Eph signalling functions downstream of Val to regulate cell sorting and boundary formation in the caudal hindbrain

Rhombomeres are segmental units of the vertebrate hindbrain that underlie t he reiterated organisation of cranial neural crest migration and neuronal d ifferentiation. valentine (val), a zebrafish homologue of the mouse bzip tr anscription factor-encoding gene, kreisler, is required for segment boundar y formation caudal to rhombomere 4 (r4), val is normally expressed in r5/6 and is required for cells to contribute to this region, In val(-) mutants, rX, a region one rhombomere in length and of mixed identity, lies between r 4 and r7, While a number of genes involved in establishing rhombomeric identity are k nown, it is still largely unclear how segmental integrity is established an d boundaries are formed. Members of the Eph family of receptor tyrosine kin ases and their ligands, the ephrins, are candidates for functioning in rhom bomere boundary formation. Indeed, expression of the receptor ephB4a coinci des with val in r5/6, whilst ephrin-B2a, which encodes a ligand for EphB4a, is expressed in r4 and r7, complementary to the domain of val expression. Here we show that in val(-) embryos, ephB4a expression is downregulated and ephrin-B2a expression is upregulated between r4 and r7, indicating that Va l is normally required to establish the mutually exclusive expression domai ns of these two genes. We show that juxtaposition of ephB4-expressing cells and ephrin-B2a-expressing cells in the hindbrain leads to boundary formati on. Loss of the normal spatial regulation of eph/ephrin expression in val m utants correlates not only with absence of boundaries but also with the ina bility of mutant cells to contribute to wild-type r5/6. Using a genetic mos aic approach, we show that spatially inappropriate Eph signalling underlies the repulsion of val(-) cells from r5/6. We propose that Val controls eph expression and that interactions between EphB4a and Ephrin-B2a mediate cell sorting and boundary formation in the segmenting caudal hindbrain.