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Based on their expression patterns Eph receptor tyrosine kinases have been implicated as a potential candidates involved in cell interactions in cranial neural crest emanating from the hindbrain.

Receptor tyrosine kinases (RTKs) play critical roles in signal transduction pathways in which they relay signals such as growth factors from the extracellular environment across the plasma membrane. All RTKs possess an extracellular domain, a hydrophobic transmembrane domain and an intracellular tyrosine kinase domain. Upon ligand binding to the extracellular domain, receptor dimérisation occurs and subsequently there is a transient activation of the intracellular tyrosine kinase domain by autophosphorylation of specific tyrosine residues (Ullrich and Schlessinger, 1990) (van der Geer et al., 1994). The phosphorylated receptors are then free to interact with the SH2 domains on intracellular signalling proteins by means of short phosphotyrosyl-containing amino acid sequences (van der Geer et al., 1994).

RTKs can be subdivided into 14 subfamilies depending on the structure of the extracellular domain. The largest of these subfamilies is the Eph family which comprises at least 13 members. All members of the Eph family are characterised by a highly conserved extracellular domain consisting of a single immunoglobulin-like domain, two fibronectin type III repeats and 20 conserved cysteine residues (Lindberg and Hunter, 1990) (Lhotak et al., 1991) (Pasquale, 1991) (Gilardi-Hebenstreit et al., 1992) (Sajjadi and Pasquale, 1993). The cysteine residues are thought to hold the receptors in a conserved conformation. The Eph receptors have a highly conserved intracellular domain but little is known about the molecules which interact with the intracellular domain of the Eph receptors.

Members of the Eph family were initially identified as orphan receptors but recently a family of ligands have been characterised. All the ligands are membrane bound and the attachment to the cell membrane has been shown to be necessary for activation of the receptor (Davis et al., 1994). This makes them unique as RTK ligands in that they are inactive in a soluble form and this suggests that the Eph receptors and their ligands may mediate cell contact dependent signalling. Artificially clustering the soluble ligands results in receptor activation, indicating that attachment to the cell surface may facilitate ligand oligomerisation (Davis et al., 1994).

The ligands can be divided into two subclasses depending on their method of attachment to the cell surface. They can be anchored to the cell membrane either by a transmembrane

domain or a glycosylphosphatidylinositol (GPI) linkage. There are three transmembrane ligands, Elk-L, Htk-L and Elk-L3 and these have strongest homology in the C-terminal portion of their cytoplasmic regions (Bergemann et a i, 1995) (Gale et al., 1996a). Based on this strong conservation it has been postulated that this region may have a role (Brambilla et at., 1995).

B61, EHKl-L, RAGS, Elf-1, and LERK4 constitute a separate class of ligands, which are attached to the cell surface via a GPI anchor attached at the C-terminal tail. These ligands share greater homology with each other than with members of the transmembrane subclass of ligands (Gale e ta l, 1996b) and the region of greatest homology within this subclass is the putative receptor binding region which includes four conserved cysteine residues (Kozlosky etal., 1995).

Functional binding studies have shown that the all the ligands for the Eph receptors can be divided into two subclasses (Gale et al., 1996b) (Figure 1.4) that correspond to the two structural subclasses. Transmembrane ligands, Elk-L, Htk-L and Elk-L3, have been shown to interact exclusively with four of the Eph receptors, termed the Elk subgroup, consisting of Sek-3, Sek-4, Elk and Htk (Gale et al., 1996b) (Beckmann et al., 1994) (Brambilla et al., 1995). The GPI-anchored ligands, B61, EHKI-L, RAGS, Elf-1, and LERK4, bind to a different subset of the Eph receptors known as the Eck subgroup (Gale et al., 1996b). There is a level of promiscuity within each group with various receptors able to bind to distinct ligands with different affinities (Kozlosky et al., 1995) (Cheng and Flanagan,

1994) (Gale et al., 1996b). The receptor Sekl is the only exception, with the ability to interact with all the GPI-anchored ligands and Htk-l and Elk-L3 of the transmembrane ligands (Gale et ah, 1996a) (Gale et al., 1996b).

Fusing of the extracellular portion of the receptors or ligands to the Fc domain of IgG and has been used to analyse the distribution of the different classes of receptors and ligands in whole mouse embryos. Detection of transmembrane or GPI-anchored ligands using the IgG Fc fusion proteins shows complementary expression patterns to their corresponding subclass of receptor in embryos at embryonic day 10.5 (Gale et al., 1996b) and in embryos at embryonic day 9.5 (Flenniken et al., 1996) in a variety of tissues. In addition, whole mount in situ hybridisation has also shown this complimentary in the hindbrain where receptors and ligands are expressed in alternating rhombomeres (Figure 1.5). S e k l, Sek-3

and Sek-4, are expressed at high levels in rhombomeres 3 and 5 whereas the ligands Elk-L, Elf-2, and Elk-L3 are expressed at high levels in even numbered rhombomeres.

The Eph family of receptors and their ligands have been implicated in a number of different functions including establishing a topographic map from the retina to the tectum. Expression patterns and binding studies using receptor or ligand affinity probes show that

Figure 1.4: Binding specificity subclasses of the Eph RTKs and their ligands.

The Eph RTKs and their ligands are divided into two subfamilies depending on the binding specificities. This also correlates with their structural homologies. Members of one receptor subfamily can bind to all of the transmembrane ligands whereas members of the other subfamily bind to the GPI-anchored ligands. The exception is Sekl which can bind to the GPI-anchored ligands and two of the transmembrane ligands. Based on (Gale et a i, 1996).

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Figure 1.5: Segmental expression patterns of Eph RTKs and their ligands.

The diagram shows the segment restricted expression pattern of the Eph RTKs and their ligands in the hindbrain of a 9.5 day mouse embryo. The shading depicts the relative levels of transcripts. Note the complimentary expression patterns of the transmembrane ligands and their receptors.

ri r2 r3 r4 r5 r6 r7 Sek-2 (eck) ebk Sek-1 Sek-3 (nuk) Sek-4 Receptors Elk-L Elf-2 Elk-L3 Transm em brane Ligands

there are complementary gradients of the ligands Elf-1 and RAGS in the developing tectum with Mek4 in the developing retina (Drescher et a i, 1995) (Cheng et al., 1995). RAGS and

Elf-1 are expressed in decreasing posterior to anterior gradients in the tectum with the highest levels in the posterior tectum, whereas Mek4 has a complementary gradient of expression in the retina with the highest concentration in the temporal retinal ganglion axons (Cheng et a i, 1995). These axons project to the anterior tectum where the ligands are expressed at low levels. Nasal retinal ganglion axons, which are expressing low levels of the receptor project to the posterior tectum where there is high levels of ligand expression. Whereas, temporal retinal ganglion axons, which are expressing high levels of the receptor project to the anterior tectum where there is low levels of ligand expression. The migration of retinal axons to the tectum has been analysed in vitro using a “membrane stripe assay” (Walter et al., 1987). In this assay, retinal axons are given the choice of growing along anterior or posterior tectal membranes. Although temporal axons can grow on membranes from the posterior tectum they grow preferentially along stripes of membranes from the anterior tectum. If posterior membranes are added to growing cultures of temporal axons their growth cones are immediately paralysed and retract but if anterior membranes are added, no effect is observed (Walter et al., 1987). These in vitro assays have shown that Elf-1 is repellent for temporal and not nasal axon guidance, whereas, RAGS is repellent for both types of axons, though to different degrees (Monschau et al.,

1996) (Nakamoto et al., 1996). Additional experiments involving retroviral overexpression of Elf-1 in the chick tectum leads to retinal axons avoiding ectopic patches of Elf-1

expression and their subsequent mapping to abnormally anterior positions (Nakamoto et al., 1996). Based on these data a high level of Mek4 receptor expression in the temporal retinal ganglion cells appears to cause the axons to be repelled away from high levels of expression of the ligands RAGS and Elf-1 in the tectum.

There appears to be a similar role for RAGS (AL-7) in axon fasciculation (Winslow et al.,

1995). It has been observed in vitro that rat cortical neurones bundle together as they grow across astrocytes (Winslow etal., 1995) and that RAGS is expressed on astrocytes during development and a putative receptor Ehkl (RekT) is expressed on the cortical neurones. Receptor blocking experiments have been performed where the extracellular domain of

Ehkl has been fused to the constant region of the IgG domain and added to cultures of cortical neurones growing across astrocytes. The chimeric protein inhibits binding of the ligand to the receptor and prevents the growing neurones from bundling together suggesting that RAGS is required for axon bundling. Taken together with studies in the retinotectal system, it has been proposed that this is due to repulsion by the astrocytes.

The receptor Sekl has been shown to play a role in the developing hindbrain. S e k l was originally identified by screening a mouse cDNA library in a search for tyrosine kinases which are segmentally expressed in the hindbrain (Nieto et a l, 1992) and is expressed at high levels in rhombomeres 3 and 5. Homologues of Sekl have been identified in zebrafish (Xu et al., 1994), chick (Sajjadi and Pasquale, 1993) (Patel et al., 1996) and Xenopus

(Winning and Sargent, 1994) (Xu et al., 1995). In vivo functional studies on S ekl have been performed in zebrafish and Xenopus by a dominant negative approach (Xu et al.,

1995) (Xu et al., 1996). Cleavage stage zebrafish embryos, or one cell of a two cell

Xenopus embryos were injected with mRNA encoding truncated dominant negative Sekl

lacking the intracellular kinase domain. In the injected embryos display disrupted hindbrain segmentation. This is observed by KroxlO expressing cells in even numbered rhombomeres in a domain contiguous with the KroxlO expression in rhombomeres 3 and 5. Since Krox20 is normally expressed in rhombomeres 3 and 5 this demonstrates that the expression of dominant negative Sekl results in the presence of odd numbered rhombomere cells in even numbered rhombomere territory (Xu et al., 1995). Cell lineage labelling experiments of rhombomeric cells have shown that there is little crossing of rhombomere boundaries by cells once the boundaries have formed (Birgbauer and Fraser,

1994) (Fraser et al., 1990). Therefore, the phenotypes caused by expression of dominant negative Sekl may indicate that the cells of rhombomeres 3 and 5 are now able to cross rhombomere boundaries and enter even numbered rhombomere territory. Alternatively, these phenotypes may indicate that Sekl is required to switch rhombomere cell identity. During hindbrain development cells are able to cross between rhombomeres prior to the formation of boundaries, but analysis using molecular markers shows that ectopic odd or even numbered rhombomeric cells are never observed in inappropriate territories. This suggests that Sekl could be involved in regulating cell fate changes (Xu et al., 1995). The hindbrain phenotypes can be rescued by coinjecting full length S ekl with dominant negative Sekl, indicating a need for the intracellular kinase domain in the normal function of Sekl (Xu et al., 1995). In the zebrafish embryo the expression of dominant negative

Sekl also causes a forebrain phenotype which involves an expansion of the retina and loss of diencephalic structures (Xu et al., 1996). As with the hindbrain phenotypes this suggests a role for Sekl in regulating cell fate change or cell movement in the forebrain.