Cell death and the developing enteric nervous system

This review discusses current knowledge about cell death in the developing enteric nervous system (ENS). It also includes findings about the molecular mechanisms by which such death is mediated. Additional consideration is given to trophic factors that contribute to survival of the precursors and neurons and glia of the ENS, as well to genes that, when mutated or deleted, trigger their death. Although further confirmation is needed, present observations support the view that enteric neural crest-derived precursor cells en route to the gut undergo substantial levels of apoptotic death, but that once these cells colonize the gut, there is relatively little death of precursor cells or of neurons and glia during the fetal period. There are also indications that normal neuron loss occurs in the ENS, but at times beyond the perinatal stage. Taken together, these findings suggest that ENS development is similar is some ways, but different in others from extra-enteric areas of the vertebrate central and peripheral nervous systems, in which large-scale apoptotic death of precursor neurons and glia occurs during the fetal and perinatal periods. Potential reasons for these differences are discussed such as a fetal enteric microenvironment that is especially rich in trophic support. In addition to the cell death that occurs during normal ENS development, this review discusses mechanisms of experimentally-induced ENS cell death, such as those that are associated with defective glial cell-line derived neurotrophic factor/Ret signaling, which are an animal model of human congenital megacolon (aganglionosis; Hirschsprung’s disease). Such considerations underscore the importance of understanding cell death in the developing ENS, not just from a curiosity-driven point of view, but also because the pathophysiology behind many disorders of human gastrointestinal function may originate in abnormalities of the mechanisms that govern cell survival and death during ENS development.     

GDNF availability determines enteric neuron number by controlling precursor proliferation

To clarify the role of Ret signaling components in enteric nervous system (ENS) development, we evaluated ENS anatomy and intestinal contractility in mice heterozygous for Ret, GFRalpha1 and Ret ligands. These analyses demonstrate that glial cell line-derived neurotrophic factor (GDNF) and neurturin are important for different aspects of ENS development. Neurturin is essential for maintaining the size of mature enteric neurons and the extent of neuronal projections, but does not influence enteric neuron number. GDNF availability determines enteric neuron number by controlling ENS precursor proliferation. However, we were unable to find evidence of programmed cell death in the wild type ENS by immunohistochemistry for activated caspase 3. In addition, enteric neuron number is normal in Bax(-/-) and Bid(-/-) mice, suggesting that, in contrast to most of the rest of the nervous system, programmed cell death is not important for determining enteric neuron numbers. Only mild reductions in neuron size and neuronal fiber counts occur in Ret(+/-) and Gfra1(+/-) mice. All of these heterozygous mice, however, have striking problems with intestinal contractility and neurotransmitter release, demonstrating that Ret signaling is critical for both ENS structure and function.     

Conditional deletion of Hand2 reveals critical functions in neurogenesis and cell type-specific gene expression for development of neural crest-derived noradrenergic sympathetic ganglion neurons

Neural crest-derived structures that depend critically upon expression of the basic helix-loop-helix DNA binding protein Hand2 for normal development include craniofacial cartilage and bone, the outflow tract of the heart, cardiac cushion, and noradrenergic sympathetic ganglion neurons. Loss of Hand2 is embryonic lethal by E9.5, obviating a genetic analysis of its in-vivo function. We have overcome this difficulty by specific deletion of Hand2 in neural crest-derived cells by crossing our line of floxed Hand2 mice with Wnt1-Cre transgenic mice. Our analysis of Hand2 knock-out in neural crest-derived cells reveals effects on development in all neural crest-derived structures where Hand2 is expressed. In the autonomic nervous system, conditional disruption of Hand2 results in a significant and progressive loss of neurons as well as a significant loss of TH expression. Hand2 affects generation of the neural precursor pool of cells by affecting both the proliferative capacity of the progenitors as well as affecting expression of Phox2a and Gata3, DNA binding proteins important for the cell autonomous development of noradrenergic neurons. Our data suggest that Hand2 is a multifunctional DNA binding protein affecting differentiation and cell type-specific gene expression in neural crest-derived noradrenergic sympathetic ganglion neurons. Hand2 has a pivotal function in a non-linear cross-regulatory network of DNA binding proteins that affect cell autonomous control of differentiation and cell type-specific gene expression.     

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.     

The endothelin receptor-B is required for the migration of neural crest-derived melanocyte and enteric neuron precursors

Mutations in the genes encoding endothelin receptor-B (Ednrb) and its ligand endothelin-3 (Edn3) affect the development of two neural crest-derived cell types, melanocytes and enteric neurons. EDNRB signaling is exclusively required between E10.5 and E12.5 during the migratory phase of melanoblast and enteric neuroblast development. To determine the fate of Ednrb-expressing cells during this critical period, we generated a strain of mice with the bacterial beta-galactosidase (lacZ) gene inserted downstream of the endogenous Ednrb promoter. The expression of the lacZ gene was detected in melanoblasts and precursors of the enteric neuron system (ENS), as well as other neural crest cells and nonneural crest-derived lineages. By comparing Ednrb(lacZ)/+ and Ednrb(lacZ)/Ednrb(lacZ) embryos, we determined that the Ednrb pathway is not required for the initial specification and dispersal of melanoblasts and ENS precursors from the neural crest progenitors. Rather, the EDNRB-mediated signaling is required for the terminal migration of melanoblasts and ENS precursors, and this pathway is not required for the survival of the migratory cells.     

Lineage-restricted neural precursors can be isolated from both the mouse neural tube and cultured ES cells.

We have previously identified multipotent neuroepithelial (NEP) stem cells and lineage-restricted, self-renewing precursor cells termed NRPs (neuron-restricted precursors) and GRPs (glial-restricted precursors) present in the developing rat spinal cord (A. Kalyani, K. Hobson, and M. S. Rao, 1997, Dev. Biol. 186, 202-223; M. S. Rao and M. Mayer-Proschel, 1997, Dev. Biol. 188, 48-63; M. Mayer-Proschel, A. J. Kalyani, T. Mujtaba, and M. S. Rao, 1997, Neuron 19, 773-785). We now show that cells identical to rat NEPs, NRPs, and GRPs are present in mouse neural tubes and that immunoselection against cell surface markers E-NCAM and A2B5 can be used to isolate NRPs and GRPs, respectively. Restricted precursors similar to NRPs and GRPs can also be isolated from mouse embryonic stem cells (ES cells). ES cell-derived NRPs are E-NCAM immunoreactive, undergo self-renewal in defined medium, and differentiate into multiple neuronal phenotypes in mass culture. ES cells also generate A2B5-immunoreactive cells that are similar to E9 NEP-cell-derived GRPs and can differentiate into oligodendrocytes and astrocytes. Thus, lineage restricted precursors can be generated in vitro from cultured ES cells and these restricted precursors resemble those derived from mouse neural tubes. These results demonstrate the utility of using ES cells as a source of late embryonic precursor cells.     

Pleiotropic effects of the bone morphogenetic proteins on development of the enteric nervous system

Formation of the enteric nervous system (ENS) from migratory neural crest-derived cells that colonize the primordial gut involves a complex interplay among different signaling molecules. The bone morphogenetic proteins (BMPs), specifically BMP2 and BMP4, play a particularly important role in virtually every stage of gut and ENS development. BMP signaling helps to pattern both the anterior-posterior axis and the radial axis of the gut prior to colonization by migratory crest progenitor cells. BMP signaling then helps regulate the migration of enteric neural crest-derived precursors as they colonize the fetal gut and form ganglia. BMP2 and -4 promote differentiation of enteric neurons in early fetal ENS development and glia at later stages. A major role for BMP signaling in the ENS is regulation of responses to other growth factors. Thus BMP signaling first regulates neurogenesis by modulating responses to GDNF and later gliogenesis through its effects on GGF-2 responses. Furthermore, BMPs promote growth factor dependency for survival of ENS neurons (on NT-3) and glia (on GGF-2) by inducing TrkC (neurons) and ErbB3 (glia). BMP signaling limits total neuron numbers, favoring the differentiation of later born neuronal phenotypes at the expense of earlier born ones thus influencing the neuronal composition of the ENS and the glia/neuron ratio. BMP2 and -4 also promote gangliogenesis via modification of neural cell adhesion molecules and promote differentiation of the circular and then longitudinal smooth muscles. Disruption of BMP signaling leads to defects in the gut and in ENS function commensurate with these complex developmental roles.     

BMP signaling regulates murine enteric nervous system precursor migration, neurite fasciculation, and patterning via altered Ncam1 polysialic acid addition

The enteric nervous system (ENS) forms from migrating neural crest-derived precursors that differentiate into neurons and glia, aggregate into ganglion cell clusters, and extend neuronal processes to form a complex interacting network that controls many aspects of intestinal function. Bone morphogenetic proteins (BMPs) have diverse roles in development and influence the differentiation, proliferation, and survival of ENS precursors. We hypothesized that BMP signaling might also be important for the ENS precursor migration, ganglion cell aggregation, and neurite fasciculation necessary to form the enteric nervous system. We now demonstrate that BMP signaling restricts murine ENS precursors to the outer bowel wall during migration. In addition, blocking BMP signaling causes faster colonization of the murine colon, reduces ganglion cell aggregation, and reduces neurite fasciculation. BMP signaling also influences patterns of neurite extension within the developing bowel wall. These effects on ENS precursor migration and neurite fasciculation appear to be mediated at least in part by increased polysialic acid addition to neural cell adhesion molecule (Ncam1) in response to BMP. Removing PSA enzymatically reverses the BMP effects on ENS precursor migration and neurite fasciculation. These studies demonstrate several novel roles for BMP signaling and highlight new functions for sialyltransferases in the developing ENS.     

Expression of EGF receptor and FGF receptor isoforms during neuroepithelial stem cell differentiation

To characterize the role of epidermal growth factor (EGF) and fibroblast growth factor (FGF) in regulating neuroepithelial stem cells differentiation, we have examined the expression of FGF, EGF, and their receptors by neuroepithelial (NEP) cells and their derivatives. Our results indicate that undifferentiated NEP cells express a subset of FGF receptor (FGFR) isoforms, but do not express platelet-derived growth factor receptors (PDGFRs) or epidermal growth factor receptor (EGFR). The FGFR pattern of expression by differentiated neuron and glial cells differs from that found on NEP stem cells. FGFR-4 is uniquely expressed on NEP cells, while FGFR-1 is expressed by both NEP cells and neurons, and FGFR-2 is down-regulated during neuronal differentiation. FGFRs present on astrocytes and oligodendrocytes also represent a subset of those present on NEP cells. Expression of FGF and EGF by NEP cells and their progeny was also examined. NEP cells synthesize detectable levels of both FGF-1 and FGF-2, and EGF. FGF-1 and FGF-2 synthesis is likely to be biologically relevant, as cells grown at high density do not require exogenous FGF for their survival and cells grown in the presence of neutralizing antibodies to FGF show a reduction in cell survival and division. Thus, neuroepithelial cells synthesize and respond to FGF, but not to EGF, and are therefore distinct from other neural stem cells (neurospheres). The unique pattern of expression of FGF isoforms may serve to distinguish NEP cells from their more differentiated progeny.     

Neural crest and placode roles in formation and patterning of cranial sensory ganglia in lamprey

Vertebrates possess paired cranial sensory ganglia derived from two embryonic cell populations, neural crest and placodes. Cranial sensory ganglia arose prior to the divergence of jawed and jawless vertebrates, but the developmental mechanisms that facilitated their evolution are unknown. Using gene expression and cell lineage tracing experiments in embryos of the sea lamprey, Petromyzon marinus, we find that in the cranial ganglia we targeted, development consists of placode‐derived neuron clusters in the core of ganglia, with neural crest cells mostly surrounding these neuronal clusters. To dissect functional roles of neural crest and placode cell associations in these developing cranial ganglia, we used CRISPR/Cas9 gene editing experiments to target genes critical for the development of each population. Genetic ablation of SoxE2 and FoxD‐A in neural crest cells resulted in differentiated cranial sensory neurons with abnormal morphologies, whereas deletion of DlxB in cranial placodes resulted in near‐total loss of cranial sensory neurons. Taken together, our cell‐lineage, gene expression, and gene editing results suggest that cranial neural crest cells may not be required for cranial ganglia specification but are essential for shaping the morphology of these sensory structures. We propose that the association of neural crest and placodes in the head of early vertebrates was a key step in the organization of neurons and glia into paired sensory ganglia.     

RGMa inhibits neurite outgrowth of neuronal progenitors from murine enteric nervous system via the neogenin receptor in vitro

The enteric nervous system (ENS) in vertebrate embryos is formed by neural crest-derived cells. During development, these cells undergo extensive migration from the vagal and sacral regions to colonize the entire gut, where they differentiate into neurons and glial cells. Guidance molecules like netrins, semaphorins, slits, and ephrins are known to be involved in neuronal migration and axon guidance. In the CNS, the repulsive guidance molecule (RGMa) has been implicated in neuronal differentiation, migration, and apoptosis. Recently, we described the expression of the subtypes RGMa and RGMb and their receptor neogenin during murine gut development. In the present study, we investigated the influence of RGMa on neurosphere cultures derived from fetal ENS. In functional in vitro assays, RGMa strongly inhibited neurite outgrowth of differentiating progenitors via the receptor neogenin. The repulsive effect of RGMa on processes of differentiated enteric neural progenitors could be demonstrated by collapse assay. The influence of the RGM receptor on ENS was also analyzed in neogenin knockout mice. In the adult large intestine of mutants we observed disturbed ganglia formation in the myenteric plexus. Our data indicate that RGMa may be involved in differentiation processes of enteric neurons in the murine gut.     

Migration and differentiation of transplanted enteric neural crest-derived cells in murine model of Hirschsprung’s disease

Stem cell therapy offers the potential of rebuilding the enteric nervous system (ENS) in the aganglionic bowel of patients with Hirschsprung’s disease. P0-Cre/Floxed-EGFP mice in which neural crest-derived cells express EGFP were used to obtain ENS stem/progenitor cells. ENS stem/progenitor cells were transplanted into the bowel of Ret^−/− mouse, an animal model of Hirschsprung’s disease. Immunohistochemical analysis was performed to determine whether grafted cells gave rise to neurons in the recipient bowel. EGFP expressing neural crest-derived cells accounted for 7.01 ± 2.52 % of total cells of gastrointestinal tract. ENS stem/progenitor cells were isolated using flow cytometry and expanded as neurosphere-like bodies (NLBs) in a serum-free culture condition. Some cells in NLBs expressed neural crest markers, p75 and Sox10 and neural stem/progenitor cells markers, Nestin and Musashi1. Multipotency of isolated ENS stem/progenitor cells was determined as they differentiated into neurons, glial cells, and myofibloblasts in culture. When co-cultured with explants of hindgut of Ret^−/− mice, ENS stem/progenitor cells migrated into the aganglionic bowel and gave rise to neurons. ENS stem/progenitor cells used in this study appear to be clinically relevant donor cells in cell therapy to treat Hirschsprung’s disease capable of colonizing the affected bowel and giving rise to neurons.