Migration and differentiation of transplanted enteric neural crest-derived cells in murine model of Hirschsprung’s disease

Stem cell therapy offers the potential of rebuilding the enteric nervous system (ENS) in the aganglionic bowel of patients with Hirschsprung’s disease. P0-Cre/Floxed-EGFP mice in which neural crest-derived cells express EGFP were used to obtain ENS stem/progenitor cells. ENS stem/progenitor cells were transplanted into the bowel of Ret^−/− mouse, an animal model of Hirschsprung’s disease. Immunohistochemical analysis was performed to determine whether grafted cells gave rise to neurons in the recipient bowel. EGFP expressing neural crest-derived cells accounted for 7.01 ± 2.52 % of total cells of gastrointestinal tract. ENS stem/progenitor cells were isolated using flow cytometry and expanded as neurosphere-like bodies (NLBs) in a serum-free culture condition. Some cells in NLBs expressed neural crest markers, p75 and Sox10 and neural stem/progenitor cells markers, Nestin and Musashi1. Multipotency of isolated ENS stem/progenitor cells was determined as they differentiated into neurons, glial cells, and myofibloblasts in culture. When co-cultured with explants of hindgut of Ret^−/− mice, ENS stem/progenitor cells migrated into the aganglionic bowel and gave rise to neurons. ENS stem/progenitor cells used in this study appear to be clinically relevant donor cells in cell therapy to treat Hirschsprung’s disease capable of colonizing the affected bowel and giving rise to neurons.     

Divergent fate and origin of neurosphere-like bodies from different layers of the gut

Enteric neural stem cells (ENSCs) are a population of neural crest-derived multipotent stem cells present in postnatal gut that may play an important role in regeneration of the enteric nervous system. In most studies, these cells have been isolated from the layer of the gut containing the myenteric plexus. However, a recent report demonstrated that neurosphere-like bodies (NLBs) containing ENSCs could be isolated from mucosal biopsy specimens from children, suggesting that ENSCs are present in multiple layers of the gut. The aim of our study was to assess whether NLBs isolated from layers of gut containing either myenteric or submucosal plexus are equivalent. We divided the mouse small intestine into two layers, one containing myenteric plexus and the other submucosal plexus, and assessed for NLB formation. Differences in NLB density, proliferation, apoptosis, neural crest origin, and phenotype were investigated. NLBs isolated from the myenteric plexus layer were present at a higher density and demonstrated greater proliferation, lower apoptosis, and higher expression of nestin, p75, Sox10, and Ret than those from submucosal plexus. Additionally, they contained a higher percentage of neural crest-derived cells (99.4 ± 1.5 vs. 0.7 ± 1.19% of Wnt1-cre:tdTomato cells; P < 0.0001) and produced more neurons and glial cells than those from submucosal plexus. NLBs from the submucosal plexus layer expressed higher CD34 and produced more smooth muscle-like cells. NLBs from the myenteric plexus layer contain more neural crest-derived ENSCs while those from submucosal plexus appear more heterogeneous, likely containing a population of mesenchymal stem cells.     

Enteric Neurospheres Are Not Specific to Neural Crest Cultures: Implications for Neural Stem Cell Therapies

Objectives

Enteric neural stem cells provide hope of curative treatment for enteric neuropathies. Current protocols for their harvesting from humans focus on the generation of ‘neurospheres’ from cultures of dissociated gut tissue. The study aims to better understand the derivation, generation and composition of enteric neurospheres.

Design

Gut tissue was obtained from Wnt1-Cre;Rosa26^Yfp/Yfp transgenic mice (constitutively labeled neural crest cells) and paediatric patients. Gut cells were cultured either unsorted (mixed neural crest/non-neural crest), or following FACS selection into neural crest (murine-YFP+ve/human-p75+ve) or non-neural crest (YFP-ve/p75-ve) populations. Cultures and resultant neurospheres were characterized using immunolabelling in vitro and following transplantation in vivo.

Results

Cultures of (i) unsorted, (ii) neural crest, and (iii) non-neural crest cell populations generated neurospheres similar in numbers, size and morphology. Unsorted neurospheres were highly heterogeneous for neural crest content. Neural crest-derived (YFP+ve/p75+ve) neurospheres contained only neural derivatives (neurons and glia) and were devoid of non-neural cells (i.e. negative for SMA, c-Kit), with the converse true for non-neural crest-derived (YFP-ve/p75-ve) ‘neurospheres’. Under differentiation conditions only YFP+ve cells gave rise to neural derivatives. Both YFP+ve and YFP-ve cells displayed proliferation and spread upon transplantation in vivo, but YFP-ve cells did not locate or integrate within the host ENS.

Conclusions

Spherical accumulations of cells, so-called ‘neurospheres’ forming in cultures of dissociated gut contain variable proportions of neural crest-derived cells. If they are to be used for ENS cell replacement therapy then improved protocols for their generation, including cell selection, should be sought in order to avoid inadvertent transplantation of non-therapeutic, non-ENS cells.     

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.     

Isolation of Enteric Nervous System Progenitor Cells from the Aganglionic Gut of Patients with Hirschsprung’s Disease

Enteric nervous system progenitor cells isolated from postnatal human gut and cultured as neurospheres can then be transplanted into aganglionic gut to restore normal patterns of contractility. These progenitor cells may be of future use to treat patients with Hirschprung’s disease, a congenital condition characterized by hindgut dysmotility due to the lack of enteric nervous system ganglia. Here we demonstrate that progenitor cells can also be isolated from aganglionic gut removed during corrective surgery for Hirschsprung’s disease. Although the enteric nervous system marker calretinin is not expressed in the aganglionic gut region, de novo expression is initiated in cultured neurosphere cells isolated from aganglionic Hirschsprung bowel. Furthermore, expression of the neural markers NOS, VIP and GFAP also increased during culture of aganglionic gut neurospheres which we show can be transplantation into cultured embryonic mouse gut explants to restore a normal frequency of contractility. To determine the origin of the progenitor cells in aganglionic region, we used fluorescence-activated cell sorting to demonstrate that only p75-positive neural crest-derived cells present in the thickened nerve trunks characteristic of the aganglionic region of Hirschsprung gut gave rise to neurons in culture. The derivation of enteric nervous system progenitors in the aganglionic gut region of Hirschprung’s patients not only means that this tissue is a potential source of cells for future autologous transplantation, but it also raises the possibility of inducing the differentiation of these endogenous cells in situ to compensate for the aganglionosis.     

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.     

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.     

Vascularisation is not necessary for gut colonisation by enteric neural crest cells

The vasculature and nervous system share striking similarities in their networked, tree-like architecture and in the way they are super-imposed in mature organs. It has previously been suggested that the intestinal microvasculature network directs the migration of enteric neural crest cells (ENCC) along the gut to promote the formation of the enteric nervous system (ENS). To investigate the inter-relationship of migrating ENCC, ENS formation and gut vascular development we combined fate-mapping of ENCC with immunolabelling and intravascular dye injection to visualise nascent blood vessel networks. We found that the enteric and vascular networks initially had very distinct patterns of development. In the foregut, ENCC migrated through areas devoid of established vascular networks. In vessel-rich areas, such as the midgut and hindgut, the distribution of migrating ENCC did not support the idea that these cells followed a pre-established vascular network. Moreover, when gut vascular development was impaired, either genetically in Vegfa^120/120 or Tie2-Cre;Nrp1^fl/− mice or using an in vitro Wnt1-Cre;Rosa26^Yfp/+ mouse model of ENS development, ENCC still colonised the entire length of the gut, including the terminal hindgut. These results demonstrate that blood vessel networks are not necessary to guide migrating ENCC during ENS development. Conversely, in miRet⁵¹ mice, which lack ENS in the hindgut, the vascular network in this region appeared to be normal suggesting that in early development both networks form independently of each other.     

Non-cell-autonomous effects of Ret deletion in early enteric neurogenesis

Neural crest cells (NCCs) form at the dorsal margin of the neural tube and migrate along distinct pathways throughout the vertebrate embryo to generate multiple cell types. A subpopulation of vagal NCCs invades the foregut and colonises the entire gastrointestinal tract to form the enteric nervous system (ENS). The colonisation of embryonic gut by NCCs has been studied extensively in chick embryos, and genetic studies in mice have identified genes crucial for ENS development, including Ret. Here, we have combined mouse embryo and organotypic gut culture to monitor and experimentally manipulate the progenitors of the ENS. Using this system, we demonstrate that lineally marked intestinal ENS progenitors from E11.5 mouse embryos grafted into the early vagal NCC pathway of E8.5 embryos colonise the entire length of the gastrointestinal tract. By contrast, similar progenitors transplanted into Ret-deficient host embryos are restricted to the proximal foregut. Our findings establish an experimental system that can be used to explore the interactions of NCCs with their cellular environment and reveal a previously unrecognised non-cell-autonomous effect of Ret deletion on ENS development.     

Cell death and the developing enteric nervous system

This review discusses current knowledge about cell death in the developing enteric nervous system (ENS). It also includes findings about the molecular mechanisms by which such death is mediated. Additional consideration is given to trophic factors that contribute to survival of the precursors and neurons and glia of the ENS, as well to genes that, when mutated or deleted, trigger their death. Although further confirmation is needed, present observations support the view that enteric neural crest-derived precursor cells en route to the gut undergo substantial levels of apoptotic death, but that once these cells colonize the gut, there is relatively little death of precursor cells or of neurons and glia during the fetal period. There are also indications that normal neuron loss occurs in the ENS, but at times beyond the perinatal stage. Taken together, these findings suggest that ENS development is similar is some ways, but different in others from extra-enteric areas of the vertebrate central and peripheral nervous systems, in which large-scale apoptotic death of precursor neurons and glia occurs during the fetal and perinatal periods. Potential reasons for these differences are discussed such as a fetal enteric microenvironment that is especially rich in trophic support. In addition to the cell death that occurs during normal ENS development, this review discusses mechanisms of experimentally-induced ENS cell death, such as those that are associated with defective glial cell-line derived neurotrophic factor/Ret signaling, which are an animal model of human congenital megacolon (aganglionosis; Hirschsprung’s disease). Such considerations underscore the importance of understanding cell death in the developing ENS, not just from a curiosity-driven point of view, but also because the pathophysiology behind many disorders of human gastrointestinal function may originate in abnormalities of the mechanisms that govern cell survival and death during ENS development.     

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.     

Phactr4

The enteric nervous system (ENS) is critically important for many intestinal functions such as peristalsis and secretion. Defects in the embryonic formation of the ENS cause Hirschsprung disease (HSCR) or megacolon, a severe birth defect that affects approximately 1 in 5,000 newborns. One of the least understood aspects of ENS development are the cellular and molecular mechanisms that control chain migration of the ENS cells during their migration into and along the embryonic gut. We recently reported a mouse model of HSCR in which mutant embryos carrying a hypomorphic allele of the Phactr4 gene show an embryonic gastrointestinal defect due to loss of enteric neurons in the colon. We found that Phactr4 modulates integrin signaling and cofilin activity to coordinate the forces that drive enteric neural crest cell (ENCC) migration in the mammalian embryo. In this extra view, we briefly summarize the current knowledge on integrin signaling in ENCC migration and introduce the Phactr protein family. Employing the ENS as a model, we shed some light on the mechanisms by which Phactr4 regulates integrin signaling and controls the cell polarity required for directional ENCC migration in the mouse developing gut.     

From neural crest to bowel: Development of the enteric nervous system

The ENS resembles the brain and differs both physiologically and structurally from any other region of the PNS. Recent experiments in which crest cell migration has been studied with DiI, a replication-deficient retrovirus, or antibodies that label cells of neural crest origin, have confirmed that both the avian and mammalian bowel are colonized by émigrés from the sacral as well as the vagal level of the neural crest. Components of the extracellular matrix, such as laminin, may play roles in enteric neural and glial development. The observation that an overabundance of laminin develops in the presumptive aganglionic region of the gut in Is/Is mutant mice and is associated with the inability of crest-derived cells to colonize this region of the bowel has led to the hypothesis that laminin promotes the development of crest-derived cells as enteric neurons. Premature expression of a neuronal phenotype would cause crest-derived cells to cease migrating before they complete the colonization of the gut. The acquisition by crest-derived cells of a nonintegrin, nervespecific, 110 kD laminin-binding protein when they enter the bowel may enable these cells to respond to laminin differently from their pre-enteric migrating predecessors. Crest-derived cells migrating along the vagal pathway to the mammalian gut are transiently catecholaminergic (TC). This phenotype appears to be lost rapidly as the cells enter the bowel and begin to follow their program of terminal differentiation. The appearance and disappearance of TC cells may thus be an example of the effects of the enteric microenvironment on the differentiation of crest-derived cells in situ. Crest-derived cells can be isolated from the enteric microenvironment by immunoselection, a method that takes advantage of the selective expression on the surfaces of crest-derived cells of certain antigens. One neurotrophin, NT-3, promotes the development of enteric neurons and glia in vitro. Because trkC is expressed in the developing and mature gut, it seems likely that NT-3 plays a critical role in the development of the ENS in situ. Although the factors that are responsible for the development of the unique properties of the ENS remain unknown, progress made in understanding enteric neuronal development has recently accelerated. The application of new techniques and recently developed probes suggest that the accelerated pace of discovery in this area can be expected to continue. © 1993 John Wiley & Sons, Inc.     

The receptor tyrosine kinase RET regulates hindgut colonization by sacral neural crest cells

The enteric nervous system (ENS) is formed from vagal and sacral neural crest cells (NCC). Vagal NCC give rise to most of the ENS along the entire gut, whereas the contribution of sacral NCC is mainly limited to the hindgut. This, and data from heterotopic quail-chick grafting studies, suggests that vagal and sacral NCC have intrinsic differences in their ability to colonize the gut, and/or to respond to signalling cues within the gut environment. To better understand the molecular basis of these differences, we studied the expression of genes known to be essential for ENS formation, in sacral NCC within the chick hindgut. Our results demonstrate that, as in vagal NCC, Sox10, EdnrB, and Ret are expressed in sacral NCC within the gut. Since we did not detect a qualitative difference in expression of these ENS genes we performed DNA microarray analysis of vagal and sacral NCC. Of 11 key ENS genes examined from the total data set, Ret was the only gene identified as being highly differentially expressed, with a fourfold increase in expression in vagal versus sacral NCC. We also found that over-expression of RET in sacral NCC increased their ENS developmental potential such that larger numbers of cells entered the gut earlier in development, thus promoting the fate of sacral NCC towards that of vagal NCC.     

Endoderm-derived Sonic hedgehog and mesoderm Hand2 expression are required for enteric nervous system development in zebrafish

The zebrafish enteric nervous system (ENS), like those of all other vertebrate species, is principally derived from the vagal neural crest cells (NCC). The developmental controls that govern the migration, proliferation and patterning of the ENS precursors are not well understood. We have investigated the roles of endoderm and Sonic hedgehog (SHH) in the development of the ENS. We show that endoderm is required for the migration of ENS NCC from the vagal region to the anterior end of the intestine. We show that the expression of shh and its receptor ptc-1 correlate with the development of the ENS and demonstrate that hedgehog (HH) signaling is required in two phases, a pre-enteric and an enteric phase, for normal ENS development. We show that HH signaling regulates the proliferation of vagal NCC and ENS precursors in vivo. We also show the zebrafish hand2 is required for the normal development of the intestinal smooth muscle and the ENS. Furthermore we show that endoderm and HH signaling, but not hand2, regulate gdnf expression in the intestine, highlighting a central role of endoderm and SHH in patterning the intestine and the ENS.     

Phactr4: A new integrin modulator required for directional migration of enteric neural crest cells

The enteric nervous system (ENS) is critically important for many intestinal functions such as peristalsis and secretion. Defects in the embryonic formation of the ENS cause Hirschsprung disease (HSCR) or megacolon, a severe birth defect that affects approximately 1 in 5,000 newborns. One of the least understood aspects of ENS development are the cellular and molecular mechanisms that control chain migration of the ENS cells during their migration into and along the embryonic gut. We recently reported a mouse model of HSCR in which mutant embryos carrying a hypomorphic allele of the Phactr4 gene show an embryonic gastrointestinal defect due to loss of enteric neurons in the colon. We found that Phactr4 modulates integrin signaling and cofilin activity to coordinate the forces that drive enteric neural crest cell (ENCC) migration in the mammalian embryo. In this extra view, we briefly summarize the current knowledge on integrin signaling in ENCC migration and introduce the Phactr protein family. Employing the ENS as a model, we shed some light on the mechanisms by which Phactr4 regulates integrin signaling and controls the cell polarity required for directional ENCC migration in the mouse developing gut.     

The emergence of neural activity and its role in the development of the enteric nervous system

The enteric nervous system (ENS) is a vital part of the autonomic nervous system that regulates many gastrointestinal functions, including motility and secretion. All neurons and glia of the ENS arise from neural crest-derived cells that migrate into the gastrointestinal tract during embryonic development. It has been known for many years that a subpopulation of the enteric neural crest-derived cells expresses pan-neuronal markers at early stages of ENS development. Recent studies have demonstrated that some enteric neurons exhibit electrical activity from as early as E11.5 in the mouse, with further maturation of activity during embryonic and postnatal development. This article discusses the maturation of electrophysiological and morphological properties of enteric neurons, the formation of synapses and synaptic activity, and the influence of neural activity on ENS development.     

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.