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Keywords:

  • development;
  • enteric nervous system;
  • neural crest cells;
  • gut;
  • neural stem cells

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Migration of neural crest cells to and within the gut
  5. Differentiation of enteric neural crest-derived cells
  6. Neural stem cells as replacement therapy
  7. Acknowledgments
  8. References

The enteric nervous system arises from two regions of the neural crest; the vagal neural crest which gives rise to the vast majority of enteric neurones throughout the gastrointestinal tract, and the sacral neural crest which contributes a smaller number of cells that are mainly distributed within the hindgut. The migration of vagal neural crest cells into, and along the gut is promoted by GDNF, which is expressed by the gut mesenchyme and is the ligand for the Ret/GFRα1 signalling complex present on migrating vagal-derived crest cells. Sacral neural crest cells enter the gut after it has been colonized by vagal neural crest cells, but the molecular control of sacral neural crest cell development has yet to be elucidated. Under the influence of both intrinsic and extrinsic cues, neural crest cells differentiate into glia and different types of enteric neurones at different developmental stages. Recently, the potential for neural stem cells to form an enteric nervous system has been examined, with the ultimate aim of using neural stem cells as a therapeutic strategy for some gut disorders where enteric neurones are reduced or absent.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Migration of neural crest cells to and within the gut
  5. Differentiation of enteric neural crest-derived cells
  6. Neural stem cells as replacement therapy
  7. Acknowledgments
  8. References

Recently the enteric nervous system (ENS) has received much attention from developmental biologists, due mainly to insights gained following analysis of the ENS in mice with spontaneous or targeted mutations. Such studies have unravelled some of the major signalling pathways involved in ENS development, although without doubt, much remains to be elucidated. This focus on ENS development has also led to the investigation of stem cells as a replacement therapy for treatment of ENS defects, where numbers of enteric neurones are reduced or absent. This review summarizes how neural crest cells migrate to and within the gut, the factors controlling their subsequent differentiation into the appropriate neuronal and glial phenotypes, and the potential for neuronal stem cells to form the ENS.

Migration of neural crest cells to and within the gut

  1. Top of page
  2. Abstract
  3. Introduction
  4. Migration of neural crest cells to and within the gut
  5. Differentiation of enteric neural crest-derived cells
  6. Neural stem cells as replacement therapy
  7. Acknowledgments
  8. References

All of the neurones and glial cells that comprise the ENS are derived from the neural crest (NC). The anatomical location of the NC, on the dorsal aspect of the neural tube, has facilitated numerous experimental interventions in order to trace neural crest cell (NCC) lineages as they migrate to and within the gut. Such techniques have involved: (i) microsurgical ablation of segments of neural tube, and subsequent examination of embryos for loss of cell types; (ii) interspecies grafting in the avian embryo, where sections of neural tube from quail embryos are microsurgically transplanted into chick embryos (quail cells can subsequently be detected within the developing chick using antiquail cell antibodies); and (iii) microinjection of cell tracers, such as the fluorescent dye, Dil, or replication-deficient retroviruses. Using such methods, two specific regions of the NC have been shown to form the ENS; the vagal NC, which contributes the majority of ENS precursors along the entire length of the gut, and the sacral NC, which contributes a smaller number of cells, that are mainly restricted to the hindgut.1

In the chick embryo, vagal NCC leave the neural tube and migrate towards the developing foregut at approximately embryonic day (E)1.5 of development. These cells initially accumulate in the caudal branchial arches, move into the foregut and migrate in a rostro-caudal direction, colonizing the entire length of the gut by E8.5 (Fig. 1).2 In the mouse and human, the gut is colonized by vagal NCC at E143 and week 7 (AJB, unpublished), respectively. Although the factors that control this unidirectional migration have yet to be fully elucidated, among the most influential are members of the Ret signalling pathway. Ret and its coreceptor GFRα1 are expressed in migrating vagal NCC and both are essential for ENS development. The ligand for the Ret/GFRα1 signalling complex, glial cell line-derived neurotrophic factor (GDNF), is expressed in the gut wall, and has been shown to act as a chemoattractant for enteric neural cells that promotes directed migration of vagal NCC along the gut.4 Recent studies have shown that GDNF protein levels are higher in regions of the gut preceding the rostro-caudal migration front of vagal NCC, i.e. firstly in the stomach, then in the caecum.5 Vagal NCC appear to migrate towards these GDNF ‘sinks’ in a concentration gradient-dependent manner. However, interactions with other signalling molecules, such as endothelin-3, have been shown to be important in controlling NCC migration/proliferation,6 while molecules such as netrins and their receptors appear to be involved in guiding migrations NCC within the gut.7

image

Figure 1. Electroporation of GFP-containing vector into the vagal neural tube of a 10 somite stage chick embryo. Panel A: The neural tube (NT) is labelled on the anode side. Panels B and C: Sections through the gut (G) reveal labelled NCC- derived ENS cells (arrowheads) external to the circular muscle layer (CM).

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In contrast to vagal NCC, which rapidly migrate into the gut, in the chick, sacral NCC initially form the nerve of Remak (a ganglionated nerve adjacent to the gut wall) and appear to lie dormant within this nerve for a number of days until the gut has been colonized by vagal NCC. At E7, nerve fibres derived from the nerve of Remak penetrate the gut wall, and sacral NCC move into the gut in close association with these nerve fibres. Sacral cells then proliferate and migrate in a caudo-rostral direction, with the rostral limit of colonization occurring at the level of the umbilicus.8 Although such a delayed entry of sacral NCC into the hindgut has also been reported in the mouse,9 the cellular fates, and extent of gut colonization of these cells remains to be determined in mammals.

Differentiation of enteric neural crest-derived cells

  1. Top of page
  2. Abstract
  3. Introduction
  4. Migration of neural crest cells to and within the gut
  5. Differentiation of enteric neural crest-derived cells
  6. Neural stem cells as replacement therapy
  7. Acknowledgments
  8. References

Once enteric NC-derived cells begin to colonize the gut, they differentiate into glial cells and diverse subsets of neurones that differ in their axon projection patterns, dendritic morphology, soma size and complements of neurotransmitters, ion channels and receptors. All enteric neurones express pan-neuronal proteins (e.g. neurofilaments, Hu antigens, SCG10, PGP9.5), and different subsets of neurones express particular type-specific markers such as neurotransmitter synthetic enzymes. During the differentiation of enteric neurones, panneuronal markers seem to be expressed prior to any of the neurotransmitter synthetic enzymes that present in mature enteric neurones. Some vagal NCC begin to express pan-neuronal markers prior to their entry into the foregut,10 and the proportion of crest-derived cells expressing pan-neuronal markers then increases after their entry into the gut (Fig. 2).11 Various types of enteric neurones and glia are generated at different developmental stages12,13 and are produced in precise ratios with respect to one another. The first neurone type-specific marker expressed by subpopulations of enteric crest-derived cells is nitric oxide synthase (NOS) [the synthetic enzyme for nitric oxide (NO)], which in embryonic mice is expressed over 2 days after the arrival of vagal crest-derived cells into the gut.14 5-HT neurones also develop early, but cholinergic and CGRP neurones develop later and are not present in significant numbers until late embryonic and early newborn stages.

image

Figure 2. Paired confocal micrographs of neural crest-derived cells, immunostained using an antibody to Phox2b, in the foregut of an E10.5 mouse (A). Two of the Phox2b + cells (arrowed) show immunostaining for the pan-neuronal protein, Hu (B). Scale bar: 10 µm.

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What regulates the generation of different types of enteric neurones and glial cells? In all parts of the nervous system investigated to date, the generation of the correct types of neurones in the appropriate location (called ‘cell fate determination’) is regulated by a combination of intrinsic and extrinsic cues. Intrinsic cues that influence neural fate determination include: (i) the expression of combinations of transcription factors that specify particular neurone types or glial cells; (ii) the expression of cell surface receptors that enable some precursors, but not others, to respond to environmental signals; and (iii) asymmetric cell division in which cytoplasmic cell fate determinants are segregated during mitosis and inherited by only one daughter cell. Extrinsic cues can be in the form of diffusible molecules, cell surface proteins or molecules in the extracellular matrix. Thus far, the only intrinsic factors identified that are associated with cell fate determination of enteric neural crest-derived cells are the transcription factors, Mash1 and Sox10. Mash1 appears to be involved in the development of enteric 5-HT neurones,15 and Sox10 is required for enteric glial cell differentiation.16 Transplantation and in vitro studies have shown that molecules within the gut environment can influence the types of neurones that develop from NC-derived cells.17,18 GDNF, neurotrophin-3 and BMP2, which are expressed by the gut mesenchyme, promote the differentiation of NCC isolated from the gut6,19,20 but it is unknown whether they promote the differentiation of specific classes of enteric neurones. Neurturin, a member of the GDNF family of neurotrophic factors, is also expressed by the gut mesenchyme, and appears to play a role in the development of cholinergic neurones.21,22 As different types of enteric neurones develop at different stages, it is possible that environmental cues within the gut change over time, and/or that the intrinsic cues expressed by enteric neural progenitors vary with developmental age, so that the cells are only competent to differentiate into a particular cell type at a particular stage of development. Recent data suggest that, like the CNS, not all NCC within the gut differentiate during development, and that a small pool of enteric neural crest stem cells persist in adult gut.23 Should such stem cells, or cells from other locations with similar developmental potency, be isolated and manipulated, they may have therapeutic potential in addressing conditions where there is a lack, or reduction in number, of enteric neurones.

Neural stem cells as replacement therapy

  1. Top of page
  2. Abstract
  3. Introduction
  4. Migration of neural crest cells to and within the gut
  5. Differentiation of enteric neural crest-derived cells
  6. Neural stem cells as replacement therapy
  7. Acknowledgments
  8. References

Neural stem cell (NSC) transplantation is an emerging technique with immense promise for the treatment of disorders of the peripheral and central nervous system. Following in vivo implantation into the brain and other structures, NSC have been shown to be surprisingly plastic: they not only generate neurones and glia and respond to local tissue-specific cues,24 but even cross traditional germ layer boundaries. They may be able to adopt a haematopoietic fate including myeloid and lymphoid cell lines25 and when injected into blastocysts, can contribute to the formation of chimeric chick and mouse embryos and give rise to cell derivatives of all germ layers, populating even liver, stomach and intestines.26 Durbec and colleagues have recently shown that cultured neurosphere-derived cells from the subventricular zone are capable of migrating along chick NC pathways and can differentiate into phenotypes similar to NCC derivatives.27

With this in mind, the use of NSC as a viable therapeutic strategy for several gastrointestinal motility disorders such as achalasia, characterized by partial or complete loss of intrinsic neurones, is currently being investigated.

There are several potential sources of neural stem cells, including: (i) the neurogenic areas of both adult and fetal rodent brain [the subventricular zone (SVZ) and the hippocampus]; (ii) totipotential human and rodent embryonic stem cells that can be induced to assume a more committed neuronal fate; (iii) human fetal and adult neuronal tissue, including the ENS;23 and possibly (iv) transdifferentiation of stem cells from other germ layers.28 In pilot studies published recently, Micci et al. have shown that central nervous system-derived NSC (CNS-NSC) from embryonic rat SVZ express nNOS and produce NO in vitro.29 When transplanted into the gut, CNS-NSC differentiate into neurones, continue to express nNOS and survive at least 8 weeks postimplantation. These authors have also shown that CNS-NSC express a functional receptor complex (RET and GFRa1) for GDNF, which, in turn, induces expansion of the RET-expressing CNS-NSC population. These studies therefore support the suggestion that transplantation of CNS-NSC is a promising cellular replacement strategy for enteric neurones.

Having established the feasibility of neuronal transplantation into the gut, the optimal conditions required for successful engraftment and functional benefit need to be investigated. The hypothesis is that CNS-NSC can survive transplantation in the gut, undergo differentiation into functional enteric neurones and form the correct synaptic connections. To test this idea, CNS-NSC derived from embryonic Green Fluorescent Protein (GFP) transgenic mice (The Jackson Laboratory, Bar Harbor, USA) are being implanted into the pylorus of mice under various conditions including immunosuppression, and the addition of various growth and chemotactic factors (PJP, unpublished). In a parallel effort, human CNS-derived NSC (hCNS-NSC) are being implanted. Of particular interest are the factors that could induce neuronal NOS expression in NSC, and ways of generating mature neurones expressing nNOS from hCNS-NSC are being investigated.30

Many issues need to be addressed and techniques optimized before the procedure of stem cell replacement in the ENS can be considered practical. These include the best source (CNS, neural crest, embryonic) as well as methods to ensure proper geographical location, adequate survival and appropriate neuronal differentiation. We also need to know more about how to ‘nudge’ these cells to assume the desired phenotype for a given therapeutic indication. Hopefully, the efforts of investigators in this field will help make this promise a reality.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Migration of neural crest cells to and within the gut
  5. Differentiation of enteric neural crest-derived cells
  6. Neural stem cells as replacement therapy
  7. Acknowledgments
  8. References

AJB is supported by the Biotechnology and Biological Sciences Research Council. PJP is supported by grants from the National Institutes of Health DK61423-01 and Stem Cells Inc. (Palo Alto, CA). HMY is supported by the National Health and Medical Research Council of Australia.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Migration of neural crest cells to and within the gut
  5. Differentiation of enteric neural crest-derived cells
  6. Neural stem cells as replacement therapy
  7. Acknowledgments
  8. References
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