Anteroposterior patterning of the zebrafish ear through Fgf- and Hh-dependent regulation of hmx3a expression

Abstract

In the zebrafish, Fgf and Hh signalling assign anterior and posterior identity, respectively, to the poles of the developing ear. Mis-expression of fgf3 or inhibition of Hh signalling results in double-anterior ears, including ectopic expression of hmx3a . To understand how this double-anterior pattern is established, we characterised transcriptional responses in Fgf gain-of-signalling or Hh loss-of-signalling backgrounds. Mis-expression of fgf3 resulted in rapid expansion of anterior otic markers, refining over time to give the duplicated pattern. Response to Hh inhibition was very different: initial anteroposterior asymmetry was retained, with de novo duplicate expression domains appearing later. We show that Hmx3a is required for normal anterior otic patterning, and that otic patterning defects in hmx3a -/- mutants are a close phenocopy to those seen in fgf3 -/- mutants. However, neither loss nor gain of hmx3a function was sufficient to generate full ear duplications. Using our data to infer a transcriptional regulatory network required for acquisition of otic anterior identity, we can recapitulate both the wild-type and the double-anterior pattern in a mathematical model.

Hmx3a Has Essential Functions in Zebrafish Spinal Cord, Ear and Lateral Line Development

“…Previous research has shown that Hmx2 and Hmx3 have crucial functions in ear development in mouse, and our recent work shows that this is also the case for Hmx3a in zebrafish ( Wang et al 1998 ; Wang et al 2001 ; Wang et al 2004 ; Wang and Lufkin 2005 ; Hartwell et al 2019 ). In mouse, both Hmx2 and Hmx3 mutants have ear defects and these are more severe in double mutants ( Wang et al 2001 ; Wang et al 2004 ).…”

“…The hmx3a SU3 allele was described previously ( Hartwell et al 2019 ). With the exception of hmx3a sa23054 (generated in the Zebrafish Mutation Project and obtained from the Zebrafish International Resource Centre), we created all of the other hmx2 , hmx3a , and hmx2;hmx3a double deletion mutants described in this paper using CRISPR mutagenesis.…”

“…With the exception of hmx3a sa23054 (generated in the Zebrafish Mutation Project and obtained from the Zebrafish International Resource Centre), we created all of the other hmx2 , hmx3a , and hmx2;hmx3a double deletion mutants described in this paper using CRISPR mutagenesis. For all alleles, other than the hmx2 MENTHU allele, we designed and synthesized single guide RNA (sgRNA) and Cas9 messenger RNA (mRNA) as in Hartwell et al (2019 ). For the hmx2 MENTHU allele, we designed the CRISPR RNA (crRNA) using the Microhomology-mediated End joining kNockout Target Heuristic Utility (MENTHU) tool (version 2.1.2), in the Gene Sculpt Suite ( Ata et al 2018 ; Mann et al 2019 ).…”

“…We identified founder fish for hmx2 SU35 , hmx2 SU36 , hmx2 SU37 , and hmx3a SU42 alleles using high-resolution melt analysis (HRMA), and the supermix and amplification programs described in Hartwell et al (2019 ). For the PCRs described below, we used Phusion High-Fidelity DNA Polymerase (M0530L; New England BioLabs Inc.) unless otherwise stated.…”

“…Four-day-old embryos generated from incrosses of heterozygous hmx2 SU39 or hmx2;hmx3a SU44 parents were fixed and processed for phalloidin staining as described in Hartwell et al (2019 ). Stained embryos were stored in DABCO [2% w/v 1,4-Diazabicyclo[2.2.2]octane (D27802; Sigma Aldrich) in 80% glycerol in sterile distilled water].…”