According to current dogma, there is little or no ongoing neurogenesis in the fully developed adult enteric nervous system. This lack of neurogenesis leaves unanswered the question of how enteric neuronal populations are maintained in adult guts, given previous reports of ongoing neuronal death. Here, we confirm that despite ongoing neuronal cell loss because of apoptosis in the myenteric ganglia of the adult small intestine, total myenteric neuronal numbers remain constant. This observed neuronal homeostasis is maintained by new neurons formed in vivo from dividing precursor cells that are located within myenteric ganglia and express both Nestin and p75NTR, but not the pan-glial marker Sox10. Mutation of the phosphatase and tensin homolog gene in this pool of adult precursors leads to an increase in enteric neuronal number, resulting in ganglioneuromatosis, modeling the corresponding disorder in humans. Taken together, our results show significant turnover and neurogenesis of adult enteric neurons and provide a paradigm for understanding the enteric nervous system in health and disease.

Background & Aims: The enteric nervous system (ENS) exists in close proximity to luminal bacteria. Intestinal microbes regulate ENS development, but little is known about their effects on adult enteric neurons. We investigated whether intestinal bacteria or their products affect the adult ENS via toll like receptors (TLRs) in mice. Methods:We performed studies with conventional C57/BL6, germ-free C57/BL6, Nestin-creER T2 :tdTomato, Nestin-GFP, and ChAT-cre:tdTomato. Mice were given drinking water with ampicillin or without (controls). Germ-free mice were given drinking water with TLR2 agonist or without (controls). Some mice were given a blocking antibody against TLR2 or a TLR4 inhibitor.We performed whole-gut transit, bead latency, and geometric center studies. Feces were collected and analyzed by 16S rRNA gene sequencing. Longitudinal muscle myenteric plexus (LMMP) tissues were collected, analyzed by immunohistochemistry, and levels of nitric oxide were measured. Cells were isolated from colonic LMMP of Nestin-creER T2 :tdTomato mice and incubated with agonists of TLR2 (receptor for Gram-positive bacteria), TLR4 (receptor for Gramnegative bacteria), or distilled water (control) andd analyzed by flow cytometry.Results: Stool from mice given ampicillin had altered composition of gut microbiota with reduced abundance of Gram-positive bacteria and increased abundance of Gram-negative bacteria, compared with mice given only water. Mice given ampicillin had reduced colon motility compared with mice given only water, and their colonic LMMP had reduced numbers of nitrergic neurons, reduced nNOS production, and reduced colonic neurogenesis. Numbers of colonic myenteric neurons increased after mice were switched from ampicillin to plain water, with increased markers of neurogenesis. Nestin-positive ENPCs expressed TLR2 and TLR4. In cells isolated from the colonic LMMP, incubation with the TLR2 agonist increased the percentage of neurons originating from ENPCs to approximately 10%, compared to approximately 0.01% in cells incubated with the TLR4 agonist or distilled water. Mice given an antibody against TLR2 had prolonged whole-gut transit times; their colonic LMMP had reduced total neurons and a smaller proportion of nitrergic neurons per ganglion, and reduced markers of neurogenesis compared with mice given saline. Colonic LMMP of mice given the TLR4 inhibitor did not have reduced markers of neurogenesis. Colonic LMMP of germ-free mice given TLR2 agonist had increased neuronal numbers compared with control germ-free mice. Conclusions:In the adult mouse colon, TLR2 promotes colonic neurogenesis, regulated by intestinal bacteria. Our findings indicate that colonic microbiota help maintain the adult ENS via

Conditional deletion of murine fibroblast growth factor receptors (Fgfrs) 1 and 2 in metanephric mesenchyme leads to renal agenesis with unbranched ureteric buds; however, there are occasionally two buds per nephric duct. Our goal was to determine whether conditional deletion of Fgfr1 or Fgfr2 alone resulted in multiple ureteric bud induction sites. Although deletion of Fgfr1 alone results in no abnormalities, loss of Fgfr2 often leads to multiple ureteric buds and anomalies including renal aplasia, misshaped kidneys, partially duplicated kidneys, duplicated ureters, and obstructed hydroureter. Deletion of Fgfr2 did not change expression domains of glial cell line-derived neurotrophic factor (GDNF), Robo2, bone morphogenetic protein 4, or Sprouty1, all of which regulate ureteric bud induction. Cultured Fgfr2 mutant nephric ducts were also not more sensitive to exogenous GDNF than controls. Whole mount in situ hybridization revealed that in mutant embryos, Fgfr2 was deleted from stromal cells around the nephric duct and ureteric bud base, which correlates well with the ureteric bud induction abnormalities. Thus, Fgfr2 is critical in ensuring that there is a single ureteric bud from the nephric duct. The plethora of later stage defects in Fgfr2 conditional knockouts is reminiscent of many human cases of genetic urogenital anomalies. (Pediatr Res 64: 592-598, 2008) F ibroblast growth factor receptors (Fgfrs) are receptor tyrosine kinases with four known signaling members and 22 ligands in mammals (1). Fgfrs are expressed throughout embryogenesis in many tissues, including the kidney (1).The metanephric kidney arises from two embryonic tissues, the metanephric mesenchyme and ureteric bud. At embryonic day (E) 10.5 in the mouse and the 5th week of gestation in humans, the metanephric mesenchyme induces a single ureteric bud from the Wolffian (nephric) duct near the hind limb (2). Subsequently, the ureteric bud elongates and branches within the metanephric mesenchyme, giving rise to the collecting ducts, pelvis, and ureter (2). At its tips, the ureteric bud induces local metanephric mesenchyme to condense and differentiate into nephron epithelia. Regions of stromal mesenchyme also surround the developing nephrons.Many studies have documented diverse actions of Fgfrs in developing kidneys. Addition of Fgf2 to isolated rat metanephric mesenchymal tissues prevented apoptosis (3,4) and promoted condensation (4). Transgenic mice with a dominant negative Fgfr fragment developed renal agenesis/severe dysgenesis (5). Mice null for Fgf7, Fgf10, or Fgfr2-IIIb (the receptor isoform for Fgf7 and Fgf10), have small kidneys with normal-appearing nephrons (6 -8). Mice with conditional deletion of Fgf8 from the metanephric mesenchyme, however, have interrupted nephron development (9,10).Although Fgfr1 and Fgfr2 null mice are early embryonic lethal (11-14), conditional knockout approaches have revealed roles for these receptors in kidney development. We observed that deletion of Fgfr2 from the ureteric bud results in aberran...

Purpose Mice with conditional deletion of fibroblast growth factor receptor 2 (Fgfr2) in metanephric mesenchyme (Fgfr2Mes−/−) have ureteric bud induction abnormalities. Our goal was to determine if Fgfr2Mes−/− mutants developed abnormally positioned ureters predisposing to vesico-ureteral reflux (VUR). Materials and Methods We measured common nephric duct (CND) lengths and assayed for apoptosis in embryonic day (E) 11.5 mice. We performed 3 dimensional (3D) reconstructions of and real time PCR and whole mount in situ hybridization for Fgfr2 in urinary tracts in E15.5 embryos. We performed cystograms followed by 3D reconstructions in postnatal animals. Results Compared with controls, Fgfr2Mes−/− embryos had increased CND lengths (with no differences in apoptosis) indicating cranially displaced ureteric buds. 3-D reconstructions at E15.5 showed low insertions of ureters into the bladder (near bladder necks) in Fgfr2Mes−/− mice. Postnatal Fgfr2Mes−/− mutants had high rates of VUR compared with controls (47.4% vs. 4.0%, p=0.00006). In postnatal mutants with unilateral reflux, the refluxing ureters inserted closer to the bladder neck than non-refluxing ureters. The external ureteral insertional angles at the outer bladder wall (formed by the ureteral insertion points and bladder neck) were greater in mutant refluxing ureters compared to contralateral non-refluxing ureters and to control ureters. At E15.5, Fgfr2 levels were decreased in Fgfr2Mes−/− kidneys compared with controls, but were not statistically different in ureters or bladders. Conclusions Fgfr2Mes−/− mice have ureteric induction abnormalities, associated with abnormal ureteral insertion in the bladder and subsequent VUR, consistent with the Mackie and Stephens hypothesis.