Emergence and development of gut motility in the chicken embryo.

The gastrointestinal tract transports the food bolus by peristalsis. Gut motility starts at an early age in the developing embryo, well before it is required for nutrition of the organism. We present a comprehensive kinematic study of the emergence and physiological development of gut motility in all regions of the lower digestive tract of the chicken embryo from embryonic days E5 through E9. We characterized motility emergence time, propagation patterns, speed, frequency and amplitude of peristalsis waves. We found that the emergence of an uninterrupted circular ring of smooth muscle correlated with the appearance of propagative contractile waves, at E6 in the hindgut and midgut, and at E9 in the caecal appendix. We show that peristalsis at these stages is critically dependent on calcium and is not mediated by neurons as gut motility is insensitive to tetrodotoxin and takes place in the hindgut in the absence of neurons. We further demonstrate that motility also matures in ex-vivo organ culture. We compare our results to existing literature on zebrafish, mouse and human motility development, and discuss their chronological relationship with other major developmental events occurring in the chicken embryonic gut at these stages. Our work sets a baseline for further investigations of motility development in this important animal model.

Vagal Fibers Form Associations With Interstitial Cells of Cajal During Fetal Development.

Vagal intramuscular arrays (IMAs) have been shown to form complexes with intramuscular interstitial cells of Cajal (ICC). We tested the hypothesis that associations between vagal nerve endings and ICC arise in fetal development. Intraganglionic laminar endings (IGLEs) and IMAs were identified by applying 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanineperchlorate (DiI) to vagal nerve trunks and myenteric plexus (MP) and intramuscular (IM) ICC were immunolabeled with antibodies to c-Kit in fetal and early postnatal mice (E16-P7). At E16, c-Kit immunoreactive cells were abundant in the primordial smooth muscle, with early ICC networks discernable by E18 and ongoing organization at P1 and P7. The distribution of vagal endings was found to change during the course of development, with significantly more putative IGLEs in the prenatal compared to the postnatal period and less IMAs in the prenatal compared to postnatal period. Associations of ICC with both IGLEs and IMAs were detected as early as E16 and were maintained into postnatal life. These findings suggest that vagal fibers begin to associate with ICC during prenatal development. Future studies will be needed to determine the mechanisms through which vagal endings and ICC interact.

Embryonic hematopoietic stem cells and interstitial Cajal cells in the hindgut of late stage human embryos: evidence and hypotheses.

There have been few studies on human embryos describing a specific pattern of hindgut colonization by hematopoietic stem cells (HSCs) and interstitial Cajal cells (ICCs). We aimed to study CD34, CD45 and CD117/c-kit expression in late stage human embryos, to attain observational data that could be related to studies on the aorta-gonad-mesonephros (AGM)-derived HSCs, and data on hindgut ICCs. Antibodies were also applied to identify alpha-smooth muscle actin and neurofilaments. Six human embryos of 48-56 days were used. In the 48 day embryo, the hindgut was sporadically populated by c-kit+ ICCs, but, in all other embryos, a layer of myenteric ICCs had been established. Intraneural c-kit+ cells were found in pelvic nerves and vagal trunks, suggesting that the theory of Ramon y Cajal assuming that ICCs may be primitive neurons may not be so invalid. Also in the 48 day embryo, c-kit+/CD45+ perivascular cells were found along the pelvic neurovascular axes, suggesting that not only liver, but also other organs could be seeded with HSCs from the AGM region. CD45+ cells with dendritic morphologies were found in all hindgut layers, including the epithelium. This last evidence is suggestive of an AGM contribution to the tissue resident macrophages and could be related to processes of sprouting angiogenesis which, in turn, have been found to be guided by filopodia of endothelial tip cells. Further studies on human embryonic and fetal material should be performed to attempt to clarify whether the hindgut colonization with HSCs is a transitory or definitive process.

Appearance of cholinergic myenteric neurons during enteric nervous system development: comparison of different ChAT fluorescent mouse reporter lines.

BACKGROUND: Cholinergic neurons have been identified with the acetylcholine synthetic enzyme choline acetyltransferase (ChAT). However, ChAT is difficult to localize in newly differentiated peripheral neurons making the study of cholinergic neuronal development problematic. Consequently, researchers have used mouse reporter lines to indicate the presence of ChAT. METHODS: Our objective was to determine which ChAT reporter line was the most sensitive indicator of ChAT expression. We utilized two different fluorescent ChAT reporter lines (ChAT-GFP and ChAT-Cre;R26R:floxSTOP:tdTomato) together with immunolocalization of ChAT protein (ChAT-IR) to characterize the spatial and temporal expression of ChAT in myenteric neurons throughout enteric nervous system (ENS) development. KEY RESULTS: ChAT-IR cells were first seen in the intestine at E10.5, even within the migration wavefront of neural precursors. Myenteric neurons within the distal small intestine (dSI) and proximal colon were first labeled by ChAT-IR, then ChAT-GFP, and finally ChAT-Cre tdTomato. The percentage of ChAT-IR neurons is equivalent to adult levels in the dSI by E13.5 and proximal colon by P0. After these stages, the percentages remained relatively constant throughout development despite dramatic changes in neuronal density. CONCLUSIONS & INFERENCES: These observations indicate that neurotransmitter expression occurs early and there is only a brief gap between neurogenesis and neurotransmitter expression. Our finding that the proportion of ChAT myenteric neurons reached adult levels during embryonic development suggests that the fate of cholinergic neurons is tightly regulated and that their differentiation might influence further neuronal development. ChAT-GFP is a more accurate indicator of early ENS cholinergic neuronal differentiation than the ChAT-Cre;R26R:floxSTOP:tdTomato reporter mouse.

Birthdating of myenteric neuron subtypes in the small intestine of the mouse.

There are many different types of enteric neurons. Previous studies have identified the time at which some enteric neuron subtypes are born (exit the cell cycle) in the mouse, but the birthdates of some major enteric neuron subtypes are still incompletely characterized or unknown. We combined 5-ethynynl-2'-deoxyuridine (EdU) labeling with antibody markers that identify myenteric neuron subtypes to determine when neuron subtypes are born in the mouse small intestine. We found that different neurochemical classes of enteric neuron differed in their birthdates; serotonin neurons were born first with peak cell cycle exit at E11.5, followed by neurofilament-M neurons, calcitonin gene-related peptide neurons (peak cell cycle exit for both at embryonic day [E]12.5-E13.5), tyrosine hydroxylase neurons (E15.5), nitric oxide synthase 1 (NOS1) neurons (E15.5), and calretinin neurons (postnatal day [P]0). The vast majority of myenteric neurons had exited the cell cycle by P10. We did not observe any EdU+/NOS1+ myenteric neurons in the small intestine of adult mice following EdU injection at E10.5 or E11.5, which was unexpected, as previous studies have shown that NOS1 neurons are present in E11.5 mice. Studies using the proliferation marker Ki67 revealed that very few NOS1 neurons in the E11.5 and E12.5 gut were proliferating. However, Cre-lox-based genetic fate-mapping revealed a small subpopulation of myenteric neurons that appears to express NOS1 only transiently. Together, our results confirm a relationship between enteric neuron subtype and birthdate, and suggest that some enteric neurons exhibit neurochemical phenotypes during development that are different from their mature phenotype.

Development of myenteric cholinergic neurons in ChAT-Cre;R26R-YFP mice.

Cholinergic neurons are the major excitatory neurons of the enteric nervous system (ENS), and include intrinsic sensory neurons, interneurons, and excitatory motor neurons. Cholinergic neurons have been detected in the embryonic ENS; however, the development of these neurons has been difficult to study as they are difficult to detect prior to birth using conventional immunohistochemistry. In this study we used ChAT-Cre;R26R-YFP mice to examine the development of cholinergic neurons in the gut of embryonic and postnatal mice. Cholinergic (YFP+) neurons were first detected at embryonic day (E)11.5, and the proportion of cholinergic neurons gradually increased during pre- and postnatal development. At birth, myenteric cholinergic neurons comprised less than half of their adult proportions in the small intestine (25% of myenteric neurons were YFP+ at P0 compared to 62% in adults). The earliest cholinergic neurons appear to mainly project anally. Projections into the presumptive circular muscle were first observed at E14.5. A subpopulation of cholinergic neurons coexpress calbindin through embryonic and postnatal development, but only a small proportion coexpressed neuronal nitric oxide synthase. Our study shows that cholinergic neurons in the ENS develop over a protracted period of time.

Connective tissue in gut development: a key player in motility and in intestinal desmosis.

INTRODUCTION: Efficient intestinal peristalsis is a function of intact enteric nervous system, muscle, and connective muscularis propria tissue. Malfunction of any component results in impaired peristalsis. Hirschsprung disease (HD) as prototypic enteric neural migration disorder is increasingly well characterized. More recently, intestinal myopathies and particularly defects of myenteric collagenization have entered the focus of attention. However, detailed development of muscularis propria connective tissue is not well known. The aim of this study was to morphologically characterize intestinal connective tissue in fetal and postnatal development and intestinal pseudo-obstruction. MATERIALS AND METHODS: In this study, 130 archival specimens of fetal autopsies, intestinal resections, and biopsies were analyzed. Patients' age was 10th gestational week (gw) to 70 years. Muscularis mucosae, muscle layers, collagen tissue, and enteric plexus were analyzed. Picrosirius red stains, enzyme histochemistry, and immunohistochemistry for collagens I, III, and IV were performed. RESULTS: Total 89 normal intestinal specimens were from fetal autopsies or intestinal resections; 41 patients showed a primary structural colon wall defect (HD, desmosis). Our results showed a constant increase in tunica muscularis propria thickness with age. Separation into circular and longitudinal muscle layer first occurred in the 11th gw. A tendinous collagen plexus layer first arose in the 10th gw and showed a steady caliber increase. Muscularis mucosae first appeared in the 10th gw and grew independent of any primary gastrointestinal disease. In the 11th gw, enteric ganglia were fully developed. In desmosis, a collagen plexus layer was absent. In contrast, in HD, muscularis mucosae showed hypertrophy, but the collagen plexus layer was intact in the aganglionic segment. In intestinal neuronal dysplasia and hypoganglionosis, nerve cell development was disturbed; connective tissue and muscle layers were well developed. CONCLUSION: Our comprehensive study of intestinal connective tissue development in comparison to neural intestinal wall components in normal and pathological conditions showed that tendinous tissue develops parallel to muscularis propria and arises early in embryogenesis. In enteric nervous system disorders, ganglionic lesions develop independently of impaired collagen network, whereas mucosal biopsies serve for diagnosis of HD, seromuscular biopsies are required to prove desmosis in gastrointestinal dysmotility disorders.

Abnormal development of intrinsic innervation in murine embryos with anorectal malformations.

INTRODUCTION: Constipation, soiling, and incontinence are common problems after definitive repair of anorectal malformations (ARMs) in children. We studied the expression of substance P (SP), vasoactive intestinal peptide (VIP), and c-kit in the rectum of murine embryos with or without ARMs at later developmental stages. METHODS: On the 9th embryonic day (E9), pregnant Institute of Cancer Research mice were fed etretinate, a synthetic vitamin A analogue (60 mg/kg), whereas controls were fed only with sesame oil. Embryos were excised between E14 and E18, and prepared for histological examination. The SP, VIP, and c-kit expressions were examined by immunohistochemical staining for the SP, VIP, and c-kit antigens, respectively. RESULTS: On E14 and E15, the expression levels of the anti-SP and anti-VIP antibodies in the rectum did not differ between the control and etretinate-treated group. However, as compared to the controls, a decreased SP and VIP immunoreactivity was observed in the circular muscle layer of the rectum between E16 and E18. On the other hand, on E14 and E15, the expression of anti-c-kit antibody in the rectum did not differ between the etretinate-treated and control group. However, c-kit immunoreactivity was slightly higher in the circular muscle layer of the rectum in the controls on E16 and E17, and considerably higher on E18 than that of the muscle layer in the etretinate-treated group. CONCLUSION: At later developmental stages, the expression levels of SP, VIP, and c-kit reduced in the circular muscle layer of the rectum in mice with etretinate-induced ARMs. This result indicates that reduced SP, VIP, and c-kit expression levels in the circular muscle layer may cause severe constipation in children who develop severe ARMs after definitive surgery.

Abnormal innervation patterns in the anorectum of ETU-induced fetal rats with anorectal malformations.

To investigate whether anorectal malformations (ARMs) were associated with a global neuromuscular maldevelopment of the lower gastrointestinal (GI) tract and anorectum, the distribution of neuronal markers protein gene product (PGP9.5), nitric oxide synthases (NOs), neuromuscular junction markers (synaptophysin, SYP), interstitial cells of Cajal (ICC) marker (c-kit) within the terminal rectum were analyzed by immunohistochemistry and Western blot in rat embryos with ethylenethiourea (ETU) induced ARMs. From Gestational day16 (Gd16) to Gd21, neural crest-derived cells (NCC) migrated from the proximal gut into the terminal colon, colonising it along its entire length, gradually proliferated and differentiated to innervate the distal gut. From Gd19 to Gd21, significant gross-morphological differences of the anorectum of normal (n=90) and ARMs (n=90) embryos were found. Different myenteric plexus (MPs) development of the anorectum suggested that ARMs were associated with a global abnormal innervation patterns in the anorectum in gestational course and might have some postoperative effect.

The timing and location of glial cell line-derived neurotrophic factor expression determine enteric nervous system structure and function.

Ret signaling is critical for formation of the enteric nervous system (ENS) because Ret activation promotes ENS precursor survival, proliferation, and migration and provides trophic support for mature enteric neurons. Although these roles are well established, we now provide evidence that increasing levels of the Ret ligand glial cell line-derived neurotrophic factor (GDNF) in mice causes alterations in ENS structure and function that are critically dependent on the time and location of increased GDNF availability. This is demonstrated using two different strains of transgenic mice and by injecting newborn mice with GDNF. Furthermore, because different subclasses of ENS precursors withdraw from the cell cycle at different times during development, increases in GDNF at specific times alter the ratio of neuronal subclasses in the mature ENS. In addition, we confirm that esophageal neurons are GDNF responsive and demonstrate that the location of GDNF production influences neuronal process projection for NADPH diaphorase-expressing, but not acetylcholinesterase-, choline acetyltransferase-, or tryptophan hydroxylase-expressing, small bowel myenteric neurons. We further demonstrate that changes in GDNF availability influence intestinal function in vitro and in vivo. Thus, changes in GDNF expression can create a wide variety of alterations in ENS structure and function and may in part contribute to human motility disorders.

Age-related changes in the myenteric plexus of the porcine bowel.

INTRODUCTION: Myenteric plexus (MP) is well recognized as an important regulator of peristaltic activity. Knowledge regarding prenatal and postnatal normal morphological changes is important when interpreting histopathologic findings in motility disorders of childhood. The aim of this study was to determine the neuronal density and morphology of the myenteric plexus (MP) of the porcine bowel from fetal life to adulthood. METHOD: Small and large bowel whole-mount preparations of the MP were stained using NADPH diaphorase histochemistry in animals from 6 different age groups (60 and 90 days of gestation, newborn, 4-week and 12-week-old, and adult pigs). Using light microscopy, above parameters was quantified, and cell/nucleus sizes were measured. Results were analyzed using 1-way analysis of variance test. RESULTS: There were significant regional and age-related differences in cell numbers per ganglia noted in MP throughout the lifetime of a pig. There was an abrupt increase in cell numbers per ganglia from the newborn to 4-week-old animals, which then stabilized in most parts of the bowel, except in the distal large bowel, where it continued to increase. Ganglion density and ganglia cell density both decreased steadily with advancing age. Cell size increased with age, mostly secondary to increase in the cytoplasm. CONCLUSION: Our results show that significant changes occur in the MP in relation to age and the region of the bowel. These changes are most evident in the immediate period after birth but continue throughout life. Such age-related changes must be taken into account during morphological evaluation of biopsy specimens taken from infants who had constipation.

Myenteric plexus alterations downstream from a prenatal intestinal obstruction in a rat model.

The enteric nervous system maturation occurs during embryonic life and continues after birth. Some prenatal events on the digestive tract such as intestinal atresia have been shown to dramatically alter this maturation. Thus, we developed a fetal rat model of intestinal atresia by surgically obstructing the small bowel at embryonic day E18. Fetuses were removed at day E21, and small bowels sections were examined by immuno-histochemistry. Synaptophysin and smooth muscle actin staining was used to define the cellular aspect. Labeling revealed marked alterations of the myenteric plexus in the lower extremity of the occluded small bowel. At day E21, the myenteric plexus of the lower part and the 2 muscle layers surrounding it, retained the staining pattern observed at day E17. This cellular pattern was classified as: immature (aspect at day E17) vs. mature (aspect of day E21) by 3 pathologists not familiar with the study. The number of samples with an immature cellular pattern at the lower end of the occluded bowel was different from that observed for the upper end (Mac Nemar test, p<0.008). Our study suggests that a prenatal obstruction induces a maturation delay of the myenteric plexus downstream of the obstruction. This might be important for pediatric purposes.

Postnatal changes in enteric plexus axonal thickness.

PURPOSE: Chronological age has not always been an accurate predictor of gut motility in newborns. We hypothesized that the enteric plexus is immature at birth. We studied whole-mount preparations of the myenteric plexus in pigs from mid-point of gestation to adulthood. METHODS: Distal large bowel whole-mount preparations of the myenteric plexus were stained using ACHE histochemistry in five pigs from six different age groups (60 and 90 days gestation, newborn, 4 and 12 weeks old, and adult pigs). With the aid of light micrographs and Image J program the axonal diameter, distance between ganglia, neuronal size and neuronal nucleus size were measured. Statistical significance was measured using one-way ANOVA test. RESULTS: There was significant increase in axonal diameter and interganglionic distance, with increasing age in the myenteric plexus of rectum in pigs (P < 0.05). Neuronal cell size and nucleus size of the enteric neurons also increased with age, but was not statistically significant. CONCLUSION: This study shows for the first time that the axonal thickness in the enteric myenteric plexus undergoes striking changes during the first 12 weeks of life in piglets. Assessment of axonal thickness in rectal biopsies may be a valuable morphological feature in diagnosing functional intestinal obstruction in infants.

Developmental pattern of three vesicular glutamate transporters in the myenteric plexus of the human fetal small intestine.

Three vesicular glutamate transporters (VGLUT1-3) have previously been identified in the central nervous system, where they define the glutamatergic phenotype, and their expression is tightly regulated during brain development. In the present study we applied immunocytochemistry to examine the distribution of the immunoreactivity of all three VGLUTs during prenatal development of the myenteric plexus in the human small intestine. We also investigated changes in their localization in the different segments of the small intestine and in the different compartments of the developing myenteric ganglia. Immunoreactivity against all three VGLUTs was found predominantly in the ganglionic neuropil, interganglionic varicose fibers and perisomatic puncta, but cytoplasmic labeling with different intensities also occurred. Each transporter displayed a characteristic spatiotemporal expression pattern, with the transient increase or decrease of immunoreactive cell bodies, varicosities or perisomatic puncta, depending on the fetal age, the gut segment or the ganglionic compartment. Throughout gestational weeks 14-23, VGLUT1 immunoreactivity always predominated over VGLUT2 immunoreactivity, though both peaked around week 20. VGLUT3 immunoreactivity was less abundant in the developing myenteric plexus than those of VGLUT1 and VGLUT2 immunoreactivity. It was mainly expressed in the ganglionic neuropil and in the perisomatic puncta throughout the examined gestational period. Neuronal perikarya immunoreactive for VGLUT3 were restricted to between weeks 18 and 20 of gestation and exclusively to the oral part of the small intestine.

The developmental stage and cell type dependent phosphorylation of eNOS in murine enteric mucosa and myenteric plexus.

In order to clarify the developmental regulation of the eNOS activity in intestine by phosphorylation, we examined the immunohistochemical localizations of the eNOS phosphorylation sites at Ser(1177), Ser(116) and at Thr(495) in cells of the mouse enteric mucosa and myenteric plexus at E13.5, E14.5, E16.5, E18.5, E20.5 and P3. In addition, in cells of the E16.5 stage the protein levels of eNOS and the phosphorylation sites of eNOS at Ser(1177), Ser(116) and at Thr(495) were investigated by immunoblot. From E14.5 to P3, phosphorylation residues of eNOS at Ser(1177) and at Ser(116) were detected with different staining intensities in the enteric mucosa epithelium. In ganglion cells of the myenteric plexus Ser(116) was identified at E18.5 to P3. The absence of phosphorylated Thr(495) in cells of intestine during all developmental stages, was confirmed by immunoblot at E16.5. The immunoblot levels of eNOS and eNOS phosphorylated at Ser(1177) and at Ser(116) were comparable with the immunohistochemical results of E16.5 mouse intestine. It was concluded that development of epithelial cells of the enteric mucosa may be modulated by phosphorylation of eNOS at Ser(1177) and at Ser(116). The phosphorylation of eNOS in cells of the myenteric plexus is modulated at Ser(116). These data suggest that there is a developmental stage and cell type dependent phosphorylation of eNOS.

Anterograde tracing method using DiI to label vagal innervation of the embryonic and early postnatal mouse gastrointestinal tract.

The mouse is an extremely valuable model for studying vagal development in relation to strain differences, genetic variation, gene manipulations or pharmacological manipulations. Therefore, a method using 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) was developed for labeling vagal innervation of the gastrointestinal (GI) tract in embryonic and postnatal mice. DiI labeling was adapted and optimized for this purpose by varying several facets of the method. For example, insertion and crushing of DiI crystals into the nerve led to faster DiI diffusion along vagal axons and diffusion over longer distances as compared with piercing the nerve with a micropipette tip coated with dried DiI oil. Moreover, inclusion of EDTA in the fixative reduced leakage of DiI out of nerve fibers that occurred with long incubations. Also, mounting labeled tissue in PBS was superior to glycerol with n-propyl gallate, which resulted in reduced clarity of DiI labeling that may have been due to DiI leaking out of fibers. Optical sectioning of flattened wholemounts permitted examination of individual tissue layers of the GI tract wall. This procedure aided identification of nerve ending types because in most instances each type innervates a different tissue layer. Between embryonic day 12.5 and postnatal day 8, growth of axons into the GI tract, formation and patterning of fiber bundles in the myenteric plexus and early formation of putative afferent and efferent nerve terminals were observed. Thus, the DiI tracing method developed here has opened up a window for investigation during an important phase of vagal development.

Kit-like immunoreactivity in the zebrafish gastrointestinal tract reveals putative ICC.

Gastrointestinal (GI) motility results from the coordinated actions of enteric neurons, interstitial cells of Cajal (ICC), and smooth muscle cells. The GI tract of the zebrafish has a cellular anatomy that is essentially similar to humans. Although enteric nerves and smooth muscle cells have been described, it is unknown if ICC are present in the zebrafish. Immunohistochemistry and PCR were used determine expression for the zebrafish Kit orthologue in the zebrafish gastrointestinal tract. Cells displaying Kit-like immunoreactivity were identified in the muscular layers of the adult zebrafish gastrointestinal tract. Two layers of Kit-positive cells were identified, one with multipolar cells located between the longitudinal and circular smooth muscle layers and one with simple bipolar cells located deep in the circular muscle layer. Primers specifically designed to amplify mRNA coding for two zebrafish kit genes, kita and kitb, and two kit ligands, kitla and kitlb, amplified the expected transcript from total RNA isolated from zebrafish GI tissues. The Sparse mutant, a kita null mutant, showed reduced contraction frequency and increased size of the GI tract indicating a functional role for kita. These data establish the presence of a cellular network with Kit-like immunoreactivity in the myenteric plexus region of the zebrafish GI tract, adjacent to enteric neurons. Expression of kita and kitb, and the ligands kitla and kitlb, were verified in the adult GI tract. The anatomical arrangement of the Kit-positive cells strongly suggests that they are ICC.

Intestinal coelomic transplants: a novel method for studying enteric nervous system development.

Normal development of the enteric nervous system (ENS) requires the coordinated activity of multiple proteins to regulate the migration, proliferation, and differentiation of enteric neural crest cells. Much of our current knowledge of the molecular regulation of ENS development has been gained from transgenic mouse models and cultured neural crest cells. We have developed a method for studying the molecular basis of ENS formation complementing these techniques. Aneural quail or mouse hindgut, isolated prior to the arrival of neural crest cells, was transplanted into the coelomic cavity of a host chick embryo. Neural crest cells from the chick host migrated to and colonized the grafted hindgut. Thorough characterization of the resulting intestinal chimeras was performed by using immunohistochemistry and vital dye labeling to determine the origin of the host-derived cells, their pattern of migration, and their capacity to differentiate. The formation of the ENS in the intestinal chimeras was found to recapitulate many aspects of normal ENS development. The host-derived cells arose from the vagal neural crest and populated the graft in a rostral-to-caudal wave of migration, with the submucosal plexus being colonized first. These crest-derived cells differentiated into neurons and glial cells, forming ganglionated plexuses grossly indistinguishable from normal ENS. The resulting plexuses were specific to the grafted hindgut, with quail grafts developing two ganglionated plexuses, but mouse grafts developing only a single myenteric plexus. We discuss the advantages of intestinal coelomic transplants for studying ENS development.

Effects of pre- and postnatal protein deprivation and postnatal refeeding on myenteric neurons of the rat small intestine: a quantitative morphological study.

We investigated weight gain, the size of the small intestine and numbers and sizes of enteric neurons in rats whose mothers had been deprived of protein during pregnancy and who themselves were deprived postpartum. Postnatally, protein deprivation was for 42 days, or for 21 days with refeeding for a further 21 days. Control animals received normal nourishment. Neurons were located by nicotinamide adenine dinucleotide (NADH) diaphorase staining, by acetylcholinesterase (AChE) activity and immunoreactivity for choline acetyltransferase (ChAT). The collagen and elastic fibers in the myenteric ganglia were evaluated histologically. The myenteric ganglia were regular and uniform in the nourished and refed groups. In the undernourished group, the myenteric ganglia were irregularly arranged and the cytoplasm of most of the neurons showed less intense staining for NADH diaphorase, AChE and ChAT. AChE activity and ChAT immunoreactivity showed that most ganglionic neurons were stained in nourished and refed groups, but the neurons of undernourished rats were unstained or moderately stained. The distribution of the connective tissue of the ganglionic capsule was similar in the three groups. There was a decrease in weight of undernourished rats, which was restored in refed rats. The size of the small intestine of the undernourished group was smaller than in the normally fed group, by about 45%, but it was similar in nourished and refed rats. After 42 days of protein deprivation the numbers of neurons that were revealed by NADH diaphorase were fewer than in well nourished rats, but numbers were not different between nourished and refed rats. These observations indicate that protein deprivation alters histological features and acetylcholinesterase activity of neurons and also reduces body weight but these were restored by refeeding.

Differences in nitrergic innervation of the developing chick cloaca and colorectum.

The intrinsic innervation of the developing gut has long been a subject of investigation, but little is known regarding that of the embryonic cloaca. The cloaca, like the rest of the gastrointestinal tract, is intrinsically innervated by the enteric nervous system. Nitrergic neurons and fibres make up a large part of this system, thus, their distribution provides us with a useful insight into its development. Cloacal and colorectal tissue specimens were removed from chick embryos at embryonic days 11 (E11), E15 and E19. NADPH-diaphorase (NADPH-d) histochemistry was carried out using whole mount tissue preparations. Ganglia density, the number of NADPH-d-positive cells per ganglia in the myenteric plexus and cell size were calculated and statistical analysis was performed to compare both regions of the gut (P<0.001). There were significant differences in the ganglia density in the cloaca compared to the colorectum at E11 (P<0.05) and E15 (P<0.01), with the colorectum having a much denser network. In both the cloaca and the colorectum, ganglia density significantly decreased with age (P<0.001), while significant differences were observed in the number of NADPH-d-positive cells per ganglia in both regions through development. Total cell size was similar in both the cloaca and colorectum at each stage and increased in both regions through development, predominantly due to an increase in the cytoplasm. Results reveal striking differences in innervation between the chick embryo cloaca and colorectum. The sparse network of innervation evident within the cloaca in contrast to the dense network within the colorectum emphasizes the individuality of both regions. These results highlight the need for a further in-depth analysis of the enteric nervous system's development within the embryonic cloaca.