Inhibition of cell death results in hyperganglionosis: implications for enteric nervous system development

Abstract  The enteric nervous system (ENS) is derived from vagal and sacral neural crest cells (NCC) that delaminate from the neural tube and undergo extensive migration and proliferation in order to colonize the entire length of the gut and differentiate into many millions of neurons and glial cells. Although apoptotic programmed cell death is an essential physiological process during development of the majority of the vertebrate nervous system, apoptosis within early ENS development has not been comprehensively investigated. The aim of this study was to determine the presence and extent of apoptosis within the vagal NCC population that gives rise to most of the ENS in the chick embryo. We demonstrated that apoptotic cells, as shown by terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling and active caspase-3 immunoreactivity, are present within an electroporated green fluorescent protein (GFP) and human natural killer-1 (HNK-1) immunopositive NCC population migrating from the vagal region of the neural tube to the developing foregut. Inhibition of caspase activity in vagal NCC, by electroporation with a dominant-negative form of caspase-9, increased the number of vagal NCC available for ENS formation, as shown by 3-dimensional reconstruction of serial GFP or HNK-1 labelled sections, and resulted in hyperganglionosis within the proximal foregut, as shown by NADPH-diaphorase whole gut staining. These findings suggest that apoptotic cell death may be a normal process within the precursor pool of pre-enteric NCC that migrates to the gut, and as such it may play a role in the control of ENS formation.     

Choices choices: regulation of precursor differentiation during enteric nervous system development

Background The enteric nervous system (ENS) is the largest subdivision of the peripheral nervous system and forms a complex circuit of neurons and glia that controls the function of the gastrointestinal (GI) tract. Within this circuit, there are multiple subtypes of neurons and glia. Appropriate differentiation of these various cell subtypes is vital for normal ENS and GI function. Studies of the pediatric disorder Hirschprung's Disease (HSCR) have provided a number of important insights into the mechanisms and molecules involved in ENS development; however, there are numerous other GI disorders that potentially may result from defects in development/differentiation of only a subset of ENS neurons or glia. Purpose Our understanding of the mechanisms and molecules involved in enteric nervous system differentiation is far from complete. Critically, it remains unclear at what point the fates of enteric neural crest cells (ENCCs) become committed to a specific subtype cell fate and how these cell fate choices are made. We will review our current understanding of ENS differentiation and highlight key questions that need to be addressed to gain a more complete understanding of this biological process.     

Functional development of the enteric nervous system – from migration to motility

Abstract  The enteric nervous system (ENS) consists of many different types of enteric neurones forming complex reflex circuits that underlie or regulate many gut functions. Studies of humans with Hirschsprung's disease (distal aganglionosis), and of animal models of Hirschsprung's disease, have led to the identification of many of the genetic, molecular and cellular mechanisms responsible for the colonization of the gut by enteric neurone precursors. However, later events in the ENS development are still poorly understood, including the development of functioning ENS circuits. This article is a personal view of the current state of play in our understanding of the ENS development and of the future of the field.     

Genetic model system studies of the development of the enteric nervous system, gut motility and Hirschsprung's disease

Abstract  The enteric nervous system (ENS) is the largest and most complicated subdivision of the peripheral nervous system. Its action is necessary to regulate many of the functions of the gastrointestinal tract including its motility. Whilst the ENS has been studied extensively by developmental biologists, neuroscientists and physiologists for several decades it has only been since the early 1990s that the molecular and genetic basis of ENS development has begun to emerge. Central to this understanding has been the use of genetic model organisms. In this article, we will discuss recent advances that have been achieved using both mouse and zebrafish model genetic systems that have led to new insights into ENS development and the genetic basis of Hirschsprung's disease.     

Heterogeneity and phenotypic plasticity of glial cells in the mammalian enteric nervous system

Enteric glial cells are vital for the autonomic control of gastrointestinal homeostasis by the enteric nervous system. Several different functions have been assigned to enteric glial cells but whether these are performed by specialized subtypes with a distinctive phenotype and function remains elusive. We used Mosaic Analysis with Double Markers and inducible lineage tracing to characterize the morphology and dynamic molecular marker expression of enteric GLIA in the myenteric plexus. Functional analysis in individually identified enteric glia was performed by Ca²⁺ imaging. Our experiments have identified four morphologically distinct subpopulations of enteric glia in the gastrointestinal tract of adult mice. Marker expression analysis showed that the majority of glia in the myenteric plexus co‐express glial fibrillary acidic protein (GFAP), S100β, and Sox10. However, a considerable fraction (up to 80%) of glia outside the myenteric ganglia, did not label for these markers. Lineage tracing experiments suggest that these alternative combinations of markers reflect dynamic gene regulation rather than lineage restrictions. At the functional level, the three myenteric glia subtypes can be distinguished by their differential response to adenosine triphosphate. Together, our studies reveal extensive heterogeneity and phenotypic plasticity of enteric glial cells and set a framework for further investigations aimed at deciphering their role in digestive function and disease. GLIA 2015;63:229–241     

L1cam acts as a modifier gene for members of the endothelin signalling pathway during enteric nervous system development

Background The enteric nervous system originates from neural crest cells that migrate into the embryonic foregut and then sequentially colonize the midgut and hindgut. Defects in neural crest migration result in regions of the gut that lack enteric ganglia, a condition in humans called Hirschsprung’s disease. The high degree of phenotypic variability reported in Hirschsprung’s disease suggests the involvement of modifier genes. Methods We used a two‐locus complementation approach to screen for genetic interactions between L1cam and members of the endothelin signalling pathway. Immunohistochemistry was used to label PGP9.5⁺ enteric neurons and Sox10⁺ neural crest‐derived cells in wholemount preparations of embryonic gut. Key Results Loss or haploinsufficiency of L1cam significantly increased the severity of aganglionosis in Et‐3 and Ednrb null mutant embryos. Furthermore, the colonization of the developing gut by neural crest‐derived cells was significantly delayed in L1cam^?/y; Et‐3^?/? and L1cam^?/y;Ednrb^sl/sl embryos. Conclusions & Inferences We have identified the X‐linked gene, L1cam, as the first modifier gene for members of the endothelin signalling pathway during development of the enteric nervous system. Mutations in L1CAM may act to modulate the severity of aganglionosis in some cases of Hirschsprung’s disease.     

Tissue culture analysis of neurite outgrowth in the presence and absence of serum: possible relevance for central nervous system regeneration

A tissue culture model has been developed to examine the hypothesis that axons can only regenerate when their growing tips are surrounded by extracellular fluid containing proteins derived from the blood. Fetal rat cerebral explants were cultured in serum medium for 10 days, followed by serum-free (SF) medium (from which serum had been removed) until 18 days in vitro (DIV). All explants cultured in serum medium for 0-10 DIV exhibited greater than 77% neurite viability (neurite viability ratio, NVR, 3.10). This degree of neurite viability was maintained for those explants exposed to serum until 18 DIV (NVR 2.82 at 18 DIV). By contrast, explants maintained in SF medium from 10-18 DIV had a much lower NVR, which, by 18 DIV, had declined to 0.30 (7.5% viability). Transmission electron microscopic analysis of explants fixed at 18 DIV confirmed these phase-contrast results and also showed a predominance of axonal profiles within the neurite population. In the center of explants, tissue viability was in excess of 75% in both the serum and SF media, suggesting that serum is of primary importance for axonal extension rather than neuronal survival. These data strengthen the hypothesis that blood-derived proteins may be needed for prolonged regeneration.     

A peptide derived from a trans‐homophilic binding site in neural cell adhesion molecule induces neurite outgrowth and neuronal survival

The neural cell adhesion molecule (NCAM) plays a key role in neural development, regeneration, and synaptic plasticity. The crystal structure of a fragment of NCAM comprising the three N‐terminal immunoglobulin (Ig)‐like modules indicates that the first and second Ig modules bind to each other, thereby presumably mediating dimerization of NCAM molecules expressed on the same cell surface (cis‐interactions), whereas the third Ig module, through interactions with the first or second Ig module, mediates interactions between NCAM molecules expressed on the surface of opposing cells (trans‐interactions). We have designed a new potent peptide ligand of NCAM, termed plannexin, based on a discontinuous sequence in the second NCAM Ig module that represents a homophilic binding site for an opposing third Ig module. The peptide was found by surface plasmon resonance analysis to bind the third NCAM Ig module. It promoted survival of cultured cerebellar granule neurons (CGNs) and also induced neurite extension in cultures of dopaminergic neurons and CGNs; the latter effect was shown to be dependent on NCAM expression, indicating that plannexin mimics the neuritogenic effect of homophilic NCAM binding. © 2010 Wiley‐Liss, Inc.     

Development of the enteric nervous system: bringing together cells, signals and genes

Abstract  The enteric nervous system (ENS), the intrinsic innervation of the gastrointestinal tract that controls essential functions such as motility, secretion and blood flow, comprises a vast number of neurons and glial cells that are organized into complex networks of interconnected ganglia distributed throughout the entire length of the gut wall. Enteric neurons and glia are derived from neural crest cells that undergo extensive migration, proliferation, differentiation and survival in order to form a functional ENS. Investigations of the developmental processes that underlie ENS formation in animal models, and of the common human congenital ENS abnormality Hirschsprung's disease, have been intimately related and recently led to major advances in the field. This review touches on some of these advances and introduces two topics that are elaborated upon in this journal issue: (i) genome wide approaches for profiling gene expression in wild type and mutant ENS that have been used to identify novel molecules with important roles in enteric neurogenesis, and (ii) the use of multilineage ENS progenitors isolated from embryonic or postnatal gut as novel cell replacement therapies for Hirschsprung's disease. Such studies will not only unravel the mechanisms underlying ENS development, but will also shed light on the pathogenesis of ENS developmental disorders and help to establish novel therapeutic strategies for restoring or repairing malfunctioning enteric neural circuits prevalent in numerous gastrointestinal diseases.     

Neuronal migration in cerebellar microcultures is inhibited by antibodies against a neurite outgrowth domain of laminin.

The functional role of laminin in neuronal migration was investigated by using polyclonal antibodies or their divalent (Fab')2 fragments to a neurite outgrowth promoting domain of the B2 chain of laminin in a cerebellar microculture system widely recognized as a model for neuronal migration. We show here that these antibodies or their (Fab')2 fragments totally inhibit migration of the mouse cerebellar granule cells along the glial and other neuronal cell processes. Antibodies to native laminin or other control antibodies have no inhibitory effect. Immunocytochemical analysis of the cerebellar microcultures indicates that the functional role of these antibodies may relate to the fact that the punctate deposits of laminin and its neurite outgrowth promoting domain accumulate in between the migrating neurons and the glial cells. These data provide the first direct evidence for the functional role of laminin and its neurite outgrowth domain in neuronal migration in the mammals. They further suggest that a neuronal cell surface contact with the extracellular deposits of a neurite outgrowth domain of the B2 chain of laminin may mediate neuronal-glial interactions.     

Differentiation of glial cells and neurite outgrowth obtained from embryonic locust central nervous system explants

Up to the present it has not been possible to obtain viable glial cells from dissociated insect nervous system cultures. We report here that the use of explant culture of locust embryo central nervous system (CNS) has been successful in allowing the proliferation of glial cells derived from glial precursors located at the periphery of the embryonic CNS. In such cultures, maintained for 3 months under specific conditions, 4 cell types at intermediate stages of differentiation can be distinguished around the explants after 2 weeks in vitro. They have been identified by scanning and transmission electron microscopy. The first type (stage 1) consists of flat epithelioid cells with an abundant and lucent cytoplasm containing little granular endoplasmic reticulum. These earliest cells subsequently develop flat ameboid prolongations forming an intermediate cell type (stage 2) which then differentiates into protuberant bipolar cells (stage 3) in which appear well-organized cisterns of granular endoplasmic reticulum. The last stage of differentiation (stage 4) is composed of multipolar cells with an electron-dense cytoplasm and well-defined processes characterized by the presence of ribosomes and granular endoplasmic reticulum. These (stage 4) differentiated cells resemble mature glial cells. In the same in vitro system, neurites, growing from neurons originating in the explants, form a network lying upon the glial cells and in close relationship with them. Neurites present growth cones and lack ribosomes and granular endoplasmic reticulum. We conclude that this model is a potentially useful system for use in in vitro studies of insect glia and neurite-glia interactions.     

Acetylcholinesterase antibody treatment results in neurite detachment and reduced outgrowth from cultured neurons: Further evidence for a cell adhesive role for neuronal acetylcholinesterase

Data from our laboratory and others demonstrate that acetylcholinesterase (AChE) is expressed transiently by neurons during periods of neurite outgrowth preceding synaptogenesis, suggesting an extrasynaptic function for this molecule. These findings, along with reports that AChE shares amino acid sequence homology and structural similarities with known cell adhesion molecules, have led to the theory that, during development, AChE may exert a morphogenic effect through cell adhesion. To further test this hypothesis, we have examined the effects of an AChE monoclonal antibody (MAB304) on neurite outgrowth in primary cultures of rat dorsal root ganglion (DRG) neurons. Short-term, high-concentration antibody treatment produced a rapid detachment of established DRG neurites, which was followed by regrowth upon removal of the antibody from the culture medium. This effect appeared to be site-specific, because other AChE antibodies that were able to detect AChE immunocytochemically failed to produce this disadhesion. Long-term, low-concentration antibody exposure produced a 50% reduction in total area of outgrowth, in which neurites were more densely packed and interlaced compared with the neurites in control cultures. These results extend our previous observations on the outgrowth perturbing effects of AChE inhibitor treatment and provide further evidence that AChE may support neurite outgrowth through a cell adhesive role. J. Neurosci. Res. 53:454–464, 1998. © 1998 Wiley-Liss, Inc.     

The development of a rat in vitro model of spinal cord injury demonstrating the additive effects of Rho and ROCK inhibitors on neurite outgrowth and myelination

It is currently thought that treatment for spinal cord injury (SCI) will involve a combined pharmacological and biological approach; however, testing their efficacy in animal models of SCI is time-consuming and requires large animal cohorts. For this reason we have modified our myelinating cultures as an in vitro model of SCI and studied its potential as a prescreen for combined therapeutics. This culture comprises dissociated rat embryonic spinal cord cells plated onto a monolayer of astrocytes, which form myelinated axons interspaced with nodes of Ranvier. After cutting the culture, an initial cell-free area appears persistently devoid of neurites, accompanied over time by many features of SCI, including demyelination and reduced neurite density adjacent to the lesion, and infiltration of microglia and reactive astrocytes into the lesioned area. We tested a range of concentrations of the Rho inhibitor C3 transferase (C3) and ROCK inhibitor Y27632 that have been shown to promote SCI repair in vivo. C3 promoted neurite extension into the lesion and enhanced neurite density in surrounding areas but failed to induce remyelination. In contrast, while Y27632 did not induce significant neurite outgrowth, myelination adjacent to the lesion was dramatically enhanced. The effects of the inhibitors were concentration-dependent. Combined treatment with C3 and Y27632 had additive affects with an enhancement of neurite outgrowth and increased myelination adjacent to the lesion, demonstrating neither conflicting nor synergistic effects when coadministered. Overall, these results demonstrate that this culture serves as a useful tool to study combined strategies that promote CNS repair.     

CD9 of mouse brain is implicated in neurite outgrowth and cell migration in vitro and is associated with the α6/β1 integrin and the neural adhesion molecule L1

We describe here a novel monoclonal antibody (mab INTRODUCTION H6) which recognizes CD9, an integral cell surface constituent previously described in cells of the hematopoietic lineage and involved in the aggregation of platelets. Mab H6 was raised against membranes of immature mouse astrocytes and reacted with a protein of 25–27 kD in detergent extracts of adult mouse brain membranes. Sequence analysis of the N-terminal amino acids revealed an identity of 96% with CD9 from mouse kidney. CD9 was localized in the central and peripheral mouse nervous systems: in the spinal cord of 11-day-old mouse embryos, CD9 was strongly expressed in the floor and roof plates. In the adult mouse sciatic nerve, myelin sheaths were highly CD9immunoreactive. Mab H6 reacted with the cell surfaces of both glial cells and neurons in culture and inhibited migration of neuronal cell bodies, neurite fasciculation and outgrowth of astrocytic processes from cerebellar microexplants. Neurite outgrowth from isolated small cerebellar neurons was increased in the presence of mab H6 on substrate-coated laminin, but not on substrate-coated poly-L-lysine. Addition of mab H6 elicited an increase in intracellular Ca²⁺ concentration in these cells on substrate-coated laminin. Immunoprecipitates of CD9 from cultured mouse neuroblastoma N2A cells contained the α6/β1 integrin. Moreover, preparations of CD9 immunoaffinity-purified from adult mouse brain using a mab H6 column contained the neural adhesion molecule L1, but not other neural adhesion molecules. CD9 bound to L1, but not to NCAM or MAG. Both the α6/β1 integrin and L1 could be induced to coredistribute with CD9 on the surface of cultured neuroblastoma N2A cells. The combined observations suggest that CD9 can associate with L1 and α6/β1 integrin to influence neural cell interactions in vitro. © 1996 Wiley-Liss, Inc.     

Lysosomal storage results in impaired survival but normal neurite outgrowth in dorsal root ganglion neurones from a mouse model of Sandhoff disease

Sandhoff disease is a heritable lysosomal storage disease resulting from impaired degradation of GM2 ganglioside and related substrates. A mouse model of Sandhoff disease created by gene targeting displays progressive neurological manifestations, similar to patients with the disease. In the present in vivo and in vitro studies, we examined morphological and functional abnormalities of dorsal root ganglion (DRG) neurones in Sandhoff disease mice at an asymptomatic stage (approximately 1 month of age). Light microscopic studies with Nissl staining and immunocytochemistry suggested extensive intracytoplasmic storage of GM2 ganglioside in the Sandhoff mouse DRG neurones. These findings were consistent with the results of electron microscopy, in which a huge number of pleomorphic inclusion bodies immunoreactive for GM2 ganglioside were present in the cytoplasm of the neurones. The inclusion bodies were also identified in satellite cells and Schwann cells in the Sandhoff mouse DRG. The survival ratios of DRG neurones after 1, 2, 4 and 6 days in culture were significantly lower in the Sandhoff mice than in the age-matched heterozygous mice. The ratio of neurite-bearing cells on poly-l-lysine-coated dishes after 2 days in culture was also lower by approximately 10% in the Sandhoff mice compared to the heterozygotes, but additional coating of laminin onto poly-l-lysine dramatically enhanced the neurite extension from the neurones in both groups of mice. These results indicate that accumulation of GM2 ganglioside in DRG neurones impairs the capability of the neurones to survive in vitro, although viable neurones from the Sandhoff mice in culture can regenerate neurites nearly as well as unaffected neurones.