Mathematical and experimental insights into the development of the enteric nervous system and Hirschsprung's disease

The vertebrate enteric nervous system is formed by a rostro-caudally directed invasion of the embryonic gastrointestinal mesenchyme by neural crest cells. Failure to complete this invasion results in the distal intestine lacking intrinsic neurons. This potentially fatal condition is called Hirschsprung's Disease. A mathematical model of cell invasion incorporating cell motility and proliferation of neural crest cells to a carrying capacity predicted invasion outcomes to imagined manipulations, and these manipulations were tested experimentally. Mathematical and experimental results agreed. The results show that the directional invasion is chiefly driven by neural crest cell proliferation. Moreover, this proliferation occurs in a small region at the wavefront of the invading population. These results provide an understanding of why many genes implicated in Hirschsprung's Disease influence neural crest population size. In addition, during in vivo development the underlying gut tissues are growing simultaneously as the neural crest cell invasion proceeds. The interactions between proliferation, motility and gut growth dictate whether or not complete colonization is successful. Mathematical modeling provides insights into the conditions required for complete colonization or a Hirschsprung's-like deficiency. Experimental evidence supports the hypotheses suggested by the modeling.     

Differentiation of the zebrafish enteric nervous system and intestinal smooth muscle

Development of the enteric nervous system is critical for normal functioning of the digestive system. In vertebrates, enteric precursors originate from the neural crest and migrate into the digestive system. Enteric neurons enable the digestive system to sense and respond to local conditions without the need for central nervous system input. Here we describe major steps in differentiation of the zebrafish enteric nervous system. During migration and neural differentiation of enteric precursors, we identify regions of the enteric nervous system in different phases of differentiation. Early in migration, a small group of anterior enteric neurons are first to form. This is followed by an anterior to posterior wave of enteric neural differentiation later in the migratory phase. Enteric precursors continue proliferating and differentiating into the third day of embryogenesis. nNOS neurons form early while serotonin neurons form late toward the end of enteric neural differentiation. Numbers of enteric neurons increase gradually except during periods of circular and longitudinal intestinal smooth muscle differentiation.     

The migratory behavior of immature enteric neurons

While they are migrating caudally along the developing gut, around 10%-20% of enteric neural crest-derived cells start to express pan-neuronal markers and tyrosine hydroxylase (TH). We used explants of gut from embryonic TH-green fluorescence protein (GFP) mice and time-lapse microscopy to examine whether these immature enteric neurons migrate and their mode of migration. In the gut of E10.5 and E11.5 TH-GFP mice, around 50% of immature enteric neurons (GFP(+) cells) migrated, with an average speed of around 15 mum/h. This is slower than the speed at which the population of enteric neural crest-derived cells advances along the developing gut, and hence neuronal differentiation seems to slow, but not necessarily halt, the caudal migration of enteric neural crest cells. Most migrating immature enteric neurons migrated caudally by extending a long-leading process followed by translocation of the cell body. This mode of migration is different from that of non-neuronal enteric neural crest-derived cells and neural crest cells in other locations, but resembles that of migrating neurons in many regions of the developing central nervous system (CNS). In migrating immature enteric neurons, a swelling often preceded the movement of the nucleus in the direction of the leading process. However, the centrosomal marker, pericentrin, was not localized to either the leading process or swelling. This seems to be the first detailed report of neuronal migration in the developing mammalian peripheral nervous system.     

Activity‐dependent secretion of alpha‐synuclein by enteric neurons

There is growing evidence supporting a role of extracellular alpha‐synuclein in the spreading of Parkinson's disease (PD) pathology. Recent pathological studies have raised the possibility that the enteric nervous system (ENS) is one of the initial sites of alpha‐synuclein pathology in PD. We therefore undertook this survey to determine whether alpha‐synuclein can be secreted by enteric neurons. Alpha‐synuclein secretion was assessed by immunoblot analysis of the culture medium from primary culture of ENS. We show that alpha‐synuclein is physiologically secreted by enteric neurons via a conventional, endoplasmic reticulum/Golgi‐dependent exocytosis, in a neuronal activity‐regulated manner. Our study is the first to evidence that enteric neurons are capable of secreting alpha‐synuclein, thereby providing new insights into the role of the ENS in the pathophysiology of PD.     

The α1 subunit of laminin-1 promotes the development of neurons by interacting with LBP110 expressed by neural crest-derived cells immunoselected from the fetal mouse gut

A plasmalemmal protein, LBP110, which binds to the α1 chain of laminin-1, is acquired by the neural crest-derived precursors of enteric neurons after they colonize the gut. We tested the hypothesis that laminin-1 interacts with LBP110 to promote enteric neuronal development. The effects of laminin-1 on neuronal development were studied in cultures of cells immunoselected from fetal mouse gut (E14–15) with antibodies to LBP110 or p75^NTR, a marker for enteric crest—derived cells. No matter which antibody was used, the development of cells expressing neuronal markers was increased three- to fourfold by culturing the cells on a laminin-1—containing substrate. To determine whether this effect of laminin-1 is due to the selective adherence of a neurocompetent subset of precursors, immunoselected cells were permitted to preadhere to poly-D-lysine. Addition of soluble laminin-1 24 h later promoted neuronal but not glial development. The laminin-1 induced increment in neuronal development was abolished both by a peptide containing the sequence of the LBP110-binding domain, IKVAV, and by antibodies to laminin α1 that recognize the IKVAV domain. Neither reagent affected the total number of cells. In contrast, the response to laminin-1 was not affected by control peptides, preimmune sera, or antibodies to laminin β1. Laminin-1 transiently induced the expression of nuclear Fos immunoreactivity; this action was blocked specifically by the IKVAV peptide. These data are consistent with the hypothesis that LBP110 interacts with the IKVAV domain of laminin α1 to promote the differentiation of neurons from enteric crest—derived precursors. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 118–138, 1997     

Different neural crest populations exhibit diverse proliferative behaviors

The rate of proliferation of cells depends on the proportion of cycling cells and the frequency of cell division. Here, we describe in detail methods for quantifying the proliferative behavior of specific cell types in situ, and use the method to examine cell cycle dynamics in two neural crest derivatives—dorsal root ganglia (DRG) using frozen sections, and the enteric nervous system (ENS) using wholemount preparations. In DRG, our data reveal a significant increase in cell cycle length and a decrease in the number of cycling Sox10+ progenitor cells at E12.5–E13.5, which coincides with the commencement of glial cell generation. In the ENS, the vast majority of Sox10+ cells remain proliferative during embryonic development, with only relatively minor changes in cell cycle parameters. Previous studies have identified proliferating cells expressing neuronal markers in the developing ENS; our data suggest that most cells undergoing neuronal differentiation in the developing gut commence expression of neuronal markers during G2 phase of their last division. Combined with previous studies, our findings show that different populations of neural crest‐derived cells show tissue‐specific patterns of proliferation. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 287–301, 2015     

Development of LBP110 expression by neural crest-derived enteric precursors: Migration and differentiation potential in ls/ls mutant mice

Neural crest-derived cells acquire a 110-kD laminin-binding protein (LBP110) when they colonize the murine bowel. Laminin stimulates LBP110-expressing cells to develop as neurons. We have followed the development of LBP110 by neural crest-derived cells as they enter the gut of control and ls/ls mutant mice. The expression of neurofilament and choline acetyltransferase was used as markers of a neuronal phenotype. Tyrosine hydroxylase was used as a marker for the mash-1-dependent lineage of enteric precursors, while calcitonin gene-related peptide was used as a marker for the mash-1-independent lineage of crest-derived cells. A subset of cells expressing LBP110 was located along the vagi at E10 at cervical and thoracic levels. At E12, cells expressing LBP110 extended from the foregut to the midgut. The expression of neurofilament protein lagged behind that of LBP110 by about 0.5 day and then became coincident with LBP110 immunoreactivity. By E15, cells doubly labeled with antibodies to LBP110 and neurofilament protein were located along the entire extent of the bowel up to but not including the terminal colon. By E16, both the proximal and terminal colon contained cells expressing LBP110 and neurofilaments. The pattern of immunoreactivity could not be distinguished between ls/ls and control animals prior to E16. By E16, when the terminal colon of control animals contained many cells expressing LBP110 and neurofilaments, the terminal colon of ls/ls animals lacked cells expressing these proteins; nevertheless, structures outside of the terminal colon were heavily endowed with cells expressing LBP110 and neurofilaments. These ectopically located cells derived from both mash-1-dependent and -independent lineages of crest-derived precursors. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 341–354, 1998     

Development of interstitial cells of Cajal in the human digestive tract as the result of reciprocal induction of mesenchymal and neural crest cells

Neural crest cells (NCC) can migrate into different parts of the body and express their strong inductive potential. In addition, they are multipotent and are able to differentiate into various cell types with diverse functions. In the primitive gut, NCC induce differentiation of muscular structures and interstitial cells of Cajal (ICC), and they themselves differentiate into the elements of the enteric nervous system (ENS), neurons and glial cells. ICC develop by way of mesenchymal cell differentiation in the outer parts of the primitive gut wall around the myenteric plexus (MP) ganglia, with the exception of colon, where they appear simultaneously also at the submucosal border of the circular muscular layer around the submucosal plexus (SMP) ganglia. However, in a complex process of reciprocal induction of NCC and local mesenchyma, c‐kit positive precursors are the first to differentiate, representing probably the common precursors of ICC and smooth muscle cells (SMC). C‐kit positive precursors could represent a key impact factor regarding the final differentiation of NCC into neurons and glial cells with neurons subsequently excreting stem cell factor (SCF) and other signalling molecules. Under the impact of SCF, a portion of c‐kit positive precursors lying immediately around the ganglia differentiate into ICC, while the rest differentiate into SMC.     

Inhibition of protein kinase A in murine enteric neurons causes lethal intestinal pseudo‐obstruction

A number of in vitro studies suggest that many important developmental and functional events in the enteric nervous system are regulated by the intracellular signaling enzyme cAMP protein kinase A (PKA). To evaluate the in vivo significance of these observations, a Cre‐inducible, dominant‐negative, mutant regulatory subunit (RIαB) of PKA was activated in enteric neurons by either a Proteolipid protein‐Cre transgene or a Hox11L1‐Cre “knock‐in” allele. In both models, RIαB activation resulted consistently in profound distension of the proximal small intestine within 2 weeks after birth. Intestinal transit of radio‐opaque tracers was severely retarded in the double‐transgenic animals, which died shortly after weaning. In the enteric nervous system, recombination was restricted to neurons as demonstrated by histochemical analysis and confocal microscopic colocalization of a Cre recombinase‐dependent reporter gene with the neuronal marker Hu(C/D), in contrast with the glial marker S100. Histochemical analysis of β‐galactosidase expression and acetylcholinesterase activity, as well as neuronal counts, demonstrated that intestinal dysmotility was not associated with obvious malformation of the myenteric plexus. However, inhibition of PKA activity in enteric neurons disrupted the major motor complexes of isolated intestinal segments in vitro. These results provide strong evidence that PKA activity plays a critical role in enteric neurotransmission in vivo, and highlight neuronal PKA or related signaling molecules as potential therapeutic targets in gastrointestinal motility disorders. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2006     

From neural crest to bowel: Development of the enteric nervous system

The ENS resembles the brain and differs both physiologically and structurally from any other region of the PNS. Recent experiments in which crest cell migration has been studied with DiI, a replication-deficient retrovirus, or antibodies that label cells of neural crest origin, have confirmed that both the avian and mammalian bowel are colonized by émigrés from the sacral as well as the vagal level of the neural crest. Components of the extracellular matrix, such as laminin, may play roles in enteric neural and glial development. The observation that an overabundance of laminin develops in the presumptive aganglionic region of the gut in Is/Is mutant mice and is associated with the inability of crest-derived cells to colonize this region of the bowel has led to the hypothesis that laminin promotes the development of crest-derived cells as enteric neurons. Premature expression of a neuronal phenotype would cause crest-derived cells to cease migrating before they complete the colonization of the gut. The acquisition by crest-derived cells of a nonintegrin, nervespecific, 110 kD laminin-binding protein when they enter the bowel may enable these cells to respond to laminin differently from their pre-enteric migrating predecessors. Crest-derived cells migrating along the vagal pathway to the mammalian gut are transiently catecholaminergic (TC). This phenotype appears to be lost rapidly as the cells enter the bowel and begin to follow their program of terminal differentiation. The appearance and disappearance of TC cells may thus be an example of the effects of the enteric microenvironment on the differentiation of crest-derived cells in situ. Crest-derived cells can be isolated from the enteric microenvironment by immunoselection, a method that takes advantage of the selective expression on the surfaces of crest-derived cells of certain antigens. One neurotrophin, NT-3, promotes the development of enteric neurons and glia in vitro. Because trkC is expressed in the developing and mature gut, it seems likely that NT-3 plays a critical role in the development of the ENS in situ. Although the factors that are responsible for the development of the unique properties of the ENS remain unknown, progress made in understanding enteric neuronal development has recently accelerated. The application of new techniques and recently developed probes suggest that the accelerated pace of discovery in this area can be expected to continue. © 1993 John Wiley & Sons, Inc.     

hPSC-derived sacral neural crest enables rescue in a severe model of Hirschsprung’s disease

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BMP signaling is necessary for neural crest cell migration and ganglion formation in the enteric nervous system

The enteric nervous system (ENS) is derived from neural crest cells that migrate along the gastrointestinal tract to form a network of neurons and glia that are essential for regulating intestinal motility. Despite the number of genes known to play essential roles in ENS development, the molecular etiology of congenital disorders affecting this process remains largely unknown. To determine the role of bone morphogenetic protein (BMP) signaling in ENS development, we first examined the expression of bmp2, bmp4, and bmprII during hindgut development and find these strongly expressed in the ENS. Moreover, functional BMP signaling, demonstrated by the expression of phosphorylated Smad1/5/8, is present in the enteric ganglia. Inhibition of BMP activity by noggin misexpression within the developing gut, both in ovo and in vitro, inhibits normal migration of enteric neural crest cells. BMP inhibition also leads to hypoganglionosis and failure of enteric ganglion formation, with crest cells unable to cluster into aggregates. Abnormalities of migration and ganglion formation are the hallmarks of two human intestinal disorders, Hirschsprung's disease and intestinal neuronal dysplasia. Our results support an essential role for BMP signaling in these aspects of ENS development and provide a basis for further investigation of these proteins in the etiology of neuro-intestinal disorders.     

Dual embryonic origin of the mammalian enteric nervous system

The enteric nervous system is thought to originate solely from the neural crest. Transgenic lineage tracing revealed a novel population of clonal pancreatic duodenal homeobox-1 (Pdx1)-Cre lineage progenitor cells in the tunica muscularis of the gut that produced pancreatic descendants as well as neurons upon differentiation in vitro. Additionally, an in vivo subpopulation of endoderm lineage enteric neurons, but not glial cells, was seen especially in the proximal gut. Analysis of early transgenic embryos revealed Pdx1-Cre progeny (as well as Sox-17-Cre and Foxa2-Cre progeny) migrating from the developing pancreas and duodenum at E11.5 and contributing to the enteric nervous system. These results show that the mammalian enteric nervous system arises from both the neural crest and the endoderm. Moreover, in adult mice there are separate Wnt1-Cre neural crest stem cells and Pdx1-Cre pancreatic progenitors within the muscle layer of the gut.     

The enteric nervous system relays psychological stress to intestinal inflammation

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Endothelial cells promote migration and proliferation of enteric neural crest cells via β1 integrin signaling

Enteric neural crest-derived cells (ENCCs) migrate along the intestine to form a highly organized network of ganglia that comprises the enteric nervous system (ENS). The signals driving the migration and patterning of these cells are largely unknown. Examining the spatiotemporal development of the intestinal neurovasculature in avian embryos, we find endothelial cells (ECs) present in the gut prior to the arrival of migrating ENCCs. These ECs are patterned in concentric rings that are predictive of the positioning of later arriving crest-derived cells, leading us to hypothesize that blood vessels may serve as a substrate to guide ENCC migration. Immunohistochemistry at multiple stages during ENS development reveals that ENCCs are positioned adjacent to vessels as they colonize the gut. A similar close anatomic relationship between vessels and enteric neurons was observed in zebrafish larvae. When EC development is inhibited in cultured avian intestine, ENCC migration is arrested and distal aganglionosis results, suggesting that ENCCs require the presence of vessels to colonize the gut. Neural tube and avian midgut were explanted onto a variety of substrates, including components of the extracellular matrix and various cell types, such as fibroblasts, smooth muscle cells, and endothelial cells. We find that crest-derived cells from both the neural tube and the midgut migrate avidly onto cultured endothelial cells. This EC-induced migration is inhibited by the presence of CSAT antibody, which blocks binding to β1 integrins expressed on the surface of crest-derived cells. These results demonstrate that ECs provide a substrate for the migration of ENCCs via an interaction between β1 integrins on the ENCC surface and extracellular matrix proteins expressed by the intestinal vasculature. These interactions may play an important role in guiding migration and patterning in the developing ENS.