Activin receptor-like kinase 2 can mediate atrioventricular cushion transformation

The development of enteric and sympathetic neurons from neural crest precursor cells is regulated by signals produced by the embryonic environments to which the cells migrate. Bone morphogenetic proteins (BMPs) are present in the developing embryo and act to induce neuronal differentiation and noradrenergic properties of neural crest cells. We have investigated the role of BMP2 in regulating the appearance of distinct populations of autonomic neurons from postmigratory, HNK-1-positive neural crest precursor cells. BMP2 promotes neuronal differentiation of sympathetic and enteric precursor cells isolated from E14.5 rat. The effects of BMP2 change over time, resulting in a decrease in neuron number that can be attributed to apoptotic cell death. BMP2-dependent neuron death is rescued by gut-derived factors that provide trophic support to maturing neurons, indicating that BMP2 regulates the acquisition of trophic dependence of developing peripheral neurons. In addition to regulating neuron number, BMP2 promotes both panneuronal maturation and the acquisition of an enteric phenotype, as measured by lineage-specific changes in the expression of tyrosine hydroxylase and MASH-1. While BMP2 is sufficient to induce neuronal differentiation and panneuronal development, these results suggest that additional factors in the environment must collaborate with BMP2 to promote the final noradrenergic phenotype of sympathetic neurons.     

In vivo transplantation of mammalian neural crest cells into chick hosts reveals a new autonomic sublineage restriction.

The study of mammalian neural crest development has been limited by the lack of an accessible system for in vivo transplantation of these cells. We have developed a novel transplantation system to study lineage restriction in the rodent neural crest. Migratory rat neural crest cells (NCCs), transplanted into chicken embryos, can differentiate into sensory, sympathetic, and parasympathetic neurons, as shown by the expression of neuronal subtype-specific and pan-neuronal markers, as well as into Schwann cells and satellite glia. In contrast, an immunopurified population of enteric neural precursors (ENPs) from the fetal gut can also generate neurons in all of these ganglia, but only expresses appropriate neuronal subtype markers in Remak's and associated pelvic parasympathetic ganglia. ENPs also appear restricted in the kinds of glia they can generate in comparison to NCCs. Thus ENPs have parasympathetic and presumably enteric capacities, but not sympathetic or sensory capacities. These results identify a new autonomic lineage restriction in the neural crest, and suggest that this restriction preceeds the choice between neuronal and glial fates.     

Restriction of developmental potential during divergence of the enteric and sympathetic neuronal lineages.

In the peripheral nervous system, enteric and sympathetic neurons develop from multipotent neural crest cells. While local environmental signals in the gut and in the region of the sympathetic ganglia play a role in the choice of cell fate, little is known about the mechanisms that underlie restriction to specific neuronal phenotypes. We investigated the divergence and restriction of the enteric and sympathetic neuronal lineages using immuno-isolated neural crest-derived cells from the gut and sympathetic ganglia. Analysis of neuronal and lineage-specific mRNAs and proteins indicated that neural crest-derived cells from the gut and sympathetic ganglia had initiated neuronal differentiation and phenotypic divergence by E14.5 in the rat. We investigated the developmental potential of these cells using expression of tyrosine hydroxylase as a marker for a sympathetic phenotype. Tyrosine hydroxylase expression was examined in neurons that developed from sympathetic and enteric neuroblasts under the following culture conditions: culture alone; coculture with gut monolayers to promote enteric differentiation; or coculture with dorsal aorta monolayers to promote noradrenergic differentiation. Both enteric and sympathetic neuroblasts displayed developmental plasticity at E14.5. Sympathetic neuroblasts downregulated tyrosine hydroxylase in response to signals from the gut environment and enteric neuroblasts increased expression of tyrosine hydroxylase when grown on dorsal aorta or in the absence of other cell types. Tracking of individual sympathetic cells displaying a neuronal morphology at the time of plating indicated that neuroblasts retained phenotypic plasticity even after initial neuronal differentiation had occurred. By E19.5 both enteric and sympathetic neuroblasts had undergone a significant loss of their developmental potential, with most neuroblasts retaining their lineage-specific phenotype in all environments tested. Together our data indicate that the developmental potential of enteric and sympathetic neuroblasts becomes restricted over time and that this restriction takes place not as a consequence of initial neuronal differentiation but during the period of neuronal maturation. Further, we have characterized a default pathway of adrenergic differentiation in the enteric nervous system and have defined a transient requirement for gut-derived factors in the maintenance of the enteric neuronal phenotype.     

The sympathoadrenal lineage in avian embryos. II. Effects of glucocorticoids on cultured neural crest cells

The neural crest-derived precursors of the sympathoadrenal lineage depend on environmental cues to differentiate as sympathetic neurons and pheochromocytes. We have used the monoclonal antibody A2B5 as a marker for neuronal differentiation and antisera against catecholamine synthesis enzymes to investigate the differentiation of catecholaminergic cells in cultures of quail neural crest cells. Cells corresponding phenotypically to sympathetic neurons and pheochromocytes can be identified in neural crest cell cultures after 5-6 days in vitro. Expression of the A2B5 antigen precedes expression of immunocytochemically detectable levels of tyrosine hydroxylase in cultured neural crest cells. Glucocorticoid treatment decreases the proportion of TH+ neural crest cells that express neuronal traits. We conclude that environmental cues normally encountered by sympathoadrenal precursors in vivo can influence the differentiation of a subpopulation of cultured neural crest cells in the sympathoadrenal lineage.     

Sequential specification of neurons and glia by developmentally regulated extracellular factors

Cortical progenitor cells give rise to neurons during embryonic development and to glia after birth. While lineage studies indicate that multipotent progenitor cells are capable of generating both neurons and glia, the role of extracellular signals in regulating the sequential differentiation of these cells is poorly understood. To investigate how factors in the developing cortex might influence cell fate, we developed a cortical slice overlay assay in which cortical progenitor cells are cultured over cortical slices from different developmental stages. We find that embryonic cortical progenitors cultured over embryonic cortical slices differentiate into neurons and those cultured over postnatal cortical slices differentiate into glia, suggesting that the fate of embryonic progenitors can be influenced by developmentally regulated signals. In contrast, postnatal progenitor cells differentiate into glial cells when cultured over either embryonic or postnatal cortical slices. Clonal analysis indicates that the postnatal cortex produces a diffusible factor that induces progenitor cells to adopt glial fates at the expense of neuronal fates. The effects of the postnatal cortical signals on glial cell differentiation are mimicked by FGF2 and CNTF, which induce glial fate specification and terminal glial differentiation respectively. These observations indicate that cell fate specification and terminal differentiation can be independently regulated and suggest that the sequential generation of neurons and glia in the cortex is regulated by a developmental increase in gliogenic signals.     

ADAM17 Is Critical for Multipolar Exit and Radial Migration of Neuronal Intermediate Progenitor Cells in Mice Cerebral Cortex

The radial migration of neuronal progenitor cells is critical for the development of cerebral cortex layers. They go through a critical step transforming from multipolar to bipolar before outward migration. A Disintegrin and Metalloprotease 17 (ADAM17) is a transmembrane protease which can process many substrates involved in cell-cell interaction, including Notch, ligands of EGFR, and some cell adhesion molecules. In this study, we used in utero electroporation to knock down or overexpress ADAM17 at embryonic day 14.5 (E14.5) in neuronal progenitor cells to examine the role of ADAM17 in cortical embryonic neurogenesis. Our results showed that the radial migration of ADAM17-knocked down cells were normal till E16.5 and reached the intermediate zone (IZ). Then most transfected cells stopped migration and stayed at the IZ to inner cortical plate (CP) layer at E18.5, and there was higher percentage of multipolar cells at IZ layer in the ADAM17-knocked down group compared to the cells in control group. Marker staining revealed that those ADAM17-knocked down cells differentiated normally from neural stem cells (NSCs) to neuronal intermediate progenitor cells (nIPCs) but did not differentiate into mature neurons. The migration and multipolar exit defects caused by ADAM17 knockdown could be partially rescued by over-expressing an shRNA resistant ADAM17, while overexpressing ADAM17 alone did not affect the radial migration. Taken together, our results showed for the first time that, ADAM17 is critical in regulating the multipolar-stage exit and radial migration of the nIPCs during telencephalon cortex development in mice.     

MAH Cells and the Development of Rat Sympathetic Neurons

The generation of cell lines in the sympathoadrenal lineage has greatly facilitated our understanding of how precursor cells that do not respond to NGF give rise to mature NGF-dependent neurons. The neuronal developmental pathway in this lineage has been worked out by studying both primary precursor cells in culture and the v-myc-immortalized MAH cell line. MAH cells were established by retroviral infection of immunoisolated rat sympathoadrenal precursor cells. These cells have many of the characteristics of primary progenitor cells including neural precursor morphology, antigenic profile, and response to growth factors. MAH cells are able to recapitulate sympathetic development, giving rise to mature, postmitotic, NGF-dependent neurons. These cells have provided a model system for studying the factors, receptors, and modulating influences that play a role in the development of sympathetic neurons.     

中脑神经前体细胞的体外分离、培养和特性

为了解中脑神经前体细胞的体外培养特性和建立中脑神经前体细胞的体外分化调控机制提供细胞模型。本实验采用含有丝分裂源表皮生长因子(EGF)的无血清培养基培养来源于大鼠胚胎E14.5天的中脑神经前体细胞,应用免疫细胞化学方法了解其前体细胞特性。结果发现中脑神经前体细胞呈神经前体细胞特征性标记Nestin免疫染色阳性,无分化细胞标记;细胞克隆实验证实中脑神经前体细 胞有自我更新能力;在EGF刺激下增殖迅速;当撤去EGF后置于含胎牛血清的培养基和被覆多聚赖氨酸(PLL)的培养皿内,中脑神经前体细胞可分化成神经元和星形胶质细胞。本试验证明我们培养的中脑神经前体细胞具有增殖、自我更新能力和多向分化潜能特性。     

Neurofilament expression in vagal neural crest-derived precursors of enteric neurons

In order to gain insight into the potential role of the enteric microenvironment in the neuronal determination of the neural crest-derived precursor cells of enteric neurons, an attempt was made to ascertain when and where along the migratory route of these cells that they first express neuronal properties. The immunocytochemical detection of the 160-kDa component of the triplet of the chick neurofilament peptides served as a neuronal marker. In addition, neurogenic potential was assessed by growing explants of tissue suspected of containing presumptive neuroblasts in culture or as grafts on the chorioallantoic membrane of chick embryonic hosts. Neurofilament immunoreactivity was first detected in the foregut by Day 4 of development and spread to the hindgut by Day 7. Within the hindgut, development was more advanced within the colorectum than within the more proximal terminal ileum and caecal appendages. This probably reflects the distal-proximal migration of sacral neural crest cells in the postumbilical bowel. The ability of enteric explants to show neuronal development in vitro correlated with whether or not cells containing neurofilament immunoreactivity had reached that segment of gut at the age of explantation. These data suggest that enteric neuronal precursors have already begun to differentiate as neurons by the time they colonize the gut. Prior to the appearance of fibrillar neurofilament immunoreactivity in the foregut, cells that express this marker were found transiently within the mesenchyme of branchial arches 3, 4, and 5. These cells had disappeared from this region by developmental Day 6. The neurogenic potential of branchial arches 3 and 4 was demonstrated by the correlation that was found between the ability of explants of these arches to show neuronal development in vitro and the presence within them of cells that display neurofilament immunoreactivity. No similar neurogenic potential was found in the more rostral branchial arches which lacked the masses of neurofilament-immunoreactive cells. The location of the caudal branchial arches below the migrating vagal neural crest, the transience of the neurofilament immunoreactivity in them, and the coincident transience of their neurogenic potential in vitro, suggested that the masses of neurofilament immunoreactive cells in the caudal branchial arches might be vagal neural crest-derived neuronal precursor cells en route to the pharynx and the rest of the gut. This possibility was supported by the observation of neurofilament immunoreactivity in a subset of cells of the premigratory and early migratory neural crest in the vagal, but not other, regions of the neuraxis prior to the appearance of neurofilament immunoreactivity in the branchial arches. Proliferative expansion of cells with neurofilament immunoreactivity was indicated by the observation of mitotic figures in them. It is suggested that the vagal neural crest cells that populate the ENS are already committed to the neuronal lineage while still in the vagal region of the neuraxis. It is therefore not likely that the enteric microenvironment plays a role in this process.     

A v-myc-immortalized sympathoadrenal progenitor cell line in which neuronal differentiation is initiated by FGF but not NGF

Sympathetic neurons differentiate from a developmentally restricted progenitor cell in the neural crest-derived sympathoadrenal lineage. We have isolated these progenitors by fluorescence-activated cell sorting and immortalized them using a v-myc-containing retrovirus. The complement of antigenic markers expressed by these lines suggests that they have retained many of the properties of their normal counterparts. These lines initiate neuronal differentiation in response to basic FGF, but not to NGF, and do not contain NGF receptor mRNA. In NGF plus FGF, however, a small percentage of the cells differentiate to NGF-dependent postmitotic neurons. Furthermore, an induction of NGF receptor mRNA can be observed in response to FGF. Thus, the development of sympathetic neurons may involve a relay, in which FGF both initiates differentiation and induces the NGF receptor, which in turn controls further maturation and survival.     

Embryonic stem cell-derived neural progenitors as non-tumorigenic source for dopaminergic neurons

AIM:To find a safe source for dopaminergic neurons,we generated neural progenitor cell lines from human embryonic stem cells.METHODS:The human embryonic stem(hES)cell line H9 was used to generate human neural progenitor(HNP)cell lines.The resulting HNP cell lines were differentiated into dopaminergic neurons and analyzed by quantitative real-time polymerase chain reaction and immunofluorescence for the expression of neuronal differentiation markers,including beta-III tubulin(TUJ1)and tyrosine hydroxylase(TH).To assess the risk of teratoma or other tumor formation,HNP cell lines and mouse neuronal progenitor(MNP)cell lines were injected subcutaneously into immunodeficient SCID/beige mice.RESULTS:We developed a fairly simple and fast protocol to obtain HNP cell lines from hES cells.These cell lines,which can be stored in liquid nitrogen for several years,have the potential to differentiate in vitro into dopaminergic neurons.Following day 30 of differentiation culture,the majority of the cells analyzed expressed the neuronal marker TUJ1 and a high proportion of these cells were positive for TH,indicating differentiation into dopaminergic neurons.In contrast to H9 ES cells,the HNP cell lines did not form tumors in immunodeficient SCID/beige mice within 6 mo after subcutaneous injection.Similarly,no tumors developed after injection of MNP cells.Notably,mouse ES cells or neuronal cells directly differentiated from mouse ES cells formed teratomas in more than 90%of the recipients.CONCLUSION:Our findings indicate that neural progenitor cell lines can differentiate into dopaminergic neurons and bear no risk of generating teratomas or other tumors in immunodeficient mice.     

Origins of the insect enteric nervous system: differentiation of the enteric ganglia from a neurogenic epithelium.

The enteric nervous system (ENS) of the moth Manduca sexta is organized into two distinct cellular domains: an anterior domain that includes several small ganglia on the surface of the foregut, and a more posterior domain consisting of a branching nerve plexus (the enteric plexus) that spans the foregut-midgut boundary. Previously, we showed that the neurons of the posterior domain, the enteric plexus, are generated from a large placode that invaginates from the caudal lip of the foregut; subsequently, the cells become distributed throughout the enteric plexus by a sequence of active migration. We now demonstrate that the neurons of the anterior domain, the cells of the enteric ganglia, arise via a distinct developmental sequence. Shortly after the foregut has begun to form, three neurogenic zones differentiate within the foregut epithelium and give rise to chains of cells that emerge onto the foregut surface. The three zones are not sites of active mitosis, as indicated by the absence of labelling with a thymidine analogue and by clonal analyses using intracellularly injected dyes. Rather, the zones serve as loci through which epithelial cells are recruited into a sequence of delamination and neuronal differentiation. As they emerge from the epithelium, the cells briefly become mitotically active, each cell dividing once or twice. In this manner, they resemble the midline precursor class of neural progenitors in the insect central nervous system more than neuroblast stem cells. The progeny of these zone-derived precursors then gradually coalesce into the ganglia and nerves of the anterior ENS. Although this reorganization results in some variability in the precise configuration of neurons within the ganglia, the overall morphology of the ganglia is highly stereotyped, consisting of cortical layers of cells that surround a ventral neuropil. In addition, a number of the neurons within the frontal and hypocerebral ganglia express identifiable phenotypes in a manner that is similar to many cells of the insect central nervous system. These observations indicate that the differentiation of the enteric ganglia in Manduca involves an unusual combination of features seen during the formation of other regions of the nervous system and, as such, constitutes a distinct program of neurogenesis.