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.     

FGF2 deficit during development leads to specific neuronal cell loss in the enteric nervous system

The largest part of the peripheral nervous system is the enteric nervous system (ENS). It consists of an intricate network of several enteric neuronal subclasses with distinct phenotypes and functions within the gut wall. The generation of these enteric phenotypes is dependent upon appropriate neurotrophic support during development. Glial cell line-derived neurotrophic factor (GDNF) and fibroblast growth factor-2 (FGF2) play an important role in the differentiation and function of the ENS. A lack of GDNF or its receptor (Ret) causes intestinal aganglionosis in mice, while fibroblast growth factor receptor signaling antagonist is identified as regulating proteins in the GDNF/Ret signaling in the developing ENS. Primary myenteric plexus cultures and wholemount preparations of wild type (WT) and FGF2-knockout mice were used to analyze distinct enteric subpopulations. Fractal dimension (D) as a measure of self-similarity is an excellent tool to analyze complex geometric shape and was applied to classify the subclasses of enteric neurons concerning their individual morphology. As a consequence of a detailed analysis of subpopulation variations, wholemount preparations were stained for the calcium binding proteins calbindin and calretinin. The fractal analysis showed a reliable consistence of subgroups with different fractal dimensions (D) in each culture investigated. Seven different neuronal subtypes could be differentiated according to a rising D. Within the same D, the neurite length revealed significant differences between wild type and FGF2-knockout cultures, while the subclass distribution was also altered. Depending on the morphological characteristics, the reduced subgroup was supposed to be a secretomotor neuronal type, which could be confirmed by calbindin and calretinin staining of the wholemount preparations. These revealed a reduction up to 40 % of calbindin-positive neurons in the FGF2-knockout mouse. We therefore consider FGF2 playing a more important role in the fine-tuning of the ENS during development as previously assumed.     

RGMa inhibits neurite outgrowth of neuronal progenitors from murine enteric nervous system via the neogenin receptor in vitro

The enteric nervous system (ENS) in vertebrate embryos is formed by neural crest-derived cells. During development, these cells undergo extensive migration from the vagal and sacral regions to colonize the entire gut, where they differentiate into neurons and glial cells. Guidance molecules like netrins, semaphorins, slits, and ephrins are known to be involved in neuronal migration and axon guidance. In the CNS, the repulsive guidance molecule (RGMa) has been implicated in neuronal differentiation, migration, and apoptosis. Recently, we described the expression of the subtypes RGMa and RGMb and their receptor neogenin during murine gut development. In the present study, we investigated the influence of RGMa on neurosphere cultures derived from fetal ENS. In functional in vitro assays, RGMa strongly inhibited neurite outgrowth of differentiating progenitors via the receptor neogenin. The repulsive effect of RGMa on processes of differentiated enteric neural progenitors could be demonstrated by collapse assay. The influence of the RGM receptor on ENS was also analyzed in neogenin knockout mice. In the adult large intestine of mutants we observed disturbed ganglia formation in the myenteric plexus. Our data indicate that RGMa may be involved in differentiation processes of enteric neurons in the murine gut.     

Targeted mutation of serine 697 in the Ret tyrosine kinase causes migration defect of enteric neural crest cells

The RET receptor tyrosine kinase plays a critical role in the development of the enteric nervous system (ENS) and the kidney. Upon glial-cell-line-derived neurotrophic factor (GDNF) stimulation, RET can activate a variety of intracellular signals, including the Ras/mitogen-activated protein kinase, phosphatidylinositol 3-kinase (PI3K)/AKT, and RAC1/JUN NH(2)-terminal kinase (JNK) pathways. We recently demonstrated that the RAC1/JNK pathway is regulated by serine phosphorylation at the juxtamembrane region of RET in a cAMP-dependent manner. To determine the importance of cAMP-dependent modification of the RET signal in vivo, we generated mutant mice in which serine residue 697, a putative protein kinase A (PKA) phosphorylation site, was replaced with alanine (designated S697A mice). Homozygous S697A mutant mice lacked the ENS in the distal colon, resulting from a migration defect of enteric neural crest cells (ENCCs). In vitro organ culture showed an impaired chemoattractant response of the mutant ENCCs to GDNF. JNK activation by GDNF but not ERK, AKT and SRC activation was markedly reduced in neurons derived from the mutant mice. The JNK inhibitor SP600125 and the PKA inhibitor KT5720 suppressed migration of the ENCCs in cultured guts from wild-type mice to comparable degrees. Thus, these findings indicated that cAMP-dependent modification of RET function regulates the JNK signaling responsible for proper migration of the ENCCs in the developing gut.     

Pleiotropic effects of the bone morphogenetic proteins on development of the enteric nervous system

Formation of the enteric nervous system (ENS) from migratory neural crest-derived cells that colonize the primordial gut involves a complex interplay among different signaling molecules. The bone morphogenetic proteins (BMPs), specifically BMP2 and BMP4, play a particularly important role in virtually every stage of gut and ENS development. BMP signaling helps to pattern both the anterior-posterior axis and the radial axis of the gut prior to colonization by migratory crest progenitor cells. BMP signaling then helps regulate the migration of enteric neural crest-derived precursors as they colonize the fetal gut and form ganglia. BMP2 and -4 promote differentiation of enteric neurons in early fetal ENS development and glia at later stages. A major role for BMP signaling in the ENS is regulation of responses to other growth factors. Thus BMP signaling first regulates neurogenesis by modulating responses to GDNF and later gliogenesis through its effects on GGF-2 responses. Furthermore, BMPs promote growth factor dependency for survival of ENS neurons (on NT-3) and glia (on GGF-2) by inducing TrkC (neurons) and ErbB3 (glia). BMP signaling limits total neuron numbers, favoring the differentiation of later born neuronal phenotypes at the expense of earlier born ones thus influencing the neuronal composition of the ENS and the glia/neuron ratio. BMP2 and -4 also promote gangliogenesis via modification of neural cell adhesion molecules and promote differentiation of the circular and then longitudinal smooth muscles. Disruption of BMP signaling leads to defects in the gut and in ENS function commensurate with these complex developmental roles.     

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 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     

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.     

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.     

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.     

In ovo transplantation of enteric nervous system precursors from vagal to sacral neural crest results in extensive hindgut colonisation

The enteric nervous system (ENS) is derived from vagal and sacral neural crest cells (NCC). Within the embryonic avian gut, vagal NCC migrate in a rostrocaudal direction to form the majority of neurons and glia along the entire length of the gastrointestinal tract, whereas sacral NCC migrate in an opposing caudorostral direction, initially forming the nerve of Remak, and contribute a smaller number of ENS cells primarily to the distal hindgut. In this study, we have investigated the ability of vagal NCC, transplanted to the sacral region of the neuraxis, to colonise the chick hindgut and form the ENS in an experimentally generated hypoganglionic hindgut in ovo model. Results showed that when the vagal NC was transplanted into the sacral region of the neuraxis, vagal-derived ENS precursors immediately migrated away from the neural tube along characteristic pathways, with numerous cells colonising the gut mesenchyme by embryonic day (E) 4. By E7, the colorectum was extensively colonised by transplanted vagal NCC and the migration front had advanced caudorostrally to the level of the umbilicus. By E10, the stage at which sacral NCC begin to colonise the hindgut in large numbers, myenteric and submucosal plexuses in the hindgut almost entirely composed of transplanted vagal NCC, while the migration front had progressed into the pre-umbilical intestine, midway between the stomach and umbilicus. Immunohistochemical staining with the pan-neuronal marker, ANNA-1, revealed that the transplanted vagal NCC differentiated into enteric neurons, and whole-mount staining with NADPH-diaphorase showed that myenteric and submucosal ganglia formed interconnecting plexuses, similar to control animals. Furthermore, using an anti-RET antibody, widespread immunostaining was observed throughout the ENS, within a subpopulation of sacral NC-derived ENS precursors, and in the majority of transplanted vagal-to-sacral NCC. Our results demonstrate that: (1) a cell autonomous difference exists between the migration/signalling mechanisms used by sacral and vagal NCC, as transplanted vagal cells migrated along pathways normally followed by sacral cells, but did so in much larger numbers, earlier in development; (2) vagal NCC transplanted into the sacral neuraxis extensively colonised the hindgut, migrated in a caudorostral direction, differentiated into neuronal phenotypes, and formed enteric plexuses; (3) RET immunostaining occurred in vagal crest-derived ENS cells, the nerve of Remak and a subpopulation of sacral NCC within hindgut enteric ganglia.     

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.     

Neurturin is a neuritogenic but not a survival factor for developing and adult central noradrenergic neurons

Noradrenergic neurons of the locus coeruleus (LC) express the receptor tyrosine kinase c-ret, which binds ligands of the glial cell line-derived neurotrophic factor (GDNF) family. In the present study, we evaluated the function of neurturin (NTN), a GDNF family ligand whose function on LC neurons is unknown. Interestingly, we found that tyrosine hydroxylase (TH)-positive neurons in the LC express both GFRalpha1 and 2 receptors in a developmentally regulated fashion, suggesting a function for their preferred ligands: GDNF and NTN, respectively. Moreover, our results show that NTN mRNA expression is developmentally down-regulated in the LC and peaks in the postnatal hippocampus and cerebral cortex, during the target innervation period. In order to examine the function of NTN, we next performed LC primary cultures, and found that neither GDNF nor NTN promoted the survival of TH-positive neurons. However, both factors efficiently induced neurite outgrowth in noradrenergic neurons (147% and 149% over controls, respectively). Similarly, grafting of fibroblast cell lines engineered to express high levels of NTN did not prevent the loss of LC noradrenergic neurons in a 6-hydroxydopamine (6-OHDA) lesion model, but induced the sprouting of TH-positive cells. Thus our findings show that NTN does not promote the survival of LC noradrenergic neurons, but induces neurite outgrowth in developing noradrenergic neurons in vitro and in a model of neurodegeneration in vivo. These data, combined with data in the literature, suggest that GDNF family ligands are able to independently regulate neuronal survival and/or neuritogenesis.     

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.     

Expression of PROKR1 and PROKR2 in human enteric neural precursor cells and identification of sequence variants suggest a role in HSCR

Background

The enteric nervous system (ENS) is entirely derived from neural crest and its normal development is regulated by specific molecular pathways. Failure in complete ENS formation results in aganglionic gut conditions such as Hirschsprung''s disease (HSCR). Recently, PROKR1 expression has been demonstrated in mouse enteric neural crest derived cells and Prok-1 was shown to work coordinately with GDNF in the development of the ENS.

Principal Findings

In the present report, ENS progenitors were isolated and characterized from the ganglionic gut from children diagnosed with and without HSCR, and the expression of prokineticin receptors was examined. Immunocytochemical analysis of neurosphere-forming cells demonstrated that both PROKR1 and PROKR2 were present in human enteric neural crest cells. In addition, we also performed a mutational analysis of PROKR1, PROKR2, PROK1 and PROK2 genes in a cohort of HSCR patients, evaluating them for the first time as susceptibility genes for the disease. Several missense variants were detected, most of them affecting highly conserved amino acid residues of the protein and located in functional domains of both receptors, which suggests a possible deleterious effect in their biological function.

Conclusions

Our results suggest that not only PROKR1, but also PROKR2 might mediate a complementary signalling to the RET/GFRα1/GDNF pathway supporting proliferation/survival and differentiation of precursor cells during ENS development. These findings, together with the detection of sequence variants in PROKR1, PROK1 and PROKR2 genes associated to HSCR and, in some cases in combination with RET or GDNF mutations, provide the first evidence to consider them as susceptibility genes for HSCR.     

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.     

Site-specific gene expression and localization of growth factor ligand receptors RET, GFRα1 and GFRα2 in human adult colon

Two of the glial-cell-line-derived neurotrophic factor (GDNF) family ligands (GFLs), namely GDNF and neurturin (NRTN), are essential neurotropic factors for enteric nerve cells. Signal transduction is mediated by a receptor complex composed of GDNF family receptor alpha 1 (GFRα1) for GDNF or GFRα2 for NRTN, together with the tyrosine kinase receptor RET (rearranged during transfection). As both factors and their receptors are crucial for enteric neuron survival, we assess the site-specific gene expression of these GFLs and their corresponding receptors in human adult colon. Full-thickness colonic specimens were obtained after partial colectomy for non-obstructing colorectal carcinoma. Samples were processed for immunohistochemistry and co-localization studies. Site-specific gene expression was determined by real-time quantitative polymerase chain reaction in enteric ganglia and in circular and longitudinal muscle harvested by microdissection. Protein expression of the receptors was mainly localized in the myenteric and submucosal plexus. Dual-label immunohistochemistry with PGP 9.5 as a pan-neuronal marker detected immunoreactivity of the receptors in neuronal somata and ganglionic neuropil. RET immunoreactivity co-localized with neuronal GFRα1 and GFRα2 signals. The dominant source of receptor mRNA expression was in myenteric ganglia, whereas both GFLs showed higher expression in smooth muscle layers. The distribution and expression pattern of GDNF and NRTN and their corresponding receptors in the human adult enteric nervous system indicate a role of both GFLs not only in development but also in the maintenance of neurons in adulthood. The data also provide a basis for the assessment of disturbed signaling components of the GDNF and NRTN system in enteric neuropathies underlying disorders of gastrointestinal motility.