Loss of Sprouty2 partially rescues renal hypoplasia and stomach hypoganglionosis but not intestinal aganglionosis in Ret Y1062F mutant mice

The glial cell line-derived neurotrophic factor (GDNF)/RET tyrosine kinase signaling pathway plays crucial roles in the development of the enteric nervous system (ENS) and the kidney. Tyrosine 1062 (Y1062) in RET is an autophosphorylation residue that is responsible for the activation of the PI3K/AKT and RAS/MAPK signaling pathways. Mice lacking signaling via Ret Y1062 show renal hypoplasia and hypoganglionosis of the ENS although the phenotype is milder than the Gdnf- or Ret-deficient mice. Sprouty2 (Spry2) was found to be an antagonist for fibroblast growth factor receptor (FGFR) and acts as an inhibitory regulator of ERK activation. Spry2-deficient mice exhibit hearing loss and enteric nerve hyperplasia. In the present study, we generated Spry2-deficient and Ret Y1062F knock-in (tyrosine 1062 is replaced with phenylalanine) double mutant mice to see if abnormalities of the ENS and kidney, caused by loss of signaling via Ret Y1062, are rescued by a deficiency of Spry2. Double mutant mice showed significant recovery of ureteric bud branching and ENS development in the stomach. These results indicate that Spry2 regulates downstream signaling mediated by GDNF/RET signaling complex in vivo.     

Novel mechanism of regulation of Rac activity and lamellipodia formation by RET tyrosine kinase

Rac activation in neuronal cells plays an important role in lamellipodia formation that is a critical event for neuritogenesis. It is well known that the Rac activity is regulated via activation of phosphatidylinositol 3-kinase (PI3K) by a variety of receptor tyrosine kinases. Here we show that increased serine phosphorylation on RET receptor tyrosine kinase following cAMP elevation promotes lamellipodia formation of neuronal cells induced by glial cell line-derived neurotrophic factor (GDNF). We identified serine 696 in RET as a putative phosphorylation site by protein kinase A and found that mutation of this serine almost completely inhibited lamellipodia formation by GDNF without affecting activation of the PI3K/AKT signaling pathway. Mutation of tyrosine 1062 in RET, whose phosphorylation is crucial for activation of PI3K, also inhibited lamellipodia formation by GDNF. Inhibition of lamellipodia formation by mutation of either serine 696 or tyrosine 1062 was associated with decrease of the Rac1-guanine nucleotide exchange factor (GEF) activity, suggesting that this activity is regulated by two different signaling pathways via serine 696 and tyrosine 1062 in RET. Moreover, in the presence of serine 696 mutation, lamellipodia formation was rescued by replacing tyrosine 687 with phenylalanine. These findings propose a novel mechanism that receptor tyrosine kinase modulates actin dynamics in neuronal cells via its cAMP-dependent phosphorylation.     

Signalling by the RET receptor tyrosine kinase and its role in the development of the mammalian enteric nervous system.

RET is a member of the receptor tyrosine kinase (RTK) superfamily, which can transduce signalling by glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) in cultured cells. In order to determine whether in addition to being sufficient, RET is also necessary for signalling by these growth factors, we studied the response to GDNF and NTN of primary neuronal cultures (peripheral sensory and central dopaminergic neurons) derived from wild-type and RET-deficient mice. Our experiments show that absence of a functional RET receptor abrogates the biological responses of neuronal cells to both GDNF and NTN. Despite the established role of the RET signal transduction pathway in the development of the mammalian enteric nervous system (ENS), very little is known regarding its cellular mechanism(s) of action. Here, we have studied the effects of GDNF and NTN on cultures of neural crest (NC)-derived cells isolated from the gut of rat embryos. Our findings suggest that GDNF and NTN promote the survival of enteric neurons as well as the survival, proliferation and differentiation of multipotential ENS progenitors present in the gut of E12.5-13.5 rat embryos. However, the effects of these growth factors are stage-specific, since similar ENS cultures established from later stage embryos (E14. 5-15.5), show markedly diminished response to GDNF and NTN. To examine whether the in vitro effects of RET activation reflect the in vivo function(s) of this receptor, the extent of programmed cell death was examined in the gut of wild-type and RET-deficient mouse embryos by TUNEL histochemistry. Our experiments show that a subpopulation of enteric NC undergoes apoptotic cell death specifically in the foregut of embryos lacking the RET receptor. We suggest that normal function of the RET RTK is required in vivo during early stages of ENS histogenesis for the survival of undifferentiated enteric NC and their derivatives.     

Requirement of signalling by receptor tyrosine kinase RET for the directed migration of enteric nervous system progenitor cells during mammalian embryogenesis

The majority of neurones and glia of the enteric nervous system (ENS) are derived from the vagal neural crest. Shortly after emigration from the neural tube, ENS progenitors invade the anterior foregut and, migrating in a rostrocaudal direction, colonise in an orderly fashion the rest of the foregut, the midgut and the hindgut. We provide evidence that activation of the receptor tyrosine kinase RET by glial cell line-derived neurotrophic factor (GDNF) is required for the directional migration of ENS progenitors towards and within the gut wall. We find that neural crest-derived cells present within foetal small intestine explants migrate towards an exogenous source of GDNF in a RET-dependent fashion. Consistent with an in vivo role of GDNF in the migration of ENS progenitors, we demonstrate that Gdnf is expressed at high levels in the gut of mouse embryos in a spatially and temporally regulated manner. Thus, during invasion of the foregut by vagal-derived neural crest cells, expression of Gdnf was restricted to the mesenchyme of the stomach, ahead of the invading NC cells. Twenty-four hours later and as the ENS progenitors were colonising the midgut, Gdnf expression was upregulated in a more posterior region - the caecum anlage. In further support of a role of endogenous GDNF in enteric neural crest cell migration, we find that in explant cultures GDNF produced by caecum is sufficient to attract NC cells residing in more anterior gut segments. In addition, two independently generated loss-of-function alleles of murine Ret, Ret.k- and miRet51, result in characteristic defects of neural crest cell migration within the developing gut. Finally, we identify phosphatidylinositol-3 kinase and the mitogen-activated protein kinase signalling pathways as playing crucial roles in the migratory response of enteric neural crest cells to GDNF.     

Prokineticin-1 (Prok-1) works coordinately with glial cell line-derived neurotrophic factor (GDNF) to mediate proliferation and differentiation of enteric neural crest cells

Enteric neural crest cells (NCC) are multipotent progenitors which give rise to neurons and glia of the enteric nervous system (ENS) during fetal development. Glial cell line-derived neurotrophic factor (GDNF)/RET receptor tyrosine kinase (Ret) signaling is indispensable for their survival, migration and differentiation. Using microarray analysis and isolated NCCs, we found that 45 genes were differentially expressed after GDNF treatment (16 h), 29 of them were up-regulated including 8 previously undescribed genes. Prokineticin receptor 1 (PK-R1), a receptor for Prokineticins (Prok), was identified in our screen and shown to be consistently up-regulated by GDNF in enteric NCCs. Further, PK-R1 was persistently expressed at a lower level in the enteric ganglions of the c-Ret deficient mice when compared to that of the wild-type littermates. Subsequent functional analysis showed that GDNF potentiated the proliferative and differentiation effects of Prok-1 by up-regulating PK-R1 expression in enteric NCCs. In addition, expression analysis and gene knock-down experiments indicated that Prok-1 and GDNF signalings shared some common downstream targets. More importantly, Prok-1 could induce both proliferation and expression of differentiation markers of c-Ret deficient NCCs, suggesting that Prok-1 may also provide a complementary pathway to GDNF signaling. Taken together, these findings provide evidence that Prok-1 crosstalks with GDNF/Ret signaling and probably provides an additional layer of signaling refinement to maintain proliferation and differentiation of enteric NCCs.     

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout the developing vertebrate embryo. In addition to providing peripheral glia and neurons, NCC generate melanocytes as well as most of the cranio-facial skeleton. NCC migration and differentiation is controlled by a combination of their axial origin along the neural tube and their exposure to regionally distinct extracellular cues. Such contribution of extracellular ligands is especially evident during the formation of the enteric nervous system (ENS), a complex interconnected network of neural ganglia that locally controls (among other things) gut muscle movement and intestinal motility. Most of the ENS is derived from a small initial pool of NCC that undertake a long journey in order to colonize - in a rostral to caudal fashion - the entire length of the prospective gut. Among several signaling pathways known to influence enteric NCC colonization, GDNF/RET signaling is recognized as the most important. Indeed, spatiotemporally controlled secretion of the RET ligand GDNF by the gut mesenchyme is chiefly responsible for the attraction and guidance of RET-expressing enteric NCC to and within the embryonic gut. Here, we describe an ex vivo cell migration assay, making use of a transgenic mouse line possessing fluorescently labeled NCC, which allows precise quantification of enteric NCC migration potential in the presence of various growth factors, including GDNF.     

Enteric neural crest-derived cells promote their migration by modifying their microenvironment through tenascin-C production

The enteric nervous system (ENS) is derived from vagal and sacral neural crest cells that migrate, proliferate, and differentiate into enteric neurons and glia within the gut wall. The mechanisms regulating enteric neural crest-derived cell (ENCC) migration are poorly characterized despite the importance of this process in gut formation and function. Characterization of genes involved in ENCC migration is essential to understand ENS development and could provide targets for treatment of human ENS disorders. We identified the extracellular matrix glycoprotein tenascin-C (TNC) as an important regulator of ENCC development. We find TNC dynamically expressed during avian gut development. It is absent from the cecal region just prior to ENCC arrival, but becomes strongly expressed around ENCCs as they enter the ceca and hindgut. In aganglionic hindguts, TNC expression is strong throughout the outer mesenchyme, but is absent from the submucosal region, supporting the presence of both ENCC-dependent and independent expression within the gut wall. Using rat–chick coelomic grafts, neural tube cultures, and gut explants, we show that ENCCs produce TNC and that this ECM protein promotes their migration. Interestingly, only vagal neural crest-derived ENCCs express TNC, whereas sacral neural crest-derived cells do not. These results demonstrate that vagal crest-derived ENCCs actively modify their microenvironment through TNC expression and thereby help to regulate their own migration.     

Enteric neuroblasts require the phosphatidylinositol 3‐kinase pathway for GDNF‐stimulated proliferation

The enteric nervous system (ENS) develops from neural crest cells that enter the gut, migrate, proliferate, and differentiate into neurons and glia. The growth factor glial‐derived neurotrophic factor (GDNF) stimulates the proliferation and survival of enteric crest‐derived cells. We investigated the intracellular signaling pathways activated by GDNF and their involvement in proliferation. We found that GDNF stimulates the phosphorylation of both the PI 3‐kinase downstream substrate Akt and the MAP kinase substrate ERK in cultures of immunoaffinity‐purified embryonic avian enteric crest‐derived cells. The selective PI 3‐kinase inhibitor LY‐294002 blocked GDNF‐stimulated Akt phosphorylation in purified crest cells, and reduced proliferation in cultures of dissociated quail gut. The ERK kinase (MEK) inhibitors PD 98059 and UO126 did not reduce GDNF‐stimulated proliferation, although PD 98059 blocked GDNF‐stimulated phosphorylation of ERK. We conclude that the PI 3‐kinase pathway is necessary for the GDNF‐stimulated proliferation of enteric neuroblasts. © 2001 John Wiley & Sons, Inc. J Neurobiol 47: 306–317, 2001     

Phosphorylation of serine 43 is not required for inhibition of c-Raf kinase by the cAMP-dependent protein kinase

The activity of the serine/threonine kinase c-Raf (Raf) is inhibited by increased intracellular cAMP. This is believed to require phosphorylation with the cAMP-dependent protein kinase (PKA), although the mechanism by which PKA inhibits Raf is controversial. We investigated the requirement for PKA phosphorylation using Raf mutants expressed in HEK293 or NIH 3T3 cells. Phosphopeptide mapping of (32)P-labeled Raf (WT) or a mutant lacking a putative PKA phosphorylation site (serine to alanine, S43A) confirmed that serine 43 (Ser(43)) was the major cAMP (forskolin)-stimulated phosphorylation site in vivo. Interestingly, the EGF-stimulated Raf kinase activity of the S43A mutant was inhibited by forskolin equivalently to that of the WT Raf. Forskolin also inhibited the activation of an N-terminal deletion mutant Delta5-50 Raf completely lacking this phosphorylation site. Although WT Raf was phosphorylated by PKA, phosphorylation did not inhibit Raf catalytic activity in vitro, nor did forskolin treatment inhibit the activity of an N-terminally truncated Raf protein (Raf 22W) or a full-length Raf protein (Raf-CAAX) expressed in NIH 3T3 cells. In contrast, forskolin inhibited the EGF-dependent activation of a Raf isoform (B-Raf), lacking an analogous phosphorylation site to Ser(43). Thus, these results demonstrate that PKA exerts its inhibitory effects independently of direct Raf phosphorylation and suggests instead that PKA prevents an event required for the EGF-dependent activation of Raf.     

Ureteric bud outgrowth in response to RET activation is mediated by phosphatidylinositol 3-kinase

The c-ret gene encodes a receptor tyrosine kinase (RET) essential for the development of the kidney and enteric nervous system. Activation of RET requires the secreted neurotrophin GDNF (glial cell line-derived neurotrophic factor) and its high affinity receptor, a glycosyl phosphatidylinositol-linked cell surface protein GFRalpha1. In the developing kidney, RET, GDNF, and GFRalpha1 are all required for directed outgrowth and branching morphogenesis of the ureteric bud epithelium. Using MDCK renal epithelial cells as a model system, activation of RET induces cell migration, scattering, and formation of filopodia and lamellipodia. RET-expressing MDCK cells are able to migrate toward a localized source of GDNF. In this report, the intracellular signaling mechanisms regulating RET-dependent migration and chemotaxis are examined. Activation of RET resulted in increased levels of phosphatidylinositol 3-kinase (PI3K) activity and Akt/PKB phosphorylation. This increase in PI3K activity is essential for regulating the GDNF response, since the specific inhibitor, LY294002, blocks migration and chemotaxis of MDCK cells. Using an in vitro organ culture assay, inhibition of PI3K completely blocks the GDNF-dependent outgrowth of ectopic ureter buds. PI3K is also essential for branching morphogenesis once the ureteric bud has invaded the kidney mesenchyme. The data suggest that activation of RET in the ureteric bud epithelium signals through PI3K to control outgrowth and branching morphogenesis.     

Enteric nervous system progenitors are coordinately controlled by the G protein-coupled receptor EDNRB and the receptor tyrosine kinase RET

The enteric nervous system (ENS) in vertebrates is derived mainly from vagal neural crest cells that enter the foregut and colonize the entire wall of the gastrointestinal tract. Failure to completely colonize the gut results in the absence of enteric ganglia (Hirschsprung's disease). Two signaling systems mediated by RET and EDNRB have been identified as critical players in enteric neurogenesis. We demonstrate that interaction between these signaling pathways controls ENS development throughout the intestine. Activation of EDNRB specifically enhances the effect of RET signaling on the proliferation of uncommitted ENS progenitors. In addition, we reveal novel antagonistic roles of these pathways on the migration of ENS progenitors. Protein kinase A is a key component of the molecular mechanisms that integrate signaling by the two receptors. Our data provide strong evidence that the coordinate and balanced interaction between receptor tyrosine kinases and G protein-coupled receptors controls the development of the nervous system in mammals.     

Endothelial cells promote migration and proliferation of enteric neural crest cells via β1 integrin signaling

Enteric neural crest-derived cells (ENCCs) migrate along the intestine to form a highly organized network of ganglia that comprises the enteric nervous system (ENS). The signals driving the migration and patterning of these cells are largely unknown. Examining the spatiotemporal development of the intestinal neurovasculature in avian embryos, we find endothelial cells (ECs) present in the gut prior to the arrival of migrating ENCCs. These ECs are patterned in concentric rings that are predictive of the positioning of later arriving crest-derived cells, leading us to hypothesize that blood vessels may serve as a substrate to guide ENCC migration. Immunohistochemistry at multiple stages during ENS development reveals that ENCCs are positioned adjacent to vessels as they colonize the gut. A similar close anatomic relationship between vessels and enteric neurons was observed in zebrafish larvae. When EC development is inhibited in cultured avian intestine, ENCC migration is arrested and distal aganglionosis results, suggesting that ENCCs require the presence of vessels to colonize the gut. Neural tube and avian midgut were explanted onto a variety of substrates, including components of the extracellular matrix and various cell types, such as fibroblasts, smooth muscle cells, and endothelial cells. We find that crest-derived cells from both the neural tube and the midgut migrate avidly onto cultured endothelial cells. This EC-induced migration is inhibited by the presence of CSAT antibody, which blocks binding to β1 integrins expressed on the surface of crest-derived cells. These results demonstrate that ECs provide a substrate for the migration of ENCCs via an interaction between β1 integrins on the ENCC surface and extracellular matrix proteins expressed by the intestinal vasculature. These interactions may play an important role in guiding migration and patterning in the developing ENS.     

Enteric neuroblasts require the phosphatidylinositol 3-kinase pathway for GDNF-stimulated proliferation

The enteric nervous system (ENS) develops from neural crest cells that enter the gut, migrate, proliferate, and differentiate into neurons and glia. The growth factor glial-derived neurotrophic factor (GDNF) stimulates the proliferation and survival of enteric crest-derived cells. We investigated the intracellular signaling pathways activated by GDNF and their involvement in proliferation. We found that GDNF stimulates the phosphorylation of both the PI 3-kinase downstream substrate Akt and the MAP kinase substrate ERK in cultures of immunoaffinity-purified embryonic avian enteric crest-derived cells. The selective PI 3-kinase inhibitor LY-294002 blocked GDNF-stimulated Akt phosphorylation in purified crest cells, and reduced proliferation in cultures of dissociated quail gut. The ERK kinase (MEK) inhibitors PD 98059 and UO126 did not reduce GDNF-stimulated proliferation, although PD 98059 blocked GDNF-stimulated phosphorylation of ERK. We conclude that the PI 3-kinase pathway is necessary for the GDNF-stimulated proliferation of enteric neuroblasts.     

The contribution of serine residues 1588 and 1755 to phosphorylation of the type I inositol 1,4,5-trisphosphate receptor by PKA and PKG

Type I inositol 1,4,5-trisphosphate receptors can be phosphorylated by cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG). To define the site-specificity of these events we analyzed the phosphorylation of mutant receptors expressed in intact cells. These studies showed that S(1588) and S(1755), the serine residues within kinase consensus sequences, are equally sensitive to PKA, that phosphorylation events at these sites are independent of each other, and that PKG predominantly phosphorylates S(1588). These findings provide the basis for understanding the functional consequences of type I inositol 1,4,5-trisphosphate receptor phosphorylation.     

Drosophila Cyclin J is a mitotically stable Cdk1 partner without essential functions

Enteric neural crest-derived cells (ENCCs) migrate along the intestine to form a highly organized network of ganglia that comprises the enteric nervous system (ENS). The signals driving the migration and patterning of these cells are largely unknown. Examining the spatiotemporal development of the intestinal neurovasculature in avian embryos, we find endothelial cells (ECs) present in the gut prior to the arrival of migrating ENCCs. These ECs are patterned in concentric rings that are predictive of the positioning of later arriving crest-derived cells, leading us to hypothesize that blood vessels may serve as a substrate to guide ENCC migration. Immunohistochemistry at multiple stages during ENS development reveals that ENCCs are positioned adjacent to vessels as they colonize the gut. A similar close anatomic relationship between vessels and enteric neurons was observed in zebrafish larvae. When EC development is inhibited in cultured avian intestine, ENCC migration is arrested and distal aganglionosis results, suggesting that ENCCs require the presence of vessels to colonize the gut. Neural tube and avian midgut were explanted onto a variety of substrates, including components of the extracellular matrix and various cell types, such as fibroblasts, smooth muscle cells, and endothelial cells. We find that crest-derived cells from both the neural tube and the midgut migrate avidly onto cultured endothelial cells. This EC-induced migration is inhibited by the presence of CSAT antibody, which blocks binding to β1 integrins expressed on the surface of crest-derived cells. These results demonstrate that ECs provide a substrate for the migration of ENCCs via an interaction between β1 integrins on the ENCC surface and extracellular matrix proteins expressed by the intestinal vasculature. These interactions may play an important role in guiding migration and patterning in the developing ENS.     

Coordinated activation of autophosphorylation sites in the RET receptor tyrosine kinase: importance of tyrosine 1062 for GDNF mediated neuronal differentiation and survival.

The catalytic and signaling activities of RET, a tyrosine kinase receptor for glial cell line-derived neurotrophic factor (GDNF), are controlled by the autophosphorylation of several tyrosine residues in the RET cytoplasmic domain. To analyze the phosphorylation state of individual tyrosines, we generated antibodies recognizing specific phosphotyrosine sites involved in the catalytic (Tyr(905)) and downstream signaling (Tyr(1015), Tyr(1062), and Tyr(1096)) activities of this receptor. Stimulation with GDNF induced coordinated phosphorylation of the 4 tyrosine residues in neuronal cell lines and in primary cultures of sympathetic neurons isolated from rat superior cervical ganglia. Neurturin and artemin, two other members of the GDNF ligand family, also induced synchronized phosphorylation of RET tyrosines with kinetics comparable to those observed with GDNF. Tyrosine phosphorylation was maximal 15 min after ligand stimulation, decaying thereafter with similar kinetics in all 4 residues. Co-stimulation with a soluble form of the GFRalpha1 co-receptor potentiated ligand-dependent phosphorylation of different intracellular tyrosines to a similar extent and increased the survival of superior cervical ganglion neurons compared with treatment with GDNF alone. In vivo, high levels of phosphorylated Tyr(905), Tyr(1015), and Tyr(1062) were detected in embryonic mouse dorsal root ganglia, with a sharp decline at early postnatal stages. Protein transduction of anti-Tyr(P)(1062) antibodies into cultured cells reduced activation of MAPKs ERK1 and ERK2 and the AKT kinase in response to GDNF and diminished GDNF-dependent neuronal differentiation and survival of embryonic sensory neurons from the nodose ganglion. These results demonstrate synchronized utilization of individual RET tyrosine residues in neurons in vivo and reveal an important role for RET Tyr(1062) in mediating neuronal survival by GDNF.     

Phosphorylation of Snapin by PKA modulates its interaction with the SNARE complex

cAMP-dependent protein kinase A (PKA) can modulate synaptic transmission by acting directly on unknown targets in the neurotransmitter secretory machinery. Here we identify Snapin, a protein of relative molecular mass 15,000 that is implicated in neurotransmission by binding to SNAP-25, as a possible target. Deletion mutation and site-directed mutagenetic experiments pinpoint the phosphorylation site to serine 50. PKA-phosphorylation of Snapin significantly increases its binding to synaptosomal-associated protein-25 (SNAP-25). Mutation of Snapin serine 50 to aspartic acid (S50D) mimics this effect of PKA phosphorylation and enhances the association of synaptotagmin with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. Furthermore, treatment of rat hippocampal slices with nonhydrolysable cAMP analogue induces in vivo phosphorylation of Snapin and enhances the interaction of both Snapin and synaptotagmin with the SNARE complex. In adrenal chromaffin cells, overexpression of the Snapin S50D mutant leads to an increase in the number of release-competent vesicles. Our results indicate that Snapin may be a PKA target for modulating transmitter release through the cAMP-dependent signal-transduction pathway.     

Ret isoform function and marker gene expression in the enteric nervous system is conserved across diverse vertebrate species

The enteric nervous system (ENS) derives from migratory neural crest cells that colonize the developing gut tube, giving rise to an integrated network of neurons and glial cells, which together regulate important aspects of gut function, including coordinating the smooth muscle contractions of the gut wall. The absence of enteric neurons in portions of the gut (aganglionosis) is the defining feature of Hirschsprung’s disease (HSCR) and has been replicated in a number of mouse models. Mutations in the RET tyrosine kinase account for over half of familial cases of HSCR and mice mutant for Ret exhibit aganglionosis. RET exists in two main isoforms, RET9 and RET51 and studies in mouse have shown that RET9 is sufficient to allow normal development of the ENS. In the last several years, zebrafish has emerged as a model of vertebrate ENS development, having been supported by a number of demonstrations of conservation of gene function between zebrafish, mouse and human. In this study we further analyse the potential similarities and differences between ENS development in zebrafish, mouse and human. We demonstrate that zebrafish Ret is required in a dose-dependent manner to regulate colonization of the gut by neural crest derivatives, as in human. Additionally, we show that as in mouse and human, zebrafish ret is produced as two isoforms, ret9 and ret51. Moreover, we show that, as in mouse, the Ret9 isoform is sufficient to support colonization of the gut by enteric neurons. Finally, we identify zebrafish orthologues of genes previously identified to be expressed in the mouse ENS and demonstrate that these genes are expressed in the developing zebrafish ENS, thereby identifying useful ENS markers in this model organism. These studies reveal that the similarities between gene expression and gene function across vertebrate species is more extensive than previously appreciated, thus supporting the use of zebrafish as a general model for vertebrate ENS development and the use of zebrafish genetic screens as a way to identify candidate genes mutated in HSCR cases.     

GDNF availability determines enteric neuron number by controlling precursor proliferation

To clarify the role of Ret signaling components in enteric nervous system (ENS) development, we evaluated ENS anatomy and intestinal contractility in mice heterozygous for Ret, GFRalpha1 and Ret ligands. These analyses demonstrate that glial cell line-derived neurotrophic factor (GDNF) and neurturin are important for different aspects of ENS development. Neurturin is essential for maintaining the size of mature enteric neurons and the extent of neuronal projections, but does not influence enteric neuron number. GDNF availability determines enteric neuron number by controlling ENS precursor proliferation. However, we were unable to find evidence of programmed cell death in the wild type ENS by immunohistochemistry for activated caspase 3. In addition, enteric neuron number is normal in Bax(-/-) and Bid(-/-) mice, suggesting that, in contrast to most of the rest of the nervous system, programmed cell death is not important for determining enteric neuron numbers. Only mild reductions in neuron size and neuronal fiber counts occur in Ret(+/-) and Gfra1(+/-) mice. All of these heterozygous mice, however, have striking problems with intestinal contractility and neurotransmitter release, demonstrating that Ret signaling is critical for both ENS structure and function.     

Phosphorylation of syntaphilin by cAMP-dependent protein kinase modulates its interaction with syntaxin-1 and annuls its inhibitory effect on vesicle exocytosis

cAMP-dependent protein kinase (PKA) can modulate synaptic transmission by acting directly on the neurotransmitter secretory machinery. Here, we identify one possible target: syntaphilin, which was identified as a molecular clamp that controls free syntaxin-1 and dynamin-1 availability and thereby regulates synaptic vesicle exocytosis and endocytosis. Deletion mutation and site-directed mutagenesis experiments pinpoint dominant PKA phosphorylation sites to serines 43 and 56. PKA phosphorylation of syntaphilin significantly decreases its binding to syntaxin-1A in vitro. A syntaphilin mutation of serine 43 to aspartic acid (S43D) shows similar effects on binding. To characterize in vivo phosphorylation events, we generated antisera against a peptide of syntaphilin containing a phosphorylated serine 43. Treatment of rat brain synaptosomes or syntaphilin-transfected HEK 293 cells with the cAMP analogue BIMPS induces in vivo phosphorylation of syntaphilin and inhibits its interaction with syntaxin-1 in neurons. To determine whether PKA phosphorylation of syntaphilin is involved in the regulation of Ca(2+)-dependent exocytosis, we investigated the effect of overexpression of syntaphilin and its S43D mutant on the regulated secretion of human growth hormone from PC12 cells. Although expression of wild type syntaphilin in PC12 cells exhibits significant reduction in high K(+)-induced human growth hormone release, the S43D mutant fails to inhibit exocytosis. Our data predict that syntaphilin could be a highly regulated molecule and that PKA phosphorylation could act as an "off" switch for syntaphilin, thus blocking its inhibitory function via the cAMP-dependent signal transduction pathway.