Development of colonic motility in the neonatal mouse-studies using spatiotemporal maps

Colonic migrating motor complexes (CMMCs) are spontaneous, anally propagating constrictions, repeating every 3-5 min in mouse colon in vitro. They are regulated by the enteric nervous system and may be equivalent to mass movement contractions. We examined postnatal development of CMMCs and circular muscle innervation to gain insight into mechanisms regulating transit in the maturing colon. Video recordings of mouse colon in vitro were used to construct spatiotemporal maps of spontaneous contractile patterns. Development of nitric oxide synthase (NOS) and cholinergic nerve terminals in the circular muscle was examined immunohistochemically. In adults, CMMCs appeared regularly at 4.6 +/- 0.9-min intervals (n = 5). These intervals were reduced by inhibition of NOS (2.7 +/- 0.2 min; n = 5; P < 0.05). CMMCs were abolished by tetrodotoxin (n = 4). CMMCs at postnatal day (P)10 were indistinguishable from adult. At birth and P4, CMMCs were absent. Instead, small constrictions that propagated both orally and anally, "ripples," were seen. Ripples were unaffected by tetrodotoxin or inhibition of NOS and were present in Ret(-/-) mice (which lack enteric neurons) at embryonic day 18.5. In P6 mice, only ripples were seen in control, but NOS inhibition induced CMMCs (n = 8). NOS terminals were abundant in the circular muscle at birth; cholinergic terminals were sparse but were common by P10. In mouse, myogenic ripples are the only mechanism available to produce colonic transit at birth. At P6, neural circuits that generate CMMCs are present but are inhibited by tonic activity of nitric oxide. Adult patterns appear by P10.     

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.     

Cooperation between GDNF/Ret and ephrinA/EphA4 signals for motor-axon pathway selection in the limb

Establishment of limb innervation by motor neurons involves a series of hierarchical axon guidance decisions by which motor-neuron subtypes evaluate peripheral guidance cues and choose their axonal trajectory. Earlier work indicated that the pathway into the dorsal limb by lateral motor column (LMC[l]) axons requires the EphA4 receptor, which mediates repulsion elicited by ephrinAs expressed in ventral limb mesoderm. Here, we implicate glial-cell-line-derived neurotrophic factor (GDNF) and its receptor, Ret, in the same guidance decision. In Gdnf or Ret mutant mice, LMC(l) axons follow an aberrant ventral trajectory away from dorsal territory enriched in GDNF, showing that the GDNF/Ret system functions as an instructive guidance signal for motor axons. This phenotype is enhanced in mutant mice lacking Ret and EphA4. Thus, Ret and EphA4 signals cooperate to enforce the precision of the same binary choice in motor-axon guidance.     

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.     

Neural cells in the esophagus respond to glial cell line-derived neurotrophic factor and neurturin, and are RET-dependent

Glial cell line-derived neurotrophic factor (GDNF) is expressed in the gastrointestinal tract of the developing mouse and appears to play an important role in the migration of enteric neuron precursors into and along the small and large intestines. Two other GDNF family members, neurturin and artemin, are also expressed in the developing gut although artemin is only expressed in the esophagus. We examined the effects of GDNF, neurturin, and artemin on neural crest cell migration and neurite outgrowth in explants of mouse esophagus, midgut, and hindgut. Both GDNF and neurturin induced neural crest cell migration and neurite outgrowth in all regions examined. In the esophagus, the effect of GDNF on migration and neurite outgrowth declined with age between E11.5 and E14.5, but neurturin still had a strong neurite outgrowth effect at E14.5. Artemin did not promote neural migration or neurite outgrowth in any region investigated. The effects of GDNF family ligands are mediated by the Ret tyrosine kinase. We examined the density of neurons in the esophagus of Ret-/- mice, which lack neurons in the small and large intestines. The density of esophageal neurons in Ret-/- mice was only about 4% of the density of esophageal neurons in Ret+/- and Ret+/+ mice. These results show that GDNF and neurturin promote migration and neurite outgrowth of crest-derived cells in the esophagus as well as the intestine. Moreover, like intestinal neurons, the development of esophageal neurons is largely Ret-dependent.     

Interleukin-1beta activates specific populations of enteric neurons and enteric glia in the guinea pig ileum and colon

Fos expression was used to assess whether the proinflammatory cytokine interleukin-1beta (IL-1beta) activated specific, chemically coded neuronal populations in isolated preparations of guinea pig ileum and colon. Whether the effects of IL-1beta were mediated through a prostaglandin pathway and whether IL-1beta induced the expression of cyclooxygenase (COX)-2 was also examined. Single- and double-labeling immunohistochemistry was used after treatment of isolated tissues with IL-1beta (0.1-10 ng/ml). IL-1beta induced Fos expression in enteric neurons and also in enteric glia in the ileum and colon. For enteric neurons, activation was concentration-dependent and sensitive to indomethacin, in both the myenteric and submucosal plexuses in both regions of the gut. The maximum proportion of activated neurons differed between the ileal (approximately 15%) and colonic (approximately 42%) myenteric and ileal (approximately 60%) and colonic (approximately 75%) submucosal plexuses. The majority of neurons activated in the myenteric plexus of the ileum expressed nitric oxide synthase (NOS) or enkephalin immunoreactivity. In the colon, activated myenteric neurons expressed NOS. In the submucosal plexus of both regions of the gut, the majority of activated neurons were vasoactive intestinal polypeptide (VIP) immunoreactive. After treatment with IL-1beta, COX-2 immunoreactivity was detected in the wall of the gut in both neurons and nonneuronal cells. In conclusion, we have found that the proinflammatory cytokine IL-1beta specifically activates certain neurochemically defined neural pathways and that these changes may lead to disturbances in motility observed in the inflamed bowel.     

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.     

Video Imaging and Spatiotemporal Maps to Analyze Gastrointestinal Motility in Mice

The enteric nervous system (ENS) plays an important role in regulating gastrointestinal (GI) motility and can function independently of the central nervous system. Changes in ENS function are a major cause of GI symptoms and disease and may contribute to GI symptoms reported in neuropsychiatric disorders including autism. It is well established that isolated colon segments generate spontaneous, rhythmic contractions known as Colonic Migrating Motor Complexes (CMMCs). A procedure to analyze the enteric neural regulation of CMMCs in ex vivo preparations of mouse colon is described. The colon is dissected from the animal and flushed to remove fecal content prior to being cannulated in an organ bath. Data is acquired via a video camera positioned above the organ bath and converted to high-resolution spatiotemporal maps via an in-house software package. Using this technique, baseline contractile patterns and pharmacological effects on ENS function in colon segments can be compared over 3-4 hr. In addition, propagation length and speed of CMMCs can be recorded as well as changes in gut diameter and contraction frequency. This technique is useful for characterizing gastrointestinal motility patterns in transgenic mouse models (and in other species including rat and guinea pig). In this way, pharmacologically induced changes in CMMCs are recorded in wild type mice and in the Neuroligin-3^R451C mouse model of autism. Furthermore, this technique can be applied to other regions of the GI tract including the duodenum, jejunum and ileum and at different developmental ages in mice.     

Effects of pharmacological inhibition of small GTPases on axon extension and migration of enteric neural crest-derived cells

In the developing enteric nervous system, there is a close association between migrating neural crest-derived cells and the axons of early differentiating neurons. We used pharmacological inhibitors of small GTPases to determine if crest cell migration and axon growth could be uncoupled in cultured intact explants of embryonic mouse gut and slices of embryonic gut grown on collagen gels containing GDNF. Inhibition of the Rho effectors, ROCKI/II, or Rac/Cdc42 inhibited both cell migration and neurite growth in intact explants of embryonic gut. The effects of both ROCKI/II and Rac/Cdc42 inhibitors were more severe on cell migration and axon extension in gut explants from Ret(+/-) mice than in explants from wildtype mice, indicating that Rho GTPases probably act downstream of the receptor tyrosine kinase, Ret. Inhibition of ROCKI/II had different effects on migration and axon extension in gut slices grown on collagen gels containing GDNF from that seen in intact explants of gut. We conclude that ROCKI/II and Rac/Cdc42 are required for both neural crest-derived cell migration and axon growth in the developing gut.     

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.     

Neurochemical identification of enteric neurons expressing P2X(3) receptors in the guinea-pig ileum

It was hypothesised that P2X(3) receptors, predominantly labelling spinal and cranial sensory ganglionic neurons, are also expressed in intrinsic sensory enteric neurons, although direct evidence is lacking. The aim of this study was to localise P2X(3) receptors in the enteric nervous system of the guinea-pig ileum, and to neurochemically identify the P2X(3)-expressing neurons. In the submucous plexus, cholinergic neurons expressing calretinin (CRT), were immunostained for P2X(3). These neurons made up about 12% of the submucous neurons. In the myenteric plexus, approximately 36% of the neurons expressed P2X(3). Half of the latter neurons were immunoreactive for CRT, whereas about 20% were immunoreactive for nitric oxide synthase (NOS). Based on earlier neurochemical analysis of enteric neurons in the guinea-pig, the myenteric neurons exhibiting P2X(3)/CRT immunoreactivity were identified as longitudinal muscle motor neurons, and those expressing P2X(3)/NOS immunoreactivity as short inhibitory circular muscle motor neurons. In both plexuses, no colocalisation was observed between P2X(3) and calbindin, a marker for intrinsic sensory neurons. Multiple staining with antisera raised against somatostatin, neuropeptide Y, substance P or neurofilament protein did not reveal any costaining. It can be concluded that in the guinea-pig ileum, intrinsic sensory neurons do not express P2X(3) receptors. However, this does not negate the possibility that extrinsic sensory nerves expressing P2X(3) are involved in a purinergic mechanosensory transduction pathway as demonstrated in other organs.     

Gene targeting reveals a critical role for neurturin in the development and maintenance of enteric, sensory, and parasympathetic neurons

Neurturin (NTN) is a neuronal survival factor that activates the Ret tyrosine kinase in the presence of a GPI-linked coreceptor (either GFR alpha1 or GFR alpha2). Neurturin-deficient (NTN-/-) mice generated by homologous recombination are viable and fertile but have defects in the enteric nervous system, including reduced myenteric plexus innervation density and reduced gastrointestinal motility. Parasympathetic innervation of the lacrimal and submandibular salivary gland is dramatically reduced in NTN-/- mice, indicating that Neurturin is a neurotrophic factor for parasympathetic neurons. GFR alpha2-expressing cells in the trigeminal and dorsal root ganglia are also depleted in NTN-/- mice. The loss of GFR alpha2-expressing neurons, in conjunction with earlier studies, provides strong support for GFR alpha2/Ret receptor complexes as the critical mediators of NTN function in vivo.     

Nitric oxide synthase immunoreactivity in the enteric nervous system of the developing human digestive tract

We have investigated indirectly the presence of nitric oxide in the enteric nervous system of the digestive tract of human fetuses and newborns by nitric oxide synthase (NOS) immunocytochemistry and nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry. In the stomach, NOS immunoactivity was confined to the myenteric plexus and nerve fibres in the outer smooth musculature; few immunoreactive nerve cell bodies were found in ganglia of the outer submucous plexus. In the pyloric region, a few nitrergic perikarya were seen in the inner submucous plexus and some immunoreactive fibres were found in the muscularis mucosae. In the small intestine, nitrergic neurons clustered just underneath or above the topographical plane formed by the primary nerve strands of the myenteric plexus up to the 26th week of gestation, after which stage, they occurred throughout the ganglia. Many of their processes contributed to the dense fine-meshed tertiary nerve network of the myenteric plexus and the circular smooth muscle layer. NOS-immunoreactive fibres directed to the circular smooth muscle layer originated from a few NOS-containing perikarya located in the outer submucous plexus. In the colon, caecum and rectum, labelled nerve cells and fibres were numerous in the myenteric plexus; they were also found in the outer submucous plexus. The circular muscle layer had a much denser NOS-immunoreactive innervation than the longitudinally oriented taenia. The marked morphological differences observed between nitrergic neurons within the developing human gastrointestinal tract, together with the typical innervation pattern in the ganglionic and aganglionic nerve networks, support the existenc of distinct subpopulations of NOS-containing enterice neurons acting as interneurons or (inhibitory) motor neurons.     

Cannabinoid receptor type 1 modulates excitatory and inhibitory neurotransmission in mouse colon

The effects of cannabinoid receptor agonists and antagonists on smooth muscle resting membrane potentials and on membrane potentials following electrical neuronal stimulation in a myenteric neuron/smooth muscle preparation of wild-type and cannabinoid receptor type 1 (CB1)-deficient mice were investigated in vitro. Double staining for CB1 and nitric oxide synthase (neuronal) was performed to identify the myenteric CB1-expressing neurons. Focal electrical stimulation of the myenteric plexus induced a fast (f) excitatory junction potential (EJP) followed by a fast and a slow (s) inhibitory junction potential (IJP). Treatment of wild-type mice with the endogenous CB1 receptor agonist anandamide reduced EJP while not affecting fIJP and sIJP. EJP was significantly higher in CB1-deficient mice than in wild-type littermate controls, and anandamide induced no effects in CB1-deficient mice. N-arachidonoyl ethanolamide (anandamide), R-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3,-de]- 1,4-benzoxazin-6-yl]-1-naphtalenylmethanone, a synthetic CB1 receptor agonist, nearly abolished EJP and significantly reduced the fIJP in wild-type mice. N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-caroxamide (SR141716A), a CB1-specific receptor antagonist, was able to reverse the agonist effects induced in wild-type mice. SR141716A, when given alone, significantly increased EJP in wild-type mice without affecting IJP in wild-type and EJP in CB1-deficient mice. Interestingly, SR141716A reduced fIJP in CB1-deficient mice. In the mouse colon, nitrergic myenteric neurons do not express CB1, implying that CB1 is expressed in cholinergic neurons, which is in line with the functional data. Finally, excitatory and inhibitory neurotransmission in the mouse colon is modulated by activation of CB1 receptors. The significant increase in EJP in CB1-deficient mice strongly suggests a physiological involvement of CB1 in excitatory cholinergic neurotransmission.     

Gdnf Haploinsufficiency Causes Hirschsprung-Like Intestinal Obstruction and Early-Onset Lethality in Mice

Hirschsprung disease (HSCR) is a common congenital disorder that results in intestinal obstruction and lethality, as a result of defective innervation of the gastrointestinal (GI) tract. Despite its congenital origin, the molecular etiology of HSCR remains elusive for >70% of patients. Although mutations in the c-RET receptor gene are frequently detected in patients with HSCR, mutations in the gene encoding its ligand (glial cell line-derived neurotrophic factor [GDNF]), are rarely found. In an effort to establish a possible link between human HSCR and mutations affecting the Gdnf locus, we studied a large population of mice heterozygous for a Gdnf null mutation. This Gdnf(+/-) mutant cohort recapitulates complex features characteristic of HSCR, including dominant inheritance, incomplete penetrance, and variable severity of symptoms. The lack of one functioning Gdnf allele causes a spectrum of defects in gastrointestinal motility and predisposes the mutant mice to HSCR-like phenotypes. As many as one in five Gdnf(+/-) mutant mice die shortly after birth. Using a transgenic marking strategy, we identified hypoganglionosis of the gastrointestinal tract as a developmental defect that renders the mutant mice susceptible to clinical symptoms of HSCR. Our findings offer a plausible way to link an array of seemingly disparate features characteristic of a complex disease to a much more narrowly defined genetic cause. These findings may have general implications for the genetic analysis of cause and effect in complex human diseases.     

The GDNF System Is Altered in Diverticular Disease – Implications for Pathogenesis

Background & Aims

Absence of glial cell line-derived neurotrophic factor (GDNF) leads to intestinal aganglionosis. We recently demonstrated that patients with diverticular disease (DD) exhibit hypoganglionosis suggesting neurotrophic factor deprivation. Thus, we screened mRNA expression pattern of the GDNF system in DD and examined the effects of GDNF on cultured enteric neurons.

Methods

Colonic specimens obtained from patients with DD (n = 21) and controls (n = 20) were assessed for mRNA expression levels of the GDNF system (GDNF, GDNF receptors GFRα1 and RET). To identify the tissue source of GDNF and its receptors, laser-microdissected (LMD) samples of human myenteric ganglia and intestinal muscle layers were analyzed separately by qPCR. Furthermore, the effects of GDNF treatment on cultured enteric neurons (receptor expression, neuronal differentiation and plasticity) were monitored.

Results

mRNA expression of GDNF and its receptors was significantly down-regulated in the muscularis propria of patients with DD. LMD samples revealed high expression of GDNF in circular and longitudinal muscle layers, whereas GDNF receptors were also expressed in myenteric ganglia. GDNF treatment of cultured enteric neurons increased mRNA expression of its receptors and promoted neuronal differentiation and plasticity revealed by synaptophysin mRNA and protein expression.

Conclusions

Our results suggest that the GDNF system is compromised in DD. In vitro studies demonstrate that GDNF enhances expression of its receptors and promotes enteric neuronal differentiation and plasticity. Since patients with DD exhibit hypoganglionosis, we propose that the observed enteric neuronal loss in DD may be due to lacking neurotrophic support mediated by the GDNF system.