BAP1 is required prenatally for differentiation and maintenance of postnatal murine enteric nervous system.

Epigenetic regulatory mechanisms are underappreciated, yet are critical for enteric nervous system (ENS) development and maintenance. We discovered that fetal loss of the epigenetic regulator Bap1 in the ENS lineage caused severe postnatal bowel dysfunction and early death in Tyrosinase-Cre Bap1fl/fl mice. Bap1-depleted ENS appeared normal in neonates; however, by P15, Bap1-deficient enteric neurons were largely absent from the small and large intestine of Tyrosinase-Cre Bap1fl/fl mice. Bowel motility became markedly abnormal with disproportionate loss of cholinergic neurons. Single-cell RNA sequencing at P5 showed that fetal Bap1 loss in Tyrosinase-Cre Bap1fl/fl mice markedly altered the composition and relative proportions of enteric neuron subtypes. In contrast, postnatal deletion of Bap1 did not cause enteric neuron loss or impaired bowel motility. These findings suggest that BAP1 is critical for postnatal enteric neuron differentiation and for early enteric neuron survival, a finding that may be relevant to the recently described human BAP1-associated neurodevelopmental disorder.

Three-Dimensional Imaging of the Enteric Nervous System in Human Pediatric Colon Reveals New Features of Hirschsprung's Disease.

BACKGROUND & AIMS: Hirschsprung's disease is defined by the absence of the enteric nervous system (ENS) from the distal bowel. Primary treatment is "pull-through" surgery to remove bowel that lacks ENS, with reanastomosis of "normal" bowel near the anal verge. Problems after pull-through are common, and some may be due to retained hypoganglionic bowel (ie, low ENS density). Testing this hypothesis has been difficult because counting enteric neurons in tissue sections is unreliable, even for experts. Tissue clearing and 3-dimensional imaging provide better data about ENS structure than sectioning. METHODS: Regions from 11 human colons and 1 ileal specimen resected during Hirschsprung's disease pull-through surgery were cleared, stained with antibodies to visualize the ENS, and imaged by confocal microscopy. Control distal colon from people with no known bowel problems were similarly cleared, stained, and imaged. RESULTS: Quantitative analyses of human colon, ranging from 3 days to 60 years old, suggest age-dependent changes in the myenteric plexus area, ENS ganglion area, percentage of myenteric plexus occupied by ganglia, neurons/mm2, and neuron Feret's diameter. Neuron counting using 3-dimensional images was highly reproducible. High ENS density in neonatal colon allowed reliable neuron counts using 500-µm2 × 500-µm2 regions (36-fold smaller than in adults). Hirschsprung's samples varied 8-fold in proximal margin enteric neuron density and had diverse ENS architecture in resected bowel. CONCLUSIONS: Tissue clearing and 3-dimensional imaging provide more reliable information about ENS structure than tissue sections. ENS structure changes during childhood. Three-dimensional ENS anatomy may provide new insight into human bowel motility disorders, including Hirschsprung's disease.

Glial Cell-Derived Neurotrophic Factor Induces Enteric Neurogenesis and Improves Colon Structure and Function in Mouse Models of Hirschsprung Disease.

BACKGROUND & AIMS: Hirschsprung disease (HSCR) is a life-threatening birth defect in which the distal colon is devoid of enteric neural ganglia. HSCR is treated by surgical removal of aganglionic bowel, but many children continue to have severe problems after surgery. We studied whether administration of glial cell derived neurotrophic factor (GDNF) induces enteric nervous system regeneration in mouse models of HSCR. METHODS: We performed studies with four mouse models of HSCR: Holstein (HolTg/Tg, a model for trisomy 21-associated HSCR), TashT (TashTTg/Tg, a model for male-biased HSCR), Piebald-lethal (Ednrbs-l//s-l, a model for EDNRB mutation-associated HSCR), and Ret9/- (with aganglionosis induced by mycophenolate). Mice were given rectal enemas containing GDNF or saline (control) from postnatal days 4 through 8. We measured survival times of mice, and colon tissues were analyzed by histology, immunofluorescence, and immunoblots. Neural ganglia regeneration and structure, bowel motility, epithelial permeability, muscle thickness, and neutrophil infiltration were studied in colon tissues and in mice. Stool samples were collected, and microbiomes were analyzed by 16S rRNA gene sequencing. Time-lapse imaging and genetic cell-lineage tracing were used to identify a source of GDNF-targeted neural progenitors. Human aganglionic colon explants from children with HSCR were cultured with GDNF and evaluated for neurogenesis. RESULTS: GDNF significantly prolonged mean survival times of HolTg/Tg mice, Ednrbs-l//s-l mice, and male TashTTg/Tg mice, compared with control mice, but not Ret9/- mice (which had mycophenolate toxicity). Mice given GDNF developed neurons and glia in distal bowel tissues that were aganglionic in control mice, had a significant increase in colon motility, and had significant decreases in epithelial permeability, muscle thickness, and neutrophil density. We observed dysbiosis in fecal samples from HolTg/Tg mice compared with feces from wild-type mice; fecal microbiomes of mice given GDNF were similar to those of wild-type mice except for Bacteroides. Exogenous luminal GDNF penetrated aganglionic colon epithelium of HolTg/Tg mice, inducing production of endogenous GDNF, and new enteric neurons and glia appeared to arise from Schwann cells within extrinsic nerves. GDNF application to cultured explants of human aganglionic bowel induced proliferation of Schwann cells and formation of new neurons. CONCLUSIONS: GDNF prolonged survival, induced enteric neurogenesis, and improved colon structure and function in 3 mouse models of HSCR. Application of GDNF to cultured explants of aganglionic bowel from children with HSCR induced proliferation of Schwann cells and formation of new neurons. GDNF might be developed for treatment of HSCR.

Down syndrome mouse models have an abnormal enteric nervous system.

Children with trisomy 21 (Down syndrome [DS]) have a 130-fold increased incidence of Hirschsprung Disease (HSCR), a developmental defect where the enteric nervous system (ENS) is missing from distal bowel (i.e., distal bowel is aganglionic). Treatment for HSCR is surgical resection of aganglionic bowel, but many children have bowel problems after surgery. Post-surgical problems like enterocolitis and soiling are especially common in children with DS. To determine how trisomy 21 affects ENS development, we evaluated the ENS in two DS mouse models, Ts65Dn and Tc1. These mice are trisomic for many chromosome 21 homologous genes, including Dscam and Dyrk1a, which are hypothesized to contribute to HSCR risk. Ts65Dn and Tc1 mice have normal ENS precursor migration at E12.5 and almost normal myenteric plexus structure as adults. However, Ts65Dn and Tc1 mice have markedly reduced submucosal plexus neuron density throughout the bowel. Surprisingly, the submucosal neuron defect in Ts65Dn mice is not due to excess Dscam or Dyrk1a, since normalizing copy number for these genes does not rescue the defect. These findings suggest the possibility that the high frequency of bowel problems in children with DS and HSCR may occur because of additional unrecognized problems with ENS structure.

Loss of Tbx3 in murine neural crest reduces enteric glia and causes cleft palate, but does not influence heart development or bowel transit.

Transcription factors that coordinate migration, differentiation or proliferation of enteric nervous system (ENS) precursors are not well defined. To identify novel transcriptional regulators of ENS development, we performed microarray analysis at embryonic day (E) 17.5 and identified many genes that were enriched in the ENS compared to other bowel cells. We decided to investigate the T-box transcription factor Tbx3, which is prominently expressed in developing and mature ENS. Haploinsufficiency for TBX3 causes ulnar-mammary syndrome (UMS) in humans, a multi-organ system disorder. TBX3 also regulates several genes known to be important for ENS development. To test the hypothesis that Tbx3 is important for ENS development or function, we inactivated Tbx3 in all neural crest derivatives, including ENS progenitors using Wnt1-Cre and a floxed Tbx3 allele. Tbx3 fl/fl; Wnt1-Cre conditional mutant mice die shortly after birth with cleft palate and difficulty feeding. The ENS of mutants was well-organized with a normal density of enteric neurons and nerve fiber bundles, but small bowel glial cell density was reduced. Despite this, bowel motility appeared normal. Furthermore, although Tbx3 is expressed in cardiac neural crest, Tbx3 fl/fl; Wnt1-Cre mice had structurally normal hearts. Thus, loss of Tbx3 within neural crest has selective effects on Tbx3-expressing neural crest derivatives.

Muscularis macrophage development in the absence of an enteric nervous system.

The nervous system of the bowel regulates the inflammatory phenotype of tissue resident muscularis macrophages (MM), and in adult mice, enteric neurons are the main local source of colony stimulating factor 1 (CSF1), a protein required for MM survival. Surprisingly, we find that during development MM colonize the bowel before enteric neurons. This calls into question the requirement for neuron-derived CSF1 for MM colonization of the bowel. To determine if intestinal innervation is required for MM development, we analyzed MM of neonatal Ret-/- (Ret KO) mice that have no enteric nervous system in small bowel or colon. We found normal numbers of well-patterned MM in Ret KO bowel. Similarly, the abundance and distribution of MM in aganglionic human colon obtained from Hirschsprung disease patients was normal. We also identify endothelial cells and interstitial cells of Cajal as the main sources of CSF1 in the developing bowel. Additionally, MM from neonatal Ret KOs do not differ from controls in baseline activation status or cytokine-production in response to lipopolysaccharide. Unexpectedly, these data demonstrate that the enteric nervous system is dispensable for MM colonization and patterning in the bowel, and suggest that modulatory interactions between MM and the bowel nervous system are established postnatally.

Hirschsprung disease - integrating basic science and clinical medicine to improve outcomes.

Hirschsprung disease is defined by the absence of enteric neurons at the end of the bowel. The enteric nervous system (ENS) is the intrinsic nervous system of the bowel and regulates most aspects of bowel function. When the ENS is missing, there are no neurally mediated propulsive motility patterns, and the bowel remains contracted, causing functional obstruction. Symptoms of Hirschsprung disease include constipation, vomiting, abdominal distension and growth failure. Untreated disease usually causes death in childhood because bloodstream bacterial infections occur in the context of bowel inflammation (enterocolitis) or bowel perforation. Current treatment is surgical resection of the bowel to remove or bypass regions where the ENS is missing, but many children have problems after surgery. Although the anatomy of Hirschsprung disease is simple, many clinical features remain enigmatic, and diagnosis and management remain challenging. For example, the age of presentation and the type of symptoms that occur vary dramatically among patients, even though every affected child has missing neurons in the distal bowel at birth. In this Review, basic science discoveries are linked to clinical manifestations of Hirschsprung disease, including partial penetrance, enterocolitis and genetics. Insights into disease mechanisms that might lead to new prevention, diagnostic and treatment strategies are described.

White paper on guidelines concerning enteric nervous system stem cell therapy for enteric neuropathies.

Over the last 20 years, there has been increasing focus on the development of novel stem cell based therapies for the treatment of disorders and diseases affecting the enteric nervous system (ENS) of the gastrointestinal tract (so-called enteric neuropathies). Here, the idea is that ENS progenitor/stem cells could be transplanted into the gut wall to replace the damaged or absent neurons and glia of the ENS. This White Paper sets out experts' views on the commonly used methods and approaches to identify, isolate, purify, expand and optimize ENS stem cells, transplant them into the bowel, and assess transplant success, including restoration of gut function. We also highlight obstacles that must be overcome in order to progress from successful preclinical studies in animal models to ENS stem cell therapies in the clinic.

Neural crest requires Impdh2 for development of the enteric nervous system, great vessels, and craniofacial skeleton.

Mutations that impair the proliferation of enteric neural crest-derived cells (ENCDC) cause Hirschsprung disease, a potentially lethal birth defect where the enteric nervous system (ENS) is absent from distal bowel. Inosine 5' monophosphate dehydrogenase (IMPDH) activity is essential for de novo GMP synthesis, and chemical inhibition of IMPDH induces Hirschsprung disease-like pathology in mouse models by reducing ENCDC proliferation. Two IMPDH isoforms are ubiquitously expressed in the embryo, but only IMPDH2 is required for life. To further understand the role of IMPDH2 in ENS and neural crest development, we characterized a conditional Impdh2 mutant mouse. Deletion of Impdh2 in the early neural crest using the Wnt1-Cre transgene produced defects in multiple neural crest derivatives including highly penetrant intestinal aganglionosis, agenesis of the craniofacial skeleton, and cardiac outflow tract and great vessel malformations. Analysis using a Rosa26 reporter mouse suggested that some or all of the remaining ENS in Impdh2 conditional-knockout animals was derived from cells that escaped Wnt1-Cre mediated DNA recombination. These data suggest that IMPDH2 mediated guanine nucleotide synthesis is essential for normal development of the ENS and other neural crest derivatives.

Hepatocyte Growth Factor and MET Support Mouse Enteric Nervous System Development, the Peristaltic Response, and Intestinal Epithelial Proliferation in Response to Injury.

Factors providing trophic support to diverse enteric neuron subtypes remain poorly understood. We tested the hypothesis that hepatocyte growth factor (HGF) and the HGF receptor MET might support some types of enteric neurons. HGF and MET are expressed in fetal and adult enteric nervous system. In vitro, HGF increased enteric neuron differentiation and neurite length, but only if vanishingly small amounts (1 pg/ml) of glial cell line-derived neurotrophic factor were included in culture media. HGF effects were blocked by phosphatidylinositol-3 kinase inhibitor and by MET-blocking antibody. Both of these inhibitors and MEK inhibition reduced neurite length. In adult mice, MET was restricted to a subset of calcitonin gene-related peptide-immunoreactive (IR) myenteric plexus neurons thought to be intrinsic primary afferent neurons (IPANs). Conditional MET kinase domain inactivation (Met(fl/fl); Wnt1Cre+) caused a dramatic loss of myenteric plexus MET-IR neurites and 1-1'-dioctodecyl-3,3,3',3'-tetramethylindocarbocyamine perchlorate (DiI) labeling suggested reduced MET-IR neurite length. In vitro, Met(fl/fl); Wnt1Cre+ mouse bowel had markedly reduced peristalsis in response to mucosal deformation, but normal response to radial muscle stretch. However, whole-bowel transit, small-bowel transit, and colonic-bead expulsion were normal in Met(fl/fl); Wnt1Cre+ mice. Finally, Met(fl/fl); Wnt1Cre+ mice had more bowel injury and reduced epithelial cell proliferation compared with WT animals after dextran sodium sulfate treatment. These results suggest that HGF/MET signaling is important for development and function of a subset IPANs and that these cells regulate intestinal motility and epithelial cell proliferation in response to bowel injury. SIGNIFICANCE STATEMENT: The enteric nervous system has many neuronal subtypes that coordinate and control intestinal activity. Trophic factors that support these neuron types and enhance neurite growth after fetal development are not well understood. We show that a subset of adult calcitonin gene-related peptide (CGRP)-expressing myenteric neurons produce MET, the receptor for hepatocyte growth factor, and that loss of MET activity affects peristalsis in response to mucosal stroking, reduces MET-immunoreactive neurites, and increases susceptibility to dextran sodium sulfate-induced bowel injury. These observations may be relevant for understanding and treating intestinal motility disorders and also suggest that enhancing the activity of MET-expressing CGRP neurons might be a useful strategy to reduce bowel inflammation.

Building a second brain in the bowel.

The enteric nervous system (ENS) is sometimes called the "second brain" because of the diversity of neuronal cell types and complex, integrated circuits that permit the ENS to autonomously regulate many processes in the bowel. Mechanisms supporting ENS development are intricate, with numerous proteins, small molecules, and nutrients that affect ENS morphogenesis and mature function. Damage to the ENS or developmental defects cause vomiting, abdominal pain, constipation, growth failure, and early death. Here, we review molecular mechanisms and cellular processes that govern ENS development, identify areas in which more investigation is needed, and discuss the clinical implications of new basic research.

Vascular and neural stem cells in the gut: do they need each other?

Enteric neurons and blood vessels form intricate networks throughout the gastrointestinal tract. To support the hypothesis of a possible interaction of both networks, we investigated whether primary mesenteric vascular cells (MVCs) and enteric nervous system (ENS)-derived cells (ENSc) depend on each other using two- and three-dimensional in vitro assays. In a confrontation assay, both cell types migrated in a target-oriented manner towards each other. The migration of MVCs was significantly increased when cultured in ENSc-conditioned medium. Co-cultures of ENSc with MVCs resulted in an improved ENSc proliferation and differentiation. Moreover, we analysed the formation of the vascular and nervous system in developing mice guts. It was found that the patterning of newly formed microvessels and neural stem cells, as confirmed by nestin and SOX2 stainings, is highly correlated in all parts of the developing gut. In particular in the distal colon, nestin/SOX2-positive cells were found in the tissues adjacent to the capillaries and in the capillaries themselves. Finally, in order to provide evidences for a mutual interaction between endothelial and neural cells, the vascular patterns of a RET((-/-)) knockout mouse model as well as human Hirschsprung's cases were analysed. In the distal colon of postnatal RET((-/-)) knockout mice, the vascular and neural networks were similarly disrupted. In aganglionic zones of Hirschsprung's patients, the microvascular density was significantly increased compared with the ganglionic zone within the submucosa. Taken together, these findings indicate a strong interaction between the enteric nervous and vascular system.

Retinoblastoma protein prevents enteric nervous system defects and intestinal pseudo-obstruction.

The retinoblastoma 1 (RB1) tumor suppressor is a critical regulator of cell cycle progression and development. To investigate the role of RB1 in neural crest-derived melanocytes, we bred mice with a floxed Rb1 allele with mice expressing Cre from the tyrosinase (Tyr) promoter. TyrCre+;Rb1fl/fl mice exhibited no melanocyte defects but died unexpectedly early with intestinal obstruction, striking defects in the enteric nervous system (ENS), and abnormal intestinal motility. Cre-induced DNA recombination occurred in all enteric glia and most small bowel myenteric neurons, yet phenotypic effects of Rb1 loss were cell-type specific. Enteric glia were twice as abundant in mutant mice compared with those in control animals, while myenteric neuron number was normal. Most myenteric neurons also appeared normal in size, but NO-producing myenteric neurons developed very large nuclei as a result of DNA replication without cell division (i.e., endoreplication). Parallel studies in vitro found that exogenous NO and Rb1 shRNA increased ENS precursor DNA replication and nuclear size. The large, irregularly shaped nuclei in NO-producing neurons were remarkably similar to those in progeria, an early-onset aging disorder that has been linked to RB1 dysfunction. These findings reveal a role for RB1 in the ENS.

Ret heterozygous mice have enhanced intestinal adaptation after massive small bowel resection.

Intestinal adaptation is an important compensatory response to massive small bowel resection (SBR) and occurs because of a proliferative stimulus to crypt enterocytes by poorly understood mechanisms. Recent studies suggest the enteric nervous system (ENS) influences enterocyte proliferation. We, therefore, sought to determine whether ENS dysfunction alters resection-induced adaptation responses. Ret+/- mice with abnormal ENS function and wild-type (WT) littermates underwent sham surgery or 50% SBR. After 7 days, ileal morphology, enterocyte proliferation, apoptosis, and selected signaling proteins were characterized. Crypt depth and villus height were equivalent at baseline in WT and Ret+/- mice. In contrast after SBR, Ret+/- mice had longer villi (Ret+/- 426.7 ± 46.0 µm vs. WT 306.5 ± 7.7 µm, P < 0.001) and deeper crypts (Ret+/- 119 ± 3.4 µm vs. WT 82.4 ± 3.1 µm, P < 0.001) than WT. Crypt enterocyte proliferation was higher in Ret+/- (48.8 ± 1.3%) than WT (39.9 ± 2.1%; P < 0.001) after resection, but apoptosis rates were similar. Remnant bowel of Ret+/- mice also had higher levels of glucagon-like peptide 2 (6.2-fold, P = 0.005) and amphiregulin (4.6-fold, P < 0.001) mRNA after SBR, but serum glucagon-like peptide 2 protein levels were equal in WT and Ret+/- mice, and there was no evidence of increased c-Fos nuclear localization in submucosal neurons. Western blot confirmed higher crypt epidermal growth factor receptor (EGFR) protein levels (1.44-fold; P < 0.001) and more phosphorylated EGFR (2-fold; P = 0.003) in Ret+/- than WT mice after SBR. These data suggest that Ret heterozygosity enhances intestinal adaptation after massive SBR, likely via enhanced EGFR signaling. Reducing Ret activity or altering ENS function may provide a novel strategy to enhance adaptation attenuating morbidity in patients with short bowel syndrome.

Organotypic specificity of key RET adaptor-docking sites in the pathogenesis of neurocristopathies and renal malformations in mice.

The receptor tyrosine kinase ret protooncogene (RET) is implicated in the pathogenesis of several diseases and in several developmental defects, particularly those in neural crest-derived structures and the genitourinary system. In order to further elucidate RET-mediated mechanisms that contribute to these diseases and decipher the basis for specificity in the pleiotropic effects of RET, we characterized development of the enteric and autonomic nervous systems in mice expressing RET9 or RET51 isoforms harboring mutations in tyrosine residues that act as docking sites for the adaptors Plcgamma, Src, Shc, and Grb2. Using this approach, we found that development of the genitourinary system and the enteric and autonomic nervous systems is dependent on distinct RET-stimulated signaling pathways. Thus, mutation of RET51 at Y1062, a docking site for multiple adaptor proteins including Shc, caused distal colon aganglionosis reminiscent of Hirschsprung disease (HSCR). On the other hand, this mutation in RET9, which encodes an isoform that lacks the Grb2 docking site present in RET51, produced severe abnormalities in multiple organs. Mutations that abrogate RET-Plcgamma binding, previously shown to produce features reminiscent of congenital anomalies of kidneys or urinary tract (CAKUT) syndrome, produced only minor abnormalities in the nervous system. Abrogating RET51-Src binding produced no major defects in these systems. These studies provide insight into the basis of organotypic specificity and redundancy in RET signaling within these unique systems and in diseases such as HSCR and CAKUT.

Vitamin A facilitates enteric nervous system precursor migration by reducing Pten accumulation.

Hirschsprung disease is a serious disorder of enteric nervous system (ENS) development caused by the failure of ENS precursor migration into the distal bowel. We now demonstrate that retinoic acid (RA) is crucial for GDNF-induced ENS precursor migration, cell polarization and lamellipodia formation, and that vitamin A depletion causes distal bowel aganglionosis in serum retinol-binding-protein-deficient (Rbp4(-/-)) mice. Ret heterozygosity increases the incidence and severity of distal bowel aganglionosis induced by vitamin A deficiency in Rbp4(-/-) animals. Furthermore, RA reduces phosphatase and tensin homolog (Pten) accumulation in migrating cells, whereas Pten overexpression slows ENS precursor migration. Collectively, these data support the hypothesis that vitamin A deficiency is a non-genetic risk factor that increases Hirschsprung disease penetrance and expressivity, suggesting that some cases of Hirschsprung disease might be preventable by optimizing maternal nutrition.

The timing and location of glial cell line-derived neurotrophic factor expression determine enteric nervous system structure and function.

Ret signaling is critical for formation of the enteric nervous system (ENS) because Ret activation promotes ENS precursor survival, proliferation, and migration and provides trophic support for mature enteric neurons. Although these roles are well established, we now provide evidence that increasing levels of the Ret ligand glial cell line-derived neurotrophic factor (GDNF) in mice causes alterations in ENS structure and function that are critically dependent on the time and location of increased GDNF availability. This is demonstrated using two different strains of transgenic mice and by injecting newborn mice with GDNF. Furthermore, because different subclasses of ENS precursors withdraw from the cell cycle at different times during development, increases in GDNF at specific times alter the ratio of neuronal subclasses in the mature ENS. In addition, we confirm that esophageal neurons are GDNF responsive and demonstrate that the location of GDNF production influences neuronal process projection for NADPH diaphorase-expressing, but not acetylcholinesterase-, choline acetyltransferase-, or tryptophan hydroxylase-expressing, small bowel myenteric neurons. We further demonstrate that changes in GDNF availability influence intestinal function in vitro and in vivo. Thus, changes in GDNF expression can create a wide variety of alterations in ENS structure and function and may in part contribute to human motility disorders.

Tissue-type plasminogen activator deficiency exacerbates cholestatic liver injury in mice.

UNLABELLED: Recent studies demonstrating a role for plasminogen activator inhibitor (PAI)-1 in cholestatic liver disease in mice suggested that tissue-type plasminogen activator (tPA) or urokinase plasminogen activator (uPA) might be important after biliary tract obstruction. We now demonstrate that blocking tPA exacerbates liver injury after bile duct ligation (BDL). tPA deficient mice have increased bile infarcts, increased TUNEL positive cells, increased neutrophil infiltration, reduced hepatocyte proliferation and reduced ductular reaction 72 hours after BDL compared to wild type mice. In addition, the protective and proliferative effects of plasminogen activator inhibitor 1 (PAI-1) deficiency after BDL are dramatically blocked by the tPA inhibitor tPA-STOP. One potential mechanism for these effects is that both tPA deficiency and tPA-STOP reduce hepatocyte growth factor (HGF) activation and c-Met phosphorylation in the liver after BDL. In support of this hypothesis, HGF treatment reverses the effects of tPA deficiency in mice. Furthermore, preferential tPA activation in areas of injury after BDL might occur because fibrin accumulates in bile infarcts and activates tPA. CONCLUSION: tPA inactivation accelerates liver injury after BDL and reduces HGF activation. These data suggest that strategies to increase HGF activation might be protective in liver diseases with biliary tract obstruction even without increased HGF production.

Reduced endothelin converting enzyme-1 and endothelin-3 mRNA in the developing bowel of male mice may increase expressivity and penetrance of Hirschsprung disease-like distal intestinal aganglionosis.

Hirschsprung disease (distal intestinal aganglionosis, HSCR) is a multigenic disorder with incomplete penetrance, variable expressivity, and a strong male gender bias. Recent studies demonstrated that these genetic patterns arise because gene interactions determine whether enteric nervous system (ENS) precursors successfully proliferate and migrate into the distal bowel. We now demonstrate that male gender bias in the extent of distal intestinal aganglionosis occurs in mice with Ret dominant-negative mutations (RetDN) that mimic human HSCR. We hypothesized that male gender bias could result from reduced expression of a gene already known to be essential for ENS development. Using quantitative real-time polymerase chain reaction (PCR) we demonstrated reduced levels of endothelin converting enzyme-1 and endothelin-3 mRNA in the male mouse bowel at the time that ENS precursors migrate into the colon. Other HSCR-associated genes are expressed at comparable levels in male and female mice. Testosterone and Mullerian inhibiting substance had no deleterious effect on ENS precursor development, but adding EDN3 peptide to E11.5 male RetDN heterozygous mouse gut explants in organ culture significantly increased the rate of ENS precursor migration through the bowel.

BMP signaling regulates murine enteric nervous system precursor migration, neurite fasciculation, and patterning via altered Ncam1 polysialic acid addition.

The enteric nervous system (ENS) forms from migrating neural crest-derived precursors that differentiate into neurons and glia, aggregate into ganglion cell clusters, and extend neuronal processes to form a complex interacting network that controls many aspects of intestinal function. Bone morphogenetic proteins (BMPs) have diverse roles in development and influence the differentiation, proliferation, and survival of ENS precursors. We hypothesized that BMP signaling might also be important for the ENS precursor migration, ganglion cell aggregation, and neurite fasciculation necessary to form the enteric nervous system. We now demonstrate that BMP signaling restricts murine ENS precursors to the outer bowel wall during migration. In addition, blocking BMP signaling causes faster colonization of the murine colon, reduces ganglion cell aggregation, and reduces neurite fasciculation. BMP signaling also influences patterns of neurite extension within the developing bowel wall. These effects on ENS precursor migration and neurite fasciculation appear to be mediated at least in part by increased polysialic acid addition to neural cell adhesion molecule (Ncam1) in response to BMP. Removing PSA enzymatically reverses the BMP effects on ENS precursor migration and neurite fasciculation. These studies demonstrate several novel roles for BMP signaling and highlight new functions for sialyltransferases in the developing ENS.