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.

Loss of Baz1b in mice causes perinatal lethality, growth failure, and variable multi-system outcomes.

BAZ1B is one of 25-27 coding genes deleted in canonical Williams syndrome, a multi-system disorder causing slow growth, vascular stenosis, and gastrointestinal complaints, including constipation. BAZ1B is involved in (among other processes) chromatin organization, DNA damage repair, and mitosis, suggesting reduced BAZ1B may contribute to Williams syndrome symptoms. In mice, loss of Baz1b causes early neonatal death. 89.6% of Baz1b-/- mice die within 24 h of birth without vascular anomalies or congenital heart disease (except for patent ductus arteriosus). Some (<50%) Baz1b-/- were noted to have prolonged neonatal cyanosis, patent ductus arteriosus, or reduced lung aeration, and none developed a milk spot. Meanwhile, 35.5% of Baz1b+/- mice die over the first three weeks after birth. Surviving Baz1b heterozygotes grow slowly (with variable severity). 66.7% of Baz1b+/- mice develop bowel dilation, compared to 37.8% of wild-type mice, but small bowel and colon transit studies were normal. Additionally, enteric neuron density appeared normal in Baz1b-/- mice except in distal colon myenteric plexus, where neuron density was modestly elevated. Combined with several rare phenotypes (agnathia, microphthalmia, bowel dilation) recovered, our work confirms the importance of BAZ1B in survival and growth and suggests that reduced copy number of BAZ1B may contribute to the variability in Williams syndrome phenotypes.

Generation of CHOPi012-A iPSC line from a patient with visceral myopathy-related chronic intestinal pseudo-obstruction.

Visceral myopathies are debilitating conditions characterized by dysfunction of smooth muscle in visceral organs (bowel, bladder, and uterus). Individuals affected by visceral myopathy experience feeding difficulties, growth failure, life-threatening abdominal distension, and may depend on intravenous nutrition for survival. Unfortunately, our limited understanding of the pathophysiology of visceral myopathies means that current therapies remain supportive, with no mechanism-based treatments. We developed a patient-derived iPSC line with a c.769C > T p.R257C/+ mutation, the most common genetic cause of visceral myopathy. This cell line will facilitate studies of how the ACTG2 R257C heterozygous variant affects smooth muscle development and function.

Multi-disciplinary Insights from the First European Forum on Visceral Myopathy 2022 Meeting.

Visceral myopathy is a rare, life-threatening disease linked to identified genetic mutations in 60% of cases. Mostly due to the dearth of knowledge regarding its pathogenesis, effective treatments are lacking. The disease is most commonly diagnosed in children with recurrent or persistent disabling episodes of functional intestinal obstruction, which can be life threatening, often requiring long-term parenteral or specialized enteral nutritional support. Although these interventions are undisputedly life-saving as they allow affected individuals to avoid malnutrition and related complications, they also seriously compromise their quality of life and can carry the risk of sepsis and thrombosis. Animal models for visceral myopathy, which could be crucial for advancing the scientific knowledge of this condition, are scarce. Clearly, a collaborative network is needed to develop research plans to clarify genotype-phenotype correlations and unravel molecular mechanisms to provide targeted therapeutic strategies. This paper represents a summary report of the first 'European Forum on Visceral Myopathy'. This forum was attended by an international interdisciplinary working group that met to better understand visceral myopathy and foster interaction among scientists actively involved in the field and clinicians who specialize in care of people with visceral myopathy.

Generation of CHOPe003-A ESC line to study an ACTG2 variant affecting smooth muscle development and function.

Dysfunction of visceral smooth muscle ("visceral myopathy") impairs bowel, bladder, and uterine function. Symptoms of this life-threatening condition include massive intestinal distension with slow transit, vomiting, feeding intolerance, growth failure, poor bladder emptying, and difficult vaginal delivery. The most common genetic cause of visceral myopathy is a heterozygous point mutation (R257C) in gamma smooth muscle actin (ACTG2). We genetically modified the WAe0009-A human embryonic stem cell line to carry the c.769C>T p.R257C/+ mutation. This cell line will facilitate studies of how the ACTG2 R257C heterozygous variant affects smooth muscle development and function.

The enteric nervous system relays psychological stress to intestinal inflammation.

Mental health profoundly impacts inflammatory responses in the body. This is particularly apparent in inflammatory bowel disease (IBD), in which psychological stress is associated with exacerbated disease flares. Here, we discover a critical role for the enteric nervous system (ENS) in mediating the aggravating effect of chronic stress on intestinal inflammation. We find that chronically elevated levels of glucocorticoids drive the generation of an inflammatory subset of enteric glia that promotes monocyte- and TNF-mediated inflammation via CSF1. Additionally, glucocorticoids cause transcriptional immaturity in enteric neurons, acetylcholine deficiency, and dysmotility via TGF-ß2. We verify the connection between the psychological state, intestinal inflammation, and dysmotility in three cohorts of IBD patients. Together, these findings offer a mechanistic explanation for the impact of the brain on peripheral inflammation, define the ENS as a relay between psychological stress and gut inflammation, and suggest that stress management could serve as a valuable component of IBD care.

Single Nucleus Sequencing of Human Colon Myenteric Plexus-Associated Visceral Smooth Muscle Cells, Platelet Derived Growth Factor Receptor Alpha Cells, and Interstitial Cells of Cajal.

BACKGROUND AND AIMS: Smooth muscle cells (SMCs), interstitial cells of Cajal (ICCs), and platelet-derived growth factor receptor alpha (PDGFRα+) cells (PαCs) form a functional syncytium in the bowel known as the "SIP syncytium." The SIP syncytium works in concert with the enteric nervous system (ENS) to coordinate bowel motility. However, our understanding of individual cell types that form this syncytium and how they interact with each other remains limited, with no prior single-cell RNAseq analyses focused on human SIP syncytium cells. METHODS: We analyzed single-nucleus RNA sequencing data from 10,749 human colon SIP syncytium cells (5572 SMC, 372 ICC, and 4805 PαC nuclei) derived from 15 individuals. RESULTS: Consistent with critical contractile and pacemaker functions and with known enteric nervous system interactions, SIP syncytium cell types express many ion channels, including mechanosensitive channels in ICCs and PαCs. PαCs also prominently express extracellular matrix-associated genes and the inhibitory neurotransmitter receptor for vasoactive intestinal peptide (VIPR2), a novel finding. We identified 2 PαC clusters that differ in the expression of many ion channels and transcriptional regulators. Interestingly, SIP syncytium cells co-express 6 transcription factors (FOS, MEIS1, MEIS2, PBX1, SCMH1, and ZBTB16) that may be part of a combinatorial signature that specifies these cells. Bowel region-specific differences in SIP syncytium gene expression may correlate with regional differences in function, with right (ascending) colon SMCs and PαCs expressing more transcriptional regulators and ion channels than SMCs and PαCs in left (sigmoid) colon. CONCLUSION: These studies provide new insights into SIP syncytium biology that may be valuable for understanding bowel motility disorders and lead to future investigation of highlighted genes and pathways.

Sequencing Reveals miRNAs Enriched in the Developing Mouse Enteric Nervous System.

The enteric nervous system (ENS) is an essential network of neurons and glia in the bowel wall. Defects in ENS development can result in Hirschsprung disease (HSCR), a life-threatening condition characterized by severe constipation, abdominal distention, bilious vomiting, and failure to thrive. A growing body of literature connects HSCR to alterations in miRNA expression, but there are limited data on the normal miRNA landscape in the developing ENS. We sequenced small RNAs (smRNA-seq) and messenger RNAs (mRNA-seq) from ENS precursor cells of mid-gestation Ednrb-EGFP mice and compared them to aggregated RNA from all other cells in the developing bowel. Our smRNA-seq results identified 73 miRNAs that were significantly enriched and highly expressed in the developing ENS, with miR-9, miR-27b, miR-124, miR-137, and miR-488 as our top 5 miRNAs that are conserved in humans. However, contrary to prior reports, our follow-up analyses of miR-137 showed that loss of Mir137 in Nestin-cre, Wnt1-cre, Sox10-cre, or Baf53b-cre lineage cells had no effect on mouse survival or ENS development. Our data provide important context for future studies of miRNAs in HSCR and other ENS diseases and highlight open questions about facility-specific factors in development.

A solution to the long-standing problem of actin expression and purification.

Actin is the most abundant protein in the cytoplasm of eukaryotic cells and interacts with hundreds of proteins to perform essential functions, including cell motility and cytokinesis. Numerous diseases are caused by mutations in actin, but studying the biochemistry of actin mutants is difficult without a reliable method to obtain recombinant actin. Moreover, biochemical studies have typically used tissue-purified α-actin, whereas humans express six isoforms that are nearly identical but perform specialized functions and are difficult to obtain in isolation from natural sources. Here, we describe a solution to the problem of actin expression and purification. We obtain high yields of actin isoforms in human Expi293F cells. Experiments along the multistep purification protocol demonstrate the removal of endogenous actin and the functional integrity of recombinant actin isoforms. Proteomics analysis of endogenous vs. recombinant actin isoforms confirms the presence of native posttranslational modifications, including N-terminal acetylation achieved after affinity-tag removal using the actin-specific enzyme Naa80. The method described facilitates studies of actin under fully native conditions to determine differences among isoforms and the effects of disease-causing mutations that occur in all six isoforms.

Environmental perception and control of gastrointestinal immunity by the enteric nervous system.

The enteric nervous system (ENS) forms a versatile sensory system along the gastrointestinal tract that interacts with most cell types in the bowel. Herein, we portray host-environment interactions at the intestinal mucosal surface through the lens of the enteric nervous system. We describe local cellular interactions as well as long-range circuits between the enteric, central, and peripheral nervous systems. Additionally, we discuss recently discovered mechanisms by which enteric neurons and glia respond to biotic and abiotic environmental changes and how they regulate intestinal immunity and inflammation. The enteric nervous system emerges as an integrative sensory system with manifold immunoregulatory functions under both homeostatic and pathophysiological conditions.

Cell-autonomous retinoic acid receptor signaling has stage-specific effects on mouse enteric nervous system.

Retinoic acid (RA) signaling is essential for enteric nervous system (ENS) development, since vitamin A deficiency or mutations in RA signaling profoundly reduce bowel colonization by ENS precursors. These RA effects could occur because of RA activity within the ENS lineage or via RA activity in other cell types. To define cell-autonomous roles for retinoid signaling within the ENS lineage at distinct developmental time points, we activated a potent floxed dominant-negative RA receptor α (RarαDN) in the ENS using diverse CRE recombinase-expressing mouse lines. This strategy enabled us to block RA signaling at premigratory, migratory, and postmigratory stages for ENS precursors. We found that cell-autonomous loss of RA receptor (RAR) signaling dramatically affected ENS development. CRE activation of RarαDN expression at premigratory or migratory stages caused severe intestinal aganglionosis, but at later stages, RarαDN induced a broad range of phenotypes including hypoganglionosis, submucosal plexus loss, and abnormal neural differentiation. RNA sequencing highlighted distinct RA-regulated gene sets at different developmental stages. These studies show complicated context-dependent RA-mediated regulation of ENS development.

Visceral myopathy: clinical syndromes, genetics, pathophysiology, and fall of the cytoskeleton.

Visceral smooth muscle is a crucial component of the walls of hollow organs like the gut, bladder, and uterus. This specialized smooth muscle has unique properties that distinguish it from other muscle types and facilitate robust dilation and contraction. Visceral myopathies are diseases where severe visceral smooth muscle dysfunction prevents efficient movement of air and nutrients through the bowel, impairs bladder emptying, and affects normal uterine contraction and relaxation, particularly during pregnancy. Disease severity exists along a spectrum. The most debilitating defects cause highly dysfunctional bowel, reduced intrauterine colon growth (microcolon), and bladder-emptying defects requiring catheterization, a condition called megacystis-microcolon-intestinal hypoperistalsis syndrome (MMIHS). People with MMIHS often die early in childhood. When the bowel is the main organ affected and microcolon is absent, the condition is known as myopathic chronic intestinal pseudo-obstruction (CIPO). Visceral myopathies like MMIHS and myopathic CIPO are most commonly caused by mutations in contractile apparatus cytoskeletal proteins. Here, we review visceral myopathy-causing mutations and normal functions of these disease-associated proteins. We propose molecular, cellular, and tissue-level models that may explain clinical and histopathological features of visceral myopathy and hope these observations prompt new mechanistic studies.

scRNA-Seq Reveals New Enteric Nervous System Roles for GDNF, NRTN, and TBX3.

BACKGROUND AND AIMS: Bowel function requires coordinated activity of diverse enteric neuron subtypes. Our aim was to define gene expression in these neuron subtypes to facilitate development of novel therapeutic approaches to treat devastating enteric neuropathies, and to learn more about enteric nervous system function. METHODS: To identify subtype-specific genes, we performed single-nucleus RNA-seq on adult mouse and human colon myenteric plexus, and single-cell RNA-seq on E17.5 mouse ENS cells from whole bowel. We used immunohistochemistry, select mutant mice, and calcium imaging to validate and extend results. RESULTS: RNA-seq on 635 adult mouse colon myenteric neurons and 707 E17.5 neurons from whole bowel defined seven adult neuron subtypes, eight E17.5 neuron subtypes and hundreds of differentially expressed genes. Manually dissected human colon myenteric plexus yielded RNA-seq data from 48 neurons, 3798 glia, 5568 smooth muscle, 377 interstitial cells of Cajal, and 2153 macrophages. Immunohistochemistry demonstrated differential expression for BNC2, PBX3, SATB1, RBFOX1, TBX2, and TBX3 in enteric neuron subtypes. Conditional Tbx3 loss reduced NOS1-expressing myenteric neurons. Differential Gfra1 and Gfra2 expression coupled with calcium imaging revealed that GDNF and neurturin acutely and differentially regulate activity of ∼50% of myenteric neurons with distinct effects on smooth muscle contractions. CONCLUSION: Single cell analyses defined genes differentially expressed in myenteric neuron subtypes and new roles for TBX3, GDNF and NRTN. These data facilitate molecular diagnostic studies and novel therapeutics for bowel motility disorders.

Sympathetic Input to Multiple Cell Types in Mouse and Human Colon Produces Region-Specific Responses.

BACKGROUND & AIMS: The colon is innervated by intrinsic and extrinsic neurons that coordinate functions necessary for digestive health. Sympathetic input suppresses colon motility by acting on intrinsic myenteric neurons, but the extent of sympathetic-induced changes on large-scale network activity in myenteric circuits has not been determined. Compounding the complexity of sympathetic function, there is evidence that sympathetic transmitters can regulate activity in non-neuronal cells (such as enteric glia and innate immune cells). METHODS: We performed anatomical tracing, immunohistochemistry, optogenetic (GCaMP calcium imaging, channelrhodopsin), and colon motility studies in mice and single-cell RNA sequencing in human colon to investigate how sympathetic postganglionic neurons modulate colon function. RESULTS: Individual neurons in each sympathetic prevertebral ganglion innervated the proximal or distal colon, with processes closely opposed to multiple cell types. Calcium imaging in semi-intact mouse colon preparations revealed changes in spontaneous and evoked neural activity, as well as activation of non-neuronal cells, induced by sympathetic nerve stimulation. The overall pattern of response to sympathetic stimulation was unique to the proximal or distal colon. Region-specific changes in cellular activity correlated with motility patterns produced by electrical and optogenetic stimulation of sympathetic pathways. Pharmacology experiments (mouse) and RNA sequencing (human) indicated that appropriate receptors were expressed on different cell types to account for the responses to sympathetic stimulation. Regional differences in expression of α-1 adrenoceptors in human colon emphasize the translational relevance of our mouse findings. CONCLUSIONS: Sympathetic neurons differentially regulate activity of neurons and non-neuronal cells in proximal and distal colon to promote distinct changes in motility patterns, likely reflecting the distinct roles played by these 2 regions.

Pseudo-obstruction-inducing ACTG2R257C alters actin organization and function.

Actin γ 2, smooth muscle (ACTG2) R257C mutation is the most common genetic cause of visceral myopathy. Individuals with ACTG2 mutations endure prolonged hospitalizations and surgical interventions, become dependent on intravenous nutrition and bladder catheterization, and often die in childhood. Currently, we understand little about how ACTG2 mutations cause disease, and there are no mechanism-based treatments. Our goal was to characterize the effects of ACTG2R257C on actin organization and function in visceral smooth muscle cells. We overexpressed ACTG2WT or ACTG2R257C in primary human intestinal smooth muscle cells (HISMCs) and performed detailed quantitative analyses to examine effects of ACTG2R257C on (a) actin filament formation and subcellular localization, (b) actin-dependent HISMC functions, and (c) smooth muscle contractile gene expression. ACTG2R257C resulted in 41% fewer, 13% thinner, 33% shorter, and 40% less branched ACTG2 filament bundles compared with ACTG2WT. Curiously, total F-actin probed by phalloidin and a pan-actin antibody was unchanged between ACTG2WT- and ACTG2R257C-expressing HISMCs, as was ultrastructural F-actin organization. ACTG2R257C-expressing HISMCs contracted collagen gels similar to ACTG2WT-expressing HISMCs but spread 21% more and were 11% more migratory. In conclusion, ACTG2R257C profoundly affects ACTG2 filament bundle structure, without altering global actin cytoskeleton in HISMCs.