Immortalized Hensen's node cells secrete a factor that regulates avian neural crest cell fates in vitro.

The derivatives of the neural crest are regionally specified with respect to the anterior-posterior axis of the avian embryo. We have shown previously that young Hensen's node can act in vitro to regulate the expression of certain region-specific phenotypes in trunk neural crest cells. To study potential factors acting on the neural crest, we have generated an immortalized cell line from young Hensen's node. Here we show that a factor produced by these cells stimulates the expression of two cranial-specific phenotypes (fibronectin and smooth muscle actin) in trunk neural crest cells and decreases their expression of a trunk-specific phenotype (melanin). The active factor is a secreted protein with a molecular weight >30 kDa. Clonal studies suggest that the factor acts by changing the phenotypic fates of individual neural crest cells, rather than by selective effects on cell proliferation or survival. Previous work has shown that TGF-betas can mimic the effects of Hensen's node cells on neural crest differentiation. Results from the present study suggest that the factor in the conditioned medium of the immortalized node cell line is not a TGF-beta isoform. However, the cranial phenotype-inducing activity of the conditioned medium factor requires the presence of neural crest cell-derived TGF-betas.

Hensen's node regulates avian neural crest differentiation in vitro.

Different anteroposterior (AP) regions of the neural crest normally produce different cell types, both in vivo and in vitro. AP differences in neural crest cell fates appear to be specified in part by mechanisms that act prior to neural crest cell migration. We, therefore, examined the possibility that the fates of neural crest cells, like those of neural tube cells, can be regulated by interactions with Hensen's node. Using a transfilter co-culture system, we found that young (stage 3+ to 4) Hensen's node up-regulates the expression of two cranial-specific phenotypes (fibronectin and smooth muscle actin immunoreactivities) in mass cultures of trunk neural crest cells, and down-regulates the expression of a trunk-specific phenotype (melanin synthesis). The changes in phenotype produced by exposure to young Hensen's node were not accompanied by changes in the proliferation of either fibronectin immunoreactive cells or melanocytes. The capacity of Hensen's node to elicit changes in trunk neural crest cell phenotype decreased as the developmental age of the node increased and was lost by stage 6. In addition, old Hensen's node did not stimulate the expression of trunk-specific phenotypes in cranial neural crest cells, suggesting that cranial- and trunk-specific phenotypes are induced by different mechanisms.

Role of the transforming growth factor-beta family in the expression of cranial neural crest-specific phenotypes.

Cranial and trunk neural crest cells produce different derivatives in vitro. Cranial neural crest cultures produce large numbers of cells expressing fibronectin (FN) and procollagen I (PCol I) immunoreactivities, two markers expressed by mesenchymal derivatives in vivo. Trunk neural crest cultures produce relatively few FN or PCol I immunoreactive cells, but they produce greater numbers of melanocytes than do cranial cultures. Treatment of trunk neural crest cultures with transforming growth factor-beta 1 (TGF-beta 1) stimulates them to express both FN and PCol I immunoreactivities at levels comparable to those normally seen in cranial cultures and simultaneously decreases their expression of melanin. These observations raised the possibility that endogenous TGF-beta is involved in specifying differences in the phenotypes expressed by cranial and trunk neural crest cells in vitro. Consistent with this idea, we found that treatment of cranial cultures with a function-blocking TGF-beta antiserum inhibits the development of FN immunoreactive cells and stimulates the development of melanocytes. Cranial and trunk neural crest cells express approximately equal levels of TGF-beta mRNA. However, trunk neural crest cells are significantly less sensitive to the FN-inducing effect of TGF-beta 1 than are cranial neural crest cells. These results suggest that: (1) endogenous TGF-beta is required for the expression of mesenchymal phenotypes by cranial neural crest cells, and (2) differences in the phenotypes expressed by cranial and trunk neural crest cells in vitro result in part from differences in the sensitivities of these two cell populations to TGF-beta.

Rostrocaudal differences in the expression of extracellular matrix proteins by avian neural crest cells in vitro.

Cranial and trunk neural crest cells developing in vitro differed in their patterns of expression of two major extracellular matrix proteins, fibronectin and collagen I. Cranial neural crest cells showed two distinct phases of fibronectin expression: the first occurred during the initial migration of cells from explants onto the culture dish; the second was associated with the differentiative period of in vitro development. Fibronectin-immunoreactive cells eventually represented one of the most abundant cell types in cranial cultures. Large numbers of procollagen I-immunoreactive cells also developed in cranial cultures, and procollagen I was colocalized with fibronectin in individual cranial neural crest cells. Neither fibronectin nor procollagen I immunoreactivities were seen in either neurons or melanocytes, consistent with the idea that the fibronectin-immunoreactive cells in cranial neural crest cultures are committed to the mesenchymal lineage. In contrast to cranial neural crest, trunk neural crest produced very few fibronectin-immunoreactive cells at any time in vitro. Trunk neutral crest also produced smaller proportions of procollagen I-immunoreactive cells than did cranial explants. Mitotic labelling experiments showed that the differing proportions of fibronectin- and procollagen I-immunoreactive cells in cranial versus trunk cultures did not result from differences in rates of cell proliferation.

Coexpression of sensory and autonomic neurotransmitter traits by avian neural crest cells in vitro.

This study shows that explants of quail neural crest cultured in a medium containing serum and chick embryo extract give rise to large numbers of cells expressing immunoreactivity for substance P (SP), a neuropeptide found in sensory neurons. These cells arise from cycling precursors, but do not appear to divide after expressing SP. The SP-positive cells in cranial neural crest cultures express both neurofilament and the Q211 antigen, but those in trunk cultures express only the Q211 antigen. In both cranial and trunk cultures, large subpopulations of the SP-positive cells express tyrosine hydroxylase and/or choline acetyltransferase, neurotransmitter markers characteristic of autonomic neurons. This finding argues against the idea that SP expression necessarily indicates commitment to the sensory neuron lineage. I further show that embryonic dorsal root ganglion (DRG) cells retain the ability to coexpress SP and tyrosine hydroxylase in vitro, although to a lesser extent than do neural crest cells.

Differential development of cholinergic neurons from cranial and trunk neural crest cells in vitro.

Several studies have suggested that the development of cholinergic properties in cranial parasympathetic neurons is determined by these cells' axial level of origin in the neural crest. All cranial parasympathetic neurons normally derive from cranial neural crest. Trunk neural crest cells give rise to sympathetic neurons, most of which are noradrenergic. To determine if there is an intrinsic difference in the ability of cranial and trunk neural crest cells to form cholinergic neurons, we have compared the development of choline acetyltransferase (ChAT)-immunoreactive cells in explants of quail cranial and trunk neural crest in vitro. Both cranial and trunk neural crest explants gave rise to ChAT-immunoreactive cells in vitro. In both types of cultures, some of the ChAT-positive cells also expressed immunoreactivity for the catecholamine synthetic enzyme tyrosine hydroxylase. However, several differences were seen between cranial and trunk cultures. First, ChAT-immunoreactive cells appeared two days earlier in cranial than in trunk cultures. Second, cranial cultures contained a higher proportion of ChAT-immunoreactive cells. Finally, a subpopulation of the ChAT-immunoreactive cells in cranial cultures exhibited neuronal traits, including neurofilament immunoreactivity. In contrast, neurofilament-immunoreactive cells were not seen in trunk cultures. These results suggest that premigratory cranial and trunk neural crest cells differ in their ability to form cholinergic neurons.

Differentiation of noradrenergic traits in the principal neurons and small intensely fluorescent cells of the parasympathetic sphenopalatine ganglion of the rat.

Catecholamine synthetic enzymes are found in many cranial parasympathetic principal neurons, and in the small intensely fluorescent (SIF) cells that populate parasympathetic as well as sympathetic ganglia. While there is evidence that the acquisition of noradrenergic properties in sympathetic neuron precursors depends on factors that these cells encounter in the trunk environment, the mechanisms that direct the development of noradrenergic traits in cranial parasympathetic neurons and SIF cells are not understood. The present study examines the time course of appearance of tyrosine hydroxylase (TH) immunoreactivity in the principal neurons and SIF cells of the rat sphenopalatine ganglion. We show that the sphenopalatine ganglion of normal adult rats contains both a small population of TH-immunoreactive principal neurons and many SIF cells. The TH-immunoreactive principal neurons do not synthesize or store detectable catecholamines, even though the majority of sphenopalatine ganglion neurons do contain 1-amino acid decarboxylase catalytic activity. Sphenopalatine ganglion principal neurons do not accumulate detectable levels of exogenous catecholamines. This observation suggests that they lack a high affinity norepinephrine uptake system. In contrast to what has been observed previously for sympathetic neurons, the appearance of TH immunoreactivity in sphenopalatine neurons is not temporally correlated with the cessation of neural crest cell migration. The first TH-immunoreactive neurons do not appear in the sphenopalatine ganglion until Embryonic Day 16.5, 2 days after the ganglion has condensed and process outgrowth has begun. The number of sphenopalatine neurons that express TH immunoreactivity increases dramatically between Embryonic Day 18.5 and Postnatal Day 1, but then decreases. In fact, the percentage of sphenopalatine neurons that express TH immunoreactivity is almost fivefold higher in newborn than in adult rats. SIF cells cannot be definitively identified in the sphenopalatine ganglion until after Embryonic Day 18.5. The time course of appearance of TH immunoreactivity in sphenopalatine ganglion cells raises the possibility that TH expression is stimulated in these cells by factors encountered either at their condensation site or at their target, such as glucocorticoids or nerve growth factor. The relatively late appearance of SIF cells in the sphenopalatine ganglion argues against the hypothesis that SIF cells are the precursors of all autonomic neurons.

Target specificity of neuropeptide Y-immunoreactive cranial parasympathetic neurons.

We recently showed that neuropeptide Y (NPY)-like immunoreactivity occurs in subpopulations of neurons in 3 cranial parasympathetic ganglia: the otic, sphenopalatine, and ciliary. The present work identifies the target tissues innervated by cranial parasympathetic NPY-immunoreactive neurons. Plexuses of NPY-immunoreactive fibers were observed in the parotid gland, the target of the otic ganglion, and in the intraorbital lacrimal gland and palate, targets of the sphenopalatine ganglion. NPY-immunoreactive fibers of apparent parasympathetic origin innervated glandular acini in all 3 structures and were also present around small blood vessels in the parotid and intraorbital lacrimal glands. These fibers were presumed to be parasympathetic because they were not affected by removal of the superior cervical ganglion and because their distribution was coextensive with that of vasoactive intestinal polypeptide (VIP) immunoreactivity, which we have previously shown to be colocalized with NPY in the cell bodies of otic and sphenopalatine ganglion neurons. In contrast, no NPY-immunoreactive fibers were observed in the iris or ciliary body of acutely sympathectomized rats, suggesting that NPY-immunoreactive neurons in the ciliary ganglion do not normally transport detectable levels of NPY to their terminals. The target specificities of cranial parasympathetic NPY-immunoreactive neurons are different from those of sympathetic NPY-immunoreactive neurons. Sympathetic NPY-immunoreactive fibers innervated the iris and ciliary body, and the blood vessels but not the parenchymal cells of all the glands examined. In contrast, parasympathetic NPY-immunoreactive fibers primarily innervated glandular acini. NPY-immunoreactive neurons in the sphenopalatine ganglion displayed an additional level of specificity in their projection pattern in that they innervated only a subset of the ganglion's array of target glands: they innervated the intraorbital lacrimal gland and the seromucous glands of the palate but not the exorbital lacrimal gland or the glands of the nasal mucosa. The finding that NPY immunoreactivity is present in the parasympathetic innervation of secretory acini in several craniofacial glands raises the possibility that NPY plays a role in the parasympathetic control of glandular secretion. The observed overlap in the distributions of NPY- and VIP-immunoreactive fibers in these glands further suggests that NPY may interact with VIP to stimulate secretion.

Neuropeptide Y-like immunoreactivity in rat cranial parasympathetic neurons: coexistence with vasoactive intestinal peptide and choline acetyltransferase.

Neuropeptide Y (NPY) is widely distributed in the sympathetic nervous system, where it is colocalized with norepinephrine. We report here that NPY-immunoreactive neurons are also abundant in three cranial parasympathetic ganglia, the otic, sphenopalatine, and ciliary, in the rat. High-performance liquid chromatographic analysis of the immunoreactive material present in the otic ganglion indicates that this material is very similar to porcine NPY and indistinguishable from the NPY-like immunoreactivity present in rat sympathetic neurons. These findings raise the possibility that NPY acts as a neuromodulator in the parasympathetic as well as the sympathetic nervous system. In contrast to what has been observed for sympathetic neurons, NPY-immunoreactive neurons in cranial parasympathetic ganglia do not contain detectable catecholamines or tyrosine hydroxylase (EC 1.14.16.2) immunoreactivity, and many do contain immunoreactivity for vasoactive intestinal peptide and/or choline acetyltransferase (EC 2.3.1.6). These findings suggest that there is no simple rule governing coexpression of NPY with norepinephrine, acetylcholine, or vasoactive intestinal peptide in autonomic neurons. Further, while functional studies have indicated that NPY exerts actions on the peripheral vasculature which are antagonistic to those of acetylcholine and vasoactive intestinal peptide, the present results raise the possibility that these three substances may have complementary effects on other target tissues.

alpha-Noradrenergic potentiation of neurotransmitter-stimulated cAMP production in rat striatal slices.

The present study examines the possible involvement of alpha-adrenergic receptors in catecholamine-stimulated cAMP production in intact slices of rat striatum. Norepinephrine (NE) produces a greater stimulation of cAMP levels than does the beta-adrenergic agonist, isoproterenol (ISO), and the NE response is inhibited by both the beta-adrenergic antagonist, propranolol, and the alpha-adrenergic antagonist, phentolamine. The alpha-adrenergic agonist, 6-fluoronorepinephrine (6-FNE), has little or no effect on basal cAMP levels; however, 6-FNE causes a marked potentiation of the cAMP response to ISO. Hence, NE stimulation of cAMP levels in striatal slices appears to involve a synergistic interaction between alpha- and beta-adrenergic receptors. alpha-Receptors also potentiate adenosine stimulation of cAMP levels in striatal slices. However, in contrast to results previously reported in cerebral cortical slices, the alpha-adrenergic component of the NE response in striatal slices is not dependent on endogenous adenosine. Finally, 6-FNE interactions with adenylate cyclase in striatal homogenates differ from those observed in the slice preparation. In homogenates, 6-FNE appears to directly stimulate adenylate cyclase through a D-1 receptor. D-1 receptor involvement in catecholamine responses in the striatal slice preparation, on the other hand, appears to be minimal.

Regulation of rat pineal hydroxyindole-O-methyltransferase: evidence of S-adenosylmethionine-mediated glucocorticoid control.

Rat pineal hydroxyindole-O-methyltransferase is controlled similarly to adrenal medullary phenylethanolamine N-methyltransferase. S-adenosylmethionine (SAM), the in vivo cofactor utilized by the enzyme to convert N-acetylserotonin to melatonin, protects this methyltransferase against tryptic proteolysis in vitro. Furthermore, in vivo studies suggest that the nucleoside itself is controlled by glucocorticoids. Hypophysectomy decreases hydroxyindole-O-methyltransferase levels as compared with control animals, while dexamethasone and SAM administration restore enzyme levels toward control values. In vitro proteolytic studies further demonstrate that, although N-acetylserotonin does not stabilize the enzyme against trypsinization, this substrate acts synergistically with SAM to confer greater stabilization than observed with SAM alone.