Dlx3b/4b is required for early-born but not later-forming sensory hair cells during zebrafish inner ear development.

Morpholino-mediated knockdown has shown that the homeodomain transcription factors Dlx3b and Dlx4b are essential for proper induction of the otic-epibranchial progenitor domain (OEPD), as well as subsequent formation of sensory hair cells in the developing zebrafish inner ear. However, increasing use of reverse genetic approaches has revealed poor correlation between morpholino-induced and mutant phenotypes. Using CRISPR/Cas9-mediated mutagenesis, we generated a defined deletion eliminating the entire open reading frames of and () and investigated a potential phenotypic difference between mutants and morpholino-mediated knockdown. Consistent with previous findings obtained by morpholino-mediated knockdown of Dlx3b and Dlx4b, mutants display compromised otic induction, the development of smaller otic vesicles and an elimination of all indications of otic specification when combined with loss of , a second known OEPD competence factor in zebrafish. Furthermore, sensorigenesis is also affected in mutants. However, we find that only early-born sensory hair cells (tether cells), that seed and anchor the formation of otoliths, are affected. Later-forming sensory hair cells are present, indicating that two genetically distinct pathways control the development of early-born and later-forming sensory hair cells. Finally, impairment of early-born sensory hair cell formation in mutant embryos reverses the common temporal sequence of neuronal and sensory hair cell specification in zebrafish, resembling the order of cell specification in amniotes; expression before expression. We conclude that the Dlx3b/4b-dependent pathway has been either acquired newly in the fish lineage or lost in other vertebrate species during evolution, and that the events during early inner ear development are remarkably similar in fish and amniotes in the absence of this pathway.