Eph receptor tyrosine kinases, nervous system development and angiogenesis: Cloning and characterization of Eph receptors from zebrafish and mice

Eph receptors and their membrane-associated ephrin ligands regulate cell-cell interactions during development. The biochemical and biological functions of this receptor tyrosine kinase family are still being elucidated, but include roles in nervous system segmentation and development, axon pathfinding, and angiogenesis. In a search for novel tyrosine kinases involved in vertebrate development, cDNAs corresponding to three distinct members of the Eph-family of receptor tyrosine kinases, zek1, zek2 and zek3 , have been isolated from zebrafish. RNA in situ hybridization analysis on whole-mounted zebrafish embryos revealed distinct, yet overlapping expression of all three gene products within the developing nervous system. zek2 is the zebrafish ortholog of EphA4, but zek1, and zek3 appear to be novel members of the Eph receptor family. Immunoprecipitation and blotting with anti-Zek1 antibodies determined recombinant and native Zek1 protein to be approximately 140 kDa. Pull-down experiments with various ephrin proteins determined that Zek1 associates with ephrinA4 and A5 which, in other organisms, are involved in axon pathfinding in many neural systems. A degenerate PCR-based cloning method was designed to isolate Eph-family member cDNAs. Murine EphA2, A3, A4, A7, B1, B2 and B4 cDNAs were used to generate RNA in situ hybridization probes to examine the developmental expression patterns of these genes in 9.5d mouse embryos. In addition to the expected abundant expression of these Eph receptors in the developing CNS, several of these genes were expressed in discrete regions of the developing vascular system. Recently, EphBs and ephrinBs have been implicated in the processes of capillary sprouting and boundary formation in the circulatory system. We predict that both EphA and B receptors and their ephrin ligands are involved in angiogenesis. While the degenerate PCR method proved effective at isolation of Eph receptor cDNAs, our results suggested that zek1 may not have a murine ortholog. The zebrafish genome has undergone an extra round of duplication after they diverged from mammals, therefore zek1 may have no mammalian ortholog. Since zek1 is widely expressed within the nervous system of the zebrafish, it will be interesting to see what role this receptor plays in the development and maintenance of this organism.