Targeted germ line disruptions reveal general and species-specific roles for paralog group 1 hox genes in zebrafish

The developing vertebrate hindbrain is transiently segmented into rhombomeres by a process requiring Hox activity. Hox genes control specification of rhombomere fates, as well as the stereotypic differentiation of rhombomere-specific neuronal populations. Accordingly, germ line disruption of the paralog group 1 (PG1) Hox genes Hoxa1 and Hoxb1 causes defects in hindbrain segmentation and neuron formation in mice. However, antisense-mediated interference with zebrafish hoxb1a and hoxb1b (analogous to murine Hoxb1 and Hoxa1, respectively) produces phenotypes that are qualitatively and quantitatively distinct from those observed in the mouse. This suggests that PG1 Hox genes may have species-specific functions, or that anti-sense mediated interference may not completely inactivate Hox function in zebrafish.

Using zinc finger and TALEN technologies, we disrupted hoxb1a and hoxb1b in the zebrafish germ line to establish mutant lines for each gene. We find that zebrafish hoxb1a germ line mutants have a more severe phenotype than reported for Hoxb1a antisense treatment. This phenotype is similar to that observed in Hoxb1 knock out mice, suggesting that Hoxb1/hoxb1a have the same function in both species. Zebrafish hoxb1b germ line mutants also have a more severe phenotype than reported for hoxb1b antisense treatment (e.g. in the effect on Mauthner neuron differentiation), but this phenotype differs from that observed in Hoxa1 knock out mice (e.g. in the specification of rhombomere 5 (r5) and r6), suggesting that Hoxa1/hoxb1b have species-specific activities. We also demonstrate that Hoxb1b regulates nucleosome organization at the hoxb1a promoter and that retinoic acid acts independently of hoxb1b to activate hoxb1a expression.

We generated several novel germ line mutants for zebrafish hoxb1a and hoxb1b. Our analyses indicate that Hoxb1 and hoxb1a have comparable functions in zebrafish and mouse, suggesting a conserved function for these genes. In contrast, while Hoxa1 and hoxb1b share functions in the formation of r3 and r4, they differ with regards to r5 and r6, where Hoxa1 appears to control formation of r5, but not r6, in the mouse, whereas hoxb1b regulates formation of r6, but not r5, in zebrafish. Lastly, our data reveal independent regulation of hoxb1a expression by retinoic acid and Hoxb1b in zebrafish.