MICROSCOPY RESEARCH AND TECHNIQUE 47:303–308 (1999) Embryological Origin of Interstitial Cells of Cajal H.M. YOUNG* Department of Anatomy and Cell Biology, University of Melbourne, Parkville, 3052, VIC, Australia KEY WORDS Kit; neural crest; mesenchyme; developmental origin ABSTRACT Until recently, the embryological origin of the interstitial cells of Cajal (ICC) within the intestine was unclear. An origin from the neural crest or from the mesenchyme was considered possible because ICC possess some characteristics in common with neural crest-derived cells, and some characteristics in common with cells derived from the mesenchyme. Experiments in both mammalian and avian species, in which segments of embryonic gut were removed prior to the arrival of neural crest cells and grown in organ culture, have now shown that ICC do not arise from the neural crest. It appears that ICC and smooth muscle cells arise from common mesenchymal precursor cells. From mid-embryonic stages, ICC precursors express Kit, which is a receptor tyrosine kinase. Both ICC and many smooth muscle cell precursors initially express Kit, and then the cells destined to become smooth muscle cells down-regulate Kit and up-regulate the synthesis of myofilament proteins, whereas cells destined to differentiate into ICC maintain their expression of Kit. Adult mice with mutations that block the activity of Kit have disrupted arrays of ICC, whereas normal ICC are present until shortly after birth in such mice. It, therefore, appears that the Kit signalling pathway in not necessary for the embryonic development of ICC, but rather the post-natal proliferation of ICC. Microsc. Res. Tech. 47:303–308, 1999. r 1999 Wiley-Liss, Inc. INTRODUCTION The gastrointestinal tract is formed from cells derived from all three embryonic germ layers. The endoderm gives rise to the epithelial cells lining the lumen and the glands opening into the lumen, the mesoderm gives rise to blood and lymphatic vessels, smooth muscle cells, and connective tissue, and the ectoderm is the source of neural crest cells that migrate into the gastrointestinal tract and give rise to enteric neurons and glial cells. Although the interstitial cells of Cajal (ICC) were first described toward the end of the last century, until recently the embryological origin of ICC was uncertain. Because ICC have some characteristics in common with cells of neural crest origin (neurons and glial cells) and some characteristics in common with cells of mesenchymal origin (fibroblasts and smooth muscle cells; see below), it was unclear whether ICC arise from the neural crest or from the mesenchyme. Finally, in 1996, studies using chickens and quail (Lecoin et al., 1996) and mice (Young et al., 1996) demonstrated that ICC do not arise from the neural crest, and it was proposed that they arise from the mesenchyme. More recent studies in mice have shown that ICC appear to have a common developmental origin with smooth muscle cells (Klüppel et al., 1998; Torihashi et al., 1997). SIMILARITIES OF ICC WITH BOTH CELLS OF NEURAL CREST ORIGIN AND CELLS OF MESENCHYMAL ORIGIN Although cells with similar characteristics do not necessarily have similar developmental origins, similarities between ICC and neurons, neuronal support cells, fibroblasts and smooth muscle cells had raised the possibilities that ICC could be either of neural crest or mesenchymal origin. Cajal thought that ICC were r 1999 WILEY-LISS, INC. neural in nature because, like neurons, they could be stained with methylene blue or with silver chromate, and they also formed intimate relationships with nerves (see Christensen, 1992). Early ultrastructural studies suggested that ICC could be either specialised neuronal support cells, fibroblasts, or smooth muscle cells (see Thuneberg, 1982, 1989). More recent studies have shown that ICC in different locations differ in their appearance and the expression of particular antigens (Christensen, 1992; Thuneberg, 1989; Torihashi et al., 1994). However, ICC in all locations can be distinguished from other cell types by their ultrastructure and the expression of cell-specific markers (Christensen, 1992; Thuneberg, 1989;). There have been no reports of ICC in any location expressing glial fibrillary acidic protein (GFAP) or S100, which are selective markers of glial and Schwann cells, respectively, or pan-neuronal markers, such as neuron-specific enolase or PGP9.5 (protein gene product 9.5). Although ICC at the level of the myenteric plexus show some similarities to fibroblasts, they can be clearly distinguished from fibroblasts using ultrastructural criteria, such as inconspicuous Golgi apparatus, incomplete or no basal lamina, and numerous surface caveolae (Thuneberg, 1989). ICC associated with the inner margin of the circular muscle layer show some features in common with smooth muscle cells, such as smooth muscle actinand myosin light chain-immunoreactivity, but they differ from smooth muscle cells in that contractile filaments are less abundant and desmin is not ex- Contract grant sponsor: NHMRC (Australia). *Correspondence to: Dr. H.M. Young, Department of Anatomy and Cell Biology, University of Melbourne, Parkville, 3052, VIC, Australia. E-mail: firstname.lastname@example.org Received 20 December 1999; accepted in revised form 29 April 1999 304 H.M. YOUNG pressed at detectable levels (Torihashi et al., 1993, 1994). Hence, ICC represent a cell type clearly different from neurons, glia, fibroblasts, and smooth muscle cells, and whose developmental origin cannot be assumed from their morphological and histological characteristics. STUDIES SHOWING THAT ICC DO NOT ARISE FROM THE NEURAL CREST Like studies of many aspects of mature ICC, studies of the embryological origin of ICC were hampered by a lack of specific, reliable markers of ICC. It was only following the discovery that ICC can be specifically localised within the gastrointestinal tract using probes or antibodies to the receptor tyrosine kinase, Kit (Huizinga et al., 1995; Maeda et al., 1992; Torihashi et al., 1995; Ward et al., 1994), that studies were performed to examine the developmental origin of ICC. There is now strong evidence in both avian and mammalian species, and using a variety of experimental approaches, that ICC do not arise from the neural crest. Enteric neurons and glial cells arise from the neural crest (Yntema and Hammond, 1954). The vast majority of enteric neurons in the stomach and intestine arise from the ‘‘vagal’’ level (somites 1–7) neural crest cells, which enter the foregut and migrate rostrocaudally (Le Douarin and Teillet, 1973, 1974; Yntema and Hammond, 1954). In mice and birds, there is an interval of 3–4 days between the time that the neural crest cells first enter the foregut and when they reach the most caudal part of the hindgut (Allan and Newgreen, 1980; Burns and Le Douarin, 1998; Kapur et al., 1992; Young et al., 1998a). In birds, cells from sacral-level neural crest (caudal to somite 28) also give rise to some enteric neurons in the hindgut, but they do not enter the hindgut until approximately the same time that vagallevel neural crest cells reach the hindgut (Burns and Le Douarin, 1998). In mice, it is unclear whether sacrallevel neural crest cells give rise to some enteric neurons in the hindgut. However, if sacral-level neural crest cells do populate the hindgut, it appears that they either do not enter the hindgut, or do not differentiate into enteric neurons, until after the vagal neural crest cells have reached the hindgut (Kapur et al., 1992; Young et al., 1998a). Lecoin et al. (1996) were the first to show that ICC in the avian intestine do not arise from the neural crest. They performed two types of experiments. In the first set of experiments, they exploited the fact the nuclei of quail cells can be distinguished from those of chicken cells (Le Douarin, 1973); quail-chick chimeras were constructed in which the vagal-level neural tubes of chick embryos were replaced by vagal-level neural tubes from equivalent stage quail embryos, and the chimeras were left to grow for about a week before the gut was sectioned and processed to determine whether any of the quail cells within the intestine were positive to a c-kit nucleic acid probe. Although there were numerous c-kit-positive cells (ICC) within the gut surrounding the myenteric ganglia of the chimeras, none of the quail cells within the myenteric plexus (which were all presumably enteric neurons and glial cells) expressed c-kit. In the second series of experiments, Lecoin et al. (1996) utilised the knowledge that neural crest–derived cells do not reach the hindgut until 2–3 days after they colonise the fore- and mid-guts (see above). While they are migrating through the gut mesenchyme, neural crest–derived cells do not express a neuronal or glial phenotype, and thus to determine if neuronal or glial cell precursors are present in a particular segment of embryonic gut, it must be removed and grown in isolation for several days before being analysed for the presence of neurons or glial cells. In both chick (Lecoin et al., 1996) and mouse (Torihashi et al., 1997) embryos, ICC precursors do not express c-kit until after neural crest-derived cells have almost colonised the entire gut, and thus the chronological appearance of c-kit-positive cells cannot be used to ascertain whether ICC arise from the neural crest. Lecoin and her colleagues removed segments of hindgut from embryonic day 3.5 (E3.5) chick embryos prior to the arrival of neural crest-derived cells and grew them on the chorioallantoic membrane (CAM) of host embryonic chicks for 7 days, before the explants were processed for in situ hybridisation or electron microscopy. Confirming previous studies (Allan and Newgreen, 1980; Smith et al., 1977), no enteric neurons were found within the hindgut explants grown on the CAM. However, c-kit-positive cells, and cells with the ultrastructural characteristics of ICC were observed in the explants lacking neural crest-derived cells, thus confirming the results from the chick-quail chimeras, that ICC do not arise from the neural crest. Shortly afterwards, it was also shown that ICC in the mouse gut do not arise from the neural crest (Young et al., 1996). This study involved the removal of segments of embryonic mouse gut either before or after the arrival of neural crest-derived cells; the explants were grown under the kidney capsule of adult host mice for 3–6 weeks before being examined for the presence of neurons and Kit-immunoreactive ICC. These experiments were, therefore, very similar to the experiments of Lecoin et al. (1996) described above, in which segments of embryonic chick gut were removed and grown on the CAM. As in chick embryos, the presence of ICC in the explants of embryonic mouse gut grown under the kidney capsule was independent of the presence of enteric neurons, and Kit-immunoreactive ICC were present in all explants, regardless of the developmental stage and region from which they were removed. In contrast, enteric neurons were only present in explants containing neural crest–derived cells at the time of removal (Kapur et al., 1992 Nishijima et al., 1990). Since ICC develop in explants of embryonic chicken or mouse gut lacking neural crest–derived cells, it was concluded that ICC arise from the local mesenchyme. Recently a technique has been devised in which segments of embryonic mouse intestine can be grown in organ culture under conditions in which it retains its 3-dimensional shape, and growth and differentiation of the different cell types proceed (Fig. 1A–C; Hearn et al., 1999; Young et al., 1998a). Consistent with the results obtained when segments of embryonic chicken hindgut are grown on the CAM of host chicken embryos (Lecoin et al., 1996) or when segments of embryonic mouse intestine are grown under the kidney capsule of host adult mice (Young et al., 1996), when segments of hindgut from embryonic mouse hindgut are removed prior to the arrival of neural crest cells and grown in EMBRYOLOGICAL ORIGIN OF ICC organ culture for 6 days, Kit-immunoreactive ICC are present in the explants (Fig. 1D,E; Hearn et al., 1999). There are a number of spontaneous mouse mutants, and also mice that have been generated by genetargetting techniques, which lack neural crest-derived cells in parts of the gastrointestinal tract (see Gershon, 1997, 1998 for reviews). The presence of ICC has been examined in the intestine of mice lacking Ret and in ls/ls mice (Wu et al., 1996). Ret is a receptor tyrosine kinase, and is the receptor for glial-derived neurotrophic factor (GDNF), and in ret-/- mice there are no neural crest-derived cells in the gastrointestinal tract caudal to the stomach (Schuchardt et al., 1994). In ls/ls mice, the gene encoding endothelin 3 is disrupted and there are no neural crest–derived cells in the caudal part of the large intestine (Baynash et al., 1994). However, Kit-immunoreactive ICC are present in both the intestine of embryonic ret-/- mice and in the large intestine of ls/ls mice (Wu et al., 1996), again indicating that ICC do not arise from the neural crest. Additional evidence corroborating a non-neural crest origin of ICC is that ICC do not express any of the currently known markers of neural crest-derived cells. Neural crest-derived cells within the developing gut express a number of different markers including Ret (Durbec et al., 1996; Pachnis et al., 1993, 1998; Tsuzuki et al., 1995; Watanabe et al., 1997), the low affinity neurotrophin receptor, p75 (Chalazonitis et al., 1994; Lo and Anderson, 1995), and the transcription factor, Phox2b (Pattyn et al., 1997; Young et al., 1998a). However, ICC do not express Ret (Torihashi et al., 1997), p75 (Figs. 1F, F’), or Phox2b (Young et al., 1998a). ICC APPEAR TO HAVE A COMMON DEVELOPMENTAL ORIGIN WITH SMOOTH MUSCLE CELLS Faussone-Pellegrini (1984, 1985) examined the ultrastructural histogenesis of ICC at the level of the myenteric plexus and the deep muscular plexus in the mouse small intestine. She identified ICC precursor cells in early post-natal mice that were closely associated with nerve fibres of the myenteric plexus, but she was unable to determine whether the precursors cells were of neural crest or of mesenchymal origin. The results from two more recent studies (Klüppel et al., 1998; Torihashi et al., 1997) suggest that ICC arise from mesenchymal cells and have common precursors with smooth muscle cells. Torihashi et al. (1997) looked at the development of immunoreactivity to Kit, smooth muscle markers (actin, myosin, desmin) and a marker of neural crestderived cells (Ret) in the small intestine of embryonic and neonatal mice. They found that Kit-immunoreactive cells were first detected in the outer layers of the gut of E12.5 mice, and these cells were undifferentiated in appearance and lacked the morphological feature of both ICC and muscle cells. Kit-positive cells were peripheral to, and did not overlap with, the developing, Ret-immunoreactive myenteric ganglion cells. At E15, cells in the putative circular muscle layer, which was internal to the myenteric plexus, started to show actinand myosin-immunoreactivity, whereas cells in the location of the future longitudinal muscle layer were Kit-positive, but actin- and myosin-negative. By late embryonic stages, it appeared that a sub-population of 305 Kit-positive cells began to develop into smooth muscle cells (and showed immunoreactivity to the myofilament proteins), and subsequently no longer showed detectable levels of Kit immunoreactivity. The authors speculated that ICCs and longitudinal smooth muscle cells develop from common, Kit-positive precursor cells in the peripheral layers of the gut, and that those cells in which the Kit receptor is activated by contact with stem cell factor-expressing neural cells (see below) will differentiate into ICCs, whereas those cells without direct appositions with neural cells will differentiate into longitudinal smooth muscle cells and down-regulate the expression of Kit. The study by Klüppel et al. (1998) also suggests strongly that ICCs and smooth muscle cells arise from common precursor cells. In this study, the expression of mRNA for c-kit and smooth muscle myosin heavy chain (SMMHC) in the embryonic and post-natal mouse gut was examined. Unlike Torihashi et al. (1997), they found co-expression of c-kit and SMMHC by cells of the circular muscle layer and the future longitudinal muscle layer, and then c-kit expression was lost first from circular smooth muscle cells, and then subsequently also from longitudinal smooth muscle cells, whereas cells destined to become ICCs retained their expression of c-kit, but down-regulated expression of SMMHC. Thus, although the results of the studies by Torihashi et al. (1997) and Klüppel et al. (1998) differ somewhat, probably due to technical differences, the main conclusions are the same, that is, ICCs have a common precursor with smooth muscle cells. IS THE SCF-KIT SIGNALLING PATHWAY NECESSARY FOR THE DEVELOPMENT OR THE SURVIVAL OF ICC? In mice, all ICC appear to express c-kit. W/Wv mice, which have a mutation that reduces but does not abolish the tyrosine kinase activity of Kit, lack particular sub-populations of ICC (Huizinga et al., 1995; Ward et al., 1994). Moreover, when the normal action of the Kit receptor is blocked with neutralising antibodies during a critical post-natal period, there is a dramatic reduction in the number of myenteric ICC (Maeda et al., 1992; Torihashi et al., 1995). The natural ligand for Kit is stem cell factor (SCF, also known as steel factor, Kit ligand, or mast cell growth factor), and mice with mutations affecting the gene encoding SCF display many phenotypic changes similar to W mutants (Ward et al., 1995). It was, therefore, suggested that the SCF-Kit signalling pathway is critical for the development and/or survival of ICC (Sanders, 1996; Torihashi et al., 1995; Ward et al., 1995). Kit expression can be detected in cells in the outer mesenchyme of the gut of mid-stage embryonic mice (Bernex et al., 1996; Klüppel et al., 1998; Torihashi et al., 1997). However, electrophysiological experiments in the small intestine of late embryonic mice have shown that slow waves, which are mediated by myenteric ICC (Huizinga et al., 1995; Ward et al., 1994), cannot be detected until about one day before birth (Torihashi et al., 1997), and using ultrastructural criteria, ICC at the level of the myenteric plexus cannot be identified until the first post-natal week (FaussonePellegrini, 1985). Hence, the Kit-positive cells observed during the embryonic period are ICC precursors, and 306 H.M. YOUNG Fig. 1. 307 EMBRYOLOGICAL ORIGIN OF ICC cells functionally and morphologically recognisable as myenteric ICC do not develop until around birth. Similarly, melanocyte precursor cells express Kit before they begin to differentiate into melanocytes (Bernex et al., 1996). The differentiation of ICC at the inner margin of the circular muscle (whose function is still uncertain) lags behind the myenteric ICC by several days (Torihashi et al., 1997). Since mice with mutations in the genes encoding Kit or its ligand, SCF, lack particular sub-populations of ICC (see above), it was proposed that the SCF-Kit signalling pathway is necessary for the development of ICC (Sanders, 1996; Torihashi et al., 1995). However, there is now evidence from studies of WlacZ/WlacZ mice (Bernex et al., 1996) and Wbd/Wbd mice (Klüppel et al., 1998) that suggests that Kit is not necessary for the development of ICC precursors, but rather for the post-natal survival and proliferation of ICC. WlacZ/ WlacZ mice have been genetically engineered to express the lacZ reporter gene under the control of c-kit, so the expression of lacZ reflects normal expression of c-kit (Bernex et al., 1996). Mice homozygous for the mutation (WlacZ/WlacZ mice) do not express c-kit mRNA or Kit protein and die shortly after birth. However, ICC are present in the gut of newborn WlacZ/ WlacZ mice with a similar distribution to that observed in WlacZ/⫹ newborn mice, indicating that the SCF-Kit signalling pathway is not necessary for the survival of ICC precursors during embryonic development. Wbd/ Wbd mice have a regulatory W mutation, which affects c-kit expression (Klüppel et al., 1997). There is no expression of c-kit within the mesenchyme of the intestine of embryonic Wbd/Wbd mice, and adult Wbd/ Wbd mice possess only sparse ICC (Klüppel et al., 1998). Surprisingly, however, post-natal day-5 Wbd/ Wbd mice do possess a normal network of ICC. Unfortunately, the presence of slow waves has not been examined in these early post-natal Wbd/Wbd mice. It was Fig. 1. A: Explant of midgut from an E11.5 mouse after growth in catenary organ culture for 7 days. The explant was suspended between the 2 sides of a ‘V’ cut into a small piece of filter paper, and during the time in culture the explant increased 2.5⫻ in length, underwent gross morphological changes including coiling, and the cells continued to differentiate and proliferate (see Hearn et al., 1999). Scale bar ⫽ 500 mm. B,C: After growth in organ culture for several days, PGP9.5immunoreactive neurons (arrows) can be observed in segments of midgut from E11.5 mice (B), indicating that neural crest cells were present in the midgut at the time of explantation. However, neurons cannot be detected in explants of E11.5 hindgut (C), even after growth in organ culture for up to 2 weeks. Thus, neural crest–derived cells are not present in the hindgut of E11.5 mice (Young et al., 1998a; Hearn et al., 1999). Scale bars (B, C) ⫽ 20 mm. D: Although Kit-immunoreactive cells cannot be detected in the hindgut of E11.5 mice, following growth in organ culture for 3–10 days, Kit-immunoreactive cells (arrows) are found in the cultured explants. Since there are no neural crest–derived cells in the hindgut of E11.5 mice, the Kit-positive cells cannot arise from the neural crest. The Kit-positive cells present in the organcultured explants of E11.5 gut (D) are similar in appearance to the Kit-positive cells observed in the gut of control E15.5 mice (E) (see also Hearn et al., 1999). Scale bars (D, E) ⫽ 20 mm. F, F8: Paired micrographs of a wholemount preparation of circular muscle from an adult mouse that had been processed for both p75- and Kitimmunoreactivity. Within the circular muscle, Kit-immunoreactive cells (F8) are ICC, whereas the p75 antibody labels glial cells (F, asterisk), which are not labelled by the Kit antiserum. The asterisk in F8 indicates the location of the p75-positive glial cell. The processes of the glial cells and ICC often run in the same bundles. Scale bar ⫽ 20 mm. calculated that, unlike wild-type mice in which there is an 11-fold increase in the number of ICC after postnatal day 5, there is no increase in the number of ICC in post-natal Wbd/Wbd mice. Thus, it appears that only the post-natal proliferation of ICC is dependent on the SCF-Kit signalling pathway, and the Kit-positive ICC precursor cells present in embryonic mice do not require this pathway for survival, or to differentiate along the ICC lineage (Bernex et al., 1996: Klüppel et al., 1998). DO ICC REQUIRE ENTERIC NEURONS FOR THEIR DEVELOPMENT? The ligand for Kit is SCF (see above). To date, there are no antisera available that specifically localise SCF in mice. However, in mice that have been genetically engineered to express lacZ under the regulation of the cell-specific promotor for SCF, enteric neurons appear to be the sole source of SCF (Torihashi et al., 1996; Young et al., 1998b). If enteric neurons are the sole source of SCF, and the SCF-Kit signalling pathway is essential for ICC proliferation, then ICC should not develop normally in segments of intestine lacking enteric neurons. However, apparently normal numbers of Kit-positive cells, which have the morphological characteristics of ICC, do develop in segments of both chicken and mouse intestine that lack enteric neurons (Lecoin et al., 1996; Wu et al., 1996; Young et al., 1996; see above), suggesting that enteric neurons may not be the only source of SCF. Interestingly, however, aganglionic segments of colon of humans with Hirschsprung’s disease have reduced the numbers of ICC compared to the normal colon (Vanderwinden et al., 1996; Yamataka et al., 1995). Although the reduction in the number of ICC in the aganglionic segments of Hirschsprung’s patients is not necessarily a direct result of an absence of enteric neurons, there is also a high incidence of Hirschsprung’s disease in humans with the pigment disorder, piebaldism (see Kapur, 1993). Since piebaldism results from the mutations in the c-kit gene (Fleishman, 1992; Spritz et al., 1992), it appears that sometimes there can be a correlation between abnormalities in c-kit expression and neural crest derivatives. SUMMARY ICC do not arise from the neural crest, but from precursor cells in the outer mesenchyme of the embryonic gut that express the receptor tyrosine kinase, Kit. However, some of the Kit-positive mesenchymal cells are destined to become smooth muscle cells, and such cells down-regulate the expression of Kit and upregulate the expression of myofilament proteins. ICC maintain their expression of Kit, even in mature animals. 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