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.     

In ovo transplantation of enteric nervous system precursors from vagal to sacral neural crest results in extensive hindgut colonisation

The enteric nervous system (ENS) is derived from vagal and sacral neural crest cells (NCC). Within the embryonic avian gut, vagal NCC migrate in a rostrocaudal direction to form the majority of neurons and glia along the entire length of the gastrointestinal tract, whereas sacral NCC migrate in an opposing caudorostral direction, initially forming the nerve of Remak, and contribute a smaller number of ENS cells primarily to the distal hindgut. In this study, we have investigated the ability of vagal NCC, transplanted to the sacral region of the neuraxis, to colonise the chick hindgut and form the ENS in an experimentally generated hypoganglionic hindgut in ovo model. Results showed that when the vagal NC was transplanted into the sacral region of the neuraxis, vagal-derived ENS precursors immediately migrated away from the neural tube along characteristic pathways, with numerous cells colonising the gut mesenchyme by embryonic day (E) 4. By E7, the colorectum was extensively colonised by transplanted vagal NCC and the migration front had advanced caudorostrally to the level of the umbilicus. By E10, the stage at which sacral NCC begin to colonise the hindgut in large numbers, myenteric and submucosal plexuses in the hindgut almost entirely composed of transplanted vagal NCC, while the migration front had progressed into the pre-umbilical intestine, midway between the stomach and umbilicus. Immunohistochemical staining with the pan-neuronal marker, ANNA-1, revealed that the transplanted vagal NCC differentiated into enteric neurons, and whole-mount staining with NADPH-diaphorase showed that myenteric and submucosal ganglia formed interconnecting plexuses, similar to control animals. Furthermore, using an anti-RET antibody, widespread immunostaining was observed throughout the ENS, within a subpopulation of sacral NC-derived ENS precursors, and in the majority of transplanted vagal-to-sacral NCC. Our results demonstrate that: (1) a cell autonomous difference exists between the migration/signalling mechanisms used by sacral and vagal NCC, as transplanted vagal cells migrated along pathways normally followed by sacral cells, but did so in much larger numbers, earlier in development; (2) vagal NCC transplanted into the sacral neuraxis extensively colonised the hindgut, migrated in a caudorostral direction, differentiated into neuronal phenotypes, and formed enteric plexuses; (3) RET immunostaining occurred in vagal crest-derived ENS cells, the nerve of Remak and a subpopulation of sacral NCC within hindgut enteric ganglia.     

Vital dye analysis of cranial neural crest cell migration in the mouse embryo.

The spatial and temporal aspects of cranial neural crest cell migration in the mouse are poorly understood because of technical limitations. No reliable cell markers are available and vital staining of embryos in culture has had limited success because they develop normally for only 24 hours. Here, we circumvent these problems by combining vital dye labelling with exo utero embryological techniques. To define better the nature of cranial neural crest cell migration in the mouse embryo, premigratory cranial neural crest cells were labelled by injecting DiI into the amniotic cavity on embryonic day 8. Embryos, allowed to develop an additional 1 to 5 days exo utero in the mother before analysis, showed distinct and characteristic patterns of cranial neural crest cell migration at the different axial levels. Neural crest cells arising at the level of the forebrain migrated ventrally in a contiguous stream through the mesenchyme between the eye and the diencephalon. In the region of the midbrain, the cells migrated ventrolaterally as dispersed cells through the mesenchyme bordered by the lateral surface of the mesencephalon and the ectoderm. At the level of the hindbrain, neural crest cells migrated ventrolaterally in three subectodermal streams that were segmentally distributed. Each stream extended from the dorsal portion of the neural tube into the distal portion of the adjacent branchial arch. The order in which cranial neural crest cells populate their derivatives was determined by labelling embryos at different stages of development. Cranial neural crest cells populated their derivatives in a ventral-to-dorsal order, similar to the pattern observed at trunk levels. In order to confirm and extend the findings obtained with exo utero embryos, DiI (1,1-dioctadecyl-3,3,3',3'-tetramethylindo-carbocyanine perchlorate) was applied focally to the neural folds of embryos, which were then cultured for 24 hours. Because the culture technique permitted increased control of the timing and location of the DiI injection, it was possible to determine the duration of cranial neural crest cell emigration from the neural tube. Cranial neural crest cell emigration from the neural folds was completed by the 11-somite stage in the region of the rostral hindbrain, the 14-somite stage in the regions of the midbrain and caudal hindbrain and not until the 16-somite stage in the region of the forebrain. At each level, the time between the earliest and latest neural crest cells to emigrate from the neural tube appeared to be 9 hours.(ABSTRACT TRUNCATED AT 400 WORDS)     

Endothelin-3 regulates neural crest cell proliferation and differentiation in the hindgut enteric nervous system

Neural crest cells (NCC) migrate, proliferate, and differentiate within the wall of the gastrointestinal tract to give rise to the neurons and glial cells of the enteric nervous system (ENS). The intestinal microenvironment is critical in this process and endothelin-3 (ET3) is known to have an essential role. Mutations of this gene cause distal intestinal aganglionosis in rodents, but its mechanism of action is poorly understood. We find that inhibition of ET3 signaling in cultured avian intestine also leads to hindgut aganglionosis. The aim of this study was to determine the role of ET3 during formation of the avian hindgut ENS. To answer this question, we created chick-quail intestinal chimeras by transplanting preganglionic quail hindguts into the coelomic cavity of chick embryos. The quail grafts develop two ganglionated plexuses of differentiated neurons and glial cells originating entirely from the host neural crest. The presence of excess ET3 in the grafts results in a significant increase in ganglion cell number, while inhibition of endothelin receptor-B (EDNRB) leads to severe hypoganglionosis. The ET3-induced hyperganglionosis is associated with an increase in enteric crest cell proliferation. Using hindgut explants cultured in collagen gel, we find that ET3 also inhibits neuronal differentiation in the ENS. Finally, ET3, which is strongly expressed in the ceca, inhibits the chemoattraction of NCC to glial-derived neurotrophic factor (GDNF). Our results demonstrate multiple roles for ET3 signaling during ENS development in the avian hindgut, where it influences NCC proliferation, differentiation, and migration.     

Pathways of trunk neural crest cell migration in the mouse embryo as revealed by vital dye labelling

Analysis of neural crest cell migration in the mouse has been difficult due to the lack of reliable cell markers. Recently, we found that injection of DiI into the chick neural tube marks premigratory neural crest cells whose endfeet are in contact with the lumen of the neural tube (Serbedzija et al. Development 106, 809-819 (1989)). In the present study, this technique was applied to study neural crest cell migratory pathways in the trunk of the mouse embryo. Embryos were removed from the mother between the 8th and the 10th days of development and DiI was injected into the lumen of the neural tube. The embryos were then cultured for 12 to 24 h, and analyzed at the level of the forelimb. We observed two predominant pathways of neural crest cell migration: (1) a ventral pathway through the rostral portion of the somite and (2) a dorsolateral pathway between the dermamyotome and the epidermis. Neural crest cells were observed along the dorsolateral pathway throughout the period of migration. The distribution of labelled cells along the ventral pathway suggested that there were two overlapping phases of migration. An early ventrolateral phase began before E9 and ended by E9.5; this pathway consisted of a stream of cells within the rostral sclerotome, adjacent to the dermamyotome, that extended ventrally to the region of the sympathetic ganglia and the dorsal aorta.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Critical numbers of neural crest cells are required in the pathways from the neural tube to the foregut to ensure complete enteric nervous system formation

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.     

PTEN基因对早期胚胎神经嵴细胞迁移的作用研究

目的 初步探讨PTEN基因在早期神经嵴细胞迁移中的作用.方法 首先胚胎整体的原位杂交和免疫荧光方法检测鸡胚胎内源性的PTEN基因及蛋白水平的表达情况;其次,利用鸡胚胎体内半侧神经管转染的方法,使神经管一侧PTEN基因过表达,对侧神经管为正常对照侧;最后,通过Pax7的整体胚胎免疫荧光表达观察PTEN基因对其标记的部分神经嵴细胞迁移的影响.结果 内源性PTEN基因在mRNA和蛋白水平表达显示,其在早期胚胎HH4期的神经板即开始明显的表达;通过半侧过表达PTEN基因后观察到过表达PTEN基因侧的头部神经嵴细胞迁移与对照侧相比明显受到抑制,但对躯干部的影响并不明显.结论 PTEN基因可能抑制早期胚胎头部神经嵴细胞的迁移.     

Specification of neural crest cell formation and migration in mouse embryos

Of all the model organisms used to study human development, rodents such as mice most accurately reflect human craniofacial development. Collective advances in mouse embryology and mouse genetics continue to shape our understanding of neural crest cell development and by extrapolation the etiology of human congenital head and facial birth defects. The aim of this review is to highlight the considerable progress being made in our understanding of cranial neural crest cell patterning in mouse embryos.     

Primary culture of chick, mouse or human neural crest cells

A highly enriched population of neural crest cells (NCCs) from amniote embryos, such as from chicks, mice and humans, is desirable for experiments in fate determination. NCCs are also useful for testing the functional effects of molecular changes underlying numerous human diseases of neural crest derivatives and for investigating their potential for therapeutic compensation. This protocol details embryonic microdissection followed by neural tube explantation. Conditions favoring NCC expansion and the maintenance of their stem cell-like properties are described. Although neural crest-like cells can be derived from a number of sites in the mature organism, full potential is best ensured by their purification from their source tissue at the outset of migration. Going from embryo to established cell line takes 4 d; the first is the most labor-intensive day, but minimal intervention is required thereafter.     

The neural crest: basic biology and clinical relationships in the craniofacial and enteric nervous systems

The highly migratory, mesenchymal neural crest cell population was discovered over 100 years ago. Proposals of these cells' origin within the neuroepithelium, and of the tissues they gave rise to, initiated decades-long heated debates, since these proposals challenged the powerful germ-layer theory. Having survived this storm, the neural crest is now regarded as a pluripotent stem cell population that makes vital contributions to an astounding array of both neural and non-neural organ systems. The earliest model systems for studying the neural crest were amphibian, and these pioneering contributions have been ably refined and extended by studies in the chick, mouse, and more recently the fish to provide detailed understanding of the cellular and molecular mechanisms regulating and regulated by the neural crest. The key questions regarding control of craniofacial morphogenesis and innervation of the gut illustrate the wide range of developmental contexts in which the neural crest plays an important role. These questions also focus attention on common issues such as the role of growth factor signaling in neural crest cell development and highlight the central role of the neural crest in human congenital disease.     

The migratory pathway of neural crest cells into the skin of mouse embryos

The migration of neural crest derived melanoblasts into the skin of mice was studied by the ectoderm-mesoderm recombination technique. Dorsolateral skin from albino and black mouse embryos at the time of initial melanoblast invasion was separated into ectoderm and mesoderm components, recombined with each other, and grown in the chick embryo coelom for a sufficient period to allow melanin formation. Recombined skin from embryos 11 days old formed pigment only when the mesodermal component was from a genetically black embryo. The black ectoderm-albino mesoderm recombinations failed to produce pigment in all cases. At this critical age when melanoblasts were first entering the skin, they were present exclusively in the mesodermal component. Skin recombinations made from 12-day mouse embryos showed a spread of melanoblasts into the ectodermal component, and by 13 and 14 days both dermal mesoderm and epidermal ectoderm were populated by melanoblasts.     

Migration of neural crest-derived enteric nervous system precursor cells to and within the gastrointestinal tract

The enteric nervous system, the intrinsic innervation of the gastrointestinal tract, consists of large numbers of phenotypically diverse neurons and glial cells, arranged in complex interconnecting plexuses situated between the smooth muscle layers of the gut wall. Recently, the enteric nervous system has attracted much attention from developmental biologists whose efforts have focused on analysing the cellular origins of enteric nervous system precursor cells, how these cells migrate to and within the gut and the identification of signalling mechanisms which cause migrating cells to differentiate into the appropriate phenotypes in the appropriate locations. This review summarises the state of knowledge concerning the early stages of enteric nervous system development and concentrates on: (i) the embryological origins of the neural crest cells which colonise the gastrointestinal tract, (ii) their spatiotemporal migration within the gut, (iii) the possible pre-specification of neural crest cells as enteric nervous system precursors and (iv) factors influencing their directional migration within the gut.     

Cell division in the ciliary ganglion of quail embryos in situ and after back-transplantation into the neural crest migration pathways of chick embryos

Embryonic 4- to 15-day-old quail ciliary ganglia (CG) were grafted into the neural crest migration pathway of 2-day-old chick embryos at the adrenomedullary level of the neural axis. This back-transplantation results in dispersion of cells of the implanted ganglion, their migration in the host embryo, and subsequent promotion of their differentiation into a variety of neural-crest-derived cell types including adrenergic cells of the sympathetic ganglia and adrenal medulla. These cells can be recognized in the host through the nuclear marker that they carry. Here, we have analyzed quantitatively the expansion of CG-derived cell population after the graft, and compared cell division in CG after back-transplantation and during normal in situ development over the same period of time. Tritiated-thymidine [( 3H]TdR) incorporation showed that grafted CG cells proliferated during their migration and, to a greater extent, after they had homed to the host structures. Furthermore, proliferative activity of quail cells in the graft was found to be significantly higher than the growth rate of the CG cells in situ during the same period of development. In the quail donor embryo, the birthdate of the CG neurons occurred early in development; from 6 days onward, only nonneuronal cells were still dividing. When back-transplanted, the 4- to 5-day-old CG provided numerous quail cells located in autonomic structures of the host embryo. However, this increase of the total quail cell population and of cell division was reduced when CG were taken from quail donors at progressively later developmental stages. Postmitotic neurons from mature CG were found not to survive under the graft conditions. It is proposed that back-transplantation of the CG stimulates cell division and modifies the developmental programme of still undifferentiated precursor cells which then can give rise to a variety of cell types belonging either to the glial or the autonomic nerve and paraganglionic cell phenotypes, to the exclusion of sensory neurons which never derive from CG grafts.     

Intrinsic differences among spatially distinct neural crest stem cells in terms of migratory properties, fate determination, and ability to colonize the enteric nervous system

We have systematically examined the developmental potential of neural crest stem cells from the enteric nervous system (gut NCSCs) in vivo to evaluate their potential use in cellular therapy for Hirschsprung disease and to assess differences in the properties of postmigratory NCSCs from different regions of the developing peripheral nervous system (PNS). When transplanted into developing chicks, flow-cytometrically purified gut NCSCs and sciatic nerve NCSCs exhibited intrinsic differences in migratory potential and neurogenic capacity throughout the developing PNS. Most strikingly, gut NCSCs migrated into the developing gut and formed enteric neurons, while sciatic nerve NCSCs failed to migrate into the gut or to make enteric neurons, even when transplanted into the gut wall. Enteric potential is therefore not a general property of NCSCs. Gut NCSCs also formed cholinergic neurons in parasympathetic ganglia, but rarely formed noradrenergic sympathetic neurons or sensory neurons. Supporting the potential for autologous transplants in Hirschsprung disease, we observed that Endothelin receptor B (Ednrb)-deficient gut NCSCs engrafted and formed neurons as efficiently in the Ednrb-deficient hindgut as did wild-type NCSCs. These results demonstrate intrinsic differences in the migratory properties and developmental potentials of regionally distinct NCSCs, indicating that it is critical to match the physiological properties of neural stem cells to the goals of proposed cell therapies.