Int J Clin Exp Pathol 2016;9(2):1416-1423 www.ijcep.com /ISSN:1936-2625/IJCEP0019807 Original Article Neuregulin 1 is required for the development of enteric neurons and vagus in zebrafish Meng Jiang*, Jia-Rui Pu*, De-Hua Yang, Xi Zhang, Shao-Tao Tang Department of Pediatric Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China. *Equal contributors. Received November 15, 2015; Accepted January 10, 2016; Epub February 1, 2016; Published February 15, 2016 Abstract: Hirschsprung’s disease (HSCR) is a congenital malformation of the colon characterised by the absence of enteric ganglion in the submucosal and myenteric plexuses. Recently genome-wide association studies have identified that neuregulin 1 (nrg1) gene might be a susceptibility locus for HSCR. This study aimed to investigate whether nrg1 is involved in the development of the enteric nervous system. By in situ hybridization, we revealed that nrg1 was mainly expressed in the mucous layer of the adult zebrafish gut. After been injected with nrg1 morpholino antisense oligonucleotides, zebrafish embryos showed delayed hatching, small head, pericardial edema, shortened- twisted trunk, and impaired motor ability. Some of these defects could be partially ameliorated by the injection of nrg1 mRNA. Further, we observed that knock down of nrg1 resulted in reduced number of enteric neurons (29.5 ± 5.8 vs 51.5 ± 8.3, P<0.01) and aganglionosis (an HSCR-like phenotype), and also affected the development of the vagal innervation of the gut. Taken together, our findings indicated that nrg1 played a critical role in the development of the enteric neurons as well as vagus nerve. Therefore, this work provides an evidence of nrg1 in the pathogenesis of HSCR. Keywords: Neuregulin 1, Hirschsprung’s disease, enteric neuron, vagus, zebrafish, morpholino Introduction could not relax and pass stool through the bowel, leading to an obstruction. According to Hirschsprung’s disease (HSCR), also named the extent of aganglionosis, HSCR can be clas- aganglionic megacolon, is a congenital disorder sified into short segment (S-HSCR; 80% of the characterized by the absence of the enteric cases), long-segment (L-HSCR; 15%) and total ganglia along a variable length of the intestine, colonic aganglionosis (TCA; 5%). The incidence which can lead to tonic contraction of the of HSCR varies greatly by gender and racial, and affected segment, intestinal obstruction, and is most prevalent among Asians (2.8 per 10,000 massive distension of the bowel [1]. HSCR can live births) [3]. be contributed to a failure in the migration of enteric neural crest-derived cells (ENCCS) into Mounting evidence show that HSCR has a com- the intestine during embryonic development. plex genetic etiology. A number of susceptible During normal prenatal development, cells orig- genes, such as RET, GDNF, SOX10, PHOX2B, inrated from the neural crest migrate into the SIP1, GFRA1, NRTN, NKX2.1, KIAA1279, large intestine to form the enteric nervous sys- EDNRB, NTRK3, EDN3, ECE-1, ZFXH1B, and tem (ENS), which regulates the state of the wall L1CAM have been identified by candidate gene and lumen of the gut and activates intrinsic study [4-9]. Considerable literature shows that reflexes that generate complicated and propul- the RET gene, which encoding a tyrosine-kinase sive peristaltic movements [2]. However, in receptor, is the critical HSCR causative gene Hirschsprung’s disease, this process is incom- and its normal expression is crucial for the plete and part of the colon lacks these enteric development of the ENS [10, 11]. Deletion of neurons that regulate the activity of the colon. RET in the mouse or knockdown of RET in So the affected segment of the large intestine zebrafish resulted in complete intestinal agan- Neuregulin 1 for the development of enteric neurons and vagus glionosis [12, 13]. However, only 50% of the ved by the Animal Care and Use Committee of HSCR familial cases and 5-30% of sporadic Huazhong University of science and tech- cases can be attributed to mutations in the nology. RET coding region, and mutations in other HSCR causative genes mentioned above Whole mount in situ hybridization account for about 7% of HSCR sporadic cases, indicating that there must be additional uniden- We performed the whole-mount in situ hybrid- tified loci responsible for HSCR [14]. ization and immunohistochemistry according to standard protocols. The cDNA clones of neuro- Recently genome-wide association studies genin 1 were used to prepare riboprobes. (GWAS) have identified that neuregulin 1 (nrg1) Primers for amplification of the template cDNA may be a susceptibility locus for HSCR [15-17]. were designed by using the software of Primer Nrg1 and its receptors, the ErbB family of tyro- Premier 5.0 and NCBI-Primer BLAST. Digo- sine kinase receptors, are expressed in adult xigenin-labeled RNA probes were synthesized intestinal epithelia of both humans and mice, in vitro using a DIG RNA labeling kit (Roche). and the loss of ErbB2 is linked with postnatal Embryos were fixed in 4% paraformaldehyde colonic aganglionosis in mice [18]. However, (containing 1‰ DEPC), then the embryos were whether loss-of-function of nrg1 can lead to digested with proteinase K, embryos were pre- HSCR has not been confirmed in an animal hybridized and incubated in HYB+ containing model. In view of there are many advantages of RNA probe (Promega), then blocked with BSA using zebrafish as a model organism, such as and incubated with anti-DIG antibody and visu- rapid life cycle, optical clarity, low cost to main- alized with NBT/BCIP. The chromogenic embry- tain, and rapid external embryonic develop- os were preserved in PBST with 30% glycerol. ment, zebrafish were selected in our research. What’s more important is that zebrafish nrg1 Morpholino injections shared 67% nucleotide identity with human Nrg1 morpholino antisense oligonucleotides nrg1, the high homology indicted that it might (nrg1-MO) were synthesized by Gene Tools have the same function in zebrafish as it has in (Philomath, USA). Morpholino oligonucleotides human. (MO) were diluted in 1× Danieau buffer (58 mM In this study, we identified the expression of NaCl, 0.7 mM KCl, 0.4 mM MgSO4, 0.6 mM nrg1 in zebrafish colon and, based on it, we Ca(NO3)2, 5.0 mM HEPES pH 7.6) for injection at analyzed its function in the development of the concentrations shown below. Approximately 1 ENS by knocking down it with antisense mor- nl of MO solutions was injected into one- to pholino in zebrafish embryos. Deformities, dis- two-cell stage embryos. For comparison, the turbed growth and development as well as same amount of control-MO was injected. The death can be observed by interfering nrg1 sequences and the concentrations for injec- expression of zebrafish embryos. In addition, tions are shown below: nrg1-MO (5’-CTT- nrg1 can affect the survival, proliferation, TGCCTGCTTTCACCTCAGCCAT-3’) at 0.5 ng/μl, migration of enteric neurons. Further study control-MO (5’-CCTCTTACCTCAGTTACAATTTATA- showed that nrg1 is not only involved in the 3’) at 0.5 ng/μl. Control/nrg1-morpholino and development of enteric neurons, but also in the EGFP-nrg1 lineared vector were co-injected into development of vagal innervation of the gut. zebrafish embryos; the strength of green fluo- rescence was used to evaluate knockdown Materials and methods efficiency. Animals Assessment of enteric neurons Wild-type zebrafish (AB type) were maintained, Immunohistochemistry of the neuronal marker and embryos were raised according to the HuC/D was conducted as described to assess Zebrafish Book. Zebrafish embryos were kept in the enteric neurons in the distal intestine [20]. a 28.5°C embryo medium (5 mM NaCl, 0.17 Embryos were fixed in 4% paraformaldehyde mM KCl, 0.33 mM CaCl2, and 0.33 mM MgSO4) (containing 1‰ DEPC), then the embryos were and were staged by hours post-fertilisation digested and washed, anti-Hu MAb 16A11 anti- (hpf) or days post fertilisation (dpf) as described body (Molecular Probes) was added, at last the [19]. All experimental procedures were appro- embryos were incubated by fluorescent sec- 1417 Int J Clin Exp Pathol 2016;9(2):1416-1423 Neuregulin 1 for the development of enteric neurons and vagus Figure 1. Expression of nrg1 in zebrafish gut. A. Nrg1 expressed in mucous layer, no expression was detected in muscularis and serosa. B. Nrg1 started to express in blastocyst stage (4 hpf). Strong expression was detected in gastrulation stage (8 hpf) and somite stage (12 hpf), mainly located in the animal pole. In pharyngula period (24 hpf, 36 hpf, 48 hpf), nrg1 expression was weak; the nrg1 expression level increased from hatching period (72 hpf, 96 hpf), and started to decrease from 120 hpf. Scale bars = 200 μm. ondary antibody. Images were captured using a muscularis and serosa (Figure 1A). As Figure confocal microscope (Nikon). Finally HuC/D 1B shows, nrg1 started to express in blastocyst positive cells were counted in the 30 μm length stage. Strong expression was detected in gas- of distal-most intestine. trulation stage and somite stage, which mainly located in the animal pole. In pharyngeal peri- Staining of intestinal vagal nerve fibers od, the expression of nrg1 was weak, after that the nrg1 expression level increased from hatch- Acetylated tubulin (AcT) was used as a marker ing period, and started to decrease from 120 of the vagal nerve fibers as described [21]. hpf. Embryos were fixed in 4% paraformaldehyde (containing 1‰ DEPC), then the embryos were Function of nrg1 in the development of zebraf- digested with proteinase K, after washed by ish embryo PTD (PBS, triton X, DMSO) , acetylated α-tubulin antibody (Sigma) was added, 4°C overnight, at In this study, control/nrg1-morpholino and last the embryos were incubated by fluorescent EGFP-nrg1 lineared vector were co-injected into secondary antibody.