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Introduction: Parenteral nutrition (PN) is a necessary therapy used to feed patients with gastrointestinal dysfunction. Unfortunately, PN results in intestinal atrophy and changes to host immune function. PN may also induce additional effects on gut motility that we hypothesized would result from changes in the enteric nervous system., Methods: Mice received an intravenous (i.v.) catheter and were randomized to chow (n = 5), i.v. PN (n = 6), or i.v. PN + bombesin (BBS, 15 μg/kg, 3×/d) (n = 6) for 5 d. Colons were removed and dissected to measure the length and circumference. Enteric neuronal density and neurotransmitter expression were determined by co-immunostaining whole-mount tissue with Hu and neuronal nitric oxide synthase (nNOS)., Results: The number of myenteric neurons expressing Hu and nNOS increased per unit length in the mid-colon during PN treatment compared with chow. This increase was abrogated by the addition of BBS to the PN regimen. However, the percentage of nNOS-expressing neurons was not significantly altered by PN. Morphometric analysis revealed a decrease in the length and circumference of the colon during PN administration that was partially normalized by supplementation of PN with BBS. A significant reduction in total fecal output was observed in PN animals compared with chow and was increased by mice receiving BBS in addition to PN., Conclusions: PN causes a constriction of the bowel wall, reducing not only the length but also the circumference of the colon. These changes cause a condensation of enteric neurons but no difference in neurotransmitter expression. BBS supplementation partially restores the constriction and increases the fecal output during PN treatment compared with PN treatment alone., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Hirschsprung disease (HSCR) is a human congenital disorder, defined by the absence of ganglia from variable lengths of the colon. These ganglia comprise the enteric nervous system (ENS) and are derived from migratory neural crest cells (NCCs). The inheritance of HSCR is complex, often non-Mendelian and characterized by variable penetrance. Although extensive research has identified many key players in the pathogenesis of Hirschsprung disease, a large number of cases remain genetically undefined. Therefore, additional unidentified genes or modifiers must contribute to the etiology and pathogenesis of Hirschsprung disease. We have discovered that Tcof1 may be one such modifier. Haploinsufficiency of Tcof1 in mice results in a reduction of vagal NCCs and their delayed migration along the length of the gut during early development. This alone, however, is not sufficient to cause colonic aganglionosis as alterations in the balance of NCC proliferation and differentiation ensures NCC colonize the entire length of the gut of Tcof1(+/-) mice by E18.5. In contrast, Tcof1 haploinsufficiency is able to sensitize Pax3(+/-) mice to colonic aganglionosis. Although, Pax3 heterozygous mice do not show ENS defects, compound Pax3;Tcof1 heterozygous mice exhibit cumulative apoptosis which severely reduces the NCC population that migrates into the foregut. In addition, the proliferative capacity of these NCC is also diminished. Taken together with the opposing effects of Pax3 and Tcof1 on NCC differentiation, the synergistic haploinsufficiency of Tcof1 and Pax3 results in colonic aganglionosis in mice and may contribute to the pathogenesis of Hirschsprung disease.

The enteric nervous system (ENS) comprises a complex neuronal network that regulates peristalsis of the gut wall and secretions into the lumen. The ENS is formed from a multipotent progenitor cell population called the neural crest, which is derived from the neuroepithelium. Neural crest cells (NCCs) migrate over incredible distances to colonize the entire length of the gut and during their migration they must survive, proliferate and ultimately differentiate. The absence of an ENS from variable lengths of the colon results in Hirschsprung's disease (HSCR) or colonic aganglionosis. Mutations in about 12 different genes have been identified in HSCR patients but the complex pattern of inheritance and variable penetrance suggests that additional genes or modifiers must be involved in the etiology and pathogenesis of this disease. We discovered that Tcof1 haploinsufficiency in mice models many of the early features of HSCR. Neuroepithelial apoptosis diminished the size of the neural stem cell pool resulting in reduced NCC numbers and their delayed migration along the gut from E10.5 to E14.5. Surprisingly however, we observe continued and complete colonization of the entire colon throughout E14.5-E18.5, a period in which the gut is considered to be non- or less-permissive to NCC. Thus, we reveal for the first time that reduced NCC progenitor numbers and delayed migration do not unequivocally equate with a predisposition for the pathogenesis of HSCR. In fact, these deficiencies can be overcome by balancing NCC intrinsic processes of proliferation and differentiation with extrinsic influences of the gut microenvironment.

Background & Aims: Enteric nervous system stem cells (ENSSCs) provide potential therapeutic tools to replenish absent ganglia in Hirschsprung's disease. Although full-thickness human postnatal gut tissue can be used to generate ENSSCs, reliance on its harvesting from surgical resection poses significant practical limitations. This study aimed to explore whether gut tissue obtained utilizing minimally invasive routine endoscopy techniques could be used to generate ENSSCs and whether such cells retain the potential to generate an ENS upon transplantation into aganglionic gut., Methods: Postnatal human gut mucosal tissue obtained from children undergoing gastrointestinal endoscopy was used to generate cell cultures in which ENSSCs were contained within neurosphere-like bodies (NLBs). These NLBs were characterized by immunostaining, and their potential to generate components of the ENS, in vitro and upon transplantation into models of aganglionic gut, was examined., Results: Gut mucosal biopsy specimens were obtained from 75 children (age, 9 months-17 years). The biopsy specimens contained neural cells and ENSSCs and, on culturing, generated characteristic NLBs at all ages examined. Postnatal mucosa-derived NLBs contained cells that, akin to their embryonic counterparts, were proliferating, expressed ENSSC markers, were bipotent, and capable of generating large colonies in clonogenic cultures and multiple ENS neuronal subtypes. Upon transplantation, cells from NLBs colonized cultured recipient aganglionic chick and human hindgut to generate ganglia-like structures and enteric neurons and glia., Conclusions: The results represent a significant practical advance toward the development of definitive cell replenishment therapies for ENS disorders such as Hirschsprung's disease.

The enteric nervous system (ENS) is mainly derived from vagal neural crest cells (NCC) that arise at the level of somites 1-7. To understand how the size and composition of the NCC progenitor pool affects ENS development, we reduced the number of NCC by ablating the neural tube adjacent to somites 3-6 to produce aganglionic gut. We then back-transplanted various somite lengths of quail neural tube into the ablated region to determine the 'tipping point', whereby sufficient progenitors were available for complete ENS formation. The addition of one somite length of either vagal, sacral or trunk neural tube into embryos that had the neural tube ablated adjacent to somites 3-6, resulted in ENS formation along the entire gut. Although these additional cells contributed to the progenitor pool, the quail NCC from different axial levels retained their intrinsic identities with respect to their ability to form the ENS; vagal NCC formed most of the ENS, sacral NCC contributed a limited number of ENS cells, and trunk NCC did not contribute to the ENS. As one somite length of vagal NCC was found to comprise almost the entire ENS, we ablated all of the vagal neural crest and back-transplanted one somite length of vagal neural tube from the level of somite 1 or somite 3 into the vagal region at the position of somite 3. NCC from somite 3 formed the ENS along the entire gut, whereas NCC from somite 1 did not. Intrinsic differences, such as an increased capacity for proliferation, as demonstrated in vitro and in vivo, appear to underlie the ability of somite 3 NCC to form the entire ENS.

The formation of neural tissue, in association with airway smooth muscle (ASM), is a feature of normal lung development and function. Intrinsic neuronal tissue has recently been shown, in animal models, to be derived from neural crest cells (NCC). Since defects in NCC development underlie a range of disease states (neurocristopathies), it is important to determine the spatiotemporal development of NCC in the human lung, as defects in their development could have pathophysiologic implications. The aims of this study were to: (1) establish a time course for the formation of ASM and neural tissue within the embryonic and fetal human lung, (2) investigate whether intrinsic neural tissue within the lung is derived from NCC, and (3) gain insight into the possible signaling mechanisms underlying the development of the intrinsic lung innervation. Using human lung tissue from Weeks 6 to 12 of gestation, we analyzed the formation of ASM, NCC, neuronal and glial tissue, and the expression of Gfralpha1, a receptor component of the RET (rearranged during transfection) tyrosine kinase signaling pathway. Our results showed that NCC accumulated along the branching airways, in close association with the ASM, and differentiated into neurons and glia. Neural crest-derived neural tissue within the lung strongly expressed membrane-bound Gfralpha1, and soluble Gfralpha1 was expressed within the lung mesenchyme, but only at early developmental stages. Together these findings indicate that the intrinsic innervation of the human lung is derived from the neural crest.

The enteric nervous system (ENS) is formed from vagal and sacral neural crest cells (NCC). Vagal NCC give rise to most of the ENS along the entire gut, whereas the contribution of sacral NCC is mainly limited to the hindgut. This, and data from heterotopic quail-chick grafting studies, suggests that vagal and sacral NCC have intrinsic differences in their ability to colonize the gut, and/or to respond to signalling cues within the gut environment. To better understand the molecular basis of these differences, we studied the expression of genes known to be essential for ENS formation, in sacral NCC within the chick hindgut. Our results demonstrate that, as in vagal NCC, Sox10, EdnrB, and Ret are expressed in sacral NCC within the gut. Since we did not detect a qualitative difference in expression of these ENS genes we performed DNA microarray analysis of vagal and sacral NCC. Of 11 key ENS genes examined from the total data set, Ret was the only gene identified as being highly differentially expressed, with a fourfold increase in expression in vagal versus sacral NCC. We also found that over-expression of RET in sacral NCC increased their ENS developmental potential such that larger numbers of cells entered the gut earlier in development, thus promoting the fate of sacral NCC towards that of vagal NCC.

The transcriptional regulator SOX10 and the signalling molecule endothelin 3 have important roles in the development of the mammalian enteric nervous system (ENS). Using a clonal cell culture system, we show that SOX10 inhibits overt neuronal and glial differentiation of multilineage ENS progenitor cells (EPCs), without interfering with their neurogenic commitment. We also demonstrate that endothelin 3 inhibits reversibly the commitment and differentiation of EPCs along the neurogenic and gliogenic lineages, suggesting a role for this factor in the maintenance of multilineage ENS progenitors. Consistent with such a role, the proportion of Sox10-expressing progenitors in the total population of enteric neural crest cells is reduced in the gut of endothelin 3-deficient embryos. This reduction may be related to the requirement of endothelin signalling for the proliferation of ENS progenitors. The dependence of ENS progenitors on endothelin 3 is more pronounced at the migratory front of enteric neural crest cells, which is associated with relatively high levels of endothelin 3 mRNA. Our findings indicate that SOX10 and endothelin 3 have a crucial role in the maintenance of multilineage enteric nervous system progenitors.

The enteric nervous system (ENS) in vertebrates is derived mainly from vagal neural crest cells that enter the foregut and colonize the entire wall of the gastrointestinal tract. Failure to completely colonize the gut results in the absence of enteric ganglia (Hirschsprung's disease). Two signaling systems mediated by RET and EDNRB have been identified as critical players in enteric neurogenesis. We demonstrate that interaction between these signaling pathways controls ENS development throughout the intestine. Activation of EDNRB specifically enhances the effect of RET signaling on the proliferation of uncommitted ENS progenitors. In addition, we reveal novel antagonistic roles of these pathways on the migration of ENS progenitors. Protein kinase A is a key component of the molecular mechanisms that integrate signaling by the two receptors. Our data provide strong evidence that the coordinate and balanced interaction between receptor tyrosine kinases and G protein-coupled receptors controls the development of the nervous system in mammals.

Background/Aims: Hepatic stellate cells (HSC) have been hypothesised to derive from the neural crest, based on their expression of multiple neural/neuroendocrine features and their contacts with autonomic nerve endings. Methods: We studied the emergence of HSC in the liver during embryonic development in a transgenic mouse line expressing yellow fluorescent protein (YFP) in all neural crest cells and their derivatives. Cellular YFP expression in these mice was compared with desmin expression between embryonic day (E) 11.5 and adulthood. Results: YFP was abundantly expressed in neural crest cells delaminating from the neural tube and in all known neural crest-derived structures and cell populations. In particular, YFP expressing cells perfectly mimicked the spatial and temporal pattern of enteric nervous system development from neural crest cells migrating from the postotic region. Cells within the adrenal medulla were also YFP positive. Analysis of the liver showed that desmin-expressing, stellate-shaped, perisinusoidally located HSC were evident from E11.5 onwards. However, no detectable YFP expression was seen in the developing liver or in HSC, from E11.5 until adulthood. Conclusions: These findings suggest HSC do not descend from the neural crest, and therefore may derive from the septum transversum mesenchyme, from endoderm or from the mesothelial liver capsule. [Copyright &y& Elsevier]