Enteric nervous system development: analysis of the selective developmental potentialities of vagal and sacral neural crest cells using quail-chick chimeras

The majority of the enteric nervous system (ENS) is derived from vagal neural crest cells (NCC). For many years, the contribution from a second region of the neuraxis (the sacral neural crest) to the ENS has been less clear, with conflicting reports appearing in the literature. To resolve this longstanding issue, we documented the spatiotemporal migration and differentiation of vagal and sacral-derived NCC within the developing chick embryo using quail-chick grafting and antibody labelling. Results showed that vagal NCC colonised the entire length of the gut in a rostrocaudal direction. The hindgut, the region of the gastrointestinal tract most frequently affected in developmental disorders, was found to be colonised in a complex manner. Vagal NCC initially migrated within the submucosa, internal to the circular muscle layer, before colonising the myenteric plexus region. In contrast, sacral NCC, which colonised the hindgut in a caudorostral direction, were primarily located in the myenteric plexus region from where they subsequently migrated to the submucosa. We also observed that sacral NCC migrated into the hindgut in significant numbers only after vagal-derived cells had colonised the entire length of the gut. This suggested that to participate in ENS formation, sacral cells may require an interaction with vagal-derived cells, or with factors or signalling molecules released by them or their progeny. To investigate this possible inter-relationship, we ablated sections of vagal neural crest (NC) to prevent the rostrocaudal migration of ENS precursors and, thus, create an aganglionic hindgut model. In the same NC ablated animals, quail-chick sacral NC grafts were performed. In the absence of vagal-derived ganglia, sacral NCC migrated and differentiated in an apparently normal manner. Although the numbers of sacral cells within the hindgut was slightly higher in the absence of vagal-derived cells, the increase was not sufficient to compensate for the lack of enteric ganglia. As vagal NCC appear to be more invasive than sacral NCC, since they colonise the entire length of the gut, we investigated the ability of transplanted vagal cells to colonise the hindgut by grafting the vagal NC into the sacral region. We found that when transplanted, vagal cells retained their invasive capacity and migrated into the hindgut in large numbers. Although sacral-derived cells normally contribute a relatively small number of precursors to the post-umbilical gut, many heterotopic vagal cells were found within the hindgut enteric plexuses at much earlier stages of development than normal. Heterotopic grafting of invasive vagal NCC into the sacral neuraxis may, therefore, be a means of rescuing an aganglionic hindgut phenotype.     

Enteric nervous system patterning in the avian hindgut.

The enteric nervous system (ENS) is principally derived from vagal and sacral neural crest cells that migrate throughout the gastrointestinal tract before differentiating into neurons and glia. These cells form two concentric rings of ganglia and regulate intestinal motility, absorption, and secretion. Abnormalities of ENS development can lead to disorders of intestinal function, including Hirschsprung's disease. These disorders are generally limited to the distal hindgut, suggesting unique features to development of this region. This study characterized the normal spatiotemporal development of the ENS within the avian hindgut. Neural crest cells begin to populate the hindgut at E8, with patterning of both plexuses complete by embryonic day 9. Crest-derived cells arrive in the submucosal layer before the myenteric layer, as well as differentiate to a neuronal phenotype first. The cloaca demonstrates a unique pattern, characterized by a disorganized myenteric plexus and a flattened nerve of Remak. Detailed understanding of normal avian hindgut ENS development will allow better utilization of this model system to study abnormalities of the intestinal nervous system.     

Hoxb3 vagal neural crest-specific enhancer element for controlling enteric nervous system development.

The neural and glial cells of the intrinsic ganglia of the enteric nervous system (ENS) are derived from the hindbrain neural crest at the vagal level. The Hoxb3 gene is expressed in the vagal neural crest and in the enteric ganglia of the developing gut during embryogenesis. We have identified a cis-acting enhancer element b3IIIa in the Hoxb3 gene locus. In this study, by transgenic mice analysis, we examined the tissue specificity of the b3IIIa enhancer element using the lacZ reporter gene, with emphasis on the vagal neural crest cells and their derivatives in the developing gut. We found that the b3IIIa-lacZ transgene marks only the vagal region and not the trunk or sacral region. Using cellular markers, we showed that the b3IIIa-lacZ transgene was expressed in a subset of enteric neuroblasts during early development of the gut, and the expression was maintained in differentiated neurons of the myenteric plexus at later stages. The specificity of the b3IIIa enhancer in directing gene expression in the developing ENS was further supported by genetic analysis using the Dom mutant, a spontaneous mouse model of Hirschsprung's disease characterized by the absence of enteric ganglia in the distal gut. The colonization of lacZ-expressing cells in the large intestine was incomplete in all the Dom/b3IIIa-lacZ hybrid mutants we examined. To our knowledge, this is the only vagal neural crest-specific genetic regulatory element identified to date. This element could be used for a variety of genetic manipulations and in establishing transgenic mouse models for studying the development of the ENS.     

Lumbo-sacral neural crest contributes to the avian enteric nervous system independently of vagal neural crest.

Most of the avian enteric nervous system is derived from the vagal neural crest, but a minority of the neural cells in the hindgut, and to an even lesser extent in the midgut, are of lumbo-sacral crest origin. Since the lumbo-sacral contribution was not detected or deemed negligible in the absence of vagal cells, it had been hypothesised that lumbo-sacral neural crest cells require vagal crest cells to contribute to the enteric nervous system. In contrast, zonal aganglionosis, a rare congenital human bowel disease led to the opposite suggestion, that lumbo-sacral cells could compensate for the absence of vagal cells to construct a complete enteric nervous system. To test these notions, we combined E4 chick midgut and hindgut, isolated prior to arrival of neural precursors, with E1. 7 chick vagal and/or E2.7 quail lumbo-sacral neural tube as crest donors, and grafted these to the chorio-allantoic membrane of E9 chick hosts. Double and triple immuno-labelling for quail cells (QCPNA), neural crest cells (HNK-1), neurons and neurites (neurofilament) and glial cells (GFAP) indicated that vagal crest cells produced neurons and glia in large ganglia throughout the entire intestinal tissues. Lumbo-sacral crest contributed small numbers of neurons and glial cells in the presence or absence of vagal cells, chiefly in colorectum, but not in nearby small intestinal tissue. Thus for production of enteric neural cells the avian lumbo-sacral neural crest neither requires the vagal neural crest, nor significantly compensates for its lack. However, enteric neurogenesis of lumbo-sacral cells requires the hindgut microenvironment, whereas that of vagal cells is not restricted to a particular intestinal region.     

In vitro formation of enteric neural network structure in a gut-like organ differentiated from mouse embryonic stem cells

Using an embryoid body (EB) culture system, we developed a functional organ-like cluster--a "gut"--from mouse embryonic stem (ES) cells (ES gut). Each ES gut exhibited spontaneous contractions but did not exhibit distinct peristalsis-like movements. In these spontaneously contracting ES guts, dense distributions of interstitial cells of Cajal (c-kit [a transmembrane receptor that has tyrosine kinase activity]-positive cells; gut pacemaker cells) and smooth muscle cells were discernibly identified; however, enteric neural ganglia were absent in the spontaneously differentiated ES gut. By adding brain-derived neurotrophic factor (BDNF) only during EB formation, we for the first time succeeded in in vitro formation of enteric neural ganglia with connecting nerve fiber tracts (enteric nervous system [ENS]) in the ES gut. The ES gut with ENS exhibited strong peristalsis-like movements. During EB culture in BDNF(+) medium, we detected each immunoreactivity associated with the trk proto-oncogenes (trkB; BDNF receptors) and neural crest marker, proto-oncogene tyrosine-protein kinase receptor ret precursor (c-ret), p75, or sox9. These results indicated that the present ENS is differentiated from enteric neural crest-derived cells. Moreover, focal stimulation of ES guts with ENS elicited propagated increases in intracellular Ca(2+) concentration ([Ca(2+)](i)) at single or multiple sites that were attenuated by atropine or abolished by tetrodotoxin. These results suggest in vitro formation of physiologically functioning enteric cholinergic excitatory neurons. We for the first time succeeded in the differentiation of functional neurons in ENS by exogenously adding BDNF in the ES gut, resulting in generation of distinct peristalsis-like movements.     

Trans-mesenteric neural crest cells are the principal source of the colonic enteric nervous system

Cell migration is fundamental to organogenesis. During development, the enteric neural crest cells (ENCCs) that give rise to the enteric nervous system (ENS) migrate and colonize the entire length of the gut, which undergoes substantial growth and morphological rearrangement. How ENCCs adapt to such changes during migration, however, is not fully understood. Using time-lapse imaging analyses of mouse ENCCs, we show that a population of ENCCs crosses from the midgut to the hindgut via the mesentery during a developmental time period in which these gut regions are transiently juxtaposed, and that such 'trans-mesenteric' ENCCs constitute a large part of the hindgut ENS. This migratory process requires GDNF signaling, and evidence suggests that impaired trans-mesenteric migration of ENCCs may underlie the pathogenesis of Hirschsprung disease (intestinal aganglionosis). The discovery of this trans-mesenteric ENCC population provides a basis for improving our understanding of ENS development and pathogenesis.     

Extrinsic innervation of the pelvic organs in the lesser pelvis of human embryos

Realistic models to understand the developmental appearance of the pelvic nervous system in mammals are scarce. We visualized the development of the inferior hypogastric plexus and its preganglionic connections in human embryos at 4–8 weeks post-fertilization, using Amira 3D reconstruction and Cinema 4D-remodelling software. We defined the embryonic lesser pelvis as the pelvic area caudal to both umbilical arteries and containing the hindgut. Neural crest cells (NCCs) appeared dorsolateral to the median sacral artery near vertebra S1 at ~5 weeks and had extended to vertebra S5 1 day later. Once para-arterial, NCCs either formed sympathetic ganglia or continued to migrate ventrally to the pre-arterial region, where they formed large bilateral inferior hypogastric ganglionic cell clusters (IHGCs). Unlike more cranial pre-aortic plexuses, both IHGCs did not merge because the 'pelvic pouch', a temporary caudal extension of the peritoneal cavity, interposed. Although NCCs in the sacral area started to migrate later, they reached their pre-arterial position simultaneously with the NCCs in the thoracolumbar regions. Accordingly, the superior hypogastric nerve, a caudal extension of the lumbar splanchnic nerves along the superior rectal artery, contacted the IHGCs only 1 day later than the lumbar splanchnic nerves contacted the inferior mesenteric ganglion. The superior hypogastric nerve subsequently splits to become the superior hypogastric plexus. The IHGCs had two additional sources of preganglionic innervation, of which the pelvic splanchnic nerves arrived at ~6.5 weeks and the sacral splanchnic nerves only at ~8 weeks. After all preganglionic connections had formed, separate parts of the inferior hypogastric plexus formed at the bladder neck and distal hindgut.     

Lineage and development of the parasympathetic nervous system of the embryonic chick heart

 We were interested in the contribution of the cardiac neural crest to the complete anterior and posterior nerve plexus of the chick heart. This includes the pathways by which these cardiac neural crest-derived neuronal precursors enter the heart. As lineage techniques we used the traditional quail-chick chimera in combination with the newly introduced technique of retroviral reporter gene transfer to premigratory cardiac neural crest cells. Retrovirally infected embryos (n=23) and quail-chick chimeras (n=19) between stages HH27 and 40, were immunohistochemically evaluated, using the lineage markers LacZ (retroviral reporter) and QCPN (anti-quail nuclear marker), respectively and the neuronal differentiation markers HNK-1, RMO-270 and DO-170. Between stages HH27 and 33, quail-derived and LacZ positive cells were situated around the arterial cardiac vagal branches at the arterial pole, and vagal branches along the anterior cardinal veins and the sinal vagal branch at the venous pole. From stage HH35 onward, QCPN/LacZ-positive cardiac ganglia were observed throughout the anterior and posterior plexus and were mainly concentrated in the subepicardium near the distal ends of the arterial cardiac vagal branches and the sinal cardiac vagal branch respectively. From stage HH36 both the anterior and posterior plexus contained a population of large cardiac ganglion cells and a population of smaller cells along nerve branches as well as in the cardiac ganglia, which means that differentiation starts in both plexus at the same time. Furthermore only nerve fiber connections between the anterior and posterior plexus were observed. These results show that the cardiac neural crest contributes to the cardiac ganglion cells from both the entire anterior and posterior plexus. Furthermore these results suggest that these precursor cells enter the arterial pole via the arterial cardiac vagal branches and the venous pole via the sinal cardiac vagal branch without intermixing. Finally we show that in addition to the cardiac ganglia, the cardiac neural crest contributes to small myocardial glia or undifferentiated cells along nerve fibers, and some myocardial nerve fibers as well as nerve tissue in the adventitia of the large veins at the venous pole and in the adventitia of the coronary arteries. Accepted: 30 March 1998     

Expression of Hand2 is sufficient for neurogenesis and cell type-specific gene expression in the enteric nervous system.

The basic helix-loop-helix DNA binding protein Hand2 is expressed in neural crest-derived precursors of enteric neurons and has been shown to affect both neurogenesis and neurotransmitter specification of noradrenergic sympathetic ganglion neurons. In the current study, our goal was to determine whether Hand2 affects neurogenesis and/or expression of vasoactive intestinal polypeptide and choline acetyltransferase in developing enteric neurons. Gain-of-function of Hand2 in HNK-1(+) immmunoselected precursor cells resulted in increased neurogenesis. The number of neurons expressing vasoactive intestinal polypeptide increased in response to Hand2 overexpression although choline acetyltransferase was not affected. Targeted deletion of Hand2 in neural crest cells resulted in loss of all neurons expressing vasoactive intestinal polypeptide along the length of the gastrointestinal tract, patterning defects in the myenteric plexus of the stomach, and altered number and morphology of neurons expressing TH. Our data demonstrate that expression of Hand2 is sufficient and necessary for neurogenesis and expression of a subset of cell type-specific markers in the developing enteric nervous system.     

Colonization of the developing pancreas by neural precursors from the bowel.

Neurons in ganglia of the myenteric plexus of the duodenum and stomach have recently been demonstrated to innervate pancreatic ganglia and transsynaptically to excite acinar and islet cells. The hypothesis that crest-derived cells first colonize the foregut and secondarily enter the pancreas by way of the pancreatic buds was tested. Studies were done with fetal rats (days E11-E15). Pancreatic rudiments and foregut were explanted separately and in co-culture. The development of neurons in the explants, identified by demonstrating the immunoreactivities of neurofilaments and growth-associated protein-43 (GAP-43), provided an indirect assay for the presence of neural precursors in the tissue at the time of explantation. Cells of putative neural crest origin were visualized immunocytochemically using the monoclonal antibody, NC-1. Additional markers included the immunoreactivities of dopamine-beta-hydroxylase (DBH), which is expressed by vagal crest-derived cells that colonize the bowel, neuropeptides (substance P and neuropeptide Y [NPY]) found in mature pancreatic neurons, and serotonin (5-HT), which is located in the cell bodies of enteric but not pancreatic neurons. Neurons were detected in cultures of foregut, but not pancreas, when these tissues were explanted by themselves at days E11 and E12. At E11 neural precursors did not leave explants of bowel or migrate into co-cultured pancreatic rudiments. When the foregut was explanted at E12, however, neural precursors migrated away from the bowel, giving rise both to distant ganglia and to neurons within co-cultured pancreatic rudiments. Intrapancreatic ganglia developed in the co-cultures even when the pancreatic attachment to the bowel was severed. Neurons appeared in pancreatic rudiments explanted by themselves on day E13. Neurons developing in pancreatic explants expressed the immunoreactivities of DBH, substance P, and NPY, but not 5-HT. These observations support the idea that pancreatic ganglia develop from crest-derived cells that first colonize the fetal rat foregut and there acquire the ability to colonize the pancreas. A later migration into the pancreatic rudiments of a subset of the original émigrés or their progeny between days E12 and E13 gives rise to a network of pancreatic ganglia that can be regarded as an extension of the enteric nervous system.     

Intestinal smooth muscle is required for patterning the enteric nervous system

The development of the enteric nervous system (ENS) and intestinal smooth muscle occurs in a spatially and temporally correlated manner, but how they influence each other is unknown. In the developing mid‐gut of the chick embryo, we find that α‐smooth muscle actin expression, indicating early muscle differentiation, occurs after the arrival of migrating enteric neural crest‐derived cells (ENCCs). In contrast, hindgut smooth muscle develops prior to ENCC arrival. Smooth muscle development is normal in experimentally aganglionic hindguts, suggesting that proper development and patterning of the muscle layers does not rely on the ENS. However, inhibiting early smooth muscle development severely disrupts ENS patterning without affecting ENCC proliferation or apoptosis. Our results demonstrate that early intestinal smooth muscle differentiation is required for patterning the developing ENS.     

Contributions of placodal and neural crest cells to avian cranial peripheral ganglia

The method of embryonic tissue transplantation was used to confirm the dual origin of avian cranial sensory ganglia, to map precise locations of the anlagen of these sensory neurons, and to identify placodal and neural crest-derived neurons within ganglia. Segments of neural crest or strips of presumptive placodal ectoderm were excised from chick embryos and replaced with homologous tissues from quail embryos, whose cells contain a heterochromatin marker. Placode-derived neurons associated with cranial nerves V, VII, IX, and X are located distal to crest-derived neurons. The generally larger, embryonic placodal neurons are found in the distal portions of both lobes of the trigeminal ganglion, and in the geniculate, petrosal and nodose ganglia. Crest-derived neurons are found in the proximal trigeminal ganglion and in the combined proximal ganglion of cranial nerves IX and X. Neurons in the vestibular and acoustic ganglia of cranial nerve VIII derive from placodal ectoderm with the exception of a few neural crest-derived neurons localized to regions within the vestibular ganglion. Schwann sheath cells and satellite cells associated with all these ganglia originate from neural crest. The ganglionic anlagen are arranged in cranial to caudal sequence from the level of the mesencephalon through the third somite. Presumptive placodal ectoderm for the VIIIth, the Vth, and the VIIth, IXth, and Xth ganglia are located in a medial to lateral fashion during early stages of development reflecting, respectively, the dorsolateral, intermediate, and epibranchial positions of these neurogenic placodes.     

Contribution of the cervical sympathetic ganglia to the innervation of the pharyngeal arch arteries and the heart in the chick embryo

In the chick heart, sympathetic innervation is derived from the sympathetic neural crest (trunk neural crest arising from somite level 10–20). Since the trunk neural crest gives rise to sympathetic ganglia of their corresponding level, it suggests that the sympathetic neural crest develops into cervical ganglia 4–14. We therefore tested the hypothesis that, in addition to the first thoracic ganglia, the cervical ganglia might contribute to cardiac innervation as well. Putative sympathetic nerve connections between the cervical ganglia and the heart were demonstrated using the differentiation markers tyrosine hydroxylase and HNK‐1. In addition, heterospecific transplantation (quail to chick) of the cardiac and trunk neural crest was used to study the relation between the sympathetic neural crest and the cervical ganglia. Quail cells were visualized using the quail nuclear antibody QCPN. The results by immunohistochemical study show that the superior and the middle cervical ganglia and possibly the carotid paraganglia contribute to the carotid nerve. This nerve subsequently joins the nodose ganglion of the vagal nerve via which it contributes to nerve fibers in cardiac vagal branches entering the arterial and venous pole of the heart. In addition, the carotid nerve contributes to nerve fibers connected to putative baro‐ and chemoreceptors in and near the wall of pharyngeal arch arteries suggesting a role of the superior and middle cervical ganglia and the paraganglia of the carotid plexus in sensory afferent innervation. The lower cervical ganglia 13 and 14 contribute predominantly to nerve branches entering the venous pole via the anterior cardinal veins. We did not observe a thoracic contribution. Heterospecific transplantation shows that the cervical ganglia 4–14 as well as the carotid paraganglia are derived from the sympathetic neural crest. The cardiac neural crest does not contribute to the neurons of the cervical ganglia. We conclude that the cervical ganglia contribute to cardiac innervation which explains the contribution of the sympathetic neural crest to the innervation of the chick heart. Anat Rec 255:407–419, 1999. © 1999 Wiley‐Liss, Inc.     

Contribution of the cervical sympathetic ganglia to the innervation of the pharyngeal arch arteries and the heart in the chick embryo.

In the chick heart, sympathetic innervation is derived from the sympathetic neural crest (trunk neural crest arising from somite level 10-20). Since the trunk neural crest gives rise to sympathetic ganglia of their corresponding level, it suggests that the sympathetic neural crest develops into cervical ganglia 4-14. We therefore tested the hypothesis that, in addition to the first thoracic ganglia, the cervical ganglia might contribute to cardiac innervation as well. Putative sympathetic nerve connections between the cervical ganglia and the heart were demonstrated using the differentiation markers tyrosine hydroxylase and HNK-1. In addition, heterospecific transplantation (quail to chick) of the cardiac and trunk neural crest was used to study the relation between the sympathetic neural crest and the cervical ganglia. Quail cells were visualized using the quail nuclear antibody QCPN. The results by immunohistochemical study show that the superior and the middle cervical ganglia and possibly the carotid paraganglia contribute to the carotid nerve. This nerve subsequently joins the nodose ganglion of the vagal nerve via which it contributes to nerve fibers in cardiac vagal branches entering the arterial and venous pole of the heart. In addition, the carotid nerve contributes to nerve fibers connected to putative baro- and chemoreceptors in and near the wall of pharyngeal arch arteries suggesting a role of the superior and middle cervical ganglia and the paraganglia of the carotid plexus in sensory afferent innervation. The lower cervical ganglia 13 and 14 contribute predominantly to nerve branches entering the venous pole via the anterior cardinal veins. We did not observe a thoracic contribution. Heterospecific transplantation shows that the cervical ganglia 4-14 as well as the carotid paraganglia are derived from the sympathetic neural crest. The cardiac neural crest does not contribute to the neurons of the cervical ganglia. We conclude that the cervical ganglia contribute to cardiac innervation which explains the contribution of the sympathetic neural crest to the innervation of the chick heart.     

Embryonic development of the ganglion plexuses and the concentric layer structure of human gut: a topographical study

In this study, we performed a detailed topographical study on the development of ganglion plexuses and the smooth muscle layers of human embryonic and fetal gut. Neuron and glia differentiation was investigated with anti-PGP9.5 and anti-S100 antibodies respectively. The differentiation of smooth muscle and interstitial cells of Cajal (ICC) was studied with anti-smooth muscle -actin and anti-C-Kit antibodies respectively. By week 7, rostro-caudal neural crest cell (NCC) colonization of the gut was complete, and NCCs have differentiated into neurons and glia. At the foregut, neurons and glia were aggregated into ganglion plexus in the myenteric region, and the longitudinal and circular muscle layers have started to differentiate; however, neurons and glia were not found in the submucosa. At the hindgut, neurons and glia were dispersed within the mesenchyme. Myenteric plexus, longitudinal and circular muscle layers formed along the entire gut by week 9. Scattered and individual neurons and glia, and small ganglion plexuses were detected in the foregut and midgut submucosa by week 12. Ganglion plexus was not seen in the hindgut submucosa until week 14. Muscularis mucosae was formed at the foregut and midgut by week 12 but was only discernible at the hindgut 2 weeks later. As the gut wall developed, ganglion plexus increased in size with more neurons and glia, and the formation of intra-plexus nerve fascicle. ICCs were localized in the ganglion plexus as early as week 7. ICCs were initially dispersed in the plexus and were preferentially localized at the periphery of the plexus by week 20. The specification of the annular layers of human embryonic and fetal gut follows a strict spatio-temporal pattern in a rostro-caudal and centripetal manner suggesting that interaction between (1) homotypic and/or heterotypic cells; and (2) cells and the extracellular matrix is critical for the embryonic development of the gut mesenchyme and the enteric nervous system.     

How to innervate a simple gut: familiar themes and unique aspects in the formation of the insect enteric nervous system.

Like the vertebrate enteric nervous system (ENS), the insect ENS consists of interconnected ganglia and nerve plexuses that control gut motility. However, the insect ENS lies superficially on the gut musculature, and its component cells can be individually imaged and manipulated within cultured embryos. Enteric neurons and glial precursors arise via epithelial-to-mesenchymal transitions that resemble the generation of neural crest cells and sensory placodes in vertebrates; most cells then migrate extensive distances before differentiating. A balance of proneural and neurogenic genes regulates the morphogenetic programs that produce distinct structures within the insect ENS. In vivo studies have also begun to decipher the mechanisms by which enteric neurons integrate multiple guidance cues to select their pathways. Despite important differences between the ENS of vertebrates and invertebrates, common features in their programs of neurogenesis, migration, and differentiation suggest that these relatively simple preparations may provide insights into similar developmental processes in more complex systems.     

Expression profile of some neuronal and glial cell markers in the ovine ileal enteric nervous system during prenatal development

The enteric nervous system (ENS) is a network of neurons and glia found in the gut wall and governs this gastrointestinal function independently from the central nervous system (CNS). ENS comprises the myenteric plexus (MP) and the submucous plexus (SP). In this study, we examined the expression profile of neurofilament heavy chain (NF-H), neuron-specific enolase (NSE), calcyclin (S100A6), vimentin and glial fibril acidic protein (GFAP) in ovine ileal enteric neurons and enteric glia cells (EGCs) during prenatal development using an immunohistochemical method. The material of the study consisted of 15 different fetal ileum tissues obtained between days 60 and 150 of pregnancy. NF-H was observed in the majority of ganglion cells in SP and MP throughout the fetal period. It was determined that there was no NF-H reaction in some ganglion cells in Peyer’s patches of internal submucosal plexus (ISPF). In the early stage of pregnancy (60–90 days), there was no expression of NSE and S1006 in ileum. After this period, NSE and S1006 were expressed in the ganglion cells of the plexus, indicating an increase in the amount of expression towards the end of pregnancy. In the early period, vimentin expression was only detected in intramuscular interstitial cells (ICs) (60–90 days), but later (90–150 days) it was also seen in the cells around the ganglion cells in the plexus. On days 60–90 of gestation, GFAP expression only occurred in MP, but in later stages, staining was also detected in SP. In the plexus, an immunoreactivity was present in EGCs forming a network around the ganglion cell. During the last period of gestation (120–150 days), the number of GFAP-positive plexus increased, with the majority of these stained cells being observed in MP. Interestingly, weak staining or reaction did not occur in ISPF, unlike other plexuses. In conclusion, this is the first study that demonstrated the expression of NF-H, vimentin, S100A6, NSE and glial fibril acidic protein (GFAP) in ovine ileal ENS in the prenatal period. In the last period of gestation (120–150 days), the expression profile of ENS was similar to that of adult animals. The expression of the used markers increased toward the end of pregnancy. Our results suggest that neurons and EGCs show heterogeneity, and GFAP and NF-H cannot be used as panenteric glial or panneuronal markers, respectively. We also demonstrated, for the first time, the prenatal expression of S100A6 in enteric neurons and the possibility of using this protein for the identification of enteric neurons.     

Organization of lumbar spinal outflow to distal colon and pelvic organs

The lumbar sympathetic outflow projects through the lumbar splanchnic, lumbar colonic, and hypogastric nerves (and to a lesser degree through the sacral sympathetic chain and pelvic nerves). It is thought to be involved in the regulation of the storage and evacuation functions of the following three organ systems: lower urinary tract, hindgut, and reproductive organs. In addition, it controls vascular resistance and capacitance. Thus the target tissues of the postganglionic neurons are vascular smooth muscle, visceral smooth muscles, probably secretory epithelia, and also neurons in the enteric nervous system and the pelvic ganglia. The preganglionic neurons are situated in the caudal part of the spinal representation, neurons associated with the colon being located rostral to those associated with the pelvic organs. Most lie medial to the classical intermediolateral cell column that may contain mainly vasoconstrictor neurons. Most (if not all) preganglionic neurons are cholinergic; some also contain an identified peptide. Most of the postganglionic neurons are situated in the inferior mesenteric ganglion (or equivalent structures); again, those projecting to the colon lie rostral to those projecting to the pelvic organs. Others lie in intercalated prevertebral ganglia, in the pelvic plexus, and in sacral paravertebral ganglia. The majority is noradrenergic, and most also contain one or several peptides, the topographical distribution of which appears to characterize functional subgroups of neurons. The terminations of noradrenergic axons in many pelvic organs probably make close contact with both vascular and nonvascular effectors. In the colon, most endings are located in the enteric plexuses. The responses of these organs to electrical stimulation of visceral nerves, and their reflex responses (together with those observed in the efferent axons of visceral nerve trunks) to electrical and natural stimulation of afferent fibers, lead to the general conclusion that several distinct classes of pre- and postganglionic neurons exist. 1) Vasoconstrictor neurons demonstrate ongoing activity with cardiac rhythm and appropriate reflexes to stimulation of cardiovascular afferent receptors and respond only weakly to natural stimulation of visceral receptors. 2) MR neurons respond to visceral stimuli but are not influenced from arterial baro- and chemoreceptors. These show at least two different response patterns consistent with their separate involvement in the reciprocal behavior of the colon and bladder. 3) Other neurons are silent in anesthetized animals and do not respond to any stimuli used thus far.(ABSTRACT TRUNCATED AT 400 WORDS)