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ABSTRACT: The enteric nervous system (ENS), the intrinsic innervation of the gastrointestinal tract, is an essential component of the gut neuromusculature and controls many aspects of gut function, including coordinated muscular peristalsis. The ENS is entirely derived from neural crest cells (NCC) which undergo a number of key processes, including extensive migration into and along the gut, proliferation, and differentiation into enteric neurons and glia, during embryogenesis and fetal life. These mechanisms are under the molecular control of numerous signaling pathways, transcription factors, neurotrophic factors and extracellular matrix components. Failure in these processes and consequent abnormal ENS development can result in so-called enteric neuropathies, arguably the best characterised of which is the congenital disorder Hirschsprung disease (HSCR), or aganglionic megacolon. This review focuses on the molecular and genetic factors regulating ENS development from NCC, the clinical genetics of HSCR and its associated syndromes, and recent advances aimed at improving our understanding and treatment of enteric neuropathies.
Clinical Genetics 11/2012; · 3.13 Impact Factor
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ABSTRACT: Neural crest cells (NCC) are multipotent progenitors that migrate extensively throughout the developing embryo and generate a diverse range of cell types. Vagal NCC migrate from the hindbrain into the foregut and from there along the gastrointestinal tract to form the enteric nervous system (ENS), the intrinsic innervation of the gut, and into the developing lung buds to form the intrinsic innervation of the lungs. The aim of this study was to determine the developmental potential of vagal NCC that had already colonised the gut or the lungs. We used transgenic chicken embryos that ubiquitously express green fluorescent protein (GFP) to permanently mark and fate-map vagal NCC using intraspecies grafting. This was combined with back-transplantation of gut and lung segments, containing GFP-positive NCC, into the vagal region of a second recipient embryo to determine, using immunohistochemical staining, whether gut or lung NCC are competent of re-colonising both these organs, or whether their fate is restricted. Chick(GFP)-chick intraspecies grafting efficiently labelled NCC within the gut and lung of chick embryos. When segments of embryonic day (E)5.5 pre-umbilical midgut containing GFP-positive NCC were back-transplanted into the vagal region of E1.5 host embryos, the GFP-positive NCC remigrated to colonise both the gut and lungs and differentiated into neurons in stereotypical locations. However, GFP-positive lung NCC did not remigrate when back-transplanted. Our studies suggest that gut NCC are not restricted to colonising only this organ, since upon back-transplantation GFP-positive gut NCC colonised both the gut and the lung.
The International journal of developmental biology 01/2012; 56(4):245-54. · 2.16 Impact Factor
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ABSTRACT: Currently available therapies for gastrointestinal motility conditions are often inadequate. Recent scientific advances, however, have facilitated the identification of neural stem cells as novel tools for cellular replenishment. Such cells can be generated from a number of tissue sources including the gut itself. Neural stem cells can readily be harvested from postnatal human gut including by conventional endoscopy, and in experimental transplantation studies appear capable of generating a neo-Enteric Nervous System. Current initiatives are addressing pre-clinical proof of concept studies in vivo utilising animal models of disease. Although definitive cell replenishment therapies for gut motility disorders appear to be an exciting and realistic prospect, even in the short-term, a number of challenges remain to be addressed before definitive clinical application.
Current Opinion in Pharmacology 11/2011; 11(6):617-23. · 6.86 Impact Factor
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Caroline Rooryck,
Anna Diaz-Font,
Daniel P S Osborn,
Elyes Chabchoub,
Victor Hernandez-Hernandez,
Hanan Shamseldin,
Joanna Kenny,
Aoife Waters,
Dagan Jenkins,
Ali Al Kaissi, [......],
Bruno Dallapiccola,
Franco Carnevale,
Maria Bitner-Glindzicz,
Melissa Lees,
Raoul Hennekam,
Philip Stanier, Alan J Burns,
Hilde Peeters,
Fowzan S Alkuraya,
Philip L Beales
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ABSTRACT: 3MC syndrome has been proposed as a unifying term encompassing the overlapping Carnevale, Mingarelli, Malpuech and Michels syndromes. These rare autosomal recessive disorders exhibit a spectrum of developmental features, including characteristic facial dysmorphism, cleft lip and/or palate, craniosynostosis, learning disability and genital, limb and vesicorenal anomalies. Here we studied 11 families with 3MC syndrome and identified two mutated genes, COLEC11 and MASP1, both of which encode proteins in the lectin complement pathway (collectin kidney 1 (CL-K1) and MASP-1 and MASP-3, respectively). CL-K1 is highly expressed in embryonic murine craniofacial cartilage, heart, bronchi, kidney and vertebral bodies. Zebrafish morphants for either gene develop pigmentary defects and severe craniofacial abnormalities. Finally, we show that CL-K1 serves as a guidance cue for neural crest cell migration. Together, these findings demonstrate a role for complement pathway factors in fundamental developmental processes and in the etiology of 3MC syndrome.
Nature Genetics 01/2011; 43(3):197-203. · 35.53 Impact Factor
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ABSTRACT: Within the embryonic lung, intrinsic nerve ganglia, which innervate airway smooth muscle, are required for normal lung development and function. We studied the development of neural crest-derived intrinsic neurons within the embryonic mouse lung by crossing Wnt1-Cre mice with R26R-EYFP reporter mice to generate double transgenic mice that express yellow fluorescent protein (YFP) in all neural crest cells (NCCs) and their derivatives. In addition to utilizing conventional immunohistochemistry on frozen lung sections, the complex organization of lung innervation was visualized in three dimensions by combining the genetic labelling of NCCs with optical projection tomography, a novel imaging technique that is particularly useful for the 3D examination of developing organs within embryos. YFP-positive NCCs migrated into the mouse lung from the oesophagus region at embryonic day 10.5. These cells subsequently accumulated around the bronchi and epithelial tubules of the lung and, as shown by 3D lung reconstructions with optical projection tomography imaging, formed an extensive, branching network in association with the developing airways. YFP-positive cells also colonized lung maintained in organotypic culture, and responded in a chemoattractive manner to the proto-oncogene, rearranged during transfection (RET) ligand, glial-cell-line-derived neurotrophic factor (GDNF), suggesting that the RET signalling pathway is involved in neuronal development within the lung. However, when the lungs of Ret(-/-) and Gfrα1(-/-) embryos, deficient in the RET receptor and GDNF family receptor α 1 (GFRα1) co-receptor respectively, were examined, no major differences in the extent of lung innervation were observed. Our findings demonstrate that intrinsic neurons of the mouse lung are derived from NCCs and that, although implicated in the development of these cells, the role of the RET signalling pathway requires further investigation.
Journal of Anatomy 12/2010; 217(6):651-64. · 2.37 Impact Factor
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ABSTRACT: Two noncoding variations in RET-the T allele of the single nucleotide polymorphism (SNP) rs2435357 (Enh1:C>T) and the A allele of the SNP rs2506004 (Enh2:C>A)-are associated with Hirschsprung's disease. These SNPs are in strong linkage disequilibrium and located in an enhancer element in intron 1 of the RET gene. The T allele of the Enh1 variant results in reduced expression of RET, compared with the C allele, because the T allele disrupts binding to the transcription factor SOX10. We studied whether the A allele of Enh2 (Enh2-A) also affects RET gene expression.
We evaluated the function of Enh1 and Enh2 using luciferase reporter assays with constructs that contained each allele, separately or in combination. We performed in silico analysis to identify transcription activators or repressors that bind to Enh2-C.
The Enh1-T and the Enh2-A alleles reduced expression of the luciferase reporter gene. In silico analysis identified the sequence of Enh2-C and its surrounding sequence (ACGTG) as a potential binding site for the NXF-ARNT2 and SIM2-ARNT2 transcription factor heterodimers. The affinity of NXF-ARNT2 for Enh2-C was confirmed by electrophoresis mobility shift and supershift assays. Transfection of neuroblastoma cell lines with NXF-ARNT2 or SIM2-ARNT2 increased and decreased expression of RET, respectively.
More than one SNP on an associated haplotype can influence gene expression and ultimately disease phenotype. Binding of the transcription factors NXF, ARNT2, and SIM2 to RET depend on the RET polymorphism of Enh2 and affect RET expression and the development of Hirschsprung's disease.
Gastroenterology 10/2010; 140(2):572-582.e2. · 11.68 Impact Factor
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ABSTRACT: The aim of this study was to assess the angiogenic potential of chitosan-glycerol phosphate (GP)-hydroxyethyl cellulose (HEC) binder for injectable bone tissue engineering applications. The angiogenic response of chitosan-GP-HEC combined with and without human bone marrow-derived mesenchymal stem cells (hMSCs) was examined using the chick chorioallantoic membrane (CAM) assay. Chitosan-GP-HEC gel did not show any angiogenic potential, whereas the presence of hMSCs gave rise to an enhanced angiogenic response when placed on the CAM for 3 days. Quantitatively, significantly more blood vessel formation was observed for the stem cell-containing group as compared to all other groups (p < 0.05), except for the b-FGF-positive control. The results indicate that the chitosan-GP-HEC binder does not contribute to enhanced angiogenesis and that the presence of hMSCs enhances angiogenesis.
Journal of Tissue Engineering and Regenerative Medicine 06/2010; 4(4):309-15. · 3.28 Impact Factor
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01/2010: pages 263 - 300; , ISBN: 9780470090121
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ABSTRACT: Motility patterns in the mature intestine require the coordinated interaction of enteric neurons, gastrointestinal smooth muscle, and interstitial cells of Cajal. In Hirschsprung's disease, the aganglionic segment causes functional obstruction, and thus the enteric nervous system (ENS) is essential for gastrointestinal motility after birth. Here we review the development of the ENS. We then focus on motility patterns in the small intestine and colon of fetal mice and larval zebrafish, where recent studies have shown that the first intestinal motility patterns are not neurally mediated. Finally, we review the development of gastrointestinal motility in humans.
Seminars in Pediatric Surgery 11/2009; 18(4):196-205. · 2.93 Impact Factor
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ABSTRACT: Collagen is an abundant protein found in the extracellular matrix of many tissues. Due to its biocompatibility, it is a potentially ideal biomaterial for many tissue engineering applications. However, harvested collagen often requires restructuring into a three-dimensional matrix to facilitate applications such as implantation into poorly accessible tissue cavities. The aim of the current study was to produce a conformable collagen-based scaffold material capable of supporting tissue regeneration for use in wound repair applications. Microporous collagen spheres were prepared using a thermally induced phase separation (TIPS) technique and their biocompatibility was assessed. The collagen spheres were successfully cross-linked with glutaraldehyde vapour, rendering them mechanically more stable. When cultured with myofibroblasts the collagen spheres stimulated a prolonged significant increase in secretion of the angiogenic growth factor, vascular endothelial growth factor (VEGF), compared with cells alone. Control polycaprolactone (PCL) spheres failed to stimulate a similar prolonged increase in VEGF secretion. An enhanced angiogenic effect was also seen in vivo using the chick embryo chorioallantoic membrane assay, where a significant increase in the number of blood vessels converging towards collagen spheres was observed compared with control PCL spheres. The results from this study indicate that microporous collagen spheres produced using TIPS are biologically active and could offer a novel conformable scaffold for tissue regeneration in poorly accessible wounds.
Acta biomaterialia 10/2009; 6(3):1158-66. · 3.98 Impact Factor
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ABSTRACT: Enteric nervous system stem cells (ENSSCs) provide potential therapeutic tools to replenish absent ganglia in Hirschsprung's disease. Although full-thickness human postnatal gut tissue can be used to generate ENSSCs, reliance on its harvesting from surgical resection poses significant practical limitations. This study aimed to explore whether gut tissue obtained utilizing minimally invasive routine endoscopy techniques could be used to generate ENSSCs and whether such cells retain the potential to generate an ENS upon transplantation into aganglionic gut.
Postnatal human gut mucosal tissue obtained from children undergoing gastrointestinal endoscopy was used to generate cell cultures in which ENSSCs were contained within neurosphere-like bodies (NLBs). These NLBs were characterized by immunostaining, and their potential to generate components of the ENS, in vitro and upon transplantation into models of aganglionic gut, was examined.
Gut mucosal biopsy specimens were obtained from 75 children (age, 9 months-17 years). The biopsy specimens contained neural cells and ENSSCs and, on culturing, generated characteristic NLBs at all ages examined. Postnatal mucosa-derived NLBs contained cells that, akin to their embryonic counterparts, were proliferating, expressed ENSSC markers, were bipotent, and capable of generating large colonies in clonogenic cultures and multiple ENS neuronal subtypes. Upon transplantation, cells from NLBs colonized cultured recipient aganglionic chick and human hindgut to generate ganglia-like structures and enteric neurons and glia.
The results represent a significant practical advance toward the development of definitive cell replenishment therapies for ENS disorders such as Hirschsprung's disease.
Gastroenterology 06/2009; 136(7):2214-25.e1-3. · 11.68 Impact Factor
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Jonathan L Tobin,
Matt Di Franco,
Erica Eichers,
Helen May-Simera,
Monica Garcia,
Jiong Yan,
Robyn Quinlan,
Monica J Justice,
Raoul C Hennekam,
James Briscoe,
Masazumi Tada,
Roberto Mayor, Alan J Burns,
James R Lupski,
Peter Hammond,
Philip L Beales
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ABSTRACT: Facial recognition is central to the diagnosis of many syndromes, and craniofacial patterns may reflect common etiologies. In the pleiotropic Bardet-Biedl syndrome (BBS), a primary ciliopathy with intraflagellar transport dysfunction, patients have a characteristic facial "gestalt" that dysmorphologists have found difficult to characterize. Here, we use dense surface modeling (DSM) to reveal that BBS patients and mouse mutants have mid-facial defects involving homologous neural crest-derived structures shared by zebrafish morphants. These defects of the craniofacial (CF) skeleton arise from aberrant cranial neural crest cell (NCC) migration. These effects are not confined to the craniofacial region, but vagal-derived NCCs fail to populate the enteric nervous system, culminating in disordered gut motility. Furthermore, morphants display hallmarks of disrupted Sonic Hedgehog (Shh) signaling from which NCCs take positional cues. We propose a model whereby Bbs proteins modulate NCC migration, contributing to craniofacial morphogenesis and development of the enteric nervous system. These migration defects also explain the association of Hirschsprung's disease (HD) with BBS. Moreover, this is a previously undescribed method of using characterization of facial dysmorphology as a basis for investigating the pathomechanism of CF development in dysmorphic syndromes.
Proceedings of the National Academy of Sciences 06/2008; 105(18):6714-9. · 9.68 Impact Factor
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ABSTRACT: The enteric nervous system (ENS) is mainly derived from vagal neural crest cells (NCC) that arise at the level of somites 1-7. To understand how the size and composition of the NCC progenitor pool affects ENS development, we reduced the number of NCC by ablating the neural tube adjacent to somites 3-6 to produce aganglionic gut. We then back-transplanted various somite lengths of quail neural tube into the ablated region to determine the 'tipping point', whereby sufficient progenitors were available for complete ENS formation. The addition of one somite length of either vagal, sacral or trunk neural tube into embryos that had the neural tube ablated adjacent to somites 3-6, resulted in ENS formation along the entire gut. Although these additional cells contributed to the progenitor pool, the quail NCC from different axial levels retained their intrinsic identities with respect to their ability to form the ENS; vagal NCC formed most of the ENS, sacral NCC contributed a limited number of ENS cells, and trunk NCC did not contribute to the ENS. As one somite length of vagal NCC was found to comprise almost the entire ENS, we ablated all of the vagal neural crest and back-transplanted one somite length of vagal neural tube from the level of somite 1 or somite 3 into the vagal region at the position of somite 3. NCC from somite 3 formed the ENS along the entire gut, whereas NCC from somite 1 did not. Intrinsic differences, such as an increased capacity for proliferation, as demonstrated in vitro and in vivo, appear to underlie the ability of somite 3 NCC to form the entire ENS.
Development 06/2008; 135(9):1681-91. · 6.60 Impact Factor
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ABSTRACT: The formation of neural tissue, in association with airway smooth muscle (ASM), is a feature of normal lung development and function. Intrinsic neuronal tissue has recently been shown, in animal models, to be derived from neural crest cells (NCC). Since defects in NCC development underlie a range of disease states (neurocristopathies), it is important to determine the spatiotemporal development of NCC in the human lung, as defects in their development could have pathophysiologic implications. The aims of this study were to: (1) establish a time course for the formation of ASM and neural tissue within the embryonic and fetal human lung, (2) investigate whether intrinsic neural tissue within the lung is derived from NCC, and (3) gain insight into the possible signaling mechanisms underlying the development of the intrinsic lung innervation. Using human lung tissue from Weeks 6 to 12 of gestation, we analyzed the formation of ASM, NCC, neuronal and glial tissue, and the expression of Gfralpha1, a receptor component of the RET (rearranged during transfection) tyrosine kinase signaling pathway. Our results showed that NCC accumulated along the branching airways, in close association with the ASM, and differentiated into neurons and glia. Neural crest-derived neural tissue within the lung strongly expressed membrane-bound Gfralpha1, and soluble Gfralpha1 was expressed within the lung mesenchyme, but only at early developmental stages. Together these findings indicate that the intrinsic innervation of the human lung is derived from the neural crest.
American Journal of Respiratory Cell and Molecular Biology 04/2008; 38(3):269-75. · 5.13 Impact Factor
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ABSTRACT: The enteric nervous system (ENS) is formed from vagal and sacral neural crest cells (NCC). Vagal NCC give rise to most of the ENS along the entire gut, whereas the contribution of sacral NCC is mainly limited to the hindgut. This, and data from heterotopic quail-chick grafting studies, suggests that vagal and sacral NCC have intrinsic differences in their ability to colonize the gut, and/or to respond to signalling cues within the gut environment. To better understand the molecular basis of these differences, we studied the expression of genes known to be essential for ENS formation, in sacral NCC within the chick hindgut. Our results demonstrate that, as in vagal NCC, Sox10, EdnrB, and Ret are expressed in sacral NCC within the gut. Since we did not detect a qualitative difference in expression of these ENS genes we performed DNA microarray analysis of vagal and sacral NCC. Of 11 key ENS genes examined from the total data set, Ret was the only gene identified as being highly differentially expressed, with a fourfold increase in expression in vagal versus sacral NCC. We also found that over-expression of RET in sacral NCC increased their ENS developmental potential such that larger numbers of cells entered the gut earlier in development, thus promoting the fate of sacral NCC towards that of vagal NCC.
Developmental Biology 02/2008; 313(1):279-92. · 4.07 Impact Factor
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Alan J Burns
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ABSTRACT: Interstitial cells of Cajal (ICCs) have, in the past 2 decades, been recognised as important elements in the regulation of gastrointestinal motility. Specifically, they have been shown to be critical for the generation and propagation of electrical slow waves that regulate the phasic contractile activity of gastrointestinal smooth muscle, and for mediating neurotransmission from enteric motor neurons to smooth muscle cells. These different functional roles are carried out by different phenotypic classes of ICC that have discrete distributions within the tunica muscularis. Identifying the functional roles of ICC within the gut has been facilitated by studying mutant mice deficient in ICC, either as a consequence of loss of the tyrosine kinase receptor, Kit, or its ligand, stem cell factor, both of which are necessary for normal ICC development. In humans, under certain pathophysiological conditions, loss or defects in ICC networks appear to play a role in the generation of certain motility disorders. Alterations in ICC distribution have been reported in conditions such as achalasia, chronic intestinal pseudoobstruction, Hirschsprung disease, inflammatory bowel diseases, and slow transit constipation. Molecular and genetic techniques are helping researchers to determine whether defects in ICC networks are the cause of motility disorders, or whether the disrupted ICC networks are a consequence of gut dysfunction.
Journal of pediatric gastroenterology and nutrition 01/2008; 45 Suppl 2:S103-6. · 2.18 Impact Factor
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ABSTRACT: The homeobox gene Hesx1 is an essential repressor that is required within the anterior neural plate for normal forebrain development in mouse and humans. Combining genetic cell labelling and marker analyses, we demonstrate that the absence of Hesx1 leads to a posterior transformation of the anterior forebrain (AFB) during mouse development. Our data suggest that the mechanism underlying this transformation is the ectopic activation of Wnt/beta-catenin signalling within the Hesx1 expression domain in the AFB. When ectopically expressed in the developing mouse embryo, Hesx1 alone cannot alter the normal fate of posterior neural tissue. However, conditional expression of Hesx1 within the AFB can rescue the forebrain defects observed in the Hesx1 mutants. The results presented here provide new insights into the function of Hesx1 in forebrain formation.
Development 05/2007; 134(8):1499-508. · 6.60 Impact Factor
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ABSTRACT: The generation of functional neuromuscular activity within the pre-natal gastrointestinal tract requires the coordinated development of enteric neurons and glial cells, concentric layers of smooth muscle and interstitial cells of Cajal (ICC). We investigated the genesis of these different cell types in human embryonic and fetal gut material ranging from weeks 4-14. Neural crest cells (NCC), labelled with antibodies against the neurotrophin receptor p75NTR, entered the foregut at week 4, and migrated rostrocaudally to reach the terminal hindgut by week 7. Initially, these cells were loosely distributed throughout the gut mesenchyme but later coalesced to form ganglia along a rostrocaudal gradient of maturation; the myenteric plexus developed primarily in the foregut, then in the midgut, and finally in the hindgut. The submucosal plexus formed approximately 2-3 weeks after the myenteric plexus, arising from cells that migrated centripetally through the circular muscle layer from the myenteric region. Smooth muscle differentiation, as evidenced by the expression of alpha-smooth muscle actin, followed NCC colonization of the gut within a few weeks. Gut smooth muscle also matured in a rostrocaudal direction, with a large band of alpha-smooth muscle actin being present in the oesophagus at week 8 and in the hindgut by week 11. Circular muscle developed prior to longitudinal muscle in the intestine and colon. ICC emerged from the developing gut mesenchyme at week 9 to surround and closely appose the myenteric ganglia by week 11. By week 14, the intestine was invested with neural cells, longitudinal, circular and muscularis mucosae muscle layers, and an ICC network, giving the fetal gut a mature appearance.
Cell and Tissue Research 04/2005; 319(3):367-82. · 3.11 Impact Factor
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Alan J Burns
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ABSTRACT: The enteric nervous system, the intrinsic innervation of the gastrointestinal tract, consists of large numbers of phenotypically diverse neurons and glial cells, arranged in complex interconnecting plexuses situated between the smooth muscle layers of the gut wall. Recently, the enteric nervous system has attracted much attention from developmental biologists whose efforts have focused on analysing the cellular origins of enteric nervous system precursor cells, how these cells migrate to and within the gut and the identification of signalling mechanisms which cause migrating cells to differentiate into the appropriate phenotypes in the appropriate locations. This review summarises the state of knowledge concerning the early stages of enteric nervous system development and concentrates on: (i) the embryological origins of the neural crest cells which colonise the gastrointestinal tract, (ii) their spatiotemporal migration within the gut, (iii) the possible pre-specification of neural crest cells as enteric nervous system precursors and (iv) factors influencing their directional migration within the gut.
The International Journal of Developmental Biology 02/2005; 49(2-3):143-50. · 2.82 Impact Factor
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ABSTRACT: The development of intrinsic ganglia, comprised of neurons and glia cells that innervate airway smooth muscle, is a recognized component of the growing lung. However, the embryological origin of these neurons and glia is unclear. The lung buds develop as an outgrowth of the foregut, which contains migrating neural crest cells (NCC) that ultimately give rise to the enteric nervous system (ENS) along the entire length of the gut. It has therefore been proposed that the intrinsic ganglia of the lung arise from a subset of NCC that leave the gut and migrate into the lung buds during early development. We have tested this hypothesis using quail-chick interspecies grafting to selectively label the hindbrain-derived neural crest cell population that colonizes the gut. In conjunction with antibody labeling and in situ hybridization, we demonstrate that: (i) lung ganglia arise from vagal NCC that migrate from the foregut into the lung buds; (ii) like ENS precursors, these NCC express the transcription factor Sox10, and the receptors EDNRB and RET; (iii) the co-receptor for RET, GFRalpha1, is expressed in the lung mesenchyme and in ganglia; (iv) ganglia persist within the lung throughout development and contain cells immunopositive for the pan-neuronal markers ANNA-1 and PGP9.5, the inhibitory neurotransmitter NO, as shown by NADPH-diaphorase staining, and the glial marker GFAP.
Developmental Biology 02/2005; 277(1):63-79. · 4.07 Impact Factor
