Abstract
Hirschsprung disease (HSCR) is a complex genetic disorder of neural crest development resulting in incomplete formation of the enteric nervous system (ENS). This life-threatening neurocristopathy affects 1/5000 live births, with a currently unexplained male-biased ratio. To address this lack of knowledge, we took advantage of the TashT mutant mouse line, which is the only HSCR model to display a robust male bias. Our prior work revealed that the TashT insertional mutation perturbs a Chr.10 silencer-enriched non-coding region, leading to transcriptional dysregulation of hundreds of genes in neural crest-derived ENS progenitors of both sexes. Here, through sex-stratified transcriptome analyses and targeted overexpression in ENS progenitors, we show that male-biased ENS malformation in TashT embryos is not due to upregulation of Sry-the murine ortholog of a candidate gene for the HSCR male bias in humans-but instead involves upregulation of another Y-linked gene, Ddx3y. This discovery might be clinically relevant since we further found that the DDX3Y protein is also expressed in the ENS of a subset of male HSCR patients. Mechanistically, other data including chromosome conformation captured-based assays and CRISPR/Cas9-mediated deletions suggest that Ddx3y upregulation in male TashT ENS progenitors is due to increased transactivation by p53, which appears especially active in these cells yet without triggering apoptosis. Accordingly, in utero treatment of TashT embryos with the p53 inhibitor pifithrin-α decreased Ddx3y expression and abolished the otherwise more severe ENS defect in TashT males. Our data thus highlight novel pathogenic roles for p53 and DDX3Y during ENS formation in mice, a finding that might help to explain the intriguing male bias of HSCR in humans.
Highlights
Hirschsprung disease (HSCR), known as aganglionic megacolon, is a severe male-biased congenital malformation where ganglia of the enteric nervous system (ENS) are missing from the rectum and over a variable length of the distal bowel [1, 2]
We found DDX3Y to be expressed in enteric neurons of a subset of male HSCR patients, suggesting that manipulation of the putative p53-DDX3Y pathway might eventually have therapeutic value in humans as well
As per our prior work [29], TashTTg/Tg enteric neural crest cells (ENCCs) were recovered from e12.5 stomachs and small intestines by FACS owing to the presence of a SRY promoter-driven fluorescent reporter that labels neural crest derivatives [28]
Summary
Hirschsprung disease (HSCR), known as aganglionic megacolon, is a severe male-biased congenital malformation where ganglia of the enteric nervous system (ENS) are missing from the rectum and over a variable length of the distal bowel [1, 2]. Current treatment of HSCR is surgical removal of the distal aganglionic segment followed by anastomosis between a proximal region with ENS ganglia and the anal verge. This approach is usually life saving, there is a great need for alternative treatments since many of these children continue to have severe problems after surgery [3]. This calls for a better understanding of the pathogenic mechanisms underlying HSCR, which are not fully defined.
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