S

loxP I I o a h b X X bpA I e I I p I o a a S b h b X X X IRES-hPLAP-bpA I I I I V I t I e R d I I a l o o n c p b i a c a S X H E S S N

Three way ligation Excise with SalI/NotI pBst(KS)

Figure 5.2 Preparation of the reporter cassette. A three way ligation of three separate plasmids was performed. The multiple cloning steps are described in the text.

Initially the pBst_λ4.2 genomic clone was test digested with BamHI and HpaI to confirm the absence of these restriction sites. The BamHI site in Exon 2 was used as the insertion or knock-in site of the reporter cassette. The enzyme HpaI was used to linearise the reporter construct. A modification of the standard pBluescript plasmid was prepared by designing a polylinker and inserting it into SacI/KpnI double digested pBluescript (SK-) and designated pBst_SacI_HpaI_XbaI_KpnI. The 7.5 kB XbaI fragment of the λ4.2 genomic clone was ligated into XbaI digested pBst_SacI HpaI XbaI KpnI. The pBst_SacI_HpaI_XbaI_ KpnI_λ4.2 plasmid was partially digested with

126 Queensland Institute of Medical Research Chapter 5: Generation and analysis of the EphA1 reporter mice

BamHI, because there is another BamHI site 600 bp upstream of Exon 2, such that the polylinker (BamHI_SalI_NotI_BamHI)[Sigma-Genosys] was inserted into the BamHI site within Exon 2.

Finally, the SalI and NotI digested IRES_hPLAP_bpA_floxed_PGK_neo reporter cassette was inserted into the pBst_λ4.2_(BamHI_SalI_NotI_BamHI) plasmid to complete the targeting vector (Figure 5.3). At each step of preparing the targeting construct, direct sequencing of mini-prep DNA was used to confirm the success of the cloning step before proceeding. The mini-prep DNA was then expanded using the Qiagen Midi-Prep technique (see Section 2.2.1b) in order to produce sufficient DNA for the subsequent cloning step. The reporter-targeting vector was linearised with HpaI for electroporation into ES cells.

5.3.4 Embryonic stem cell culture

The murine 129 s/v derived embryonic stem (ES) cell line was routinely passaged on an underlayer of irradiated primary mouse embryonic fibroblasts (PMEF) on gelatinised tissue culture dishes in ES cell culture medium. The ES cell culture medium was Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 15% fetal bovine serum [HyClone] (which had been heat inactivated at 56ºC for 30 minutes), 1X non- essential amino acids [Gibco-BRL], 1,000 U/ml leukaemia inhibitory factor [AMRAD], 2 mM glutamine, 100 mM β-mercaptoethanol, 25 mM each of adenosine, guanosine, cytosine, uridine and thymidine, 60 mg/L penicillin and 100 mg/L streptomycin. ES cell cultures were incubated at 37ºC in a humidified 5% CO2 atmosphere.

5.3.5 Generation of targeted EphA1 reporter ES cell lines

The targeting vector was linearised with HpaI, and 25 µg of linearised vector was added to a 0.4 cm cuvette containing 1 x 107 ES cells in 900 µl of PBS. The ES cells were pulsed with 250 V at a capacitance of 500 µF in a Bio-Rad GenePulser, then resuspended in 60 ml ES cell culture medium and divided between three 90 mm tissue- culture dishes over a layer of PMEFs at 37ºC in a humidified 5% CO2 atmosphere. Antibiotic selection with neomycin (G418) at a concentration of 0.2 mg/L was commenced after 24 hours of recovery and continued for 10 days. ES cells were re-fed daily with G418 ES cell medium.

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IRES - hPLAP-bpA Floxed -PGK-neo Sal I HIndIII EcoRV Not I Sal I Bgl II Not I BamH1 Linker BamHI

Exon 1 Exon 2 XbaI BamHI Sac I Kpn I Hpa I λ4.2 7.5kB Xba I KI linker

Total size 14.8 kB

pBst (SK) Linearise HpaI

Figure 5.3 Preparation of the complete EphA1 reporter targeting construct. The multiple cloning steps are described in the text.

After 10 days, single colonies of G418-resistant cells had become visible to the naked eye. The ES cell colonies were washed with PBS, and then single colonies were transferred to a 96-well round bottom plate previously prepared with 25 µl of trypsin- EDTA and incubated at 37ºC for 30 minutes to allow dissociation of the colonies. The trypsin was inactivated by the addition of ES cell medium, and the cell suspension was transferred to a 96-well plate (flat bottomed) with previously prepared PMEFs and 150 µl of ES cell medium. These cells were grown with daily refeeding until they achieved confluence. The cells were split and plated onto a duplicate 96-well feeder plate and duplicate 96-well gelatinised plate without PMEFs for subsequent genomic DNA extraction.

The ES cells grown on gelatinised plates without feeders were used to extract genomic DNA for analysis for homologous recombinants. The medium was aspirated and the cells treated with 50 µl ES cell DNA lysis buffer (Appendix 5) and 100 ug/ml Proteinase K. The plates were sealed and incubated overnight at 65ºC in a humidified incubator. The next day the plates were allowed to cool down to room temperature and

128 Queensland Institute of Medical Research Chapter 5: Generation and analysis of the EphA1 reporter mice the genomic DNA was precipitated by the addition of 75 mM NaCl in cold 100% ethanol (100 µl per well). The plates were allowed to stand for at least 30 minutes at room temperature, then the ethanol was removed by careful inversion, and the wells were washed three times with 150 µl of 70% ethanol. The plates were air dried and stored at –20ºC until required.

The genomic DNA was digested in the 96-well plates. A master mix of the restriction enzyme EcoRV was prepared, including the recommended buffer NEB1, 100 µg/ml BSA, 1 mM Spermidine and 100 µg/ml RnaseA. A 30 µl aliquot of master mix was added to each well, and the plates were vortexed and incubated overnight at 37ºC in a humidified incubator. The digested ES cell genomic DNA was analysed by Southern blot (see Section 2.2.16) and probed with the 672 bp Exon 3ab probe to identify homologous recombinant ES cell clones.

5.3.6 ES cell micro-injection and production of chimaeras

The blastocyst injections were performed by staff of the QIMR transgenic facility at the Herston Medical Research Centre, under the supervision of Dr Graham Kay (QIMR), using the standardised techniques. The blastocysts were isolated at 3.5 days post coitus from super-ovulated four-week old C57BL/6J females which had been mated with C57BL/6J stud males. The donor ES cell clones carrying the EphA1 reporter allele were injected into blastocysts and these chimaeric blastocyts were implanted into outbred pseudo-pregnant foster mothers (ARC), which had been mated with vasectomised stud males. Chimaeric offspring were identified by the presence of patchy coat colour that indicated a contribution from both the 129T2/SvEms ES cell clones (agouti coat colour) and the host C57BL/J6 (black coat colour) blastocyst. The chimaeras were subsequently mated with wild type C57BL/J6 mice. If the chimaeric blastocyst had contributed to the germ line then the F1 offspring would demonstrate the dominant agouti coat colour; in other words, they would be heterozygous for the 129T2/SvEms genome, which had been carried through the germ cells. The agouti coloured F1 progeny were genotyped by Southern blot analysis of genomic DNA extracted from tail tips for the presence of the EphA1 reporter allele using the EcoRV digest and Exon 3ab probe.

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5.3.7 Generation of homozygous EphA1 reporter knockout mice

Heterozygote EphA1 reporter males (+/neo) identified by Southern blot analysis were mated with ubiquitous Cre-deleter females to produce offspring with the PGK-neo cassette excised from the EphA1 reporter allele. The Cre-recombinase gene is situated on the X chromosome, and in order to produce both male and female pups with the PGK_neo cassette excised, only heterozygote EphA1 reporter males were mated with Cre-deleter females. The EphA1 reporter allele of heterozygous mice included the 4.3 kB IRES_hPLAP_bpA_floxed PGK-neo reporter cassette inserted into the BamHI site of Exon 2. Although there are two EcoRV restriction sites within the reporter cassette, digestion of genomic DNA with EcoRV and subsequent probing with the λ1.1 Sac-Sac probe would be informative. The λ1.1 Sac-Sac 5’ probe was prepared from the SacI digestion of the pBst_λ1.1 genomic clone to release the 550 bp probe which was gel purified. A Southern blot of EcoRV digested wild type genomic DNA produced a 9.8 kB band, whereas the reporter allele resulted in a 9.0 kB band, allowing discrimination of homologous recombination (Figure 5.4). A heterozygote male (+/neo) was then mated with a Cre-deleter female with the subsequent removal of the 1.5 kB floxed PGK_neo cassette by Cre-recombinase and resulted in a 7.2 kB band, which is distinguished from both the wild type and heterozygous alleles. This is demonstrated in Figure 5.4. Mice heterozygous for the EphA1 reporter allele with the PGK_neo cassette excised (+/PLAP) were mated to produce homozygous males and females, which were used to establish a stable breeding colony from the two separate ES cell lines (designated Ethan and Emery).

5.3.8 Northern blot analysis of EphA1 reporter knockout mice

Poly(A)+ mRNA was prepared from the livers of wild type (C57BL6) (EA1+/+), heterozygous (EA1+/PLAP) and homozygous EphA1 reporter (EA1PLAP/PLAP) mice derived from two distinct ES cell lines as described in Section 2.2.17b. At least 5 µg of poly(A)+ mRNA was run on a formaldehyde gel and transferred as described in Section 2.2.18.

The filter was sequentially probed with the Exon 3ab probe, hPLAP probe, and a β-actin probe. The hPLAP probe was prepared by XbaI digestion of the IRES-hPLAP plasmid, releasing the entire 1.8 kB IRES_hPLAP fragment. The β-actin probe was prepared

130 Queensland Institute of Medical Research Chapter 5: Generation and analysis of the EphA1 reporter mice from a standard laboratory plasmid. The filter was washed at 60°C with 2X SSPE/ 0.1% SDS and exposed to KODAK X-OMAT AR film at –80°C from overnight to seven nights. The filter was stripped between probes with boiling 0.1% SDS.

5.3.9 PCR genotyping of EphA1 reporter knockout mice

A strategy for genotyping the EphA1 reporter mice by PCR was devised with the primers listed in Table 5.1. All primers were designed using the Primer3 software. The expected size of the wild type (+/+) amplicon (Intron 1FX + Intron 2RX) was 315 bp and the knockout (EA1PLAP/PLAP) amplicon (Intron 1FX + IRES MCS 2RX) was 214 bp. The thermocycler was programmed with activation for 10 minutes at 95°C, followed by 30 step cycles of melting at 95°C for 30 seconds, annealing at 50°C for 30 seconds, then extension at 72 °C for 1 minute.

Table 5.1 Primers used for genotyping the EphA1 reporter mice

F12946 IRES-MCS-R R13260

Ex o n 15’ Exon 2 I RES- PLAP- p o l y A PGK- NEO 3’ Exon 2

loxP loxP

Primer Name Sequence (5’ – 3’)

Wild type mEphA1-F12946 GGAGTCCCAGAATACCCAGTG (Intron-1FX) (amplicon = 315 bp) mEphA1-R13260 CCCAGCTCCCTAGGAGTACC (Intron-2FX)

Knockout mEphA1-F12946 GGAGTCCCAGAATACCCAGTG (Intron-1FX) (amplicon = 160 bp) mEphA1-IRES-MCS- GGGCTGCAGGAATTCGATA R (IRESMCS-2RX)

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5.3.10 Quantitative PCR

First-strand cDNA synthesis was performed from poly(A)+ mRNA derived from mouse liver. The PCR reaction was performed as indicated in Section 2.2.19 with the following primer pairs:- EphA1, EphA2, ephrin A1, hPLAP and “housekeeping genes”, including β-Actin and HMBS to serve as controls. All primers were designed with the Primer3 software (Table 5.2).

Table 5.2 Primers used for Q-PCR

Primer Name Sequence (5’ – 3’)

EphA1_sense GTGGACACTGTCATAGGAGAAGG

EphA1_antisense GGTCTTAATGGCCACAGTCTTG

Ephrin A1_ sense CCGGAGAAGCTGTCTGAGAA

Ephrin A1_antisense GGTTTGGAGATGTAGTAGTAGCTGTG

EphA2_sense GGGACCTGATGCAGAACATC

EphA2_antisense AGTTGGTGCGGAGCCAGT

HMBS_sense TGCACGATCCTGAAACTCTG

HMBS_antisense TGCATGCTATCTGAGCCATC

β-Actin-F561 CACACTGTGCCCATCTACGA

β-Actin-R688 GTGGTGGTGAAGCTGTAGCC

5.3.11 EphA2 knockout mice

The EphA2 knockout mice were imported from the Animal Research Institute, Japan. The generation method and phenotype of this mouse have been described (Naruse- Nakajima et al., 2001). These mice were used for controls in the Q-PCR experiments.

132 Queensland Institute of Medical Research Chapter 5: Generation and analysis of the EphA1 reporter mice

5.3.12 Phenotypic analysis of the EphA1 reporter mice

EphA1 reporter mice with the PGK_NEO cassette deleted (EA1PLAP/PLAP) were identified by Southern blot analysis (Figure 5.4) and all results refer to these mice. The PLAP/PLAP EA1 mice were euthanised by asphyxiation in CO2, and the organs were removed. The tissue was either fixed in formalin and paraffin-embedded for haematoxylin and eosin (H&E) staining or frozen sections were cut and stained to identify hPLAP activity. (Section 2.3.2). Mouse tails were decalcified in 15% EDTA solution for 24 hours prior to paraffin embedding.

The health, welfare, sex ratio and fertility of the EphA1 reporter mice was monitored for more than two years. 5.4 Results

5.4.1 General comments

The EphA1 reporter mutant mice, both heterozygotes and homozygotes, have been observed for over two years, and several hundred mice (>300) have been examined and approximately 100 dissected for further examination. In general, EphA1 reporter null mice are physically normal in appearance (except for the kinky tail discussed later), viable and fertile (discussed in more detail below).

In a series of heterozygote matings which produced 104 pups, there were 57 males and 47 females (P = 0.21). The inheritance of the hPLAP mutant allele was determined in a cohort of 61 pups:- wild type +/+ (n = 13), heterozygote EA1+/PLAP (n = 36) and homozygous knockout EA1PLAP/PLAP (n = 12). These results confirm a normal Mendelian inheritance for the hPLAP mutant allele (Chi-squared test with 2 degrees of freedom P = 0.60). In conclusion the sex ratio of EphA1 reporter knockout mice is normal and the hPLAP reporter (mutant) allele is inherited in a normal Mendelian ratio.

The subsequent results described below refer to mice mated with Cre-deleter mice to remove the neomycin cassette and the strong PGK promoter.

5.4.2 Southern blot analysis of tail tip genomic DNA digests

Southern blot analysis was initially used as the method of genotyping during the generation of the EphA1 reporter mice. The results of a typical Southern blot analysis

Queensland Institute of Medical Research 133 RV 9.8kB RV RV 1.8kB 7.2kB

probe Exon 1 Exon 2 targeting construct RV Eco RV λH3 wt +/neo +/PLAP PLAP/PLAP

23 cross +/neo with Cre-deleter Lox P 9.4 6.6 IRES-hPLAP-bpA cassette

4.4 wt = 9.8kB PGK – neo cassette 2.3 +/-neo = 7.2 + 1.8 = 9.0kB 2.0 PLAP/PLAP = 7.2kB +/neo = +/PGK-neo-PLAP +/PLAP = following Cre-mediated excision

Figure 5.4 Diagram of Southern blot to genotype EphA1 reporter mice. Genomic DNA was digested with EcoRV and subjected to Southern hybridisation with a λ 1.1 SacI-excised probe. Band sizes are EA1wt 9.8kB, EA1+/neo 9.0kB and EA1PLAP/PLAP 7.2kB. - Cre Mating of EA1 +/neo male -/- +/- A: = heterozygote following +/- +/PLAP 1.1 Sac-Sac probe; B λ wt Cre-mediated PGK_neo cassette deletion. +/– = EA1 . 23 9.4 6.6 4.4 homozygote following PLAP/PLAP EA1 RVEco and the filter was probed with the +/- +/- EA1 heterozygote with PGK_neo cassette, wt wt +/neo : Mating of EA1 +/– male and female. neo B EA1 A = +/ +/neo 6.6 2.0 23 9.4 2.3 4.4 = wild type, wt with Cre-deleter female, mediated PGK_neo cassette deletion, –/– = Figure 5.5 Southern blot of EphA1 reporter tail tip genomic DNA Genomic tail tip DNA was digested with Chapter 5: Generation and analysis of the EphA1 reporter mice are shown in Figure 5.5. These results confirmed the calculated sizes of the bands. It was straightforward to differentiate between EphA1 heterozygous reporter cassette knocked-in (EA1+/–), EphA1 (EA1+/PLAP) heterozygous Cre-deleted (with the PGK-neo cassette removed) and EphA1 reporter null (EA1PLAP/PLAP) alleles.

5.4.3 Genotyping the EphA1 reporter mutant mice by PCR

The PCR was undertaken as described in Chapter 2.2.14. The following primer pairs according to Genbank AC153915, Mus musculus 10 BAC RP23-329D23 were designed for testing (Table 5.1).

The PCR genotyping strategy was successful with the wild type band (Intron 1FX + Intron 2RX) of the expected size (315 bp) and the reporter knockout band (EA1PLAP/PLAP) (Intron 1FX + IRESMCS 2RX) also of the expected size (214 bp) (Figure 5.6). This will facilitate genotyping when the mixed background (129 s/v X C57Bl/6) mice are crossed onto a C57 Bl/6 background for future analysis.

1 2 3 4 5

315bp EA1 +/+ 187bp EA1PLAP/PLAP

Figure 5.6 PCR genotyping EphA1 reporter KO mice. Duplex PCR (after 35 cycles). Lane 1: 100 bp Ladder; Lane 2: Neg control; Lane 3: wt control C57/Bl6; Lane 4: EA1+/PLAP; Lane 5: EA1PLAP/PLAP.

5.4.4 Northern blot results

The Northern blot analysis of liver RNA confirmed that EphA1 reporter null mice (–/–) do not produce the previously described 3.2 kB EphA1 transcript, but in fact have switched transcription to the hPLAP mRNA (Figure 5.7). The heterozygote mice (+/–) produced an intermediate amount of both EphA1 and hPLAP transcript. Although not indicated in the figure, the size of the RNA bands for EphA1, hPLAP and β-actin, when compared with the

136 Queensland Institute of Medical Research Chapter 5: Generation and analysis of the EphA1 reporter mice positions of the 18S and 28S ribosomal bands, were consistent with previous results from this laboratory. wt +/- -/-

28S (a) Exon 3ab 18S

28S (b) 18S hPLAP

28S

(c) 18S β-Actin

Figure 5.7 Northern blot. poly(A)+ mRNA was extracted from adult mouse liver of wild type (+/+), heterozygote (+/–) and knockout mice (–/– = EA1PLAP/PLAP) and 5 µg/lane was loaded. Hybridisation with 32P labelled probes (a) Exon 3ab (672 bp), (b) hPLAP (1.8 kB) and (c) β-actin (540 bp) as indicated ; wt = wild type, +/– = EA1+/PLAP EA1 heterozygote with deleted PGK_neo cassette, –/– = EA1PLAP/PLAP homozygote with Cre deleted PGK_neo cassette.

5.4.5 Quantitative PCR

The results of the Q-PCR from mRNA extracted from liver are shown in Figure 5.8. These results confirm the previous findings of Northern blot analysis that the EphA1 reporter homozygous mice (EA1PLAP/PLAP) were in fact null mice and there was background transcription only. These results also indicate as expected that the heterozgyote (EA1+/PLAP) mice transcribe approximately half the amount of hPLAP RNA as the null mice (EA1PLAP/PLAP). The “housekeeping genes”, β-actin and hydroxymethybilane synthase, (data shown for β-actin only) and EphA2 null mice were used as positive and negative controls respectively for the Q-PCR in separate

Queensland Institute of Medical Research 137 Chapter 5: Generation and analysis of the EphA1 reporter mice experiments. The two-fold difference between hPLAP and EphA1 message is not considered significant in Q-PCR in the small sample (n = 2) studied. (This finding may be due to differences in the stability of the relevant transcripts.) The difference in the quantity of EphA1 message relative to β-actin is within the limits of experimental tolerance for Q-PCR. This experiment was performed to confirm that the EA1PLAP/PLAP mice were null mice.

125 EphA1R KO EphA1R Ht

-actin) 100 Wild-type β

75

50

25

rncitnme (per 1000 number Transcript 0

1 2 1 P A A A A h h n L p p ri P E E h ep

Figure 5.8 Quantitative real-time PCR. Eph/ephrin expression in liver tissue in EphA1 reporter KO (= EA1PLAP/PLAP), heterozygote (= EA1+/PLAP) and wild type mice.

5.4.6 Haematoxylin and eosin stained paraffin sections

H&E stained paraffin sections were prepared from all major organs from adult male and female wild type, heterozygotes and null mice, and were examined by an experienced pathologist (Dr Gael Phillips). The tissues examined included liver, gall bladder, spleen, kidney, pancreas, oesophagus, stomach, small intestine, colon, coagulation gland, testes, ovary, thymus, lymph node, heart, lung and brain. No specific abnormalities of tissue architecture were identified in these organs (data not shown).

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5.4.7 Immunohistochemistry

The paraffin embedded tissue sections were stained with an hPLAP monoclonal antibody [Dako] (see Section 2.3.4) and the protocol optimised including the incorporation of an antigen retrieval step. However, there was always significant background staining in the wild type tissues (skin, kidney, small intestine). This work is still in progress and the method requires further optimisation; consequently the results will not be reported in this thesis.

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A B

C D

E F

G H

Figure 5.9 hPLAP staining of frozen section mouse tissues. (wt = wild type, null = EA1PLAP/PLAP). (A) wt, (B) null tail skin; (C) wt, (D) null back skin; (E) wt, (F) null vagina; (G) wt, (H) null renal pelvis. Magnification X 80. Scale bars 100 µm.

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5.4.8 hPLAP expression

All the major organs were collected from a total of 40 mice, including heterozygotes and homozygous knockout adult mice between the ages of 3 months and 12 months which included a similar numbers of males and females. The frozen sections were stained to detect EphA1 expression through hPLAP reporter activity. The results showed functional hPLAP activity, hence EphA1 expression, in skin, kidney, small intestine, uterus and vagina. As there was very strong hPLAP reporter activity in skin, frozen sections of skin were used as positive controls in all subsequent experiments. There was no expression detected in liver, gall bladder, stomach, spleen, lymph nodes, bone marrow, colon, brain, heart or lungs with this technique.

In Figure 5.9, hPLAP staining of tissue sections of skin, vagina and renal pelvis at X 80 magnification are shown for wild type mice (A, C, E, G) and null mice (= EA1PLAP/PLAP) (B, D, F, H). The frozen sections of uterus and small intestine were of sufficient quality to allow preliminary confirmation of hPLAP reporter activity; however, tissue morphology was poorly preserved and these sections are not shown. The sections of skin indicate that EphA1 is widely expressed in the skin and is associated with hair follicles. The EphA1-hPLAP expression in the renal pelvis was previously not reported and expression in the vagina was also unexpected.

To improve the morphology, the tissues were paraffin embedded prior to staining. However, paraffin embedded tissue sections did not retain hPLAP reporter activity despite optimising the protocol and lengthening the duration of the incubation step with the BM purple substrate. The HOPE fixation method (see Section 2.3.3) was subsequently used in an attempt to improve the morphology of tissues and retain hPLAP reporter activity. However, despite great care with this technique, several repetitions of the protocol and discussions with the manufacturer, hPLAP reporter activity was not preserved in the HOPE fixed tissues sections.

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wt EA1 PLAP/PLAP

Figure 5.10 Kinky tail phenotype in EphA1 reporter null mice. The EphA1 null (EA1PLAP/PLAP) mice have a variable kink in the distal tail (Magnification X3).

wt EA1PLAP/PLAP EA1PLAP/PLAP

Figure 5.11 Haematoxylin and eosin stained sections of the tail. There is considerable variation in the severity of the kinky tail (Magnification X20).

+ EA1 /PLAP

EA1PLAP/PLAP

Figure 5.12 Plain radiographs of the EphA1 reporter mice. EphA1 heterozygote (EA1+/PLAP) and EphA1 null (EA1PLAP/PLAP).

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5.4.9 Developmental defects in the tail

The EphA1 reporter null mice demonstrated abnormalities in tail development. This was manifested by a range of malformations from slight kinks to severe perturbations in the positioning of the most caudal vertebrae (Figure 5.10). The characteristics of the tail malformations were similar in mice derived from both ES cell lines; however, there was a difference in the penetrance. The “Ethan” derived ES cell line mice had a kinky tail penetrance of ~80%, whereas the “Emery” derived mice displayed a penetrance of ~50% from a total of ~300 mice examined.

The H&E sections of decalcified tails confirm the disorganisation of the caudal tail vertebrae (Figure 5.11). There were no abnormalities in tail formation observed in either heterozygous or wild type mice which resulted from the heterozygous matings which were used to produce the homozygous EA1PLAP/PLAP mice. There were no gross bony defects noted in other skeletal elements; this was confirmed by radiographs (Figure 5.12) and suggests that the kinky tail phenomenon in the EphA1 reporter null mice is due to the specific role of EphA1 in the formation of the most caudal tail structures.

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wt EA1 PLAP/PLAP

A B

C D

R R

V U

E F

Figure 5.13 Imperforate vagina in EphA1 reporter null mice. Appearance of wild type and EA1PLAP/PLAP (null) mice affected with the genital tract phenotype. (A) Wild type mouse with a normal vaginal (V) entrance as indicated by the arrow. (B) Appearance of an imperforate vagina (V). Note the swollen vaginal region (arrow). (C) Dissected reproductive tract of a wild type mouse with a normal uterus and vagina (V), rectum (R). (D) Dissected reproductive tract of an EA1PLAP/PLAP mouse with a fluid filled distended uterus with closed vagina. (E) H&E normal vagina in transverse section (F) imperforate vagina resulting in distended uterus (U) (Magnification X20).

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5.4.10 Imperforate vagina phenotype of the EphA1 reporter null mice

On continued analysis some mice were noted to have a defect in the development of the female genital tract (Figure 5.13). A failure of the normal process of vaginal opening, or the imperforate vagina phenotype, was observed in ~18% of EphA1 reporter null females (n >100) which was not seen in heterozygous or wild type litter mates. The imperforate vagina phenotype was observed in mice derived from both ES cell lines. The affected female mice were infertile and had a completely closed vaginal entrance, which could not be probed. In some cases, the mice developed a swollen abdomen due to the enlarged uterus distended by the accumulation of clear intrauterine fluid, a condition termed hydrometrocolpos. There was no apparent increase in mortality associated with the imperforate vagina phenotype and affected mice did not display any obvious signs of discomfort. The abnormality was found at a similar frequency in females from breeding stock housed with males and females housed alone.

5.5 VDiscussion V This chapter describes the generation of a reporter mutant mouse in which EphA1 has been knocked out by the introduction of an IRES-hPLAP cassette into Exon 2 of the mouse EphA1 gene. This prevents production of a functional EphA1 mRNA and it was confirmed that the EphA1 promoter drives hPLAP expression through the IRES cassette. In the adult mice, the previously reported expression of EphA1 in skin, liver, thymus and kidney and the expression pattern of hPLAP largely corresponded to this pattern (Coulthard et al., 2001). The hPLAP expression in the skin was the most striking, and expression was strongest in the basal layers of the epidermis and was also associated with the deeper structures of the hair follicles. In addition EphA1-hPLAP expression was demonstrated in small intestine, uterus and vagina, which was previously unknown. Regrettably hPLAP activity could not be detected in paraffin embedded tissues, which suggests that the hPLAP enzyme function is lost during the processes of fixation and paraffin embedding. Further attempts to detect hPLAP immunohistochemically using an anti-hPLAP monoclonal antibody from a single manufacturer [Dako] were unsuccessful. The loss of hPLAP enzyme activity during fixation must be considered by other investigators in future, who may be choosing between the hPLAP and the β-galactosidase reporter enzyme systems. However, the use

Queensland Institute of Medical Research 145 Chapter 5: Generation and analysis of the EphA1 reporter mice of an EphA1 polyclonal hPLAP antibody may prove to be successful and this approach will be attempted in future experiments.

The technique of Q-PCR detected hPLAP transcription in both liver (Figure 5.8) and kidney (data not shown); however, the BM purple staining was not sufficiently sensitive to detect hPLAP enzyme activity visually in either liver or kidney. Nevertheless, the simplicity of the staining technique provides a major advantage in detecting EphA1 expression. The previously reported expression of EphA1 in the corticomedullary region of the thymus could not be confirmed by this technique because there was staining in thymic medullary cells of both wild type and null mice. The reasons for this are unclear, but may imply unexpected hPLAP expression in thymic stromal elements.

However, the EphA1 reporter null mice have an identifiable physical phenotype. A high proportion (~ 20–80%, dependent on the derived ES cell line) of the knockout mice displayed the kinky tail phenotype (discussed in more detail later) and nearly 20% of null females demonstrated the imperforate vagina phenotype (also discussed in more detail later). These obvious phenotypic differences allow us to concentrate future research efforts investigating the function of EphA1 towards a putative role in caudal tail formation and the opening of the vaginal entrance.

The analysis of mutant mice (genetic studies) has been extremely valuable in accumulating the current body of knowledge about Eph/ephrin receptor-ligand function and signalling mechanisms (Blits-Huizinga et al., 2004), and some of the subtleties and complexities of Eph/ephrin protein signalling have been elucidated in genetic studies in mice. Functional analysis of the Eph/ephrin system is challenging, firstly, because of the overlapping functions such that loss of one receptor can be partially compensated by another Eph receptor with a similar expression pattern and ligand binding specificity and, secondly, because the Eph/ephrin proteins display bi-directional signalling and can act as both a ligand and as a receptor.

The analysis of the EphB2 and EphB3 knockout mice gave the first clues that the Eph/ephrin proteins had both a repulsive and adhesive function (Henkemeyer et al., 1996; Orioli et al., 1996). Further studies of knockout mice established that Eph receptors had both kinase-dependent and kinase-independent functions and confirmed bi-directional signalling (Kullander et al., 2001b). In another example, it was determined that the cytoplasmic domain of ephrin-B2 participates in bi-directional

146 Queensland Institute of Medical Research Chapter 5: Generation and analysis of the EphA1 reporter mice signalling and is required for vascular development, but not for the migrating cranial neural crest cells (Adams et al., 2001).

In other instances, the phenotype of a gene knockout mouse has correlated with a particular human phenotype, which has led to identifying mutations in the human orthologue as the cause of the human phenotype. The phenotype of the ephrin-B1 knockout mice (Compagni et al., 2003) led to the suggestion that craniofrontonasal syndrome (CFNS, MIM 304110) in humans was due to mutations in EFNB1 which has since been confirmed (Twigg et al., 2004; Wieland et al., 2004). The biological functions of Eph/ephrins as presumed from the study of gene knockout mice has confirmed much earlier in vitro receptor-ligand binding analysis (Blits-Huizinga et al., 2004). For example, the binding affinity of EphA4 and ephrin-B3 is low to moderate (Kd >6 nM) (Bergemann et al., 1998; Gale et al., 1996). The EphA4 knockout mice display a hopping gait due to midline guidance defects of the central pattern generator neurons in the spinal cord, and the ephrin-B3 knockout mice have a very similar phenotype (Kullander et al., 2003). The binding studies indicate that EphB4 binds only with ephrin-B2. The EphB4 and ephrin-B2 knockout mice have an almost identical cardiovascular phenotype (Gerety et al., 1999). The development of gene knockout mice has allowed the development of mouse models that mimic human diseases, with the implication that the model system can be used to investigate novel methods of diagnosis and treatment (Bockamp et al., 2002).

Following neurulation, in situ hybridisation detects EphA1 expression in the tail bud mesenchyme and the posterior presomitic mesoderm (Duffy et al., 2006). In fact, EphA1, ephrin-A1 and ephrin-A3 form an expression domain in the presomitic tail mesoderm, which is mutually exclusive of EphA2, which is expressed at the tip of the tail notochord (Naruse-Nakajima et al., 2001). We speculate that the balance of adhesive signalling between EphA1 and ephrin-A1/ephrin-A3 and the repulsive signalling between EphA2 and ephrin-A1/ephrin-A3 is responsible for cell movement in the tailbud.

In developing mouse models of human disease, the genetic background needs to be carefully considered (Brown et al., 2006). The gold standard for comparing the phenotype of a knockout mouse is with a genetically identical animal. In general, six to 10 generations of backcrosses onto a defined strain are required to achieve 99%

Queensland Institute of Medical Research 147 Chapter 5: Generation and analysis of the EphA1 reporter mice background homogeneity (Joyner, 2000). A targeted mutation of the epidermal growth factor receptor (Egfr) results in three non-overlapping phenotypes depending on the genetic background:- (1) peri-implantation lethality on a 129/Sv-CF-1 background; (2) mid-gestation lethality on an inbred 129/Sv background and (3) peri-natal lethality on a 129/Sv-CD-1 or C57BL/6J background (Strunk et al., 2004; Threadgill et al., 1995). Changing the genetic backgrounds can also have a profound effect on the penetrance of the phenotype. The cystic fibrosis transmembrane regulator (Cftr)-deficient mice also fall into three distinct phenotypes:- (1) lethality within 10 days of birth (129/Sv- DBA/2J); (2) lethality around weaning (129/Sv-CD1, C57Nl/J6, or BALB/cJ) or (3) prolonged survival beyond six weeks of age (Joyner, 2000). In EphA4 knockout mice on a mixed C57BL6/DBA background backcrossed for four generations onto a C57BL/6 background, the thymic hypoplasia phenotype was less penetrant (Munoz et al., 2006). Similarly EphA2 mutant mice on a 129/Svj and C57BL/6J mixed background displayed a kinky tail phenotype (Naruse-Nakajima et al., 2001); however, EphA2 mutant mice on a C57BL/6J background had no observed phenotype (Chen et al., 1996).

In fact, backcrossing the EphA2 mutant kinky tail mice for four generations on to C57BL/6J alleviated the severity of the phenotype (Naruse-Nakajima et al., 2001). The ephrin-B2∆V/∆V mice (terminal valine of the PDZ domain deleted to prevent interaction with PDZ proteins) on a C57BL/6 background die at perinatal day 6 of massive chylothorax; however, on a CD1 background, the ephrin-B2∆V/∆V mice survive for several months and show relatively minor lymphatic defects (Makinen et al., 2005). The presence of phenotypic variation in mutant mice with different genetic backgrounds allows the opportunity to map and clone modifier genes (Rozmahel et al., 1996).

There are approximately 100 currently catalogued mutant genes and polymorphic loci responsible for abnormalities of the mouse tail (Doolittle et al., 1996). Some of the many candidate genes responsible for tail formation have been elucidated from the results of insertional mutagenesis programs and the outcome of homologous recombination techniques, because the genes responsible for the mutant tail phenotypes are being mapped (Schrick et al., 1995). In mutant mice homozygous for Brachury (T), the embryos die at around embryonic day 10 and display abnormalities of mesodermal structures including the notochord and somites. The heterozygous mice (T/+) have malformed sacral vertebrae and varying reductions in tail length with a kink (Willison,

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1990). Mice with a targeted disruption of int-2 (Fgf-3) do not survive to adulthood, and homozygous mutant mice display defects of development of the tail and inner ear. The shortened, kinky tail phenotype has a 100% penetrance (Mansour et al., 1993). Mice heterozygous for a targeted disruption of Wnt-3a also have shortened tails (Takada et al., 1994). The mouse Fused (Fu) locus was identified over 60 years ago as a result of a dominant mutation with a kinky tail phenotype (Vasicek et al., 1997). The Tss (Tail- short Shionogi) mutation found in BALB/CMs mice maps closely to the previously identified Ts locus on mouse chromosome 11 (Tsukahara et al., 2000). A selective breeding program for aggressive behaviours generated a mutant mouse with a short trunk and kinky tail. This phenotype is due to a substitution of Cys
Gly in the delta- like 3 (Dll3) gene (Shinkai et al., 2004). The mouse Fu locus encodes axin, an inhibitor of the Wnt signalling pathway, and thus regulates embryonic axis formation (Zeng et al., 1997). Recently increased DNA methylation at the AXIN1 gene in a monozygotic twin for a pair discordant for a caudal duplication anomaly was reported (Oates et al., 2006). These examples and other results of the analysis of mutant mice with obvious physical phenotypes, like a kinky tail, will continue to be fruitful in the search for other genetic causes of congenital malformations in humans, as it already has in the clinical presentation of congenital and idiopathic scoliosis (Giampietro et al., 2003).

The vagina of the mouse is closed at birth and does not open until the female is 24 to 28 days of age around the onset of sexual maturity, and can be induced in sexually immature females by β-oestradiol injections (Maronpot et al., 1999; Rugh, 1968). The imperforate vagina or septated vagina phenotype in mice has been described as an incidental finding in inbred strains of laboratory mice (Cunliffe-Beamer and Feldman, 1976; Sundberg and Brown, 1994). However, more recently, the imperforate vagina (ipv) phenotype has been described as an autosomal recessive mutation with high penetrance in a line of mice selected for low lean tissue mass and has also been observed in several strains of inbred laboratory mice including loop-tail (Lp), dominant hemimelia (Dh) and cloud gray (Slcg), an allele at the steel locus (a ligand for c-kit) (Eisen et al., 1989). The degree of penetrance of the imperforate vagina phenotype in these latter three mutants varied from low frequency to about one-third. The presence of imperforate vagina leads to accumulation of secretions resulting in dilatation of the vagina and uterus, termed mucometra, and marked distension of the abdomen and perineum with infertility (Eisen et al., 1989).

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The Jackson laboratory has records of 44 cases of imperforate vagina (http://jaxmice.jax.org/library/notes/JaxmiceNotes_1_8597_8597.html) with mucometra submitted between 1987 and 1989. The condition is therefore quite rare in female mice, and all the affected mice displayed extreme distension of the abdomen (Sundberg and Brown, 1994). The precise mechanism of the formation of the mouse vagina remains the subject of investigation. It has previously been thought that the outer two-fifths of the mouse vagina formed from the urogenital sinus and the inner three-fifths of the vagina formed from the fusion of the paired Müllerian ducts (Forsberg and Olivecrona, 1965). However, studies of mice with androgen receptor insensitivity (Drews, 1975) suggested that the vagina is formed from a downgrowth of both Wolffian and Müllerian ducts (Mauch et al., 1985).

Embryogenesis is characterised by the remodelling of numerous tissues and involves programmed cell death or apoptosis, for example, interdigital tissue regression, palate fusion and central nervous system formation (Gilbert, 2003). In the mouse, some definitive tissue remodelling, which occurs only after birth, includes opening of the eyes, the ears and the vaginal cavity. In transgenic mice overexpressing the apoptosis inhibitor protein Bcl2, the eyes and ears open, but not the vagina (Rodriguez et al., 1997). Analysis of vaginal tissue by the TUNEL method (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling) (Gavrieli et al., 1992) confirmed apoptosis in the area of the vaginal opening in the wild type but not in the Bcl2 transgenic mice. This finding suggests that mouse vaginal cavity development at the time of sexual maturity is a hormone triggered apoptotic event (Rodriguez et al., 1997). In Drosophila, ovo is expressed in all germ cells and is required for female sex differentiation and oogenesis. The movo1 null mice display abnormalities in hair formation, defects in spermatogenesis and 60% of females have an imperforate vagina (Dai et al., 1998). The deletion of the pro-apoptotic genes Bak and Bax on a 129 X C57Bl/6 background results in all females with imperforate vagina (Lindsten et al., 2000). The follicle-stimulating hormone receptor null mice have low circulating levels of oestrogen, with elevated circulating levels of both follicle-stimulating hormone and luteinising hormone. The females are infertile with smaller ovaries, atretic uterus and imperforate vagina secondary to a lack of oestrogen (Abel et al., 2000). A proportion of the glypican-3 deficient mice, which exhibit overgrowth, perinatal death, cystic dysplastic kidneys, abnormal lung development and mandibular hypoplasia, also display

150 Queensland Institute of Medical Research Chapter 5: Generation and analysis of the EphA1 reporter mice an imperforate vagina (Cano-Gauci et al., 1999). Glypican-3 is a heparan sulfate proteoglycan, which negatively regulates insulin-like growth factor II, induces apoptosis in vitro (Gonzalez et al., 1998) and is mutated in patients with the Simpson-Golabi- Behmel syndrome (OMIM #312870). The MMTV-trBrca1 mice display defective mammary gland development on a C57Bl/6 x CBA background with no other defects; however, there is failure of vaginal opening (11.5%) on a C57Bl/6 x CBA x BALB/C background. BRCA1 is implicated in the regulation of apoptosis in fibroblasts and tumour-derived epithelial cells (Simpson et al., 2004).

Although the imperforate vagina phenotype in mice has been described as an incidental finding in inbred strains of laboratory mice (Cunliffe-Beamer and Feldman, 1976; Sundberg and Brown, 1994), and is associated with recessive inheritance (ipv) and several other phenotypes, the imperforate vagina phenotype is specifically associated with defects in hormone, hormone receptor and apoptotic pathways, (Abel et al., 2000; Cano-Gauci et al., 1999; Rodriguez et al., 1997; Simpson et al., 2004). The high incidence with which the EphA1 knockout mice display the imperforate vagina phenotype suggests that EphA1 does play a specific role in the pathway responsible for opening of the vaginal cavity in mice.

The human correlation of the mouse imperforate vagina phenotype is not immediately obvious, although imperforate hymen, transverse vaginal septum (as part of McKusick- Kaufman syndrome) and congenital absence of the vagina and uterus (Mayer- Rokitansky-Kuster-Hauser syndrome) all result in vaginal and uterine dilatation with infertility (Creighton, 2005). The McKusick-Kaufman syndrome [OMIM #236700] comprises polydactyly, congenital heart disease and hydrometrocolpos due to a vaginal septum. This syndrome is diagnosed most frequently in the Old Order Amish population, but physical and gene mapping has not yet identified the candidate gene, although all affected individuals were homozygous for the 20p12 region, which includes the jagged1 gene (Stone et al., 1998). The possible mouse model, ipv (imperforate vagina), has not yet been mapped but the candidate gene is neither the loop tail (Lp) nor dominant hemimelia (Dh) genes, which both map to chromosome 1 (Eisen et al., 1989). The Mayer-Rokitansky-Kuster-Hauser syndrome [OMIM #277000], which affects approximately 1 in 4,500 women, is characterised by congenital absence of the upper part of the vagina and uterus, renal abnormalities and skeletal malformations, and may be due to a mutation in the Wnt4 gene or the HOX genes (Guerrier et al., 2006).

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The congenital abnormality of the human female reproductive tract, imperforate hymen, occurs in 1 in 1,000 live female births, and usually presents around puberty with cyclical abdominal pain and amenorrhoea, but can present in the neonatal period as an abdominal mass (Shaw et al., 1983). Imperforate hymen may be difficult to diagnose and may result in serious morbidity and infertility (Bajaj et al., 2006). The condition is most commonly sporadic although some familial cases have been reported (Lim et al., 2003; McIlroy and Ward, 1930; Stelling et al., 2000; Usta et al., 1993). An imperforate hymen may be an isolated anomaly in an otherwise normal female, although it is had been observed with other genitourinary tract developmental abnormalities, including hand-foot-uterus syndrome, caused by a mutation in the HOXA13 gene [OMIM #140000] (Mortlock and Innis, 1997), and ulnar-mammary syndrome, due to a mutation in TBX3 [OMIM #181450] (Bamshad et al., 1997).

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Chapter 6: Generation and analysis of the EphA1 conditional knockout mice

6 THE GENERATION OF EPHA1 CONDITIONAL

KNOCKOUT MICE

6.1 Introduction

6.1.1 General comments

The analysis of gene function by reverse genetics or “knocking out” the gene of interest and examining the resulting phenotype to imply the function of the gene will occasionally produce an embryonic lethal phenotype. Although this would suggest that the gene of interest has an extremely important developmental function, it does not allow complete characterisation of gene function in the adult. Subsequently, methods of removing the gene function conditionally, or at the discretion of the investigator, in a tissue specific manner have been developed. This allows the gene of interest to function normally in the embryo, then following birth the investigator can “knock out” or turn off the gene of interest in order to analyse gene function in the intact adult animal (Lewandoski, 2001).

The Cre-loxP system, which was discussed earlier, allows the removal of the sequence flanked (“floxed”) by loxP sites in the same orientation, when the genetically modified mouse with loxP sites is crossed with a mouse strain expressing the Cre recombinase. As described in the previous chapter, this allows the removal of the antibiotic selection cassette and its associated strong promoter which could potentially influence the expression of the targeted gene, or the expression of nearby genes, and hence influence the phenotype of the mutant mouse. Similarly the Flp-e system allows the removal of sequence flanked by FRT (flp recognition target) sites in the same orientation when the mouse with FRT sites is crossed with a deleter strain expressing the Flp-e recombinase. The incorporation of both site-specific sequences in the same orientation into a targeting construct allows the flexibility of removing the antibiotic selection cassette as a separate step in the process of silencing the gene under investigation. This gives the scientist more confidence that the mutant phenotype is only due to removal of the gene of interest and is not due to some perturbation in the actual process of generating the genetically modified mouse itself (Dymecki, 2000).

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6.1.2 The Flp-e FRT system

The Flp recombinase is derived from Saccharomyces cerevisiae and recognises a 34 bp target sequence with an 8 bp spacer, which determines orientation (Figure 6.1). The activity of the Flp recombinase at 37°C has been improved by changing four amino acids in the recombinase (hence Flp-e) (Dymecki, 2000).

5' G A A G T T C C T A T T C T C T A G A A A G T A T A G G A A C T T C 3'

3' C T T C A A G G A T T A G A G A T C T T A C A T A T C C T T G A A G 5'

Figure 6.1 Structure of the 34 bp FRT site. There are two inverted repeats 13 bp in length separated by an 8 bp spacer which determines the orientation. 6.2 Aims

(1) Construct a replacement type gene-targeting vector.

(2) Design a method of detecting homologous recombination by Southern blot and PCR.

(3) Generate EphA1 conditional knockout mice. 6.3 Materials and methods

6.3.1 Generation of the EphA1 conditional targeting construct

A replacement type targeting vector was constructed using a multi-step cloning strategy such that Exon 3ab, the ligand binding domain of the endogenous mEphA1, would be replaced and, following Flp-e mediated excision of the PGK_neo cassette, the replacement Exon 3ab would be flanked by loxP sites in the same orientation. This would allow Cre recombinase mediated excision of the ligand binding domain and the subsequent creation of a null allele in the whole mouse when crossed with a Cre-deleter strain or tissue specific removal of Exon 3ab, following crossing with conditional Cre- deleter mouse strains. This strategy was chosen as the laboratory had previous experience with generating a knockout mouse in which the ligand binding domain was the target.

Prior to embarking on the multi-step cloning strategy, the plasmids containing the fragments of genomic DNA were test digested with all enzymes to be used in the

156 Queensland Institute of Medical Research Chapter 6: Generation and analysis of the EphA1 conditional knockout mice strategy. At each step of the cloning strategy, success was confirmed by restriction digestion and direct sequencing. Appropriate clones were expanded and plasmid DNA was extracted by a Qiagen Midi-preparation [Qiagen]. As the targeting construct was very large (> 10 kB) the E.coli strain XL-10 GOLD was preferred for transformation using a heat shock method.

The initial step involved preparation of a pBst plasmid in which the standard multiple cloning site was replaced by a multiple cloning site that contained the following restriction sites:- KpnI, SalI, NdeI, XbaI, XhoI, NotI, SacI. This was achieved by double digestion of pBluescript (KS-) [Stratagene] with KpnI and SacI and inserting the polylinker below. The polylinker was prepared by annealing two oligonucleotides:-

Forward: 5’ –GG GGTACC GTCGAC ATATG TCTAGA CTCGAG–3’

Reverse:5’ – GG GAGCTC GCGGCCGC TCGAG TCTAGA CAT ATG–3’

The oligonucleotides were resuspended in Milli-Q water and annealed at 50°C, then digested with both KpnI and SacI to expose the restriction sites and ligated into the KpnI/SacI double digested pBst (KS-).

The genomic fragment containing Exon 3ab (middle fragment) was moved into the modified pBst_Linker (KS-). The plasmid pBst λ1.2_Exon 3ab, which carries a 3.1 kB XbaI genomic fragment which includes Exon 3ab, was XbaI digested then ligated into XbaI digested pBst_Linker (KS-) and the orientation of the insert checked by digestion with NdeI.

A XhoI _loxP_BamHI NotI linker was designed, annealed, phosphorylated then ligated into XhoI/NotI digested pBst (KS-) Linker λ1.2 Exon 3ab. (The 8 bp spacer which gives the loxP site direction is shown in red.) A BamHI digest was used to check for recombinant clones (Figure 6.2).

Forward 5’– CTCGAG ATAACTTCGTATA GCATACAT TATACGAAGTTAT GGATCC ATAAGAAT GC – 3’

Reverse 5’– GGCCGC ATTCTTAT GGATCC ATAACTTCGTATA ATGTATGC TATACGAAGTTAT C – 3’

Queensland Institute of Medical Research 157 Not I Spe I Xba I Xba I

BamH I 3’ fragment λBst1.2 (3’ fragment) Hind III Hind Kpn I Pst I Hind III Hind Xba I Nde I EcoRV Xba I Sac I Sac Kpn I Xho I Sph I BamH I Xho I I Sac Xba I III Hind Sac I Xba I III Hind Nde I Sal I BamH I Not I Not F F lox:p transcription FRT2-PGK-neo pBstλ1.2-LExon3 (middle fragment) Sal I Xho I I Not Kpn I Nde I Xba I Sac I

pBST-Kpn-Sac_LKA

Figure 6.2 The construction of the targeting vector – middle and 3' fragments of the targeting vector. The multiple cloning steps are described in detail in the text. (Genomic sequence is denoted by bold lines, not drawn to scale). Chapter 6: Generation and analysis of the EphA1 conditional knockout mice

The 3’ genomic fragment of the replacement vector was released by sequential digestion with BamHI then NotI from pBst (KS-)_λ1.2, and ligated into BamHI/NotI digested pBst (KS-) Linker_ λ1.2 Exon 3_ loxP from the previous cloning step.

The pBst (λ1.2 Exon_loxP_XhoI_NotI_3’ fragment) plasmid was partially NdeI digested to specifically open the NdeI site in the linker, because there is also an NdeI site in the λ1.2 genomic fragment, then SalI digested to make the SalI and NdeI sites in the linker available (Figure 6.2). The recombinant clones were test digested with SpeI.

The plasmid pBst_FRT2_PGK_neo which has FRT sites flanking the PGK_neo cassette was provided by Dr Ian Tonks (QIMR). The SalI/NdeI fragment which includes the FRT sites and PGK_neo cassette was ligated into the SalI/NdeI digested pBst (λ1.2 Exon_ loxP_ XhoI_ NotI_3’ fragment) from above.

The plasmid pBst loxP:KS3 (provided by Dr Ian Tonks QIMR) contains a modified multiple cloning site into which the loxP sequence has been ligated (Figure 6.3). An additional NotI site was inserted into the multiple cloning site by preparing a BamHI_NotI_KpnI linker (see below) which was ligated into KpnI, then BamHI sequentially digested plasmid (pBst loxP:KS3).

Forward 5’ – GG GGATCC GCGGCCGC GGTACC – 3’

Reverse 5’ – GG GGTACC GCGGCCGC GGATCC – 3’

The BamHI_NotI_KpnI linker was annealed and sequentially digested with KpnI then BamHI to expose the restriction sites (Figure 6.3).

The modified plasmid pBst loxP:KS3 Bam_Not_Kpn was digested sequential with XbaI then SacI, then the 5’ genomic fragment was released from pBst λ1.1 by XbaI, then SacI sequential digestion was ligated into the plasmid (Figure 6.3). The recombinant clones were test digested with HindIII to check for insert.

In the final cloning step, the plasmid pBst λ1.1 loxP:KS3_BamHI_NotI_KpnI containing the 5’ genomic fragment was digested with SalI and NotI. The plasmid pBst Linker FRT2_PGK_neo_λ1.2 (middle genomic fragment)_loxP_λ1.3 (3’ genomic fragment) was SalI and NotI digested and the ~8 kB fragment released was ligated into

Queensland Institute of Medical Research 159 Xba I Nsp I Bgl II Nde I Hind III Pvu II Sac I Not I pBst- λ1.1 BamH I Kpn I (5’ fragment) Kpn I Not I Apa I Sac II BstX I Xho I I Sal BamH I Pst I Sac I Hind III Xba I lox:p

lox p:KS3

Figure 6.3 The construction of the targeting vector – the 5' fragment of the targeting vector. The construction of the targeting vector was a multiple step process described in the text. (Genomic sequence is denoted by bold lines, not drawn to scale). I ba I BamHI Not I Not I Nsp I Spe I Xba I Bgl II Pvu II Xba I Xba I Hind III Hind Sac I Sac I Xba I Hind III Sac I X III Hind Nde I BamH I Xho I Kpn I III Hind Nde I Pst I EcoRV Sac II BamH I Sph I Xho I Kpn I Nde I (Nco I) Sal I Sal SpeI BamH I Not F F lox:p

pBst- λ1.1 λpBst1.2 FRT2-PGK-neo λpBst1.2-Lexon3 5’ fragment 3’ fragment Exon 3 I BamH I Xho I Hind III Hind Sac I Pst I Xba I Kpn I Sal lox:p Sal I Kpn I Not I Sac I Nde I Xho I Xba I lox p:KS3 pBstλ1.1

pBst-Kpn-Sac_LKA

Digest lox:p KS3- pBsλ1.1 with SalI/Not I Digest pBst-Kpn-Sac_LKA - FRT2 PGK-neo- λpBst1.2-Lexon3-pBstλ1.2 with Sal I/Not I then ligate that fragment into lox p:KS3 pBstλ1.1 Linearize the vector with Not I Probe for recombinant clones following Xho I digest

Figure 6.4 The final step of construction of the targeting vector. The two plasmids from above were SalI-NotI digested then ligated. The details are described in the text. (Genomic sequence is denoted by bold lines, not drawn to scale).

I Not

b I Xba

Xho I 17,700 I Xho Xba I 17,320 I Xba

3.2kb

p I Spe

3’ fragment aHI BamH

EphA1 genomic map July 30th, 2000

lox:p

h I Xho

Sac II Sac

p I Sph

EcoRV

s I Pst

d I Nde

Hind III Hind NoI) (Nco

Exon 3 3.1kb

Middle fragment

Nde I 15,350 I Nde

p I Kpn Hind III Hind

F Xba I I Xba

Sac I Sac

aHI BamH

p I Spe Hind III Hind

-PGK-neo

b I Xba 2 lox p:KS3 Sac I Sac

FRT h I Xho F Genomic sequence is denoted by bold lines, not drawn to scale, map numbers

. p I Kpn

Sal I Sal

h I Xho

s I Pst lox:p

Hind III Hind

Xba I 14,010 I Xba

Nde I 13,950 I Nde Bgl II 13,460 II Bgl 2.7kb

5’ fragment

v II Pvu

s I Nsp Hind III Hind HI /external probe

I /external probe

II/ internal probe Sac I 11,310 I Sac wt = (? -10,385) ~ht 9kb = {(11,310 -10,385)} + 2700 = 3,625 + neo) ~5kb Bam wt = 8,800bp ht = 5,100bp (11,310 - 8,880 + 2700) Xho wt = 20,310 - 13,460 = 6.8kb Bgl ht = (6.8 + neo) = ~8.6kb BamHI 10,385 XhoI 8,880 refer to idiosyncratic laboratory sequence map (see Appendix 1). Figure 6.5 Overview of the targeting construct pBst λ1.1 loxP:KS3_BamHI_NotI_KpnI from the previous step (Figure 6.4). The colonies were tested for recombinant clones by BglII/NotI digestion.

The EphA1 conditional targeting vector was sequenced and expanded with a CsCl Maxi DNA preparation and passed over a NAP-5 column as described in Section 2.2.1c. The EphA1 conditional targeting vector was linearised with NotI.

6.3.2 In vitro Cre testing of the targeting construct

An aliquot of the BS591 bacterial strain which is stably transfected with Cre recombinase was provided by Dr Ian Tonks (QIMR) and transfected with 100 ng of the EphA1 reporter construct and plated out on LB-ampicillin plates at 37ºC overnight. Individual colonies were picked and mini-prep DNA prepared using the “EasyPrep” method. If the construct had been correctly prepared, then the genomic DNA in the middle fragment flanked by loxP sites would be excised and the plasmid retrieved by mini-prep DNA would be smaller by 3.2 kB, and could be determined by restriction digestion and confirmed by direct sequencing.

The mini-prep DNA was amplified with AmpliTaq Gold® using the following primer pairs which were 5’ and 3’ of the loxP sites respectively. If the construct was prepared as expected with correctly orientated loxP sites, which was then excised through transfection in the BS591 bacterial cell line, then there would be amplification of an approximately 100 bp PCR fragment. These primers could not amplify if the sequence between the loxP sites was not excised as the extension time was only 60 seconds.

Forward 5’ – TGGAAGAACAGTCAGTGCTC – 3’

Reverse 5’ – TTTCCTCTCACCCAAGCCAC – 3’.

6.3.3 Generation of EphA1 conditional mutant mice

The generation of targeted EphA1 conditional ES cell lines was undertaken by the QIMR transgenic facility as described in Chapter 5.3.5. The subsequent ES cell micro- injection and production of chimaeras was also performed by the QIMR transgenic facility as decribed in Chapter 5.3.6. The chimaeras were mated with wild type C57BL/J6 mice and germ-line transmission confirmed by brown coat colour in F1 progeny. Chapter 6: Generation and analysis of the EphA1 conditional knockout mice

6.3.4 Generation of EphA1 conditional knockout mice

The generation of heterozygous EphA1 conditional mutant mice (designated EA1C+ /loxP PGK-neo) resulted in a wild type phenotype as expected. The mutant allele comprises the ligand binding domain flanked by loxP sites and the PGK-neo antibiotic selection cassette. The generation of a null genotype subsequently required in vivo Cre- mediated excision to remove the PGK-neo antibiotic selection cassette and excise the ligand binding domain, which introduces a frameshift mutation and an early stop codon thus resulting in failure of EphA1 transcription (EA1C-/-). This was achieved by mating EphA1 conditional males (EA1CloxP PGK-neo/loxP PGK-neo) with Cre-deleter females.

Alternatively Flp-e recombinase mediated removal of the PGK-neo cassette only produced a line of EphA1 conditional mice (EA1CloxP/loxP) with the antibiotic selection cassette removed but with the EphA1 ligand binding domain intact but flanked by loxP sites. These mice would be suitable for tissue specific Cre-mediated excision of the ligand binding domain which would result in complete cessation of EphA1 transcription and knock out EphA1 gene function.

6.3.5 Detecting the recombinant allele in ES cell homologous recombinants and genomic tail tip Southern blots

The ES cell homologous recombinants were detected using the following strategies.

1) A XhoI digest and λ1.1 Sac 660 bp probe (Figure 6.6). The λ1.1 Sac probe was prepared by PCR from the pBluescript λ1.1 genomic fragment using the primers below:-

Forward 5’– CTGTGCTGAGGCTTGGTAGA – 3’

Reverse 3’ – TCCACATCAGGGACTCAACT – 3’.

2) A BamHI digest and Exon 3ab probe (Figure 6.7). The Exon 3ab probe was prepared by PCR amplification of EphA1 cDNA with the primers described in Section 3.1.1.

3) A BglII digest and 3’ fragment probe (Figure 6.8). The 3’ fragment (FR) probe was prepared by PCR amplification of the pBst λ1.2 genomic fragment. The primers were:-

164 Queensland Institute of Medical Research Chapter 6: Generation and analysis of the EphA1 conditional knockout mice

Forward 5 – ’CTGTGCTGAGGCTTGGTAGA – 3’

Reverse 3’ – TCCACATCAGGGACTCAACT – 3’.

6.3.6 Genotyping the EphA1 conditional mutant mice by PCR

The following primer pairs were designed for testing. The primers are according to Genbank AC153915, Mus musculus 10 BAC RP23-329D23 (Table 6.1).

The PCR was undertaken as described in Chapter 2.2.14. The annealling temperature of 50°C was held for 30 seconds and the thermocycling reaction was run for a total of 30–35 cycles.

Table 6.1 Primers for genotyping EphA1 conditional knockout mice. Primers numbered according to GB:AC153915, Mus Musculus 10BAC RP23-329D23. (There is a possible 1 kB amplicon from mEphA1-F19118 and –R20118, however, with the short extension times this should not arise.) F16670 F19931 R20118

F19118 R19399

Intron 2 PGK-Neo Cassette EphA1 Exon 3 Intron 3

loxP/ FRT FRT loxP

Primer Nam Sequence (5’ – 3’)

Wild type (wt) mEphA1-F19118 ACCATCAGAACCTGGCAT

(amplicon = 282 bp) mEphA1-R19399 GTGCATTCGTGGTGTACCTG

Floxed mEphA1-F19931 AATGGAGACCCAGGTAGTCAG

(amplicons of wt-187 bp, mEphA1-R20118 GGTGTCCATTTCCTCTCACC loxP-227 bp)

Knockout (KO) mEphA1-F16670 CTCAGAAGGCTTTGAAAGTGG

(amplicon = 214 bp) mEphA1-R20118 GGTGTCCATTTCCTCTCACC

Queensland Institute of Medical Research 165

9.0kB Xho 1 Xho 1 Xho 1 8,900 4.5kB 17,900

1.1 Sac probe

λH3 wt +/- -/- +/+ -/- λH3 targeting construct

lox:P 9.4

6.6 Exon 3

4.4 PGK-neo

2.3 2.0

Figure 6.6 Strategy for determining ES cell homologous recombination. XhoI digestion of wild type genomic DNA releases a 9.0 kB fragment. XhoI digestion of the homologously recombined allele releases a 4.5 kB fragment detected with the λ1.1 Sac 660 bp probe. Heterozygotes have both a wild type (9.0 kB band) and a knockout (4.5 kB band); homozygous knockout mice have only the 4.5 kB band.

BamHI BamHI ?12kB ??22,000 10,200 BamHI , BamHI 5kB 17,400 ?5 kB

1.1 Sac probe

Exon 3

λH3 wt +/- flp/ + fp/flp λH3 targeting construct

lox:P 9.4

6.6 Exon 3

4.4 PGK-neo

2.3 2.0

Figure 6.7 BamHI digestion and λ1.1 Sac probe. BamHI digest of wild type genomic DNA releases a 20 kB fragment detectable with the λ1.1 Sac probe. BamHI digestion of the homologously recombined allele releases a 7.4 kB fragment detectable with the λ1.1 Sac probe. BamHI digestion of the Cre deleted recombined allele releases a 3.2 kB fragment which is detected with the 3’ fragment probe. flp/+ indicates a heterozygote for the mutant allele with flp-e mediated excision of the PGK-neo cassette; flp/flp indicates a homozygote for the mutant allele with flp-e mediated excision of the PGK-neo cassette.

BglII 6.8kB BglII 13,460 20,310

3.3kB

Exon 3 FR probe λH3 wt +/- -/- Cre/ +Cre/ Cre λH3

Crossed with Cre - deleter targeting construct

lox:P 9.4

6.6 Exon 3

4.4 PGK-neo

2.3 2.0

Figure 6.8 BglII digestion and 3’ (FR) probe. BglII digestion of wild type genomic DNA releases a 6.8 kB fragment detectable with the 3’ (FR) probe. BglII digestion of the homologously recombined allele (without Cre-mediated deletion) releases a 8.6 kB fragment. BglII digestion following Cre-mediated deletion of Exon 3ab releases a 3.3 kB fragment. Cre/+ indicates a heterozygote for the mutant allele with Cre- mediated excision of the PGK-neo cassette; Cre/Cre indicates a homozygote for the mutant allele with Cre- mediated excision of the PGK-neo cassette.

Chapter 6: Generation and analysis of the EphA1 conditional knockout mice

6.4 Results

6.4.1 In vitro Cre testing of the targeting construct

The mini-prep DNA recovered from BS591 E.coli (stably transfected with Cre- recombinase) was ~3.0 kB smaller than control targeting construct DNA, consistent with Cre-mediated excision of the 3.2 kB middle fragment of the targeting construct.

λH3 BS591/Cre Control λH3 NotI Uncut

23

9.4 6.6

4.4

Figure 6.9 Mini-prep DNA recovered following transfection of BS591 E. coli with the targeting construct. The plasmid recovered from BS 591 Cre E. coli is smaller than the control NotI linearised complete targeting vector.

The mini-prep DNA recovered from BS591 E.coli transfected with targeting construct amplified an approximately 100 bp fragment by PCR, whereas the tail tip genomic DNA samples of wild type control mice at various concentrations did not amplify (Figure 6.10). λH3 λH3 Tail Tip genomic DNA samples BS591 Easyprep DNA 2,300 bp 2,000 bp 560bp

Figure 6.10 PCR amplification to detect Cre deletion. The BS591 mini-prep samples amplify an approximately 150 bp fragment as expected. The tail tip genomic DNA control samples do not amplify.

Queensland Institute of Medical Research 173 Chapter 6: Generation and analysis of the EphA1 conditional knockout mice

5’ fragment

13981 XbaI loxP

XbaI 17340 3’ fragment

Figure 6.11 Sequencing of mini-prep DNA recovered from BS591 transfected cells. The genomic DNA between the loxP sites has been excised as indicated by the apposition of the targeting vector 5' fragment and 3' fragment sequence. The map numbers (13981 & 17340) correspond to positions on the EphA1 genomic contiguous DNA sequence (see Appendix 1). The non excised loxP site with orientation is shown. Also the sequence shows apposition of the targeting vector 5' fragment (13981) with the 3' fragment (17340) sequence confirming the exision of the middle fragment sequence. Therefore, the targeting vector demonstrates in vitro Cre-mediated excision, which was important to confirm before proceeding with the ES cell micro- injections.

6.4.2 Southern blot results

The Southern blots were probed as outlined above in Figures 6.6, 6.7 & 6.8 and confirmed that homologous recombination had taken place (Figure 6.12). The BglII digest releases a wild type (wt) band of 6.8 kB and the homologously recombined allele (+/–) without Flp-e or Cre-mediated deletion resulted in a slightly larger band due the inserted PGK-neo cassette. However, Cre-mediated exision deleted both the PGK-neo cassette and Exon 3a and Exon 3b which resulted in the release of a 3.2 kB fragment (Cre +/–).

174 Queensland Institute of Medical Research Chapter 6: Generation and analysis of the EphA1 conditional knockout mice

wt cre/+ +/- wt cre/+ +/- +/- cre/+ +/- wt 9.4

6.6

4.4

2.3 2.0

Figure 6.12 Southern blot of genomic tail tip digested with BglII and probed with 3’ (FR) probe. BglII digestion of wild type genomic DNA releases a 6.8 kB fragment detectable with the 3’ (FR) probe. BglII digestion of the homologously recombined allele (without Cre-mediated deletion) releases a 8.6 kB fragment. BglII digestion of the Cre-mediated deletion of Exon 3ab releases a 3.3 kB fragment. (For explanation see Figure 6.8.)

6.4.3 PCR genotyping results

The results below confirm that the EphA1 conditional heterozygote and homozygous null mice can be differentiated on the basis of this convenient PCR reaction (Figure 6.12).

1 2 3 4 5 6 7 8 9

282bp EA1+/- 214bp EA1-/-

Figure 6.13 PCR genotyping EphA1 conditional KO mice. Duplex PCR (after 35 cycles). Lane 1: 100 bp Ladder; Lane 2: Neg control wt primers; Lane 3: Neg control KO primers; Lane 4: C57/BL6 wt primers; Lane 5: C57Bl/6 KO primers; Lane 6: EA1 +/- wt primers; Lane 7: EA1 +/- KO primers; Lane 8: EphA1-/- wt primers; Lane 9: EphA1 -/- KO primers.

Queensland Institute of Medical Research 175 Chapter 6: Generation and analysis of the EphA1 conditional knockout mice

6.5 Discussion

The EphA1 conditional KO project was commenced prior to the EphA1 hPLAP reporter knockout project. The decision to generate a conditional knockout mouse was justified because the EphB2/EphB3 double knockout mouse, which was lethal in the perinatal period, had been published just prior to the commencement of the project (Orioli et al., 1996). We could not be certain that the EphA1 gene deletion would not be embryonically lethal and as the generation of a conditional KO only requires (1) some additional cloning steps, (2) breeding with Cre-deleter mice and (3) these knockout projects take several years to complete, then the additional effort spent in generating a conditional knockout seemed worthwhile at the time.

The EphA1 conditional knockout mouse was generated and a polymerase chain reaction method was developed for convenient genotyping. A complete phenotypic analysis of these mice is scheduled in the near future. The kinky tail phenotype which is typical of the EphA1 reporter knockout mouse has been an incidental finding in the EphA1 conditional knockout null mouse. The importance of the genetic background on the expression of phenotype has been reviewed previously and I speculate that it is an important reason that the EphA1 hPLAP reporter knockout mouse has a different phenotype from the EphA1 conditional knockout mouse. For example there are currently at least four distinct EphA2 knockout mouse strains (Chen et al., 1996; Michael et al., 1999); however, only one strain has the kinky tail phenotype (Naruse-Nakajima et al., 2001). A further EphA2 knockout mouse was produced using the secretory-trap vector method, and this EphA2 null mouse is fertile and develops normally (Mitchell et al., 2001). X-Gal staining for the β-geo transgene under control of the EphA2 promoter reveals EphA2 expression in the interfollicular epidermis, hair follicles and sebaceous glands. In a classic two-stage carcinogenesis protocol to induce skin tumours, EphA2 null mice are more susceptible to skin tumour development with dramatically increased tumour growth, suggesting that EphA2 is a tumour suppressor gene (Guo et al., 2006). So although the EphA1 conditional null mouse does not display the same frequency of the kinky tail phenotype as the EphA1 reporter null mouse, this mutant mouse does result in conditional silencing of EphA1 and will be a useful laboratory tool.

EphA1, the first described Eph receptor, was overexpressed in an erythropoietin producing hepatocarcinoma cell line (Hirai et al., 1987). Over expression of EphA1 was subsequently found in a subset of colon, lung, liver and mammary carcinomas (Maru et al., 1988), and when EphA1 was highly expressed in NIH3T3 cells, the cells acquired

176 Queensland Institute of Medical Research Chapter 6: Generation and analysis of the EphA1 conditional knockout mice tumourigenic ability in nude mice (Maru et al., 1990). There is continued evidence to suggest that EphA1 does play a role in cancer development and progression and overexpression of EphA1 has been described in prostate cancer (Robinson et al., 1996) and gastric cancers (Kao et al., 2003a). A recent report determined Eph/ephrin expression by quantitative real-time PCR in advanced ovarian cancer, and there was a greater than 10-fold overexpression of EphA1 which correlated with ephrin-A1 expression (Herath et al., 2006). The expression of EphA1 in non-melanoma skin cancers was measured by quantitative real-time PCR and confirmed by immunohistochemistry in a series of basal cell and squamous cell carcinomas, and the degree of EphA1 downregulation correlated with skin tumour thickness (Hafner et al., 2006). The EphA1 conditional knockout mouse with the facility to silence EphA1 expression in the epidermis by mating EphA1 homozygous floxed mice with a deleter mouse strain carrying the Cre-recombinase under the control of the Keratin 14 promoter will allow further analysis of the role of EphA1 in the development of skin tumours (Kuraguchi et al., 2006).

In conclusion, an EphA1 conditional mutant mouse has been generated, and future experiments will provide valuable insight into the role of EphA1 in normal and malignant tissues.

Queensland Institute of Medical Research 177

Chapter7: Future directions

7 FUTURE DIRECTIONS

7.1 Role of EphAs in cancer

The role of the Eph/ephrin proteins in cancer is still not clear, although up-regulation generally supports a role in tumour progression (Wimmer-Kleikamp and Lackmann, 2005). Human EphA1 was originally isolated from a carcinoma cell line (Hirai et al., 1987), and overexpression was also found in a subset of colon, lung, liver and mammary carcinomas (Maru et al., 1990). There is high level expression of EphA1, EphA2 and ephrin-A1 in the normal colon, and EphA1 and EphA2 are overexpressed in some colorectal carcinomas (Maru et al., 1990; Saito et al., 2004). Soluble EphA2-Fc fusion protein blocks angiogenesis and tumour progression (Dobrzanski et al., 2004). However, recent results from our laboratory suggest that in colorectal cancer, down- regulation of EphA1, EphA2 and ephrin-A1 correlates with tumour invasiveness and metastasis (unpublished observations, NI Herath and AW Boyd). In advanced ovarian cancer, overexpression of ephrin-A1 and ephrin-A5 in the presence of high expression of EphA1 and EphA2 correlated with poor prognosis (Herath et al., 2006). As ephrin-A1 is implicated is these cancers, the generation of EphA1 null mice (EphA1 is the cognate receptor for ephrin-A1) will be a valuable tool to further investigating the role of these specific Eph/ephrin proteins.

Indeed, our laboratory has planned experiments to specifically define the role of EphA1 in colorectal carcinoma progression and prognosis using mutant mouse models to elucidate the pathways involved. The EphA1 conditional mouse will be crossed with the Villin-Cre deleter mouse to generate a mutant mouse with EphA1 knocked-out in the gastrointestinal tract only (el Marjou et al., 2004). Our laboratory has also produced a transgenic EphA1 mouse, in which EphA1 has been targeted to the ROSA26 locus of C57Bl/6 mice. When these mice are crossed with Villin-Cre deleters, the loxP flanked β-geo cassette is removed, thus allowing overexpression of EphA1 in the gut (unpublished). These two strains of mice, one with EphA1 knocked-out in the gut and the other overexpressing EphA1, will be treated with azoxymethane and dextran sodium sulphate to induce colorectal tumours (Greten et al., 2004; Wirtz et al., 2007). These mouse models will be used to investigate the role of EphAs in the pathogenesis of colorectal cancer.

Queensland Institute of Medical Research 179 Chapter7: Future directions

7.2 Role in inflammation

The role of Eph/ephrin proteins in injury and inflammation is particularly intriguing (Ivanov and Romanovsky, 2006; Ivanov et al., 2005). The key pathogenic process of the systemic inflammatory response syndrome (SIRS) (Bone et al., 1992; Consensus Conference, 1992) to sepsis, trauma or injury, is an altered endothelium which is “leaky” to both fluid and inflammatory cells and is pro-adhesive for leukocytes and platelets (Hotchkiss and Karl, 2003). These processes require increased activity of adhesion molecules, reorganisation of the cytoskeleton and intercellular de-adhesion (gap formation), all functions regulated by Eph/ephrin signalling (Klein, 2004).

We hypothesise that the Eph/ephrin proteins, in particular EphA1, EphA2 and ephrin-A1, may play a prominent role in the pathogenesis of sepsis and multiple organ failure by:- (1) initiating signalling events at endothelial paracellular junctions leading to increased endothelial cell permeability and altered adhesive properties and (2) promoting the adhesion of leukocyte and leukocyte-platelet aggregates to activated vascular endothelium. We plan to investigate this hypothesis by in vitro analysis of the Eph/ephrin signalling pathways responsible for the increased vascular permeability and adhesion of leukocytes and platelets to activated vascular endothelium in SIRS.

A key mediator of SIRS is tumour necrosis factor alpha (TNF-α) (Bone, 1991), and activation of macrophages and monocytes in response to infection/injury promotes release of TNF-α, which induces increased ephrin-A1 expression in normal vascular endothelial cells (Pandey et al., 1995b). Ephrin-A1 was first isolated as an early response gene from a TNF-α-activated human umbilical vein endothelial cell cDNA library, and was later shown to be a ligand for EphA2 (Dixit et al., 1990; Holzman et al., 1990). Vascular expression of ephrin-A1 was demonstrated by whole mount in situ hybridisation in mouse embryos (Flenniken et al., 1996; McBride and Ruiz, 1998). The HoxB3 “knock-down” endothelial cells, which did not display normal three dimensional capillary-like morphology, had the ability to form branching networks resembling capillaries partially restored by the addition of clustered mouse ephrin-A1/human Fc fusion protein, which induced EphA2 phosphorylation (Myers et al., 2000). These studies confirm the presence of EphA2 and ephrin-A1 on tumour vascular endothelium and suggest a role in neovascularisation. In fact, the endothelial barrier function is dependent on cell-cell junctions in which transmembrane adhesive proteins are

180 Queensland Institute of Medical Research Chapter7: Future directions anchored to the actin cytoskeleton of the cell (Bazzoni and Dejana, 2004; Tanaka et al., 2005a).

The vascular endothelium normally provides a non-thrombogenic barrier between the blood and the tissues, and disruption of this barrier is an important step in the pathophysiology of SIRS and septic shock (Hotchkiss and Karl, 2003). During the development of SIRS there are changes in the expression of a large number of genes in the endothelial cell. In particular, there is upregulation of adhesion molecules, resulting in adhesion of lymphocytes, neutrophils and monocytes and their subsequent diapedesis, along with a flux of fluid, into the interstitial tissue space (Cook-Mills and Deem, 2005). Human leukocytes express ephrin-B2, which activates microvascular endothelial cells (Zamora et al., 2006). Platelets express EphA4, EphB1 and ephrin-B1, and Eph/ephrin clustering and signalling promotes continuing platelet aggregation, allowing stabilisation of the haemostatic plug (Prevost et al., 2002). Platelet microthrombi occluding the microcirculation are a feature of fatal septic shock (Hotchkiss and Karl, 2003). These endothelial changes result in the capillary leak and hypovolaemia which characterise septic shock (Lopez-Bojorquez et al., 2004).

Our laboratory has produced EphA1 and EphA4 knockout mice, has imported EphA2 knockout mice and generated EphA1/EphA2 double knockout mice. We have also generated a novel anti-mouse EphA2 antibody by immunising EphA2 knockout mice. We plan to investigate the expression of Eph/ephrin proteins and key endothelial junctional proteins in vitro in response to inflammation. Specifically, we will investigate the Eph/ephrin regulation of endothelial cell cytoskeletal dynamics, barrier function and signalling molecule activation. Furthermore, we will also investigate Eph/ephrin interactions, which promote leukocyte/platelet aggregation and increased adhesion to activated vascular endothelium. In addition, if the in vitro studies confirm that Eph/ephrin proteins are involved in the pathogenesis of SIRS, then we will explore the contribution of Eph/ephrin proteins in a mouse model of sepsis. Specifically, we will analyse knockout mice by testing the effect of various Eph/ephrin inhibitors with or without agents shown to have some therapeutic effect (e.g. activated protein C, anti- TNF-α) in the mouse model.

Finally, if endothelial expressed Eph/ephrin proteins are key mediators of the vascular permeability and leukocyte adhesion in SIRS, then we can use various molecules to

Queensland Institute of Medical Research 181 Chapter7: Future directions downregulate the inflammatory signalling pathways, for example EphA2-Fc, ephrin- A1-Fc, or, specifically designed EphA2 kinase inhibitors, and potentially reverse the vascular permeability and inflammation. The ability to reverse the increased vascular inflammation of SIRS will allow us to treat patients more specifically and successfully and improve their outcome in terms of both morbidity and mortality. 7.3 Role in development

The EphA1 reporter mouse allows us to examine the expression of EphA1 during embryological development. Although this work is not reported in this thesis, the developmental expression of EphA1 in the mouse embryo is being carried out in this laboratory concurrently with analysis of EphA1 expression in the adult and investigation of the phenotype reported here. Consequently, the role of EphA1 in the normal development of the female genital tract will be more completely investigated. As discussed in Chapter 5, the imperforate vagina phenotype is:- (1) described infrequently in inbred strains of laboratory mice (Cunliffe-Beamer and Feldman, 1976; Sundberg and Brown, 1994); (2) recessively inherited in some cases (Eisen et al., 1989); (3) associated with knockout mice involving hormone receptors (Abel et al., 2000; Rodriguez et al., 1997) and (4) associated with knockout mice silencing pro-apoptotic proteins (Cano-Gauci et al., 1999; Lindsten et al., 2000; Simpson et al., 2004). The high penetrance (~20%) in the EphA1 reporter null mice does suggest a role for EphA1 in the development of the vagina and a possible role in the pro-apoptotic pathway. In fact, several Ephs have been implicated in the apoptotic pathways. EphA2 has been identified as a target for the p53 tumour suppressor protein, and increased EphA2 expression results in increased apoptosis. EphA2 disrupts focal adhesions and may sensitise cells to pro-apoptotic pathways (Dohn et al., 2001). EphA7 regulates the size of the brain through influencing apoptosis in forebrain neural progenitors (Depaepe et al., 2005). In EphA4 null mice, the thymus is smaller and deficient in thymocytes (Munoz et al., 2006). The EphA1 reporter null mouse with the imperforate vagina phenotype will serve as a useful model to further investigate the complexities of Eph/ephrin protein signalling and to examine the development of the female genital tract.

Lastly, the experiments described in this thesis have produced original findings which have advanced our understanding of the biological role of the Eph/ephrin proteins,

182 Queensland Institute of Medical Research Chapter7: Future directions particularly in the adult animal. The knockout mice generated during the undertaking of this project are valuable resources and are likely to contribute towards future discoveries in the field of Eph/ephrin protein biology with long-term implications for both science and medicine.

Queensland Institute of Medical Research 183

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Appendix 1: EphA1 genomic sequence contig

APPENDIX 1 EPHA1 GENOMIC SEQUENCE CONTIG

The EphA1 λ genomic clones were sub-cloned into pBluescript for sequencing and manipulation. The sequence was joined into the following contig using the SEQED function of the GCG program. The exon-intron boundaries have been determined from a line-up with the EphA1 cDNA sequence and the current published Ensembl intron-exon boundaries. The important restriction enzymes sites used for the construction of the EphA1 reporter targeting vector and the EphA1 conditional targeting vector are indicated. The contig sequence numbering is an idiosyncratic laboratory numbering and for brevity the 5’-UTR sequence (nearly 9 kb) has been omitted. The exons are marked in green and the single letter amino acid sequence is bold.

X h o I AATGTCTCCCACTCCTAAATTTCCAAGTGAGCATGACAAGTCTCGAGTCCACAGAAAAGA 8941 ------+------+------+------+------+------+ 9000 TTACAGAGGGTGAGGATTTAAAGGTTCACTCGTACTGTTCAGAGCTCAGGTGTCTTTTCT

B E g c l o I R I I AAGGGAGTAAAACGGAGATCTTTTTTAAGTCAAGCTGAATTCGATTGAAAGCTGGATCAA 9001 ------+------+------+------+------+------+ 9060 TTCCCTCATTTTGCCTCTAGAAAAAATTCAGTTCGACTTAAGCTAACTTTCGACCTAGTT

AACTGGGTTGGGTTTAGGATACATTTAAGAATGTCTGTCTAGTCAATTCCTAGCAACACC 9061 ------+------+------+------+------+------+ 9120 TTGACCCAACCCAAATCCTATGTAAATTCTTACAGACAGATCAGTTAAGGATCGTTGTGG

CCACTAGGCGGCGCCTTCAGTCTCATTCCAACCAAGACGCCTCCAGGTTTTAAAGTTTAT 9121 ------+------+------+------+------+------+ 9180 GGTGATCCGCCGCGGAAGTCAGAGTAAGGTTGGTTCTGCGGAGGTCCAAAATTTCAAATA

TACCTGTCTAACAAGTTTCCTTCCTGTCCTCCTGTCCCTTTAAGAAGGTGAACCAGGTGA 9181 ------+------+------+------+------+------+ 9240 ATGGACAGATTGTTCAAAGGAAGGACAGGAGGACAGGGAAATTCTTCCACTTGGTCCACT

Queensland Institute of Medical Research 229 Appendix 1: EphA1 genomic sequence contig

GGCTGAGGCCCCGCCCCTCATCTGGCCCCGCCCTCCGCCCCCGCCTAGCCGGCCTGGATC 9241 ------+------+------+------+------+------+ 9300 CCGACTCCGGGGCGGGGAGTAGACCGGGGCGGGAGGCGGGGGCGGATCGGCCGGACCTAG

GCCCAGTCCCTCGCGACCTGGCCCAGCCGTAGGGGCCGTGGTCCCAGGTCCCGGCCGGCG 9301 ------+------+------+------+------+------+ 9360 CGGGTCAGGGAGCGCTGGACCGGGTCGGCATCCCCGGCACCAGGGTCCAGGGCCGGCCGC

N c o I CCATGGAGCGGCGCTGGCCCCTGGGGCTTGCATTGCTGCTGCTGCTGCTCTGCGCCCCGC 9361 ------+------+------+------+------+------+ 9420 GGTACCTCGCCGCGACCGGGGACCCCGAACGTAACGACGACGACGACGAGACGCGGGGCG EXON I M E R R W P L G L A L L L L L L C A P L-

K p n I TGCCCCCGGGGGCGCGCGCCGAGGAAGGTACCGACCCCCGCCCTCTCCCTTTCCCGGCTG 9421 ------+------+------+------+------+------+ 9480 ACGGGGGCCCCCGCGCGCGGCTCCTTCCATGGCTGGGGGCGGGAGAGGGAAAGGGCCGAC P P G A R A E E

ACGGGCAGCAGCCTGCAAGAGCCGGCATTCCTGAGGAGGCCCTCTTCCCCGAGCCCCACT 9481 ------+------+------+------+------+------+ 9540 TGCCCGTCGTCGGACGTTCTCGGCCGTAAGGACTCCTCCGGGAGAAGGGGCTCGGGGTGA C l a I ACTTCTCCCTGGCTTCTCCCCAGTAGCGGGAAGATCGATTGGCCAGGCCAATCTTTGGTC 9541 ------+------+------+------+------+------+ 9600 TGAAGAGGGACCGAAGAGGGGTCATCGCCCTTCTAGCTAACCGGTCCGGTTAGAAACCAG

TTTAACCCCAGACTCCTGCCCACGGGATCAGTCACCCCTACTCTGTTGGCCAGCACTATT 9601 ------+------+------+------+------+------+ 9660 AAATTGGGGTCTGAGGACGGGTGCCCTAGTCAGTGGGGATGAGACAACCGGTCGTGATAA

230 Queensland Institute of Medical Research Appendix 1: EphA1 genomic sequence contig

TGCTGCGGTTGGAGAGGGAGTTCCTGACCACAGCCAGGAAGCTGCATTCCTACTGCAGCA 9661 ------+------+------+------+------+------+ 9720 ACGACGCCAACCTCTCCCTCAAGGACTGGTGTCGGTCCTTCGACGTAAGGATGACGTCGT

TCATGGGACCCTCCAAATGCCCACCCCTGACTTTGCAGCAGCACTCAGAAGTCTGAGGCG 9721 ------+------+------+------+------+------+ 9780 AGTACCCTGGGAGGTTTACGGGTGGGGACTGAAACGTCGTCGTGAGTCTTCAGACTCCGC

GAAAGCCTCCTTCTGCCAGCCTAACCGGAGCACAAATAATCCTCCAGTGGTGGAAGGATT 9781 ------+------+------+------+------+------+ 9840 CTTTCGGAGGAAGACGGTCGGATTGGCCTCGTGTTTATTAGGAGGTCACCACCTTCCTAA

AGAACTTTTGGGATTGCTAGGTGTAGGGGTCCAATCTTCCACCTAAAGTTTTGCACACTC 9841 ------+------+------+------+------+------+ 9900 TCTTGAAAACCCTAACGATCCACATCCCCAGGTTAGAAGGTGGATTTCAAAACGTGTGAG

TGTCCATGCCCCTCCCACTTCCCAAAATCATAGCTTCACCTAAGGCCCCACCTCCTCTAC 9901 ------+------+------+------+------+------+ 9960 ACAGGTACGGGGAGGGTGAAGGGTTTTAGTATCGAAGTGGATTCCGGGGTGGAGGAGATG

B a m H I CCTCACCTACCAGAGCAGGCCACTGACAACTGGAGTCAAAATCCAGCAGGATCCCCCTGC 9961 ------+------+------+------+------+------+ 10020 GGAGTGGATGGTCTCGTCCGGTGACTGTTGACCTCAGTTTTAGGTCGTCCTAGGGGGACG

CAGGGTCCCACTCAGGACTACAAAGGCTTAGCTTCCCTGACGTCCGCCCACGGCATCCTT 10021 ------+------+------+------+------+------+ 10080 GTCCCAGGGTGAGTCCTGATGTTTCCGAATCGAAGGGACTGCAGGCGGGTGCCGTAGGAA

GCAGATGTTCTCCAGGGCTCCGTGGGTGCCACGTCTGGGGTTGCTCTTCCAACGCTTGCC 10081 ------+------+------+------+------+------+ 10140 CGTCTACAAGAGGTCCCGAGGCACCCACGGTGCAGACCCCAACGAGAAGGTTGCGAACGG

Queensland Institute of Medical Research 231 Appendix 1: EphA1 genomic sequence contig

GTCCTGACTCTGGTTCAGGCACCAGTCCCCCAGCCCTTGCCCCACTCTGCCTTTCTCCTC 10141 ------+------+------+------+------+------+ 10200 CAGGACTGAGACCAAGTCCGTGGTCAGGGGGTCGGGAACGGGGTGAGACGGAAAGAGGAG

TGCCTCAGGATGTCACACTGTTAGGAAGACGTTCTGGGCAGCCTCGGACTGGGGAGTCCC 10201 ------+------+------+------+------+------+ 10260 ACGGAGTCCTACAGTGTGACAATCCTTCTGCAAGACCCGTCGGAGCCTGACCCCTCAGGG

K p n I AGAATACCCAGTGTAGGGAGTTGGGTACCACCACCCCAGGGATGACATCGTTCTTTTTCT 10261 ------+------+------+------+------+------+ 10320 TCTTATGGGTCACATCCCTCAACCCATGGTGGTGGGGTCCCTACTGTAGCAAGAAAAAGA

CCCCCATTCAGTCACTCTAATGGACACAAGCACAGCACAAGGAGAGCTTGGGCTGGCTTT 10321 ------+------+------+------+------+------+ 10380 GGGGGTAAGTCAGTGAGATTACCTGTGTTCGTGTCGTGTTCCTCTCGAACCCGACCGAAA EXON II V T L M D T S T A Q G E L G L A F -

B a m H I CTGGGATCCCCCAGAGACTGGGGTAAGTATTTGAAGGACCTGGGAAAGTTCAAGTTTGAA 10381 ------+------+------+------+------+------+ 10440 GACCCTAGGGGGTCTCTGACCCCATTCATAAACTTCCTGGACCCTTTCAAGTTCAAACTT W D P P E T G

GGCCAGTGGGAAATGGGGTGGAGAGGTGGGTGGAGGAACAGAAAAGAGCAGGAAAAACTG 10441 ------+------+------+------+------+------+ 10500 CCGGTCACCCTTTACCCCACCTCTCCACCCACCTCCTTGTCTTTTCTCGTCCTTTTTGAC

GAAGTGTCTCCTCAAAGAGGGGGAGGGGAGCTGAGTCAGAGGCTCCTGAATGGTACTCCT 10501 ------+------+------+------+------+------+ 10560 CTTCACAGAGGAGTTTCTCCCCCTCCCCTCGACTCAGTCTCCGAGGACTTACCATGAGGA

232 Queensland Institute of Medical Research Appendix 1: EphA1 genomic sequence contig

AGGGAGCTGGGGGGTGTCAAACTCCACTCAGTGGGACTATCGCTACTCCCTCACCCTCCT 10561 ------+------+------+------+------+------+ 10620 TCCCTCGACCCCCCACAGTTTGAGGTGAGTCACCCTGATAGCGATGAGGGAGTGGGAGGA

S a c I ACCATAGGGTTGGGGGTGGGGGTATGAGCTCTCGACTCCCAGGGAGAGTATGCCTATGCT 10621 ------+------+------+------+------+------+ 10680 TGGTATCCCAACCCCCACCCCCATACTCGAGAGCTGAGGGTCCCTCTCATACGGATACGA

AAGGAAGGCTGGATGAGAGCTGAGACACCTGGAAAGCATGCCCCAGTCTCAGCTCCGTTC 10681 ------+------+------+------+------+------+ 10740 TTCCTTCCGACCTACTCTCGACTCTGTGGACCTTTCGTACGGGGTCAGAGTCGAGGCAAG

CTTACAGTGGAGTTATGTGGATGGTTTTCTGAACATTAGAGAACTCCATAAAAGGGTAAA 10741 ------+------+------+------+------+------+ 10800 GAATGTCACCTCAATACACCTACCAAAAGACTTGTAATCTCTTGAGGTATTTTCCCATTT

TGATGGATGGATTTTTTTTTTCTTTCTGAAGAAGAGAGCCAAAACTTTCATCTAAGTCTT 10801 ------+------+------+------+------+------+ 10860 ACTACCTACCTAAAAAAAAAAGAAAGACTTCTTCTCTCGGTTTTGAAAGTAGATTCAGAA

CAAAGGTATTTTGACTCCCTCAAACTTAAAAGCTGCGCCCCTCCATGGGCCCTGCACACT 10861 ------+------+------+------+------+------+ 10920 GTTTCCATAAAACTGAGGGAGTTTGAATTTTCGACGCGGGGAGGTACCCGGGACGTGTGA

CTCCATCCCTNCCAGGCAGGCTTNCACAAACACCATGGATTCCAGCTGTTTTGAGAAAGA 10921 ------+------+------+------+------+------+ 10980 GAGGTAGGGANGGTCCGTCCGAANGTGTTTGTGGTACCTAAGGTCGACAAAACTCTTTCT

GAAAGGGAAAAAAAAAAAAAAAAACAGCTCCCAAGTTGCTAACTTCCTTAGGTGTGGTGT 10981 ------+------+------+------+------+------+ 11040 CTTTCCCTTTTTTTTTTTTTTTTTGTCGAGGGTTCAACGATTGAAGGAATCCACACCACA

Queensland Institute of Medical Research 233 Appendix 1: EphA1 genomic sequence contig

CACTTGAAACCCATTGAGCNANAGGTGGACNGTTAGTGTGTCCTACANACTNTTCANACC 11041 ------+------+------+------+------+------+ 11100 GTGAACTTTGGGTAACTCGNTNTCCACCTGNCAATCACACAGGATGTNTGANAAGTNTGG

CAAAGTCCAGAGACAACCTTGCTGCAACGCACTAGAGCTGTCCTGAGAACCCCAGAACGG 11101 ------+------+------+------+------+------+ 11160 GTTTCAGGTCTCTGTTGGAACGACGTTGCGTGATCTCGACAGGACTCTTGGGGTCTTGCC

K p n I TACCTCTAGGATTGGAGACTTACTACTGGTCCTGGACCTATTCTCTGGCTTCCTTCCTCC 11161 ------+------+------+------+------+------+ 11220 ATGGAGATCCTAACCTCTGAATGATGACCAGGACCTGGATAAGAGACCGAAGGAAGGAGG

AATACCCAAGACATGTGCTGCTGCTGTTTCTTGGGAGTAGGGTTATTGCCTTCTTTCTCT 11221 ------+------+------+------+------+------+ 11280 TTATGGGTTCTGTACACGACGACGACAAAGAACCCTCATCCCAATAACGGAAGAAAGAGA

S a c I CTTGGACTGGAGGGTCGAGGCTCCCCTTTGAGCTCTTCCACAGAGTGGCCTATAATTAGA 11281 ------+------+------+------+------+------+ 11340 GAACCTGACCTCCCAGCTCCGAGGGGAAACTCGAGAAGGTGTCTCACCGGATATTAATCT

GTTAATAGACTAAACCATAATGTATCCTCCAGGAGGGGCCATTTGGAAAATCCCAGATTA 11341 ------+------+------+------+------+------+ 11400 CAATTATCTGATTTGGTATTACATAGGAGGTCCTCCCCGGTAAACCTTTTAGGGTCTAAT

GGCCAGCTTTAATAGGTGCCTCATCTCATAAACCAGCTAATCACCCACAACCCAAGGCAG 11401 ------+------+------+------+------+------+ 11460 CCGGTCGAAATTATCCACGGAGTAGAGTATTTGGTCGATTAGTGGGTGTTGGGTTCCGTC

CAGGACCATCTGCTGGCCTCCACTGGCTCTTCCTTAAGCTCCACCAGCCCCACCCAAATA 11461 ------+------+------+------+------+------+ 11520 GTCCTGGTAGACGACCGGAGGTGACCGAGAAGGAATTCGAGGTGGTCGGGGTGGGTTTAT

234 Queensland Institute of Medical Research Appendix 1: EphA1 genomic sequence contig

ACTCGTCCCTAACTGAGCCCCAATGGAAGCAAGGCCCTTTTCCTGTCTGGTGCTAGAAGC 11521 ------+------+------+------+------+------+ 11580 TGAGCAGGGATTGACTCGGGGTTACCTTCGTTCCGGGAAAAGGACAGACCACGATCTTCG

GCAGTGCACCCCCTGCAACTGTACTCCCAGTCTCTTTCATGTCCCTTCCCTCTTCTAAAA 11581 ------+------+------+------+------+------+ 11640 CGTCACGTGGGGGACGTTGACATGAGGGTCAGAGAAAGTACAGGGAAGGGAGAAGATTTT

CAGATTTAAGTCCTGGAGCCAAGCTCCCAGAATACACCCTATGCTGGAGTAGCAACCAGC 11641 ------+------+------+------+------+------+ 11700 GTCTAAATTCAGGACCTCGGTTCGAGGGTCTTATGTGGGATACGACCTCATCGTTGGTCG

AGCTCCTGAGTGTCAAGTGAAAGGACCCTCAGTGCTGTTTCTGGCCCCAGAGATGTCCCC 11701 ------+------+------+------+------+------+ 11760 TCGAGGACTCACAGTTCACTTTCCTGGGAGTCACGACAAAGACCGGGGTCTCTACAGGGG

ATCTGTCACTGCTGGAGCTGGAAGAGGCAGTCACAGCTGGGAGCATCTGTTTCCCTCTGC 11761 ------+------+------+------+------+------+ 11820 TAGACAGTGACGACCTCGACCTTCTCCGTCAGTGTCGACCCTCGTAGACAAAGGGAGACG

P s t I TCCAGCTGCAGCCTTGGCAGTCTGCTTAGTGCTCTGCCGCACAGTACCACAACCACCCTG 11821 ------+------+------+------+------+------+ 11880 AGGTCGACGTCGGAACCGTCAGACGAATCACGAGACGGCGTGTCATGGTGTTGGTGGGAC

TCTACCTGCCTGGGCCCTATGCCTTGCCTCTTAGGCTTCCAGCAAAGTTTCCCCTCAGTA 11881 ------+------+------+------+------+------+ 11940 AGATGGACGGACCCGGGATACGGAACGGAGAATCCGAAGGTCGTTTCAAAGGGGAGTCAT

GGCTCCAAGGTGTCCCTTCATTCCAGCATTGTGGCCTTTGGACACCCCTCTTGATCTTTG 11941 ------+------+------+------+------+------+ 12000 CCGAGGTTCCACAGGGAAGTAAGGTCGTAACACCGGAAACCTGTGGGGAGAACTAGAAAC

Queensland Institute of Medical Research 235 Appendix 1: EphA1 genomic sequence contig

AACTCTATGACATAATACAGAATTATGTGCATCAAAAGGGCCCCCGCCTACCAATTCTTA 12001 ------+------+------+------+------+------+ 12060 TTGAGATACTGTATTATGTCTTAATACACGTAGTTTTCCCGGGGGCGGATGGTTAAGAAT

ATTAGTGATAGTGNTTTTTGNTTTGGTTTTTTAAGTCATGAGGGCCACTCACTGGACTGG 12061 ------+------+------+------+------+------+ 12120 TAATCACTATCACNAAAAACNAAACCAAAAAATTCAGTACTCCCGGTGAGTGACCTGACC

AATAAAATCTCAGAATGAACTTTATTAGGACCTCAAGTCTCTTCTCCATCTAGTGATGGG 12121 ------+------+------+------+------+------+ 12180 TTATTTTAGAGTCTTACTTGAAATAATCCTGGAGTTCAGAGAAGAGGTAGATCACTACCC

ATGGCATCCCAAATATAGTAAACTATTTTGTGAAATCTGTGGGCTTTCATAACTATATCT 12181 ------+------+------+------+------+------+ 12240 TACCGTAGGGTTTATATCATTTGATAAAACACTTTAGACACCCGAAAGTATTGATATAGA

GGCCAAGAAAATACTTCAGCGAAAGATGTAGTACGGTATCTGTTCCACCAACAGATAATC 12241 ------+------+------+------+------+------+ 12300 CCGGTTCTTTTATGAAGTCGCTTTCTACATCATGCCATAGACAAGGTGGTTGTCTATTAG

AGAACATCGACAGTAATTAAAACCTGGAATGATGACAAGAAACCCAGGCCAAGGGAACAG 12301 ------+------+------+------+------+------+ 12360 TCTTGTAGCTGTCATTAATTTTGGACCTTACTACTGTTCTTTGGGTCCGGTTCCCTTGTC

AGTAGAAAGTCCAAACACAGAATCCACATGTCCGATACTGTCACGTGGATGGCCACGGAA 12361 ------+------+------+------+------+------+ 12420 TCATCTTTCAGGTTTGTGTCTTAGGTGTACAGGCTATGACAGTGCACCTACCGGTGCCTT

TTTCTAGTCAATGTGAGAAGTATTGCTGGGGACAGCAGTACTTCCTACCCTCCCCCTAAG 12421 ------+------+------+------+------+------+ 12480 AAAGATCAGTTACACTCTTCATAACGACCCCTGTCGTCATGAAGGATGGGAGGGGGATTC

CAAGGCTGTGAAAGTGACAACAGATGATGACTGAACAGGAGAAACACTATTTTAATTACC 12481 ------+------+------+------+------+------+ 12540 GTTCCGACACTTTCACTGTTGTCTACTACTGACTTGTCCTCTTTGTGATAAAATTAATGG

236 Queensland Institute of Medical Research Appendix 1: EphA1 genomic sequence contig

H i n d I I I TTCATTCACACAGGATGAAGTCCAGGGAAGTTCAGAGGGTTGAAGCTTGTGTATTACCCT 12541 ------+------+------+------+------+------+ 12600 AAGTAAGTGTGTCCTACTTCAGGTCCCTTCAAGTCTCCCAACTTCGAACACATAATGGGA

GAGCTTTAGAAAGGAGTGGGGGTGGGGAGGTGGTGACAGGCCCAAGTGTGAAATGAACAA 12601 ------+------+------+------+------+------+ 12660 CTCGAAATCTTTCCTCACCCCCACCCCTCCACCACTGTCCGGGTTCACACTTTACTTGTT

AGGCTGTCCTGCTGTGCCCAGTTTAAAAGTCTCTGGGGTTAAAGAAGTTTTNAGTGAGCA 12661 ------+------+------+------+------+------+ 12720 TCCGACAGGACGACACGGGTCAAATTTTCAGAGACCCCAATTTCTTCAAAANTCACTCGT

GCCTTCTTCCTGGCAGTGTTAGCTCACTAATTTAAATCCCTTCCAAAGATTTAAGTTTCT 12721 ------+------+------+------+------+------+ 12780 CGGAAGAAGGACCGTCACAATCGAGTGATTAAATTTAGGGAAGGTTTCTAAATTCAAAGA

TTTACCAAAGGGCAGCTTGTATGAGCTAATCTATAGTTTTTTCCTAGTGATCATCTCAAA 12781 ------+------+------+------+------+------+ 12840 AAATGGTTTCCCGTCGAACATACTCGATTAGATATCAAAAAAGGATCACTAGTAGAGTTT

CAATGCCACATAAATATATTGGGGGTGTTCACTGGCACCCCCCCCCAAAAGAACCATACT 12841 ------+------+------+------+------+------+ 12900 GTTACGGTGTATTTATATAACCCCCACAAGTGACCGTGGGGGGGGGTTTTCTTGGTATGA

GCCAACTGGATGTTTGTAAGTTTTGTGACAGAATGAAAGGGAGAACTCCTCTTCTTATTC 12901 ------+------+------+------+------+------+ 12960 CGGTTGACCTACAAACATTCAAAACACTGTCTTACTTTCCCTCTTGAGGAGAAGAATAAG

TATGAGCACATACAAAAAATTAATTCTAGTAAGAATAAAGTTTCAACCAAAATNAAAAAA 12961 ------+------+------+------+------+------+ 13020 ATACTCGTGTATGTTTTTTAATTAAGATCATTCTTATTTCAAAGTTGGTTTTANTTTTTT

Queensland Institute of Medical Research 237 Appendix 1: EphA1 genomic sequence contig

AATCCTACAAAAATTTCAAGCCAAAAGATAAAAAAAATTTTATGATCTCAACCAAAACAC 13021 ------+------+------+------+------+------+ 13080 TTAGGATGTTTTTAAAGTTCGGTTTTCTATTTTTTTTAAAATACTAGAGTTGGTTTTGTG

ACAGCCCAAAACTCATGGTGTTATTATGCACAAAATAAAAAATGTGTTTTCACATAAAAG 13081 ------+------+------+------+------+------+ 13140 TGTCGGGTTTTGAGTACCACAATAATACGTGTTTTATTTTTTACACAAAAGTGTATTTTC

ATGCCACTAGCAAAAATGTGGCATGGCTGAAACCATNTGAAGGCATACTTATNCAGGCAA 13141 ------+------+------+------+------+------+ 13200 TACGGTGATCGTTTTTACACCGTACCGACTTTGGTANACTTCCGTATGAATANGTCCGTT

AATAATTAGTATTTTAAANATTTTTAAAAACTCACCCAAAGTGAATAATGTTAGTCAATA 13201 ------+------+------+------+------+------+ 13260 TTATTAATCATAAAATTTNTAAAAATTTTTGAGTGGGTTTCACTTATTACAATCAGTTAT

AGCATAAAGCTNATGAAGAAAAAAAGCAANCATACAACATCCCCCTTCAAAGACNTGTGA 13261 ------+------+------+------+------+------+ 13320 TCGTATTTCGANTACTTCTTTTTTTCGTTNGTATGTTGTAGGGGGAAGTTTCTGNACACT

CTTGGNAGGGCAACGAGGCNTGCCTTGGCCATGGAACTGGCAAAATGCAAAATGAACATT 13321 ------+------+------+------+------+------+ 13380 GAACCNTCCCGTTGCTCCGNACGGAACCGGTACCTTGACCGTTTTACGTTTTACTTGTAA

AATATCTCTCNTAATGAGCTTTTTAGAAAATGAACTCTCTTGAACTTGTTTCANAAGTGT 13381 ------+------+------+------+------+------+ 13440 TTATAGAGAGNATTACTCGAAAAATCTTTTACTTGAGAGAACTTGAACAAAGTNTTCACA

B g l I I AAGCTGATAAAACCTTTGGAGATCTGTNTATTGATTCAGCAATTNTATGGAAATCTTACA 13441 ------+------+------+------+------+------+ 13500 TTCGACTATTTTGGAAACCTCTAGACANATAACTAAGTCGTTAANATACCTTTAGAATGT

238 Queensland Institute of Medical Research Appendix 1: EphA1 genomic sequence contig

TTTATCAGTGTGCTTTCCTAAAATAAAATGAAAGGAAAAAGGGCTAGAAACTAATTAGCC 13501 ------+------+------+------+------+------+ 13560 AAATAGTCACACGAAAGGATTTTATTTTACTTTCCTTTTTCCCGATCTTTGATTAATCGG

AACAACAGGTGAATGGTTAATGATTATGGTGCATCCATATGCTTAGATGCTGTTGATACA 13561------+------+------+------+------+------+ 13620 TTGTTGTCCACTTACCAATTACTAATACCACGTAGGTATACGAATCTACGACAACTATGT

CTGCAACAAGAGACAGGTCTGAATGTGTTCTTTNTNTGNTTTTGGTTTGTTTGGTTTTTT 13621 ------+------+------+------+------+------+ 13680 GACGTTGTTCTCTGTCCAGACTTACACAAGAAANANACNAAAACCAAACAAACCAAAAAA

TTTTTTTTTTAAGATTCATTTAGTTTTATTTATATGAGNACATTGTNGCTGTCTTCAGAC 13681 ------+------+------+------+------+------+ 13740 AAAAAAAAAATTCTAAGTAAATCAAAATAAATATACTCNTGTAACANCGACAGAAGTCTG

ACACCAGAAGAAGGTATCAGATCCTATNACAGATGGTTGTGAGCCACCATGTGGTGGCTG 13741 ------+------+------+------+------+------+ 13800 TGTGGTCTTCTTCCATAGTCTAGGATANTGTCTACCAACACTCGGTGGTACACCACCGAC

GGAATTGAACTCAGGACCTTTGGAAGAACAGTCAGTGCTCTTAACCGCTGAGCCATCTCT 13801 ------+------+------+------+------+------+ 13860 CCTTAACTTGAGTCCTGGAAACCTTCTTGTCAGTCACGAGAATTGGCGACTCGGTAGAGA

CCAGCCCCCTGAATGTATTCTATAGGCCTAGTTCCAGAACATTAATTGTTAAAACAAAGG 13861 ------+------+------+------+------+------+ 13920 GGTCGGGGGACTTACATAAGATATCCGGATCAAGGTCTTGTAATTAACAATTTTGTTTCC

AGGTTACAGAATAAGCCTTGTATGATATTTATTCTAACAAGGCAGGACATTAAAATAAAA 13921 ------+------+------+------+------+------+ 13980 TCCAATGTCTTATTCGGAACATACTATAAATAAGATTGTTCCGTCCTGTAATTTTATTTT X b a I TATTAGAATATTTCTCAGAAGGCTTTGAAAGTGGTCTAGACAGCTTATTTCAAACCATTA 13981 ------+------+------+------+------+------+ 14040 ATAATCTTATAAAGAGTCTTCCGAAACTTTCACCAGATCTGTCGAATAAAGTTTGGTAAT

Queensland Institute of Medical Research 239 Appendix 1: EphA1 genomic sequence contig

AGAATGGCTACCTCTCAGAATAAAGGTGGAAGGATGGGGAGACCTACCCTTTGTCAGTCG 14041 ------+------+------+------+------+------+ 14100 TCTTACCGATGGAGAGTCTTATTTCCACCTTCCTACCCCTCTGGATGGGAAACAGTCAGC

ATTCCTAAACCTGCTAGATGTGAAGTTTAAGAGCATAAATTCTGTACATGGAGTTCCTGG 14101 ------+------+------+------+------+------+ 14160 TAAGGATTTGGACGATCTACACTTCAAATTCTCGTATTTAAGACATGTACCTCAAGGACC

CTTTGTACTCCAACTCTGTCTCAGATGTTGGGCAAGCCAGTCCACACTGGCTCTATATCA 14161 ------+------+------+------+------+------+ 14220 GAAACATGAGGTTGAGACAGAGTCTACAACCCGTTCGGTCAGGTGTGACCGAGATATAGT

GTGGTCTGTGGCAAGTTATGGTAATGCCAGCCATTATGATTACTACATTTAAAAATTCTT 14221 ------+------+------+------+------+------+ 14280 CACCAGACACCGTTCAATACCATTACGGTCGGTAATACTAATGATGTAAATTTTTAAGAA

K p n I TCAATGTGGAAAAAAAGTCAAGATGGTACCTGGGAGCCCCACTAACTGTTGGGACTTTGG 14281 ------+------+------+------+------+------+ 14340 AGTTACACCTTTTTTTCAGTTCTACCATGGACCCTCGGGGTGATTGACAACCCTGAAACC

TGGTGGCTATATGTAGAAGGAATATTGGAGAGGGTAAGACCTCCTGTATACAGTTCTTTT 14341 ------+------+------+------+------+------+ 14400 ACCACCGATATACATCTTCCTTATAACCTCTCCCATTCTGGAGGACATATGTCAAGAAAA

CTGGTTACTATTTGAAAAAATAAGGCTCTAAACAAACCAGGAGATGGCTTTTGAGTGAGC 14401 ------+------+------+------+------+------+ 14460 GACCAATGATAAACTTTTTTATTCCGAGATTTGTTTGGTCCTCTACCGAAAACTCACTCG

K p n I TTTGCCTTAGTGGGTACCTGACAGTTCTGCTGACTACATAGGTCAAAATTCCTACCCATA 14461 ------+------+------+------+------+------+ 14520 AAACGGAATCACCCATGGACTGTCAAGACGACTGATGTATCCAGTTTTAAGGATGGGTAT

240 Queensland Institute of Medical Research Appendix 1: EphA1 genomic sequence contig

CTCTTGTATTTGAACAGATGAAATTAAGGGACCAACCAGGGCTGCCTAGGATCTTAAGGG 14521 ------+------+------+------+------+------+ 14580 GAGAACATAAACTTGTCTACTTTAATTCCCTGGTTGGTCCCGACGGATCCTAGAATTCCC

GTTGCTATCTTATCTGCCCCAAGTCCAAATAATGCCCCAGCCCTGGGGCTGGAAGGTCCA 14581 ------+------+------+------+------+------+ 14640 CAACGATAGAATAGACGGGGTTCAGGTTTATTACGGGGTCGGGACCCCGACCTTCCAGGT

B s t X I GTGTGGTCCATTAACCTGGGAGCTTTGACTCCAGCTCCATCTTCATCAGCTCTGGTCACT 14641 ------+------+------+------+------+------+ 14700 CACACCAGGTAATTGGACCCTCGAAACTGAGGTCGAGGTAGAAGTAGTCGAGACCAGTGA

GAGCCTGATTCTCTTCTTGCAGTGGAGTGAGGTGCAACAAATGCTAAACGGGACACCCCT 14701 ------+------+------+------+------+------+ 14760 CTCGGACTAAGAGAAGAACGTCACCTCACTCCACGTTGTTTACGATTTGCCCTGTGGGGA EXON IIIA W S E V Q Q M L N G T P L -

GTACATGTACCAAGACTGCCCAATACAGGAAGGTGGGGACACTGACCACTGGCTTCGCTC 14761 ------+------+------+------+------+------+ 14820 CATGTACATGGTTCTGACGGGTTATGTCCTTCCACCCCTGTGACTGGTGACCGAAGCGAG Y M Y Q D C P I Q E G G D T D H W L R S -

H i S n a d c I I I I I CAATTGGATCTaCCgCGGAGAGGAAGCTTCACGCATCtACGTGGAGCTGCAGTTCACCgt 14821 ------+------+------+------+------+------+ 14880 GTTAACCTAGAtGGcGCCTCTCCTTCGAAGTGCGTAGaTGCACCTCGACGTCAAGTGGca N W I Y R G E E A S R I Y V E L Q F T V -

Queensland Institute of Medical Research 241 Appendix 1: EphA1 genomic sequence contig

GCGGGACTGTAAGAGTTTCCCAGGGGGAGCTGGGCCTCTGGGATgCAAAGAGACCTTCAA 14881 ------+------+------+------+------+------+ 14940 CGCCCTGACATTCTCAAAGGGTCCCCCTCGACCCGGAGACCCTAcGTTTCTCTGGAAGTT R D C K S F P G G A G P L G C K E T F N -

CCTTTTCTACATGGAGAGTGACCAGGATGTGGGCATTCAGCTCCGACGACCTTTGTTCCA 14941 ------+------+------+------+------+------+ 15000 GGAAAAGATGTACCTCTCACTGGTCCTACACCCGTAAGTCGAGGCTGCTGGAAACAAGGT L F Y M E S D Q D V G I Q L R R P L F Q -

AAAGGTGCTGCCTTCCCTCCATGCCATCCCTTCTCTGATATAGATCCTTACTTTGGTCCT 15001 ------+------+------+------+------+------+ 15060 TTTCCACGACGGAAGGGAGGTACGGTAGGGAAGAGACTATATCTAGGAATGAAACCAGGA K V

CGCTATGATCCAGCTCAGCCAAGGAGAAACAGAGAAGAAGCTGGAAGCAAGTGCGAATGT 15061 ------+------+------+------+------+------+ 15120 GCGATACTAGGTCGAGTCGGTTCCTCTTTGTCTCTTCTTCGACCTTCGTTCACGCTTACA

AGAATAAGAAGGCAGGACCAGACACCAGTCCGGAAAGGCTGTACTGAGTACCACTGGCTG 15121 ------+------+------+------+------+------+ 15180 TCTTATTCTTCCGTCCTGGTCTGTGGTCAGGCCTTTCCGACATGACTCATGGTGACCGAC

GGCCCTGGCTCTAAGGGCATCATTTAACCTAACCTCTCTGAACATTAGTTACCTCGTCAT 15181 ------+------+------+------+------+------+ 15240 CCGGGACCGAGATTCCCGTAGTAAATTGGATTGGAGAGACTTGTAATCAATGGAGCAGTA

ATAATAACCTTATTACTTGCATCTATTGAAATTGCTCGATGCTGTTTTTGAAAGGGGAAG 15241 ------+------+------+------+------+------+ 15300 TATTATTGGAATAATGAACGTAGATAACTTTAACGAGCTACGACAAAAACTTTCCCCTTC

GATAGTGTGGAGATTTTCGGACATTTCACTTCAGTGCTCATGATTTTCATATGTGACATG 15301 ------+------+------+------+------+------+ 15360 CTATCACACCTCTAAAAGCCTGTAAAGTGAAGTCACGAGTACTAAAAGTATACACTGTAC

242 Queensland Institute of Medical Research Appendix 1: EphA1 genomic sequence contig

CCCCCAAGAGTATTGCCAGAACTACACAGGACAGTTCCTGGCTGCCAATCACCAGTCCGT 15361 ------+------+------+------+------+------+ 15420 GGGGGTTCTCATAACGGTCTTGATGTGTCCTGTCAAGGACCGACGGTTAGTGGTCAGGCA

TCTTTCCTAGCGTGGAAACATACTAGAACACAAACCAGTCTGGAGCTTATTATTATAGGA 15421 ------+------+------+------+------+------+ 15480 AGAAAGGATCGCACCTTTGTATGATCTTGTGTTTGGTCAGACCTCGAATAATAATATCCT

TGCGCTCTAATTTCTTTCCAAAGAGCGAGGGTTGTAAAGTTTGTTATTGGTAATAAATTA 15481 ------+------+------+------+------+------+ 15540 ACGCGAGATTAAAGAAAGGTTTCTCGCTCCCAACATTTCAAACAATAACCATTATTTAAT

GTTATGACATACTCTGTAACTGATCACCTCATTGGCACCAGGCTTGGAAACACCTCCCAG 15541 ------+------+------+------+------+------+ 15600 CAATACTGTATGAGACATTGACTAGTGGAGTAACCGTGGTCCGAACCTTTGTGGAGGGTC

TGATGGAAATTTTATGGCCCGTAGCTGAACTCATTCCGCTGTCTGAGTTTCACTCCTAGC 15601 ------+------+------+------+------+------+ 15660 ACTACCTTTAAAATACCGGGCATCGACTTGAGTAAGGCGACAGACTCAAAGTGAGGATCG

TCCATCATGTCATAGCTATACTATCTGTTAGAAGAGCTGGTATGCTGTAGCCTTAGTTTC 15661 ------+------+------+------+------+------+ 15720 AGGTAGTACAGTATCGATATGATAGACAATCTTCTCGACCATACGACATCGGAATCAAAG

CTTTTCTGTAAGGTAAAATGGTAAGACATCCTGTCTCATAGGGTTATTGCATGAGCTGAA 15721 ------+------+------+------+------+------+ 15780 GAAAAGACATTCCATTTTACCATTCTGTAGGACAGAGTATCCCAATAACGTACTCGACTT

AAACATGTCTATAAAATGCTCACCTGGGGACCCTGCACATCATGAGCTTTCCCATCATGG 15781 ------+------+------+------+------+------+ 15840 TTTGTACAGATATTTTACGAGTGGACCCCTGGGACGTGTAGTACTCGAAAGGGTAGTACC

CTATAGAATTGGGTGTTCCTTACCCACAATGTGTTTCAGATTTCAGGACTTGGAATATTT 15841 ------+------+------+------+------+------+ 15900 GATATCTTAACCCACAAGGAATGGGTGTTACACAAAGTCTAAAGTCCTGAACCTTATAAA

Queensland Institute of Medical Research 243 Appendix 1: EphA1 genomic sequence contig

ATATAGACATATCTTAGGAATGGGACCTAATTCCATTTTTAAAAAGATTTATTATTTTAT 15901 ------+------+------+------+------+------+ 15960 TATATCTGTATAGAATCCTTACCCTGGATTAAGGTAAAAATTTTTCTAAATAATAAAATA

ATATATGAGTACATTGTCGTTATCTTGAGACACACCANGAAAGGGCATCAGATCCCATTA 15961 ------+------+------+------+------+------+ 16020 TATATACTCATGTAACAGCAATAGAACTCTGTGTGGTNCTTTCCCGTAGTCTAGGGTAAT

CAGATGGTTGTGAGCCACCAAGTGGTTGCTGTGAATTGAACTCAGGACCTNTGGAAGAGC 16021 ------+------+------+------+------+------+ 16080 GTCTACCAACACTCGGTGGTTCACCAACGACACTTAACTTGAGTCCTGGANACCTTCTCG

AGCCAGTGCTTTTAACCACTGAGCCATCTTTCCAGCCCCTGGACCTAGTTCTAATTATGA 16081 ------+------+------+------+------+------+ 16140 TCGGTCACGAAAATTGGTGACTCGGTAGAAAGGTCGGGGACCTGGATCAAGATTAATACT

AATTCATTTATGTTCTATATATGTAGCTTGAAGGAATTTTATACAATTTTTTTAGTTTGA 16141 ------+------+------+------+------+------+ 16200 TTAAGTAAATACAAGATATATACATCGAACTTCCTTAAAATATGTTAAAAAAATCAAACT

TTCCATTCTATTCTTNNTATTTTTTTACTGTGATTAATCTCATGAGATCATATATAGAGG 16201 ------+------+------+------+------+------+ 16260 AAGGTAAGATAAGAANNATAAAAAAATGACACTAATTAGAGTACTCTAGTATATATCTCC

TTCCCAGTGTGGCAGGCTATAGGAATTGTCANGTNTGGAAACTCTTTATGTNTTAAATTT 16261 ------+------+------+------+------+------+ 16320 AAGGGTCACACCGTCCGATATCCTTAACAGTNCANACCTTTGAGAAATACANAATTTAAA

TTGGATTAGAGGTAGCCATCCTATATTAAATCAAGAGTTATTTATTATACAGGACGAAGC 16321 ------+------+------+------+------+------+ 16380 AACCTAATCTCCATCGGTAGGATATAATTTAGTTCTCAATAAATAATATGTCCTGCTTCG

ATGGCTTAACATCATCCCTTCTTCTCCCCTAGGTAACAACTGTGGCAGcAGACCAGAGCT 16381 ------+------+------+------+------+------+ 16440 TACCGAATTGTAGTAGGGAAGAAGAGGGGATCCATTGTTGACACCGTCgTCTGGTCTCGA EXON IIIB V T T V A A D Q S F -

244 Queensland Institute of Medical Research Appendix 1: EphA1 genomic sequence contig

TCACCATCAGAGACCTGGCATCTGACTCTGTAAAGCTGAATGTAGAACGCTGcTCGTTGG 16441 ------+------+------+------+------+------+ 16500 AGTGGTAGTCTCTGGACCGTAGACTGAGACATTTCGACTTACATCTTGCGACgAGCAACC T I R D L A S D S V K L N V E R C S L G -

S m a I GCCACCTCACCCGCCGTGGcCTCTaCTTAGCTTTCCACAACCCGGGTTCCTGTGTGGcGC 16501 ------+------+------+------+------+------+ 16560 CGGTGGAGTGGGCGGCACCgGAGAtGAATCGAAAGGTGTTGGGCCCAAGGACACACCgCG H L T R R G L Y L A F H N P G S C V A L -

TAGTGTCTGTAAGGGTGTTCTACCAGCGCTGTGCCGAGACCGTGCATGGCTTGGCCCACT 16561 ------+------+------+------+------+------+ 16620 ATCACAGACATTCCCACAAGATGGTCGCGACACGGCTCTGGCACGTACCGAACCGGGTGA V S V R V F Y Q R C A E T V H G L A H F -

TCCCTGACACTCTCCCTGGACCTGGAGGGTTGGTTGAAGTAGCTGGAACGTGCCTCTCCC 16621 ------+------+------+------+------+------+ 16680 AGGGACTGTGAGAGGGACCTGGACCTCCCAACCAACTTCATCGACCTTGCACGGAGAGGG P D T L P G P G G L V E V A G T C L S H A -

P s t I ATGCACAGATCAGCTTGGGGTCCTCAGGTACACCACGAATGCACTGCAGCCCTGATGGCG 16681 ------+------+------+------+------+------+ 16740 TACGTGTCTAGTCGAACCCCAGGAGTCCATGTGGTGCTTACGTGACGTCGGGACTACCGC Q I S L G S S G T P R M H C S P D G E W -

AGTGGCTGGTGCCTGTGGGTCAGTGCCAGTGCGAGCCTGGCTATGAAGAAAGCAGTGGAA 16741 ------+------+------+------+------+------+ 16800 TCACCGACCACGGACACCCAGTCACGGTCACGCTCGGACCGATACTTCTTTCGTCACCTT L V P V G Q C Q C E P G Y E E S S G N V -

ATGTGGGATGCACTGGTAAGAAACGGGGAAGCGGGAAACCCCAGGGACCACCTGACTGGC 16801 ------+------+------+------+------+------+ 16860 TACACCCTACGTGACCATTCTTTGCCCCTTCGCCCTTTGGGGTCCCTGGTGGACTGACCG G C T A

Queensland Institute of Medical Research 245 Appendix 1: EphA1 genomic sequence contig

TCTTCCAGGTCCCCAAACAAGGCTTCCAGGATTTTTAACCTGAGTGGGATGTCTGTGGGA 16861 ------+------+------+------+------+------+ 16920 AGAAGGTCCAGGGGTTTGTTCCGAAGGTCCTAAAAATTGGACTCACCCTACAGACACCCT

GTGGGGGGCAGGGGTACGGGGGGGGGGGGGGCTAAGGGTTTTGTTCATCATGTAAAAACA 16921 ------+------+------+------+------+------+ 16980 CACCCCCCGTCCCCATGCCCCCCCCCCCCCCGATTCCCAAAACAAGTAGTACATTTTTGT

AAACAACCAAAAACATGGGCTGCCTTCTCTCTCCAGCCTGTCCTACTGGTTTCTATCGAG 16981 ------+------+------+------+------+------+ 17040 TTTGTTGGTTTTTGTACCCGACGGAAGAGAGAGGTCGGACAGGATGACCAAAGATAGCTC EXON IV C P T G F Y R V –

TGGACATGAATACACTCCGTTGTCTCAAGTGCCCCCAACATAGCATAGCAGAGTCTGAGG 17041 ------+------+------+------+------+------+ 17100 ACCTGTACTTATGTGAGGCAACAGAGTTCACGGGGGTTGTATCGTATCGTCTCAGACTCC D M N T L R C L K C P Q H S I A E S E G -

GGTCTACCATCTGTACCTGTGAGAATGGACATTATCGAGCCCCTGGGGAGGGTCCCCAGG 17101 ------+------+------+------+------+------+ 17160 CCAGATGGTAGACATGGACACTCTTACCTGTAATAGCTCGGGGACCCCTCCCAGGGGTCC S T I C T C E N G H Y R A P G E G P Q V -

TAGCATGCACACGTAAGTCTTGCAGTCGGGGCCTGGCGATCTGGTAACAGGTGCTAGACC 17161 ------+------+------+------+------+------+ 17220 ATCGTACGTGTGCATTCAGAACGTCAGCCCCGGACCGCTAGACCATTGTCCACGATCTGG A C T R

CTCCTTGGGACCAGAATTTCCAGAGCTTACCAAGTAATGGAGACCCAGGTAGTCAGACAC 17221 ------+------+------+------+------+------+ 17280 GAGGAACCCTGGTCTTAAAGGTCTCGAATGGTTCATTACCTCTGGGTCCATCAGTCTGTG

X b a I TGATTATAGCCACTCCCTGCCCTCTGTCCTCTCTAGAATGCAGAGGCATGTTACATTCTA 17281 ------+------+------+------+------+------+ 17340 ACTAATATCGGTGAGGGACGGGAGACAGGAGAGATCTTACGTCTCCGTACAATGTAAGAT

246 Queensland Institute of Medical Research Appendix 1: EphA1 genomic sequence contig

CTTCCTGTTTGGTGGAAGTTTAATCTCTCATGAGGGCTTTGGGAGACATAATTGGGATGA 17341 ------+------+------+------+------+------+ 17400 GAAGGACAAACCACCTTCAAATTAGAGAGTACTCCCGAAACCCTCTGTATTAACCCTACT

TAGAAGCAGCGGTCAGTGGCTTGGGTGAGAGGAAATGGACACCTATTTGATAACTCCTCT 17401 ------+------+------+------+------+------+ 17460 ATCTTCGTCGCCAGTCACCGAACCCACTCTCCTTTACCTGTGGATAAACTATTGAGGAGA

TCTCCCACAGGTCCCCCATCGGCTCCCCAAAATCTGAGCTTCTCCACATCAGGGACTCAA 17461 ------+------+------+------+------+------+ 17520 AGAGGGTGTCCAGGGGGTAGCCGAGGGGTTTTAGACTCGAAAAGGTGTAGTCCCTGAGTT EXON V P P S A P Q N L S F S T S G T Q -

E c o R V CTCTCCCTGCGCTGGGAGCCCCCCAGAGATACAGGGGGACGCCATGATATCAGATACAGC 17521 ------+------+------+------+------+------+ 17580 GAAAGGGACGCGACCCTCGGGGGGTCTCTATGTCCCCCTGCGGTACTATAGTCTATGTCG L S L R W E P P R D T G G R H D I R Y S -

GTGGAGTGCTTGCAGTGTCGGGGCATTGCACAGGATGGGGGTCCCTGCCAACCCTGTGGA 17581 ------+------+------+------+------+------+ 17640 CACCTCACGAACGTCACAGCCCCGTAACGTGTCCTACCCCCAGGGACGGTTGGGACACCT V E C L Q C R G I A Q D G G P C Q P C G -

AAAGGTGTGCACTTTTCCCCGGCTGCTTCCGGGCTCACCACATCTACCGTGCAAGTGCAA 17641 ------+------+------+------+------+------+ 17700 TTTCCACACGTGAAAAGGGGCCGACGAAGGCCCGAGTGGTGTAGATGGCACGTTCACGTT K G V H F S P A A S G L T T S T V Q V Q - X h o I GGCCTCGAGCCTTACGCCAACTACACATTTACCGTCAAATCCCAAAACAGAGTGTCAGGA 17701 ------+------+------+------+------+------+ 17760 CCGGAGCTCGGAATGCGGTTGATGTGTAAATGGCAGTTTAGGGTTTTGTCTCACAGTCCT G L E P Y A N Y T F T V K S Q N R V S G -

Queensland Institute of Medical Research 247 Appendix 1: EphA1 genomic sequence contig

CTGGACAGTTCCAGCCCTAGCAGCGCCTCCCTCAGTATCAACATGGGGCACGCAGGTGAG 17761 ------+------+------+------+------+------+ 17820 GACCTGTCAAGGTCGGGATCGTCGCGGAGGGAGTCATAGTTGTACCCCGTGCGTCCACTC L D S S S P S S A S L S I N M G H A

GCACCTAAGACCATAGGGCTGAGTGATGGGCAGGAAGCAGCGGAGCTACAGCTGACGTTA 17821 ------+------+------+------+------+------+ 17880 CGTGGATTCTGGTATCCCGACTCACTACCCGTCCTTCGTCGCCTCGATGTCGACTGCAAT

TTCCTTCTTACCTTCCGAACTCAACCTTGTCGTTCCGGGTGTCCAGAGTCACTCTCTGGC 17881 ------+------+------+------+------+------+ 17940 AAGGAAGAATGGAAGGCTTGAGTTGGAACAGCAAGGCCCACAGGTCTCAGTGAGAGACCG EXON VI E S L S G -

CTGTCACTGAAGCTGGTGAAGAAAGAACCGAGGCAGCTGGAGCTGACTTGGGCAGGGTCC 17941 ------+------+------+------+------+------+ 18000 GACAGTGACTTCGACCACTTCTTTCTTGGCTCCGTCGACCTCGACTGAACCCGTCCCAGG

L S L K L V K K E P R Q L E L T W A G S - CGACCCCGAAATCCTGGAGGGAATCTGAGCTATGAGCTGCACGTGCTGAATCAGGTCAGA 18001 ------+------+------+------+------+------+ 18060 GCTGGGGCTTTAGGACCTCCCTTAGACTCGATACTCGACGTGCACGACTTAGTCCAGTCT R P R N P G G N L S Y E L H V L N

AAACACTTGCAGATACCATTCAGTGTTTGGGGTATAGCCTAGCTTCCTAACTTGGGGTAT 18061 ------+------+------+------+------+------+ 18120 TTTGTGAACGTCTATGGTAAGTCACAAACCCCATATCGGATCGAAGGATTGAACCCCATA

AGGATGGAGGGAAATATCCAAAACAAACTTTCTGAGGCTAAACATAACCCTGCAAAGGAT 18121 ------+------+------+------+------+------+ 18180 TCCTACCTCCCTTTATAGGTTTTGTTTGAAAGACTCCGATTTGTATTGGGACGTTTCCTA

TCTGACCTGGACTGGACAGCNCCACCCTGTCCAGGCCCTTCCATGTCTATCTTCTCTACC 18181 ------+------+------+------+------+------+ 18240 AGACTGGACCTGACCTGTCGNGGTGGGACAGGTCCGGGAAGGTACAGATAGAAGAGATGG

248 Queensland Institute of Medical Research Appendix 1: EphA1 genomic sequence contig

AAGCCTCAGCACAGCCTCATTAATGCAAGTCCTACCCACAGGACGAAGAATGGCACCAGA 18241 ------+------+------+------+------+------+ 18300 TTCGGAGTCGTGTCGGAGTAATTACGTTCAGGATGGGTGTCCTGCTTCTTACCGTGGTCT EXON VIII Q D E E W H Q M -

TGGTGTTGGAACCCAGGGTCTTGCTGACAAAACTTCAGCCAGATACCACATACATTGTCA 18301 ------+------+------+------+------+------+ 18360 ACCACAACCTTGGGTCCCAGAACGACTGTTTTGAAGTCGGTCTATGGTGTATGTAACAGT V L E P R V L L T K L Q P D T T Y I V R -

GAGTGCGAACACTGACCCCACTGGGGCCTGGCCCTTTCTCCCCTGACCATGAGTTTCGGA 18361 ------+------+------+------+------+------+ 18420 CTCACGCTTGTGACTGGGGTGACCCCGGACCGGGAAAGAGGGGACTGGTACTCAAAGCCT V R T L T P L G P G P F S P D H E F R T -

CAAGCCCACCAGGTAGGTTACCAATCTCTTGTCCCACCCGAACATACACAATTGTTATCT 18421 ------+------+------+------+------+------+ 18480 GTTCGGGTGGTCCATCCAATGGTTAGAGAACAGGGTGGGCTTGTATGTGTTAACAATAGA S P P

CCTGGAGATAGTTCAGGTCCTATTTTTAAGTGACCCTTGCTCTTCTCTCATGCACGTAAT 18481 ------+------+------+------+------+------+ 18540 GGACCTCTATCAAGTCCAGGATAAAAATTCACTGGGAACGAGAAGAGAGTACGTGCATTA

GGGCCCATTCAGTAATCTCTGTTAAGGGAAACATGGACCCAATGTCCACTTAAAAACTGG 18541 ------+------+------+------+------+------+ 18600 CCCGGGTAAGTCATTAGAGACAATTCCCTTTGTACCTGGGTTACAGGTGAATTTTTGACC

CCGTGGGTATACTTGGGACCCTACCTTTTAGCCATTATAACCTCTAACGGCTGTGAGCCC 18601 ------+------+------+------+------+------+ 18660 GGCACCCATATGAACCCTGGGATGGAAAATCGGTAATATTGGAGATTGCCGACACTCGGG

TNAGCCTTCTGAACTCTGACACCCAGTTTCCAGAAGCCTGACCGGAGGAGAGATTGTGGC 18661 ------+------+------+------+------+------+ 18720 ANTCGGAAGACTTGAGACTGTGGGTCAAAGGTCTTCGGACTGGCCTCCTCTCTAACACCG EXON IX V S R S L T G G E I V A –

Queensland Institute of Medical Research 249 Appendix 1: EphA1 genomic sequence contig

CGTCATCTTTGGATTGCTGCTTGGAATAGCTCTGCTGATCGGGATTTATGTCTTCCGTTC 18721 ------+------+------+------+------+------+ 18780 GCAGTAGAAACCTAACGACGAACCTTATCGAGACGACTAGCCCTAAATACAGAAGGCAAG V I F G L L L G I A L L I G I Y V F R S -

AAGGTGCTGGCTCTGTCTTCCCCATCCATTCATCTGGGATTCCTTTGCCTTGTTAGGCTG 18781 ------+------+------+------+------+------+ 18840 TTCCACGACCGAGACAGAAGGGGTAGGTAAGTAGACCCTAAGGAAACGGAACAATCCGAC R

TACCCCCTAATGATTCTCCTGTACCCTAAAGTTTAGACATGCCCCACACCCAGCTCTCGT 18841 ------+------+------+------+------+------+ 18900 ATGGGGGATTACTAAGAGGACATGGGATTTCAAATCTGTACGGGGTGTGGGTCGAGAGCA

S a c I GTCTGTGGCAAATTCAGCCAGAGCTCATTGGAGTTTCCCTGAGTCCCTGAAAAAGTTGCC 18901 ------+------+------+------+------+------+ 18960 CAGACACCGTTTAAGTCGGTCTCGAGTAACCTCAAAGGGACTCAGGGACTTTTTCAACGG

CTACTTCCACAGGAGAGGCCAGAGACAGAGACAGCAGAGGCAGCGTGAACGCACCACCAA 18961 ------+------+------+------+------+------+ 19020 GATGAAGGTGTCCTCTCCGGTCTCTGTCTCTGTCGTCTCCGTCGCACTTGCGTGGTGGTT EXON X R R G Q R Q R Q Q R Q R E R T T N -

TGTCGATCGAGGTGAGCCAGGAGTGTGAAGGGGGGGGGGGGGAGATTTTGTGTGTGTGTG 19021 ------+------+------+------+------+------+ 19080 ACAGCTAGCTCCACTCGGTCCTCACACTTCCCCCCCCCCCCCTCTAAAACACACACACAC V D R

TGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGACACCCAGATCAGCTCTAGG 19081 ------+------+------+------+------+------+ 19140 ACACACACACACACACACACACACACACACACACACACACTGTGGGTCTAGTCGAGATCC

AATGTGGGTGCAGTATACTGTCCCTAAGGATCTTGTGTTTTGCCACCTAATTAATTTTGG 19141 ------+------+------+------+------+------+ 19200 TTACACCCACGTCATATGACAGGGATTCCTAGAACACAAAACGGTGGATTAATTAAAACC

250 Queensland Institute of Medical Research Appendix 1: EphA1 genomic sequence contig

TTAATTAACTCTGCCCTGATAGCTGTCAGCAGCTCTGATGCAATTATCTACTAGCTCTTG 19201 ------+------+------+------+------+------+ 19260 AATTAATTGAGACGGGACTATCGACAGTCGTCGAGACTACGTTAATAGATGATCGAGAAC

K p n I GTACCAGTGCAGTTACCCTGGGCTCTGGACCATGTTCTCCATCCTGGTGGCCAGGTCTAC 19261 ------+------+------+------+------+------+ 19320 CATGGTCACGTCAATGGGACCCGAGACCTGGTACAAGAGGTAGGACCACCGGTCCAGATG

GGCTAAGACCAGGCAGGAAGAGGTTGAAGTCCTCTAAGACGTGCAGGCTTCCTGCTTGTC 19321 ------+------+------+------+------+------+ 19380 CCGATTCTGGTCCGTCCTTCTCCAACTTCAGGAGATTCTGCACGTCCGAAGGACGAACAG

ACCTCATTAGTTGTCTTTGCCAGAGGGAGCATTTGCTGTCTATCTGAATAGCTGGCACTG 19381 ------+------+------+------+------+------+ 19440 TGGAGTAATCAACAGAAACGGTCTCCCTCGTAAACGACAGATAGACTTATCGACCGTGAC

AAGGGAGGTTTCATCTGGGAGTGAATGAGCTGTAAGTGACTTGGACAGCATGGGGCCAGA 19441 ------+------+------+------+------+------+ 19500 TTCCCTCCAAAGTAGACCCTCACTTACTCGACATTCACTGAACCTGTCGTACCCCGGTCT

AGCTGGGTCACAAGTCATAGGAAGATAGGATAACTCAGAGCGGGGAATTTACTGCAGGAT 19501 ------+------+------+------+------+------+ 19560 TCGACCCAGTGTTCAGTATCCTTCTATCCTATTGAGTCTCGCCCCTTAAATGACGTCCTA

E c o R I CTGAATTCTGGCCACTGCTGGACCCTGAACAGTAGGACAGAAAGACATAGAAAATAGACA 19561 ------+------+------+------+------+------+ 19620 GACTTAAGACCGGTGACGACCTGGGACTTGTCATCCTGTCTTTCTGTATCTTTTATCTGT

Queensland Institute of Medical Research 251 Appendix 1: EphA1 genomic sequence contig

AGAAAAAGGCTGGGGTCCACGTGGAGGCTTTATTGCTAAGAATAGAGATTTTCTCTGCTT 19621 ------+------+------+------+------+------+ 19680 TCTTTTTCCGACCCCAGGTGCACCTCCGAAATAACGATTCTTATCTCTAAAAGAGACGAA

GAGCTAAAGCCAGGCTCACCTCAGAGAGCAAAGAAGTACAGGGAACGGGATCAGCTGTCT 19681 ------+------+------+------+------+------+ 19740 CTCGATTTCGGTCCGAGTGGAGTCTCTCGTTTCTTCATGTCCCTTGCCCTAGTCGACAGA

GCCCTTCTTCCTAGCCCCCCATGCTCTGATCCTTGTGCTACAGTGGCTGAGCCCCCTTGT 19741 ------+------+------+------+------+------+ 19800 CGGGAAGAAGGATCGGGGGGTACGAGACTAGGAACACGATGTCACCGACTCGGGGGAACA

CCCCCCCAGAGGACAAGCTGTGGCTAAAACCCTATGTGGACCTCCAGGCCTATGAGGACC 19801 ------+------+------+------+------+------+ 19860 GGGGGGGTCTCCTGTTCGACACCGATTTTGGGATACACCTGGAGGTCCGGATACTCCTGG EXON XI E D K L W L K P Y V D L Q A Y E D P -

CTGCACAGGGAGCCTTAGACTTTGCCCAGGAACTGGACCCAGCCTGGCTGATTGTGGACA 19861 ------+------+------+------+------+------+ 19920 GACGTGTCCCTCGGAATCTGAAACGGGTCCTTGACCTGGGTCGGACCGACTAACACCTGT A Q G A L D F A Q E L D P A W L I V D T -

S p e I CTGTCATAGGAGAAGGTGAGCCCTGCTGTGACCCCAACTAGTCCTCTCTGTGTGGTAGGT 19921 ------+------+------+------+------+------+ 19980 GACAGTATCCTCTTCCACTCGGGACGACACTGGGGTTGATCAGGAGAGACACACCATCCA V I G E G

AGCACATGGCTGCTACTAGGTTCCTTTCTTGGAGCCTTGAGTTCCCACATGGGCTAATGC 19981 ------+------+------+------+------+------+ 20040 TCGTGTACCGACGATGATCCAAGGAAAGAACCTCGGAACTCAAGGGTGTACCCGATTACG

CTGCTCTATCCTGTATTCTTGAATTTCTATGCCCTGGAGCAGCCTCTGCCTCCTACACAC 20041 ------+------+------+------+------+------+ 20100 GACGAGATAGGACATAAGAACTTAAAGATACGGGACCTCGTCGGAGACGGAGGATGTGTG

252 Queensland Institute of Medical Research Appendix 1: EphA1 genomic sequence contig

TTTGCTCTGTGGTTATGGGGTTTCTGATAAAGAATGTTAAACACACCATCCCTACCCATA 20101 ------+------+------+------+------+------+ 20160 AAACGAGACACCAATACCCCAAAGACTATTTCTTACAATTTGTGTGGTAGGGATGGGTAT

GAAGTGTCACCATGCCAAGGTGCAGGTTGTTCTCAGAGGATTCCCAAAGCAGGAGAGACT 20161 ------+------+------+------+------+------+ 20220 CTTCACAGTGGTACGGTTCCACGTCCAACAAGAGTCTCCTAAGGGTTTCGTCCTCTCTGA

CAGAGCCTCCCTAAGTTTAGAGCATTCTACCCCATCAGGGGCCCAGTGTCCTTGGGCTGC 20221 ------+------+------+------+------+------+ 20280 GTCTCGGAGGGATTCAAATCTCGTAAGATGGGGTAGTCCCCGGGTCACAGGAACCCGACG

H i B n g d l I I I I I CTCAATCCCCTGGGTCACCAAGGAAAATAGATCTCAACAGAGAACAAAGCTTGGCAGTGT 20281 ------+------+------+------+------+------+ 20340 GAGTTAGGGGACCCAGTGGTTCCTTTTATCTAGAGTTGTCTCTTGTTTCGAACCGTCACA

S a c I CTTGTCAGTGGTGCGACTGAGCCAGCAAGAGCTGTGACCAGGGAGCTCACTCACAGGGAA 20341 ------+------+------+------+------+------+ 20400 GAACAGTCACCACGCTGACTCGGTCGTTCTCGACACTGGTCCCTCGAGTGAGTGTCCCTT

CAGGCAGGCCTCCTGTTCCTTGGGCACTAGCCTTGGACCAACTTATCCTTCATAAACACA 20401 ------+------+------+------+------+------+ 20460 GTCCGTCCGGAGGACAAGGAACCCGTGATCGGAACCTGGTTGAATAGGAAGTATTTGTGT

GTATGTAGTTACTAAATACTTAACATAAGCCAAGGAGCATGCTAGCCATGGAAGACAGTA 20461 ------+------+------+------+------+------+ 20520 CATACATCAATGATTTATGAATTGTATTCGGTTCCTCGTACGATCGGTACCTTCTGTCAT

Queensland Institute of Medical Research 253 Appendix 1: EphA1 genomic sequence contig

X b a I CTGGTTAGATTGCAGAGCTTCATGCTGATTCCATAATGTCCCTTCTCTAGAAGGGCAACC 20521 ------+------+------+------+------+------+ 20580 GACCAATCTAACGTCTCGAAGTACGACTAAGGTATTACAGGGAAGAGATCTTCCCGTTGG

S a c I TGAGCTCTGTCCTTCCTCAGATTTCCAACCTTATTTTCCTCTCATCTAGGGGAGTTTGGT 20581 ------+------+------+------+------+------+ 20640 ACTCGAGACAGGAAGGAGTCTAAAGGTTGGAATAAAAGGAGAGTAGATCCCCTCAAACCA EXON XII E F G -

GAAGTGTATCGGGGAGCCCTGAGACTCCCCAGCCAAGATTGCAAGACTGTGGCCATTAAG 20641 ------+------+------+------+------+------+ 20700 CTTCACATAGCCCCTCGGGACTCTGAGGGGTCGGTTCTAACGTTCTGACACCGGTAATTC E V Y R G A L R L P S Q D C K T V A I K -

ACCTTGAAAGATACATCCCCAGATGGCTACTGGTGGAATTTCCTTCGAGAGGCAACTATC 20701 ------+------+------+------+------+------+ 20760 TGGAACTTTCTATGTAGGGGTCTACCGATGACCACCTTAAAGGAAGCTCTCCGTTGATAG T L K D T S P D G Y W W N F L R E A T I -

ATGGGCCAGTTCAACCACCCACACATTCTACGCCTAGAAGGTGTCATCACAAAAAGTATG 20761 ------+------+------+------+------+------+ 20820 TACCCGGTCAAGTTGGTGGGTGTGTAAGATGCGGATCTTCCACAGTAGTGTTTTTCATAC M G Q F N H P H I L R L E G V I T K R

TGCATTGGTCCCCAAGAACTGGAGTGAAATGTGGGGGAGGGGTAGGAGCAGTGGGGGAGG 20821 ------+------+------+------+------+------+ 20880 ACGTAACCAGGGGTTCTTGACCTCACTTTACACCCCCTCCCCATCCTCGTCACCCCCTCC

GGAGTAGGCTGNCGAGGAAACCTGGGTCTGANTGTGATGCATGTGCTTGTGTCCCAAGGA 20881 ------+------+------+------+------+------+ 20940 CCTCATCCGACNGCTCCTTTGGACCCAGACTNACACTACGTACACGAACACAGGGTTCCT EXON XII R -

254 Queensland Institute of Medical Research Appendix 1: EphA1 genomic sequence contig

AAGCCAATCATGATCATCACAGAATTTATGGAAAATGGAGCCCTGGATGCCTTTCTGAAG 20941 ------+------+------+------+------+------+ 21000 TTCGGTTAGTACTAGTAGTGTCTTAAATACCTTTTACCTCGGGACCTACGGAAAGACTTC K P I M I I T E F M E N G A L D A F L K -

X b a I GTGAGACCGGCAGAGGATCAGTTCTAGAGGGGTCACATGATGGAGCCTCATGTCTTGCAG 21001 ------+------+------+------+------+------+ 21060 CACTCTGGCCGTCTCCTAGTCAAGATCTCCCCAGTGTACTACCTCGGAGTACAGAACGTC

GAGACTTCATCTTCTGATGTATGCATTCCCCCTACTTACAGGAACGGGAGGACCAACTAG 21061 ------+------+------+------+------+------+ 21120 CTCTGAAGTAGAAGACTACATACGTAAGGGGGATGAATGTCCTTGCCCTCCTGGTTGATC EXON XIII E R E D Q L A -

CTCCTGGTCAGCTAGTGGCTATGCTACTGGGCATAGCATCAGGCATGAACTGCCTCAGTG 21121 ------+------+------+------+------+------+ 21180 GAGGACCAGTCGATCACCGATACGATGACCCGTATCGTAGTCCGTACTTGACGGAGTCAC P G Q L V A M L L G I A S G M N C L S G -

GCCACAATTATGTCCATAGAGACCTGGCTGCCAGGAACATCTTGGTGAATCAGAACCTGT 21181 ------+------+------+------+------+------+ 21240 CGGTGTTAATACAGGTATCTCTGGACCGACGGTCCTTGTAGAACCACTTAGTCTTGGACA H N Y V H R D L A A R N I L V N Q N L C -

GCTGCAAGGTGTCTGACTTTGGCTTGACCCGCCTCCTGGATGACTTTGACGGCACCTATG 21241 ------+------+------+------+------+------+ 21300 CGACGTTCCACAGACTGAAACCGAACTGGGCGGAGGACCTACTGAAACTGCCGTGGATAC c C K V S D F G L T R L L D D F D G T Y E -

AAACCCAGGTTAGAGCCCCATCCACACTCATATGAACGCTTCATCATTTGCTGACCCGCG 21301 ------+------+------+------+------+------+ 21360 TTTGGGTCCAATCTCGGGGTAGGTGTGAGTATACTTGCGAAGTAGTAAACGACTGGGCGC T Q

Queensland Institute of Medical Research 255 Appendix 1: EphA1 genomic sequence contig

S p e I CACAAGCATGAGATTTCCTTTCCAGTTTTGTACTAGTCCACGCCCTGAAACACCACTCTC 21361 ------+------+------+------+------+------+ 21420 GTGTTCGTACTCTAAAGGAAAGGTCAAAACATGATCAGGTGCGGGACTTTGTGGTGAGAG

CTGTGCAATGCCTTCTTAGGGAGGAAAGATCCCCATCCGATGGACAGCCCCAGAAGCTAT 21421 ------+------+------+------+------+------+ 21480 GACACGTTACGGAAGAATCCCTCCTTTCTAGGGGTAGGCTACCTGTCGGGGTCTTCGATA EXON XIV G G K I P I R W T A P E A I -

TGCCCATCGGATCTTCACCACAGCCAGTGATGTGTGGAGCTTTGGGATTGTAATGTGGGA 21481 ------+------+------+------+------+------+ 21540 ACGGGTAGCCTAGAAGTGGTGTCGGTCACTACACACCTCGAAACCCTAACATTACACCCT A H R I F T T A S D V W S F G I V M W E -

GGTGTTGAGTTTTGGCGACAAACCCTATGGGCCAGATGAGCAACCAAGAGGTAAACCTGG 21541 ------+------+------+------+------+------+ 21600 CCACAACTCAAAACCGCTGTTTGGGATACCCGGTCTACTCGTTGGTTCTCCATTTGGACC V L S F G D K P Y G E M S N Q E V

ACTATTTCCAGATGCCATCCGCAATACAACTCCTGTCTCTTCTAACCCCACCCCCACCCC 21601 ------+------+------+------+------+------+ 21660 TGATAAAGGTCTACGGTAGGCGTTATGTTGAGGACAGAGAAGATTGGGGTGGGGGTGGGG

CTCACACCCACAGTCTTTTCCTATGAGTATTTCAACTCCAACCTTTGGCACTATTTCCTG 21661 ------+------+------+------+------+------+ 21720 GAGTGTGGGTGTCAGAAAAGGATACTCATAAAGTTGAGGTTGGAAACCGTGATAAAGGAC

TGCTCCCTTCTCTGCTACTTCTTTGCTCTCCTGGTCAGGGCTGCTTACAGGTGACATCTC 21721 ------+------+------+------+------+------+ 21780 ACGAGGGAAGAGACGATGAAGAAACGAGAGGACCAGTCCCGACGAATGTCCACTGTAGAG

AATCATCCTGTGTAAGTGGCTTTCTAGTAGATCCATCTAACTTCCTACCTGGACATCTCT 21781 ------+------+------+------+------+------+ 21840 TTAGTAGGACACATTCACCGAAAGATCATCTAGGTAGATTGAAGGATGGACCTGTAGAGA

256 Queensland Institute of Medical Research Appendix 1: EphA1 genomic sequence contig

K p n I CTGTCTCTTGCTACAGGTAATGAAAAGCATTGAAGATGGGTACCGGTTGCCCCCTTCCTG 21841 ------+------+------+------+------+------+ 21900 GACAGAGAACGATGTCCATTACTTTTCGTAACTTCTACCCATGGCCAACGGGGGAAGGAC EXON XV V M K S I E D G Y R L P P P V -

TGGACTTGTCCTGCCCCTCTCTATGAACTCCATGAAGAACTGCTGGGCTTACGATCGTGC 21901 ------+------+------+------+------+------+ 21960 ACCTGAACAGGACGGGGAGAGATACTTGAGGTACTTCTTGACGACCCGAATGCTAGCACG W T C P A P L Y E L M K N C W A Y D R A -

S A L I CCGTCGACCCCACTTCCTCCAGCTGCAGGCACATCTGGAACAGTTGCTTACTGACCCCCA 21961 ------+------+------+------+------+------+ 22020 GGCAGCTGGGGTGAAGGAGGTCGACGTCCGTGTAGACCTTGTCAACGAATGACTGGGGGT R R P H F L Q L Q A H L E Q L L T D P H -

TTCCCTAAGGACAATTGCCAACTTTGACCCTAGGTAAATATGTTCAAGAAGGAAGGGAGT 22021 ------+------+------+------+------+------+ 22080 AAGGGATTCCTGTTAACGGTTGAAACTGGGATCCATTTATACAAGTTCTTCCTTCCCTCA S L R T I A N F D P R

TTGGAGGTCAAATCTGGAAAAATAAGATGGACATTCCATCCTATCCACTGAGTGAGTGTT 22081 ------+------+------+------+------+------+ 22140 AACCTCCAGTTTAGACCTTTTTATTCTACCTGTAAGGTAGGATAGGTGACTCACTCACAA S a c I GGGCCCCAGGGAGCTCTGGACCGAATCCAGCATGAGACAGCACCCAGTCCATGCCTTGTG 22141 ------+------+------+------+------+------+ 22200 CCCGGGGTCCCTCGAGACCTGGCTTAGGTCGTACTCTGTCGTGGGTCAGGTACGGAACAC

Queensland Institute of Medical Research 257 Appendix 1: EphA1 genomic sequence contig

GGGAAGCTCAGGCTAATGGAGGTGGGAACCTGATGAAAGGTAAGAAGCAGAGAGCACATG 22201 ------+------+------+------+------+------+ 22260 CCCTTCGAGTCCGATTACCTCCACCCTTGGACTACTTTCCATTCTTCGTCTCTCGTGTAC

ATTCAAGGAGTCAGACCTAGATTAACCACCTCTCACTGTGTTCCTCCTGGAACCTTCAGA 22261 ------+------+------+------+------+------+ 22320 TAAGTTCCTCAGTCTGGATCTAATTGGTGGAGAGTGACACAAGGAGGACCTTGGAAGTCT

GTCATTCTTCTTGTCACCCCCACTATGTTAATTAATGACTGGGACACCACCTCTGGTGTT 22321 ------+------+------+------+------+------+ 22380 CAGTAAGAAGAACAGTGGGGGTGATACAATTAATTACTGACCCTGTGGTGGAGACCACAA

X b a I CCTCTGACCATCTAGAATTTGGTATATTTTCCTTCCCCCACATATATATTTTCAAACATG 22381 ------+------+------+------+------+------+ 22440 GGAGACTGGTAGATCTTAAACCATATAAAAGGAAGGGGGTGTATATATAAAAGTTTGTAC H i n S d p I e I I I AAGGAAAGATTCACAGATGTCACATATCTTGGAGGACCTACTAGTACAAGCTTTTGGCTA 22441 ------+------+------+------+------+------+ 22500 TTCCTTTCTAAGTGTCTACAGTGTATAGAACCTCCTGGATGATCATGTTCGAAAACCGAT

CAGCCTGGAACCTACTCCTGAGAGCATAGGCAGCTAGATTAGGGTAGGAACAGGAAGGAG 22501 ------+------+------+------+------+------+ 22560 GTCGGACCTTGGATGAGGACTCTCGTATCCGTCGATCTAATCCCATCCTTGTCCTTCCTC

E S c t o u R I I TCTGAGGAGGCCTCTGACTACATGGCCAGTCGGCCAGGGCTGAGAGACGACAGATCCGAA 22561 ------+------+------+------+------+------+ 22620 AGACTCCTCCGGAGACTGATGTACCGGTCAGCCGGTCCCGACTCTCTGCTGTCTAGGCTT

258 Queensland Institute of Medical Research Appendix 1: EphA1 genomic sequence contig

S Na S X oc a b tI l a II I I TTCGTTCCCTTTAGTGAGGGTTAATTCCGCGGCCGCGTCGACAGCTCTAGAGATCCGCCT 22621 ------+------+------+------+------+------+ 22680 AAGCAAGGGAAATCACTCCCAATTAAGGCGCCGGCGCAGCTGTCGAGATCTCTAGGCGGA

ACCTTTCACGAGTTGCGCAGTTTGTCTGCAAGACTCTATGAGAAGCAGATAAGCGATAAG 22681 ------+------+------+------+------+------+ 22740 TGGAAAGTGCTCAACGCGTCAAACAGACGTTCTGAGATACTCTTCGTCTATTCGCTATTC

TTTGCTCAACATCTTCTCGGGCATAAGTCGGACACCATGGCATCACAGTATCGTGATGAC 22741 ------+------+------+------+------+------+ 22800 AAACGAGTTGTAGAAGAGCCCGTATTCAGCCTGTGGTACCGTAGTGTCATAGCACTACTG

AGAGGCAGGGAGTGGGACAAAATTGAAATCAAATAATGATTTTATTTTGACTGATAGTGA 22801 ------+------+------+------+------+------+ 22860 TCTCCGTCCCTCACCCTGTTTTAACTTTAGTTTATTACTAAAATAAAACTGACTATCACT

CCTGTCGTTGCAACAAATTGATAAGCAATGCTTTTTTATAATGNCAACTTAGTATAAAAA 22861 ------+------+------+------+------+------+ 22920 GGACAGCAACGTTGTTTAACTATTCGTTACGAAAAAATATTACNGTTGAATCATATTTTT

AAGCAGGCTTCATCCGGATTCTCTGAGCCCACCGNGGTCACCAACCACCGNGGTGCTGGT 22921 ------+------+------+------+------+------+ 22980 TTCGTCCGAAGTAGGCCTAAGAGACTCGGGTGGCNCCAGTGGTTGGTGGCNCCACGACCA

ACGTCTGGCTTTCAAGCTGAATGGTGCAGTTCTGGACCGGGTTTCCTGNGGCCGCTTTTN 22981 ------+------+------+------+------+------+ 23040 TGCAGACCGAAAGTTCGACTTACCACGTCAAGACCTGGCCCAAAGGACNCCGGCGAAAAN

AGGACTNCTNGAANCTTTACTGGCATATTCACCCACCAAAAAAGCCCACCGGTTCCCGGC 23041 ------+------+------+------+------+------+ 23100 TCCTGANGANCTTNGAAATGACCGTATAAGTGGGTGGTTTTTTCGGGTGGCCAAGGGCCG

Queensland Institute of Medical Research 259 Appendix 1: EphA1 genomic sequence contig

K p n I GGCTTGGCATAACACTTGGGGTACCTGGNTTAATCAGAATTTATAACCCGNCCCC 23101 ------+------+------+------+------+----- 23155 CCGAACCGTATTGTGAACCCCATGGACCNAATTAGTCTTAAATATTGGGCNGGGG

Enzymes that do cut:

AccI AflIII ApaI BamHI BglII Bsp106I BspHI BstXI ClaI EagI EcoRI EcoRV HincII HindIII KpnI MluI NaeI NarI NcoI NdeI NotI NruI NsiI NspI PstI PvuI PvuII SacI SacII SalI ScaI SmaI SpeI SphI SspI StuI XbaI XhoI XmnI

Enzymes that do not cut:

HpaI SplI

260 Queensland Institute of Medical Research Appendix 2: Eph/ephrin protein signalling pathways

APPENDIX 2 EPH/EPHRIN SIGNALLING PATHWAYS

Binding Signalling Eph/ephrin Signalling Outcome of the site/ Reference molecule receptor pathway signal domain

AF6 EphA3, 4, 6, 7 C-terminus Scaffold protein Mediates clustering (Buchert et six amino which recruits Ras of Ephs at sites cell- al., 1999; EphB2, 3, 5, 6 acids family proteins cell contact Hock et al., including 1998b) PDZ motif Abl/Arg EphA4 SH2 Bind to p62dok, Src Regulates axon (Harbott and family kinases & growth & guidance Nobes, 2005; EphB2 Y605, 611 p120Ras GAP; ? Yu et al., Ena/VASP proteins 2001)

Axin EphB2 SH3 Binds SH3 domain Regulates β-catenin (Cowan and of Grb4 stability → controls Henkemeyer, Wnt signalling 2001) Axon remodelling & cell de-adhesion CAP EphB2 SH3 Binds SH3 domain Regulates actin (Cowan and of Grb4 stress fibres & focal Henkemeyer, adhesion → cell de- 2001) adhesion

Cbl EphA ? T cells ↑ ubiquitination and (Sharfe et al., proteosomal 2003) Pathway includes degradation CrkII

Cdc42 EphB3 ? Rac1/Cdc42 versus ↑ formation (Miao et al., RhoA activity lamellipodia & 2005) influences cell filopodia (kinase motility & adhesion independent)

Crk EphB3 Y614/SH2 Adaptor binds GEFs ↑ cell rounding & (Hock et al., 1998a) → RhoA activation detachment (Lawrenson et EphA3/ephrin- al., 2002; Co-localises with CAS A5 ↑ membrane Nagashima et at focal complexes ruffling al., 2002)

Ephexin EphA4 DPH ↑ RhoA & ↓ ↑ axon extension, ↓ (Sahin et al., domain Rac1/Cdc42; growth cone 2005; Shamah et al., 2001; within KD collapse Winning et al., 2002)

FAK EphA2 SHP2 ↓ FAK ↑ cell rounding & de- (Carter et al., phosphatase phosphorylation & adhesion PC3 cells 2002; Miao → inactivates inactivation; ↓ paxillin FAK et al., 2000) activation; requires ↑ fibroblast adhesion p130cas & RhoA

Queensland Institute of Medical Research 261 Appendix 2: Eph/ephrin signalling pathways

Binding Signalling Eph/ephrin Outcome of the site/ Signalling pathway Reference molecule receptor signal domain

FAK EphB2 Y397 Association Regulates dendritic (Cowan and Grb2/Grb4 & Src → spine morphogenesis Henkemeyer, ↑ FAK 2001; phosphorylation & Moeller et al., association paxillin 2006) Fyn EphA3 ? SH2 Src kinases incl Fyn Regulate cell shape, (Ellis et al., downstream effectors adhesion & migration 1996; Hock EphA4 Y602/SH2 of EphB activation in neural and non et al., 1998a; neural cell types Zisch et al., EphB2 Y614/SH2 1998)

Fyn ephrin-A5 SH2 Caveolae like domain; ↑ β1-integrin (Davy et al., ↑ Fyn, ↑ β1-integrin ,↑ mediated cell 1999; Davy MAPK (ERK1, ERK2, adhesion & neurite and Robbins, & FAK) extension 2000)

Grb2 EphB1 Y929 Binds to guanine Activates Ras family (Stein et al., nucleotide exchange proteins regulating 1996) SH2 factors the cytoskeleton

Grb2 EphB2 Y667 Binds Y outside Blocks ability to (Tong et al., kinase domain; induce neurite 2003) SH2 upregulates retraction phosphorylated ERK

Grb4 ephrin-B Y298 SH2 binds ephrin-B, ↓ loss actin stress (Bong et al., SH3 binds other fibres; disassembly 2004; Cowan and Y304 proteins cytoskeletal focal adhesions Henkemeyer, regulation 2001; Su et al., SH2/SH3 2004) Grb4 ephrin-B SH2 Grb4 recruits GIT1 & ↑ dendritic spine (Segura et β-PIX morphogenesis al., 2007; (Y392 of Zhang et al., GIT1) 2005)

Grb7 EphB1 Y928 Yeast two-hybrid Regulates cell (Han et al., screen result migration 2002) SH2 confirmed immunoprecipitation

Grb10 EphB1 Y929 Yeast two-hybrid Grb10 ~ mig 10 C. (Stein et al., screen result elegans protein, 1996) (Grb2) SAM confirmed (mutants defective immunoprecipitation neuronal migration)

262 Queensland Institute of Medical Research Appendix 2: Eph/ephrin protein signalling pathways

Signalling Eph/ephrin Binding Signalling Outcome of the Reference proteins receptor site/domain pathway signal

GRIP1 ephrin-B1 PDZ Expression Scaffold proteins (Bruckner cloning/yeast-two provides link with other et al., 1999; hybrid screen signalling cascades or GRIP2 cytoskeletal remodelling Lin et al., GRIP recruited lipid 1999) raft microdomains GRIP ephrin-B1 PDZ GluR2 (AMPA Induction LTP (Contractor subtype) mossy fibre-CA3 et al., 2002; synapses Meyer et hippocampus al., 2004)

Intersectin EphB KD Link between N- ↑ dendritic spine (Irie and WASP and Cdc42; morphogenesis Yamaguchi, upregulates actin 2002) polymerisation

Jak2 EphA4 Jak homology ↑ Jak2, activates Regulates AChE (Lai et al., domain Stat1 & Stat3 expression 2004) constitutively neuromuscular associates with junction EphA4

Kalirin EphB GEF1 Activates Rac1 → ↑ ↑ dendritic spine (Penzes et PAK morphogenesis al., 2003) KD through F-actin polymerisation

LMW-PTP EphB1 Y929/SH2 LMW-PTP recruited Regulates cell (Stein et al., by tetrameric not adhesion and 1998b) (EphB2) dimeric ligand mitogenesis

LMW-PTP EphA2 ? Binding Regulates EphA2 Dephosphorylation (Kikawa et sites/domain activity through stabilises EphA2 → ↓ al., 2002) not described dephosphorylation proteolysis

MAPK EphA2 ? Binding ERK & Rap1 (not ↑ neurogenesis (Aoki et al., sites/domain Ras) activated (neuronal stem cell 2004; EphA3 not described commitment) Vindis et al., 2003)

Nck EphB1 Y594 Activates c-Jun Regulates actin (Binns et al., kinase cytoskeleton 2000; SH2 Holland et al., 1997; Binds WASP, PAK ↑ cell attachment Stein et al., and Sos 1998a)

Queensland Institute of Medical Research 263 Appendix 2: Eph/ephrin signalling pathways

Binding Signalling Eph/ephrin Signalling Outcome of the site/ Reference proteins receptor pathway signal domain

N-WASP EphB2 Intersectin Intersectin & N- ↑ dendritic spine (Irie and (GEF) WASP interact → ↑ formation Yamaguchi, associates GEF activity activates 2002; Irie and with N- Cdc42 Yamaguchi, 2004) WASP Nurr1 EphB1 ? ? via JNK pathway ↑ dopaminergic (Calo et al., neuron survival 2005b)

p120 - EphB2 SH2/JM Downregulates Ras- Induces neurite (Tong et al., RasGAP region Erk MAPK pathway retraction 2003) Y604, Y610

Paxillin EphB1 Y31/Y118 Signalling complex ↑ cell migration (Baldwin et includes paxillin, al., 2006; LD4 motif FAK & Nck Moeller et al., 2006; Vindis of paxillin et al., 2004)

PDZ-RGS3 ephrin-B2 PDZ Links ephrin-B ↑ migration (Lu et al., reverse signalling cerebellar granule 2004; Lu et Y304 with G-protein cells al., 2001; Su coupled receptor et al., 2004) signalling

PI 3-K, p85 EphA2 SH2 ?Src kinase, Akt & ↑cell migration & (Brantley- subunit ERK1/2; adhesion (endothelial, Sieders et al., EphB4 growth cone) 2004b; → ↑ Rac activity Pandey et al., 1994; Steinle et al., 2002) PI 3-K, EphA8 JM p110 subunit ↓ integrin-mediated (Gu and p110 associates EphA8 cell adhesion Park, 2001) subunit EphA5 → filopodia formation (Fukushima (cholangiocytes) et al., 2006)

PICK EphB2/ PDZ Co-localises at Regulates synaptic (Meyer et synapses & clusters function al., 2004; ephrin-B1 Eph/ephrins Torres et al., 1998)

PLCγ EphA4 SH2 ? Regulates growth of (Zisch et al., neuronal processes & 1998) EphB2 ? cell migration

264 Queensland Institute of Medical Research Appendix 2: Eph/ephrin protein signalling pathways

Binding Signalling Eph/ephrin Signalling Outcome of the site/ Reference molecule receptor pathway signal domain

PTP-BL ephrin-B PDZ Interacts with SH2 PTP-BL mediated (Erdmann, domain & binds dephosphorylation 2003; PDZ → “switches off” Src Palmer et kinase mediated al., 2002) signal Rac/PAK ephrin-A1/ Not ↓Rac/PAK → ↓ spreading vascular (Deroanne et described ↓integrin activation smooth muscle cells al., 2003) EphA2 (Brantley- Sieders et al., 2004b) RasGAP EphB2 Y605, Y611 Bind ?p190 Regulate (Binns et al., RhoGAP and p62dok cytoskeleton by 2000; Elowe EphB3 Y614 → binds Nck modulating Ras et al., 2001; SH2 family proteins Holland et al., 1997)

RhoA EphA Various RhoA → ↑ ROCK Stimulates growth (Noren and adaptor → changes actin cone collapse & Pasquale, EphB proteins & polymerisation & inhibits axon 2004) GEFs actinomyosin regeneration contractility

Syntenin ephrin-B PDZ Adaptor protein for ? (Grootjans syndecans et al., 2000)

SLAP EphA2 SH2 Adaptor protein ? (Pandey et al., 1995b)

Src family Eph SH2 Phosphorylate Regulate cell shape, (Holland et kinases Eph/ephrins upon adhesion, and al., 1996) ephrin-B SH3 receptor-ligand migration in neural binding & clustering and non-neural cell (Zisch et al., Y611 types 1998) STAT3 EphA KD Ephs activate Stats ↑ carcinoma (Lai et al., without ligand invasiveness 2004; Yuan EphB p+1 loop binding et al., 2004b)

Yes EphA3 SH2 ? Regulate cell shape, (Zisch et al., adhesion & 1998) EphA4 ? migration in neural and non-neural cell EphB2 ? types

Queensland Institute of Medical Research 265 Appendix 3: Crosstalk between Eph/ephrin and other signalling pathway

APPENDIX 3 EPH/EPHRIN RECEPTOR CROSSTALK

Eph receptor/ Signalling Binding site/ Outcome of the Reference domain proteins pathway signal

EphB4/ephrin-B2 Abl-Crk Abl-Crk pathway → ↓ ↓cell motility & (Noren et al., matrix metalloprotease migration; ? breast 2006) activity (MMP-2) tumour suppression

EphA3/ephrin-A2 ADAM 10 Cleaves Eph bound Signal attenuation (Hattori et al., ephrins in trans 2000; Janes et EphA3/ephrin-A5 al., 2005)

EphB2/EphB3 β-catenin/TCF Wnt signalling pathway Determines cell (Batlle et al., positioning along 2002; van de intestinal crypt axis Wetering et al., 2002)

EphA5/ephrin-A5 β1- integrin Fyn in caveolae-like ↑ cell adhesion (Davy et al., domains mediates 1999; Davy activation of integrin-β1 ↑ neurite extension and Robbins, 2000)

EphA2 c-Cbl Dependent on EphA2 ↓ regulation of EphA2 (Sharfe et al., kinase activity; and TKB by ubiquitination of 2003; Walker- and RING finger domains the activated receptor Daniels et al., of c-Cbl prior to lysosomal 2002; Wang et degradation al., 2002a) EphA3/ephrin-A5 CD28 ↑ IGF-1R → CrkII ↓ adhesion to (Smith et al., stimulation → recruitment (Jurkat cells) fibronectin 2004b) ↑ EphA3

EphA4/ephrin-A1 Cdk5-p35 Mediates ephexin-1 ↑ retraction of (Fu et al., phosphorylation at dendritic spines 2007) Tyr 87 → ↑RhoA activity

EphA2 claudin-4 ↑ tyrosine ↓ cell-cell adhesion (Tanaka et al., phosphorylation both at tight junctions 2005a; Tanaka ephrin-B1 claudin-1 & claudin-4 at Tyr 208 & et al., 2005b) claudin-4 EphA2/ephrin-B1 in cis

EphA2 E-Cadherin E-Cadherin ↑ cell – ECM (Orsulic and activates/regulates EphA2 adhesion; ↓ cell Kemler, 2000; phosphorylation & proliferation Zantek et al., regulates cell membrane 1999) localisation EphB4/ephrin-B2 Ena/VASP Neural crest cells — ↑ repulsion; neurite (Evans et al., Rho/ROCK mediated retraction 2006) growth collapse, modify actin cytoskeleton

266 Queensland Institute of Medical Research Appendix 3: Crosstalk between Eph/ephrin and other signalling pathway

Signalling Outcome of the Eph receptor Binding site/pathway Reference proteins signal

ephrin-B1 FGF receptor FGF-R interacts COOH ↓ ephrin-B1 cell (Chong et al., (Xenopus) terminus ephrin-B1 & dissociative effect 2000) induces phosphorylation Regulates retinal cell fate (Moore et al., ephrin-B1 2004) ephrin-A5 Fibronectin Xenopus retinal axon stripe Fibronectin → repulsive (Weinl et al., (Xenopus) assay 2003) Laminin Laminin → attractive Signalling pathway not discussed ephrin-B1 GRIP GRIP binds PDZ domain GRIP proteins provide (Bruckner et → recruit Ser/Thr protein scaffold for signalling al., 1999) kinases to lipid raft downstream of ephrin- microdomains Bs

EphA2 HGF ↓ ERK1/MAPK activity ↓ HGF-induced (Miao et al., through actin epithelial branching 2003) cytoskeleton morphogenesis

EphA4 IFN-γ IFN-γ & LIF → EphA4 ↑ astrocytic gliosis (Goldshmit phosphorylation → RhoA following spinal cord et al., 2004) LIF activation injury

EphB Laminin/L1 ↑ Rho-GTPase activity & ↑ retinal growth cone (Birgbauer et ↓ SCG10 protein collapse (pause in axon al., 2001; Suh elongation) et al., 2004)

ephrin-B2 Metabotropic ephrin-B1, mGlu1 & ↑ polyphosphoinositide (Calo et al., glutamate 1 NMDA receptors form a hydrolysis to regulate 2005a; Calo receptor membrane bound synaptic plasticity et al., 2006) complex

EphB2/ephrin-B1 NMDA NMDA receptor & ↑ NMDA receptor (Dalva et al., receptor ephrin-B1 activated clustering & ↑ Ca2+ 2000; EphB2/ephrin-B2 EphB2 extracellular influx & may regulate Takasu et al., domains interact excitatory synapse 2002) formation EphB4/ephrin-B2 Notch/Delta- ↑ ephrin-B2 and EphB4 ↑ ischaemia-induced (Limbourg et like ligand 1 dependent branching post-natal al., 2007) morphogenesis arteriogenesis

EphB2/ephrin-B1 PDGF-β PDGF directly ↓ neuron (Bruckner et receptor phosphorylates ephrin-B1 responsiveness to al., 1997) cytoplasmic domain peptide growth factors

Queensland Institute of Medical Research 267 Appendix 3: Crosstalk between Eph/ephrin and other signalling pathways

Signalling Eph receptor Binding site/pathway Outcome of the signal Reference proteins

EphA2 p53-family p53 binding element ↑ apoptosis (EphA2 (Dohn et al., proteins EphA2 promoter; DNA mediates p53-induced 2001) ephrin-A1 damage → ↑ p53, → apoptosis) ↑EphA2

ephrin-B3 Rhomboid Intramembrane Signal attenuation (Pascall and family serine cleavage of ephrin-B3 Brown, proteinases 2004)

EphB2 Ryk Binds intracellular & Limb & craniofacial (Halford et al., extracellular Eph (does development (palate 2000; Kamitori et al., 2005; EphB3 not phosphorylate ryk); ? fusion); ↓ cortical cell PDZ domain interacts Trivier and migration Ganesan, 2002) with AF-6 EphA/ephrin-A1 SDF-1 ↓ cdc42, ↑ Rho activity → ↓ ↓ T-cell chemotaxis (Sharfe et al., actin polymerisation chemokine- ↑ chemoattraction cerebellar 2002) EphB CXCR4 - ↑ PDZ-RGS3 → ↓ granule cells; regulate (Lu et al., receptor heterotrimeric G protein endothelial cell movement 2001; Salvucci signalling response SDF-1 et al., 2006) EphB2/ephrin-B2 Synaptojanin-1 EphB2 phosphorylates ↑ clathrin-mediated (Irie et al., proline-rich domain of endocytosis 2005) synaptojanin-1

EphB2 Syndecan-2 ↑ phosphorylation ↑ dendritic spine (Ethell et al., syndecan-2 at Y189 & Y201 morphogenesis & maturation 2001) hippocampal neurons EphB4/ephrin-B2 Syndecan-1 & mediated via FGFR & (Yuan et al., syntenin ↑ inflammatory angiogenesis heparanase 2004a) ephrin-B Tie2 ↑ in vitro tyrosine ↑ angiogenic sprouting & (Adams et phosphorylation of regulates endothelial cell al., 1999; ephrin-B1 survival Pfaff et al., 2006)

EphB6 T-cell ↑ p38 MAPK → ↑ ↑ T-cell response to (Luo et al., receptor lymphokines antigen 2004; Luo et al., 2002)

ephrin-B2 TrkA & p75 kinesin-1 light chain ↑ ARMS phosphorylation (Bracale et al., neurotrophin 2007; Kong et al., 2001) EphA4 receptor α-syntrophin regulates ARMS localisation at NMJ (Luo et al., 2005)

VAB-1 WRK-1 WRK-1 expressed by Prevents axons crossing (Boulin et al., embryonic motor the midline in C. elegans 2006; Quinn neuron cells and (gatekeepers) Wadsworth, 2006)

268 Queensland Institute of Medical Research Appendix 4: Eph/ephrin knockout mice

APPENDIX 4 EPH/EPHRIN KNOCKOUT MICE

Eph/ephrin Reference Method Phenotype Anatomical Comments defect

EphA2 (Chen et al., Gene trap No No anatomical No further 1996) strategy – discernible defect detected investigation U3β-geo phenotype retrovirus

EphA2 (Naruse- Gene trap Kinky tail Kinky tail & Role of EphA2 and Nakajima et vector – ectopic ephrin-A1 in tail al., 2001) ROSAN β- vertebrae due formation geo to splitting of the notocord

EphA2 (Brantley- Replacement Not studied EphA2 -/- In vitro signalling Sieders et type vector endothelial studies of EphA2 al., 2004b) from cells do not deficient endothelial Elizabeth migrate and cells Robertson undergo Lab vascular (Harvard) assembly not published

EphA3 (Vaidya et Replacement Not Motor axon EphA3 not necessary al., 2003) type vector described projections for axial muscle here; see analysed targeting of medial later retrograde motor column (Stephen et labelling neurons al., 2006) studies

EphA3 (Stephen et Replacement Perinatal Atrioventricular 75% null mice die al., 2006) type lethality endocardial due cardiac targeting cushion defect abnormality vector

Queensland Institute of Medical Research 269 Appendix 4: Eph/ephrin knockout mice

Anatomical Eph/ephrin Reference Method Phenotype Comments defect

EphA4 (Dottori et Replacement Hopping Abnormal EphA4 expressed on al., 1998) type gait corticospinal corticospinal tract targeting tracts in axons; ?Failure of vector; pgk- spinal cord; corticospinal tracts to absent neo replaces decussate at the anterior ligand medullary pyramids binding commissure domain (both pars anterior and posterior) EphA4 (Helmbacher Replacement Hindlimb Loss of EphA4 involved in et al., 2000) type phenotype dorsal direction of motor targeting (“club foot”) hindlimb axons into the dorsal vector Exon high innervation part of the hindlimb I; lacZ penetrance (peroneal reporter nerve); absent anterior commissure

EphA4 (Kullander Knock-in Hopping KD Active kinase domain et al., strategy — gait abnormal required for normal 2001b) control, kinase hopping trajectory of corticospinal dead (KD), tracts (CST) and mutated gait; absent hindlimb motor juxtamembran anterior coordination; SAM e tyrosine commissure domain not required for (Y2F), deleted but not KD anterior commissure SAM domain mutant formation

EphA4 (Coonan et EphA4 As As Ephrin-B3 expressed al., 2003) knockout described described in midline spinal cord mice from previously previously prevents EphA4 Dottori et expressing CST al., 1998 axons from re- backcrossed crossing the midline onto a C57BL/6 background

EphA4 (Grunwald EphA4-EGFP EphA4- Ephrin-B2 Ephrin-Bs have role et al., 2004) knock-in — EGFP has forebrain post-synaptic LTP ephrin-B2- cytoplasmic hopping gait null have CamK-Cre domain EphA4 –EGFP rescues replaced EGFP normal synaptic plasticity of hippocampus ephrin-B2 ephrin-B2- EphA4 null & KD mice ephrin-B3 CamK-Cre null conditional forebrain

270 Queensland Institute of Medical Research Appendix 4: Eph/ephrin knockout mice

Anatomical Eph/ephrin Reference Method Phenotype Comments defect

EphA4 (Munoz et Mice from Previously Hypocellular EphA4 required for al., 2006) (Helmbacher described thymus, correct thymic et al., 2000) increased environment (bone apoptosis marrow transplants ↓CD4+/CD8+ in SCID mice) cells

EphA7 null (Depaepe et Transgenic TGA7A5 EphA7 null Eph/ephrin EphA7– Transgenic al., 2005) null increased signalling triggers ephrin-A5 perinatal forebrain size pro-apoptotic effect over- mice expressing lethality in forebrain EphA7- BAC clone ephrin-A5 homologous mice recombination (TGA7A5)

EphA8 (Park et al., Replacement No Defect superior EphA8 involved type targeting colliculus 1997) discernible projection to axonal pathfinding vector ; phenotype contralateral from superior targeted inferior colliculus; colliculus to disruption Abnormal contralateral Exon I –lacZ projection axons fusion from ipsilateral inferior colliculus superior colliculus receptor into spinal cord

EphB1 (Williams et EphB1 β-geo EphB1KO EphB1 & Ephrin-B2 most al., 2003) replacement none ephrin-B2 inhibitory to type vector ; described mediate uncrossed RGC Also mice retinal axon axons from divergence at (Henkemeyer optic chiasm et al., 1996, Orioli et al., 1996 & Gale et al., 2001)

EphB2 (Henkemeyer Replacement No Failure of EphB2 defines the et al., 1996) vector (pgk- discernible formation of boundary for neo) & phenotype pars posterior ephrinB3 expressing extracellular of the anterior axons crossing the domain- commissure midline; normal lacZ β-gal fusion reporter mice reporter suggests that the kinase domain has no role in pathfinding of

Queensland Institute of Medical Research 271 Appendix 4: Eph/ephrin knockout mice

Anatomical Eph/ephrin Reference Method Phenotype Comments defect

EphB2 (Cowan et Mice from Hyperactive Inner ear Abnormal amount al., 2000) (Henkemeyer & circling epithelium endolymph in et al., 1996) behaviour fibres do not semicircular canals backcrossed cross into on to CD1 contralateral strain ear

EphB2 (Henderson Henkemeyer None No anatomical EphB2 regulates et al., 2001) (1996) EphB2 described defect of the post-natal synaptic β-gal mouse hippocampus function described EphB2 EphB2 null ∆SAM mice exhibit EphB2 ∆PDZ attenuation of NMDA- dependent LTP

EphB2/EphB3 (Orioli et al., Replacement No Absent corpus EphB2/EphB3 double 1996) type targeting discernible callosum (low mutants severe vector (pgk- phenotype; penetrance); phenotype of anterior commissure, corpus neo) kinase double normal callosum and other domain mutants anterior commissural cleft palate commissure projections; with EphB2/EphB3 cooperate perinatal in formation commissural connections and facial lethality structures EphB2/EphB3 (Birgbauer et EphB2/EphB3 As Abnormal EphB2/EphB3 al., 2000) double KO described pathfinding of guide RGCs; EphB2 (Orioli 1996) previously retinal kinase domain not ganglion cells required (RGCs) within the retina

EphB1/EphB2 (Henkemeyer EphB1 mice Previously Culture EphB forward double & et al., 2003) (Williams et described hippocampal signalling important EphB3 triple al., 2003); neurons from role in hippocampal knockout EphB2 KO mice neuron dendritic (Henkemeyer spine development et al., 1996), EphB3 (Orioli et al., 1996)

272 Queensland Institute of Medical Research Appendix 4: Eph/ephrin knockout mice

Anatomical Eph/ephrin Reference Method Phenotype Comments defect

EphB4 (Gerety et Replacement Embryonic Abnormal EphB4/ephrin-B2 al., 1999) type targeting lethal E9.5 cardiac reciprocal partners in vector; (high looping; angiogenesis and extracellular penetrance); essentially a cardiac development domain similar to phenocopy replaced with lacZ ephrin-B2 of the above ephrin-B2 knockout

EphB4 (Wang et ES cells Not studied EphB4 EphB4-/- ES cells al., 2004a) from essential for studied in vitro & haematopoietic, (Gerety et endothelial, DNA microarray al., 1999) haemangioblast analysis & primitive mesoderm development

EphB6 (Shimoyama Replacement No Normal fetal Overlapping EphB et al., 2002) type targeting discernible thymic expression vector phenotype organ compensates for loss cultures; of EphB6 during CD4/CD8 thymic development flow cytometry similar

EphB6 (Luo et al., Replacement No Lymphoid Compromised T-cell 2004) type targeting discernible organs function vector (GFP- phenotype normal neo)

ephrin-A2/ (Feng et al., ephrinA2/ Not Abnormal Developing skeletal 2000) described mapping of muscle expresses all ephrin-A5 ephrinA5 the phrenic ephrin-A receptors; double double KO nerve on involved topographic knockout (Frisen diaphragm mapping of motor 1998) muscle axons

Queensland Institute of Medical Research 273 Appendix 4: Eph/ephrin knockout mice

Anatomical Eph/ephrin Reference Method Phenotype Comments defect

ephrin-A2/ (Feldheim ephrin-A2/ Not described Severe EphA3/EphA5 et al., disruption of the probable ligands ephrin-A5 ephrin-A5 double 2000) antero-posterior double KO and dorsoventral knockout topographic map of retina on tectum

ephrin-A5 (Frisen et Replacement type Subpopulation Abnormal Anterograde/retrograde al., 1998) targeting vector (17%) midline topographic dye injection studies (pgk-neo) defect dorsal mapping head temporal retinal axons on tectum

ephrin-A5 (Prakash et ephrinA5 (Frisen None Abnormal Quantitative analysis al., 2000) 1998 above) described thalamocortical of whisker functional & intracortical representation; connections retrograde axon tracing

ephrin-A5 ephrin-A5 null 17% severe Failure of Splice variants may (Holmberg craniofacial neural tube mediate the switch EphA7 EphA7 null and et al., malformations; closure; from repulsion to splice variants in 2000) most lack a unresponsive attraction vitro brain and die to folate after birth supplement

ephrin-B1 (Compagni Replacement type Perinatal Oedema, Similar phenotype to et al., targeting vector; lethality defective body the EphB2/EphB3 2003) Cre-loxP wall closure, double knockout; skeletal ephrin-B1 mutations abnormalities cause of craniofrontonasal syndrome [CFNS, OMIM #304110]

274 Queensland Institute of Medical Research Appendix 4: Eph/ephrin knockout mice

Anatomical Eph/ephrin Reference Method Phenotype Comments defect

ephrin-B1 (Davy et ephrin-B1 Perinatal NCC null Heterozygous females al., 2004) conditional lethality animals cleft more severe null neural palate; phenotype; ephrin-B1 crest cells has reverse signaling heterozygous (NCC); role in neural crest females guidance ephrin-B1 polydactyly; ∆PDZ cleft palate

ephrin-B2 (Wang et Replacement Embryonic Extensive Ephrin-B2 expression al., 1998) type targeting lethal E11 disruption of arteries not veins vector; (100% angiogenesis in required for extracellular penetrance) yolk sac; remodelling of absence domain capillary networks branches replaced tau- carotid arteries; lacZ defective trabeculation of heart

ephrin-B2 (Adams et ephrin-B2- ephrin-B2 as EphB2/EphB3 EphB2/EphB3/ephrin- al., 1999) lacZ (Wang above; double KO B2 involved EphB2/EphB3 1998); double EphB2/EphB3 similar remodelling mutant double KO ephrin-B2 embryonic EphB2/EphB3 vascular vasculature and (Orioli et al., defects 30% boundary formation 1996) penetrance between arteries and veins

ephrin-B2 (Gale et al., Lac-Z reporter Embryonic ephrin-B2 Further studies in 2001) lethal expressed Lewis lung carcinoma arterial model (Shin et al., smooth 2001) muscle

ephrin-B2 (Grunwald ephrin-B2 – Normal Investigate role et al., loxP mice; hippocampal ephrin-B2 in LTP 2001) CamKIIα -cre morphology; hippocampus to remove no change in ephrin-B2 dendritic or from synapse forebrain morphology

Queensland Institute of Medical Research 275 Appendix 4: Eph/ephrin knockout mice

Anatomical Eph/ephrin Reference Method Phenotype Comments defect

ephrin-B2 (Adams et ephrin-B2-∆C- Embryonic ephrin-B2 Role of ephrin-B2 al., 2001) HA knock-in lethal essential cytoplasmic domain haemagglutinin branchial arch tag morphogenesis on vascular and branchial arch development

ephrin-B2 (Dravis et ephrin-B2- Neonatal Hypospadias EphB/ephrin-B al., 2004) lacZ & lethality in males; midline adhesive ephrin-B2 incomplete function; forward truncated septation signalling is repulsive cloaca

ephrin-B2 (Cowan et ephrin-B2- Neonatal Thickened ephrin-B2 reverse al., 2004) lacZ & lethality cardiac signalling not ephrin-B2 valve required for early truncated leaflets vascular development

ephrin-B2 (Makinen et Knock-in ephrin-B2 Lymphoedema, ephrin-B2 essential al., 2005) strategy ∆PDZ display abnormal for lymphatic perinatal valves in development ephrin-B2 lethality lymphatics ∆PDZ ephrin-B2-5F mild lymphatic ephrin-B2 phenotype only 5F

ephrin- (Foo et al., ephrin-B2 Perinatal Vascular ephrin-B2∆P/∆SMC B2∆P/∆SMC 2006) conditional lethality; defects in lung, mural cells do not get skin, intestine, mice respiratory incorporated into (Grunwald et failure; kidney; in vitro abnormal blood vessel walls al., 2004) oedema & migration crossed haemorrhage smooth muscle Pdgfrb-Cre skin cells → ephrin-B2 ll pericytes ephrin-B3 (Yokoyama Replacement ephrinB3- Defective Ephrin-B3 expressed et al., 2001) vector (ephrin- neo hopping CST middle of spinal cord B3-neo); gait (similar pathfinding prevents EphA4 extracellular expressing CST axons domain-lacZ EphA4-KO); fusion receptor; re-crossing midline; extracellular ephrinB3- ephrin-B3 forward domain lacZ normal signalling only required truncated gait for CST formation

276 Queensland Institute of Medical Research Appendix 5: Materials and reagents

APPENDIX 5 MATERIALS AND REAGENTS

Molecular biology reagents

ΦX174 Hae III digest New England Biolabs Cat No. N3026S

λHindIII digest New England Biolabs Cat No. N3012S

ABI Prism Dye Terminator Mix BD 3.1 Applied Biosystems Cat No. 4337455

Ampicillin Sigma-Aldrich Cat No. A-9518

AmpliTaq Gold™ Applied Biosystems Cat No.

Antigen Dewax Retrieval Solution Sapphire Biosciences Cat No. 200-28-ARD1 (for ImmunoKontact)

AutoSeq™ G-50 spin columns Amersham Biosciences (GE Healthcare) Cat No. 27-5340-01

BCIP Roche Cat No. 1 383 221

Bio-Rad Gene Pulser® Cuvettes Bio-Rad Laboratories 0.1 cm gap Cat No.165 2089

BM Purple AP Substrate Roche Cat No 1 442 074

Costar Cell scraper Corning Cat No. 3010

CentriSep spin columns Princeton Separations Cat No. CS-200

Conical tubes 50ml blue Greiner Bio-One Cat No.227261

Conical tubes 15ml yellow Sarstedt Cat No. 62 9924.284

Depex™ Mounting Medium Gurr BDH

Queensland Institute of Medical Research 277 Appendix 5: Materials and reagents dNTPs Fisher Scientific Cat No. BP2564-1 (dATP) Cat No. BP2564-4 (dCTP) Cat No. BP25641 (dGTP) Cat No. BP25644 (dTTP)

Dynabeads® mRNA Purification Kit Dynal Cat No.610.06

Eppendorf™ tubes (1.5ml) Eppendorf Cat No. 0030 – 125.150

Express Hyb Solution Clontech (BD Biosciences) Cat No. 8015-1

Falcon 2059 tubes BD Biosciences Cat No. 352059

G418 (neomycin) Sigma-Aldrich

Gene pulser cuvettes 0.1cm Bio-Rad Laboratories Cat No.165 2089

Glycogen Boehringer Mannheim (Roche) Cat No. 901393

Hybond N Amersham Biosciences (GE Healthcare) Cat No. RPN 203N

Kim Wipes Kimberly-Clark Cat No. 515

Megaprime Labelling Kit Amersham Biosciences (GE Healthcare) Cat No. RPN1607

Microcentrifuge tubes tubes (2ml) Edwards Instrument Company Cat No.3046 870 000

Microcentrifuge tubes (screw cap) Quality Scientific Plastics Cat No.515

MinElute gel extraction kit Qiagen Cat No. 28604

NBT Roche Cat No. 1 383 213

Nuclear Fast Red Sigma-Aldrich Cat No. N-8002

278 Queensland Institute of Medical Research Appendix 5: Materials and reagents

Nunc cryovials Nalge Nunc International Cat No. 377224

Oakridge Centrifuge Tubes Oakridge Cat No. not available pBluescript II KS- Stratagene Laboratory stock p-GEM®-T Vector System I Promega Cat No. A3600

Proteinase K Promega Cat No. 182218

QIAquick gel purification kit Qiagen Cat No. 28704

QIAEXII agarose gel extraction kit Qiagen Cat No. 20021

Qiagen Midi-Prep kit Qiagen Cat No. 12143

Quick Seal polyallomer tube (13 x 51 mm) Beckman Instruments Inc. Cat No 342412

Restriction endonucleases New England Biolabs From NEB E-freezer

RNA millennium markers Ambion Cat No. 7151

RNaseA Sigma-Aldrich Cat No. R6513

RNAasin Promega Cat No. not available

Salmon Sperm DNA Sigma-Aldrich Cat No. D-1626

SuperScript™ III Invitrogen Cat No. 18080-093

SYBR GREEN PCR mix Applied Biosystems Part No. 4309155

Thermosensitive alkaline phosphatase (TsAP) Cat No. 10534-014 GIBCO-BRL (Invitrogen)

Queensland Institute of Medical Research 279 Appendix 5: Materials and reagents

Tissue-Tek® O.C.T Compound Sakura Cat No. 4583

VectaMount™ Vector Laboratories Cat No. H-5000

Whatman filter paper Whatman International Cat No. 3030 917

Wizard Lambda DNA Purification Kit Promega Cat No.

Zeta Probe Bio-Rad Laboratories Cat No. #162-0165

Chemical reagents

Agarose Invitrogen Bacto-tryptone Becton Dickinson (BD Biosciences) Bacto yeast extract DIFCO Laboratories (BD Biosciences) β-mercaptoethanol Sigma-Aldrich Bovine serum albumin Sigma-Aldrich DTT Sigma-Aldrich EDTA APS Ajax Finechem Ethanol BDH Merck Ethidium bromide Sigma-Aldrich Ficoll 400 Sigma-Aldrich Glacial acetic acid BDH Laboratory Supplies (BDH Merck) Glucose BDH Merck Glycerol APS Ajax Finechem HCl (concentrated 33%) APS Ajax Finechem Isoflurane Laser Animal Health Pty. Ltd. Isopropanol BDH Merck KCl BDH Merck KOH BDH Merck NaCl Sigma-Aldrich

Na2HPO4 BDH Merck

NaH2PO4 BDH Merck NaOH BDH Merck

280 Queensland Institute of Medical Research Appendix 5: Materials and reagents

NaPiPi BDH Merck

MgCl2 BDH Merck

MgSO4 BDH Merck Paraformaldehyde Cat No. P-6148 Sigma-Aldrich PEG Sigma-Aldrich Sodium acetate Sigma-Aldrich Sodium citrate Sigma-Aldrich SDS Sigma-Aldrich Sucrose BDH Merck Tris-Cl Sigma-Aldrich

Solutions

6X Loading Buffer

40% (w/v) sucrose 0.025% (w/v) bromophenol blue 0.025% (w/v) xylene cyanol FF

10X RNA gel loading buffer, pH 8.0

20% (w/v) Ficoll 400 1% (w/v) SDS 0.25% (w/v) bromophenol blue 0.25% (w/v) xylene cyanol FF

5X TBE

54 g of Tris base dissolved with 27.5 g boric acid, 20 ml of 0.5 M EDTA (pH 8.0). Dilute to 0.5 X working solution (= 45 mM Tris-borate, 1 mM EDTA).

Alkaline phosphatase buffer

Prepare from stock solutions. Final concentration 100 mM Tris-Cl, pH 9.5, NaCl 10 mM, 10 mM Mg2Cl.

Queensland Institute of Medical Research 281 Appendix 5: Materials and reagents

NBT/BCIP stain

NBT (Nitroblue tetrazolium) 337 µg/ml, BCIP (5-bromo-4-chloro-3-indolyl phosphate, toluidinium salt) 225 µg/ml.

Nuclear Fast Red stain

Dissolve 5g Al2(SO4)3.18H20 in 100ml water and stir whilst heating, then add 100mg Nuclear Fast Red and allow to cool. Filter sterilise.

4% Paraformaldehyde

Dissolve 6 g of paraformaldehyde in 50 ml of Milli-Q water and then add 1 ml of 1 M NaOH. Stir gently on the heating block (~ 65ºC until dissolved) then add 10ml of 10X PBS and allow mixture to cool. Adjust the pH to 7.4 using 1M HCl (approximately 1 ml required) and adjust the final volume to 100 ml with Milli-Q water. Filter through 0.45 µm and store in aliquots at –20ºC.

Formalin in PBS

Supplied by the QIMR Histology Department.

SM buffer

NaCl 100mM, MgSO4, Tris-Cl (pH 7.5) 50mM.

NaCl 5.8g, MgSO4.7H2O 2g; 50ml Tris-Cl (pH 7.5) and 5ml gelatine 2% (2g/100ml w/v) made up to 1 litre. Autoclave.

1M MgSO4

12.3g MgSO4.7H2O made up to 50ml and filter sterilised.

20% Maltose Maltose 20g made up to 100ml in Milli-Q water. Sterilise through 0.2 µm filter. Terrific broth

Tryptone 12 g, yeast extract 24 g, glycerol 4 ml, potassium phosphate buffer (100 ml 0.17 M KH2PO4, 0.72 M K2HPO4) made up to 1 litre and autoclaved.

LB broth

Bacto-tryptone 10g, Bacto-yeast extract 10g, NaCl 10g, 1ml of 1M NaOH made up to 1 litre and autoclaved.LB “bottom agar” plates

LB “top agarose”

LB agarose 0.7% with 10 mM MgSO4.

282 Queensland Institute of Medical Research Appendix 5: Materials and reagents

LB “bottom agar”

LB agarose 1.1%

NZCYM broth

NZ Amine (10g), NaCl (5g), Casamino Acid (1g), MgSO4 (2g) make up to 1 litre. Adjust pH to7.0 and autoclave.

NZCY+

NZ amine (10g), NaCl (5g), yeast extract (5g). Adjust pH to 7.5 with NaOH and autoclave. Add the following filter sterilised supplements prior to use:- 12.5 ml of 1 M MgCl2, 12.5 ml 1M MgSO4., 20 ml of 20% (w/v/) glucose per 1 litre.

NZCYM bottom agar (1.5%)

Add 6g agarose to 400ml NZCYM broth.

NZCYM agarose (0.7%)

Add 0.5g agarose to 100ml NZCYM broth.

Medium A

LB broth with 10 mM MgSO4.7H2O and 0.2% glucose (w/v).

Medium B

36% glycerol,12% PEG7500,10 mM MgSO4.7H2O added to LB broth and sterilised by filtration.

Inoue transformation buffer

A stock solution of 0.5 M PIPES was prepared by dissolving 15.1 g of PIPES in 80 ml of Milli-Q water then adjusting the pH to 6.7 with 5 M KOH then the final volume adjusted to 100 ml. The 0.5 M PIPES, pH 6.7, was filter sterilised by passage through a pre-rinsed Nalgene™ 0.45 µm, divided into aliquots and stored at –20°C. The transformation buffer was prepared by dissolving all the solutes: 10.88 g MnCl2.4H2O, 2.20 g CaCl2.2H2O, 18.65 g KCl and 20ml PIPES (0.5 M, pH 6.7) and adjusting the total volume to 1 litre with Milli-Q water.

Bacteriophage DNA lysis buffer

100 mM EDTA, 100 mM NaCl, 50 mM Tris-Cl, pH 8.0, 1% (w/v) SDS. Add 25 µl proteinase K (10 mg/ml) per 5 ml lysis buffer (final concentration 100 µg/ml).

ES cell DNA lysis buffer

20 mM EDTA, 2 mM NaCl, 20 mMTris and 1% sarcosyl w/v and proteinase K 1mg/ml (final concentration 100 µg/ml).

Queensland Institute of Medical Research 283 Appendix 5: Materials and reagents

EasyPrep Lysis Buffer

10 mM Tris-Cl, pH 8.0, 1 mM EDTA, 15% sucrose (w/v), 2mg/ml lysozyme, 0.2mg/ml pancreatic RNase A, 0.1 mg/ml bovine serum albumin.

Mix 500µl 1 M Tris-Cl, 100µl 0.5 M EDTA, sucrose 75 g, lysosyme 100 mg, 1 ml of RNase A 10 mg/ml, 500µl BSA 10 mg/ml, made up to 50 ml in a blue conical. Aliquot 800 µl in Eppendorf™ tubes and store at –20ºC.

Maxi prep solution I 25% w/v sucrose, 50 mM Tris-Cl, pH 8.0. For 100ml, 25 g sucrose, 5ml 1 M Tris-Cl, pH 8.0. Lysozyme (10mg/ml)

Prepared freshly at a concentration of 10 mg/ml in 0.25 M Tris-Cl, pH 8.0.

MaxiPrep solution II

1% v/v Triton-X 100, 60 mM EDTA, 50 mM Tris-Cl, pH 8.0. For 100 ml, 1 ml TX- 100, 12 ml 0.5 M EDTA, 5 ml 1M Tris-Cl, pH 8.0.

MaxiPrep solution III

50 mM NaCl, 5 mM EDTA, 50 mM Tris-Cl, pH 8.0. For 100ml, 1 ml 5 M NaCl, 5 ml 1 M Tris-Cl, pH 8.0.

Tail Tip Solution II

2.0% SDS, 50 mM Tris-Cl, pH 8.0, 20 mM EDTA, pH 8.0, Proteinase K 0.5 mg/ml.

Phenol

Phenol is highly corrosive, therefore gloves, eye protection and a laboratory gown must be worn when working with phenol. The phenol must be equilibrated to a pH > 7.8 because DNA will partition into the organic phase at acidic pH. Phenol is stored at – 20°C and, when required, a convenient volume (100ml) should be melted at 68°C. Hydroxyquinolone is added to a final concentration of 0.1%, which will impart a slightly yellow colour to the solution. Then add an equal volume of 0.5 M Tris-Cl (pH 8.0) at room temperature and mix on a magnetic stirrer for 15 minutes. When the aqueous and organic phases have separated, aspirate the upper aqueous phase with a glass pipette attached to a suction trap. Add an equal volume of 0.1 M Tris-Cl (pH 8.0) to the phenol and mix on a magnetic stirrer for 15 minutes. Allow the two phases to separate and aspirate the upper aqueous phase as previous. Repeat the extractions until the pH of the phenolic phase is >7.8 as measured with pH paper. Following removal of the final aqueous phase, add 0.1 volume of 0.1 M Tris-Cl (pH 8.0) containing 0.2% β- mercaptoethanol. The phenol solution can be stored in a light protected bottle (Schott™ bottle wrapped in Alfoil™) at 4°C for several months.

284 Queensland Institute of Medical Research Appendix 5: Materials and reagents

Phenol:Chloroform

This is prepared by mixing an equal volume of equilibrated phenol with chloroform. The phenol:chloroform mixture can be stored in a light protected bottle (Schott™ bottle wrapped in Alfoil™) and shaken vigorously immediately prior to use which makes the solution milky in appearance.

1M Tris-Cl

Dissolve 121.1 g of Tris base in 800 ml of Milli-Q water. Adjust the pH to the desired value with concentrated HCl. pH HCl 7.4 70 ml 7.5 60 ml 7.8 42 ml Allow the solution to cool to room temperature before making final adjustments to the pH and adjusting the final volume to 1 litre. Autoclave.

0.5 M EDTA (pH 8.0)

Add 186.1 g of disodium EDTA [Sigma-Aldrich] to 800 ml of Milli-Q water and stir on a magnet stirrer. Adjust the pH to 8.0 with NaOH pellets. The EDTA will not dissolved until the pH of the solution approaches 8.0. Autoclave.

TBS (Tris-buffered saline)

Tris 20 mM, NaCl 137 mM, pH 7.4

Dissolve 8 g NaCl, 0.2 g KCl and 3 g Tris-Cl in 800 ml Milli-Q water and adjust pH to 7.4 with HCl. Adjust the total volume to 1 litre. Dispense in aliquots and autoclave. Store at room temperature.

TE

10 mM Tris-Cl, pH 8.0 and 1 mM EDTA.

Make 1 ml of 1M Tris (pH 8.0 and 200 µl of 0.5 M EDTA (pH 8.0) up to 100ml.

50X Denhardt’s solution

1% (w/v Ficoll 400, 1% (w/v) polyvinylpyrrolidone, 1% (w/v) bovine serum albumin (Fraction V)

5 g Ficoll 400 [Pharmacia], 5 g polyvinylpyrrolidone, 5 g bovine serum albumin (Fraction V)[Sigma-Aldrich] made up to 500 ml filter sterilised and stored –20ºC. Diluted 10-fold to prepare prehybridisation buffer.

Salmon sperm DNA (10mg/ml)

Salmon sperm DNA (Sigma type III sodium salt) is dissolved in Milli-Q water to 10 mg/ml, and dissolved by stirring for several hours at room temperature. Adjust the

Queensland Institute of Medical Research 285 Appendix 5: Materials and reagents concentration of NaCl to 0.1 M and extract the solution once with phenol and once with phenol:chloroform. Recover the aqueous phase and shear the DNA by passing the solution 12 times rapidly through a 17-gauge hypodermic needle. Ethanol precipitate the DNA and resuspend at 10 mg/ml. Boil the solution for 10 minutes then store in small aliquots in Eppendorf™ tubes at –20ºC.

Sodium pyrophosphate (250mM)

Dissolve 16.7 g of sodium pyrophosphate in Milli-Q water and make up to 150 ml. Autoclave.

5M NaCl

Dissolve 292.2 g of NaCl in 800 ml of Milli-Q water. Adjust the volume to 1 litre. Autoclave.

3M Sodium acetate (NaOAc) pH 5.2 (NaOAc)

Dissolve 408.1 g of sodium acetate.3H20 in 800 ml Milli-Q water. Adjust the pH to 5.2 with glacial acetic acid or adjust the pH to 7.2 with dilute acetic acid. Adjust the volume to 1 litre. Autoclave.

Potassium acetate (KAc, 5M,)

Prepare 100 ml of 5 M potassium acetate by dissolving 49.1 g in 100 ml water.

Mix 60 ml 5 M potassium acetate and 11.5 ml of glacial acetic acid and make up to 100 ml with water. The final solution is 3 M potassium and 5 M acetate.

10% SDS

Wear a mask whilst preparing this solution. Dissolve 100 g [Bio-Rad] in 900 ml Milli-Q water. Heat on a magnetic stirrer (~ 68ºC) to assist dissolution. Adjust the pH to 7.0 with a few drops on concentrated HCl. Adjust the volume to 1 litre and store at room temperature. Sterilisation is NOT necessary and do NOT autoclave.

10 N NaOH

This is a highly exothermic reaction. Dissolve 100 g of NaOH pellets in 150 ml of Milli-Q water whilst stirring continuously. Adjust the volume to 250 ml when the pellets have dissolved completely. Sterilisation is NOT necessary.

1 M HCl

Dilute 86 ml of concentrated HCl (w/v 36–38% is approximately 11.6 N) to 1000 ml.

Pre-hybridisation buffer

50X Denhardt’s solution diluted one in ten (final 5X), 6X SSPE, SDS 1% (w/v), 100 µg/ml sheared salmon sperm DNA, 10 mM sodium pyrophosphate.

286 Queensland Institute of Medical Research Appendix 5: Materials and reagents

20X SSC

3.0 M NaCl, 0.3 M sodium citrate. Dissolve 175.3 g NaCl and 88.2 g of sodium citrate in 800 ml of Milli-Q water. Adjust the pH to 7.0 with concentrated HCl, then adjust the total volume to 1 litre. Autoclave.

20X SSPE

3.0 M NaCl, 0.2 M sodium phosphate, 2 mM EDTA, pH 7.0. Dissolve 175.3 g NaCl, 27.6 g of NaH2PO4.H20 and 7.4 g EDTA in 800 ml of Milli-Q water. Adjust the pH to 7.4 with 10 N NaOH then adjust the total volume to 1 litre. Autoclave.

2X SSPE

Make 100 ml 20X SSPE made up to 1000 ml.

0.1X SSPE/0.1% SDS

Make 5 ml 20X SSPE and 10 ml 10% SDS, pH 7.0 made up to 1000 ml.

Denaturing solution

1.5 M NaCl, 0.5 M NaOH.

Dilute 300 ml 5 M NaCl and 50 ml 10 N NaOH up to 1 litre.

Neutralising solution

1.5 M NaCl, 0.5 M Tris-Cl pH 8.0. Dilute 300 ml 5 M NaCl and 500 ml 1 M Tris-Cl and 2 ml 0.5 M EDTA up to 1 litre.

DEPC-water

DEPC-treated ddH2O

Add diethylpyrocarbonate (DEPC) to Milli-Q water to a final concentration of 0.1% (1 ml in 1000 ml) agitate intermittently over 4 hours then autoclave.

10X MOPS buffer

0.2 M 3-[N-Morpholino]–propane-sulphonic acid, pH 7.0, 20 mM sodium acetate, 10 mM EDTA, pH 8.0.

Dissolve 41.8 g of MOPS in 700 ml of autoclaved DEPC treated water. Adjust the pH to 7.0 with 2 N NaOH. Add 20 ml of DEPC-treated 1 M sodium acetate and 20 ml of DEPC-treated 0.5 M EDTA (pH 8.0). Adjust the volume to 1 litre with DEPC-treated water. Sterilise through a 0.45 µm filter and store protected from light.

HEPES (1M) buffer

238.3 g N-(2-hydroxyethyl)piperazine-N’-(2-ethanesulphonic acid) dissolved in Milli-Q water and made up to 1000 ml. Adjust pH to 7.4 with 10 N NaOH.

Queensland Institute of Medical Research 287 Appendix 5: Materials and reagents

PIPES (0.5 M)

Dissolve 15.1 g of PIPES (piperazine-1,2-bis-[2-ethanesulphonic acid] in 80 ml of Milli-Q water and adjust the pH to 6.7 with 5 M KOH. Filter sterilise.

RNA tissue lysis buffer

5.5 M Guanidine thiocyanate, 5 mM Na citrate, pH 7.0, 100 mM β-mercaptoethanol, in DEPC water. Filter sterilise. Add β-mercaptoethanol just prior to use.

RNA tissue resuspension buffer

5 mM EDTA, 0.5% sarkosyl, 100 mM β-mercaptoethanol

RNA gel loading buffer

20% Ficoll 400 w/v, 1% SDS w/v, 0.25% w/v bromophenol blue, 0.25% w/v xylene cyanol FF, pH 8.0

Table of bacterial strains

Strain Description

BS591 Transfected by phage with Cre

DH5α High transformation efficiency for plasmid preparation

DH10β Recombination-defective strain for propagation of BACs

JM109 Recombination-defective strain supports bacteriophage growth

SOLR Suppressor negative and resistant to infection with λ phage

STBL2 High efficiency chemical transformation of unstable inserts

XL-1 Blue Plasmid preparations. Allow blue-white screening on X-gal

XL-1 Blue MRF’ Permissive for growth of vectors carrying amber mutations

XL-1 GOLD Efficient for preparation of large plasmids (>10 kB)

288 Queensland Institute of Medical Research Appendix 6: Laboratory equipment

APPENDIX 6 LABORATORY EQUIPMENT

APERIO Scansope XT system Aperio Technologies Inc.

Bio-Rad Gene Pulser Bio-Rad Laboratories

Bio-Rad UV Cross-Linker Bio-Rad Laboratories

Eppendorf™ centrifuge 5415 D Eppendorf (Micro Centrifuge)

Beckman Coulter Avanti J-25I Beckman Coulter (Midi Centrifuge)

Beckman Coulter Optima XL 100 Beckman Coulter (Ultracentrifuge)

Dupont Sorvall Tissue Homogeniser Dupont Model 17106

UV transilluminator Vilber Lourmat (supplied by Fisher Biotec)

PCR machines GeneAmp 9700 Applied Biosystems

Rotor-gene RG-3000 Corbett Research

Savant Speed Vac SPD 111V Savant

Scintillation Counter Bioscan QC 2000 Bioscan Inc.

Queensland Institute of Medical Research 289 Appendix 7: List of suppliers

APPENDIX 7 LIST OF SUPPLIERS

Aperio Technologies, Inc. 1430 Vantage Court, Suite 106 Vista, CA 92081-8545 USA http://www.aperio.com/

Applied Biosystems, Foster City, CA,USA http://www.appliedbiosystems.com/index.cfm

Applied Biosystems (Australia) Scoresby Melbourne VIC 3179 Australia

APS Ajax Finechem Auburn SA 5000 Australia http://www.apschem.com.au/

Ambion, Inc. (Headquarters) Austin, TX USA http://www.ambion.com/

Amersham Pharmacia/Amersham Biosciences Baulkham Hills Sydney NSW 2153 Australia (now GE Healthcare) http://www5.amershambiosciences.com/aptrix/upp01077.nsf/content/australia_homepage

BDH (VWR International) http://www.bdh.com

BDH Gurr/BDH Merck 207 Colchester Road Kilsyth VIC 3137 Australia

Beckman Coulter, Inc., Fullerton, CA, USA http://www.beckman.com/default.asp?bhfv=5

290 Queensland Institute of Medical Research Appendix 7: List of suppliers

Beckman Coulter Australia Pty. Ltd. PO BOX 218 Gladesville NSW 1675 Australia

Beckman Instruments Inc. Spinco Division 117 California Avenue Palo Alto, CA 94304 USA

Becton Dickinson Company (Now BD Biosciences) BD Biosciences, 1 Becton Drive Franklin Lakes, NJ USA http://www.bd.com/

BD Bioscience Australia and New Zealand http://www.bd.com/anz/

Bio-Rad Laboratories Hercules, CA USA www.biorad.com

Bio-Rad Laboratories Pty., Ltd. Australia Unit 1, Block Y Regents Park Industrial Estate 391 Park Road Regents Park, NSW 2143 Australia http://www.bio-rad.com E-mail: [email protected]

Boehringer Mannheim Gmbh, Mannheim, Germany (now a subsidiary company of Roche Applied Science)

Clontech (now a subsidiary of BD Biosciences) http://www.clontech.com/clontech/

Corbett Research Mortlake Sydney NSW 2137 Australia. http://www.corbettresearch.com

Queensland Institute of Medical Research 291 Appendix 7: List of suppliers

Corning Incorporated, Corning, NY, USA Corning Headquarters One Riverfront Plaza Corning, NY 14831 USA http://www.corning.com/

Crown Scientific 16-18 Maud Street Newstead QLD 4006 Australia http://www.crownscientific.com.au/

DakoCytomation Glostrup Denmark http://www.dakocytomation.com

DakoCytomation Pty. Ltd. Botany Sydney, NSW 2019 Australia http://www.dakocytomation.com.au/

DIFCO Laboratories Livonia, MI, USA (now a subsidiary of BD Biosciences)

DuPont (Australia) Ltd Head Office 168 Walker Street North Sydney NSW 2060 Australia http://au.dupont.com/

Dynal Biotech http://www.dynal.no/

Dynal Biotech Pty Ltd. PO Box 204 Carlton South VIC 3053 Australia

Edwards Instrument Company 5 Bellingham St Sydney NSW 2001 Australia

292 Queensland Institute of Medical Research Appendix 7: List of suppliers

Electron Microscopy Sciences P.O. Box 550 1560 Industry Road Hatfield, PA 19440 USA E-Mail: [email protected]

Eppendorf-Vertrieb GmbH Hamburg Germany http://www.Eppendorf™.com/de/ Fisher Scientific 1 Reagent Lane Fair Lawn, NJ 07410 USA www.fishersci.com/ or http://www.fisherscientific.com/

Fisher Bioscientific P.O. Box 169 SUBIACO WA 6904 2/64 Thomas Street West Perth WA 6005 Australia http://www.fisherbiotec.com

GIBCO (now a subsidiary of Invitrogen) http://www.invitrogen.com/

Grant Instruments (Cambridge) Ltd Shepreth Cambridgeshire SG8 6GB United Kingdom http://www.grant.co.uk/

Greiner Bio-One Bioscience Frickenhausen Germany http://www.gbo.com/en/

Hybaid Limited (Thermo Electron Corporation) http://www.thermo.com/BURedirect/welcomeMsg/1,5107,127,00.html

ICN Biomedicals (now MP Biomedicals) http://www.mpbio.com/

ImmunoKontact, AMS, Biotechnology Abdingdon Oxford, United Kingdom http://www.ImmunoK.com

Queensland Institute of Medical Research 293 Appendix 7: List of suppliers

DCS Innovative Diagnostik-Systeme Poppenbütteler Chaussee 36 22397 Hamburg Germany http://dcs-diagnostics.de/

Invitrogen Corporation PO Box 6482 Carlsbad, CA USA http://www.invitrogen.com/ Invitrogen Australia Pty Ltd., Mount Waverley VIC 3149 Australia http://www.invitrogen.com/content.cfm?pageid=1

Jackson Immunoresearch PO Box 9 872 West Baltimore Pike West Grove PA 19390 USA Tel: 610 869 4024 Fax: 610 869 0171 http://www.jacksonimmuno.com/

Jenway Ltd., Dunmow, Essex, UK Gransmore Green Felsted Dunmow Essex CM6 3LB, United Kingdom http://www.jenway.com/

Kimberly-Clark Corporation PO Box 2020 Neenah, WI USA http://www.kimberly-clark.com/

Kimberly-Clark Australia Pty. Ltd. PO Box 343 Milsons Point NSW 1565 Australia http://www.kca.com.au/

KODAK (Eastman-Kodak) Rochester, NY USA http://www.kodak.com

294 Queensland Institute of Medical Research Appendix 7: List of suppliers

Lab-Line (Barnstead International) 15th & Bloomingdale Avenues Melrose Park IL 60160 USA http://www.labline.com/

Laser Animal Health Pty. Ltd. Unit 6, 70 Fison Avenue West Eagle Farm QLD 4029 Australia

Leica Camera (Australia) Adeal P/L 2 Baldwin Road Altona North Melbourne VIC 3025 Australia Tel: 03 8369 4444 Fax: 03 8369 4440 E-mail: [email protected] http://www.leicacamera.com/home/

Merck Australia http://www.merck.com.au/

Molecular Dynamics (now part of GE Healthcare) www.mdyn.com/

Nalge Nunc International Rochester, NY USA http://www.nalgenunc.com/ http://www.nuncbrand.com/

New England Biolabs Incorporated Beverly, MA USA www.neb.com

Norco Co-operative Ltd. PO Box 486 Lismore NSW 2480 Australia

Orion Research Inc. (subsidiary of the ThermoElectron Corporation) 500 Cummings Center Beverly MA 01915 USA www.orionres.com

Queensland Institute of Medical Research 295 Appendix 7: List of suppliers

PerkinElmer Corporation Boston, MA USA http://www.perkinelmer.com/

Princeton Separations Inc. PO Box 300, Adelphia, NJ USA http://www.prinsep.com/html/home/

Promega Corporation PO Box 168, Annandale NSW 2038 Australia http://www.promega.com/

Qiagen,GmbH Dusseldorf, Germany http://www1.qiagen.com/

Qiagen Pty. Ltd., Australia PO Box 641, Doncaster VIC 3108 http://www1.qiagen.com/Contact/QIAGENWorldWide.aspx

Quality Scientific Plastics Porex Bio Products, Inc. 1120 Holm Road Petaluma, CA 94954 USA http://www.porex.com

Quantum Scientific 30 Finchley Street Milton, QLD 4064

Australia http://www.quantum-scientific.com.au/ Radiometer-Pacific Radiometer Pacific Pty. Ltd. Whitehorse Road Blackburn, VIC 3130 Australia http://oceania1.radiometer.com/ or http://proscience.radiometer.com/

Ratek Instruments Pty. Ltd. 60 Wadhurst Drive Boronia, VIC 3155 Australia www.ratek.com.au

296 Queensland Institute of Medical Research Appendix 7: List of suppliers

Roche Applied Science http://www.roche-applied-science.com/

Roche Australia Pty. Ltd. PO Box 955 Castle Hill, NSW 2154 Australia

Sakura Finetek Europe B.V. Hoge Rijndijk 48a 2382 AT Zoeterwoude P.O. Box 40 2380 AA Zoeterwoude The Netherlands http://www.sakuraeu.com/

Sapphire Biosciences Pty. Ltd. Crows Nest Sydney, NSW 2065 Australia www.sapphirebioscience.com

Sarstedt Australia, Technology Park Adelaide SA 5095

Sarstedt Australia Pty. Ltd. PO Box 90, Ingle Farm, SA 5098 Australia http://www.sarstedt.com/php/main.php

SCHOTT AG Hattenbergstrasse 10 55122 Mainz Germany http://www.schott.com

Selby Biolab 2 Clayton Road Clayton, VIC 3167 Australia http://www.selbybiolab.com.au

Sigma-Aldrich,Australia http://www.sigmaaldrich.com/Area_of_Interest/Asia__Pacific_Rim/Australia/Customer _Service.html

Queensland Institute of Medical Research 297 Appendix 7: List of suppliers

Sigma-Genosys Australia PO Box 970 Castle Hill, Sydney NSW 1765 Australia http://www.sigmaaldrich.com.au/

Silenus Laboratories 5 Guest Street Hawthorn VIC 3122 Australia

Starna Pty. Ltd. PO Box 113 Thornleigh NSW 2120 Australia http://www.starna.com.au/auhome.html

Thermo Electron Corporation, Rochester, NY, USA http://www.thermo.com/

Thermo Electron Corporation, Australia 189-199 Browns Road Noble Park, VIC 3174 Australia

Whatman International Brentford Middlesex UK http://www.whatman.com/

Vector Laboratories http://www.vectorlabs.com/default.asp 30 Ingold Road Burlingame, CA 94010 USA E-mail: [email protected]

Vilber Lourmat B.P. 66 - Torcy Z.I. Sud 77202 Marne-la-Vallée Cedex 1 France http://www.vilber.com/

298 Queensland Institute of Medical Research Appendix 8 Computer Software

APPENDIX 8 COMPUTER SOFTWARE

Adobe Photoshop http://www.adobe.com/au/

BioManager K 25 Medical Foundation Building The University of Sydney Sydney NSW 2006 Australia E-mail:- [email protected] www.angis.org.au

ImageQuant, Molecular Dynamics http://www.mdyn.com

Imagescope® System Aperio Technologies Inc. 1430 Vantage Court, Suite 106 Vista, CA 92081-8545 USA www.aperio.com Tel: 760.539.1100 Fax: 760.539.1116 E-mail: [email protected] Support (Americas and Asia): [email protected]

Queensland Institute of Medical Research 299

Epilogue

EPILOGUE

From Ulysses by Alfred Lord Tennyson (1842)

Tho' much is taken, much abides; and tho'

We are not now that strength which in the old days

Moved earth and heaven; that which we are, we are;

One equal-temper of heroic hearts,

Made weak by time and fate, but strong in will

To strive, to seek, to find, and not to yield.

Queensland Institute of Medical Research 301