SEGUNDO COMPONENTE DE LA UNIDAD DE INVESTIGACIÓN

As many RTKs have been found to be important in cell-cell interactions in Drosophila and vertebrates, specific searches have been employed to identify new candidate RTKs that may be involved in patterning and segmentation in the vertebrate embryo. In the hindbrain the main focus of segmental gene expression was previously transcription factors, but searches for genes involved in signal transduction have led to the identification of members of the Eph-related RTK subfamily.

A PCR screen was used to identify genes based on a segmental expression pattern as analysed by in-situ hybridisation. Redundant oligonucleotides that correspond to conserved regions of the catalytic domain were used to PCR amplify cDNA prepared from micro-dissected hindbrains of 9.5 day mouse embryos. Clones were used as probes to screen for expression in 9.5 day mouse embryo hindbrains. This identified Sek-1, a member of the Eph subfamily of RTKs, with restricted expression within the rhombomeres, at high levels in rhombomeres 3 and 5 (Gilardi- Hebenstreit et al., 1992). The PCR screen subsequently identified further Eph-related genes with segmental expression patterns, namely Sek-2j Sek-3 and Sek-4. (Becker et

Figure 1.6

Schematic representation of signal transduction pathways.

(a) Signalling through growth factor RTKs to the c~fos promoter. An example of a signalling pathway involving Ras and MEK, where the activated protein kinase is translocated to the nucleus to phosphorylate transcription factor substrates.

(b) Signalling through interferons. An example of signalling where cytoplasmic transcription factors are translocated to the nucleus upon activation by phosphorylation.

Ligand R ecep tor R a s _Tyrosine k in ase dom ain R af MEK C > ( ERK ERK ERK Phosphorylation c-fos

mm.

IFN- 7 R ecep tor

;

3

^

S T A T 1a/S T A T 1a

S T A T 1a/S T A T 1a

al., 1994).

Eph-related RTKs were also identified by several other approaches. A similar screen was employed by another group to identify RTKs from rat cDNAs enriched in the sciatic nerve, which identified 13 RTKs, 5 of which are members of the Eph subfamily: Tyro 1, 4, 5, 6 and 11 (Lai and Lemke, 1991). Another approach was to screen a lOdpc chicken cDNA expression library with anti-phosphotyrosine antibodies, to detect any phosphorylated tyrosine kinases. From this screen a number of genes encoding Eph RTKs were identified in the chicken and named Cekl to Cek8 (Sajjadi and Pasquale, 1993; Pasquale, 1991; Sajjadi et al., 1991). Members of this family have also been identified because of their expression in transformed cell lines, suggesting an oncogenic potential of unregulated activation of the receptors (Hirai et al., 1987).

The Eph subfamily of receptor tyrosine kinases is the largest known subclass of integral membrane tyrosine kinases. There is a high degree of homology between family members suggesting they may interact with a family of related ligands. A summary of expression patterns and homologues of the family members cloned to date can be found in table 1.1. Family members are all characterised by 20 conserved cysteine residues in the large extracellular domain, thought to hold the receptors in a conserved conformation. There is also an immunoglobulin-like domain and two fibronectin type III repeats. The exact function of these repeats is unclear although they are common among RTKs and neural cell adhesion molecules, and indicate a possible role in adhesion or cell-cell interactions. A schematic diagram of the basic structure of Eph-related RTKs is shown in figure 1.7. Many of the Eph related RTKs cloned to date appear to play a role during development of the embryo, by virtue of their expression patterns in early embryos. Several of the genes are expressed in the hindbrain region of the embryo and other regions of the CNS.

1.4.7 Developmental expression of Sek-1

Sek-1 is expressed in the mouse embryo in the hindbrain, in the telencephalon and dorsal diencephalon of the forebrain, neural crest, otic vesicle, notochord, somitic mesoderm and the tail mesoderm. In the hindbrain expression is first detected in the

Table 1.1 A summary of Eph receptor tyrosine kinases and their expression patterns in the hindbrain where known

mouse chick zebrafîsh Xenopus human rat hindbrain

expression

Sek-1 Cek-8 RTK-1 XSek/

Pagliaccio

TK10/HEK8 Tyro-1 r3,r5

Sek-2/Eck G42/G50 ECK r4

Sek-3/Nuk Cek-5 RTK-3 TK5/HEK5 Tyro-5 r3,rS

Sek-4 Cek-10 HEK2 Tyro-6 r3,r5

Sek-5 r3 (r2,4,5)

RTK-2 r2,r4,r6

Elk Cek-6 Xek Elk r3,r5

Mek-4 Cek-4 TK9/HEK4 Tyro-4

Bsk Cek-7 TK19/HEK7 Ehk-l/Rek-7

Cek-9 Tyro-11 Ehk-2 Mdkl HEKll Ehk-3 r3 (r2,4) Myk-1 HTK EPH E17 G51 55

Figure 1.7

Schematic representation of the structural topology of members of the Eph RTK family.

Extracellular domain

Ig-like dom ain

C ysteine-rich region

Fibronectin III repeat

Transm em brane

Cytoplasm ic domain

0 somite embryo (7.75dpc) in a broad domain, but becomes increasingly restricted to two regions of this domain that correspond to prospective rhombomeres 3 and 5. By the 8 somite stage Sek-1 is restricted to two stripes of high level expression in these prospective rhombomeres, but a low level of expression persists in prospective rhombomeres 2, 4 and 6. As the morphological rhombomere boundaries form, down- regulation of the transcript occurs in rhombomere 4. High level expression persists in rhombomeres 3 and 5 and low level expression persists in rhombomere 2 and 6. Later Sek-1 is down-regulated, first in rhombomere 5 and then in rhombomere 3 (Figure 1.8) (Nieto, A. et al., 1992; Gilardi-Hebenstreit et al., 1992).

The Sek-1 transcript is also detected by in-situ hybridisation in two stripes in the mesoderm immediately caudal to the latest somite formed, presumably corresponding to the somitomeres. Expression is found in the entire somitomere that is next but one to condense, and in the anterior part of the somitomere that is next to form a definitive somite. Expression appears to be down-regulated in an anterior- posterior gradient in this somitomere as the somite forms and is not detected in the definitive somite (Nieto, A. et al., 1992; Gilardi-Hebenstreit et al., 1992).