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

• Published in: Development (Cambridge) Vol. 128; no. 4; pp. 571 - 580
• Main Authors: Cooke, J, Moens, C, Roth, L, Durbin, L, Shiomi, K, Brennan, C, Kimmel, C, Wilson, S, Holder, N
• Format: Journal Article
• Language: English
• Published: England The Company of Biologists Limited 01.02.2001
• Subjects: Animals; Cell Differentiation; Cell Division; Cell Lineage; Danio rerio; Eph receptors; ephB4 gene; Ephrin-B2; ephrin-B2a gene; Gene Deletion; Gene Expression Regulation, Developmental; Immunohistochemistry; In Situ Hybridization; kreisler gene; MafB Transcription Factor; Membrane Proteins - genetics; Membrane Proteins - metabolism; Microinjections; Mosaicism - genetics; Nerve Tissue Proteins - genetics; Nerve Tissue Proteins - metabolism; Phenotype; Protein Binding; Receptor Protein-Tyrosine Kinases - genetics; Receptor Protein-Tyrosine Kinases - metabolism; Receptor, EphB4; Receptors, Eph Family; Recombinant Fusion Proteins; Rhombencephalon - cytology; Rhombencephalon - embryology; Rhombomeres; RNA, Messenger - metabolism; Signal Transduction; valentino gene; Zebrafish - embryology; Zebrafish Proteins
• ISSN: 0950-1991; 1477-9129
• DOI: 10.1242/dev.128.4.571

Rhombomeres are segmental units of the developing vertebrate hindbrain that underlie the reiterated organisation of cranial neural crest migration and neuronal differentiation. valentino (val), a zebrafish homologue of the mouse bzip transcription factor-encoding gene, kreisler, is required for segment boundary 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 r4 and r7. While a number of genes involved in establishing rhombomeric identity are known, it is still largely unclear how segmental integrity is established and boundaries are formed. Members of the Eph family of receptor tyrosine kinases and their ligands, the ephrins, are candidates for functioning in rhombomere boundary formation. Indeed, expression of the receptor ephB4a coincides 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 Val is normally required to establish the mutually exclusive expression domains of these two genes. We show that juxtaposition of ephB4a-expressing cells and ephrin-B2a-expressing cells in the hindbrain leads to boundary formation. Loss of the normal spatial regulation of eph/ephrin expression in val mutants correlates not only with absence of boundaries but also with the inability of mutant cells to contribute to wild-type r5/6. Using a genetic mosaic 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.