Signaling pathways underlying zebrafish habenular development and connectivity
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Recent studies have focused on the habenulae (Hb), bilateral nuclei in the epithalamus of the vertebrate forebrain due to their involvement in addiction and psychiatric disorders. The Hb consist of dorsal (dHb) and ventral (vHb) subnuclei, which are molecularly distinct and innervate different midbrain and hindbrain targets to modulate diverse behaviors. The specific neuronal populations that comprise the dHb, how these neurons arise and form appropriate connections with the principal midbrain target, the interpeduncular nucleus, are only partly understood. My entry point to the study of habenular development was the characterization of a zebrafish mutation in the wntless (wls) gene, which encodes a chaperone protein necessary for secretion of Wnt morphogens. Through analyses of the habenular phenotype of wls homozygous mutants, I uncovered an unappreciated role for Wnt signaling in the generation of dHb progenitors. Previous studies on zebrafish mutants had shown that Wnt signaling is necessary to establish left-right (L-R) asymmetry of the dHb and for formation of the vHb. Although they develop small habenulae, L-R asymmetry is preserved in wls mutants, revealing temporally distinct requirements for Wnt activity. Hedgehog (Hh) and fibroblast growth factor (Fgf) signaling pathways are also involved in specification of dHb progenitors. Epistasis experiments indicate that Hh signaling is upstream of or parallel to Wnt signaling, which, in turn, defines the domain of Fgf signaling in the habenular region. Previously, habenular progenitors were distinguished by expression of the developing brain homeobox 1b (dbx1b) and chemokine (C-X-C motif), receptor 4b (cxcr4b) genes. However, live-imaging, lineaige tracing and long-term observation indicate that dbx1b is transcribed by dHb progenitors, whereas cxcr4b transcripts localize to neural precursors located between the progenitors and mature neurons. The function of Cxcr4b/chemokine signaling in dHb development was unknown. I determined that components of this pathway are expressed in cells surrounding the developing dHb and that Fgf signaling delimits their spatial domains. Finally, analyses of zebrafish mutants reveal that chemokine signaling directs posterior outgrowth of axons from dHb neurons. Overall, my results provide new insights into the genetic network underlying dHb development and implicate chemokine signaling in the regulation of habenular axonal outgrowth.