Deletion of choline acetyltransferase in enteric neurons results in postnatal intestinal dysmotility and dysbiosis.

Acetylcholine (ACh)-synthesizing neurons are major components of the enteric nervous system (ENS). They release ACh and peptidergic neurotransmitters onto enteric neurons and muscle. However, pharmacological interrogation has proven inadequate to demonstrate an essential role for ACh. Our objective was to determine whether elimination of ACh synthesis during embryogenesis alters prenatal viability, intestinal function, the neurotransmitter complement, and the microbiome. Conditional deletion of choline acetyltransferase ( ChAT), the ACh synthetic enzyme, in neural crest-derived neurons ( ChAT-Null) was performed. Survival, ChAT activity, gut motility, and the microbiome were studied. ChAT was conditionally deleted in ENS neural crest-derived cells. Despite ChAT absence, mice were born live and survived the first 2 wk. They failed to gain significant weight in the third postnatal week, dying between postnatal d 18 and 30. Small intestinal transit of carmine red was 50% slower in ChAT-Nulls vs. WT and ChAT- Het. The colons of many neonatal ChAT-Null mice contained compacted feces, suggesting dysmotility. Microbiome analysis revealed dysbiosis in ChAT-Null mice. Developmental deletion of ChAT activity in enteric neurons results in proximal gastrointestinal tract dysmotility, critically diminished colonic transit, failure to thrive, intestinal dysbiosis, and death. ACh is necessary for sustained gut motility and survival of neonatal mice after weaning.-Johnson, C. D., Barlow-Anacker, A. J., Pierre, J. F., Touw, K., Erickson, C. S., Furness, J. B., Epstein, M. L., Gosain, A. Deletion of choline acetyltransferase in enteric neurons results in postnatal intestinal dysmotility and dysbiosis.

The Sigma-1 Receptor-A Therapeutic Target for the Treatment of ALS?

The membrane bound 223 amino acid Sigma-1 Receptor (S1R) serves as a molecular chaperone and functional regulator of many signaling proteins. Spinal cord motor neuron activation occurs, in part, via large ventral horn cholinergic synapses called C-boutons/C-terminals. Chronic excitation of motor neurons and alterations in C-terminals has been associated with Amyotrophic Lateral Sclerosis (ALS ). The S1R has an important role in regulating motor neuron function. High levels of the S1R are localized in postsynaptic endoplasmic reticulum (ER) subsurface cisternae within 10-20 nm of the plasma membrane that contain muscarinic type 2 acetylcholine receptors (M2AChR), calcium activated potassium channels (Kv2.1) and slow potassium (SK) channels. An increase in action potentials in the S1R KO mouse motor neurons indicates a critical role for the S1R as a "brake" on motor neuron function possibly via calcium dependent hyperpolarization mechanisms involving the aforementioned potassium channels. The longevity of SOD-1/S1R KO ALS mice is significantly reduced compared to SOD-1/WT ALS controls. The S1R colocalizes in C-terminals with Indole(ethyl)amine-N-methyl transferase (INMT ), the enzyme that produces the S1R agonist , N,N'- dimethyltryptamine (DMT). INMT methylation can additionally neutralize endogenous toxic sulfur and selenium derivatives thus providing functional synergism with DMT to reduce oxidative stress in motor neurons . Small molecule activation of the S1R and INMT thus provides a possible therapeutic strategy to treat ALS .

Role of the Sigma-1 receptor in Amyotrophic Lateral Sclerosis (ALS).

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease affecting spinal cord motoneurons (MN) with an associative connection to Frontotemporal Lobar Dementia (FTLD). The endoplasmic reticulum (ER) bound Sigma-1 Receptor (S1R) chaperone protein localizes to specialized ER cisternae within 10 nm of the plasma membrane in spinal cord ventral horn cholinergic post synaptic C-terminals. Removal of the S1R gene in the Superoxide Dismutase-1 (SOD-1) mouse model of ALS exacerbated the neurodegenerative condition and resulted in a significantly reduced longevity when compared to the SOD-1/S1R wild type (WT) mouse. The proposed amelioration of the ALS phenotype by the S1R is likely due to a "brake" on excitation of the MN as evidenced by a reduction in action potential generation in the MN of the WT when compared to the S1R KO mouse MN. Although the precise signal transduction pathway(s) regulated by the S1R in the MN has/have not been elucidated at present, it is likely that direct or indirect functional interactions occur between the S1R in the ER cisternae with voltage gated potassium channels and/or with muscarinic M2 receptor signaling in the post synaptic plasma membrane. Possible mechanisms for regulation of MN excitability by S1R are discussed.

Dopamine transporter gene susceptibility to methylation is associated with impulsivity in nonhuman primates.

Impulsivity, the predisposition to act without regard for negative consequences, is a characteristic of several psychiatric disorders and is thought to result in part from genetic variation in the untranslated region of the dopamine transporter (DAT) gene. As the exact link between genetic mutations and impulsivity has not been established, we used oculomotor behavior to characterize rhesus monkeys as impulsive or calm and genetic/epigenetic analysis and positron emission tomography (PET) to correlate phenotype to DAT genotype, DAT gene methylation, and DAT availability. We found three single nucleotide polymorphisms (SNPs) in the 3'-UTR of the DAT gene, one of which provided a potential site for methylation in the impulsive group. Bisulfite analysis showed that the DNA of the impulsive but not the calm subjects was methylated at one SNP. Because genetic/epigenetic modifications could lead to differences in protein expression, we measured DAT availability using [(18)F]2ß-carbomethoxy-3ß-(4-chlorophenyl)-8-(2-fluoroethyl)-nortropane ([(18)F]FECNT) PET and found higher DAT availability in the internal globus pallidus, an output nucleus of the basal ganglia, of the impulsive group. Higher DAT availability lowers dopamine levels, potentially altering neuronal circuits involved in the initiation of action, thus contributing to the impulsive phenotype. The association between increased methylation in the DAT gene and greater DAT availability suggests that mutations to the regulatory portion of the DAT gene lead to a susceptibility to epigenetic modification resulting in a discrete behavioral phenotype.

Colonic enteric nervous system analysis during parenteral nutrition.

INTRODUCTION: Parenteral nutrition (PN) is a necessary therapy used to feed patients with gastrointestinal dysfunction. Unfortunately, PN results in intestinal atrophy and changes to host immune function. PN may also induce additional effects on gut motility that we hypothesized would result from changes in the enteric nervous system. METHODS: Mice received an intravenous (i.v.) catheter and were randomized to chow (n = 5), i.v. PN (n = 6), or i.v. PN + bombesin (BBS, 15 µg/kg, 3×/d) (n = 6) for 5 d. Colons were removed and dissected to measure the length and circumference. Enteric neuronal density and neurotransmitter expression were determined by co-immunostaining whole-mount tissue with Hu and neuronal nitric oxide synthase (nNOS). RESULTS: The number of myenteric neurons expressing Hu and nNOS increased per unit length in the mid-colon during PN treatment compared with chow. This increase was abrogated by the addition of BBS to the PN regimen. However, the percentage of nNOS-expressing neurons was not significantly altered by PN. Morphometric analysis revealed a decrease in the length and circumference of the colon during PN administration that was partially normalized by supplementation of PN with BBS. A significant reduction in total fecal output was observed in PN animals compared with chow and was increased by mice receiving BBS in addition to PN. CONCLUSIONS: PN causes a constriction of the bowel wall, reducing not only the length but also the circumference of the colon. These changes cause a condensation of enteric neurons but no difference in neurotransmitter expression. BBS supplementation partially restores the constriction and increases the fecal output during PN treatment compared with PN treatment alone.

Age-dependent changes in the gut environment restrict the invasion of the hindgut by enteric neural progenitors.

The enteric nervous system (ENS) develops from neural crest cells (NCCs) that enter the foregut and hindgut to become enteric neural-crest-derived cells (ENCCs). When these cells of neural crest origin fail to colonize the terminal hindgut, this aganglionic region becomes non-functional and results in a condition in humans known as Hirschsprung's disease (HSCR). One of the genes associated with HSCR is endothelin receptor type B (Ednrb). To study the development of colonic aganglionosis we have utilized a novel knockout mouse (Ednrb(flex3/flex3)), in which the expression of a null Ednrb allele and YFP is confined to NCCs. We have identified two primary cellular defects related to defective EDNRB signaling. First, ENCC advance in Ednrb(flex3/flex3) embryos is delayed shortly after NCCs enter the gut. Apart from this early delay, Ednrb(flex3/flex3) ENCCs advance normally until reaching the proximal colon. Second, as Ednrb(flex3/flex3) ENCCs reach the colon at E14.5, they display migratory defects, including altered trajectories and reduced speed, that are not dependent on proliferation or differentiation. We constructed grafts to test the ability of donor ENCCs to invade a recipient piece of aganglionic colon. Our results indicate that the age of the recipient, and not the age or genotype of donor ENCCs, determines whether the colon is invaded. We identify changes in laminin expression that are associated with the failure of ENCCs to invade recipient tissue. Together, our data suggest that a defect in pre-enteric Ednrb(flex3/flex3) NCCs results in delayed colonic arrival, which, due to environment changes in the colon, is sufficient to cause aganglionosis.

VEGF induces sensory and motor peripheral plasticity, alters bladder function, and promotes visceral sensitivity.

BACKGROUND: This work tests the hypothesis that bladder instillation with vascular endothelial growth factor (VEGF) modulates sensory and motor nerve plasticity, and, consequently, bladder function and visceral sensitivity.In addition to C57BL/6J, ChAT-cre mice were used for visualization of bladder cholinergic nerves. The direct effect of VEGF on the density of sensory nerves expressing the transient receptor potential vanilloid subfamily 1 (TRPV1) and cholinergic nerves (ChAT) was studied one week after one or two intravesical instillations of the growth factor.To study the effects of VEGF on bladder function, mice were intravesically instilled with VEGF and urodynamic evaluation was assessed. VEGF-induced alteration in bladder dorsal root ganglion (DRG) neurons was performed on retrogradly labeled urinary bladder afferents by patch-clamp recording of voltage gated Na+ currents. Determination of VEGF-induced changes in sensitivity to abdominal mechanostimulation was performed by application of von Frey filaments. RESULTS: In addition to an overwhelming increase in TRPV1 immunoreactivity, VEGF instillation resulted in an increase in ChAT-directed expression of a fluorescent protein in several layers of the urinary bladder. Intravesical VEGF caused a profound change in the function of the urinary bladder: acute VEGF (1 week post VEGF treatment) reduced micturition pressure and longer treatment (2 weeks post-VEGF instillation) caused a substantial reduction in inter-micturition interval. In addition, intravesical VEGF resulted in an up-regulation of voltage gated Na(+) channels (VGSC) in bladder DRG neurons and enhanced abdominal sensitivity to mechanical stimulation. CONCLUSIONS: For the first time, evidence is presented indicating that VEGF instillation into the mouse bladder promotes a significant increase in peripheral nerve density together with alterations in bladder function and visceral sensitivity. The VEGF pathway is being proposed as a key modulator of neural plasticity in the pelvis and enhanced VEGF content may be associated with visceral hyperalgesia, abdominal discomfort, and/or pelvic pain.

Targeting of endothelin receptor-B to the neural crest.

Endothelin receptor B (Ednrb) plays a critical role in the development of melanocytes and neurons and glia of the enteric nervous system. These distinct neural crest-derived cell types express Ednrb and share the property of intercalating into tissues, such as the intestine whose muscle precursor cells also express Ednrb. Such widespread Ednrb expression has been a significant obstacle in establishing precise roles for Ednrb in development. We describe here the production of an Ednrb allele floxed at exon 3 and its use in excising the receptor from mouse neural crest cells by use of Cre-recombinase driven by the Wnt1 promoter. Mice born with neural crest-specific excision of Ednrb possess aganglionic colon, lack trunk pigmentation, and die within 5 weeks due to megacolon. Ednrb receptor expression in these animals is absent only in the neural crest but present in surrounding smooth muscle cells. The absence of Ednrb from crest cells also results in a compensatory upregulation of Ednrb expression in other cells within the gut. We conclude that Ednrb loss only in neural crest cells is sufficient to produce the Hirschsprungs disease phenotype observed with genomic Ednrb mutations.

Immunohistochemical analysis of neuron types in the mouse small intestine.

The definition of the nerve cell types of the myenteric plexus of the mouse small intestine has become important, as more researchers turn to the use of mice with genetic mutations to analyze roles of specific genes and their products in enteric nervous system function and to investigate animal models of disease. We have used a suite of antibodies to define neurons by their shapes, sizes, and neurochemistry in the myenteric plexus. Anti-Hu antibodies were used to reveal all nerve cells, and the major subpopulations were defined in relation to the Hu-positive neurons. Morphological Type II neurons, revealed by anti-neurofilament and anti-calcitonin gene-related peptide antibodies, represented 26% of neurons. The axons of the Type II neurons projected through the circular muscle and submucosa to the mucosa. The cell bodies were immunoreactive for choline acetyltransferase (ChAT), and their terminals were immunoreactive for vesicular acetylcholine transporter (VAChT). Nitric oxide synthase (NOS) occurred in 29% of nerve cells. Most were also immunoreactive for vasoactive intestinal peptide, but they were not tachykinin (TK)-immunoreactive, and only 10% were ChAT-immunoreactive. Numerous NOS terminals occurred in the circular muscle. We deduced that 90% of NOS neurons were inhibitory motor neurons to the muscle (26% of all neurons) and 10% (3% of all neurons) were interneurons. Calretinin immunoreactivity was found in a high proportion of neurons (52%). Many of these had TK immunoreactivity. Small calretinin neurons were identified as excitatory neurons to the longitudinal muscle (about 20% of neurons, with ChAT/calretinin/+/- TK chemical coding). Excitatory neurons to the circular muscle (about 10% of neurons) had the same coding. Calretinin immunoreactivity also occurred in a proportion of Type II neurons. Thus, over 90% of neurons in the myenteric plexus of the mouse small intestine can be currently identified by their neurochemistry and shape.

APEX2-enhanced electron microscopy distinguishes sigma-1 receptor localization in the nucleoplasmic reticulum.

The sigma-1 receptor (Sig1R) is an endoplasmic reticulum chaperonin that is attracting tremendous interest as a potential anti-neurodegenerative target. While this membrane protein is known to reside in the inner nuclear envelope (NE) and influences transcription, apparent Sig1R presence in the nucleoplasm is often observed, seemingly contradicting its NE localization. We addressed this confounding issue by applying an antibody-free approach of electron microscopy (EM) to define Sig1R nuclear localization. We expressed APEX2 peroxidase fused to Sig1R-GFP in a Sig1R-null NSC34 neuronal cell line generated with CRISPR-Cas9. APEX2-catalyzed gold/silver precipitation markedly improved EM clarity and confirmed an apparent intra-nuclear presence of Sig1R. However, serial sectioning combined with APEX2-enhanced EM revealed that Sig1R actually resided in the nucleoplasmic reticulum (NR), a specialized nuclear compartment formed via NE invagination into the nucleoplasm. NR cross-sections also indicated Sig1R in ring-shaped NR membranes. Thus, this study distinguishes Sig1R in the NR which could otherwise appear localized in the nucleoplasm if detected with low-resolution methods. Our finding is important for uncovering potential Sig1R regulations in the nucleus.

Sigma-1 receptor expression in the dorsal root ganglion: Reexamination using a highly specific antibody.

Sigma-1 receptor (S1R) is a unique pluripotent modulator of living systems and has been reported to be associated with a number of neurological diseases including pathological pain. Intrathecal administration of S1R antagonists attenuates the pain behavior of rodents in both inflammatory and neuropathic pain models. However, the S1R localization in the spinal cord shows a selective ventral horn motor neuron distribution, suggesting the high likelihood of S1R in the dorsal root ganglion (DRG) mediating the pain relief by intrathecally administered drugs. Since primary afferents are the major component in the pain pathway, we examined the mouse and rat DRGs for the presence of the S1R. At both mRNA and protein levels, quantitative RT-PCR (qRT-PCR) and Western confirmed that the DRG contains greater S1R expression in comparison to spinal cord, cortex, or lung but less than liver. Using a custom-made highly specific antibody, we demonstrated the presence of a strong S1R immuno-fluorescence in all rat and mouse DRG neurons co-localizing with the Neuron-Specific Enolase (NSE) marker, but not in neural processes or GFAP-positive glial satellite cells. In addition, S1R was absent in afferent terminals in the skin and in the dorsal horn of the spinal cord. Using immuno-electron microscopy, we showed that S1R is detected in the nuclear envelope and endoplasmic reticulum (ER) of DRG cells. In contrast to other cells, S1R is also located directly at the plasma membrane of the DRG neurons. The presence of S1R in the nuclear envelope of all DRG neurons suggests an exciting potential role of S1R as a regulator of neuronal nuclear activities and/or gene expression, which may provide insight toward new molecular targets for modulating nociception at the level of primary afferent neurons.

Immunostaining to visualize murine enteric nervous system development.

The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Following colonization, neural crest cells must then differentiate into neurons with markers specific for their neurotransmitter phenotype. Cholinergic neurons, a major neurotransmitter phenotype in the enteric nervous system, are identified by staining for choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine. Historical efforts to visualize cholinergic neurons have been hampered by antibodies with differing specificities to central nervous system versus peripheral nervous system ChAT. We and others have overcome this limitation by using an antibody against placental ChAT, which recognizes both central and peripheral ChAT, to successfully visualize embryonic enteric cholinergic neurons. Additionally, we have compared this antibody to genetic reporters for ChAT and shown that the antibody is more reliable during embryogenesis. This protocol describes a technique for dissecting, fixing and immunostaining of the murine embryonic gastrointestinal tract to visualize enteric nervous system neurotransmitter expression.

Impaired Cellular Immunity in the Murine Neural Crest Conditional Deletion of Endothelin Receptor-B Model of Hirschsprung's Disease.

Hirschsprung's disease (HSCR) is characterized by aganglionosis from failure of neural crest cell (NCC) migration to the distal hindgut. Up to 40% of HSCR patients suffer Hirschsprung's-associated enterocolitis (HAEC), with an incidence that is unchanged from the pre-operative to the post-operative state. Recent reports indicate that signaling pathways involved in NCC migration may also be involved in the development of secondary lymphoid organs. We hypothesize that gastrointestinal (GI) mucosal immune defects occur in HSCR that may contribute to enterocolitis. EdnrB was deleted from the neural crest (EdnrBNCC-/-) resulting in mutants with defective NCC migration, distal colonic aganglionosis and the development of enterocolitis. The mucosal immune apparatus of these mice was interrogated at post-natal day (P) 21-24, prior to histological signs of enterocolitis. We found that EdnrBNCC-/- display lymphopenia of their Peyer's Patches, the major inductive site of GI mucosal immunity. EdnrBNCC-/- Peyer's Patches demonstrate decreased B-lymphocytes, specifically IgM+IgDhi (Mature) B-lymphocytes, which are normally activated and produce IgA following antigen presentation. EdnrBNCC-/- animals demonstrate decreased small intestinal secretory IgA, but unchanged nasal and bronchial airway secretory IgA, indicating a gut-specific defect in IgA production or secretion. In the spleen, which is the primary source of IgA-producing Mature B-lymphocytes, EdnrBNCC-/- animals display decreased B-lymphocytes, but an increase in Mature B-lymphocytes. EdnrBNCC-/- spleens are also small and show altered architecture, with decreased red pulp and a paucity of B-lymphocytes in the germinal centers and marginal zone. Taken together, these findings suggest impaired GI mucosal immunity in EdnrBNCC-/- animals, with the spleen as a potential site of the defect. These findings build upon the growing body of literature that suggests that intestinal defects in HSCR are not restricted to the aganglionic colon but extend proximally, even into the ganglionated small intestine and immune cells.

Intestinal dysbiosis and bacterial enteroinvasion in a murine model of Hirschsprung's disease.

BACKGROUND/PURPOSE: Hirschsprung's disease (HSCR), characterized by the absence of ganglia in the distal colon, results in functional obstruction. Despite surgical resection of the aganglionic segment, around 40% of patients suffer recurrent life threatening Hirschsprung's-associated enterocolitis (HAEC). The aim of this study was to investigate whether gut microbiota and intestinal immunity changes contribute to the HAEC risk in an HSCR model. METHODS: Mice with neural crest conditional deletion of Endothelin receptor B (EdnrB) and their littermate controls were used (EdnrB-null and EdnrB-het). Bacterial DNA was prepared from cecal contents of P16-18 and P21-24 animals and pyrosequencing employed for microbiome analysis. Ileal tissue was isolated and secretory phospholipase A2 (sPLA2) expression and activity determined. Enteroinvasion of Escherichia coli into ileal explants was measured using an ex vivo organ culture system. RESULTS: EdnrB-het and EdnrB-nulls displayed similar flora, sPLA2 expression and activity at P16-18. However, by P21-24, EdnrB-hets demonstrated increased Lactobacillus and decreased Bacteroides and Clostridium, while EdnrB-nulls exhibited reciprocal changes. EdnrB-nulls also showed reduced sPLA2 expression and luminal activity at this stage. Functionally, EdnrB-nulls were more susceptible to enteroinvasion with E. coli ex vivo and released less sPLA2 than EdnrB-hets. CONCLUSIONS: Initially, EdnrB-het and EdnrB-nulls contain similar cecal flora but then undergo reciprocal changes. EdnrB-nulls display dysbiosis, demonstrate impaired mucosal defense, decreased luminal sPLA2 and increased enteroinvasion of E. coli just prior to robust colonic inflammation and death. These findings suggest a role for the intestinal microbiome in the development of HAEC.

Sacral neural crest-derived cells enter the aganglionic colon of Ednrb-/- mice along extrinsic nerve fibers.

Both vagal and sacral neural crest cells contribute to the enteric nervous system in the hindgut. Because it is difficult to visualize sacral crest cells independently of vagal crest, the nature and extent of the sacral crest contribution to the enteric nervous system are not well established in rodents. To overcome this problem we generated mice in which only the fluorescent protein-labeled sacral crest are present in the terminal colon. We found that sacral crest cells were associated with extrinsic nerve fibers. We investigated the source, time of appearance, and characteristics of the extrinsic nerve fibers found in the aganglionic colon. We observed that the pelvic ganglion neurons contributed a number of extrinsic fibers that travel within the hindgut between circular and longitudinal muscles and within the submucosa and serosa. Sacral crest-derived cells along these fibers diminished in number from fetal to postnatal stages. A small number of sacral crest-derived cells were found between the muscle layers and expressed the neuronal marker Hu. We conclude that sacral crest cells enter the hindgut by advancing on extrinsic fibers and, in aganglionic preparations, they form a small number of neurons at sites normally occupied by myenteric ganglia. We also examined the colons of ganglionated preparations and found sacral crest-derived cells associated with both extrinsic nerve fibers and nascent ganglia. Extrinsic nerve fibers serve as a route of entry for both rodent and avian sacral crest into the hindgut.

Use of RNAlater in fluorescence-activated cell sorting (FACS) reduces the fluorescence from GFP but not from DsRed.

BACKGROUND: Flow cytometry utilizes signals from fluorescent markers to separate targeted cell populations for gene expression studies. However, the stress of the FACS process could change normal gene expression profiles. RNAlater could be used to stop such changes in original gene expression profiles through its ability to denature RNase and other proteins. The normal conformational structure of fluorescent proteins must be maintained in order to fluoresce. Whether or not RNAlater would affect signals from different types of intrinsic fluorescent proteins is crucial to its use in flow cytometry; this question has not been investigated in detail. FINDINGS: To address this question, we analyzed the effect of RNAlater on fluorescence intensity of GFP, YFP, DsRed and small fluorescent molecules attached to secondary antibodies (Cy2 and Texas-Red) when used in flow cytometry. FACS results were confirmed with fluorescence microscopy. Our results showed that exposure of YFP and GFP containing cells to RNAlater reduces the intensity of their fluorescence to such an extent that separation of such labeled cells is difficult if not impossible. In contrast, signals from DsRed2, Cy2 and Texas-Red were not affected by RNAlater treatment. In addition, the background fluorescence and clumping of dissociated cells are altered by RNAlater treatment. CONCLUSIONS: When considering gene expression studies using cell sorting with RNAlater, DsRed is the fluorescent protein of choice while GFP/YFP have severe limitations because of their reduced fluorescence. It is necessary to examine the effects of RNAlater on signals from fluorescent markers and the physical properties (e.g., clumping) of the cells before considering its use in cell sorting.

Behavior of enteric neural crest-derived cells varies with respect to the migratory wavefront.

Neural crest-derived cells colonize the entire gastrointestinal tract. The migration of these enteric neural crest-derived cells (ENCCs) occurs by their formation of cellular strands that extend into the intestinal mesenchyme. We have studied the behavior of crest cells that underlies the formation and extension of these strands by time-lapse microscopy. ENCCs expressing fluorescent marker molecules were visualized in situ in the embryonic mouse and chick gut. The major contributor to strand extension is from cells located within a region approximately 300 microm behind (rostral to) the most caudal cells in the migratory wavefront. Cells in the region immediately behind the leading cell of the strand either move intermittently in parallel with the leading cell, or advance caudally toward the wavefront over other ENCCs. Another addition to the strands arises from isolated cells located caudal to the wavefront. These cells showed a range of behavior including attachment and separation from the strands. The extending strands converged to form nodes, and then diverged along independent paths to form new strands, a behavior suggestive of attraction and repulsion. This behavior is probably responsible for the unique reticulated arrangement of ganglia in the enteric nervous system. As cells become positioned farther behind the wavefront, they exhibit more restricted movement and varied trajectories. We conclude that ENCCs exhibit different behaviors, depending on their position with respect to the wavefront. These different behaviors suggest a critical role for cell-cell interaction in the migratory process.

The pattern of neural crest advance in the cecum and colon.

Neural crest cells leave the hindbrain, enter the gut mesenchyme at the pharynx, and migrate as strands of cells to the terminal bowel to form the enteric nervous system. We generated embryos containing fluorescent enteric neural crest-derived cells (ENCCs) by mating Wnt1-Cre mice with Rosa-floxed-YFP mice and investigated ENCC behavior in the intact gut of mouse embryos using time-lapse fluorescent microscopy. With respect to the entire gut, we have found that ENCCs in the cecum and proximal colon behave uniquely. ENCCs migrating caudally through either the ileum, or caudal colon, are gradually advancing populations of strands displaying largely unpredictable local trajectories. However, in the cecum, advancing ENCCs pause for approximately 12 h, and then display an invariable pattern of migration to distinct regions of the cecum and proximal colon. In addition, while most ENCCs migrating through other regions of the gut remain interconnected as strands; ENCCs initially migrating through the cecum and proximal colon fragment from the main population and advance as isolated single cells. These cells aggregate into groups isolated from the main network, and eventually extend strands themselves to reestablish a network in the mid-colon. As the advancing network of ENCCs reaches the terminal bowel, strands of sacral crest cells extend, and intersect with vagal crest to bridge the small space between. We found a relationship between ENCC number, interaction, and migratory behavior by utilizing endogenously isolated strands and by making cuts along the ENCC wavefront. Depending on the number of cells, the ENCCs aggregated, proliferated, and extended strands to advance the wavefront. Our results show that interactions between ENCCs are important for regulating behaviors necessary for their advancement.

GDNF and insulin cooperate to enhance the proliferation and differentiation of enteric crest-derived cells.

Previously we have shown that glial derived neurotrophic factor (GDNF) stimulates modest increases in the proliferation of avian enteric crest-derived cells and similar increases in the phosphorylation of the phosphoinositide 3-kinase (PI3K) downstream substrate Akt (Akt-P). In the present study we tested whether GDNF-independent increases in PI3K activation would be sufficient to support proliferation. We found that insulin induces a large increase in the phosphorylation of Akt and can initiate DNA synthesis in avian enteric crest-derived cells, but is unable to maintain proliferation over time in culture, measured by BrdU incorporation. GDNF can also initiate DNA synthesis, but it too is unable to maintain BrdU incorporation in cultured enteric crest-derived cells. Sustained incorporation of BrdU after 16-48 h in culture is shown to be dependent on a combination of GDNF and insulin. Using a phospho-specific antibody, we found Akt-P levels to be similar in the proliferating (BrdU incorporation maintained from 16-48 h in culture) and nonproliferating populations, suggesting that Akt-P levels were not solely controlling the extent of BrdU incorporation. A minimum level of PI3K activation, however, is required, as shown by the dose-dependent reduction in proliferation with the PI3K inhibitor LY-294002. We conclude that the integrity of the PI3K pathway is essential for enteric crest-derived cell proliferation, but that the absolute levels of Akt-P do not determine the extent of proliferation. The enhanced proliferation in cultures containing both GDNF and insulin suggests that other pathways are involved, including the possibility that PI3K downstream effectors other than Akt are important in the regulation of avian enteric crest-derived cell proliferation.

Appearance of neurons and glia with respect to the wavefront during colonization of the avian gut by neural crest cells.

The enteric nervous system is formed by neural crest cells that migrate, proliferate, and differentiate into neurons and glia distributed in ganglia along the gastrointestinal tract. In the developing embryo some enteric crest cells cease their caudal movements, whereas others continue to migrate. Subsequently, the enteric neurons form a reticular network of ganglia interconnected by axonal projections. We studied the developing avian gut to characterize the pattern of migration of the crest cells, and the relationship between migration and differentiation. Crest cells at the leading edge of the migratory front appear as strands of cells; isolated individual crest cells are rarely seen. In the foregut and midgut, these strands are located immediately beneath the serosa. In contrast, crest cells entering the colon appear first in the deeper submucosal mesenchyme and later beneath the serosa. As the neural crest wavefront passes caudally, the crest cell cords become highly branched, forming a reticular lattice that presages the mature organization of the enteric nervous system. Neurons and glia first appear within the strands at the advancing wavefront. Later neurons are consistently located at the nodes where branches of the lattice intersect. In the most rostral foregut and in the colon, some neurons initially appear in close association with extrinsic nerve fibers from the vagus and Remak's nerve, respectively. We conclude that crest cells colonize the gut as chains of cells and that, within these chains, both neurons and glia appear close to the wavefront.