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

Abstract

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 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.

An Assay for Systematically Quantifying the Vestibulo-Ocular Reflex to Assess Vestibular Function in Zebrafish Larvae

“…Since the vestibular function in zebrafish larvae fundamentally relies on the normal development of the vestibular organ, knocking down the regulatory genes that control vestibular organ formation should lead to an abnormal vestibular function (Torres and Giráldez, 1998 ; Baker and Bronner-Fraser, 2001 ; Solomon and Fritz, 2002 ). Previous studies indicated that dlx3b and dlx4b have redundant function to regulate otolith formation in zebrafish; knocking down dlx3b , but not dlx4b , led to single-otolith formation in the ear while knocking down/knocking out both genes resulted in no-otolith/small ear vesicle phenotype (Solomon and Fritz, 2002 ; Liu et al, 2003 ; Schwarzer et al, 2017 ). Consistent with the previous studies (Mo et al, 2010 ; Cameron et al, 2013 ), the results of our dlx3b morphant larvae, which had only saccular otolith in their otic vesicles, suggest that utricular otolith is required for zebrafish LVOR.…”

Transcriptome analysis reveals an Atoh1b-dependent gene set downstream of Dlx3b/4b during early inner ear development in zebrafish

“…Moreover, OEPD formation depends on Dlx3b and Dlx4b (Dlx3b/4b) in zebrafish and combined loss of foxi1 and dlx3b/4b eliminates all indications of otic specification ( Hans et al, 2004 ; Solomon and Fritz, 2002 ). At vesicle stages, absence of Dlx3b/4b causes a loss of early-born hair cells (also known as tether cells) and otoliths, large, solidified bio-crystals that mediate vestibular function ( Millimaki et al, 2007 ; Schwarzer et al, 2017 ). Since the loss of Dlx3 results in early embryonic lethality in mice, the role of Dlx3, which is dynamically expressed in the OEPD in amniotes, has not been addressed so far ( Brown et al, 2005 ; Chen et al, 2017 ; Morasso et al, 1999 ).…”

Morphological, behavioral and molecular studies on CRISPR/Cas9-mediated knockout of the NOMO1 gene in zebrafish

“…Researchers have established detection and analytical methods to measure the characteristic behavioral phenotypes, including locomotion, thigmotaxis, social behavior and aggressive behavior, in zebra sh [20,21]. The CRISPR/Cas9 technique is widely used for gene editing, and a number of transgenic zebra sh models have been developed [13,[22][23][24]. Notably, zebra sh models exhibit ASD-like phenotypes similar to those observed in human diseases.…”