|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online May 23, 2006
doi: 10.1242/10.1242/dev.02418
Meeting Review |
1 Washington University School of Medicine, Department of Pediatrics, 660 South
Euclid Avenue, Box 8208, St Louis, MO 63021, USA.
2 Division of Molecular Neurobiology, MRC National Institute for Medical
Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.
* Author for correspondence (e-mail: vpachni{at}nimr.mrc.ac.uk)
SUMMARY
Scientists from around the world gathered in New York City recently to discuss the latest research on enteric nervous system development at a meeting organised by Alan Burns and Heather Young. The participants enjoyed 3 days of presentations that spurred active conversations and highlighted the rapidly advancing research in this field.
Introduction
The enteric nervous system (ENS) is a complex network of neurons and glia
within the bowel wall that controls many aspects of intestinal function,
including motility, epithelial secretion and blood flow. To perform these
complex tasks, there are many distinct subtypes of enteric neurons that differ
in neurotransmitter expression, morphology, electrophysiology and function
(Gershon et al., 1994
). The
molecular mechanisms that control ENS development were the focus of this
meeting.
Although this conference focused mainly on the developmental biology of the
ENS, a large fraction of participants were clinicians. This is because
developmental defects of the ENS result in Hirschsprung's disease (HSCR), a
congenital disorder that is characterised by aganglionosis (the absence of
enteric neurons) in the distal colon and affects roughly 1 in 5000 infants
(Chakravarti, 2001). This
defect causes tonic contraction of the affected bowel segment, resulting in
mechanical obstruction. Infants with HSCR often have severe constipation,
growth failure and are at risk of dying from the complications of toxic
megacolon (Swenson, 2002). The
current treatment for HSCR is surgical resection of the aganglionic segment of
the bowel, but intestinal dysfunction may persist after surgery. It is
presently unclear whether the postoperative morbidity is related to the
surgery or to the potentially abnormal function of the residual ENS associated
with the primary defect that initially caused aganglionosis. Although absence
of enteric neurons is usually restricted to the distal colon, some children
have much more extensive aganglionosis, which requires long-term parenteral
nutrition for survival. Other less well-understood defects in ENS function
include intestinal pseudo-obstruction syndromes and recent evidence indicates
that a subset of individuals with irritable bowel syndrome may have primary
defects within the ENS. Furthermore, the ENS can be damaged in some forms of
chronic disease, especially diabetes. For all of these reasons, understanding
the developmental mechanisms that control the migration, survival,
proliferation, differentiation and function of ENS precursors and of mature
enteric neurons and glia is our best hope of developing novel strategies to
diagnose, prevent and treat ENS defects.
Cellular and molecular mechanisms of ENS development
Colonisation of the gut by neural crest cells
The majority of enteric neurons and glia arise from a small population of
cells that originate in the vagal neural crest, invade the foregut and migrate
in a rostrocaudal direction through the developing bowel wall (Le Douarin,
1999). Additional crest-derived cells from the sacral region of the neural
tube contribute to the post-umbilical ENS and migrate in the opposite
direction through the distal bowel (Burns,
2005). To form the mature ENS, precursors must migrate away from
the gut entry points and spread uniformly throughout the entire bowel,
increase in number and undergo sequential lineage restriction before
differentiating into many distinct subtypes of interconnected neurons and
glia. The end result of all this is the formation of an integrated neuronal
network within the myenteric and submucosal plexi
(Grundy and Schemann, 2005)
(Fig. 1). Because of the
considerable length of the gut and the relatively long period required for its
colonisation by ENS progenitors, subpopulations of enteric neural crest cells,
at a given moment and place along the gut, face different challenges and have
different priorities. The main challenge in the field of ENS development is to
identify the molecular signals that control the migration, survival,
proliferation, differentiation and connectivity of ENS progenitors and enteric
neurons and to understand the mechanisms that co-ordinate these cellular
processes in time and in space. Given the complexity of the ENS, it is not
surprising that many molecules have already been implicated in its development
and organisation, and many more are waiting to be discovered.
Previous reports have suggested that ENS precursors migrate along the bowel
primarily in response to the chemoattractant effect of glial cell line-derived
neurotrophic factor (GDNF) (Natarajan et
al., 2002; Young et al.,
2001). Although this view is consistent with the phenotype of
RET-, GFR
1- and GDNF-deficient mice, it is based solely on in vitro
assays, and to date no clear evidence is available to prove an in vivo role
for the GDNF/RET signalling pathway in enteric neural crest cell migration.
Moreover, two papers presented at this meeting challenged the idea that GDNF
is the driving force for the rostrocaudal migration of neural crest cells, and
put forward the view that proliferation is the main factor that sustains
migration. A combination of mathematical modelling and organ culture
experiments suggest that `population pressure' resulting from proliferation
alone (and in the absence of chemotactic signals) could drive ENS precursor
migration down the length of the bowel (Don Newgreen, Royal Children's
Hospital, Melbourne, Australia). Consistent with these findings, Alan Burns
(Institute of Child Health, London, UK) showed in work performed with Amanda
Barlow (Institute of Child Health, London, UK) that ablation of neural crest
at the level of somites (S)3-6 reduced the number of ENS precursors in the
bowel and resulted in distal aganglionosis. Assuming that the main effect of
the ablation is a reduction in the number of ENS progenitors that invade the
foregut, these findings highlight the requirement for a critical number of ENS
progenitors for the normal colonisation of the bowel, and indicate that vagal
neural crest cells derived from levels S1, S2 and S7 have limited capacity to
compensate for the ablated segments. This analysis does not, however,
eliminate the possibility that proliferating ENS precursors compete for
limited local sources of GDNF, thus creating gradients that, in turn, have
chemotactic effects on migrating ENS precursors. The caveat of multiple
interpretations notwithstanding, these reports make a significant conceptual
advance, in that they ask us to look at the migrating ENS precursors as a
population with properties that do not simply represent the sum of the
individual cells.
|
(as presented by Bhupinder Vohra and Robert Heuckeroth, Washington University
School of Medicine, St Louis, MO, USA).
New players in ENS development
Despite significant progress having been made in recent years, many aspects
of ENS development remain poorly understood. For example, the mechanisms that
control ENS precursor differentiation into specific neuron subtypes or that
regulate patterns of neurite extension are largely unknown. The trophic
factors that support subsets of mature enteric neurons have also yet to be
completely evaluated. Even when all of the molecules currently known to
control specific aspects of ENS development are considered, the complexity of
the ENS cannot be adequately explained by the available molecular tools. Two
presentations by Robert Heuckeroth and Tiffany Heanue (National Institute of
Medical Research, London, UK) provided new data that were generated by
comparing gene expression patterns in the gut of wild-type and RET-deficient
mice, which are aganglionic. These studies led to the identification of
numerous genes, many of which were not previously known to be expressed in the
ENS. Among these newly identified genes are those that probably function in
the migration of ENS progenitors, in axon outgrowth and pathfinding, in
synaptic function, in vesicle trafficking, and in transcriptional regulation.
Genetic manipulations in mice and zebrafish, together with the ability to
modulate gene expression in cultured ENS progenitors and in enteric neurons,
promise to uncover the specific roles of many of these genes over the next few
years. The power of this genome-wide approach transcends the compilation of
more or less complete lists of genes important for ENS cell function and has
implications for medical genetics, as human homologues of genes identified in
these screens map to previously identified HSCR susceptibility loci. The
potential role of such genes as modifiers of the HSCR phenotype remains to be
established. The importance of identifying new candidate genes for HSCR in
medical genetics was further highlighted by the recent identification of
KIAA1279, which is mutated in Goldberg-Shprintzen megacolon syndrome
(Alice Brooks, ErasmusMC, Rotterdam, The Netherlands; Jean-Marie Delalande,
Emory University, Atlanta, GA, USA). Finally, novel and exciting proteomic
approaches to identify molecules expressed in the ENS were also presented
(Cornelia Hagl, University of Heidelberg, Mannheim, Germany).
Although much of our current understanding of ENS development and HSCR is based on experiments with chick embryos (an ideal system for manipulations of the neural crest) and on mouse and human genetics, it became clear at this meeting that other vertebrate model organisms, such as the zebrafish and the frog, offer new opportunities for the field of ENS development, which stem from the ability to combine powerful genetics (as in zebrafish) with embryonic gene expression manipulation and the live imaging of fluorescently tagged (ENS) progenitor cells. Using real-time video microscopy, it is now possible to examine the motility of the zebrafish gut within the intact organism, a task that in other species can be achieved only in short-term organotypic cultures of intestinal segments. At this meeting, we were given only a relatively small taste of the exciting new field of zebrafish ENS biology. Two speakers (Judith Eisen and Julie Kuhlman, University of Oregon, OR, USA) reported forward genetic screens and the identification of several loci that control various aspects of vagal neural crest biology, from their origin in the CNS to their arrival within the gut. The ability to examine in detail the peristaltic activity of the gut in these mutants offers the exciting opportunity to correlate directly the underlying neuronal deficit and the resulting dysmotility. One of the mutant loci (lessen, lsn) (discussed by Iain Shepherd, Emory University, Atlanta, GA, USA) encodes a subunit of the transcriptional mediator complex (TRAP), which, when mutated, results in a significant reduction of enteric neurons and in defects of other vagal neural crest derivatives. Interestingly, analysis of lessen mutants indicates that the gut endoderm plays a crucial role in the early development of the zebrafish ENS, thus providing additional evidence for the interdependence of the different cell types that form the gut during organogenesis (Iain Shepherd). We are confident that by the time of the next meeting (provisionally scheduled for 2008), further analysis of zebrafish ENS mutants will provide new and exciting insights into the developmental mechanisms of this branch of the autonomic nervous system. In parallel with the zebrafish experiments, similar screens were reported in mice. One of them (presented by William Pavan, NIH, Bethesda, MD, USA) was carried out in a sensitised genetic background (Sox10lacZ), and involved two mouse strains that facilitate mapping of both dominant and recessive phenotypes. Several novel loci were uncovered that affect various aspects of reporter (lacZ) expression. The molecular and phenotypic characterisation of these mutations is in progress, but this report demonstrates the feasibility of this approach for identifying novel loci regulating the development of neural crest derivatives.
Ret and Sox10 in ENS development
Many of the talks provided new insight into the role of Ret, Sox10
and related genes in the developing ENS. These talks included
structure-function analyses suggesting that the RET transmembrane domain plays
an important role in receptor dimerisation in the setting of
MEN2A-activating mutations (RET - Human Gene Nomenclature
Database) (Carlos Ibáñez, Karolinska Institute, Stockholm,
Sweden). Evidence was also presented that the RET ligand GDNF binds the
extracellular matrix molecule N-Syndecan and that this interaction is
important for mediating the role of GDNF in the tangential migration of
cortical interneurons (Mart Saarma, University of Helsinki, Helsinki,
Finland). Novel insight into the role of GDNF/GFR1/RET signalling
pathway in ENS development was also provided by new hypomorphic or conditional
mutations in RET signalling. This is important because many aspects of RET
function have been difficult to evaluate in mice with null Ret
mutations, as these mutations almost completely eliminate the ENS at early
stages of development (Newgreen and Young,
2002). The first example of what the future holds in this area
came from the use of the Cre-LoxP system to inactivate Gfra1 at
different stages of embryogenesis (Hideki Enomoto, Riken Center for
Development Biology, Kobe, Japan). This analysis demonstrated that
Gfra1 activity is required after E14.5 for ENS precursor migration,
proliferation and survival in the distal bowel. These studies also revealed a
clear and selective effect of the Gfra1 deletion on the
differentiation of ENS progenitors into neurons. Consistent with this view,
analysis of mouse embryos homozygous for a hypomorphic Ret mutation
(Ret51/51) indicated an in vivo requirement for RET
signalling in neuronal differentiation and axonogenesis (Vassilis Pachnis,
National Institute of Medical Research, London, UK). The significance of these
presentations goes beyond the understanding of the role of GDNF signalling in
ENS development and suggests that the phenotype and complications of HSCR, at
least in those cases associated with deficits of RET signalling, result from
the combined effect of the distal aganglionosis and the defects in neuronal
circuitry in the proximal bowel. Exciting results have also started to emerge
from the analysis of mouse strains expressing variants of RET with specific
amino acid substitutions that affect distinct intracellular signalling
pathways (Masahide Takahashi, Nagoya University, Nagoya, Japan). The
importance of understanding the role of intracellular signalling pathways in
ENS function was also highlighted by the description of a new mutant mouse
strain with abnormal PKA signalling and intestinal dysmotility.
Understanding the role of RET signalling in human HSCR is an ongoing
challenge (Emison et al.,
2005). Although RET is the most commonly identified
HSCR-associated gene in humans, mutations in other genes involved in ENS
development have also been described (including ECE1, EDN3, EDNRB, GDNF,
NRTN, SOX10 and ZFHX1B). However, HSCR does not follow the
conventional norms of Mendelian genetics and is a multifactorial condition
with as yet unknown genetic and environmental factors implicated
(Chakravarti, 2001). Despite
this, one theme emerging over the years and reinforced at this meeting is the
central role of RET signalling in the development of this condition
`possibly in every HSCR patient' (Aravinda Chakravarti, Johns Hopkins
University School of Medicine, Baltimore, MD, USA). This hypothesis is further
supported by the realisation that relatively common non-coding variants in the
RET locus are associated with HSCR susceptibility and make
significant contributions to risk. Such variants are either within conserved
enhancer-like sequences (Aravinda Chakravarti) or within other non-coding
regions (Isabella Ceccherini, Istituto Giannina Gaslini, Genova, Italy), and
can influence the levels of RET protein. In light of this, it was very
interesting to hear about animal model systems (mice and zebrafish) that could
be used for the evaluation of RET promoter mutations (as presented by
Elizabeth Grice and Andrew McCallion, Johns Hopkins University School of
Medicine, Baltimore, MD, USA). These models promise quick advancements in the
study of RET expression and highlight the potential implications of
promoter mutations in the development of HSCR.
Despite the key role of RET signalling in the ENS, interactions between different signalling pathways (including those of endothelin 3 and Sox10) crucially influence the development of the vertebrate ENS and the pathogenesis of HSCR. Several presentations were devoted to dissecting in more detail interactions between known genes or to identifying novel partners that increase HSCR susceptibility. Thus, genetic interactions between Sox10 and Edn3 were shown to influence not only the development of the ENS but also other neural crest derivatives such as melanocytes (Nadege Bondurand, Hôpital Henri Mondor, Creteil, France). The crosstalk between Sox10 and Sox8 was also presented using state-of-the-art gene targeting technology in mice (Michael Wegner, Universität Erlangen-Nürnberg, Erlangen, Germany). It has emerged from these studies that Sox10 and Sox8 display equivalent functions, but contribute differentially to ENS development in vivo in accordance with their corresponding levels of expression. Finally, participants heard evidence of genetic interactions between Sox10, Gdnf, Gfra1 and other genetic loci mapped in congenic strains that show different phenotypic effects of Sox10 mutations (Michelle Southard-Smith, Vanderbilt University School of Medicine, Nashville, TN, USA). The identification and characterisation of the products of these loci will provide new clues as to how Sox10 regulates the transition of multi-lineage neural crest progenitors to lineage commitment and differentiation in the peripheral nervous system (Robert Kelsh, University of Bath, Bath, UK).
In parallel with these genetic studies were reports that Sox10 regulates the expression of several small RhoGTPases and that neural crest-specific deletions of both Cdc42 and Rac1 cause a reduction in all neural crest derivatives, including the ENS (Lukas Sommer, Swiss Federal Institute of Technology, Zürich, Switzerland). The role of the small GTPase signalling network in enteric neural crest cell migration was also the subject of a poster, in which pharmacological inhibitors were used to block the activity of specific GTPases or their downstream mediators (Richard Anderson and Heather Young). Interestingly, this approach allowed the uncoupling of the axonal growth and neural crest cell migration, suggesting that the two processes are controlled independently by the gut microenvironment. The crucial role of the Cdc42 GTPase was also elegantly demonstrated by combining in vivo, four-dimensional (3D+time) confocal imaging with targeted molecular perturbation to demonstrate that chick vagal neural crest cells migrate to the gut in a programmed manner. Analysis of the migratory pattern showed that the initial group of emerging vagal neural crest cells form a wide stream that maintains very directed cell trajectories. By contrast, later emerging cells form follow-the-leader chain-like arrays and maintain contact with neighbours through filopodial extensions. Interestingly, when the ability of the neural crest cell to form filopodia and make cell-cell contacts is inhibited by perturbation of Cdc42 function, the chain assemblies are disrupted, individual cell trajectories are less directional and cells are delayed in reaching the branchial arches (Paul Kulesa, Stowers Institute for Medical Research, Kansas City, KS, USA).
|
Stunning views of migrating enteric neural crest have been made possible by
the expression of fluorescent proteins in early ENS progenitors
(Druckenbrod and Epstein, 2005;
Young et al., 2004). So far,
most of these studies have focused on imaging embryonic gut from wild-type
embryos and understanding the normal patterns of migration of vagal neural
crest. However, comparative video microscopy of guts derived from wild-type
and mutant embryos promises to provide new and exciting information regarding
specific cellular changes associated with mutations affecting the ENS (Miles
Epstein, University of Wisconsin, Madison, USA; Richard Anderson and Hideki
Enomoto) (Fig. 2). The
long-term goal of course is to understand how the building blocks of the ENS
come together to form functional reflexes and circuits that control the
complex activity of the bowel. Work presented at this meeting on the potential
roles of neurexins and neuroligins promises to provide significant advances in
this area (Michael Gershon, Columbia University, New York, NY, USA).
Establishing improved methods for assessing ENS function in humans, mice and
other model organisms is also crucially important, especially in light of the
ongoing efforts to restore ENS function by transplantation. Impressive
combinations of video imaging and sophisticated mathematical approaches in
conjunction with chemical inhibitors now allow the analysis of intestinal
motor function in mice as early as E18 (Joel Bornstein, University of
Melbourne, Australia). Other studies in organotypic gut cultures demonstrated
that intestinal muscle function can be assessed as early as E15 in mice (Kent
Sanders, University of Nevada School of Medicine, Reno, NV, USA). As many
important mutations are perinatal lethal, these analytical methods should
provide novel insight into ENS and intestinal function in mice that could not
previously be evaluated.
Although the ongoing basic research into ENS development is fascinating in
its own right, one of the long-term goals of this work is to provide new
methods to reduce human morbidity and mortality associated with digestive
diseases (Young, 2005). For
this reason, we were delighted to see a significant effort by several groups
of investigators to understand the behaviour of neural crest stem cells and to
determine whether transplantation of ENS or other neural progenitors into the
aganglionic or abnormally innervated bowel will allow the restoration of ENS
function (specifically, the laboratories of Mai Har Sham; Pankaj Jay Pasricha;
Nikhil Thapar; Gudrun Gossrau and Oliver Brustle; Jack Mosher and Sean
Morrison; Ulrich Rauch and Karl-Herbert Schäfer; and Richard Lindley and
Simon Kenney). Although there are many hurdles to overcome for this to be
successful, the work provides new hope that we will some day have novel
treatment options for people with serious defects in ENS structure and
function.
Conclusion
This is an exciting time for researchers interested in ENS development. Whereas the past decade has brought tremendous insight into the molecular and cellular mechanisms needed to form the ENS, the research presented at meeting in New York City provided new hope for the future and set forth challenges for the next 10 years.
ACKNOWLEDGMENTS
We apologise to researchers whose work was not highlighted in this short meeting summary and to those whose contributions were not fully acknowledged. We are indebted to Dr Heather Young whose tireless effort made this meeting a success. R.O.H. is supported by the NIH, the Digestive Disease Research Center Core and a grant from the March of Dimes. V.P. is supported by the MRC, the NIH and the EU.
REFERENCES
Anderson, R. B., Turner, K. N., Nikonenko, A. G., Hemperly, J., Schachner, M. and Young, H. M. (2006). The cell adhesion molecule l1 is required for chain migration of neural crest cells in the developing mouse gut. Gastroenterology 130,1221 -1232.[Medline]
Burns, A. J. (2005). Migration of neural crest-derived enteric nervous system precursor cells to and within the gastrointestinal tract. Int. J. Dev. Biol. 49,143 -150.[CrossRef][Medline]
Chakravarti, A. L. S. (2001). Hirschsprung's disease. In The Metabolic and Molecular Bases of Inherited Diseases (ed. C. R. Scriver, A. L. Beaudet, D. Valle, W. S. Sly, B. Childs, K. Kinzler and B. Vogelstein), pp.6231 -6255. New York: McGraw-Hill.
Druckenbrod, N. R. and Epstein, M. L. (2005). The pattern of neural crest advance in the cecum and colon. Dev. Biol. 287,125 -133.[CrossRef][Medline]
Emison, E. S., McCallion, A. S., Kashuk, C. S., Bush, R. T., Grice, E., Lin, S., Portnoy, M. E., Cutler, D. J., Green, E. D. and Chakravarti, A. (2005). A common sex-dependent mutation in a RET enhancer underlies Hirschsprung disease risk. Nature 434,857 -863.[CrossRef][Medline]
Gershon, M. D., Kirchgessner, A. L. and Wade, P. R. (1994). Functional anatomy of the enteric nervous system. In Physiology of the Gastrointestinal Tract. Vol.1 (ed. L. R. Johnson), pp.381 -422. New York: Raven Press.
Grundy, D. and Schemann, M. (2005). Enteric nervous system. Curr. Opin. Gastroenterol. 21,176 -182.[CrossRef][Medline]
Le Douarin, N. and Kalcheim, C. (1999). The Neural Crest. Cambridge: Cambridge University Press.
Natarajan, D., Marcos-Gutierrez, C., Pachnis, V. and De Graaff, E. (2002). Requirement of signalling by receptor tyrosine kinase RET for the directed migration of enteric nervous system progenitor cells during mammalian embryogenesis. Development 129,5151 -5160.[Medline]
Newgreen, D. and Young, H. M. (2002). Enteric nervous system: development and developmental disturbances - part 1. Pediatr. Dev. Pathol. 5,224 -247.[Medline]
Swenson, O. (2002). Hirschsprung's disease: a
review. Pediatrics 109,914
-918.
Young, H. M. (2005). Neural stem cell therapy and gastrointestinal biology. Gastroenterology 129,2092 -2095.[Medline]
Young, H. M., Hearn, C. J., Farlie, P. G., Canty, A. J., Thomas, P. Q. and Newgreen, D. F. (2001). GDNF is a chemoattractant for enteric neural cells. Dev. Biol. 229,503 -516.[CrossRef][Medline]
Young, H. M., Bergner, A. J., Anderson, R. B., Enomoto, H., Milbrandt, J., Newgreen, D. F. and Whitington, P. M. (2004). Dynamics of neural crest-derived cell migration in the embryonic mouse gut. Dev. Biol. 270,455 -473.[CrossRef][Medline]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
