The hypothesis that BMPs (bone morphogenetic proteins), which act early in gut morphogenesis, also regulate specification and differentiation in the developing enteric nervous system (ENS) was tested. Expression of BMP-2 and BMP-4, BMPR-IA (BMP receptor subunit), BMPR-IB, and BMPR-II, and the BMP antagonists, noggin, gremlin, chordin, and follistatin was found when neurons first appear in the primordial bowel at embryonic day 12 (E12). Agonists, receptors, and antagonists were detected in separated populations of neural crestand noncrest-derived cells. When applied to immunopurified E12 ENS precursors, BMP-2 and BMP-4 induced nuclear translocation of phosphorylated Smad-1 (Sma and Mad-related protein). The number of neurons developing from these cells was increased by low concentrations and decreased by high concentrations of BMP-2 or BMP-4. BMPs induced the precocious appearance of TrkC-expressing neurons and their dependence on neurotrophin-3 for survival. BMP-4 interacted with glial cell line-derived neurotrophic factor (GDNF) to enhance neuronal development but limited GDNF-driven expansion of the precursor pool. BMPs also promoted development of smooth muscle from mesenchymal cells immunopurified at E12. To determine the physiological significance of these observations, the BMP antagonist noggin was overexpressed in the developing ENS of transgenic mice under the control of the neuron-specific enolase promoter. Neuronal numbers in both enteric plexuses and smooth muscle were increased throughout the postnatal small intestine. These increases were already apparent by E18. In contrast, TrkC-expressing neurons decreased in both plexuses of postnatal nogginoverexpressing animals, again an effect detectable at E18. BMP-2 and/or BMP-4 thus limit the size of the ENS but promote the development of specific subsets of enteric neurons, including those that express TrkC.
Maintaining constant CO2 and H+ concentrations in the arterial blood is critical for life. The principal mechanism through which this is achieved in mammals is the respiratory chemoreflex whose circuitry is still elusive. A candidate element of this circuitry is the retrotrapezoid nucleus (RTN), a collection of neurons at the ventral medullary surface that are activated by increased CO2 or low pH and project to the respiratory rhythm generator. Here, we use intersectional genetic strategies to lesion the RTN neurons defined by Atoh1 and Phox2b expression and to block or activate their synaptic output. Photostimulation of these neurons entrains the respiratory rhythm. Conversely, abrogating expression of Atoh1 or Phox2b or glutamatergic transmission in these cells curtails the phrenic nerve response to low pH in embryonic preparations and abolishes the respiratory chemoreflex in behaving animals. Thus, the RTN neurons expressing Atoh1 and Phox2b are a necessary component of the chemoreflex circuitry.DOI: http://dx.doi.org/10.7554/eLife.07051.001
Taste and most sensory inputs required for the feedback regulation of digestive, respiratory, and cardiovascular organs are conveyed to the central nervous system by so-called "visceral" sensory neurons located in three cranial ganglia (geniculate, petrosal, and nodose) and integrated in the hindbrain by relay sensory neurons located in the nucleus of the solitary tract. Visceral sensory ganglia and the nucleus of the solitary tract all depend for their formation on the pan-visceral homeodomain transcription factor Phox2b, also required in efferent neurons to the viscera. We show here, by genetically tracing Phox2b + cells, that in the absence of the protein, many visceral sensory neurons (first-and second-order) survive. However, they adopt a fate-including molecular signature, cell positions, and axonal projections-akin to that of somatic sensory neurons (first-and second-order), located in the trigeminal, superior, and jugular ganglia and the trigeminal sensory nuclei, that convey touch and pain sensation from the orofacial region. Thus, the cranial sensory pathways, somatic and visceral, are related, and Phox2b serves as a developmental switch from the former to the latter.
The basic helix-loop-helix (bHLH) transcription factor Hand2 has been shown to play a role in the development of the mammalian sympathetic nervous system (SNS); however, its precise role could not be uncovered because Hand2 is required for early embryonic survival. We therefore generated a conditional Hand2 knockout mouse line by excising Hand2 in Wnt1-Cre-expressing neural crest-derived cells. These mice die at 12.5 dpc with embryos showing severe cardiovascular and facial defects. Crest-derived cells, however, populate sites of SNS development and proliferate normally. Sympathetic precursors differentiate into neurons and express the pan-neuronal markers, beta3-tubulin (Tuj1) and Hu showing that Hand2 is not essential for SNS neuronal differentiation. To determine whether Hand2 regulates noradrenergic differentiation, the levels of the norepinephrine biosynthetic enzymes, tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) was examined. Both enzymes were dramatically reduced in mutant embryos suggesting that the primary role of Hand2 in the SNS is determination of neuronal phenotype. Loss of Hand2 did not affect the expression of other members of the transcriptional circuit regulating SNS development, including Phox2a/b, Mash1 and Gata2/3; however, Hand2 was required for Hand1 expression. Our data suggest that the major role of Hand2 during SNS development is to permit sympathetic neurons to acquire a catecholaminergic phenotype.
Vagal sensory axons and migrating neural crest-derived precursor cells follow similar pathways to reach the gut. The crest-derived cells express the netrin receptor deleted in colorectal cancer (DCC) and migrate toward netrins expressed by the intestinal mucosa and pancreas; this attraction is required for the formation of submucosal and pancreatic ganglia. We tested the hypothesis that enteric netrins also attract vagal sensory fibers. These axons were located as a function of age in fetal mice by applying the lipophilic tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) bilaterally to nodose ganglia. DiI-labeled axons were found in the esophagus and proximal stomach by E12 and, more distally, in the small bowel at E14-E16. Transcripts encoding DCC were expressed in the nodose ganglia of mice from E12 to adulthood but were developmentally regulated. Paraesophageal anterior and posterior vagal trunks were DCC immunoreactive from E12 to E16. Transcripts encoding netrin-1 were expressed in the developing foregut and midgut; netrin-1 immunoreactivity was detected in the outer gut mesenchyme and mucosal epithelium. Neurites from explanted E14 nodose ganglia grew selectively toward cocultured E14 distal foregut explants (P < 0.01). Antibodies to DCC specifically abolished this preferential outgrowth (P < 0.05). Nodose axons also grew selectively toward cocultured netrin-secreting 293-EBNA cells (P < 0.005); antibodies to DCC again blocked this preferential outgrowth (P < 0.05). These data suggest that netrins, which are expressed in the bowel, attract DCC-expressing vagal sensory axons.
Background & Aims Enteric neurons have been reported to be increased in inflamed regions of the bowel in patients with inflammatory bowel disease (IBD) or intestinal neurogangliomatosis. It is impossible to determine whether this hyperinnervation predates intestinal inflammation, results from it, or contributes to its severity in humans, so we studied this process in mice. Methods To determine whether the density of enteric neurons determines the severity of inflammation, we studied transgenic mice that have greater-than-normal (Hand2+/− mice) or fewer-than-normal (NSE-noggin mice, which overexpress noggin under the control of the neuron-specific enolase promoter) numbers of neurons in the enteric nervous system (ENS). Colitis was induced with trinitrobenzene sulfonic acid or dextran sulfate sodium and the intensity of the resulting inflammation in Hand2+/− and NSE-noggin mice was compared with that of wild-type littermates. Results Severity of each form of colitis (based on survival, symptom, and histologic scores; intestinal expression of genes that encode proinflammatory molecules; and levels of neutrophil elastase and p50 NF- Hand2+/− mice and significantly increased in NSE-noggin animals. Neither mouse differed from wild-type in the severity of delayed-type hypersensitivity (edema, T-cell and neutrophil infiltration, or expression of interleukin- - - -dinitro-1-fluorobenzene. Transgene effects on inflammation were therefore restricted to the gastrointestinal tract. Conclusion The severity of intestinal inflammation is associated with the density of the enteric innervation in mice. Abnormalities in ENS development might therefore contribute to the pathogenesis of IBD.
Hand genes encode basic helix-loop-helix transcription factors that are expressed in the developing gut, where their function is unknown. We now report that enteric Hand2 expression is limited to crest-derived cells, whereas Hand1 expression is restricted to muscle and interstitial cells of Cajal. Hand2 is developmentally regulated and is intranuclear in precursors but cytoplasmic in neurons. Neurons develop in explants from wild-type but not Hand2 -/-bowel, although, in both, crest-derived cells are present and glia arise. Similarly, small interfering RNA (siRNA) silencing of Hand2 in enteric crest-derived cells prevents neuronal development. Terminally differentiated enteric neurons do not develop after conditional inactivation of Hand2 in migrating crest-derived cells; nevertheless, conditional Hand2 inactivation does not prevent precursors from expressing early neural markers. We suggest that enteric neuronal development occurs in stages and that Hand2 expression is required for terminal differentiation but not for precursors to enter the neuronal lineage.
The neural crest-derived cell population that colonizes the bowel (ENCDC) contains proliferating neural/glial progenitors. We tested the hypothesis that bone morphogenetic proteins (BMPs 2 and 4), which are known to promote enteric neuronal differentiation at the expense of proliferation, function similarly in gliogenesis. Enteric gliogenesis was analyzed in mice that overexpress the BMP antagonist, noggin, or BMP4 in the primordial ENS. Noggin-induced loss-of-function decreased, while BMP4-induced gain-of-function increased the glial density and glia/neuron ratio. When added to immunoisolated ENCDC, BMPs provoked nuclear translocation of phosphorylated SMAD proteins and enhanced both glial differentiation and expression of the neuregulin receptor ErbB3. ErbB3 transcripts were detected in E12 rat gut, before glial markers are expressed; moreover, expression of the ErbB3 ligand, glial growth factor 2 (GGF2) escalated rapidly after its first detection at E14. ErbB3-immunoreactive cells were located in the ENS of fetal and adult mice. GGF2 stimulated gliogenesis and proliferation and inhibited glial cell derived neurotrophic factor (GDNF)-promoted neurogenesis. Enhanced glial apoptosis occurred following GGF2 withdrawal; BMPs intensified this GGF2-dependence and reduced GGF2-stimulated proliferation. These observations support the hypotheses that BMPs are required for enteric gliogenesis and act by promoting responsiveness of ENCDC to ErbB3 ligands such as GGF2.
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