Paracervical ganglion in the female pig during prenatal development: Morphology and immunohistochemical characteristics.

The present study investigated the development of the paracervical ganglion in 5-, 7- and 10-week-old porcine foetuses using double labelling immunofluorescence method. In 5-week-old foetuses single PGP-positive perikarya were visible only along the mesonephric ducts. They contained DßH or VAChT, and nerve fibres usually were PGP/VAChT-positive. The perikarya were mainly oval. In 7-week-old foetuses, a compact group of PGP-positive neurons (3144±213) was visible on both sides and externally to the uterovaginal canal mesenchyme of paramesonephric ducts. Nerve cell bodies contained only DßH (36.40±1.63%) or VAChT (17.31±1.13%). In the 10-week-old foetuses, the compact group of PGP-positive neurons divided into several large and many small clusters of nerve cells and also became more expanded along the whole uterovaginal canal mesenchyme reaching the initial part of the uterine canal of the paramesonephric duct. The number of neurons located in these neuronal structures increased to 4121±259. Immunohistochemistry revealed that PGP-positive nerve cell bodies contained DßH (40.26±0,73%) and VAChT (30.73±1.34%) and were also immunoreactive for NPY (33.24±1,27%), SOM (23.6±0,44%) or VIP (22.9±1,13%). Other substances studied (GAL, NOS, CGRP, SP) were not determined at this stage of the development. In this study, for the first time, the morphology of PCG formation in the porcine foetus has been described in three stages of development. Dynamic changes in the number of neurons and their sizes were also noted, as well as the changes in immunochistochemical coding of maturing neurons.

Targeted deletion of Hand2 in enteric neural precursor cells affects its functions in neurogenesis, neurotransmitter specification and gangliogenesis, causing functional aganglionosis.

Targeted deletion of the bHLH DNA-binding protein Hand2 in the neural crest, impacts development of the enteric nervous system (ENS), possibly by regulating the transition from neural precursor cell to neuron. We tested this hypothesis by targeting Hand2 deletion in nestin-expressing neural precursor (NEP) cells. The mutant mice showed abnormal ENS development, resulting in lethal neurogenic pseudo-obstruction. Neurogenesis of neurons derived from NEP cells identified a second nestin non-expressing neural precursor (NNEP) cell in the ENS. There was substantial compensation for the loss of neurons derived from the NEP pool by the NNEP pool but this was insufficient to abrogate the negative impact of Hand2 deletion. Hand2-mediated regulation of proliferation affected both neural precursor and neuron numbers. Differentiation of glial cells derived from the NEP cells was significantly decreased with no compensation from the NNEP pool of cells. Our data indicate differential developmental potential of NEPs and NNEPs; NNEPs preferentially differentiate as neurons, whereas NEPs give rise to both neurons and glial cells. Deletion of Hand2 also resulted in complete loss of NOS and VIP and a significant decrease in expression of choline acetyltransferase and calretinin, demonstrating a role for Hand2 in neurotransmitter specification and/or expression. Loss of Hand2 resulted in a marked disruption of the developing neural network, exemplified by lack of a myenteric plexus and extensive overgrowth of fibers. Thus, Hand2 is essential for neurogenesis, neurotransmitter specification and neural network patterning in the developing ENS.

Plasticity of neural crest-placode interaction in the developing visceral nervous system.

The reciprocal relationship between rhombomere (r)-derived cranial neural crest (NC) and epibranchial placodal cells derived from the adjacent branchial arch is critical for visceral motor and sensory gangliogenesis, respectively. However, it is unknown whether the positional match between these neurogenic precursors is hard-wired along the anterior-posterior (A/P) axis. Here, we use the interaction between r4-derived NC and epibranchial placode-derived geniculate ganglion as a model to address this issue. In Hoxa1(-/-) b1(-/-) embryos, r2 NC compensates for the loss of r4 NC. Specifically, a population of r2 NC cells is redirected toward the geniculate ganglion, where they differentiate into postganglionic (motor) neurons. Reciprocally, the inward migration of the geniculate ganglion is associated with r2 NC. The ability of NC and placodal cells to, respectively, differentiate and migrate despite a positional mismatch along the A/P axis reflects the plasticity in the relationship between the two neurogenic precursors of the vertebrate head.

Factors controlling cardiac neural crest cell migration.

Cardiac neural crest cells originate as part of the postotic caudal rhombencephalic neural crest stream. Ectomesenchymal cells in this stream migrate to the circumpharyngeal ridge and then into the caudal pharyngeal arches where they condense to form first a sheath and then the smooth muscle tunics of the persisting pharyngeal arch arteries. A subset of the cells continue migrating into the cardiac outflow tract where they will condense to form the aorticopulmonary septum. Cell signaling, extracellular matrix and cell-cell contacts are all critical for the initial migration, pauses, continued migration, and condensation of these cells. This review elucidates what is currently known about these factors.

Prenatal development of the human epicardiac Ganglia.

The aim of this study was to determine the developmental anatomy of intrinsic cardiac ganglia with respect to epicardiac ganglionated nerve plexus in the human fetuses at different gestation stages. Twenty fetal hearts were investigated applying a technique of histochemistry for acetylcholinesterase to visualize the epicardiac neural ganglionated plexus with its subsequent examinations on total (non-sectioned) hearts. Most epicardiac ganglia embodied multilayered neurons and were oval in shape, but some ganglia involved neurons lying in one layer or had the irregular appearance because of their extensions along inter-ganglionic nerves. The mean ganglion area of fetuses at gestation stages of 15-40 weeks was 0.03 +/- 0.008 mm(2). The largest epicardiac ganglia, reaching in area 0.4 mm(2), were concentrated on the dorsal surface of both atria. The particular fused or "dual" ganglia were identified at the gestation stages of 23-40 weeks, but they composed only 2.3 +/- 0.7% of all found epicardiac ganglia. A direct positive correlation was determined between the fetal age and the ganglion area (mm(2)) as well as between the fetal age and the number of inter-ganglionic nerves. The revealed appearance of epicardiac ganglia in the human fetuses at 15-40 weeks of gestation confirms their prenatal development and presumable intrinsic remodelling.

Gangliogenesis in the enteric nervous system: roles of the polysialylation of the neural cell adhesion molecule and its regulation by bone morphogenetic protein-4.

The neural crest-derived cells that colonize the fetal bowel become patterned into two ganglionated plexuses. The hypothesis that bone morphogenetic proteins (BMPs) promote ganglionation by regulating neural cell adhesion molecule (NCAM) polysialylation was tested. Transcripts encoding the sialyltransferases, ST8Sia IV (PST) and ST8Sia II (STX), which polysialylate NCAM, were detectable in fetal rat gut by E12 but were downregulated postnatally. PSA-NCAM-immunoreactive neuron numbers, but not those of NCAM, were developmentally regulated similarly. Circular smooth muscle was transiently (E16-20) PSA-NCAM-immunoreactive when it is traversed by migrating precursors of submucosal neurons. Neurons developing in vitro from crest-derived cells immunoselected at E12 with antibodies to p75(NTR) expressed NCAM and PSA-NCAM. BMP-4 promoted neuronal NCAM polysialylation and clustering. N-butanoylmannosamine, which blocks NCAM polysialylation, but not N-propanoylmannosamine, which does not, interfered with BMP-4-induced neuronal clustering. Observations suggest that BMP signaling enhances NCAM polysialylation, which allows precursors to migrate and form ganglionic aggregates during the remodeling of the developing ENS.

Pelvic plexus contributes ganglion cells to the hindgut enteric nervous system.

The hindgut enteric nervous system (ENS) contains cells originating from vagal and sacral neural crest. In avians, the sacral crest gives rise to the nerve of Remak (NoR) and pelvic plexus. Whereas the NoR has been suggested to serve as the source of sacral crest-derived cells to the gut, the contribution of the pelvic ganglia is unknown. The purpose of this study was to test the hypothesis that the pelvic ganglia contribute ganglion cells to the hindgut ENS. We observed that the quail pelvic plexus develops from neural crest-derived cells that aggregate around the cloaca at embryonic day 5. Using chick-quail tissue recombinations, we found that hindgut grafts did not contain enteric ganglia unless the pelvic plexus was included. Neurofibers extended from the NoR into the intestine, but no ganglion cell contribution from the NoR was identified. These results demonstrate that the pelvic plexus, and not the NoR, serves as the staging area for sacral crest-derived cells to enter the avian hindgut, confirming the evolutionary conservation of this important embryologic process.

Delays in neuronal differentiation in Mash1/Ascl1 mutants.

The inactivation of a developmental transcription factor may lead to the complete absence of a specific cell type. More commonly, though, it only partially impairs its generation. The modalities of this partial effect have rarely been documented in any detail. Here, we report a novel function for the bHLH transcription factor Ascl1/Mash1 in the generation of the nucleus of the solitary tract (nTS). In Mash1(-/-) late embryos, the nTS is markedly atrophic. Tracing back the origin of this atrophy, we show that nTS precursors appear in the mutants 1 day later than in the wild type and then accumulate at a slower pace. We also show that the previously reported atrophy of the sympathetic chain in Mash1 mutants is similarly preceded by a delay of 1 to 2 days in the appearance of differentiated ganglionic cells. Finally, we provide evidence that the acceleration imposed by Mash1, regardless of the production of post-mitotic cells, affects differentiation itself, both generic and type-specific.

Guidance cues involved in the development of the peripheral autonomic nervous system.

All peripheral autonomic neurons arise from neural crest cells that migrate away from the neural tube and navigate to the location where ganglia will form. After differentiating into neurons, their axons then navigate to a variety of targets. During the development of the enteric nervous system, GDNF appears to play a role in inducing vagal neural crest cells to enter the gut, in retaining neural crest cells within the gut and in promoting the migration of neural crest cells along the gut. Sema3A regulates the entry of extrinsic axons into the distal hindgut, netrin-DCC signaling is responsible for the centripetal migration of cells to form the submucosal ganglia within the gut, Slit-Robo signaling prevents trunk level neural crest cells from entering the gut, and neurturin plays a role in the innervation of the circular muscle layer. During the development of the sympathetic nervous system, the migration of trunk neural crest cells through the somites is influenced by ephrin-Bs, Sema3A and F-spondin. The migration of neural crest cells ventrally beyond the somites requires neuregulin signaling and the clumping of cells into columns adjacent to the dorsal aorta is regulated by Sema3A. The rostral migration of cells to form the superior cervical ganglion (SCG) and the extension of axons along blood vessels involves artemin signaling through Ret and GFRalpha3, and the entry of sympathetic axons into target tissues involves neurotrophins and GDNF. Relatively little is known about the development of parasympathetic ganglia, but GDNF appears to play a role in the migration of some cranial ganglion precursors to their correct location, and both GDNF and neurturin are involved in the growth of parasympathetic axons into particular targets.

The proximal 2-kb of the Hoxa3 promoter directs gene expression in distinct branchial compartments and cranial ganglia.

Developing structures such as hindbrain, neural crest cells or spinal cord express Hoxa3. Here, we have investigated the regulatory role of a 2-kb fragment spanning the proximal promoter of Hoxa3 by a reporter-based approach in mice. We show that this fragment promotes reporter activity in ganglionic and branchial compartments known to express Hoxa3 but for which no cis-regulatory elements have been identified so far. We also show that the 2-kb promoter fragment is active in rhombomere 4 and in the ganglion of the cranial nerve complex VII/VIII that are devoid of Hoxa3 expression.

Dual function of Slit2 in repulsion and enhanced migration of trunk, but not vagal, neural crest cells.

Neural crest precursors to the autonomic nervous system form different derivatives depending upon their axial level of origin; for example, vagal, but not trunk, neural crest cells form the enteric ganglia of the gut. Here, we show that Slit2 is expressed at the entrance of the gut, which is selectively invaded by vagal, but not trunk, neural crest. Accordingly, only trunk neural crest cells express Robo receptors. In vivo and in vitro experiments demonstrate that trunk, not vagal, crest cells avoid cells or cell membranes expressing Slit2, thereby contributing to the differential ability of neural crest populations to invade and innervate the gut. Conversely, exposure to soluble Slit2 significantly increases the distance traversed by trunk neural crest cells. These results suggest that Slit2 can act bifunctionally, both repulsing and stimulating the motility of trunk neural crest cells.

N-terminal Slit2 promotes survival and neurite extension in cultured peripheral neurons.

To investigate the effect of the N-terminal Slit2 protein on neuronal survival and development, recombinant human N-terminal Slit2 (N-Slit2) was assayed against isolated embryonic chick dorsal root ganglion sensory, ciliary ganglion and paravertebral sympathetic neurons. N-Slit2 promoted significant levels of neuronal survival and neurite extension in all of these populations. The protein was also assayed against postnatal mouse dorsal root ganglion neurons and found to promote neuronal survival in a similar manner. These findings suggest the Slit proteins may play an important role during development of the nervous system, mediating cellular survival in addition to the well documented role these proteins play in axonal and neuronal chemorepulsion.

Retinoic acid influences the expression of the neuronal regulatory genes Mash-1 and c-ret in the developing rat heart.

We analyzed the expression of neuronal regulatory genes Mash-1 and c-ret by immunohistochemistry and reverse transcriptase-polymerase chain reaction in the developing heart of rat embryos following exogenous retinoic acid (RA) treatment of the pregnant dams. On E12, expression of Mash-1 and c-ret was confined to cells migrating via the common cardinal vein. On E16.5, Mash-1 and c-ret expression were restricted to cardiac ganglia around the great vessels and posterior atrial wall. While Mash-1 expression was down-regulated at birth, that of c-Ret was maintained. RA-treated hearts showed a down-regulation of both Mash-1 and c-Ret at the mRNA as well as at the protein level on E16.5. The present results show that differentiation of cardiac ganglionic cells is affected after RA treatment, by the down-regulation of Mash-1 and c-Ret.

Autonomic neurogenesis and apoptosis are alternative fates of progenitor cell communities induced by TGFbeta.

The question of how appropriate cell types are generated in correct numbers during development of the peripheral nervous system has become particularly intriguing with the identification of multipotent progenitor cells in postmigratory targets of the neural crest. Recently, we have provided evidence that community effects in response to factors of the TGFbeta family might represent a mechanism to suppress inappropriate nonneural fates from multipotent progenitors in developing peripheral ganglia. In culture, BMP2 and TGFbeta promote neurogenesis at the expense of a smooth-muscle-like fate in clusters of neural-crest-derived multipotent progenitor cells. We now show that the neurons generated by TGFbeta factors belong to the autonomic lineage and that cells within the developing sympathetic ganglia express TGFbeta-type II receptor. In addition to its neurogenic activity, TGFbeta but not BMP2 also induces apoptosis as an alternative fate in cultured progenitor communities. Interestingly, these fate decisions are controlled by graded changes in TGFbeta concentrations: lower doses of TGFbeta promote neurogenesis while slightly higher doses induce predominantly apoptosis. These effects of TGFbeta are specific for an early developmental stage since progenitor cells lose their competence to respond to the proapoptotic activity of TGFbeta upon neuronal differentiation. In vivo, the expression of TGFbeta3 in differentiated neurons suggests that the signal concentration gradually increases with the number of neurons formed in the autonomic ganglia. We propose that TGFbeta functions in a biphasic manner during autonomic gangliogenesis to control both neurogenesis and subsequently the number of neurons generated from progenitor cells.

Nicotinic acetylcholine receptor gene expression in developing chick autonomic ganglia.

The developmental expression patterns of ten genes encoding nicotinic acetylcholine receptor subunits were analyzed using Northern blots and in situ hybridization in chick peripheral ganglia of neural crest, placodal and dual embryonic origin. The superior cervical and ciliary ganglia were investigated in detail because they accumulated relatively abundant transcripts of the alpha3, beta4, alpha5 and alpha7 genes. In the superior cervical ganglion, these four mRNA species had similar developmental time-courses. They appeared at embryonic day 8 (E8), increased steadily until E16 and maintained a rather high plateau level until E18. In the ciliary ganglion, alpha7 transcripts were already abundant at E6, increased until E10, and considerably decreased thereafter. High-resolution in situ hybridization showed that alpha7 transcripts were present in all cell types of the E6 ciliary ganglion, whereas they were restricted to large neuronal somas at E16. Transfections with a reporter gene under the control of the alpha7 promoter demonstrated that a sharp developmental divide occurred at E11-12, after which stage the promoter was activatable in neurons exclusively.

The homeobox gene Phox2b is essential for the development of autonomic neural crest derivatives.

The sympathetic, parasympathetic and enteric ganglia are the main components of the peripheral autonomic nervous system, and are all derived from the neural crest. The factors needed for these structures to develop include the transcription factor Mash1, the glial-derived neurotrophic factor GNDF and its receptor subunits, and the neuregulin signalling system, each of which is essential for the differentiation and survival of subsets of autonomic neurons. Here we show that all autonomic ganglia fail to form properly and degenerate in mice lacking the homeodomain transcription factor Phox2b, as do the three cranial sensory ganglia that are part of the autonomic reflex circuits. In the anlagen of the enteric nervous system and the sympathetic ganglia, Phox2b is needed for the expression of the GDNF-receptor subunit Ret and for maintaining Mash1 expression. Mutant ganglionic anlagen also fail to switch on the genes that encode two enzymes needed for the biosynthesis of the neurotransmitter noradrenaline, dopamine-beta-hydroxylase and tyrosine hydroxylase, demonstrating that Phox2b regulates the noradrenergic phenotype in vertebrates.

Synaptogenesis and structure of the autonomic ganglia.

The present study summarises the current data dealing with processes leading to the establishing of the synaptic architecture and connections with both the preganglionic neurons and target tissues of autonomic ganglia Starting from the migration point of the neural crest cells, the factors and mechanisms driving the development of the autonomic nervous system, axonal pathfinding and establishing of ganglionic connections, as well as formation and plasticity of autonomic synapses are reviewed with regard to their functional relevance in these processes. Furthermore, the chemical neuroanatomy and somatotopic arrangement of either the preganglionic, sympathetic or parasympathetic division of the autonomic nervous system are discussed from the morphological ¿and functional point of view.

Role of cell-cell interactions in the developmental regulation of Ca2+-activated K+ currents in vertebrate neurons.

The functional expression of the Ca2+-activated K+ current (IK[Ca]) is dependent on cell-cell interactions in developing chick autonomic neurons. In chick ciliary ganglion (CG) neurons, expression of macroscopic IK[Ca] coincides with the formation of synapses with target tissues. CG neurons that develop in vivo in the absence of normal target tissues fail to express functional IK[Ca], although voltage-activated Ca2+ currents and most other ionic currents are expressed at normal amplitudes and densities. CG neurons placed in cell culture prior to formation of synapses with target tissues also fail to express macroscopic IK[Ca]. However, CG neurons cultured in the presence of a heat- and trypsin-sensitive extract of target tissues express IK[Ca] at normal levels. Similarly, interactions with target tissue appear to regulate the expression of whole-cell IK[Ca] in developing chick sympathetic ganglion neurons, although the relevant trophic factors appear to be different from those required by CG neurons. In addition to target tissue interactions, an intact preganglionic innervation is required for the normal in vivo development of IK[Ca] in chick CG neurons. The trophic effects of the afferent innervation do not require synaptic activation of the CG neurons, indicating secretion of a trophic factor, possibly an isoform of beta-neuregulin. The results are consistent with the hypothesis that target- and nerve terminal-derived trophic factors interact at a posttranslational level in the regulation of a functional IK[Ca]. Together, this body of data demonstrates an essential role for cell-cell interactions in the differentiation of neuronal excitability.

Embryonic expression of pituitary adenylate cyclase-activating polypeptide in sensory and autonomic ganglia and in spinal cord of the rat.

Pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide that is related structurally to vasoactive intestinal polypeptide (VIP), has been shown to stimulate neuronal growth and differentiation, indicating a possible function in the development of the nervous system. Studies have indicated that the PACAP receptor is expressed during development, but data on PACAP expression are limited mainly to postnatal development. In the present study, we used immunohistochemistry and in situ hybridization histochemistry to examine the expression of PACAP in autonomic and sensory ganglia and spinal cord of rat fetuses at embryonic days 12-21 (E12-E21). PACAP immunoreactivity was visualized by using a specific monoclonal anti-PACAP antibody to detect both PACAP-38 and PACAP-27, and PACAP mRNA was visualized by using a [33P]-labeled cRNA-probe. PACAP- nerve fibers were observed in the spinal cord as early as E13. At E14, PACAP-immunoreactive nerve fibers projected to the sympathetic trunk, where few PACAP- nerve cell bodies were seen from E15. On the same embryonic day, PACAP-immunoreactive nerve cell bodies appeared in the intermediolateral column of the spinal cord. From E15 to E16, PACAP-immunoreactive nerve cell bodies were visible within sensory and autonomic ganglia, such as the dorsal root, the trigeminal, the sphenopalatine, the otic, the submandibular, and the nodose ganglia. At E16, PACAP+ nerve fibers were innervating the adrenal medulla, and immunoreactive fibers could also be observed in the superior cervical ganglion, in which PACAP-immunoreactive cell bodies were detected occasionally from E18. The synthesis of PACAP in neuronal cell bodies was confirmed by the demonstration of PACAP mRNA with in situ hybridization histochemistry. Thus, in all of the structures examined, PACAP appeared at roughly the same embryonic stage and, thereafter, increased to the adult level before birth. Because PACAP occurred with the same distribution pattern as that described in the adult rat, there is no evidence for transient expression. The early expression of PACAP suggests a possible role for the peptide in the developing nervous system.