Deletion of a conserved regulatory element required for Hmx1 expression in craniofacial mesenchyme in the dumbo rat: a novel cause of congenital ear malformation

Abstract

SUMMARYHmx1 is a homeodomain transcription factor expressed in the developing eye, peripheral ganglia, and branchial arches of avian and mammalian embryos. Recent studies have identified a loss-of-function allele at the HMX1 locus as the causative mutation in the oculo-auricular syndrome (OAS) in humans, characterized by ear and eye malformations. The mouse dumbo (dmbo) mutation, with similar effects on ear and eye development, also results from a loss-of-function mutation in the Hmx1 gene. A recessive dmbo mutation causing ear malformation in rats has been mapped to the chromosomal region containing the Hmx1 gene, but the nature of the causative allele is unknown. Here we show that dumbo rats and mice exhibit similar neonatal ear and eye phenotypes. In midgestation embryos, dumbo rats show a specific loss of Hmx1 expression in neural-crest-derived craniofacial mesenchyme (CM), whereas Hmx1 is expressed normally in retinal progenitors, sensory ganglia and in CM, which is derived from mesoderm. High-throughput resequencing of 1 Mb of rat chromosome 14 from dmbo/dmbo rats, encompassing the Hmx1 locus, reveals numerous divergences from the rat genomic reference sequence, but no coding changes in Hmx1. Fine genetic mapping narrows the dmbo critical region to an interval of ∼410 kb immediately downstream of the Hmx1 transcription unit. Further sequence analysis of this region reveals a 5777-bp deletion located ∼80 kb downstream in dmbo/dmbo rats that is not apparent in 137 other rat strains. The dmbo deletion region contains a highly conserved domain of ∼500 bp, which is a candidate distal enhancer and which exhibits a similar relationship to Hmx genes in all vertebrate species for which data are available. We conclude that the rat dumbo phenotype is likely to result from loss of function of an ultraconserved enhancer specifically regulating Hmx1 expression in neural-crest-derived CM. Dysregulation of Hmx1 expression is thus a candidate mechanism for congenital ear malformation, most cases of which remain unexplained.

A distal 594bp ECR specifies Hmx1 expression in pinna and lateral facial morphogenesis and is regulated by Hox-Pbx-Meis

“…As previously reported, endogenous Hmx1 is readily detectable in the trigeminal, geniculate and statoacoustic ganglia (Quina et al, 2012b), but the dmECR is not active in these regions (Fig. 5C,E,K), consistent with the retention of Hmx1 expression in the cranial sensory ganglia in the dumbo rat (Quina et al, 2012a). At the level of the embryonic eye, dmECR activity is present in the anterior mesoderm, whereas endogenous Hmx1 is expressed in the eye itself (Fig.…”

“…At the level of the embryonic eye, dmECR activity is present in the anterior mesoderm, whereas endogenous Hmx1 is expressed in the eye itself (Fig. 5B,D,L-M), consistent with the dumbo rat findings (Quina et al, 2012a). However, in the anterior craniofacial mesenchyme of BA2 at E11.5, dmECR activity and endogenous Hmx1 expression overlapped only in a band of cells at the interface between the expression domains (Fig.…”

“…Despite these regions of discordance, the distribution of the dmECR-driven reporter overlapped with endogenous Hmx1 in the craniofacial mesenchyme that contributes to structures that are abnormal in the dumbo rat (Fig. 1O-R) (Quina et al, 2012a).…”

“…This condition has been termed oculo-auricular syndrome (OAS) in humans (Schorderet et al, 2008;Gillespie et al, 2015), 'dumbo' and/or 'misplaced ears' in mice, 'dumbo' in rats (Munroe et al, 2009;Kuramoto et al, 2010;Quina et al, 2012a), and 'crop ear' in cattle (Koch et al, 2013).…”

“…However, in rats, the recessive dumbo allele consists of a 5777 bp deletion ∼80 kb downstream of Hmx1. This deletion is associated with a loss of Hmx1 expression only in the first and second branchial arch (BA1 and BA2) mesenchyme (Quina et al, 2012a), suggesting that the deletion removes an important regulatory element driving Hmx1 expression in the lateral face. This deleted sequence contains a ∼600 bp evolutionarily conserved region (ECR).…”