Hirschsprung disease is associated with an L286P mutation in the fifth transmembrane domain of the endothelin-B receptor in the N-ethyl-N-nitrosourea-induced mutant line.

Hirschsprung disease (HSCR), or colonic aganglionosis, is a congenital disorder characterized by the absence of intramural ganglia along variable lengths of the colon, resulting in intestinal obstruction. It is the most common cause of congenital intestinal obstruction, with an incidence of 1 in 5,000 live births. N-ethyl-N-nitrosourea (ENU)-induced mutagenesis is a powerful tool for the study of gene function and the generation of human disease models. In the current study, a novel mutant mouse with aganglionic megacolon and coat color spotting was generated by ENU-induced mutagenesis. Histological and acetylcholinesterase (AChE) whole-mount staining analysis showed a lack of ganglion cells in the colon in mutant mice. The mutation was mapped to chromosome 14 between markers rs30928624 and D14Mit205 (Chr 14 positions 103723921 bp and 105054651 bp). The Ednrb (Chr 14 position 103814625-103844173 bp) was identified as a potential candidate gene in this location. Mutation analysis revealed a T>C missense mutation at nucleotide 857 of the cDNA encoding endothelin receptor B (EDNRB) in which a proline was substituted for the highly conserved Lys-286 residue (L286P) in the fifth transmembrane (TM V) domain of this G protein-coupled receptor. The mutant mouse was named Ednrb(m1yzcm) (Ednrb; mutation 1, Yangzhou University Comparative Medicine Center). The results of the present study implicate the structural importance of the TM V domain in Ednrb function, and the Ednrb(m1yzcm) mouse represents a valuable model for the study of HSCR in humans.

Introduction

Hirschsprung disease (HSCR), or colonic aganglionosis, is a congenital malformation characterized by the absence of intramural ganglia along variable lengths of the colon, resulting in intestinal obstruction with severe constipation in infants and adults. It is the most common cause of congenital intestinal obstruction, with an incidence of 1 in 5,000 live births, and it occurs more frequently in males than in females (4:1). HSCR has been divided into short-segment HSCR (S-HSCR, 80%), long-segment HSCR (L-HSCR, 15%), and total colonic aganglionosis (TCA, 5%) based on the length of the aganglionic tract [2, 8, 9].

Several HSCR-susceptibility genes have been identified, including the RET proto-oncogene [21], the endothelin receptor B gene (EDNRB) [1, 4, 5, 14], the endothelin-3 gene (EDN3) [7, 10, 12], GDNF [3, 13, 16], and the SOX10 gene [22].

EDNRB belongs to the superfamily of rhodopsin-like G protein-coupled receptors (GPCRs), which consist of a long extracellular N-terminus sequence, seven helical transmembrane domains (TMDs), 3 extracellular and 3 intracellular loops, and a cytoplasmic C-terminus tail. The receptor recognizes a family of small peptides known as endothelins [17, 18]. Mutations in the Ednrb gene have been linked to Hirschsprung disease in mice and humans [1, 4, 5, 14].

N-ethyl-N-nitrosourea (ENU)-induced mutagenesis is a powerful tool for the study of gene function and the generation of human disease models. In this paper, a new missense mutation in Ednrb resulted in an HSCR phenotype. This new mutant was generated in a phenotype-driven screen of mice that had been mutagenized with ENU, and it is described here along with the phenotypic characterization of the mutant mice. Mutation analysis revealed a T>C missense mutation in Ednrb exon 4 in which the highly conserved Lys-286 residue in the fifth transmembrane helix of the EDNRB was substituted with a proline (L286P). The mutant mouse was named Ednrb (Ednrb; mutation 1, Yangzhou University Comparative Medicine Center).

Materials and Methods

Mice

C57BL/6J (B6) and DBA/2J (D2) mice were obtained from the Shanghai Laboratory Animal Center (Shanghai, China). This study was conducted in strict accordance with the Guide for the Care and Use of Laboratory Animals of the National Research Council. The Animal Care and Use Committee of Yangzhou University approved all animal experiments and procedures (Approval ID: SYXK [Su] 2012–0029).

The Ednrb mouse was generated via ENU mutagenesis using B6 mice. The Ednrb heterozygotes were mated to B6 females to confirm inheritance test results. Heterozygous mutants were intercrossed to generate homozygous mutants.

Histological and acetylcholinesterase (AChE) whole-mount staining analysis

For Harris hematoxylin and eosin-Y (H&E) staining, colon tissues were dissected and fixed in 4% paraformaldehyde in phosphate-buffered saline, dehydrated, embedded in wax, sectioned at a thickness of 6 µm, and then stained with H&E.

For AChE whole-mount staining, the guts from pups at postnatal day 14 were dissected as a single piece from the proximal esophagus to the distal colon. The mesenteric attachments and pancreas were removed, and the guts were fixed in 4% paraformaldehyde for 1–2 h at 4°C. The tissues were then transferred to saturated sodium sulfate and stored overnight at 4°C. The tissues were then incubated in buffer (ethopropazine HCl [Sigma] 0.2 mM, acetylthiocholine iodide [Sigma] 4 mM, glycine 10 mM, cupric sulfate 2 mM, and sodium acetate 65 mM [pH=5.5]) for 2–4 h. Staining for AChE was done by incubation for 1.5 min in sodium sulfide (1.25%, pH=6). Tissue was rinsed extensively with water before photographing under a dissecting microscope.

Mutation mapping and identification

Ednrb heterozygotes from B6 mice were mated to D2 mice to generate F1 mice. The F1 mice were then intercrossed to generate F2 mice. DNA samples of F2 homozygous mutants were prepared from tail samples by proteinase K digestion, phenol-chloroform extraction, and ethanol precipitation. PCR was used to screen DNA samples for microsatellite markers. PCR products were separated on 4% agarose gels by electrophoresis and analyzed.

Total RNA was isolated from the heads of postnatal day 10 mice using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). cDNA was synthesized using a RevertAid First-Strand cDNA Synthesis Kit (Thermo Scientific Fermentas, St. Leon-Rot, Germany) with oligo (dT) 18 primers. RT-PCR for Ednrb was performed with the following primers: forward 5′-TTGGCTGGGGTAGCTGACTTAA-3′ and reverse 5′-CACACCTTTCTGCTAGCATGGTTT-3′. The PCR conditions consisted of one cycle of denaturation for 5 min at 94°C; 30 cycles of 30 s at 94°C, 30 s at 61°C, and 1.5 min at 72°C; and finally one cycle of elongation for 5 min at 72°C. PCR products were then purified and sequenced.

Results

Mutant mouse phenotype

The founder of the Ednrb/+ mutants was the progeny of an ENU-treated B6 male mouse and had a cluster of white spots on the abdomen. The mutant mouse was mated with wild-type B6 mice, and 9/25 of the progenies showed a similar phenotype. Homozygous mutants, generated by intercross of the heterozygous mutants, showed extensive white spotting of the coat. Well-demarcated, normally pigmented patches of coat were seen most often in the head and hip regions (Fig. 1A). The shapes of these pigmented patches were irregular and differed randomly from mouse to mouse. The eyes were dark in all cases. These mice appeared healthy during the first week after birth but had increasing abdominal distention after 1 week of age (Figs. 1B and 1C). Ultimately, all homozygotes died before postnatal 21 day.

Fig. 1.

Phenotype of Ednrb mice. (A) Note the white coat color of a homozygous Ednrb mouse (right). (B) Autopsy of the wild type (left) and homozygous Ednrb (right) mice. (C) Dissection of the entire gastrointestinal tract from homozygous Ednrb (right) mice revealed a distended colon, cecum, and ileum.

Lack of ganglion cells in the colon in Ednrbm1yzcm homozygous mice

Histological examination of longitudinal sections of the distal portion of the colon from Ednrb homozygous mice revealed a lack of myenteric (Auerbach) ganglia (Figs. 2A and 2B).

Fig. 2.

HE and AChE whole-mount staining of the intestine. (A and B) HE pathological tissue examination confirmed that no ganglia were present in the aganglionosis segment. The upper panel represents H&E staining of the colon from (A) a wild-type mouse and (B) a homozygous Ednrb mouse. HE staining of a wild-type colon, demonstrating myenteric (Auerbach) ganglionic cells (indicated by an asterisk), and homozygous Ednrb colon, in which ganglionic cells are not present. Scale bar: 50 µm. (C and D) AChE whole-mount staining to visualize the ganglia architecture in the enteric nervous system. (C) Wild-type mouse, (D) Ednrb homozygous mouse. A dense plexus of neurons is present in the wild-type colon. Although there are thick nerve fibers in the homozygous Ednrb colon, no ganglion cells are present. Scale bar: 200 µm.

To more readily visualize the absence of ganglion cells in the colon, the bowels of postnatal day 14 mice were examined using whole-mount AChE histochemistry in order to stain cell bodies and nerve fibers. AChE staining of colons from wild-type mice demonstrated a rich plexus of interconnected ganglion cells. However, in Ednrb homozygous mice, no ganglion cells were visible, although large nerve fibers, extending proximally from the distal colon, were apparent (Figs. 2C and 2D).

A mutation in the Ednrb gene of Ednrbm1yzcm mice

For initial mapping, we tested genomic DNA from 24 F2 homozygous mutants with microsatellite markers across the whole genome. We mapped the mutation to chromosome 14 and found no significant linkages with other chromosomal loci. For fine mapping, 191 F2 homozygous mutants were tested, and the mutation was determined to be between markers rs30928624 and D14Mit205(Chr 14 positions 103723921 bp and 105054651 bp) (Fig. 3). A total of 3 protein coding genes are localized within this interval. We prioritized candidate genes on the basis of expression patterns, likely function, and known human or mouse mutant phenotypes. A Hirschsprung-associated gene, Ednrb (Chr 14 position 103814625–103844173 bp), was thought to be a particularly good candidate to examine closely for mutation.

Fig. 3.

Genetic mapping places the mutant in the region between markers rs30928624 and D14Mit205. Primary markers used to refine the map position are listed on the left from the centromere (top), and the number of animals in each genotypic class is shown at the bottom.

A single nucleotide change was found in the Ednrb gene, a T/C substitution, in exon 4 at nucleotide position 857 of the Ednrb-coding region. This substitution converted the Lys-286 residue in the fifth transmembrane helix of the EDNRB to a proline residue (L286P). Sequence alignment across multiple species revealed that this aspartic acid residue is highly conserved among vertebrates (Fig. 4).

Fig. 4.

A point mutation in Ednrb. (A) Sequence analysis of the Ednrb gene in wild-type B6 and homozygous Ednrb mice. (B) Translation of wild-type and homozygous Ednrb alleles flanking the mutation site. (C) Diagram of the EDNRB protein structure with the approximate position of the L286P mutation identified in Ednrb, The 7 helical transmembrane domains are labeled within the boxes. (D) Sequence alignment of mouse EDNRB with other EDNRB amino acid sequences.

Discussion

The current study presents a novel ENU-induced mutant mouse model, Ednrb, with a coat color pigmentation anomaly and megacolon. Histological and AChE examination revealed that the megacolon of the mutant was associated with aganglionosis. The Ednrb allele was found to be produced by a T>C transition at nucleotide 857 of the Ednrb-coding region, which lead to the substitution of proline for lysine at amino acid residue 286.

Hirschsprung disease is a congenital disease that is characterized by an aganglionic megacolon with an absence of enteric ganglia and a lack of innervation to the lower gastrointestinal tract. Precursors of myenteric ganglion neurons originate in the vagal neural crest and migrate in a rostral-to-caudal direction along the developing gastrointestinal tract. The most widely accepted etiopathogenetic hypothesis for HSCR is based on a defect of craniocaudal migration of neuroblasts, which originate from the neural crest and travel to the developing gut, followed by cell proliferation and differentiation [11, 19]. Mice with a targeted null disruption of either the Ednrb or Edn3 gene exhibit an identical phenotype, indicating that the interaction of Edn3 with Ednrb is crucial for normal development of these neural crest-derived tissues [6].

Upon ligand stimulation, the EDNRB receptor activates a number of intracellular signaling mechanisms, including the phospholipase C-β pathway, via heterotrimeric G proteins, leading to an acute increase in cytoplasmic Ca2+ concentration [17]. Biochemical studies have demonstrated that different fifth transmembrane (TM V) domains contribute to ligand binding and intracellular signal transduction properties of the receptors [15, 17, 18]. W276C mutations in the TM V domain of EDNRB receptors have been identified in human patients with Hirschsprung disease. Other studies have reported that a W276C mutation affects the intracellular signal response of EDNRB [18]. A P285L mutation that causes aganglionic megacolon and white coat color has also been identified in the TM V domain of the EDNRB receptor in Ednrb27Pub mice [20]. Lys-286 is situated in the TM V domain and is highly conserved among vertebrates. The Ednrb allele harbors an L286P mutation in the TM V domain of the EDNRB receptor that causes aganglionic megacolon and white coat color, suggesting that Lys-286 is important for proper EDNRB function.

Conflicts of Interest

All authors declare that they have no conflicts of interest regarding this article.