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Background: Cells derived from the neural crest colonize the developing gut and give rise to the enteric nervous system. The rate at which the ENCC population advances along the bowel will be affected by both the speed and directionality of individual ENCCs. The aim of the study was to use time-lapse imaging and pharmacological activators and inhibitors to examine the role of several intracellular signalling pathways in both the speed and the directionality of individual enteric neural crest-derived cells in intact explants of E12.5 mouse gut. Drugs that activate or inhibit intracellular components proposed to be involved in GDNF-RET and EDN3-ETB signalling in ENCCs were used., Findings: Pharmacological inhibition of JNK significantly reduced ENCC speed but did not affect ENCC directionality. MEK inhibition did not affect ENCC speed or directionality. Pharmacological activation of adenylyl cyclase or PKA (a downstream cAMP-dependent kinase) resulted in a significant decrease in ENCC speed and an increase in caudal directionality of ENCCs. In addition, adenylyl cyclase activation also resulted in reduced cell-cell contact between ENCCs, however this was not observed following PKA activation, suggesting that the effects of cAMP on adhesion are not mediated by PKA., Conclusions: JNK is required for normal ENCC migration speed, but not directionality, while cAMP signalling appears to regulate ENCC migration speed, directionality and adhesion. Collectively, our data demonstrate that intracellular signalling pathways can differentially affect the speed and directionality of migrating ENCCs., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Time-lapse imaging of gut explants from embryonic mice in which neural crest-derived cells express fluorescent proteins allows the behavior of enteric neural crest cells to be observed and analyzed. Explants of embryonic gut are dissected, mounted on filter paper supports so the gut retains its tubular three-dimensional structure, and then placed in coverglass bottom culture dishes in tissue culture medium. A stainless steel ring is placed on top of the filter support to prevent movement. Imaging is performed using a confocal microscope in an environmental chamber. A z series of images through the network of fluorescent cells is collected every 3, 5, or 10 min. At the end of imaging, the z series are projected.

Goldberg-Shprintzen syndrome is a poorly understood condition characterized by learning difficulties, facial dysmorphism, microcephaly, and Hirschsprung disease. GOSHS is due to recessive mutations in KIAA1279, which encodes kinesin family member 1 binding protein (KIF1BP, also known as KBP). We examined the effects of inactivation of Kif1bp in mice. Mice lacking Kif1bp died shortly after birth, and exhibited smaller brains, olfactory bulbs and anterior commissures, and defects in the vagal and sympathetic innervation of the gut. Kif1bp was found to interact with Ret to regulate the development of the vagal innervation of the stomach. Although newborn Kif1bp -/- mice had neurons along the entire bowel, the colonization of the gut by neural crest-derived cells was delayed. The data show an essential in vivo role for KIF1BP in axon extension from some neurons, and the reduced size of the olfactory bulb also suggests additional roles for KIF1BP. Our mouse model provides a valuable resource to understand GOSHS.

The enteric nervous system (ENS) is an extensive network of neurons in the gut wall that arises from neural crest-derived cells. Like other populations of neural crest cells, it is known that enteric neural crest-derived cells (ENCCs) influence the behaviour of each other and therefore must communicate. However, little is known about how ENCCs communicate with each other. In this study, we used Ca 2+ imaging to examine communication between ENCCs in the embryonic gut, using mice where ENCCs express a genetically-encoded calcium indicator. Spontaneous propagating calcium waves were observed between neighbouring ENCCs, through both neuronal and non-neuronal ENCCs. Pharmacological experiments showed wave propagation was not mediated by gap junctions, but by purinergic signalling via P2 receptors. The expression of several P2X and P2Y receptors was confirmed using RT-PCR. Furthermore, inhibition of P2 receptors altered the morphology of the ENCC network, without affecting neuronal differentiation or ENCC proliferation. It is well established that purines participate in synaptic transmission in the mature ENS. Our results describe, for the first time, purinergic signalling between ENCCs during pre-natal development, which plays roles in the propagation of Ca 2+ waves between ENCCs and in ENCC network formation. One previous study has shown that calcium signalling plays a role in sympathetic ganglia formation; our results suggest that calcium waves are likely to be important for enteric ganglia development., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Cell therapy is a promising approach to generate an enteric nervous system (ENS) and treat enteric neuropathies. However, for translation to the clinic, it is highly likely that enteric neural progenitors will require manipulation prior to transplantation to enhance their ability to migrate and generate an ENS. In this study, we examine the effects of exposure to several factors on the ability of ENS progenitors, grown as enteric neurospheres, to migrate and generate an ENS. Exposure to glial-cell-line-derived neurotrophic factor (GDNF) resulted in a 14-fold increase in neurosphere volume and a 12-fold increase in cell number. Following co-culture with embryonic gut or transplantation into the colon of postnatal mice in vivo, cells derived from GDNF-treated neurospheres showed a 2-fold increase in the distance migrated compared with controls. Our data show that the ability of enteric neurospheres to generate an ENS can be enhanced by exposure to appropriate factors., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Gut motility disorders can result from an absent, damaged, or dysfunctional enteric nervous system (ENS). Cell therapy is an exciting prospect to treat these enteric neuropathies and restore gut motility. Previous studies have examined a variety of sources of stem/progenitor cells, but the ability of different sources of cells to generate enteric neurons has not been directly compared. It is important to identify the source of stem/progenitor cells that is best at colonizing the bowel and generating neurons following transplantation. The aim of this study was to compare the ability of central nervous system (CNS) progenitors and ENS progenitors to colonize the colon and differentiate into neurons. Genetically labeled CNS- and ENS-derived progenitors were cocultured with aneural explants of embryonic mouse colon for 1 or 2.5 wk to assess their migratory, proliferative, and differentiation capacities, and survival, in the embryonic gut environment. Both progenitor cell populations were transplanted in the postnatal colon of mice in vivo for 4 wk before they were analyzed for migration and differentiation using immunohistochemistry. ENS-derived progenitors migrated further than CNS-derived cells in both embryonic and postnatal gut environments. ENS-derived progenitors also gave rise to more neurons than their CNS-derived counterparts. Furthermore, neurons derived from ENS progenitors clustered together in ganglia, whereas CNS-derived neurons were mostly solitary. We conclude that, within the gut environment, ENS-derived progenitors show superior migration, proliferation, and neuronal differentiation compared with CNS progenitors., (Copyright © 2014 the American Physiological Society.)

Background: Directed cell migration is essential for normal development. In most of the migratory cell populations that have been analyzed in detail to date, all of the cells migrate as a collective from one location to another. However, there are also migratory cell populations that must populate the areas through which they migrate, and thus some cells get left behind while others advance. Very little is known about how individual cells behave to achieve concomitant directional migration and population of the migratory route. We examined the behavior of enteric neural crest-derived cells (ENCCs), which must both advance caudally to reach the anal end and populate each gut region., Results: The behavior of individual ENCCs was examined using live imaging and mice in which ENCCs express a photoconvertible protein. We show that individual ENCCs exhibit very variable directionalities and speed; as the migratory wavefront of ENCCs advances caudally, each gut region is populated primarily by some ENCCs migrating non-directionally. After populating each region, ENCCs remain migratory for at least 24 hours. Endothelin receptor type B (EDNRB) signaling is known to be essential for the normal advance of the ENCC population. We now show that perturbation of EDNRB principally affects individual ENCC speed rather than directionality. The trajectories of solitary ENCCs, which occur transiently at the wavefront, were consistent with an unbiased random walk and so cell-cell contact is essential for directional migration. ENCCs migrate in close association with neurites. We showed that although ENCCs often use neurites as substrates, ENCCs lead the way, neurites are not required for chain formation and neurite growth is more directional than the migration of ENCCs as a whole., Conclusions: Each gut region is initially populated by sub-populations of ENCCs migrating non-directionally, rather than stopping. This might provide a mechanism for ensuring a uniform density of ENCCs along the growing gut.

There are many different types of enteric neurons. Previous studies have identified the time at which some enteric neuron subtypes are born (exit the cell cycle) in the mouse, but the birthdates of some major enteric neuron subtypes are still incompletely characterized or unknown. We combined 5-ethynynl-2'-deoxyuridine (EdU) labeling with antibody markers that identify myenteric neuron subtypes to determine when neuron subtypes are born in the mouse small intestine. We found that different neurochemical classes of enteric neuron differed in their birthdates; serotonin neurons were born first with peak cell cycle exit at E11.5, followed by neurofilament-M neurons, calcitonin gene-related peptide neurons (peak cell cycle exit for both at embryonic day [E]12.5-E13.5), tyrosine hydroxylase neurons (E15.5), nitric oxide synthase 1 (NOS1) neurons (E15.5), and calretinin neurons (postnatal day [P]0). The vast majority of myenteric neurons had exited the cell cycle by P10. We did not observe any EdU+/NOS1+ myenteric neurons in the small intestine of adult mice following EdU injection at E10.5 or E11.5, which was unexpected, as previous studies have shown that NOS1 neurons are present in E11.5 mice. Studies using the proliferation marker Ki67 revealed that very few NOS1 neurons in the E11.5 and E12.5 gut were proliferating. However, Cre-lox-based genetic fate-mapping revealed a small subpopulation of myenteric neurons that appears to express NOS1 only transiently. Together, our results confirm a relationship between enteric neuron subtype and birthdate, and suggest that some enteric neurons exhibit neurochemical phenotypes during development that are different from their mature phenotype., (Copyright © 2013 Wiley Periodicals, Inc.)

Cell therapy has the potential to treat gastrointestinal motility disorders caused by diseases of the enteric nervous system. Many studies have demonstrated that various stem/progenitor cells can give rise to functional neurons in the embryonic gut; however, it is not yet known whether transplanted neural progenitor cells can migrate, proliferate, and generate functional neurons in the postnatal bowel in vivo. We transplanted neurospheres generated from fetal and postnatal intestinal neural crest-derived cells into the colon of postnatal mice. The neurosphere-derived cells migrated, proliferated, and generated neurons and glial cells that formed ganglion-like clusters within the recipient colon. Graft-derived neurons exhibited morphological, neurochemical, and electrophysiological characteristics similar to those of enteric neurons; they received synaptic inputs; and their neurites projected to muscle layers and the enteric ganglia of the recipient mice. These findings show that transplanted enteric neural progenitor cells can generate functional enteric neurons in the postnatal bowel and advances the notion that cell therapy is a promising strategy for enteric neuropathies.

In mature animals, neurons and interstitial cells of Cajal (ICC) are essential for organized intestinal motility. We investigated motility patterns, and the roles of neurons and myenteric ICC (ICC-MP), in the duodenum and colon of developing mice in vitro. Spatiotemporal mapping revealed regular contractions that propagated in both directions from embryonic day (E)13.5 in the duodenum and E14.5 in the colon. The propagating contractions, which we termed ripples, were unaffected by tetrodotoxin and were present in the intestine of embryonic Ret null mutant mice, which lack enteric neurons. Neurally mediated motility patterns were first observed in the duodenum at E18.5. To examine the possible role of ICC-MP, three approaches were used. First, intracellular recordings from the circular muscle of the duodenum did not detect slow wave activity at E16.5, but regular slow waves were observed in some preparations of E18.5 duodenum. Second, spatiotemporal mapping revealed ripples in the duodenum of E13.5 and E16.5 W/W(v) embryos, which lack KIT+ ICC-MP and slow waves. Third, KIT-immunoreactive cells with the morphology of ICC-MP were first observed at E18.5. Hence, ripples do not appear to be mediated by ICC-MP and must be myogenic. Ripples in the duodenum and colon were abolished by cobalt chloride (1 mm). The L-type Ca(2+) channel antagonist nicardipine (2.5 microm) abolished ripples in the duodenum and reduced their frequency and size in the colon. Our findings demonstrate that prominent propagating contractions (ripples) are present in the duodenum and colon of fetal mice. Ripples are not mediated by neurons or ICC-MP, but entry of extracellular Ca(2+) through L-type Ca(2+) channels is essential. Thus, during development of the intestine, the first motor patterns to develop are myogenic.

Neural crest (NC) cells are stem cells that are specified within the embryonic neuroectodermal epithelium and migrate to stereotyped peripheral sites for differentiation into many cell types. Several neurocristopathies involve a deficit of NC-derived cells, raising the possibility of stem cell therapy. In Hirschsprung's disease the distal bowel lacks an enteric nervous system caused by a failure of colonization by NC-derived cells. We have developed a robust method of producing migrating NC-like cells from human embryonic stem cell-derived neural progenitors using a coculture system of mouse embryonic fibroblasts. Significantly, subsequent exposure to Y27632, a small-molecule inhibitor of the Rho effectors ROCKI/II, dramatically increased the efficiency of differentiation into NC-like cells, identified by marker expression in vitro. NC-like cells derived by this method were able to migrate along NC pathways in avian embryos in ovo and within explants of murine bowel, and to differentiate into cells with neuronal and glial markers. This is the first study to report the use of a small molecule to induce cells with NC characteristics from embryonic stem cells that can migrate and generate neurons and support cells in complex tissue. Furthermore, this study demonstrates that small-molecule regulators of ROCKI/II signaling may be valuable tools for stem cell research aimed at treatment of neurocristopathies.

Mutations in genes encoding members of the GDNF and endothelin-3 (Et-3) signaling pathways can cause Hirschsprung's disease, a congenital condition associated with an absence of enteric neurons in the distal gut. GDNF signals through Ret, a receptor tyrosine kinase, and Et-3 signals through endothelin receptor B (Ednrb). The effects of Gdnf, Ret, and ET-3 haploinsufficiency and a null mutation in ET-3 on spontaneous motility patterns in adult and developing mice were investigated. Video recordings were used to construct spatiotemporal maps of spontaneous contractile patterns in colon from postnatal and adult mice in vitro. In Ret(+/-) and ET-3(+/-) mice, which have normal numbers of enteric neurons, colonic migrating motor complexes (CMMCs) displayed similar properties under control conditions and following inhibition of nitric oxide synthase (NOS) activity to wild-type mice. In the colon of Gdnf(+/-) mice and in the ganglionic region of ET-3(-/-) mice, there was a 50-60% reduction in myenteric neuron number. In Gdnf(+/-) mice, CMMCs were present, but abnormal, and the proportion of myenteric neurons containing NOS was not different from that of wild-type mice. In the ganglionic region of postnatal ET-3(-/-) mice, CMMCs were absent, and the proportion of myenteric neurons containing NOS was over 100% higher than in wild-type mice. Thus impairments in spontaneous motility patterns in the colon of Gdnf(+/-) mice and in the ganglionic region of ET-3(-/-) mice are correlated with a reduction in myenteric neuron density.

The enteric nervous system arises from vagal (caudal hindbrain) and sacral level neural crest-derived cells that migrate into and along the developing gut. Data from previous studies have suggested that (i) there may be gradients along the gut that induce the caudally directed migration of vagal enteric neural precursors (ENPs), (ii) exposure to the caecum might alter the migratory ability of vagal ENPs and (iii) Sema3A might regulate the entry into the hindgut of ENPs derived from sacral neural crest. Using co-cultures we show that there is no detectable gradient of chemoattractive molecules along the pre-caecal gut that specifically promotes the caudally directed migration of vagal ENPs, although vagal ENPs migrate faster caudally than rostrally along explants of hindgut. Exposure to the caecum did not alter the rate at which ENPs colonized explants of hindgut, but it did alter the ability of ENPs to colonize the midgut. The co-cultures also revealed that there is localized expression of a repulsive cue in the distal hindgut, which might delay the entry of sacral ENPs. We show that Sema3A is expressed by the hindgut mesenchyme and its receptor, neuropilin-1, is expressed by migrating ENPs. Furthermore, there is premature entry of sacral ENPs and extrinsic axons into the distal hindgut of fetal mice lacking Sema3A. These data show that Sema3A expressed by the distal hindgut regulates the entry of sacral ENPs and extrinsic axons into the hindgut. ENPs did not express neuropilin-2 and there was no detectable change in the timetable by which ENPs colonize the gut in mice lacking neuropilin-2.

The enteric nervous system arises predominantly from vagal level neural crest cells that migrate into the foregut and then colonize the entire length of the gastrointestinal tract. Previous studies have demonstrated that glial cell line-derived neurotrophic factor (GDNF) promotes the migration of enteric neural crest-derived cells (ENCs) in vitro, but a role for GDNF in the migration of ENCs in vivo has yet to be demonstrated. In this study, the effects of Gdnf haploinsufficiency on ENC rate of migration and number during mid embryonic development were examined. Although the entire gut of embryonic Gdnf(+/-) mice was colonized, a significant delay in the migration of ENCs along the embryonic hindgut was found. However, significant effects of Gdnf haploinsufficiency on ENC number were detected before the stage at which migration defects were first evident. As previous studies have shown a relationship between ENC number and migration, the effects of Gdnf haploinsufficiency on migration may be due to an indirect effect on cell number and/or a direct effect of GDNF on ENC migration. Gdnf haploinsufficiency did not cause any detectable change in the rate of neuronal differentiation of ENCs.

Neural crest cells that originate in the caudal hindbrain migrate into and along the developing gastrointestinal tract to form the enteric nervous system. While they are migrating, neural-crest-derived cells are also proliferating. Previous studies have shown that the expression of glial-derived neurotrophic factor (GDNF) and endothelin-3 is highest in the embryonic caecum, and that GDNF alone or in combination with endothelin-3 promotes the proliferation of enteric neural-crest-derived cells in vitro. However, whether neural proliferative zones, like those in the central nervous system, are found along the developing gut is unknown. We used a fluorescent nucleic acid stain to identify dividing cells or BrdU labelling (2 h after administration of BrdU to the mother), combined with antibodies specific to neural crest cells to determine the percentage of proliferating crest-derived cells in various gut regions of embryonic day 11.5 (E11.5) and E12.5 mice. The rate of proliferation of crest-derived cells did not vary significantly in different regions of the gut (including the caecum) or at different distances from the migratory wavefront of vagal crest-derived cells. The phenotype of mitotic enteric crest-derived cells was also examined. Cells expressing the pan-neuronal markers, neurofilament-M and Hu, or the glial marker, S100b, were observed undergoing mitosis. However, no evidence was found for proliferation of cells expressing neuron-type-specific markers, such as nitric oxide synthase (at E12.5) or calcitonin gene-related peptide (at E18.5). Thus, for enteric neurons, exit from the cell cycle appears to occur after the expression of pan-neuronal proteins but prior to the expression of markers of terminally differentiated neurons.

Glial cell line-derived neurotrophic factor (GDNF) is expressed in the gastrointestinal tract of the developing mouse and appears to play an important role in the migration of enteric neuron precursors into and along the small and large intestines. Two other GDNF family members, neurturin and artemin, are also expressed in the developing gut although artemin is only expressed in the esophagus. We examined the effects of GDNF, neurturin, and artemin on neural crest cell migration and neurite outgrowth in explants of mouse esophagus, midgut, and hindgut. Both GDNF and neurturin induced neural crest cell migration and neurite outgrowth in all regions examined. In the esophagus, the effect of GDNF on migration and neurite outgrowth declined with age between E11.5 and E14.5, but neurturin still had a strong neurite outgrowth effect at E14.5. Artemin did not promote neural migration or neurite outgrowth in any region investigated. The effects of GDNF family ligands are mediated by the Ret tyrosine kinase. We examined the density of neurons in the esophagus of Ret-/- mice, which lack neurons in the small and large intestines. The density of esophageal neurons in Ret-/- mice was only about 4% of the density of esophageal neurons in Ret+/- and Ret+/+ mice. These results show that GDNF and neurturin promote migration and neurite outgrowth of crest-derived cells in the esophagus as well as the intestine. Moreover, like intestinal neurons, the development of esophageal neurons is largely Ret-dependent.

Neural crest-derived cells that form the enteric nervous system undergo an extensive migration from the caudal hindbrain to colonize the entire gastrointestinal tract. Mice in which the expression of GFP is under the control of the Ret promoter were used to visualize neural crest-derived cell migration in the embryonic mouse gut in organ culture. Time-lapse imaging revealed that GFP(+) crest-derived cells formed chains that displayed complicated patterns of migration, with sudden and frequent changes in migratory speed and trajectories. Some of the leading cells and their processes formed a scaffold along which later cells migrated. To examine the effect of population size on migratory behavior, a small number of the most caudal GFP(+) cells were isolated from the remainder of the population. The isolated cells migrated slower than cells in large control populations, suggesting that migratory behavior is influenced by cell number and cell-cell contact. Previous studies have shown that neurons differentiate among the migrating cell population, but it is unclear whether they migrate. The phenotype of migrating cells was examined. Migrating cells expressed the neural crest cell marker, Sox10, but not neuronal markers, indicating that the majority of migratory cells observed did not have a neuronal phenotype.

Enteric neurons and glia arise from the neural crest. The phenotype of crest-derived cells was examined as they differentiated into neurons or glia in the mouse small and large intestine. Previous studies have shown that undifferentiated enteric crest-derived cells are Phox2b(+)/Ret(+)/p75(+)/Sox10(+), and at embryonic day (E) 10.5, about 10-15% of the crest-derived cells in the small intestine have started to differentiate into neurons. In the current study, by E12.5 and E14.5, about 25% and 47%, respectively, of Phox2b(+) cells in the small intestine were immunoreactive to the pan-neuronal protein, ubitquitin hydrolase (PGP9.5), and the percentage did not change dramatically from E14.5 onward. The differentiation of crest-derived cells into neurons in the colon lagged behind that in the small intestine by several days. Differentiating enteric neurons showed high Ret, low p75, and undetectable Sox10 immunostaining. Glial precursors were identified by the presence of brain-specific fatty acid binding protein (B-FABP) and detected first in the fore- and rostral midgut at E11.5. Glial precursors appeared to be B-FABP(+)/Sox10(+)/p75(+) but showed low Ret immunostaining. S100b was not detected until E14.5. Adult glial cells were B-FABP(+)/Sox10(+)/p75(+)/S100b(+). A nucleic acid stain (to identify all ganglion cells) was combined with immunostaining for PGP9.5 and S100b to detect neurons and glial cells, respectively, in the postnatal intestine. At postnatal day 0, fewer than 5% and 10% of cells in myenteric ganglia of the small and large intestine, respectively, were neither PGP9.5(+) nor S100b(+). Because some classes of neurons are not present in significant numbers until after birth, the expression of PGP9.5 by developing enteric neurons appeared to precede the expression of neuron type-specific markers., (Copyright 2002 Wiley-Liss, Inc.)

As neurones develop they are faced with choices as to which genes to express, to match their final phenotype to their role in the nervous system. A number of processes can guide these decisions. Within the autonomic and sensory nervous systems, there are a handful of examples that suggest that one mechanism that may match phenotype to function is the presence of target-derived differentiation factors. We tested whether the rat pineal gland controls the expression of a neuropeptide (neuropeptide Y) and a calcium-binding protein (calbindin) in sympathetic postganglionic neurones that innervate it. We first showed that the chemical phenotype of sympathetic neurones innervating the rat pineal includes the expression of both neuropeptide Y and the calcium-binding protein, calbindin. After transplanting the pineal gland of neonatal rats into the submandibular salivary gland of neonatal hosts, it was innervated by sympathetic axons from the surrounding salivary gland tissue, which do not normally express neuropeptide Y and calbindin. The presence of the pineal gland led to the appearance of neuropeptide Y and calbindin in many of the postganglionic neurones that innervated the graft. From these findings we suggest that, like the rodent sweat gland, the pineal gland generates a signal that can direct the neurochemical phenotype of innervating sympathetic neurones.

Autonomic sympathetic postganglionic neurons normally express distinct combinations of neuropeptides which are often highly correlated with the projection of the neurons. When sympathetic postganglionic neurons are axotomized, they can express quite different neuropeptides, notably substance P, vasoactive intestinal peptide or galanin. In this study, we have examined rat sympathetic postganglionic neurons in the superior cervical ganglion that project to the skin, the vasculature of the skeletal muscle or to the submandibular salivary gland, and assessed whether the neuropeptides that they express after axotomy depend on which target tissue they previously innervated. In all three populations, around half of the postganglionic neurons expressed galanin after axotomy. In contrast, only skin-projecting neurons showed a significant increase in the number of neurons that expressed substance P (22%) and vasoactive intestinal peptide (17%) following axotomy. Within the skin-projecting neurons, as judged on the basis of cell body size, substance P and vasoactive intestinal peptide were expressed predominantly in pilomotor neurons, but only rarely were the two neuropeptides present in the same nerve cell body. In conclusion, we have demonstrated that three different neuropeptides, which can be induced by axotomy in postganglionic neurons, follow quite different patterns of expression when they are viewed in relation to the function of the postganglionic neurons in the superior cervical ganglion.