Pcgf1 gene disruption reveals primary involvement of epigenetic mechanism in neuronal subtype specification in the enteric nervous system.

The enteric nervous system (ENS) regulates gut functions independently from the central nervous system (CNS) by its highly autonomic neural circuit that integrates diverse neuronal subtypes. Although several transcription factors are shown to be necessary for the generation of some enteric neuron subtypes, the mechanisms underlying neuronal subtype specification in the ENS remain elusive. In this study, we examined the biological function of Polycomb group RING finger protein 1 (PCGF1), one of the epigenetic modifiers, in the development and differentiation of the ENS by disrupting the Pcgf1 gene selectively in the autonomic-lineage cells. Although ENS precursor migration and enteric neurogenesis were largely unaffected, neuronal differentiation was impaired in the Pcgf1-deficient mice, with the numbers of neurons expressing somatostatin (Sst+ ) decreased in multiple gut regions. Notably, the decrease in Sst+ neurons was associated with the corresponding increase in calbindin+ neurons in the proximal colon. These findings suggest that neuronal subtype conversion may occur in the absence of PCGF1, and that epigenetic mechanism is primarily involved in specification of some enteric neuron subtypes.

Live visualization of a functional RET-EGFP chimeric receptor in homozygous knock-in mice.

The GDNF Family Ligands (GFLs) regulate neural development and kidney organogenesis by activating the RET receptor tyrosine kinase. Many RET-dependent developmental processes involve long-distance cell-cell communications or cell polarity, which includes cell migration and axon guidance. This suggests that spatiotemporally regulated subcellular localization of RET protein and appropriate propagation of RET signaling in cells are essential for the physiological function of the GFLs. Little is known, however, about the dynamics of RET protein in cells. Addressing this issue requires development of a system that allows visualization of RET in living cells. In this study, we report generation of a novel knock-in mouse line in which the RET-EGFP chimeric receptor is expressed under the Ret promoter. Unlike Ret-deficient mice that die after birth due to the absence of the enteric nervous system (ENS) and kidneys, RetRET-EGFP/RET-EGFP mice were viable and grew to adulthood with no overt abnormality, which indicated that RET-EGFP exerts function comparable to RET. In neurons and ENS progenitors, RET-EGFP signals were detected both on the cell membrane and in the cytoplasm, the latter of which appeared as a punctate pattern. Time-lapse imaging of cultured neural cells and embryos revealed active transport of RET-EGFP puncta in neuronal axons and cell bodies. Immunohistochemical analyses detected RET-EGFP signals in early and recycling endosomes, indicating that RET-EGFP is trafficked via the endocytic pathway. RetRET-EGFP/RET-EGFP mice enable visualization of functional RET protein in vivo for the first time and provide a unique platform to examine the dynamics and physiology of RET trafficking.

The impact of down-regulated SK3 expressions on Hirschsprung disease.

BACKGROUND: Some Hirschsprung's disease (HSCR) patients showed persistent bowel symptoms following an appropriately performed pull-through procedure. The mechanism is presumed to be down-regulated small-conductance calcium-activated potassium channel 3 (SK3) expression in the HSCR ganglionic intestines. We aimed to investigate the SK3 expression's impact in HSCR patients after a properly performed pull-through surgery in an Indonesian population, a genetically distinct group within Asia. METHODS: We assessed SK3 gene expression in both the ganglionic and aganglionic colon of HSCR patients and controls colon by quantitative real-time polymerase chain reaction (RT-PCR). RESULTS: We ascertained fourteen sporadic HSCR patients and six anorectal malformation patients as controls. Quantitative RT-PCR showed that the SK3 expression was significantly lower (23-fold) in the ganglionic colon group compared to the control group (9.9 ± 4.6 vs. 5.4 ± 3.4; p = 0.044). The expression of SK3 in the aganglionic colon group was also significantly lower (43-fold) compared to the control group (10.8 ± 4.4 vs. 5.4 ± 3.4; p = 0.015). CONCLUSION: Our study shows that the down-regulated SK3 expression in ganglionic intestines might contribute to the persistent bowel symptoms following a properly performed pull-through surgery in Indonesian HSCR patients. Furthermore, this study is the first report of SK3 expression in a sample population of Asian ancestry.

Mini-review: "Enteric glia functions in nervous tissue repair: Therapeutic target or tool?"

In the body, nerve tissue is not only present in the central nervous system, but also in the periphery. The enteric nervous system (ENS) is a highly organized intrinsic network of neurons and glial cells grouped to form interconnected ganglia. Glial cells in the ENS are a fascinating cell population: their neurotrophic role is well established, as well as their plasticity in specific circumstances. Gene expression profiling studies indicate that ENS glia retain neurogenic potential. The identification of neurogenic glial subtype(s) and the molecular basis of glia-derived neurogenesis may have profound biological and clinical implications. In this review, we discuss the potential of using gene-editing for ENS glia and cell transplantation as therapies for enteric neuropathies. Glia in the ENS: target or tool for nerve tissue repair?

A Single RET Mutation in Hirschsprung Disease Induces Intestinal Aganglionosis Via a Dominant-Negative Mechanism.

BACKGROUND & AIMS: Hirschsprung disease (HSCR) is a congenital disorder characterized by the absence of the enteric nervous system (ENS). HSCR potentially involves multiple gene aberrations and displays complex patterns of inheritance. Mutations of the RET gene, encoding the RET receptor tyrosine kinase, play a central role in the pathogenesis of HSCR. Although a wide variety of coding RET mutations have been identified, their pathogenetic significance in vivo has remained largely unclear. METHODS: We introduced a HSCR-associated RET missense mutation, RET(S811F), into the corresponding region (S812) of the mouse Ret gene. Pathogenetic impact of Ret(S812F) was assessed by histologic and functional analyses of the ENS and by biochemical analyses. Interactions of the Ret(S812F) allele with HSCR susceptibility genes, the RET9 allele and the Ednrb gene, were examined by genetic crossing in mice. RESULTS: RetS812F/+ mice displayed intestinal aganglionosis (incidence, 50%) or hypoganglionosis (50%), impaired differentiation of enteric neurons, defecation deficits, and increased lethality. Biochemical analyses revealed that Ret(S811F) protein was not only kinase-deficient but also abrogated function of wild-type RET in trans. Moreover, the Ret(S812F) allele interacted with other HSCR susceptibility genes and caused intestinal aganglionosis with full penetrance. CONCLUSIONS: This study demonstrates that a single RET missense mutation alone induces intestinal aganglionosis via a dominant-negative mechanism. The RetS812F/+ mice model HSCR displays dominant inheritance with incomplete penetrance and serves as a valuable platform for better understanding of the pathogenetic mechanism of HSCR caused by coding RET mutations.

Bilateral Rotational S Flap Technique for Preventing Restenosis in Patients With Severe Circular Anal Stenosis: A Review of 2 Cases.

Anal stenosis is a late hemorrhoidectomy complication. Sphincterotomy and various anoplasty techniques are used for treatment severe anal stenosis, such as the C flap, House flap, U flap, and rotational S flap, but no procedure is ideal for every patient. We review 2 cases of severe circular anal stenosis. Their complaints included narrow caliber of the stool and feeling unsatisfied defecation. Excision of scar tissue using the circular technique was followed by reconstruction using the bilateral rotational S flap procedure. At the 1-year follow-up, the patient had complaints about neither defecation nor pain, and no longer needed laxative agents. In conclusion, the bilateral rotational S flap technique should be considered as a viable treatment because it can also prevent the occurrence of restenosis, especially given the consideration of adequate blood supply.

Ileocolonic Transposition Esophagogastric Bypass as an Antireflux Treatment for Corrosive Esophageal Injury.

Because most surgeons perform an esophagectomy and colonic transposition as the main reconstruction method for patients with esophageal stenosis caused by swallowing corrosive materials, we report 2 cases in which ileocolonic transposition was used to treat such patients. Both patients displayed stenosis in the middle third of the esophagus. Their chief complaint was dysphagia. Ileocolonic transposition using vascularization of the Drummond and ileal arteries was followed by a prepared ileocolic graft by ligating ileocolic vessels. We performed an ileocolonic transposition esophagogastric bypass without an esophagectomy. All surgeries resulted in minimal intraoperative bleeding. Patients experienced no leakage, postoperative fistulas, dysphagia, or postoperative reflux. Three weeks after surgery, 1 patient experienced reversible hoarseness caused by extensive laryngeal-nerve manipulation. Cumulatively, ileocolonic transposition with cervical anastomosis for the treatment of patients with esophageal stenosis caused by corrosive esophageal injury can be considered to be an antireflux treatment because the ileocaecal sphincter is maintained.