vhnf1 integrates global RA patterning and local FGF signals to direct posterior hindbrain development in zebrafish

The vertebrate hindbrain is transiently divided along the anterior-posterior axis into seven morphologically and molecularly distinct segments, or rhombomeres, that correspond to Hox expression domains. The establishment of a proper 'hox code' is required for the development of unique rhombomere identities, including specification of neuronal fates. valentino (vat), the zebrafish ortholog of majB/Kreisler (Kr), encodes a bZip transcription factor that is required cell autonomously for the development of rhombomere (r) 5 and r6 and for activation of Hox group 3 gene expression. Recent work has demonstrated that the expression of vat itself depends on three factors: retinoic acid (RA) signals from the paraxial mesoderm; fibroblast growth factor (Fgf) signals from r4; and variant hepatocyte nuclear factor 1 (vhnf1, also known as tcf2), a homeodomain transcription factor expressed posterior to the r4-5 boundary. We have investigated the interactions between these inputs onto vat expression in the developing zebrafish hindbrain. We show that RA induces vat expression via activation of vhnf1 expression in the hindbrain. Fgf signals from r4, acting through the MapK pathway, then cooperate with Vhnf1 to activate vat expression and subsequent r5 and r6 development. Additionally, vhnf1 and val function as part of a multistep process required for the repression of r4 identity in the posterior hindbrain. vhnf1 acts largely independently of val to repress the r4 'hox code' posterior to the r4-5 boundary and therefore to block acquisition of r4-specific neuronal fates in the posterior hindbrain. However, vhnf1 is not able to repress all aspects of r4 identity equivalently. vat is required downstream of vhnf1 to repress r4-like cell-surface properties, as determined by an 'Eph-ephrin code', by repressing ephrin-B2a expression in r5 and r6. The different requirements for vhnf1 and vat to repress hoxb1a and ephrin-B2a, respectively, demonstrate that not all aspects of an individual rhombomere's identity are regulated coordinately.