Rhombomere boundaries and hindbrain patterning in the zebrafish
The vertebrate hindbrain is a segmented structure, divided into repeating segments called rhombomeres, at the interface of which boundary cells are induced as a specific cell type. Previous work has found that, in the zebrafish hindbrain, there is a distinctive organisation of glia and neurons along the anterior-posterior axis within each rhombomere, with specific neurons at the centre or boundary regions, separated by a glial curtain adjacent to the boundary. Using molecular markers, I have characterised the organisation of several cell types within each rhombomeres, and found that from 24 hours of development, neurogenesis occurs predominantly adjacent to the rhombomere boundaries, which suggests a role for boundaries in establishing this pattern. The aim of this work was to test a possible patterning role of boundaries, and to establish a genetic hierarchy between boundary genes by carrying out knockdowns of boundary-specific genes. The secreted factor wntl is expressed in dorsal boundaries and is a candidate mediator of a potential signalling role of boundaries. From many lines of evidence in previous work, wntl was known to promote proliferation of neural progenitors and inhibit neuronal differentiation. I found that in the zebrafish hindbrain, however, knockdown of wntl or of the Wnt pathway effector tcj3b causes expansion of boundary-specific markers and loss of neurogenesis in non- boundary regions. Knockdown of rfng, a component of the Notch pathway required for boundary expression of wntl also causes expansion of boundary markers. Wntl is necessary for the expression of ash and ngnl proneural genes, and of delta genes, which in turn block ectopic boundary marker expression. Thus, wntl mediates a lateral inhibition of boundary fate by regulating neurogenesis in hindbrain segments. Taken together with concurrent work in the lab, the model derived from this is remarkably similar to the regulatory interactions occurring at the dorso-ventral boundary of the Drosophila wing imaginal disc, and indicates recruitment of a conserved network of genes in non homologous tissues.