1 página.-- 1º Congress of the Spanish Society of Developmental Biology.-- Developmental neurobiology.Peer reviewe

Araujo, María

Nieto, M. Ángela

The International Journal of Developmental Biology

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De\'e/opmenl11/ neurobiology )57SCHARACTERISATION OF Cek-11, A MEMBER OF THE EPH-RECEPTOR FAMILY IN THE CHICK EMBRYOMaria ARAUJOand M.Angela NIETOInstituto Gajal, GSIG. Avda. de Dr.Arce, 37, 28002 Madrid, SpainEmbryonic development and pattern formation in vertebrates require coordinated growth and cell differentiation. Intercellularcommunication is crucial for development, and cells respond to many extracellular cues by signal transduction throughmembrane-bound molecules. One class of such membrane-spanning proteins is represented by receptor protein tyrosinekinases (RTKs).We have employed a screening procedure designed to identify genes encoding protein kinases (PKs) involved in the earlydevelopment of the Nervous System. RNA was purified from 1 to 3-day-old chick embryonic heads. The RNA was reversetranscribed, and Kinase catalytic domain coding sequences were PCR amplified, using two degenerate oligonucleotidescorresponding to the amino acids sequences IHRDL and DVWSFG; which are strongly conserved motifs in the catalyticdomain of the PKs.The Kinase domain amplified was 210-240 bp in length, and contains peptide sequences conserved between PKs andsequence divergences in the non conserved regions of this domain, allowing us to ident~y the PCR fragments that belong togenes encoding PKs.We have analysed over 200 PCR clones and identified sequences corresponding to fifteen genes encoding PKs. Comparisonwith GeneSank and EMSL databases revealed that, many of the clones correspond to kinases whose cDNAs have previouslybeen cloned in the chick, like EGF-R (c-erbS), cytoplasmic kinases such as c-yes, c-Src and its inhibitor Csk and themembers of the Eph family of the RTKs, CekS, Cek8 and Cek9. Other clones show a high degree of similarity to kinases clonedin other species of vertebrates, like PDGF-R and the cytoplasmic kinases Jak-1 and Jak-2.We have also identified a novel member of the Eph related receptors, that we have named Cek11, according to thenomenclature for !;hick ~mbryonic !l.inases (Cek).Antisense RNA probes, corresponding to each of the different kinase clones, were used to analyse their patterns ofexpression by whole mount in situ hybridisation, in 1 to 3-day-old chick embryos. We have obtained interesting patterns ofexpression for several clones, suggestive of their involvement in the early regionalisation of the Central Nervous System(CNS). We have chosen Cek11 for further studies.We have screened a 2-day-old chick embryo cDNA library with a probe from the Cek11 amplified fragment, and isolatedoverlapping clones that encompass the complete open reading frame. The sequence shows a very high degree of similarity, atthe nucleotide level, to the human Hek11 (88%), the mouse MDK1(84%), the rat Ehk3 (8S%) and the zebrafish ZDK1 genes.The similar~ with other members of the Eph family described in the chick is around 60%.In situ hybridisation analysis reveals that Cek11 is transiently expressed in the alar plates of several segments of the earlynervous system, including prosomeres 1 and 2 (p1, p2) of the diencephalon and all the rhombomeres (r) in the hindbrain.Transcripts can be detected before the morphological appearance of the segments, as has been described for other membersof the family, like Sek1 (the mouse homologue of Cek8). This suggests that, Cek11 might be involved in the segmentation ofthe diencephalon and the hindbrain, in agreement with the results obtained with Sek1, indicating that it is involved in theestablishment and maintenance of the rhombomeric segmentation (Nieto et aI., 1992: Xu et al.. 1995). At postsegmentationstages expression of Cek11 occurs at the presumptive location of several axonal tracts.AII.I.Fig. 1. Segmentation expression of Cekl1 in thedeveloping nervous system visualized by wholemount in situ hybridisation analysis. (A) 34-somiteembryo. Cek11 is expressed in tha alar plates of p1and p2 in the diencephalon, and rl-R) in the hinroraln.(B) Dorsal view of the hindbrain of the same embryo.Expression is detacted in the alar plate ofrhombomeres, and also draws longitudinal columnsin the basal plate, that could be related to the futurelocation of several axonal tracts.B'III--1585 Deve!opmcllwlnclirobi%gyThe expression of Cek11 in regions of segmentation and developing axonal tracts is detected before their actualappearance, suggesting a double role for Cek11 in patterning of the CNS at different stages: antero-posterior patterning inearly embryos and axonal tract formation at later stages.ReferencesNieto. M.A.. Gilarde-Hebens!reit. P., Charnay, P. and Wilkinson, D.G. (1992). A receptor protein tyrosine kinase implicated in the segmental panerning of thehindbrain and mesoderm. Development 116: 1137-1150.Xu. C.. Graham.A.. Holder,N. and Wilkinson,D.G. (1995). Expression01truncatedSek-' receptortyrosinekinase disrupts the segmental restriction of geneexpression in the Xenopus and zebrafish hindbrain. Development 121: 4005-4016.

Characterisation of Cek-11, a member of the EPH-receptor family in the chick embryo

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