Investigation of ephrin regulation during hindbrain segmentation

Early in the development of the vertebrate central nervous system, the hindbrain is transiently segmented into morphological units termed rhombomeres (r). Eph-family receptor tyrosine kinases such as EphA4 and EphB2 are expressed in rhombomeres 3 and 5, while their ligands - the ephrins - such as ephrin-B2, are expressed in r2, r4 and r6. The interaction between receptor and ligand in these complementary domains is important in the restriction of cell-mixing between rhombomeres. Hox genes are important in the parallel process of assigning axial identity to the resulting segments. These pathways are interconnected both by upstream factors such as Krox-20 and kreisler that regulate Hox genes as well as an Eph receptor, and by regulatory links between Hox genes and Ephs. In order to discover more about the regulation of ephrins during hindbrain development, two main approaches were selected. Preparatory work has been carried out for the analysis of the ephrin-B2 regulatory region and a number of candidate regulatory factors have been investigated. Analysis of kreisler mutant embryos and transgenic embryos expressing kreisler in r3 has shown that this transcription factor does not regulate ephrin-B2 or -B3 in r5/6. These data also provide new insights into the nature of the disruption to segmentation in the kreisler mutant. Analysis of Krox-20 mutant embryos indicates a role in the repression of ephrin-B2 expression in r3/5. Analysis of Hoxa1 and Hoxb1 mutant embryos has shown that the loss of either of these factors individually does not affect ephrin expression in the caudal hindbrain. Regulation of ephrin-B2 by retinoids was investigated by exposing zebrafish embryos to all-trans-retinoic acid or the retinoic acid- antagonist - BMS 493. These analyses have demonstrated a role for retinoids in ephrin regulation and have also provided further insights about the mechanisms underlying hindbrain development.