Enhanced vascular neuropeptide Y-immunoreactive innervation in two hypertensive rat strains.

Considerable evidence indicates an enhanced sympathetic innervation of resistance arterial smooth muscle in the spontaneously hypertensive rat (SHR) compared with its normotensive Wistar-Kyoto (WKY) control. In addition to sympathetic hyperinnervation, an increased vascular innervation by neuropeptide Y-containing fibers, which are known to exert a vasoconstrictive and trophic action in vascular smooth muscle, has also been described. In addition to genetic hypertension, the SHR expresses hyperactive behavior and hyperreactivity to stress. To determine whether the enhanced neuropeptide Y-immunoreactive vascular innervation is specifically associated with hypertension and/or these behavioral abnormalities, four genetically related, inbred rat strains were used: SHR, which are hypertensive and hyperactive; WKY rats, which are neither hypertensive nor hyperactive; WKHA, which are hyperactive but normotensive; and WKHT, which are hypertensive but not hyperactive. The present study demonstrated that whereas the hypertensive strains (SHR and WKHT) exhibited smooth muscle hypertrophy in both superior mesenteric and caudal arteries in adulthood (10 months) but not at a prehypertensive age (1 month), both arteries exhibited significantly increased neuropeptide Y-immunoreactive innervation at both ages. It was further observed that the mesenteric artery in WKHA, a normotensive strain, had significant smooth muscle hypertrophy at 10 months; however, neuropeptide Y innervation in this artery was no different from that of WKY controls. The findings indicate that there is a cosegregation of neuropeptide Y hyperinnervation of the vasculature with the hypertensive phenotype, evident as early as 1 month of life in the hypertensive strains, and this should be considered further as a contributory factor in genetic hypertension.(ABSTRACT TRUNCATED AT 250 WORDS)

Morphology of sympathetic preganglionic neurons in the neonatal rat spinal cord: an intracellular horseradish peroxidase study.

Understanding the central neural control of autonomic functions requires a knowledge of the morphology of the preganglionic neurons, for the location of the dendritic arborizations of these neurons will indicate which central pathways may have access to them. In the present study, individual sympathetic preganglionic neurons in the neonatal rat spinal cord have been examined by the intracellular injection of horseradish peroxidase (HRP) in an in vitro preparation. Seventeen HRP-labeled preganglionic neurons in thoracic segments T1-T3 were examined in detail; of these, 12 somata were located in the intermediolateral cell column (IML), one in the lateral funiculus (LF), two in the intercalated nucleus (IC), and two at the border between IML and IC. All of the neurons had extensive dendritic arborizations arising from an average of six primary dendrites; the average total dendritic length for these cells was 2,343 microns. The morphology of preganglionic neurons differed depending on the location of their cell bodies. Preganglionic neurons located in the IML were essentially two-dimensional: the cells had some dendrites that coursed rostrocaudally for 300-500 microns within the IML and others that coursed mediolaterally, extending to the lateral surface of the cord and close to the central canal. Axons of these cells coursed ventrally from the cell body and exited from the spinal cord at the first ventral root caudal to the cell body. No intraspinal axon branches were observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Morphology of two classes of target-specific bullfrog sympathetic preganglionic neurons.

These experiments took advantage of the unique ability to define target-specific sympathetic preganglionic neurons in the bullfrog spinal cord in order to examine the morphologies of different classes of preganglionic neurons. Sympathetic preganglionic neurons were identified by retrograde transport of fast blue from the sympathetic chain. Subsequently, fast blue-labelled sympathetic preganglionic neurons in fixed spinal cord slices were filled with lucifer yellow and processed for visualization with lucifer yellow antiserum, biotinylated secondary antiserum, and avidin peroxidase. Target specificity of sympathetic preganglionic neurons was determined by anatomical position; sympathetic preganglionic neurons that control the vasculature (C-type sympathetic preganglionic neurons) lie in a position caudal to those that control nonvascular targets [B-type sympathetic preganglionic neurons; Horn and Stofer (1988) J. Comp. Neurol. 268:71]. These two classes of sympathetic preganglionic neurons have qualitatively similar morphologies. However, they exhibit significant quantitative differences in total dendritic length and the rostrocaudal extent of dendrites. These differences are likely to be associated with differences in the number of synapses received by these two classes of sympathetic preganglionic neurons. Moreover, the segmental control of sympathetic preganglionic neurons by descending brainstem projections is likely to be finer for those involved in vascular control than for those that influence other targets.

Spinal cord development in anuran larvae: I. Primary and secondary neurons.

The spinal cord of the bullfrog (Rana catesbeiana) tadpole contains primary neurons, born during embryonic stages, and secondary neurons born for the most part during larval stages. Electrophysiological and anatomical characteristics of these two categories of neurons were examined during larval development to trace the development of secondary neurons and to determine whether primary neurons persist into adult life or are replaced by secondary neurons. Five classes of primary neuron were identified on the basis of their distinctive locations, morphologies, cytoplasmic melanin content, and presence at the earliest larval stages examined: primary motoneurons, Rohon-Beard cells, commissural cells, dorsal marginal cells, and anterolateral marginal cells. Secondary neurons of the lateral motor column and dorsal root ganglia underwent extensive developmental changes during larval life manifested both in anatomical studies with horseradish peroxidase and electrophysiological experiments on the isolated spinal cord. Primary motoneurons that innervate the tadpole tail were not found in the adult, although those innervating thoracic musculature persisted, as did at least some primary neurons projecting to other spinal segments or brainstem. Primary neurons are thus replaced or maintained through metamorphosis depending on their class and location.

Spinal cord development in anuran larvae: II. Ascending and descending pathways.

The ontogeny of ascending and descending spinal pathways was examined in bullfrog (Rana catesbeiana) tadpoles using the transported histochemical marker, horseradish peroxidase (HRP). The adult pattern of brainstem projections to lumbar spinal cord is evident as early as larval stage I (Taylor and Kollros, Anat. Rec., 94:7-24, 1946), although the number and size of projecting cells increases as the animal matures. These projections arise from presumptive hypothalamic neurons at the diencephalic-mesencephalic border as well as from neurons of the vestibular nucleus, oculomotor nucleus, and reticular formation. In contrast to the stability of the pattern of descending projections, the sources of fibers ascending to the brainstem change during larval life. In early larval stages, brainstem projections from lumbar spinal cord arise primarily from Rohon-Beard cells and neurons of the superficial dorsal horn. In later stages, neurons in the intermediate and ventral areas of the spinal gray can also be retrogradely labeled by HRP application to the brainstem at the level of the VIIIth nerve. Evidence of the existence of dorsal column and lateral cervical nuclei in adult frog and tadpoles older than stage VIII is presented. The ascending projections of embryonically born primary neurons were also investigated. Rohon-Beard cells, which are sensory neurons with their cell bodies in the spinal cord, were found to send ascending processes as least as far rostral as the level of the VIIIth nerve entry zone. Anterolateral and dorsal marginal cells, probable homologs, respectively, of mammalian spinal border cells and cells of Waldeyer (1888), were also found to project rostrally at least to the rhombencephalon. These marginal cells persisted through metamorphosis into adulthood.

Frog sympathetic ganglion cells have local axon collaterals.

Amphibian autonomic ganglia have been used as simple models for studies involving the physiology of synaptic transmission. These models assume an anatomical simplicity where the ganglion is a simple relay for central nervous system output to peripheral autonomic targets. Cholinergic preganglionic fibers innervate the soma and proximal axon of the unipolar ganglion cells, which were thought to relay the information to the periphery with little ganglionic processing. However, several different types of synaptic potentials occur in response to preganglionic stimulation. Also, a variety of neuropeptides are found in both preganglionic fibers and ganglion cells; at least one of the peptides found in preganglionic fibers is known to act as a neurotransmitter in the ganglion. Finally, there may be communication between ganglion cells. In the present study, we have explored the morphology of lumbar sympathetic chain ganglion cells by intracellular injection with horseradish peroxidase to determine whether an anatomical substrate exists for processing information within these ganglia. We have shown that 39% of these cells have axons that branch within the ganglion. While both major classes of ganglion cells (B cells and C cells) had intraganglionic axon collaterals, there was a marked difference in the frequency: 65% of the C cell axons had collaterals while only 19% of the B cell axons collateralized within the ganglion. Ultrastructural examination of labeled axon collaterals indicated that these collaterals receive synaptic input; whether the collaterals also make synapses has not been definitively established.

Galanin-like innervation of rat submandibular and sublingual salivary glands: origin and effect on acinar cell membranes.

The distribution and source of a galanin-like innervation of rat salivary glands has been examined. Additionally, submandibular and sublingual acinar cell membrane responses to galanin or a cholinergic agonist were studied. Galanin-immunoreactive fibers were observed throughout the submandibular and sublingual glands in association with ducts and acini. A subset of submandibular ganglion cells expresses galanin immunoreactivity. Parasympathectomy resulted in a marked decrease in galanin immunoreactivity in the glands. Sympathectomy resulted in marked reduction of dopamine beta-hydroxylase immunoreactivity with no appreciable change in galanin immunoreactivity. Retrograde labeling experiments demonstrated that galanin-immunoreactive sensory neurons in the trigeminal ganglion do not innervate the submandibular or sublingual gland. These results indicate that the galanin-like innervation of rat salivary glands is derived from parasympathetic nerves to the glands. Since rat sublingual glands contain largely mucous acini while rat submandibular gland acini are seromucous, electrophysiological responses to galanin and the muscarinic agonist, bethanechol, were compared. Agonist-induced voltage shifts varied between the two glands. The galanin-induced response at the level of the resting membrane potential in submandibular acinar cells was a hyperpolarization, while that in sublingual acinar cells was a depolarization. There was also a greater voltage dependence to the galanin-induced submandibular response than to the sublingual response. Differences were also noted in the acinar cell response to cholinergic stimulation between these glands. These results demonstrate the existence of a galanin-like innervation to salivary glands that may be functionally relevant. Moreover, the results challenge the idea that agonist-induced membrane responses are similar among acinar cells of different glands.

Development of differential preganglionic projections to pre- and paravertebral sympathetic ganglia.

Sympathetic preganglionic axons project to spatially distinct targets in the periphery. A precise topographic pattern exists within the thoracic preganglionic cell column relative to the direction of axonal projections within the sympathetic chain. In this study, the time course and pattern of axonal outgrowth from different populations of preganglionic neurons in the chicken embryo is examined in detail to clarify the origin of the topography in this system. Projections to prevertebral targets are established by development of the splanchnic nerves by stage 25, well after the earliest somatic motor projections at stage 19 but at least two stages before the reported onset of paravertebral projections. Further, preganglionic axons that project rostrally into the sympathetic chain may do so earlier than those that project caudally in the chain. The separation of preganglionic axons into prevertebral, rostral paravertebral or caudal paravertebral directions occurs at a common site in the ventral mesenchyme, established by the initial ventromedial projection of the splanchnic nerves. Analysis of the axonal trajectories of rostrally and caudally projecting cells reveals that preganglionic axons are not selectively fasciculated before their point of separation at the sympathetic chain. The patterning of the preganglionic cell column is specified before the establishment of functional connections within the chain, indicating that target contact is not a determinant of the segmental pattern. We suggest that the differential outgrowth of preganglionic axons to peripheral targets is determined by the unique identities of underlying subpopulations of preganglionic axons.

Mutation of low affinity nerve growth factor receptor gene is associated with the hypertensive phenotype in spontaneously hypertensive inbred rat strains.

We previously reported a missense mutation in the low affinity nerve growth factor receptor (LNGFR) gene of spontaneously hypertensive rats (SHR), proposing this gene as a promising candidate in genetic hypertension. In this study we provide further support for implicating this gene in genetic hypertension using two new inbred strains, WKHT and WKHA rats. These strains originated from crossbreeding SHR rats with normotensive Wistar-Kyoto rats (WKY): WKHT rats are hypertensive but not hyperactive, and WKHA rats are hyperactive but not hypertensive. Nucleotide sequence analysis of the LNGFR gene revealed that WKHT has the same mutation as SHR, whereas WKHA has the normal sequence, as seen in WKY. These results support our original hypothesis that the mutated LNGFR gene is linked to hypertension, since the mutation had co-segregated with the hypertensive trait, and not hyperactivity trait of SHR.

Ultrastructural analysis of the distribution of synaptic boutons from labeled preganglionic axons on rabbit ciliary neurons.

The number of preganglionic inputs that innervate rabbit ciliary ganglion cells is directly correlated with the number of dendrites arising from each ganglion cell (Purves and Hume, 1981). In general, the innervation of multiply innervated ciliary neurons by individual preganglionic axons is regionally restricted to a portion of the postsynaptic surface that usually includes the cell body and some, but not all, of the dendrites (Forehand and Purves, 1984). These observations suggest that dendrites modulate convergence to each cell by providing relatively separate postsynaptic domains for individual inputs. To examine this possibility further, I have assessed the distribution of synaptic boutons from individually labeled preganglionic axons on ciliary ganglion cells at the ultrastructural level. The results show that at least a third of the dendrites of these neurons are contacted exclusively by synaptic boutons from a single preganglionic axon. However, at least half of the dendrites (and nearly all of the cell bodies) of multiply innervated ganglion cells are innervated by at least 2 different preganglionic axons. Moreover, synapses from 2 different inputs often coexist in close proximity on the postsynaptic surface. Thus, individual preganglionic axons do not require exclusive dominion over a particular part of a postsynaptic cell in order to maintain their connection with the cell. These results suggest that competitive interactions between the inputs to these cells occur between the sets of boutons arising from different inputs, rather than at the level of individual boutons.(ABSTRACT TRUNCATED AT 250 WORDS)

Density of somatic innervation on mammalian autonomic ganglion cells is inversely related to dendritic complexity and preganglionic convergence.

I have studied superior cervical ganglion cells in mouse, hamster, rat, guinea pig, and rabbit by electron microscopy to determine how the distribution of synapses on these neurons is affected by the systematic differences in dendritic morphology and preganglionic convergence that are evident in the superior cervical ganglia of these species (Purves, D., and J. W. Lichtman (1985) Science 228: 298-302). The density of dendritic innervation is approximately the same in these animals regardless of target cell geometry (and always greater than the density of synapses on the cell soma); however, more complex ganglion cells, which are innervated by commensurately more axons, receive progressively fewer cell body synapses than geometrically simpler ganglion cells. Evidently the somatic membrane becomes a less favorable site for synapse formation as dendritic complexity and the number of inputs increase. This paradoxical decrease in the density of somatic innervation as preganglionic convergence increases may reflect competitive interactions between the axons innervating individual ganglion cells.

Segmental restriction and target specificity of bullfrog preganglionic neurons that exhibit galanin-like immunoreactivity.

These experiments were designed to examine the distribution of galanin-like peptide immunoreactivity (GAL-IR) in bullfrog sympathetic preganglionic neurons and to identify the peripheral target organs affected by these neurons. Cells expressing GAL-IR were observed in the intermediolateral column of segments 7 and 8 only. Apparent GAL-IR innervation is present, but rare, in sympathetic chain ganglia. Double-labelling with retrogradely transported fast blue and galanin antiserum demonstrated that most GAL-IR neurons project via splanchnic nerves to innervate the adrenal gland, which receives a dense plexus of GAL-IR fibers surrounding chromaffin cells. The adrenal gland is also innervated by preganglionic neurons in segments 5 and 6 that do not express GAL-IR. Because nitric oxide is expressed in sympathoadrenal preganglionic neurons in mammals (Anderson, C.R., Neurosci. Lett., 139 (1992) 280), we examined whether it is expressed in bullfrog preganglionic neurons. Nicotinamide adenine dinucleotide phosphate-diaphorase positive neurons are present in bullfrog spinal grey at segments 5 through 8. These neurons were not double-labelled with fast blue retrogradely transported from the sympathetic chain, celiac ganglion, or adrenal gland; nor were they double-labelled with GAL-antiserum. Thus nitric oxide is apparently not expressed in bullfrog sympathetic preganglionic neurons.

Regional innervation of rabbit ciliary ganglion cells by the terminals of preganglionic axons.

In the rabbit, ciliary ganglion neurons with dendrites maintain inputs from several different axons during the period of synaptic rearrangement that occurs in early postnatal life. Neurons without dendrites, on the other hand, lose the majority of their initial inputs and are innervated in maturity by the terminals of only one or two axons (Purves, D., and R.I. Hume (1981) J. Neurosci. 1: 441-452; Hume, R.I., and D. Purves (1981) Nature 293: 469-471). We have explored the basis of this phenomenon by individually marking preganglionic axons and the neurons they innervate with horseradish peroxidase. In general, the innervation of geometrically complex (multiply innervated) neurons by individual preganglionic axons is regional. That is, the synaptic contacts made by an axon on these neurons are limited to a portion of the postsynaptic surface that includes some, but not all, of the dendrites. This regional innervation of target neurons is consistent with the view that dendrites allow multiple innervation to persist by providing relatively separate postsynaptic domains for individual preganglionic axons. Such regional innervation may mitigate competitive interactions between the several axons which initially innervate the same neuron.

Development and segmental organization of rostrocaudal dendrites of rat sympathetic preganglionic neurons.

The longitudinal organization of preganglionic sympathetic neurons in the adult mammalian spinal cord takes the general form of a ladder. In the rat, the preganglionic neurons of the intermediolateral cell column (IML) have extensive dendritic arborizations in both rostrocaudal and mediolateral directions. We have studied the development of the rostrocaudal dendritic projection by retrogradely labeling single sympathetic preganglionic spinal segments with the membrane label DiI. The rostrocaudal dendrites of sympathetic preganglionic neurons in the IML begin to develop prenatally on embryonic day 19-20 (E19/20), several days after these neurons develop mediolaterally oriented dendrites. Between E19 and postnatal day 1 (P1), the rostrocaudal dendrites attain a length of approx. 200 microns. As the rostrocaudal dendrites elongate, the preganglionic neurons form distinct clusters between which dendritic bundles are seen. Following a growth spurt from E19 to P1, the average rostrocaudal dendritic length approximates twice the distance between clusters from P1 to P9.

Anatomical and behavioral recovery from the effects of spinal cord transection: dependence on metamorphosis in anuran larvae.

This study of spinal cord injury in bullfrog (Rana catesbeiana) tadpoles using the neuroanatomical tracer horseradish peroxidase (HRP) was undertaken to determine (1) whether the same anatomical regions that normally give rise to ascending or descending spinal tracts do so following complete spinal cord transection and (2) whether the course of behavioral recovery could be related to the anatomical results. The results of this study show that (1) spinal cord continuity is readily restored in tadpoles subjected to spinal cord transection, but nerve fibers crossing the site of injury end within 1 to 2 mm of the lesion site; (2) tadpoles with spinal cord transections held through metamorphosis show, as juvenile frogs, restoration of lumbar projections from all brainstem regions that normally project to the lumbar spinal cord; (3) neither long ascending projections from dorsal root ganglion cells nor those from spinal neurons caudal to the transection traverse the transection site, even after metamorphosis; and (4) consistent with the anatomical results, tadpoles show only minimal behavioral recovery, but these same animals as juvenile frogs show recovery of behaviors that are dependent upon connections to supraspinal regions. In other experiments, [3H]thymidine or [3H]apo-HRP was combined with HRP histochemistry to determine if new brainstem neurons projecting to the spinal cord are born in the metamorphic period and if, in normal animals, brainstem projections to the lumbar spinal cord persist through metamorphosis. We found no evidence that neurons with lumbar spinal cord projections are born during metamorphosis; however, evidence was found that most brainstem neurons that project to the lumbar spinal cord before metamorphosis retain this projection in the juvenile frog.

Characterization of cardiac gene cis-regulatory elements in the early stages of chicken heart morphogenesis.

To study transcriptional regulation during early stages of cardiogenesis, stage 10-17 chicken embryo hearts were transfected efficiently within the intact embryo by application of plasmid DNA complexed to liposomes. Viral regulatory sequences and the skeletal alpha-actin, SERCA2 and ANF promoters activated expression of reporter genes in the primitive heart tube. Deletion and mutation analysis of the skeletal alpha-actin promoter revealed the importance of CArG cis-regulatory elements in enhancing transcription of the gene during early heart development. These results demonstrate the utility of this method for the identification of gene regulatory elements that specify the cardiac phenotype during early stages of heart morphogenesis.

Hypertrophy of stellate ganglion cells in hypertensive, but not hyperactive, rats.

The dendritic complexity of peripheral autonomic neurons is positively matched with the size of the target they innervate, apparently by trophic interactions with the target (D. Purves, W. D. Snider, and J. T. Voyvodic. Nature Lond. 336: 123-128, 1988). We have asked whether the vascular hypertrophy associated with hypertension is accompanied by dendritic hypertrophy of sympathetic ganglion cells. To do this, we examined the morphology of stellate ganglion cells in the spontaneously hypertensive rat (SHR), its normotensive control Wistar-Kyoto rat (WKY), and two new strains derived from the SHR that independently express the hypertensive phenotype of the SHR (WKHT) and the behavioral hyperactivity present in the SHR (WKHA). Cells were examined by intracellular staining with horseradish peroxidase in in vitro preparations of the ganglia. Carotid arterial wall size was also examined. Significant hypertrophy of both the carotid arterial wall and stellate ganglion cell dendrites was observed in the two hypertensive strains (SHR and WKHT) but not in either of the normotensive strains (WKY and WKHA). This increased total dendritic length of stellate ganglion cells associated with hypertension provides a greater target area for preganglionic innervation that may result in hyperinnervation of these cells.

Differential distribution of retinoic acid synthesis in the chicken embryo as determined by immunolocalization of the retinoic acid synthetic enzyme, RALDH-2.

Retinaldehyde dehydrogenase type 2 (RALDH-2) is a major retinoic acid generating enzyme in the early embryo. Here we report the immunolocalization of this enzyme (RALDH-2-IR) in stage 6-29 chicken embryos; we also show that tissues that exhibit strong RALDH-2-IR in the embryo contain RALDH-2 and synthesize retinoic acid. RALDH-2-IR indicates dynamic and discrete patterns of retinoic acid synthesis in the embryo, particularly within the somitic mesoderm, lateral mesoderm, kidney, heart, and spinal motor neurons. Prior to somitogenesis, RALDH-2-IR is present in the paraxial mesoderm with a rostral boundary at the level of the presumptive first somite; as the somites form, they exhibit strong RALDH-2-IR. Cervical presomitic mesoderm exhibits RALDH-2-IR but thoracic presomitic mesoderm does not. Neural crest cells do not express detectable levels of RALDH-2, but migrating crest cells are associated with RALDH-2 expressing mesoderm. The developing limb mesoderm expresses little RALDH-2-IR; however, RALDH-2-IR is strongly expressed in tissues adjacent to the limb. The most lateral, earliest-projecting motor neurons at all levels of the spinal cord exhibit RALDH-2-IR. Subsequently, many additional motor neurons in the brachial and lumbar cord regions express RALDH-2-IR. Motor neuronal expression of RALDH-2-IR is present in the growing axons as they extend to the periphery, indicating a potential role of retinoic acid in nerve influences on peripheral differentiation. With the exception of a transient expression in the facial/vestibulocochlear nucleus, cranial motor neurons do not express detectable levels of RALDH-2-IR.

Segmental patterning of rat and chicken sympathetic preganglionic neurons: correlation between soma position and axon projection pathway.

The segmental organization of midthoracic rat and chicken sympathetic preganglionic neurons was examined by retrograde labeling in vivo and in vitro. The results demonstrate that individual sympathetic preganglionic neurons project only rostrally or caudally within the sympathetic chain, even though the spinal segment in which they reside provides innervation to both rostral and caudal ganglia. In addition, there is both a segmental and an intrasegmental pattern in the thoracic sympathetic column, in which the position of preganglionic neurons is related to the direction they project in the sympathetic chain. Thoracic sympathetic preganglionic neurons are organized into discrete segmental units, each of which exhibits an internal rostrocaudal polarity with respect to the direction of axon projection in the sympathetic chain. The rostrocaudal bias of this internal polarity is graded from segment to segment along the longitudinal axis. Since there is minimal overlap between thoracic segments, the transition from one segment to another entails a sharp change in the pathway choice of the preganglionic neurons. The organization of the preganglionic projections thus includes (1) segmental periodicity, (2) intrasegmental gradients of neuronal identity, and (3) an axial gradient of segment identity. The significance of these findings is twofold. First, they suggest a functional organization that may be related to the specificity of sympathetic reflex action. Second, they reveal a cellular organization that suggests an underlying segmental pattern in the developing spinal cord.

Expression and regulation of the retinoic acid synthetic enzyme RALDH-2 in the embryonic chicken wing.

Retinaldehyde dehydrogenase type 2 (RALDH-2) is a major retinoic acid (RA) generating enzyme in the embryo. Here, we report immunolocalization of this enzyme (RALDH-2-IR) in the developing wings of stage 17-30 chicken embryos. RALDH-2-IR is located in the area of the presumptive muscle masses, although it is not colocalized with developing muscle cells. RALDH-2-IR is located in tendon precursor cells and may be present in muscular connective tissue. We show that motor neurons and blood vessels, tissues showing RALDH-2-IR as they enter the limb, are capable of synthesizing and releasing RA in culture. RALDH-2-IR in the limb mesenchyme is under the control of both the vasculature and the motor innervation; it is decreased with denervation and increased with hypervascularization. RALDH-2-IR is present in the motor neuron pool of the brachial spinal cord, but this expression pattern is apparently not under the control of limb target tissues, RA in the periphery, or somitic factors. RA is known to be a potent inducer of cellular differentiation; we propose that locally synthesized RA may be involved in aspects of wing tissue specification, including cartilage condensation and outgrowth, skeletal muscle differentiation, and recruitment of smooth muscle cells to the vasculature.