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During development of the vertebrate hindbrain regulatory gene expression is confined to precise segmental domains. Studies of cell lineage and gene expression suggest that establishment of these domains may involve a dynamic regulation of cell identity and restriction of cell movement between segments. We have taken a dominant negative approach to interfere with the function of Sek-1, a member of the Eph-related receptor tyrosine kinase family expressed in rhombomeres r3 and r5. In Xenopus and zebrafish embryos expressing truncated Sek-1, lacking kinase sequences, expression of r3/r5 markers occurs in adjacent even-numbered rhombomeres, in domains contiguous with r3 or r5. This disruption is rescued by full-length Sek-1, indicating a requirement for the kinase domain in the segmental restriction of gene expression. These data suggest that Sek-1, perhaps with other Eph-related receptors, is required for interactions that regulate the segmental identity or movement of cells.
Fig. 2. Expression patterns of XSek-1 and rtk1 in the developing hindbrain. (A-F) The expression pattern of XSek-1 during Xenopus development was analysed by whole mount in situ hybridisation. Photographs were taken of either cleared whole embryos (A,D,F), or of the neural epithelium after mounting under a coverslip (B,C,E). (A) Stage 14.5. (B) Rostral neural epithelium at stage 15. (C) Higher magnification view of hindbrain at stage 15. (D) Stage 20. (E) Hindbrain at stage 20. (F) Stage 33. (G-I) The expression pattern of rtk1 during zebrafish development was analysed by whole mount in situ hybridisation. Photographs from a dorsal view were taken after mounting of the hindbrain under a coverslip. (G) 11.5 h. (H) 17 h. (I) 24 h. r, rhombomere; fb, forebrain; nc, neural crest; ol, olfactory placode; ot, otic placode; p, pronephros. Scale bars, 50 mm.
Fig. 3. Effect of truncated Sek-1 on XKrox-20 gene expression in the Xenopus hindbrain. RNA encoding truncated Sek-1 was microinjected into 1 cell of 2 cell Xenopus embryos, so that one half of the embryo received injected RNA. The embryos were allowed to develop to various neurula stages and fixed. In situ hybridisation was then carried out to analyse the expression pattern of XKrox-20, a molecular marker of r3 and r5. Photographs were taken of the mounted hindbrain. (A) Uninjected half of stage 14 embryo. (B) Injected half of stage 14 embryo. (C) Stage 15 embryo; the injected half is on the left. (D-F) Stage 16 embryos, with injected RNA present in the left (D) or right (E,F) half. r, rhombomere. The arrowheads indicate XKrox-20-expressing cells in even-numbered rhombomeres. Scale bars, 50 mm.
