Enteric Neurobiology

The ENS is a very complex structure. Development is likely to require the cooperative action of hundreds of molecules to guide precursor migration, determine the number of neurons and glia, define neuron subtypes, guide neurites to their targets and to regulate function. Our review details some of what is known (Lake and Heuckeroth, ENS Review 2013) about the molecular mechanisms of ENS development.  The full review article is here.  Additional links to our studies are below.

Our published work:

1. To identify genes expressed in the developing enteric nervous system we used a combination of microarray analysis, quantitative real-time PCR and in situ hybridization to identify hundreds of molecules produced in the developing ENS (Vohra microarray and expression paper). We also demonstrated that inhibitors of synaptic vesicle SNARE proteins inhibit ENS precursor migration and neurite growth.

2. We have generated a gene expression database from our own data and other public data that you can download (ENS gene expression database).

3. In collaboration with colleagues we identified ENS defects in mice with cohesinopathies.
Psd5a (cohesin regulatory factor) mutant mice have distal bowel aganglionosis (Zhang Psd5A/5B paper)
Psd5b mutant mice also have problems with bowel colonization by ENS precursors (Zhang Psd5B paper). Mice with mutations in Psd5A and Psd5B genes have features that resemble Cornelia de Lange syndrome.
4. We demonstrated important roles for BMP4 and NCAM1 (Fu Bmp4 paper) in the developing ENS. This paper showed that BMP4 signaling slows ENS precursor migration through the bowel, increases enteric neuron aggregation into ganglia, directs neuron positioning within the bowel wall, and and enhances neurite fasciculation by increasing polysialic acid (PSA) addition to NCAM1. We also define expression patterns for BMP4 and noggin within the developing bowel wall.
5. One of the longstanding mysteries about Hirschsprung disease is why boys are much more commonly affected than girls. In fact, for children with aganglionosis restricted to the rectum and sigmoid (short segment disease), there are 4x as many affected boys as girls. We hypothesized that females might just make more of some of the proteins that are already known to be needed for normal colonization of the end of the bowel by ENS precursors than males. We tested this idea using mice and found that there is less mRNA for endothelin-3 (Edn3) and endothelin converting enzyme (Ece1) in the male mouse fetal bowel than in females (Vohra male versus female paper). Adding EDN3 to the bowel of male mice with a Ret mutation that causes distal bowel aganglionosis increased the rate of ENS precursor migration into the end of the bowel and partially rescued bowel colonization. The reason for the difference in Edn3 and Ece1 gene expression between males and females is still not known, but this work emphasizes one area where more research is needed, since it might provide clues to preventing Hirschsprung disease.
6. We identified new roles for PKCzeta and GSK3 in ENS precursor polarity and migration (Vohra et. al.).  This paper outlines signaling from RET on the cell surface to the proteins that control the cytoskeleton.  PKCzeta and GSK3 regulate the number of axons per enteric neuron.
7. We demonstrated that a wide range of ENS structural defects can be produced by altering the timing and expression patterns for GDNF (Wang et. al.).  The ratio of neuron subtypes and pattering of NO producing neurites can also be altered by GDNF expression patterns.
8. In collaboration we demonstrated how ENS patterning is altered in a series or Ret mutant animals that have specific signaling pathways disrupted by altering intracellular tyrosine residues that are phosphorylated when RET is activated (Jain et. al.).
9. In collaboration we developed a model of intestinal pseudoobstruction syndrome that results from a mutation in Tfam (mitochondrial transcription factor A).  This work demonstrated region specific and neuron subtype specific sensitivity of the ENS to mitochondrial disease (Viader et. al.).
10. In collaboration we demonstrated that small intestine adaptation after bowel resection is enhanced in Ret heterozygous mice.  This work has interesting implications for the management of short bowel syndrome (Hitch et. al.).
11. We demonstrated interesting effects on ENS structure of mutations in RALDH1 and RALDH3 in addition to previously recognized roles for RALDH2 (Wright-Jin et. al.).
12. In collaboration we discovered a surprising intestinal pseudoobstruction phenotype in mice with mutations in RB1 (Fu et. al.).  A susbset of NO producing enteric neurons undergo endoreplicaton in the context of RB1 mutations.  We also provide evidence that NO has trophic effects on the developing ENS.
13. We discovered that the drug mycophenolate and several other medicines inhibit colonization of the distal bowel by ENS precursor cells (Lake et. al.).  Mycophenolate inhibits the enzyme inosine monophosphate dehydrogenase, the rate limiting step in de novo guanine nucleotide synthesis, effectively reducing cellular GTP and dGTP.  This work suggests that anything that reduces the efficiency of ENS precursor proliferation may increase the risk of Hirschsprung disease.
New projects: 
We continue to work to identify new genes that control specific aspects of ENS development.  We are particularly interested in:
1. Identifying gene-environment interactions that influence ENS development.
2. Determing mechanisms that control axon pathfinding in the ENS.
3. Determining how neuronal subtype identity is established in the ENS.
4. ENS stem cell biology.
5. Finding new ways to treat or prevent intestinal motility disorders.
6. Building translational research programs.