Development of the enteric nervous system, smooth muscle and interstitial cells of Cajal in the human gastrointestinal tract
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- Wallace, A.S. & Burns, A.J. Cell Tissue Res (2005) 319: 367. doi:10.1007/s00441-004-1023-2
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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 α-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 α-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.
KeywordsDevelopmentEnteric nervous systemSmooth muscleInterstitial cells of CajalHuman (embryo)
The gastrointestinal tract is formed from a primitive gut tube that arises early in development and consists of the endoderm, which gives rise to the inner epithelial lining of the gut, and splanchnic mesoderm, which gives rise to the smooth muscle (Roberts 2000). During the development of the gastrointestinal tract, neurectoderm-derived neuronal precursors colonize the lengthening gut and become distributed in concentric plexus within the gut wall to form the enteric nervous system (ENS), the intrinsic innervation of the gastrointestinal tract. All of the neurons and glial cells of the ENS are derived from the neural crest. Neural crest cell (NCC) ablation studies (Yntema and Hammond 1954; Peters-van der Sanden et al. 1993; Burns et al. 2000), quail-chick interspecies grafting (Le Douarin and Teillet 1973, 1974; Burns and Le Douarin 2001) and other cell-tracing experiments (Epstein et al. 1994) have demonstrated that the rhombencephalic (vagal) neural crest, adjacent to somites 1–7, contributes the majority of ENS cells along the entire length of the gut. A contribution from the sacral neural crest to the post-umbilical gut has also been demonstrated in the chick and mouse (Le Douarin and Teillet 1973; Pomeranz and Gershon 1990; Pomeranz et al. 1991; Serbedzija et al. 1991; Burns and Le Douarin 1998), although the extent of the contribution and the phenotypes that arise from these cells have yet to be fully elucidated in mammals.
ENS formation requires the co-ordinated migration, proliferation, differentiation and survival of NCC within the developing gut. Numerous genes/signalling molecules have recently been shown to be involved in these processes; these include transcription factors (e.g. Phox2b, Sox10, Pax3, Mash1), components of the RET and ET-3/EDNRB signalling pathways, secreted proteins (Hedgehog, BMPs), neurotrophic factors (e.g. neurotrophin-3) and extracellular matrix (ECM) molecules (e.g. laminin) (Gershon 1998, 1999a, 1999b; Taraviras and Pachnis 1999; Ramalho-Santos et al. 2000; Manie et al. 2001; Chalazonitis et al. 2004). Mutations in genes encoding some of these signalling molecules have been documented in patients with Hirschsprung disease (HSCR), a congenital defect affecting the gut of 1 in 5,000 newborn infants (Wartiovaara et al. 1998; Amiel and Lyonnet 2001). In HSCR, the hindgut (usually the distal colon) is devoid of enteric neurons and glia, a condition that results in life-threatening intestinal obstruction and megacolon (Kapur 1999).
Much evidence has been accumulated suggesting that, in order to develop normally, migrating NCC interact with signalling cues that are encountered along their migration pathways (Le Douarin and Kalcheim 1999). It is therefore not surprising that interactions between migrating NCC and the local gut environment are essential for normal ENS development. For example, in the RET and ET-3/EDNRB pathways, the receptor components (RET, GFRα1; EDNRB) are present on migrating NCC (Pachnis et al. 1993; Nataf et al. 1996), whereas the ligands (glial-cell-derived neurotrophic factor, GDNF; ET-3) are expressed in the gut mesenchyme (Hellmich et al. 1996; Leibl et al. 1999). GDNF has been shown to promote the survival, proliferation and neuronal differentiation of ENS precursor cells in vitro (Heuckeroth et al. 1998; Gianino et al. 2003) and has also been demonstrated to be a chemoattractant for vagal NCC (Young et al. 2001; Natarajan et al. 2002). The role of ET-3 is less clear but it has been shown to affect the rate of colonization and possibly the number of vagal NCC that migrate along the gut (Barlow et al. 2003). ET-3 may also affect the environment through which NCC migrate, as increased expression of the ECM molecule, laminin, is evident within the gut of mice lacking ET-3 (Rothman et al. 1996). Alterations in laminin and collagen type IV (Parikh et al. 1992) and other molecules, including tenascin and fibronectin (Parikh et al. 1994), and nidogen (Parikh et al. 1995) have been documented in patients with HSCR, thus implicating ECM molecules in enteric neurogenesis. ECM molecules have various roles in development, including the provision of matrices for navigation during cell migration, and the promotion of neurite outgrowth, neuronal differentiation and cell survival, and are produced by the gut mesenchyme and subsequently by smooth muscle and basal lamina (Tamiolakis et al. 2002; Rauch and Schafer 2003). It is therefore important to correlate the development of NCC in the gastrointestinal tract with the development of the smooth muscle layers in order to provide a basis for further studies investigating the expression of ECM molecules during human gut development.
In addition to the NCC-derived neurons and mesenchymal smooth muscle, a third class of cell type has been shown to be involved in the mature neuromuscular function of the gut. These specialized network-forming cells, the interstitial cells of Cajal (ICC), have diverse morphologies (Burns et al. 1997) and a variety of functions in the different regions of the gut (Hirst and Ward 2003). These include the generation of gut pacemaker activity (the basis for the electrical slow waves that underlie the phasic contractions of gastrointestinal muscles; Ward et al. 1994) and the transmission of nerve signals to the smooth muscle (Burns et al. 1996; Ward et al. 2000). ICC are derived from the gut mesenchyme (Lecoin et al. 1996; Young et al. 1996) and require signalling through the kit tyrosine kinase receptor (Chabot et al. 1988) and its ligand, stem cell (steel) factor (SCF; Zsebo et al. 1990) for their development (Ward et al. 1994, 1995; Huizinga et al. 1995). ICC can be immunohistochemically labelled by using anti-kit antibodies (Burns et al. 1997) and, although such antibodies have been used to good effect to study the development of ICC in animal models (Ward and Sanders 2001), little information is available regarding the early differentiation of these cells in the human gut. Investigations have instead focused on the role of ICC in a range of clinical contexts (Kenny et al. 1998; Hagger et al. 2000; Rolle et al. 2002; Feldstein et al. 2003; Tong et al. 2004).
Here, our aim has been to examine the spatiotemporal development of the three cell types necessary for gut neuromuscular activity. We have utilized human embryos and fetal material ranging from weeks 4 to 14 of development and show that the entire length of the gut is colonized by a rostrocaudal wave of migrating NCC at week 7. Smooth muscle development also occurs as a rostrocaudal wave of maturation, with distinct circular muscle layers appearing in the foregut at week 8 and in the hindgut by week 11. ICC begin to differentiate from the gut mesenchyme at week 9 and rapidly become oriented to surround the ENS cells of the myenteric plexus.
Materials and methods
Human embryonic and fetal material
Human material was obtained from voluntary medical or surgical terminations of pregnancy (MTOP/STOP) after parental consent and ethical approval from the local committee had been obtained. Staging of embryos was carried out according to the Carnegie system by using anatomical features and foot length as guides to age. Tissue was obtained in the UK from either the Human Developmental Biology Resource (HDBR), a resource funded jointly by the Medical Research Council (MRC) and the Wellcome Trust, or from the MRC-funded Tissue Bank.
Embryos aged between 4 weeks and 6 weeks were prepared intact, whereas in older specimens aged between 7 weeks and 14 weeks, the gastrointestinal tract was dissected free from surrounding tissue, straightened and pinned out in a Sylgard-coated dish (Dow Corning). Embryos or segments of gut were chemically fixed in 4% paraformaldehyde (PFA) for 3–4 h or overnight, depending on size. They were then rinsed repeatedly in phosphate-buffered saline (PBS; 3×5 min) and prepared for wax embedding or for cryosectioning. For wax embedding, tissues were dehydrated through an ascending series of alcohol (1×5 min each), cleared in Histoclear (2×5 min) and then left overnight in molten wax at 56°C prior to embedding. For cryosectioning, samples were left overnight in 15% sucrose solution in PBS and then transferred to a solution containing 15% sucrose, 5% gelatin in PBS at 37°C. Tissues were then placed in blocks, oriented appropriately in the cooling gelatin solution and frozen in isopentane, precooled in liquid nitrogen to −60°C.
Transverse sections were cut from whole embryos and dissected gut segments. Sections were obtained at a thickness of 6 μm from wax blocks and 10–15 μm from frozen blocks. All sections were placed on Superfrost Plus microscope slides (BDH Laboratories). Adjacent sections were also prepared for double immunohistochemical labelling.
α-Smooth muscle actin
Goat anti-rabbit IgG biot
Streptavidin Cy3 (1:400, Amersham) or streptavidin-HRP (1:100, Southern Biotech), then DAB (1:50, Sigma)
Rabbit anti-mouse IgG biot
Streptavidin Alexa488 (1:200, Molecular Probes)
Images were captured on a Zeiss Axiophot microscope equipped with a Hamamatsu C4742-95 or Leica DC-500 digital camera by using Improvision Openlab (v3.11) and Leica Firecam (v1.20) software, respectively. Figures were assembled and annotated by using Adobe Photoshop 7 software (Adobe).
Rostrocaudal gut colonization by p75NTR-positive neural crest cells
Development of gut smooth muscle layers positive for α-smooth muscle actin
Development of kit-positive ICC within the gastrointestinal tract
In this study, we have analysed the spatiotemporal development of three cell types necessary for the co-ordinated neuromuscular activity of the gastrointestinal tract: (1) NCC-derived precursor cells that form the neurons and glial cells of the ENS; (2) the smooth muscle cells that form the contractile component of the gut; (3) the ICC that are involved in electrical pacemaking of the smooth muscle and in the mediation of neurotransmission between enteric nerves and gut muscle. We have utilized an antibody against the low affinity neurotrophin receptor p75NTR to label migrating NCC within the developing human gastrointestinal tract. This antibody has previously been employed extensively to label NCC in species including the amphibian Xenopus laevis (Sundqvist and Holmgren 2004), the embryonic mouse (Chalazonitis et al. 1998; Young et al. 1998a, 1999, 2003), the developing human gut (Fu et al. 2003) and other various mammals including adult human, horse, cow, sheep, pig, rabbit and rat (Esteban et al. 1998). In agreement with the recent findings of Fu et al. (2003), p75NTR-positive NCC have been found to colonize the human gut in a rostrocaudal direction from week 4 to week 7 of development, when the migration front of NCC reaches the terminal hindgut. At week 4, NCC are initially located in the dorsal aspect of the foregut and, as they migrate along the gut in the subsequent weeks, they encircle the gut and become grouped into primitive ganglia. The rostrocaudal migration of NCC along the gastrointestinal tract is similar to the pattern of colonization reported in most species to date, including fish (Shepherd et al. 2004), avians (Burns and Le Douarin 2001) and mice (Young et al. 1998a) and most recently in humans (Fu et al. 2003; Wallace and Burns 2003; Fu et al. 2004). Based on comparable labelling studies, the origin of the NCC that colonise the human gut may be attributed to the vagal neural crest. Similar findings concerning the rostrocaudal colonization of the gut by NCC have also been obtained following the investigation of RET expression in the developing human gastrointestinal tract (Attie-Bitach et al. 1998). Attie-Bitach et al. (1998) have found RET-expressing ENS precursor cells in the human foregut at 26–30 days (approximately week 4) and in the midgut and hindgut at 37–42 days (5–6 weeks), i.e. at time points similar to those described in this study.
In addition to the rostrocaudal migration of crest cells along the gut, we have also demonstrated considerable variation in crest cell distribution in different gut regions as development progresses. For example, at week 7 (Fig. 3), although NCC have formed ganglia in the oesophagus and stomach, crest cells are distributed in the outer layers of the mesenchyme of the midgut and are scattered widely throughout the mesenchyme of the colon. Therefore, the maturation of the ENS, in terms of myenteric ganglia formation, also proceed in a rostrocaudal manner, with the myenteric plexus of the oesophagus being formed first and that of the colon approximately 3 weeks later.
The observation that NCC initially colonize the outer layers of the gut mesenchyme, thus forming the myenteric plexus, is consistent with the pattern of gut colonization previously described in laboratory mammals (Kapur et al. 1992; Gershon et al. 1993; McKeown et al. 2001) and in the small intestine of the chick (Burns and Le Douarin 1998). In these animals, the submucosal plexus forms later, following the secondary centripetal migration of crest cells from the myenteric plexus. In the present study, we have observed a similar rostrocaudal gradient of maturation of the submucosal plexus, with NCC occurring in the foregut beginning at week 8 and with well-developed submucosal ganglia in the midgut at week 11, approximately 3 weeks after the myenteric plexus has formed, a finding similar to that obtained by Fu et al. (2004). Fu et al. (2004) have reported scattered single neurons and a small submucosal plexus in the foregut and midgut at week 9; this plexus increases in size by week 12. However, submucosal ganglia have not been observed in the hindgut until week 14 (Fu et al. 2004). The centripetal pattern of gut colonization reported here, and previously in mice, is apparently attributable to the secondary migration of NCC from the myenteric plexus, inwards through the circular muscle layer. Although ligands such as GDNF and ET-3 (for the RET and EDNRB cell-cell signalling pathways, respectively) have been shown to be expressed in the gut mesenchyme (Suvanto et al. 1996; Leibl et al. 1999; Natarajan et al. 2002), their patterns of expression do not appear to coincide with this spatiotemporal migration of NCC within restricted areas of the gut mesenchyme and, therefore, do not explain the specific primary formation of the myenteric plexus followed by the formation of the submucosal plexus. However, netrins and netrin receptors, such as those that are deleted in colorectal cancer (DCC), have recently been shown to be involved in guiding this secondary migration of crest cells during the formation of the submucosal plexus (Jiang et al. 2003), since DCC crest cells appear to be attracted to areas of the gut at which netrin expression is high (i.e. in the submucosa).
NCC have been found to progress along the gut in a rostrocaudal direction, whereas the hindgut remains free from NCC until the migration front of vagal crest-derived cells has colonized the entire length of the gut by week 7. Therefore, as previously reported in the mouse (Young et al. 1998a; Kapur 2000) and the chick (Burns et al. 2000), there is no evidence for an early migration of NCC into the hindgut from a source that could be attributed to the sacral neural crest (Burns and Le Douarin 1998). The extent of the contribution of sacral NCC to the hindgut in the human embryo has yet to be determined and has not been addressed in this study.
We have demonstrated a rostrocaudal gradient of smooth muscle maturation as evidenced by the appearance of αSMA immunoreactivity. A broad band of αSMA staining is present in the circular muscle layer of the oesophagus at week 8 and in the hindgut at week 11. Although other immunohistochemical studies have also reported gradients of maturation in gut smooth muscle (Fu et al. 2004), the chronological correspondence between ultrastructural and histochemical development is poorly understood, as ultrastructural investigations have not yet confirmed the existence of such gradients (Gabella 2002). For example, a detailed ultrastructural and immunohistochemical study by Ward and Torihashi (1995) of the developing canine proximal colon has demonstrated that the increase in thickness of the tunica muscularis is attributable to an increase in number and size of smooth muscle cells, which also change their desmin and vimentin immuno-profile during the fetal period. However, because only the proximal colon has been examined, a correlation between a rostrocaudal change in smooth muscle cell morphology and expression of smooth muscle markers cannot be made.
Fu et al. (2004) have recently suggested that, in the human, the longitudinal and circular muscle layers appear at the same time and differentiate in a rostrocaudal manner between weeks 7 and weeks 9. However, we have found no αSMA immunoreactivity within the presumptive longitudinal muscle layer of the intestine or colon at week 8, whereas the circular muscle layer is αSMA-positive at these stages of development (see Fig. 6). This finding suggests that, in the mid- and hindgut, the longitudinal muscle layer “matures” (i.e. expresses αSMA) after the circular muscle layer. Such delayed maturation of the longitudinal muscle layer has been reported in the gut of the chick embryo by using the smooth muscle marker, 13F4 (Burns and Le Douarin 1998).
Expression of the tyrosine kinase receptor c-kit and its ligand, SCF, are essential for ICC development (Ward et al. 1994, 1995). During the establishment of the ICC network in the avian embryo, kit receptor is expressed in individual cells located at the outer border of the circular muscle layer, whereas its ligand, SCF, is expressed within the circular muscle layer (Lecoin et al. 1996). Other authors have shown that enteric neurons also express SCF (Torihashi et al. 1996; Young et al. 1998b), thus raising the question of whether ICC are capable of developing in the absence of SCF-expressing enteric neurons, an issue of particular relevance in a clinical context such as HSCR in which the absence of enteric neurons in one region of the bowel may have implications for the development of ICC, and subsequent gut function, in adjacent regions. The study by Wu et al. (2000) has shown that kit and its ligand are present in aganglionic gut obtained from ls/ls and c-ret−/− mice and that ICC develop in NCC-free cultures of ls/ls terminal colon, thus suggesting that enteric neurons are not required for the development of ICC. Similar findings have also been reported by Lecoin et al. (1996), following the appearance of ICC in cultured aganglionic chicken gut. Since Wu et al. (2000) have demonstrated that SCF immunoreactivity is coincident in cells with neuronal or smooth muscle markers, the kit ligand therefore seems to be provided by neurons and/or by cells in a smooth muscle lineage, which we show here to form before ICC in the human gut. This idea is further supported by investigations with Gdnf−/− mice, which lack ENS cells in the majority of the gut (Ward et al. 1999). Data from these animals suggest that the presence of neurons is not required for the formation of a mature ICC network and that the circular muscle layer, which develops before the ICC, can provide sufficient SCF for the formation of functional ICC networks. However, the developmental relationship between neurons and ICC is still contentious, particularly in humans, in which conflicting findings have recently been described (Huizinga et al. 2001; Rolle et al. 2002; Newman et al. 2003).
Here, we have identified kit-positive ICC in the intestine at week 9, after the colonization of the gut by NCC and following the differentiation of the circular muscle layer. Unlike these other cells, ICC do not appear to mature in a distinct rostrocaudal wave, as kit immunoreactivity is more defined in the hindgut than in the midgut at week 9. ICC rapidly mature and, by week 11, kit immunoreactivity is restricted to cells surrounding the myenteric ganglia, in a pattern that is more organized in the midgut than in the hindgut. Similar reports of ICC surrounding myenteric ganglia have been described in the human fetal small bowel (Kenny et al. 1999; Wester et al. 1999). However, Fu et al. (2004) have identified kit-positive cells within myenteric plexus at week 12; these then move to the periphery of ganglia by week 14. These authors have subsequently identified ICC preferentially located at the periphery of ganglia at week 20 (Fu et al. 2004). We have not been able to identify kit-immunopositive cells within ganglia, although we have not performed double-labelling studies with kit and neuronal markers. However, our study suggests that ICC are located on the periphery of ganglia as early as week 9 and certainly by week 11 (see Fig. 8b, c). ICC development therefore appears to lag behind that of the ENS by at least 3 weeks and slightly behind that of smooth muscle differentiation, as evidenced by αSMA immunoreactivity. A similar developmental lag for ICC has also been reported in the mouse embryo (Wu et al. 2000), as ICC forms after the gut has been colonized by neural precursors and after the development of αSMA-immunopositive muscle.
We thank the joint Medical Research Council/Wellcome Trust-funded Human Developmental Biology Resource (HDBR) for provision of human embryonic and fetal material.