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

Simone Schwarzer,Sandra Spieß,Stefan Hans,Michael Brand

doi:10.1242/bio.026211

Abstract

ABSTRACTMorpholino-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 dlx3b and dlx4b (dlx3b/4b) 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, dlx3b/4b 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 foxi1, a second known OEPD competence factor in zebrafish. Furthermore, sensorigenesis is also affected in dlx3b/4b 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 dlx3b/4b mutant embryos reverses the common temporal sequence of neuronal and sensory hair cell specification in zebrafish, resembling the order of cell specification in amniotes; Neurog1 expression before Atoh1 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.

Highlights

The vertebrate inner ear is a sensory organ mediating hearing and balance
The preplacodal region is further specified into a common otic-epibranchial progenitor domain (OEPD) that in zebrafish contains the progenitors of the anterior lateral line ganglion (Chen and Streit, 2013; McCarroll et al, 2012; Hans et al, 2013)
Competence to respond to fibroblast growth factor (Fgf) signals is conferred by Foxi3, a forkhead transcription factor, whereas in zebrafish, competence is provided by the functional homolog Foxi1 and by the distal-less homeodomain transcription factors Dlx3b and Dlx4b (Dlx3b/4b) (Hans et al, 2004; Solomon et al, 2004; Yamanishi et al, 2012; Khatri et al, 2014; Birol et al, 2016)

Summary

Introduction

The vertebrate inner ear is a sensory organ mediating hearing and balance It derives from the otic placode, a transient ectodermal thickening adjacent to the developing hindbrain, and contains a Technische Universitä t Dresden, Biotechnology Center and DFG-Center for Regenerative Therapies Dresden Cluster of Excellence, Tatzberg 47-49, 01307 Dresden, Germany. The otic placode develops into the otocyst or otic vesicle with mechanosensory hair cells generated in the sensory epithelia and neuronal precursors delaminating as neuroblasts from the ventral face of the otic vesicle (Haddon and Lewis, 1996; Rubel and Fritzsch, 2002) Both lineages require the activity of the proneural proteins Atonal homologue (Atoh1) and Neurogenin (Neurog1) (Ma et al, 1998; Bermingham et al, 1999; Andermann et al, 2002; Fritzsch et al, 2010). Amniotes do not form otoliths, but structurally similar

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