The roles of the homeobox gene Bozozok in zebrafish axis formation

The studies described in this Ph.D. dissertation demonstrate the many roles that the zebrafish bozozok (boz) locus plays in organizer induction and axis formation in zebrafish. The bozm168 mutation was isolated in a large-scale chemical (N-ethyl N-nitrosourea) mutagenesis screen for mutations that affect early development in zebrafish. boz is a recessive, zygotic, embryonic lethal mutation. At one day post fertilization (dpf) bozm168/m168 mutants exhibit a reduction or loss of the main organizer derivatives, notochord and prechordal plate, as well as deficiencies in anterior and ventral CNS structures, suggesting a key role for this gene in axis formation. Epistatic analyses demonstrated boz acts downstream or in parallel to ?-catenin and upstream or in parallel to Nodal signaling. Furthermore, boz was found to encode the homeodomain protein Dharma, which was isolated in a screen for factors with axis-inducing potential. Gene expression analyses and fate-mapping studies indicate that boz embryos exhibit reduced neural induction and that anterior neural fates are transformed to more posterior ones. Inhibition of Bmp signaling was able to suppress the neural induction defect, but not the AP patterning defect in boz mutants. In contrast, inhibition of Wnt signaling was able to suppress both the reduced neural induction and excess posteriorization in boz embryos. Therefore, these analyses indicated boz limits Bmp2/4 activity to promote neural induction and that boz negatively regulates factors such as Wnt8 to promote organizer formation and limit posteriorization of anterior neuroectoderm. Lastly, boz and din double mutant embryos exhibit a synergistic phenotype and do not form a head and trunk. bmp expression was greatly expanded in boz din mutants, resulting in an expansion of nonneural ectoderm at the expense of neural ectoderm. Furthermore, most mesendodermal precursors experienced high Bmp activity and resided vegetally in the tailbud, resulting in the subsequent contribution of these cells to the tail instead of the head and trunk. These studies indicated boz and din act in redundant, partially overlapping pathways that are essential to limit Bmp activity dorsally and allow for head and trunk formation.