Degenerate PCR-based cloning method for Eph receptors and analysis of their expression in the developing murine central nervous system and vasculature.
Eph receptors and their membrane-associated ephrin ligands regulate cell-cell interactions during development. The biochemical and biologic functions of this receptor tyrosine kinase family are still being elucidated but include roles in nervous system segmentation, axon pathfinding, and angiogenesis. To isolate murine orthologs of three zebrafish Eph family members (zek1, zek2, and zek3), we have used a degenerate RT-PCR-based cloning method specific for members of the Eph family. Although this method was effective for isolation of Eph receptor cDNAs, including members of both the A and B subfamilies, our results suggested that zek1 may not have a murine ortholog. The isolated cDNAs were also used to generate RNA in situ hybridization probes to examine the expression patterns of murine EphA2, A3, A4, A7, B1, B2, and B4 in 9.5-dpc mouse embryos. In addition to the expected abundant expression of these Eph receptors in the developing CNS and the presence of EphB receptors in vascular tissues, several of the EphA receptors were expressed in discrete regions of the developing vasculature. These results suggest a role for both EphA and EphB receptors in vascular development.
Bovenkamp
D E
DE
Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.
Greer
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PA
D000595:Amino Acid Sequence; D000818:Animals; D001808:Blood Vessels / Q000196:embryology* / Q000378:metabolism; D015139:Blotting, Southern; D002490:Central Nervous System / Q000196:embryology* / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary / Q000235:genetics; D019143:Evolution, Molecular; D005260:Female; D005326:Fetal Proteins / Q000096:biosynthesis / Q000235:genetics; D018507:Gene Expression Regulation, Developmental; D015723:Gene Library; D005865:Gestational Age; D051379:Mice; D008969:Molecular Sequence Data; D005810:Multigene Family; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000235:genetics; D009693:Nucleic Acid Hybridization; D010802:Phylogeny; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D020794:Receptor Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000235:genetics*; D036081:Receptors, Eph Family; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D014714:Vertebrates / Q000235:genetics; D015027:Zebrafish / Q000235:genetics; D029961:Zebrafish Proteins
D018076:DNA, Complementary; D005326:Fetal Proteins; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D029961:Zebrafish Proteins; D020794:Receptor Protein-Tyrosine Kinases; D036081:Receptors, Eph Family; C107358:ek1 protein, zebrafish