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Helix-loop-helix transcription factors of the Ebf/Olf1 family have previously been implicated in the control of neurogenesis in the central nervous system in both Xenopus laevis and the mouse, but their precise roles have remained unclear. We have characterised two family members in the chick, and have performed a functional analysis by gain- and loss-of-function experiments. This study revealed several specific roles for Ebf genes in the spinal cord and hindbrain regions of higher vertebrates, and enabled their precise positioning along the neurogenic cascade.During neurogenesis, cell cycle exit appears to be tightly coupled to migration to the mantle layer and to neuronal differentiation. We show that antagonizing Ebf gene activity allows the uncoupling of these processes. Ebf gene function is necessary to initiate neuronal differentiation and migration toward the mantle layer in neuroepithelial progenitors, but it is not required for cell cycle exit. Ebf genes therefore appear to be master controllers of neuronal differentiation and migration, coupling them to cell cycle exit and earlier steps of neurogenesis.Mutual activation between proneural and Ebf genes suggests that besides their involvement in the engagement of differentiation, Ebf genes may also participate in the stabilisation of the committed state. Finally,gain-of-function data raise the possibility that, in addition to these general roles, Ebf genes may be involved in neuronal subtype specification in particular regions of the CNS.

Facial branchiomotor (fbm) neurones undergo a complex migration in the segmented mouse hindbrain. They are born in the basal plate of rhombomere (r) 4, migrate caudally through r5, and then dorsally and radially in r6. To study how migrating cells adapt to their changing environment and control their pathway, we have analysed this stereotyped migration in wild-type and mutant backgrounds. We show that during their migration, fbm neurones regulate the expression of genes encoding the cell membrane proteins TAG-1, Ret and cadherin 8. Specific combinations of these markers are associated with each migratory phase in r4, r5 and r6. In Krox20 and kreisler mutant mouse embryos, both of which lack r5, fbm neurones migrate dorsally into the anteriorly positioned r6 and adopt an r6-specific expression pattern. In embryos deficient for Ebf1, a gene normally expressed in fbm neurones, part of the fbm neurones migrate dorsally within r5. Accordingly, fbm neurones prematurely express a combination of markers characteristic of an r6 location. These data suggest that fbm neurones adapt to their changing environment by switching on and off specific genes, and that Ebf1 is involved in the control of these responses. In addition, they establish a close correlation between the expression pattern of fbm neurones and their migratory behaviour, suggesting that modifications in gene expression participate in the selection of the local migratory pathway.

The developing vertebrate hindbrain is transiently subdivided along the anterior-posterior axis into metameric units, called rhombomeres (r). These segments constitute units of lineage restriction and display specific gene expression patterns. The transcription factor gene Krox-20 is restricted to r3 and r5, and is required for the development of these rhombomeres. We present evidence that Krox-20 transcriptional activity is under the control of a negative feedback mechanism in the hindbrain. This regulatory loop involves two closely related proteins, Nab1 and Nab2, previously identified as antagonists of Krox-20 transcriptional activity in cultured cells. Here we show that in the mouse hindbrain, Nab1 and Nab2 recapitulate the Krox-20 expression pattern and that their expression is dependent on Krox-20 function. Furthermore, misexpression of Nab1 or Nab2 in zebrafish embryos leads to alterations in the expression patterns of several hindbrain markers, consistent with an inhibition of Krox-20 activity. Taken together, these data indicate that Krox-20 positively regulates the expression of its own antagonists and raise the possibility that this negative feedback regulatory loop may play a role in the control of hindbrain development.