Evaluating the molecular and genetic mechanisms underlying gut motility disorders.

INTRODUCTION: Gastrointestinal (GI) motility disorders comprise a wide range of different diseases affecting the structural or functional integrity of the GI neuromusculature. Their clinical presentation and burden of disease depends on the predominant location and extent of gut involvement as well as the component of the gut neuromusculature affected. AREAS COVERED: A comprehensive literature review was conducted using the PubMed and Medline databases to identify articles related to GI motility and functional disorders, published between 2016 and 2023. In this article, we highlight the current knowledge of molecular and genetic mechanisms underlying GI dysmotility, including disorders of gut-brain interaction, which involve both GI motor and sensory disturbance. EXPERT OPINION: Although the pathophysiology and molecular mechanisms underlying many such disorders remain unclear, recent advances in the assessment of intestinal tissue samples, genetic testing with the application of 'omics' technologies and the use of animal models will provide better insights into disease pathogenesis as well as opportunities to improve therapy.

Gut Isolation from Zebrafish Larvae for Single-cell RNA Sequencing.

The gastrointestinal (GI) tract performs a range of functions essential for life. Congenital defects affecting its development can lead to enteric neuromuscular disorders, highlighting the importance to understand the molecular mechanisms underlying GI development and dysfunction. In this study, we present a method for gut isolation from zebrafish larvae at 5 days post fertilization to obtain live, viable cells which can be used for single-cell RNA sequencing (scRNA-seq) analysis. This protocol is based on the manual dissection of the zebrafish intestine, followed by enzymatic dissociation with papain. Subsequently, cells are submitted to fluorescence-activated cell sorting, and viable cells are collected for scRNA-seq. With this method, we were able to successfully identify different intestinal cell types, including epithelial, stromal, blood, muscle, and immune cells, as well as enteric neurons and glia. Therefore, we consider it to be a valuable resource for studying the composition of the GI tract in health and disease, using the zebrafish.

Promoter hypermethylation of neural-related genes is compatible with stemness in solid cancers.

BACKGROUND: DNA hypermethylation is an epigenetic feature that modulates gene expression, and its deregulation is observed in cancer. Previously, we identified a neural-related DNA hypermethylation fingerprint in colon cancer, where most of the top hypermethylated and downregulated genes have known functions in the nervous system. To evaluate the presence of this signature and its relevance to carcinogenesis in general, we considered 16 solid cancer types available in The Cancer Genome Atlas (TCGA). RESULTS: All tested cancers showed significant enrichment for neural-related genes amongst hypermethylated genes. This signature was already present in two premalignant tissue types and could not be explained by potential confounders such as bivalency status or tumor purity. Further characterization of the neural-related DNA hypermethylation signature in colon cancer showed particular enrichment for genes that are overexpressed during neural differentiation. Lastly, an analysis of upstream regulators identified RE1-Silencing Transcription factor (REST) as a potential mediator of this DNA methylation signature. CONCLUSION: Our study confirms the presence of a neural-related DNA hypermethylation fingerprint in various cancers, of genes linked to neural differentiation, and points to REST as a possible regulator of this mechanism. We propose that this fingerprint indicates an involvement of DNA hypermethylation in the preservation of neural stemness in cancer cells.

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.

Unbiased characterization of the larval zebrafish enteric nervous system at a single cell transcriptomic level.

The enteric nervous system (ENS) regulates many gastrointestinal functions including peristalsis, immune regulation and uptake of nutrients. Defects in the ENS can lead to severe enteric neuropathies such as Hirschsprung disease (HSCR). Zebrafish have proven to be fruitful in the identification of genes involved in ENS development and HSCR pathogenesis. However, composition and specification of enteric neurons and glial subtypes at larval stages, remains mainly unexplored. Here, we performed single cell RNA sequencing of zebrafish ENS at 5 days post-fertilization. We identified vagal neural crest progenitors, Schwann cell precursors, and four clusters of differentiated neurons. In addition, a previously unrecognized elavl3+/phox2bb-population of neurons and cx43+/phox2bb-enteric glia was found. Pseudotime analysis supported binary neurogenic branching of ENS differentiation, driven by a notch-responsive state. Taken together, we provide new insights on ENS development and specification, proving that the zebrafish is a valuable model for the study of congenital enteric neuropathies.

A combinatorial panel for flow cytometry-based isolation of enteric nervous system cells from human intestine.

Efficient isolation of neurons and glia from the human enteric nervous system (ENS) is challenging because of their rare and fragile nature. Here, we describe a staining panel to enrich ENS cells from the human intestine by fluorescence-activated cell sorting (FACS). We find that CD56/CD90/CD24 co-expression labels ENS cells with higher specificity and resolution than previous methods. Surprisingly, neuronal (CD24, TUBB3) and glial (SOX10) selective markers appear co-expressed by all ENS cells. We demonstrate that this contradictory staining pattern is mainly driven by neuronal fragments, either free or attached to glial cells, which are the most abundant cell types. Live neurons can be enriched by the highest CD24 and CD90 levels. By applying our protocol to isolate ENS cells for single-cell RNA sequencing, we show that these cells can be obtained with high quality, enabling interrogation of the human ENS transcriptome. Taken together, we present a selective FACS protocol that allows enrichment and discrimination of human ENS cells, opening up new avenues to study this complex system in health and disease.

The long Filamin-A isoform is required for intestinal development and motility: implications for chronic intestinal pseudo-obstruction.

Filamin A (FLNA) is a cytoplasmic actin binding protein, recently shown to be expressed as a long and short isoform. Mutations in FLNA are associated with a wide spectrum of disorders, including an X-linked form of chronic intestinal pseudo-obstruction (CIPO). However, the role of FLNA in intestinal development and function is largely unknown. In this study, we show that FLNA is expressed in the muscle layer of the small intestine from early human fetal stages. Expression of FLNA variants associated with CIPO, blocked expression of the long flna isoform and led to an overall reduction of RNA and protein levels. As a consequence, contractility of human intestinal smooth muscle cells was affected. Lastly, our transgenic zebrafish line showed that the flna long isoform is required for intestinal elongation and peristalsis. Histological analysis revealed structural and architectural changes in the intestinal smooth muscle of homozygous fish, likely triggered by the abnormal expression of intestinal smooth muscle markers. No defect in the localization or numbers of enteric neurons was observed. Taken together, our study demonstrates that the long FLNA isoform contributes to intestinal development and function. Since loss of the long FLNA isoform does not seem to affect the enteric nervous system, it likely results in a myopathic form of CIPO, bringing new insights to disease pathogenesis.

Nerves in gastrointestinal cancer: from mechanism to modulations.

Maintenance of gastrointestinal health is challenging as it requires balancing multifaceted processes within the highly complex and dynamic ecosystem of the gastrointestinal tract. Disturbances within this vibrant environment can have detrimental consequences, including the onset of gastrointestinal cancers. Globally, gastrointestinal cancers account for ~19% of all cancer cases and ~22.5% of all cancer-related deaths. Developing new ways to more readily detect and more efficiently target these malignancies are urgently needed. Whereas members of the tumour microenvironment, such as immune cells and fibroblasts, have already been in the spotlight as key players of cancer initiation and progression, the importance of the nervous system in gastrointestinal cancers has only been highlighted in the past few years. Although extrinsic innervations modulate gastrointestinal cancers, cells and signals from the gut's intrinsic innervation also have the ability to do so. Here, we shed light on this thriving field and discuss neural influences during gastrointestinal carcinogenesis. We focus on the interactions between neurons and components of the gastrointestinal tract and tumour microenvironment, on the neural signalling pathways involved, and how these factors affect the cancer hallmarks, and discuss the neural signatures in gastrointestinal cancers. Finally, we highlight neural-related therapies that have potential for the management of gastrointestinal cancers.

TFAP2B Haploinsufficiency Impacts Gastrointestinal Function and Leads to Pediatric Intestinal Pseudo-obstruction.

Background: Pediatric Intestinal Pseudo-obstruction (PIPO) is a congenital enteric disorder characterized by severe gastrointestinal (GI) dysmotility, without mechanical obstruction. Although several genes have been described to cause this disease, most patients do not receive a genetic diagnosis. Here, we aim to identify the genetic cause of PIPO in a patient diagnosed with severe intestinal dysmotility shortly after birth. Methods: Whole exome sequencing (WES) was performed in the patient and unaffected parents, in a diagnostic setting. After identification of the potential disease-causing variant, its functional consequences were determined in vitro and in vivo. For this, expression constructs with and without the causing variant, were overexpressed in HEK293 cells. To investigate the role of the candidate gene in GI development and function, a zebrafish model was generated where its expression was disrupted using CRISPR/Cas9 editing. Results: WES analysis identified a de novo heterozygous deletion in TFAP2B (NM_003221.4:c.602-5_606delTCTAGTTCCA), classified as a variant of unknown significance. In vitro studies showed that this deletion affects RNA splicing and results in loss of exon 4, leading to the appearance of a premature stop codon and absence of TFAP2B protein. Disruption of tfap2b in zebrafish led to decreased enteric neuronal numbers and delayed transit time. However, no defects in neuronal differentiation were detected. tfap2b crispants also showed decreased levels of ednrbb mRNA, a downstream target of tfap2b. Conclusion: We showed that TFAP2B haploinsufficiency leads to reduced neuronal numbers and GI dysmotility, suggesting for the first time, that this gene is involved in PIPO pathogenesis.

Autosomal Recessive ACTG2-Related Visceral Myopathy in Brothers.

Pediatric intestinal pseudo-obstruction (PIPO) is a heterogeneous condition characterized by impaired gastrointestinal propulsion, a broad clinical spectrum, and variable severity. Several molecular bases underlying primary PIPO have been identified, of which autosomal dominant ACTG2-related visceral myopathy is the most common in both familial or sporadic primary PIPO cases. We present a family with autosomal recessive ACTG2-related disease in which both parents have mild gastrointestinal symptoms and sons have severe PIPO and bladder dysfunction. Methods: Clinical genome sequencing was performed on the patients and the mother. Immunohistochemistry was performed on intestinal tissue from the patients to show expression levels of the ACTG2. Results: Genome sequencing identified a 6.8 kb 2p13.1 loss that includes the ACTG2 gene and a maternally inherited missense variant p.Val10Met in the ACTG2 gene. Discussion: This case demonstrates that monoallelic hypomorphic ACTG2 variants may underly mild primary gastrointestinal symptoms, while biallelic mild variants can cause severe diseases. The Deletions of the noncoding ACTG2 exon can be an under-recognized cause of mild gastrointestinal symptoms unidentifiable by exome sequencing, explaining some instances of interfamilial variability with an apparent autosomal dominant inheritance. Genome sequencing is recommended as a genetic work-up for primary or idiopathic PIPO because of genetic heterogeneity.

The Somatic Mutation Paradigm in Congenital Malformations: Hirschsprung Disease as a Model.

Patients with Hirschsprung disease (HSCR) do not always receive a genetic diagnosis after routine screening in clinical practice. One of the reasons for this could be that the causal mutation is not present in the cell types that are usually tested-whole blood, dermal fibroblasts or saliva-but is only in the affected tissue. Such mutations are called somatic, and can occur in a given cell at any stage of development after conception. They will then be present in all subsequent daughter cells. Here, we investigated the presence of somatic mutations in HSCR patients. For this, whole-exome sequencing and copy number analysis were performed in DNA isolated from purified enteric neural crest cells (ENCCs) and blood or fibroblasts of the same patient. Variants identified were subsequently validated by Sanger sequencing. Several somatic variants were identified in all patients, but causative mutations for HSCR were not specifically identified in the ENCCs of these patients. Larger copy number variants were also not found to be specific to ENCCs. Therefore, we believe that somatic mutations are unlikely to be identified, if causative for HSCR. Here, we postulate various modes of development following the occurrence of a somatic mutation, to describe the challenges in detecting such mutations, and hypothesize how somatic mutations may contribute to 'missing heritability' in developmental defects.

Size matters: Large copy number losses in Hirschsprung disease patients reveal genes involved in enteric nervous system development.

Hirschsprung disease (HSCR) is a complex genetic disease characterized by absence of ganglia in the intestine. HSCR etiology can be explained by a unique combination of genetic alterations: rare coding variants, predisposing haplotypes and Copy Number Variation (CNV). Approximately 18% of patients have additional anatomical malformations or neurological symptoms (HSCR-AAM). Pinpointing the responsible culprits within a CNV is challenging as often many genes are affected. Therefore, we selected candidate genes based on gene enrichment strategies using mouse enteric nervous system transcriptomes and constraint metrics. Next, we used a zebrafish model to investigate whether loss of these genes affects enteric neuron development in vivo. This study included three groups of patients, two groups without coding variants in disease associated genes: HSCR-AAM and HSCR patients without associated anomalies (HSCR-isolated). The third group consisted of all HSCR patients in which a confirmed pathogenic rare coding variant was identified. We compared these patient groups to unaffected controls. Predisposing haplotypes were determined, confirming that every HSCR subgroup had increased contributions of predisposing haplotypes, but their contribution was highest in isolated HSCR patients without RET coding variants. CNV profiling proved that specifically HSCR-AAM patients had larger Copy Number (CN) losses. Gene enrichment strategies using mouse enteric nervous system transcriptomes and constraint metrics were used to determine plausible candidate genes located within CN losses. Validation in zebrafish using CRISPR/Cas9 targeting confirmed the contribution of UFD1L, TBX2, SLC8A1, and MAPK8 to ENS development. In addition, we revealed epistasis between reduced Ret and Gnl1 expression and between reduced Ret and Tubb5 expression in vivo. Rare large CN losses-often de novo-contribute to HSCR in HSCR-AAM patients. We proved the involvement of six genes in enteric nervous system development and Hirschsprung disease.

Intestinal multicellular organoids to study colorectal cancer.

Modeling colorectal cancer (CRC) using organoids has burgeoned in the last decade, providing enhanced in vitro models to study the development and possible treatment options for this type of cancer. In this review, we describe both normal and CRC intestinal organoid models and their utility in the cancer research field. Besides highlighting studies that develop epithelial CRC organoid models, i.e. organoids without tumor microenvironment (TME) cellular components, we emphasize on the need for TME in CRC modeling, to help reduce translational disparities in this area. Also, we discuss the utilization of CRC organoids derived from pluripotent stem cells, as well as their potential to be used in cancer research. Finally, limitations and challenges in the current CRC organoids field, are discussed.

Goldberg-Shprintzen syndrome is determined by the absence, or reduced expression levels, of KIFBP.

Goldberg-Shprintzen syndrome (GOSHS) is caused by loss of function variants in the kinesin binding protein gene (KIFBP). However, the phenotypic range of this syndrome is wide, indicating that other factors may play a role. To date, 37 patients with GOSHS have been reported. Here, we document nine new patients with variants in KIFBP: seven with nonsense variants and two with missense variants. To our knowledge, this is the first time that missense variants have been reported in GOSHS. We functionally investigated the effect of the variants identified, in an attempt to find a genotype-phenotype correlation. We also determined whether common Hirschsprung disease (HSCR)-associated single nucleotide polymorphisms (SNPs), could explain the presence of HSCR in GOSHS. Our results showed that the missense variants led to reduced expression of KIFBP, while the truncating variants resulted in lack of protein. However, no correlation was found between the severity of GOSHS and the location of the variants. We were also unable to find a correlation between common HSCR-associated SNPs, and HSCR development in GOSHS. In conclusion, we show that reduced, as well as lack of KIFBP expression can lead to GOSHS, and our results suggest that a threshold expression of KIFBP may modulate phenotypic variability of the disease.

Inhibition of ROCK signaling pathway accelerates enteric neural crest cell-based therapy after transplantation in a rat hypoganglionic model.

BACKGROUND: Hirschsprung's disease (HSCR) is a congenital gastrointestinal disorder, characterized by enteric ganglia absence in part or entire of the colon, due to abnormal colonization and migration of enteric neural crest cells (ENCCs) during development. Currently, besides surgery which is the main therapy for HSCR, the potential of stem cell-based transplantation was investigated as an alternative option. Although promising, it has limitations, including poor survival, differentiation, and migration of the grafted cells. We hypothesized that modulation of extracellular factors during transplantation could promote ENCCs proliferation and migration, leading to increased transplantation efficiency. Considering that the RhoA/ROCK pathway is highly involved in cytoskeletal dynamics and neurite growth, our study explored the effect of inhibition of this pathway to improve the success of ENCCs transplantation. METHODS: Enteric neural crest cells were isolated from rat embryos and labeled with a GFP-tag. Cell viability, apoptosis, differentiation, and migration assays were performed with and without RhoA/ROCK inhibition. Labeled ENCCs were transplanted into the muscle layer of an induced hypoganglionic rat model followed by intraperitoneal injections of ROCK inhibitor. The transplanted segments were collected 3 weeks after for histological analysis. KEY RESULTS: Our results showed that inhibition of ROCK increased viable cell number, differentiation, and migration of ENCCs in vitro. Moreover, transplantation of labeled ENCCs into the hypoganglionic model showed enhanced distribution of grafted ENCCs, upon treatment with ROCK inhibitor. CONCLUSIONS AND INFERENCES: ROCK inhibitors influence ENCCs growth and migration in vitro and in vivo, and should be considered to improve the efficiency of ENCCs transplantation.

Co-cultivation of Thermoanaerobacter strains with a methanogenic partner enhances glycerol conversion.

Glycerol-rich waste streams produced by the biodiesel, bioethanol and oleochemical industries can be treated and valorized by anaerobic microbial communities to produce methane. As current knowledge of the microorganisms involved in thermophilic glycerol conversion to methane is scarce, thermophilic glycerol-degrading methanogenic communities were enriched. A co-culture of Thermoanaerobacter and Methanothermobacter species was obtained, pointing to a non-obligately syntrophic glycerol degradation. This hypothesis was further studied by incubating Thermoanaerobacter brockii subsp. finnii and T. wiegelii with glycerol (10 mM) in pure culture and with different hydrogenotrophic methanogens. The presence of the methanogen accelerated glycerol fermentation by the two Thermoanaerobacter strains up to 3.3 mM day-1 , corresponding to 12 times higher volumetric glycerol depletion rates in the methanogenic co-cultures than in the pure bacterial cultures. The catabolic pathways of glycerol conversion were identified by genome analysis of the two Thermoanaerobacter strains. NADH and reduced ferredoxin formed in the pathway are linked to proton reduction, which becomes thermodynamically favourable when the hydrogen partial pressure is kept low by the hydrogenotrophic methanogenic partner.

Zebrafish: A Model Organism for Studying Enteric Nervous System Development and Disease.

The Enteric Nervous System (ENS) is a large network of enteric neurons and glia that regulates various processes in the gastrointestinal tract including motility, local blood flow, mucosal transport and secretion. The ENS is derived from stem cells coming from the neural crest that migrate into and along the primitive gut. Defects in ENS establishment cause enteric neuropathies, including Hirschsprung disease (HSCR), which is characterized by an absence of enteric neural crest cells in the distal part of the colon. In this review, we discuss the use of zebrafish as a model organism to study the development of the ENS. The accessibility of the rapidly developing gut in zebrafish embryos and larvae, enables in vivo visualization of ENS development, peristalsis and gut transit. These properties make the zebrafish a highly suitable model to bring new insights into ENS development, as well as in HSCR pathogenesis. Zebrafish have already proven fruitful in studying ENS functionality and in the validation of novel HSCR risk genes. With the rapid advancements in gene editing techniques and their unique properties, research using zebrafish as a disease model, will further increase our understanding on the genetics underlying HSCR, as well as possible treatment options for this disease.

Neuronal Development and Onset of Electrical Activity in the Human Enteric Nervous System.

BACKGROUND & AIMS: The enteric nervous system (ENS) is the largest branch of the peripheral nervous system, comprising complex networks of neurons and glia, which are present throughout the gastrointestinal tract. Although development of a fully functional ENS is required for gastrointestinal motility, little is known about the ontogeny of ENS function in humans. We studied the development of neuronal subtypes and the emergence of evoked electrical activity in the developing human ENS. METHODS: Human fetal gut samples (obtained via the MRC-Wellcome Trust Human Developmental Biology Resource-UK) were characterized by immunohistochemistry, calcium imaging, RNA sequencing, and quantitative real-time polymerase chain reaction analyses. RESULTS: Human fetal colon samples have dense neuronal networks at the level of the myenteric plexus by embryonic week (EW) 12, with expression of excitatory neurotransmitter and synaptic markers. By contrast, markers of inhibitory neurotransmitters were not observed until EW14. Electrical train stimulation of internodal strands did not evoke activity in the ENS of EW12 or EW14 tissues. However, compound calcium activation was observed at EW16, which was blocked by the addition of 1 µmol/L tetrodotoxin. Expression analyses showed that this activity was coincident with increases in expression of genes encoding proteins involved in neurotransmission and action potential generation. CONCLUSIONS: In analyses of human fetal intestinal samples, we followed development of neuronal diversity, electrical excitability, and network formation in the ENS. These processes are required to establish the functional enteric circuitry. Further studies could increase our understanding of the pathogenesis of a range of congenital enteric neuropathies.