A collagen VI–dependent pathogenic mechanism for Hirschsprung’s disease
Rodolphe Soret, Mathilde Mennetrey, Karl F. Bergeron, Anne Dariel, Michel Neunlist, Franziska Grunder, Christophe Faure, David W. Silversides, Nicolas Pilon, for the Ente-Hirsch study group
Rodolphe Soret, Mathilde Mennetrey, Karl F. Bergeron, Anne Dariel, Michel Neunlist, Franziska Grunder, Christophe Faure, David W. Silversides, Nicolas Pilon, for the Ente-Hirsch study group
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Research Article Development Gastroenterology

A collagen VI–dependent pathogenic mechanism for Hirschsprung’s disease

Abstract

Hirschsprung’s disease (HSCR) is a severe congenital anomaly of the enteric nervous system (ENS) characterized by functional intestinal obstruction due to a lack of intrinsic innervation in the distal bowel. Distal innervation deficiency results from incomplete colonization of the bowel by enteric neural crest cells (eNCCs), the ENS precursors. Here, we report the generation of a mouse model for HSCR — named Holstein — that contains an untargeted transgenic insertion upstream of the collagen-6α4 (Col6a4) gene. This insertion induces eNCC-specific upregulation of Col6a4 expression that increases total collagen VI protein levels in the extracellular matrix (ECM) surrounding both the developing and the postnatal ENS. Increased collagen VI levels during development mainly result in slower migration of eNCCs. This appears to be due to the fact that collagen VI is a poor substratum for supporting eNCC migration and can even interfere with the migration-promoting effects of fibronectin. Importantly, for a majority of patients in a HSCR cohort, the myenteric ganglia from the ganglionated region are also specifically surrounded by abundant collagen VI microfibrils, an outcome accentuated by Down syndrome. Collectively, our data thus unveil a clinically relevant pathogenic mechanism for HSCR that involves cell-autonomous changes in ECM composition surrounding eNCCs. Moreover, as COL6A1 and COL6A2 are on human Chr.21q, this mechanism is highly relevant to the predisposition of patients with Down syndrome to HSCR.

Authors

Rodolphe Soret, Mathilde Mennetrey, Karl F. Bergeron, Anne Dariel, Michel Neunlist, Franziska Grunder, Christophe Faure, David W. Silversides, Nicolas Pilon, for the Ente-Hirsch study group

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Figure 3

The Holstein transgenic insertion upregulates Col6a4 expression in eNCCs.

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The Holstein transgenic insertion upregulates Col6a4 expression in eNCCs...
(A) Schematic representation of the Holstein transgene insertion site based on whole-genome sequencing data and adapted from the Ensembl website (www.ensembl.org). Transgenic sequences are inserted in a 153-bp deletion (Δ 153 bp) between the Col6a4 and Glyctk genes on mouse Chr.9F1, which is syntenic to human Chr.3q22. Analysis of this region with the regulation track of Ensembl revealed the presence of multiple CTCF-binding motifs (green boxes) in the vicinity of the transgene insertion site, whereas the Comparative Genomics track (Genomic Evolutionary Rate Profiling [GERD] for 39 eutherian mammals; gray boxes) revealed that highly conserved noncoding sequences are not found close to the insertion site. (B) Example of PCR-based genotyping of Holstein animals using the oligos depicted in A (see Supplemental Table 2 for primers F/R1 and F/R2). (C and D) Analysis of Col6a4 transcript levels in E12.5 embryos via semiquantitative RT-PCR. (C) In contrast to that in whole intestines, a (D) robust allele dosage-dependent increase in Col6a4 gene expression is observed in FACS-recovered eNCCs. (E) Volcano plot of RNAseq-based comparative analysis of global gene expression between HolTg/Tg and wild-type eNCCs recovered by FACS from E12.5 intestines (n = 3 groups of 5–6 intestines per genotype). The log2 fold-change is on the x axis, while the DESeq P value is on the y axis.