Enteric Nervous System Development Research Articles

Aberrant Expressions of EDNRB and EDN3 in a Multifactorial Hirschsprung Disease.

Hirschsprung disease (HSCR) is a multifactorial disorder due to the enteric nervous system (ENS) development failure. At least 35 genes have been responsible for HSCR, including EDNRB and EDN3. Here, we aimed to determine the EDRNB and EDN3 expressions effects in HSCR subjects. Our study analyzed EDNRB and EDN3 expressions in the colon of HSCR subjects and controls by a quantitative PCR. The EDNRB and EDN3 expressions were analyzed by the Livak method (2-ΔΔC T). Twenty-seven HSCR patients and 20 controls were ascertained. EDNRB and EDN3 expressions downregulated was found in ganglionic and aganglionic HSCR than control colons (EDNRB: ΔCT 6.78 ± 1.38 vs. 1.71 ± 2.79; p = 0.0001 (ganglionic); ΔCT 4.41 ± 1.63 vs. 1.71 ± 2.79; p = 0.0005 (aganglionic); and EDN3: ΔCT 7.60 ± 1.93 vs. 1.81 ± 2.89; p = 0.0001 (ganglionic); ΔCT 9.72 ± 4.32 vs. 1.81 ± 2.89; p = 0.0001 (aganglionic)). A significant difference in EDNRB and EDN3 expressions was also noted between the HSCR colon: ganglionic vs. aganglionic segment (p = 0.00002 and 0.017). We report the downregulated EDNRB and EDN3 expressions in HSCR subjects, indicating EDNRB/EDN3 expressions have a significant responsibility in HSCR pathogenesis. Nevertheless, we further clarify the complexity of the development of ENS.

The Role of de novo and Ultra-Rare Variants in Hirschsprung Disease (HSCR): Extended Gene Discovery for Risk Profiling of Patients.

Hirschsprung disease (HSCR) is a rare neurodevelopmental disorder caused by disrupted migration and proliferation of enteric neural crest cells during enteric nervous system development. Genetic studies suggest a complex etiology involving both rare and common variants, but the contribution of ultra-rare pathogenic variants (PAs) remains poorly understood. We perform whole-exome sequencing (WES) on 301 HSCR probands and 109 family trios, employing advanced statistical methods and gene prioritization strategies to identify genes carrying de novo and ultra-rare coding pathogenic variants. Multiple study designs, including case-control, de novo mutation analysis and joint test, are used to detect associated genes. Candidate genes are further prioritized based on their biological and functional relevance to disease associated tissues and onset period (i.e., human embryonic colon). We identify 19 risk genes enriched with ultra-rare coding pathogenic variants in HSCR probands, including four known genes ( RET , EDNRB , ZEB2 , SOX10 ) and 15 novel candidates (e.g., COLQ , NES , FAT3 ) functioning in neural proliferation and neuromuscular synaptic development. These genes account for 17.5% of the population-attributable risk (PAR), with novel candidates contributing 6.5%. Notably, a positive correlation between pathogenic mutational burden and disease severity is observed. Female cases exhibit at least 42% higher ultra-rare pathogenic variant burden than males (P = 0.05). This first-ever genome-wide screen of ultra-rare variants in a large, phenotypically diverse HSCR cohort highlights the substantial contribution of ultra-rare pathogenic variants to the disease risk and phenotypic variability. These findings enhance our understanding of the genetic architecture of HSCR and provide potential targets for genetic screening and personalized interventions.

Lineage-specific intersection of endothelin and GDNF signaling in enteric nervous system development

Lineage-specific intersection of endothelin and GDNF signaling in enteric nervous system development

Lineage-specific intersection of endothelin and GDNF signaling in enteric nervous system development.

Two major ligand-receptor signaling axes - endothelin Edn3 and its receptor Ednrb, and glial-derived neurotrophic factor (GDNF) and its receptor Ret - are required for migration of enteric nervous system (ENS) progenitors to the hindgut. Mutations in either component cause colonic aganglionosis, also called Hirschsprung disease. Here, we have used Wnt1Cre and Pax2Cre in mice to show that these driver lines label distinct ENS lineages during progenitor migration and in their terminal hindgut fates. Both Cre lines result in Hirschsprung disease when combined with conditional Ednrb or conditional Ret alleles. In vitro explant assays and analysis of lineage-labeled mutant embryos show that GDNF but not Edn3 is a migration cue for cells of both lineages. Instead, Edn3-Ednrb function is required in both for GDNF responsiveness albeit in different ways: by expanding the Ret+ population in the Pax2Cre lineage, and by supporting Ret function in Wnt1Cre-derived cells. Our results demonstrate that two distinct lineages of progenitors give rise to the ENS, and that these divergently utilize endothelin signaling to support migration to the hindgut.

Long-Term Implicit Epigenetic Stress Information in the Enteric Nervous System and its Contribution to Developing and Perpetuating IBS.

Psychiatric and mood disorders may play an important role in the development and persistence of irritable bowel syndrome (IBS). Previously, we hypothesized that stress-induced implicit memories may persist throughout life via epigenetic processes in the enteric nervous system (ENS), independent of the central nervous system (CNS). These epigenetic memories in the ENS may contribute to developing and perpetuating IBS. Here, we further elaborate on our earlier hypothesis. That is, during pregnancy, maternal prenatal stresses perturb the HPA axis and increase circulating cortisol levels, which can affect the maternal gut microbiota. Maternal cortisol can cross the placental barrier and increase cortisol-circulating levels in the fetus. This leads to dysregulation of the HPA axis, affecting the gut microbiota, microbial metabolites, and intestinal permeability in the fetus. Microbial metabolites, such as short-chain fatty acids (which also regulate the development of fetal ENS), can modulate a range of diseases by inducing epigenetic changes. These mentioned processes suggest that stress-related, implicit, long-term epigenetic memories may be programmed into the fetal ENS during pregnancy. Subsequently, this implicit epigenetic stress information from the fetal ENS could be conveyed to the CNS through the bidirectional microbiota-gut-brain axis (MGBA), leading to perturbed functional connectivity among various brain networks and the dysregulation of affective and pain processes.

Enhancement of enteric neural stem cell neurogenesis by glial cell-derived neurotrophic factor in experimental Hirschsprung's disease.

Stem cell therapy offers a promising solution for congenital diseases like Hirschsprung's disease (HSCR). Optimizing stem cell efficacy by modifying the cells and their environment is crucial, but in vitro culture conditions need to be further improved. Glial cell-derived neurotrophic factor (GDNF) plays an important role in neuronal survival, proliferation, migration and differentiation during enteric nervous system (ENS) development. In this study, the effects of GDNF on neurites derived from an Ednrb knockout model were investigated with the aim of enhancing the neurogenic potential of enteric neural crest cells (ENCCs). Neurospheres were generated form Ednrb+/+ (control) and Ednrb-/- mice at embryonic day13.5 (E13.5) with Sox10-green fluorescent protein (Venus) transgenic expression. These neurospheres were cultured in control media and neurospheres from Ednrb-/- were cultured with either control media or media supplemented with GDNF. ENCCs differentiation was assessed using immunofluorescence staining after 18days. GDNF-treated Ednrb-/- neurospheres showed increased size and higher density of Sox10-positive ENCCs compared to untreated Ednrb-/- neurospheres. GDNF also enhanced the distribution of both TUJ1-positive neurons and S100-positive glial cells. GDNF effectively enhanced the neurogenic potential of ENCCs from HSCR animal model. This finding is crucial for the development of cell therapy in HSCR.

BMP signaling pathway member expression is enriched in enteric neural progenitors and required for zebrafish enteric nervous system development

AbstractBackgroundThe vertebrate enteric nervous system (ENS) consists of a series of interconnected ganglia within the gastrointestinal (GI) tract, formed during development following migration of enteric neural crest cells (ENCCs) into the primitive gut tube. Much work has been done to unravel the complex nature of extrinsic and intrinsic factors that regulate processes that direct migration, proliferation, and differentiation of ENCCs. However, ENS development is a complex process, and we still have much to learn regarding the signaling factors that regulate ENCC development.ResultsHere in zebrafish, through transcriptomic, in situ transcript expression, immunohistochemical analysis, and chemical attenuation, we identified a time‐dependent role for bone morphogenetic protein (BMP) in the maintenance of Phox2bb+ enteric progenitor numbers and/or time of differentiation of the progenitor pool. In support of our in silico transcriptomic analysis, we identified expression of a novel ENS ligand‐encoding transcript, bmp5, within developmental regions of ENCCs. Through generation of a novel mutant bmp5wmr2 and bmp5 crispants, we identified a functional role for BMP5 in proper GI tract colonization, whereby phox2bb+ enteric progenitor numbers were reduced.ConclusionAltogether, this work identified time‐dependent roles for BMP signaling and a novel extrinsic factor, BMP5, that is necessary for vertebrate ENS formation.

Single Cell Profiling in the Sox10 Dom/+ Hirschsprung Mouse Implicates Hoxa6 in Enteric Neuron Lineage Allocation.

Enteric nervous system (ENS) development requires migration, proliferation, and appropriate neuronal diversification from progenitors to enable normal gastrointestinal (GI) motility. Sox10 deficit causes aganglionosis, modeling Hirschsprung disease, and disrupts ratios of postnatal enteric neurons in proximal ganglionated bowel. How Sox10 deficiency alters ratios of enteric neuron subtypes is unclear. Sox10's prominent expression in enteric neural crest-derived progenitors (ENCP) and lack of this gene in enteric neurons led us to examine Sox10 Dom effects ENS progenitors and early differentiating enteric neurons. ENS progenitors, developing neurons, and enteric glia were isolated from Sox10 +/+ and Sox10 Dom/+ littermates for single-cell RNA sequencing (scRNA-seq). scRNA-seq data was processed to identify cell type-specific markers, differentially expressed genes, cell fate trajectories, and gene regulatory network activity between genotypes. Hybridization chain reaction (HCR) validated expression changes detected in scRNA-seq. scRNA-seq profiles revealed three neuronal lineages emerging from cycling progenitors via two transition pathways accompanied by elevated activity of Hox gene regulatory networks (GRN) as progenitors transition to neuronal fates. Sox10 Dom/+ scRNA-seq profiles exhibited a novel progenitor cluster, decreased abundance of cells in transitional states, and shifts in cell distributions between two neuronal trajectories. Hoxa6 was differentially expressed in the neuronal lineages impacted in Sox10 Dom/+ mutants and HCR identified altered Hoxa6 expression in early developing neurons of Sox10 Dom/+ ENS. Sox10 Dom/+ mutation shifts enteric neuron types by altering neuronal trajectories during early ENS lineage segregation. Multiple neurogenic transcription factors are reduced in Sox10 Dom/+ scRNA-seq profiles including multiple Hox genes. This is the first report that implicates Hox genes in lineage diversification of enteric neurons.

ELP1, the Gene Mutated in Familial Dysautonomia, Is Required for Normal Enteric Nervous System Development and Maintenance and for Gut Epithelium Homeostasis.

Familial dysautonomia (FD) is a rare sensory and autonomic neuropathy that results from a mutation in the ELP1 gene. Virtually all patients report gastrointestinal (GI) dysfunction and we have recently shown that FD patients have a dysbiotic gut microbiome and altered metabolome. These findings were recapitulated in an FD mouse model and moreover, the FD mice had reduced intestinal motility, as did patients. To understand the cellular basis for impaired GI function in FD, the enteric nervous system (ENS; both female and male mice) from FD mouse models was analyzed during embryonic development and adulthood. We show here that not only is Elp1 required for the normal formation of the ENS, but it is also required in adulthood for the regulation of both neuronal and non-neuronal cells and for target innervation in both the mucosa and in intestinal smooth muscle. In particular, CGRP innervation was significantly reduced as was the number of dopaminergic neurons. Examination of an FD patient's gastric biopsy also revealed reduced and disoriented axons in the mucosa. Finally, using an FD mouse model in which Elp1 was deleted exclusively from neurons, we found significant changes to the colon epithelium including reduced E-cadherin expression, perturbed mucus layer organization, and infiltration of bacteria into the mucosa. The fact that deletion of Elp1 exclusively in neurons is sufficient to alter the intestinal epithelium and perturb the intestinal epithelial barrier highlights a critical role for neurons in regulating GI epithelium homeostasis.

Genetic association of GJA8 with long-segment Hirschsprung's disease in southern Chinese children.

Hirschsprung's disease (HSCR) is a complex congenital neurodevelopmental disorder affecting colons caused by both genetic and environmental factors. Although several genes have been identified as contributing factors in HSCR, the pathogenesis is still largely unclear, especially for the low prevalent long-segment HSCR (L-HSCR). Gap junction protein alpha 8 (GJA8) is involved in several physiological processes and has been implicated in several diseases. However, the relationship between GJA8 single nucleotide polymorphism (SNP) rs17160783 and HSCR in the southern Chinese population remains unknown. The study aimed to explore the association of genetic variants in GJA8 and HSCR susceptibility in southern Chinese. SNP rs17160783 A>G in GJA8 was genotyped by TaqMan SNP Genotyping Assay in all samples, which included 1,329 HSCR children (cases) and 1,473 healthy children (controls). Odds ratio (OR) and 95% confidence interval (CI) were used to evaluate the association of GJA8 polymorphisms with HSCR susceptibility. The GTEx database and transcription factor binding site (TFBS) prediction were used to analyze the potential regulatory function of rs17160783. Genetic association analysis illustrated that rs17160783 could increase the risk of L-HSCR (Padj=0.04, ORadj =1.48, 95% CI: 1.02-2.14). We also found that GJA8 expression was increased in HSCR and neurodevelopmentally impaired animal models. External epigenetic data revealed that GJA8 rs17160783 may have the potential to regulate the expression of the GJA8, possibly by altering the binding of transcription factors for GJA8, and consequently impacting the PI3K-Akt signaling pathway during the enteric nervous system (ENS) development. Our results suggested that rs17160783 might play a regulatory role in GJA8 expression and increase the susceptibility of L-HSCR in children from southern China.

Spontaneous enteric nervous system activity generates contractile patterns prior to maturation of gastrointestinal motility.

Spontaneous neuronal network activity is essential to the functional maturation of central and peripheral circuits, yet whether this is a feature of enteric nervous system development has yet to be established. Although enteric neurons are known exhibit electrophysiological properties early in embryonic development, no connection has been drawn between this neuronal activity and the development of gastrointestinal (GI) motility patterns. We use exvivo GI motility assays with newly developed unbiased computational analyses to identify GI motility patterns across mouse embryonic development. We find a previously unknown pattern of neurogenic contractions termed "clustered ripples" that arises spontaneously at embryonic day 16.5, an age earlier than any identified mature GI motility patterns. We further show that these contractions are driven by nicotinic cholinergic signaling. Clustered ripples are neurogenic contractile activity that arise from spontaneous ENS activity and precede all known forms of neurogenic GI motility. This earliest motility pattern requires nicotinic cholinergic signaling, which may inform pharmacology for enhancing GI motility in preterm infants.

A targeted CRISPR-Cas9 mediated F0 screen identifies genes involved in establishment of the enteric nervous system.

The vertebrate enteric nervous system (ENS) is a crucial network of enteric neurons and glia resident within the entire gastrointestinal tract (GI). Overseeing essential GI functions such as gut motility and water balance, the ENS serves as a pivotal bidirectional link in the gut-brain axis. During early development, the ENS is primarily derived from enteric neural crest cells (ENCCs). Disruptions to ENCC development, as seen in conditions like Hirschsprung disease (HSCR), lead to the absence of ENS in the GI, particularly in the colon. In this study, using zebrafish, we devised an in vivo F0 CRISPR-based screen employing a robust, rapid pipeline integrating single-cell RNA sequencing, CRISPR reverse genetics, and high-content imaging. Our findings unveil various genes, including those encoding opioid receptors, as possible regulators of ENS establishment. In addition, we present evidence that suggests opioid receptor involvement in the neurochemical coding of the larval ENS. In summary, our work presents a novel, efficient CRISPR screen targeting ENS development, facilitating the discovery of previously unknown genes, and increasing knowledge of nervous system construction.

Identification of signaling pathways that specify a subset of migrating enteric neural crest cells at the wavefront in mouse embryos

During enteric nervous system (ENS) development, pioneering wavefront enteric neural crest cells (ENCCs) initiate gut colonization. However, the molecular mechanisms guiding their specification and niche interaction are not fully understood. We used single-cell RNA sequencing and spatial transcriptomics to map the spatiotemporal dynamics and molecular landscape of wavefront ENCCs in mouse embryos. Our analysis shows a progressive decline in wavefront ENCC potency during migration and identifies transcription factors governing their specification and differentiation. We further delineate key signaling pathways (ephrin-Eph, Wnt-Frizzled, and Sema3a-Nrp1) utilized by wavefront ENCCs to interact with their surrounding cells. Disruptions in these pathways are observed in human Hirschsprung's disease gut tissue, linking them to ENS malformations. Additionally, we observed region-specific and cell-type-specific transcriptional changes in surrounding gut tissues upon wavefront ENCC arrival, suggesting their role in shaping the gut microenvironment. This work offers a roadmap of ENS development, with implications for understanding ENS disorders.

Mesenchymal cells regulate enteric neural crest cell migration via RET-GFRA1b trans-signaling

During early development, the enteric nervous system forms from the migration of enteric neural crest cells (ENCCs) from the foregut to the hindgut, where they undergo proliferation and differentiation facilitated by interactions with enteric mesenchymal cells (EMCs). This study investigates the impact on ENCC migration of EMC-ENCC communication mediated by GFRA1b expressed in EMCs. GFRA1-expressing cells in day 11–12 (E11–12) mouse embryos differentiated into smooth muscle cells from E12 onwards. Observations at E12–13.5 revealed high levels of GFRA1 expression on the anti-mesenteric side of the hindgut, correlating with enhanced ENCC migration. This indicates that GFRA1 in EMCs plays a role in ENCC migration during development. Examining GFRA1 isoforms, we found high levels of GFRA1b, which lacks amino acids 140–144, in EMCs. To assess the impact of GFRA1 isoforms on EMC-ENCC communication, we conducted neurosphere drop assays. This revealed that GFRA1b-expressing cells promoted GDNF-dependent extension and increased neurite density in ENCC neurospheres. Co-culture of ENCC mimetic cells expressing RET and GFRA1a with EMC mimetic cells expressing GFRA1a, GFRA1b, or vector alone showed that only GFRA1b-expressing co-cultured cells sustained RET phosphorylation in ENCC-mimetic cells for over 120 min upon GDNF stimulation. Our study provides evidence that GFRA1b-mediated cell-to-cell communication plays a critical role in ENCC motility in enteric nervous system development. These findings contribute to understanding the cellular interactions and signaling mechanisms that underlie enteric nervous system formation and highlight potential therapeutic targets for gastrointestinal motility disorders.

Aberrant SOX10 and RET expressions in patients with Hirschsprung disease

BackgroundHSCR is a complex genetic disorder characterized by the absence of ganglion cells in the intestine, leading to a functional obstruction. It is due to a disruption of complex signaling pathways within the gene regulatory network (GRN) during the development of the enteric nervous system (ENS), including SRY-Box Transcription Factor 10 (SOX10) and REarranged during Transfection (RET). This study evaluated the expressions of SOX10 and RET in HSCR patients in Indonesia.MethodsTotal RNA of 19 HSCR ganglionic and aganglionic colons and 16 control colons were analyzed using quantitative real-time polymerase chain reaction for SOX10 and RET with GAPDH as the reference gene. Livak’s method (2−ΔΔCT) was used to determine the expression levels of SOX10 and RET.ResultsMost patients were males (68.4%), in the short aganglionosis segment (78.9%), and had undergone transanal endorectal pull-through (36.6%). There were significant upregulated SOX10 expressions in both ganglionic (2.84-fold) and aganglionic (3.72-fold) colon of HSCR patients compared to controls’ colon (ΔCT 5.21 ± 2.04 vs. 6.71 ± 1.90; p = 0.032; and ΔCT 4.82 ± 1.59 vs. 6.71 ± 1.90; p = 0.003; respectively). Interestingly, the RET expressions were significantly downregulated in both ganglionic (11.71-fold) and aganglionic (29.96-fold) colon of HSCR patients compared to controls’ colon (ΔCT 12.54 ± 2.21 vs. 8.99 ± 3.13; p = 0.0004; and ΔCT 13.90 ± 2.64 vs. 8.99 ± 3.13; p = 0.0001; respectively).ConclusionsOur study shows aberrant SOX10 and RET expressions in HSCR patients, implying the critical role of SOX10 and RET in the pathogenesis of HSCR, particularly in the Indonesian population. Our study further confirms the involvement of SOX10-RET within the GNR during the ENS development.

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.

High incidence of EDNRB gene mutation in seven southern Chinese familial cases with Hirschsprung's disease.

Hirschsprung's disease (HSCR) is the leading cause of neonatal functional intestinal obstruction, which has been identified in many familial cases. HSCR, a multifactorial disorder of enteric nervous system (ENS) development, is associated with at least 24 genes and seven chromosomal loci, with RET and EDNRB as its major genes. We present a genetic investigation of familial HSCR to clarify the genotype-phenotype relationship. We performed whole exome sequencing (WES) on Illumina HiSeq X Ten platform to investigate genetic backgrounds of core family members, and identified the possibly harmful mutation genes. Mutation carriers and pedigree relatives were validated by Sanger sequencing for evaluating the gene penetrance. Four familial cases showed potential disease-relative variants in EDNRB and RET gene, accounting for all detection rate of 57.1%. Three familial cases exhibited strong pathogenic variants as frameshift or missense mutations in EDNRB gene. A novel c.367delinsTT mutation of EDNRB was identified in one family member. The other two EDNRB mutations, c.553G>A in family 2 and c.877delinsTT in family 5, have been reported in previous literatures. The penetrance of EDNRB variants was 33-50% according mutation carries. In family 6, the RET c.1858T>C (C620R) point mutation has previously been reported to cause HSCR, with 28.5% penetrance. We identified a novel EDNRB (deleted C and inserted TT) mutation in this study using WES. Heterozygote variations in EDNRB gene were significantly enriched in three families and RET mutations were identified in one family. EDNRB variants showed an overall higher incidence and penetrance than RET in southern Chinese families cases.

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.

Circular RNA MTCL1 targets SMAD3 by sponging miR-145-5p for regulation of cell proliferation and migration in Hirschsprung's disease.

Hirschsprung's disease (HSCR) is a congenital disorder resulting from abnormal development of the enteric nervous system (ENS). Given the complexity of its pathogenesis, it is important to investigate the role of epigenetic inheritance in its development. As Circ-MTCL1 is abundant in brain tissue and colon tissue, whether it has a significant part in the development of ENS is worth exploring. This study clarifies its role in HSCR and identifies the specific molecular mechanisms involved. Diseased and dilated segment colon tissues diagnosed as HSCR were collected for the assessment of gene expression levels using RT-PCR. EdU and CCK-8 assays were adopted to evaluate cell proliferation, and Transwell assay was adopted to assess cell migration. The interaction between Circ-MTCL1, miR-145-5p and SMAD3 was confirmed by dual luciferase reporter gene analysis, RT-PCR and Western blotting. Circ-MTCL1 was down-regulated in the aganglionic colon tissues. The decreased expression of Circ-MTCL1 associated with a reduction in cell migration and proliferation. Bioinformatics analysis and cellular experiments confirmed its role might have been associated with the inhibition of miR-145-5p. MiR-145-5p was up-regulated in HSCR diseased segment colon tissues, exhibiting a negative correlation with Circ-MTCL1. Overexpression of miR-145-5p reversed the inhibition of cell migration and proliferation associated with Circ-MTCL1 down-regulation. The expression of SMAD3 was inhibited by miR-145-5p. The overexpression of SMAD3 eliminated the miR-145-5p-associated inhibition of cell migration and proliferation. Overexpression of miR-145-5p reversed the inhibitory effects of Circ-MTCL1 down-regulation-associated inhibition of cell migration and proliferation, while suppressing SMAD3 expression. Conversely, overexpression of SMAD3 counteracted the miR-145-5p-associated inhibition of cell migration and proliferation. Circ-MTCL1 may function as a miR-145-5p sponge, regulating the expression of SMAD3 and influencing cell migration and proliferation, thus participating in the development of HSCR.

ATP5PO levels regulate enteric nervous system development in zebrafish, linking Hirschsprung disease to Down Syndrome

Hirschsprung disease (HSCR) is a complex genetic disorder characterized by the absence of enteric nervous system (ENS) in the distal region of the intestine. Down Syndrome (DS) patients have a >50-fold higher risk of developing HSCR than the general population, suggesting that overexpression of human chromosome 21 (Hsa21) genes contribute to HSCR etiology. However, identification of responsible genes remains challenging. Here, we describe a genetic screening of potential candidate genes located on Hsa21, using the zebrafish. Candidate genes were located in the DS-HSCR susceptibility region, expressed in the human intestine, were known potential biomarkers for DS prenatal diagnosis, and were present in the zebrafish genome. With this approach, four genes were selected: RCAN1, ITSN1, ATP5PO and SUMO3. However, only overexpression of ATP5PO, coding for a component of the mitochondrial ATPase, led to significant reduction of ENS cells. Paradoxically, in vitro studies showed that overexpression of ATP5PO led to a reduction of ATP5PO protein levels. Impaired neuronal differentiation and reduced mitochondrial ATP production, were also detected in vitro, after overexpression of ATP5PO in a neuroblastoma cell line. Finally, epistasis was observed between ATP5PO and ret, the most important HSCR gene. Taken together, our results identify ATP5PO as the gene responsible for the increased risk of HSCR in DS patients in particular if RET variants are also present, and show that a balanced expression of ATP5PO is required for normal ENS development.