Disorder Of Enteric Nervous System Research Articles

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.

Sacral Neural Crest-Independent Origin of the Enteric Nervous System in Mouse

The enteric nervous system (ENS), the gut's intrinsic nervous system critical for gastrointestinal function and gut-brain communication, is believed to mainly originate from vagal neural crest cells (vNCCs) and partially from sacral NCCs (sNCCs). Resolving the exact origins of the ENS is critical for understanding congenital ENS diseases buthas been confounded by the inability to distinguish between both NCC populations in situ. Here, we aimed to resolve the exact origins of the mammalian ENS. We genetically engineered mouse embryos facilitating comparative lineage-tracing of either all (pan-) NCCs including vNCCs or caudal trunk and sNCCs (s/tNCCs) excluding vNCCs. This was combined with dual-lineage tracing and 3-dimensional reconstruction of pelvic plexus and hindgut to precisely pinpoint sNCC and vNCC contributions. We further used coculture assays to determine the specificity of cell migration from different neural tissues into the hindgut. Both pan-NCCs and s/tNCCs contributed to established NCC derivatives but onlypan-NCCs contributed to the ENS. Dual-lineage tracing combined with 3-dimensional reconstruction revealed that s/tNCCs settle in complex patterns in pelvic plexus and hindgut-surrounding tissues, explaining previous confusion regarding their contributions. Coculture experiments revealed unspecific cell migration from autonomic, sensory, and neural tube explants into the hindgut. Lineage tracing of ENS precursors lastly provided complimentary evidence for an exclusive vNCC origin of the murine ENS. sNCCs do not contribute to the murine ENS, suggesting that the mammalian ENS exclusively originates from vNCCs. These results have immediate implications for comprehending (and devising treatments for) congenital ENS disorders, including Hirschsprung's disease.

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.

How to Heal the Gut's Brain: Regeneration of the Enteric Nervous System.

The neural-crest-derived enteric nervous system (ENS) is the intrinsic nervous system of the gastrointestinal (GI) tract and controls all gut functions, including motility. Lack of ENS neurons causes various ENS disorders such as Hirschsprung Disease. One treatment option for ENS disorders includes the activation of resident stem cells to regenerate ENS neurons. Regeneration in the ENS has mainly been studied in mammalian species using surgical or chemically induced injury methods. These mammalian studies showed a variety of regenerative responses with generally limited regeneration of ENS neurons but (partial) regrowth and functional recovery of nerve fibers. Several aspects might contribute to the variety in regenerative responses, including observation time after injury, species, and gut region targeted. Zebrafish have recently emerged as a promising model system to study ENS regeneration as larvae possess the ability to generate new neurons after ablation. As the next steps in ENS regeneration research, we need a detailed understanding of how regeneration is regulated on a cellular and molecular level in animal models with both high and low regenerative capacity. Understanding the regulatory programs necessary for robust ENS regeneration will pave the way for using neural regeneration as a therapeutic approach to treating ENS disorders.

Poreklo ćelija enteričkog nervnog sistema i putevi migracije tokom embrionalnog razvoja

The enteric nervous system (ENS) is represented by a complex network of neurons, glial and other cells within the wall of the digestive tract. ENS is responsible for numerous, vital functions in our body. Thus, ENS regulates motility of the digestive tract, secretion into the intestinal lumen, exchange of fluid and electrolytes through the mucosa, as well as mucosal perfusion. In order to perform these important functions, proper embryonic development of ENS is necessary. ENS cells are derived from precursor cells of the neural crest (NCCs - neural crest cells). Two cell populations that contribute to the largest number of future ENS cells are the vagal and sacral NCCs. Vagal NCCs enter the primitive gut tube in the region of the future esophagus (foregut), and begin their migration, through the midgut towards the hindgut and the future anal region. Sacral NCCs enter the hindgut region following the extrinsic nerve fibers and continue their migration rostrally, towards vagal NCCs. Along with the migration process, these cells undergo other important processes, such as proliferation, neuro-glial differentiation, gangliogenesis, axonal pathway formation and synaptogenesis. All these processes are strictly regulated by numerous signaling pathways, which are still being actively researched. Modern lineage tracing and other technologies, that enabled following of individual precursor cells through their development pathways, will significantly contribute to the better understanding of development of ENS. This may have repercussions in improving the diagnosis and treatment of some developmental (Hirschsprung disease) and other ENS disorders.

Revealing the complexity of the gut's brain.

A new study proposes an exciting new model of neuronal diversification in the developing enteric nervous system (ENS) and establishes a detailed molecular taxonomy for enteric neurons. Their findings open new horizons for ENS research and for developing cell-based therapies for ENS disorders.

An efficient approach for wholemount preparation of the myenteric plexus of rat colon

BackgroundThe nerve plexus of the enteric nervous system (ENS) plays a crucial part in regulating gastrointestinal functions, such as muscle contractile activity and nutrient absorption. Studying the nerve plexus can provide vital information for research on ENS disorders. Whole-mount preparation is an important technique for investigating the nerve plexus. However, currently available methods are time consuming and highly technical. New methodThis study describes a simple and rapid method for preparing whole mounts of the longitudinal muscle and myenteric plexuses (LMMPs) of rat colon. Integral LMMPs can be easily separated from the underlying layer by using glass rods and wet cotton swabs. ResultsThe proposed method allows the easy separation of the LMMPs in intact sheets. Immunofluorescence histochemical staining of whole mounts enable clear visualization of enteric ganglia, nerve fibers, and several subtypes of neuronal populations residing in the myenteric plexus. Comparison with existing methodsCompared with existing procedures for whole-mount preparations, the proposed method achieves a quicker and more efficient preparation of high-quality LMMPs from intestinal segments in sufficient quantity. ConclusionsThis work provides a rapid method for efficiently preparing whole mounts of the intestines. Our method can be used for in situ observation of the morphological and functional alterations of the myenteric plexus for further studies on the ENS.

Autologous Transplantation of Skin-Derived Precursor Cells in a Porcine Model

BackgroundHirschprung's disease is characterized by aganglionic bowel and often requires surgical resection. Cell-based therapies have been investigated as potential alternatives to restore functioning neurons. Skin-derived precursor cells (SKPs) differentiate into neural and glial cells in vitro and generate ganglion-like structures in rodents. In this report, we aimed to translate this approach into a large animal model of aganglionosis using autologous transplantation of SKPs. MethodsJuvenile pigs underwent skin procurement from the shoulder and simultaneous chemical denervation of an isolated colonic segment. Skin cells were cultured in neuroglial-selective medium and labeled with fluorescent dye for later identification. The cultured SKPs were then injected into the aganglionic segments of colon, and the specimens were retrieved within seven days after transplantation. SKPs in vitro and in vivo were assessed with histologic samples for various immunofluorescent markers of multipotency and differentiation. SKPs from the time of harvest were compared to those at the time of injection using PCR. ResultsPrior to transplantation, 72% of SKPs stained positive for nestin and S100b, markers of neural and glial precursor cells of neural crest origin, respectively. Markers of differentiated neurons and gliocytes, TUJ1 and GFAP, were detected in 47% of cultured SKPs. After transplantation, SKPs were identified in both myenteric and submucosal plexuses of the treated colon. Nestin co-expression was detected in the SKPs within the aganglionic colon in vivo. Injected SKPs appeared to migrate and express early neuroglial differentiation markers. ConclusionsAutologous SKPs implanted into aganglionic bowel demonstrated immunophenotypes of neuroglial progenitors. Our results suggest that autologous SKPs may be potentially useful for cell-based therapy for patients with enteric nervous system disorders. Type of StudyBasic science.

A gene regulatory network explains RET-EDNRB epistasis in Hirschsprung disease.

Disruptions in gene regulatory networks (GRNs), driven by multiple deleterious variants, potentially underlie complex traits and diseases. Hirschsprung disease (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 previously demonstrated that RET transcription in the ENS is controlled by an extensive GRN involving the transcription factors (TFs) RARB, GATA2 and SOX10 and other HSCR genes. We now demonstrate, using human and mouse cellular and animal models, that EDNRB is transcriptionally regulated in the ENS by GATA2, SOX10 and NKX2.5 TFs. Significantly, RET and EDNRB expression is regulated by their shared use of GATA2 and SOX10, and in turn, these TFs are controlled by EDNRB and RET in a dose-dependent manner. This study expands the ENS development GRN to include both RET and EDNRB, uncovers the mechanistic basis for RET-EDNRB epistasis and emphasizes how functionally different genes associated with a complex disorder can be united through a common GRN.

Alteration of the Retinoid Acid-CBP Signaling Pathway in Neural Crest Induction Contributes to Enteric Nervous System Disorder.

Hirschsprung Disease (HSCR) and/or hypoganglionosis are common pediatric disorders that arise from developmental deficiencies of enteric neural crest cells (ENCCs). Retinoid acid (RA) signaling has been shown to affect neural crest (NC) development. However, the mechanisms underlying RA deficiency-induced HSCR or hypoganglionosis are not well-defined. In this report, we found that in HSCR patient bowels, the RA nuclear receptor RARα and its interacting coregulator CREB-binding protein (CBP) were expressed in enteric neural plexuses in the normal ganglionic segment. However, the expression of these two genes was significantly inhibited in the pathological aganglionic segment. In a Xenopus laevis animal model, endogenous RARα interacted with CBP and was expressed in NC territory. Morpholino-mediated knockdown of RARα blocked expression of the NC marker genes Sox10 and FoxD3 and inhibited NC induction. The morphant embryos exhibited reduced nervous cells in the gastrointestinal anlage, a typical enteric nervous deficiency-associated phenotype. Injection of CBP mRNA rescued NC induction and reduced enteric nervous deficiency-associated phenotypes. Our work demonstrates that RARα regulates Sox10 expression via CBP during NC induction, and alteration of the RA-CBP signaling pathway may contribute to the development of enteric nervous system disorders.

Collagen VI suppresses fibronectin-induced enteric neural crest cell migration by downregulation of focal adhesion proteins

The enteric nervous system (ENS) is a network of neurons and glia that are derived from enteric neural crest cells (ENCCs) and essential for regulating peristaltic activity of the colon. ENCCs migrate along the gastrointestinal tract to form the ENS, and disruption of ENCC motility leads to ENS disorders, such as Hirschsprung's disease. Previous ENCC-transplant experiments show that ENCCs can invade into isolated mouse intestines by age E13.5, but not after E15.5. We hypothesized that altered age-specific micro-environments in the intestine are responsible for ENCC invasion/migration. Here, we compared gene expression in the intestine between at E11.5 and E15.5 and identified 1355 differentially expressed transcripts. Among these, we found that genes encoding extracellular matrix (ECM) proteins were enriched. Notably, collagen VI (ColVI) family members were upregulated in the E15.5 mouse intestine at the mRNA and protein levels, whereas fibronectin (FN) was downregulated; however, both proteins showed colocalization at E15.5. To understand the mechanisms of ColVI and FN in ENCC migration, we examined neurosphere or individual ENCC-adherence capabilities toward the ECM. ColVI suppressed FN-induced ENCC spreading/migration, whereas ColVI induced morphologically narrow ENCC spreading and weak stress-fiber formation as compared with those with FN. Additionally, in ENCCs cultured on plates containing ColVI, the expression and phosphorylation of p130Cas, a members of focal adhesion complexes, was reduced. These data indicated an inhibitory role of ColVI in ENCC migration and suggested that ColVI suppression in the intestine might represent a novel therapeutic strategy for aganglionic colonic diseases.

Gut feelings: Studying enteric nervous system development, function, and disease in the zebrafish model system.

The enteric nervous system (ENS) is the largest part of the peripheral nervous system and is entirely neural crest-derived. It provides the intrinsic innervation of the gut, controlling different aspects of gut function, such as motility. In this review, we will discuss key points of Zebrafish ENS development, genes, and signaling pathways regulating ENS development, as well as contributions of the Zebrafish model system to better understand ENS disorders. During their migration, enteric progenitor cells (EPCs) display a gradient of developmental states based on their proliferative and migratory characteristics, and show spatiotemporal heterogeneity based on gene expression patterns. Many genes and signaling pathways that regulate the migration and proliferation of EPCs have been identified, but later stages of ENS development, especially steps of neuronal and glial differentiation, remain poorly understood. In recent years, Zebrafish have become increasingly important to test candidate genes for ENS disorders (e.g., from genome-wide association studies), to identify environmental influences on ENS development (e.g., through large-scale drug screens), and to investigate the role the gut microbiota play in ENS development and disease. With its unique advantages as a model organism, Zebrafish will continue to contribute to a better understanding of ENS development, function, and disease. Developmental Dynamics 247:268-278, 2018. © 2017 Wiley Periodicals, Inc.

Pediatric Motility Disorders

Pediatric gastrointestinal motility disorders are a heterogeneous group of enteric nervous system disorders that continue to cause difficulties in children. The pathophysiology, diagnosis, and treatments of the most common disorders of the esophagus, stomach, small intestine, and colon will be discussed to understand the interplay between motility and sensory functions and the effect on gastrointestinal motility. Much remains unanswered in the pathogenesis of the majority intestinal motility disorders. Research has started to explore targeted gene mutations as potential causes of congenital motility disorders, which may be able to help better predict outcomes. An understanding of the relationship between the enteric nervous system the gastrointestinal tract, in combination with advancements in motility studies, technology, and gene therapies, will help point towards potential for novel therapies.

Inactivation of Geminin in neural crest cells affects the generation and maintenance of enteric progenitor cells, leading to enteric aganglionosis

Neural crest cells comprise a multipotent, migratory cell population that generates a diverse array of cell and tissue types, during vertebrate development. Enteric Nervous System controls the function of the gastrointestinal tract and is mainly derived from the vagal and sacral neural crest cells. Deregulation on self-renewal and differentiation of the enteric neural crest cells is evident in enteric nervous system disorders, such as Hirschsprung disease, characterized by the absence of ganglia in a variable length of the distal bowel. Here we show that Geminin is essential for Enteric Nervous System generation as mice that lacked Geminin expression specifically in neural crest cells revealed decreased generation of vagal neural crest cells, and enteric neural crest cells (ENCCs). Geminin-deficient ENCCs showed increased apoptosis and decreased cell proliferation during the early stages of gut colonization. Furthermore, decreased number of committed ENCCs in vivo and the decreased self-renewal capacity of enteric progenitor cells in vitro, resulted in almost total aganglionosis resembling a severe case of Hirschsprung disease. Our results suggest that Geminin is an important regulator of self-renewal and survival of enteric nervous system progenitor cells.

Enteric neural differentiation in innervated, physiologically functional, smooth muscle constructs is modulated by bone morphogenic protein 2 secreted by sphincteric smooth muscle cells.

The enteric nervous system (ENS) controls gastrointestinal (GI) functions, including motility and digestion, which are impaired in ENS disorders. Differentiation of enteric neurons is mediated by factors released by the gut mesenchyme, including smooth muscle cells (SMCs). SMC-derived factors involved in adult enteric neural progenitor cells (NPCs) differentiation remain elusive. Furthermore, physiologically relevant in vitro models to investigate the innervations of various regions of the gut, such as the pylorus and lower oesophageal sphincter (LES), are not available. Here, neural differentiation in bioengineered innervated circular constructs composed of SMCs isolated from the internal anal sphincter (IAS), pylorus, LES and colon of rabbits was investigated. Additionally, SMC-derived factors that induce neural differentiation were identified to optimize bioengineered construct innervations. Sphincteric and non-sphincteric bioengineered constructs aligned circumferentially and SMCs maintained contractile phenotypes. Sphincteric constructs generated spontaneous basal tones. Higher levels of excitatory and inhibitory motor neuron differentiation and secretion of bone morphogenic protein 2 (BMP2) were observed in bioengineered, innervated, sphincteric constructs compared to non-sphincteric constructs. The addition of BMP2 to non-sphincteric colonic SMC constructs increased nitrergic innervations, and inhibition of BMP2 with noggin in sphincteric constructs decreased functional relaxation. These studies provide: (a) the first bioengineered innervated pylorus and LES constructs; (b) physiologically relevant models to investigate SMCs and adult NPCs interactions; and (c) evidence of the region-specific effects of SMCs on neural differentiation mediated by BMP2. Furthermore, this study paves the way for the development of innervated bioengineered GI tissue constructs tailored to specific disorders and locations within the gut. Copyright © 2015 John Wiley & Sons, Ltd.

Recent developments in pathogenesis of Hirschsprung's disease

Hirschsprung's disease(HD) is a conginental disorder of enteric nervous system(ENS) and is associated with the abnormal migration of gut neural cerst cell(GNCC). Recent studies showed that the regulation of this migration is a complex gene network.By the development of epigenetic and stem cells, this network is becoming much more clear.Transplantation of the modified gut neural crest stem cells/gut neural crest precursors(GNCSC/GNCP) acquired from the HD bowl is becoming the potential therapy of HD.This paper summerizes the recent advances and is to promote better understanding the pathogenesis of HD. Key words: Hirschsprung's Disease; Pathogenesis; Gene network; Development

Granulocyte-colony stimulating factor: a new player for the enteric nervous system

The enteric nervous system (ENS) controls and modulates gut motility and responds to food intake and to internal and external stimuli such as toxins or inflammation. Its plasticity is maintained throughout life by neural progenitor cells within the enteric stem cell niche. Granulocyte-colony stimulating factor (G-CSF) is known to act not only on cells of the immune system but also on neurons and neural progenitors in the central nervous system (CNS). Here, we demonstrate, for the first time, that G-CSF receptor is present on enteric neurons and progenitors and that G-CSF plays a role in the expansion and differentiation of enteric neural progenitor cells. Cultured mouse ENS-neurospheres show increased expansion with increased G-CSF concentrations, in contrast to CNS-derived spheres. In cultures from differentiated ENS- and CNS-neurospheres, neurite outgrowth density is enhanced depending on the amount of G-CSF in the culture. G-CSF might be an important factor in the regeneration and differentiation of the ENS and might be a useful tool for the investigation and treatment of ENS disorders.

A Practical Guide for the Diagnosis of Primary Enteric Nervous System Disorders

Primary gastrointestinal neuropathies are a heterogeneous group of enteric nervous system (ENS) disorders that continue to cause difficulties in diagnosis and histological interpretation. Recently, an international working group published guidelines for histological techniques and reporting, along with a classification of gastrointestinal neuromuscular pathology. The aim of this article was to review and summarize the key issues for pediatric gastroenterologists on the diagnostic workup of congenital ENS disorders. In addition, we provide further commentary on the continuing controversies in the field. Although the diagnostic criteria for Hirschsprung disease are well established, those for other forms of dysganglionosis remain ill-defined. Appropriate tissue sampling, handling, and expert interpretation are crucial to maximize diagnostic accuracy and reduce interobserver variability. The absence of validated age-related normal values for neuronal density, along with the lack of correlation between clinical and histological findings, result in significant diagnostic uncertainties while diagnosing quantitative aberrations such as hypoganglionosis or ultrashort Hirschsprung disease. Intestinal neuronal dysplasia remains a histological description of unclear significance. The evaluation of cellular quantitative or qualitative abnormalities of the ENS for clinical diagnosis remains complex. Such analysis should be carried out in laboratories that have the necessary expertise and access to their own validated reference values.

Enteric neural crest-derived cells promote their migration by modifying their microenvironment through tenascin-C production

The enteric nervous system (ENS) is derived from vagal and sacral neural crest cells that migrate, proliferate, and differentiate into enteric neurons and glia within the gut wall. The mechanisms regulating enteric neural crest-derived cell (ENCC) migration are poorly characterized despite the importance of this process in gut formation and function. Characterization of genes involved in ENCC migration is essential to understand ENS development and could provide targets for treatment of human ENS disorders. We identified the extracellular matrix glycoprotein tenascin-C (TNC) as an important regulator of ENCC development. We find TNC dynamically expressed during avian gut development. It is absent from the cecal region just prior to ENCC arrival, but becomes strongly expressed around ENCCs as they enter the ceca and hindgut. In aganglionic hindguts, TNC expression is strong throughout the outer mesenchyme, but is absent from the submucosal region, supporting the presence of both ENCC-dependent and independent expression within the gut wall. Using rat–chick coelomic grafts, neural tube cultures, and gut explants, we show that ENCCs produce TNC and that this ECM protein promotes their migration. Interestingly, only vagal neural crest-derived ENCCs express TNC, whereas sacral neural crest-derived cells do not. These results demonstrate that vagal crest-derived ENCCs actively modify their microenvironment through TNC expression and thereby help to regulate their own migration.

Connective Tissue in Gut Development: A Key Player in Motility and in Intestinal Desmosis

Efficient intestinal peristalsis is a function of intact enteric nervous system, muscle, and connective muscularis propria tissue. Malfunction of any component results in impaired peristalsis. Hirschsprung disease (HD) as prototypic enteric neural migration disorder is increasingly well characterized. More recently, intestinal myopathies and particularly defects of myenteric collagenization have entered the focus of attention. However, detailed development of muscularis propria connective tissue is not well known. The aim of this study was to morphologically characterize intestinal connective tissue in fetal and postnatal development and intestinal pseudo-obstruction. In this study, 130 archival specimens of fetal autopsies, intestinal resections, and biopsies were analyzed. Patients' age was 10th gestational week (gw) to 70 years. Muscularis mucosae, muscle layers, collagen tissue, and enteric plexus were analyzed. Picrosirius red stains, enzyme histochemistry, and immunohistochemistry for collagens I, III, and IV were performed. Total 89 normal intestinal specimens were from fetal autopsies or intestinal resections; 41 patients showed a primary structural colon wall defect (HD, desmosis). Our results showed a constant increase in tunica muscularis propria thickness with age. Separation into circular and longitudinal muscle layer first occurred in the 11th gw. A tendinous collagen plexus layer first arose in the 10th gw and showed a steady caliber increase. Muscularis mucosae first appeared in the 10th gw and grew independent of any primary gastrointestinal disease. In the 11th gw, enteric ganglia were fully developed. In desmosis, a collagen plexus layer was absent. In contrast, in HD, muscularis mucosae showed hypertrophy, but the collagen plexus layer was intact in the aganglionic segment. In intestinal neuronal dysplasia and hypoganglionosis, nerve cell development was disturbed; connective tissue and muscle layers were well developed. Our comprehensive study of intestinal connective tissue development in comparison to neural intestinal wall components in normal and pathological conditions showed that tendinous tissue develops parallel to muscularis propria and arises early in embryogenesis. In enteric nervous system disorders, ganglionic lesions develop independently of impaired collagen network, whereas mucosal biopsies serve for diagnosis of HD, seromuscular biopsies are required to prove desmosis in gastrointestinal dysmotility disorders.