Neural tube patterning in vertebrates is controlled in part by locally secreted factors that act in a paracrine manner on nearby cells to regulate proliferation and gene expression. We show here by in situ hybridization that genes for the neuropeptide pituitary adenylate cyclaseactivating peptide (PACAP) and one of its high-affinity receptors (PAC 1 ) are widely expressed in the mouse neural tube on embryonic day (E) 10.5. Transcripts for the ligand are present in differentiating neurons in much of the neural tube, whereas the receptor gene is expressed in the underlying ventricular zone, most prominently in the alar region and f loor plate. PACAP potently increased cAMP levels more than 20-fold in cultured E10.5 hindbrain neuroepithelial cells, suggesting that PACAP activates protein kinase A (PKA) in the neural tube and might act in the process of patterning. Consistent with this possibility, PACAP down-regulated expression of the sonic hedgehog-and PKA-dependent target gene gli-1 in cultured neuroepithelial cells, concomitant with a decrease in DNA synthesis. PACAP is thus an early inducer of cAMP levels in the embryo and may act in the neural tube during patterning to control cell proliferation and gene expression.Recent studies suggest that phenotypic determination in the developing nervous system results from interactions of patterning genes conserved through evolution (1). For example, sonic hedgehog (shh) is one of three mammalian homologs to the segment polarity gene hedgehog (hh). shh has been implicated as a notochord-and floor plate-secreted factor that controls dorsal͞ventral patterning in the vertebrate neural tube (1-4). Abundant genetic and molecular evidence in flies (5), fish (6), and mice (7) indicates that hh and its homologs act by antagonizing cAMP-dependent protein kinase A (PKA) signaling. Although shh may act in the ventral tube by blocking constitutive PKA activity, it is possible that physiological activators of the cAMP͞PKA pathway are important in patterning.We considered that pituitary adenylate cyclase-activating peptide (PACAP) might be involved in patterning for several reasons. First, although PACAP originally was discovered as a hypothalamic factor that potently increased cAMP in the pituitary through G protein-coupled receptors (8), peptide expression later was localized to many central and peripheral neuronal populations as well as the developing embryo (9). Second, the 27-aa form of the peptide, PACAP-27, is conserved 100% in species ranging from fish to humans. Finally, our tissue culture studies indicated that PACAP and a closely related peptide vasoactive intestinal peptide stimulate cAMP production, regulating proliferation, differentiation, and͞or survival of multiple neuronal precursors (10-14). The current studies indicate that a functional PACAP ligand͞receptor cAMP signaling system is expressed in the neural tube at the onset of neurogenesis, raising the possibility that PACAP might be involved in neural tube patterning. MATERIALS AND METHODS In Situ Hybridi...

The growth rate of rodent embryonic neuroblasts and human neuroblastoma cell lines is regulated in part by autocrine or paracrine actions of neuropeptides of the family that includes vasoactive intestinal peptide (VIP), peptide histidine isoleucine (PHI), and pituitary adenylate cyclase-activating peptide (PACAP). These peptides act via seven transmembrane G-protein-linked receptors coupled to cAMP elevation, phospholipase C activation, intracellular Ca 2؉ release, and/or of mitogen-activated protein (MAP) kinase activation. Here we investigated the action of these peptides on the mouse neuroblastoma cell line Neuro2a. PHI and VIP inhibited proliferation at concentrations as low as 10 ؊13 M and 10 ؊10 M, respectively. In contrast, PACAP action was biphasic, with stimulation occurring at subnanomolar doses and inhibition at higher doses. Peptide actions were studied further by measuring cAMP and ERK1/2 MAP kinase activity and by assessing 3 H-thymidine incorporation in conjunction with a panel of signal transduction pathways inhibitors. The data obtained indicated that the PHI-inhibitory and PACAP-stimulatory activities were mediated by corresponding changes in activity of the MAP kinase pathway and independent of protein kinase A (PKA) or protein kinase C (PKC). In contrast, the inhibitory actions of VIP and PACAP were specifically blocked by antagonists of PKA. Northern blot analysis revealed gene expression for only the PACAP-preferring (PAC 1 ) receptor. However, binding experiments using 125 I-labeled PACAP27, PHI, and VIP, demonstrated the presence of PACAP-preferring sites, bivalent VIP/PACAP sites, and PHI-binding sites that did not interact with VIP. The studies demonstrate potent regulatory actions of PACAP, PHI, and VIP on neuroblastoma cell proliferation which appear to be mediated by multiple subsets of receptors which differentially couple to MAP kinase and PKA signaling pathways. The vasoactive intestinal peptide (VIP)1 neuropeptide family includes VIP, pituitary adenylate cyclase-activating peptide 27 (PACAP-27), the carboxyl-terminal extended isoform PACAP-38, peptide histidine/isoleucine (PHI), and its human homologue peptide histidine/methionine (PHM), secretin, glucagon, growth hormone releasing factor (GRF), and also analogues isolated from reptile venom (1). Expression of the VIP and PACAP genes results in the biosynthesis of peptide precursors, which also give rise to PHI/PHM and PACAP-related peptide (PRP), respectively (2, 3). The neuropeptides VIP, PHI/PHM, and PACAP are commonly expressed and secreted in neuroblastoma and certain neuroendocrine tumors (4). Moreover the finding of functional VIP and PACAP receptors expressed in neuroblastoma cell lines and freshly excised tumor resections suggests that these neuropeptides may be involved in autocrine/paracrine loops (5, 6). Although these peptides are well known to act in the processes of neurotransmission (1, 7, 8), they have been shown to regulate the proliferation rate and differentiation of numerous cell lines (9). These include thos...

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Abstract:The neuropeptide vasoactive intestinal peptide (VIP) is expressed in several distinct sites in the CNS, in cholinergic and enteric ganglia, and in a small subpopulation of neurons within sympathetic ganglia. Previous studies on the human VIP gene indicate that transcription in neural crest-derived neuroblastoma and pheochromocytoma cell lines is controlled in part by multiple regulatory elements located along 4.5 kb of upstream 5Ј flanking sequence. In the current studies, transgenic mice were created with a chimeric gene consisting of 16.5 kb of the mouse VIP gene fused to the ␤-galactosidase reporter. In situ hybridization analysis in adult mice indicated that reporter gene expression was correctly targeted to neurons in the esophagus, stomach, small intestine, and colon. No expression was observed in the brain, including regions that contain abundant VIP-expressing cells, such as the thalamus, amygdala, cerebral cortex, hippocampus, and suprachiasmatic nucleus. Analysis of transgene expression in neonatal and embryonic day 13.5 mice revealed a near perfect correlation between VIP and ␤-galactosidase gene expression in cranial cholinergic ganglia and the superior cervical ganglia, and lack of transgene expression in sensory ganglia and in nonneuronal tissue. Potential ectopic transgene expression was observed in neonates, in the cerebellar external granule layer and in a small subpopulation of neurons in the olfactory epithelium. We conclude that the 16.5 kb of VIP gene used in these studies contains sequences sufficient for directing expression specifically to VIP neurons in the PNS, and that sequences located elsewhere on the gene are required for proper CNS expression. The VIP gene sequences used here should be capable of targeting other gene products to specific populations of embryonic and adult peripheral neurons without causing significant expression in the CNS. Key Words: Vasoactive intestinal peptide -TransgenicPromoter-Cholinergic-Embryo-Neonate.