Khan, Taslima; (2001) Isolation and functional analysis of Xenopus ephrin-A3. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Segmentation is a primary requirement for the establishment of the vertebrate body plan and it is therefore of great interest to identify proteins involved in these patterning events. The Eph family is the largest sub-group of the Receptor Tyrosine Kinases (RTKs), and several Eph family members have been shown to have important roles in development, including maintenance of segmental boundaries and as guidance cues within the nervous system. Ligands of the Eph family, known as ephrins, have been identified in many vertebrate species where they have been shown to be expressed in many tissues during development and in the adult. A PCR based strategy was used to isolate members of the ephrin-A class in Xenopus. RNAase protection analysis indicated that at two of the ephrins are expressed during gastrula and/or neurula stages. A library screen isolated XLIG4, one of these putative ephrins. Sequence analysis shows that XLIG4 is an ephrin-A3 homologue. Whole mount in situ hybridisation and RNAase protection in Xenopus embryos, revealed that Xephrin-A3 expression occurs throughout gastrulation, neurulation and tailbud stages, with dynamic expression in the migrating crest. Functional analysis was carried out by overexpression of soluble and full length forms of Xephrin-A3 in the Xenopus embryo. Since Xephrin-A3 has expression within specific neural crest streams, it was possible Xephrin-A3 had a role in these cells. Utilising whole mount in situ analysis with molecular markers, it was found that both ephrin forms disrupted the migration pattern of neural crest, the severity of which depended on the concentration of ectopic Xephrin-A3 expressed. Ephrin-A class ligands interact EphA class receptors of which EphA4 is expressed in the third arch neural crest and EphA2 in the second arch neural crest in Xenopus. It is proposed that complementary and overlapping expression of ephrin-A3 and EphA receptors is involved in the targeted migration of branchial neural crest.

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