Xwnt-8 and lithium can act upon either dorsal mesodermal or neurectodermal cells to cause a loss of forebrain in Xenopus embryos.

When Xenopus gastrulae are made to misexpress Xwnt-8, or are exposed to lithium ions, they develop with a loss of anterior structures. In the current study, we have characterized the neural defects produced by either Xwnt-8 or lithium and have examined potential cellular mechanisms underlying this anterior truncation. We find that the primary defect in embryos exposed to lithium at successively earlier stages during gastrulation is a progressive rostral to caudal deletion of the forebrain, while hindbrain and spinal regions of the CNS remain intact. Misexpression of Xwnt-8 during gastrulation produces an identical loss of forebrain. Our results demonstrate that lithium and Wnts can act upon either prospective neural ectodermal cells, or upon dorsal mesodermal cells, to cause a loss of anterior pattern. Specifically, ectodermal cells isolated from lithium- or Wnt-exposed embryos are unable to form anterior neural tissue in response to inductive signals from normal dorsal mesoderm. In addition, although dorsal mesodermal cells from lithium- or Wnt-exposed embryos are specified properly, and produce normal levels of the anterior neural inducing molecules noggin and chordin, they show a greatly reduced capacity to induce anterior neural tissue in conjugated ectoderm. Taken together, our results are consistent with a model in which Wnt- or lithium-mediated signals can induce either mesodermal or ectodermal cells to produce a dominant posteriorizing morphogen which respecifies anterior neural tissue as posterior.

Glycoconjugate expression during Drosophila embryogenesis.

Glycoproteins and other glycoconjugates present on the surface of many cell types have been identified and assigned various functions. The extent of variation possible in complex glycan structures and the heterogeneity of glycoconjugate expression between two apparently similar cells has been demonstrated previously by using plant lectins to survey developmental biological models. To examine the array and extent of glycoconjugate roles in embryonic Drosophila neurogenesis, we have used plant lectins to characterize lectin receptor molecules present on the neuronal and non-neuronal cell surfaces during critical stages in axonogenesis and axon fascicle development. A collection of lectins representing a variety of hapten monocarbohydrate specificities uncovered a complex expression pattern in many glycan structures. D-Galactose-specific lectins, Bauhina purpura agglutinin (BPA) and Arachis hypogea agglutinin (peanut agglutinin, PNA), and a D-galactose/N-acetylgalactosamine-specific lectin, Glycine max agglutinin (soybean agglutinin, SBA), all recognized the surface of most cultured neurons and their axons. In the intact embryo, only the PNA and BPA receptors were found on neurons of the central and peripheral nervous systems, while SBA recognized cells of structures other than the nervous system. All three lectins recognize a high molecular weight glycoprotein when used to precipitate lectin receptor from culture homogenates. Results suggest the presence of lectin receptor glycoproteins at temporally and spatially important positions within the embryo and in culture. These glycoproteins may provide functions critical in establishing the final phenotypes of specific cells through either axon guidance/target acquisition or morphogenic adhesive events.

Characterization of a putative Drosophila GTP-binding protein.

We generated a set of monoclonal antibodies raised against Drosophila antigen eluted from a lectin affinity column. One antibody, mAb 13D5 recognizes an antigen found in the most dorsal regions of the ventral midline of Drosophila embryos at stages prior to and during axonogenesis. 13D5 recognizes cells dorsal to the ectoderm in the extended germ band beginning approximately 7 hours after fertilization and in the peripheral nervous system (PNS) at about 13 hours. In addition, 24-hour-old cultures of isolated embryonic neuroblasts possess a number of cells that express the 13D5 antigen and are not recognized by horseradish peroxidase antisera. These cells extend ramified processes with multiple growth cone-like structures or large individual processes with a broad growth cone structure. 13D5 immunoprecipitates a single band with an apparent molecular mass of 58 kDa and isolated a 1.9 kb EcoRI fragment from the lambda gt11 expression libraries. In situ hybridization to staged embryos using the digoxygenin-labeled probe reveals a pattern of expression in cells just lateral to the dorsal-most regions (mesectoderm) of the ventral midline in 8- to 9-hour embryos. In situ hybridization to cultured cells derived from whole embryos reveals several cell types with differing morphologies that express transcript recognized by the digoxygenin-labeled probe. These cells may possess either broad processes containing detectable transcript, or long thin processes with no detectable transcript. Northern analysis reveals a 2.1 kb RNA transcript detectable in all embryonic stages. Nucleotide sequence obtained from the 1.9 kb insert reveals homology with the GTP-binding regions of two signal recognition particle receptors (SNRP) isolated from canine and human tissues.

Glial interactions with neurons during Drosophila embryogenesis.

A monoclonal antibody (Mab5B12) demonstrating specificity for glial cells within the central and peripheral nervous systems of Drosophila has been used in combination with neural-specific antibodies to study the early organization of the Drosophila embryo. The embryonic central nervous system of Drosophila contains cells within the ventral midline that are recognized by monoclonal antibody 5B12. These cells are not recognized by either a polyclonal antiserum to horse radish peroxidase, which recognizes several antigens on the surface of Drosophila neurons, or Mab22C10, which recognizes an antigen specific to the peripheral nervous system. Mab5B12-positive cells lie dorsal both to the developing anterior and posterior commissures in each thoracic and abdominal segment and to the supraoesophageal commissure. They ensheath these commissures in later stage embryos. Other Mab5B12-positive cells lie dorsolateral to the CNS and send processes laterally to the lateral sensilla during axonogenesis in the PNS. These cells surround the axons of the intersegmental and segmental nerves. Other cells that line the advancing ectoderm during dorsal closure and surround the anal pads also express the Mab5B12 antigen. Neuronal cell cultures derived from Drosophila gastrulae contain cells expressing the Mab5B12 antigen. These cells can be found separate or in close association with neuronal clusters and their axons.

Catecholaminergic properties of cholinergic neurons and synapses in adult rat ciliary ganglion.

Parasympathetic neurons of the ciliary ganglion are innervated by preganglionic cholinergic neurons whose cell bodies lie in the brain stem; the ganglion cells in turn provide cholinergic innervation to the intrinsic muscles of the eye. Noradrenergic innervation of the iris is supplied by sympathetic neurons of the superior cervical ganglion. Using immunocytochemical and histochemical techniques, we have examined the ciliary ganglion of adult rats for the expression of cholinergic and noradrenergic properties. As expected, the postganglionic ciliary neurons possessed detectable levels of choline acetyltransferase immunoreactivity (ChAT-IR). Unexpectedly, many ciliary neurons also exhibited immunoreactivity for tyrosine hydroxylase (TH-IR). Some had dopamine beta-hydroxylase-like (DBH-IR) immunoreactivity, but none contained detectable catecholamines, even after treatment with nialamide and L-DOPA. A sparse plexus of fibers exhibiting faint TH-IR was present in the irises of acutely sympathectomized rats. The terminals of preganglionic axons in the ciliary ganglion exhibited not only immunoreactivity for ChAT, but also for TH and contained stores of endogenous catecholamine. Neither ciliary neurons nor their preganglionic innervation accumulated detectable stores of exogenous catecholamines. Rats sympathectomized as neonates by treatment with 6-hydroxydopamine subsequently had a greater proportion of neurons possessing detectable TH-IR in the ciliary ganglion; both the TH-IR perikarya and their axons in the iris were more intensely immunofluorescent. TH-IR was present in the ciliary neuron cell bodies of mouse, guinea pig, and ferret. These species, however, lacked detectable TH-IR or catecholamine stores in preganglionic terminals. These observations indicate that mature, functionally cholinergic neurons from 2 different embryonic origins, postganglionic ciliary neurons derived from the neural crest and preganglionic neurons derived from the neural tube, display several catecholaminergic properties.

Coexistence of calcitonin gene-related peptide and vasoactive intestinal peptide in cholinergic sympathetic innervation of rat sweat glands.

Immunoreactivity for calcitonin gene-related peptide (CGRP) has been localized with indirect immunofluorescence techniques in the cholinergic sympathetic fibers that innervate eccrine sweat glands in the rat. This innervation also contains vasoactive intestinal peptide-like immunoreactivity (VIP-IR). A small proportion of principal neurons in stellate and lumbar sympathetic ganglia which provide innervation to the sweat glands contain detectable CGRP-immunoreactivity. The CGRP-IR neurons are immunoreactive for VIP; however, many VIP-IR neurons in these ganglia do not contain detectable levels of CGRP-IR.

Neonatal treatment with nerve growth factor antiserum eliminates cholinergic sympathetic innervation of rat sweat glands.

Most mammalian sympathetic neurons are noradrenergic, and their dependence upon nerve growth factor (NGF) for survival during development is well established. A minor population of sympathetic neurons, including those that innervate sweat glands, is cholinergic. To determine whether cholinergic sympathetic neurons, like their noradrenergic counterparts, require NGF during development, neonatal rats were treated with NGF-antiserum and 3 weeks later their sweat glands were examined for the presence of innervation. Acetylcholinesterase (AChE) staining and vasoactive intestinal polypeptide-like immunoreactivity (VIP-IR) which mark the mature sweat gland innervation were absent from the sweat glands of the anti-NGF treated animals. Further, when the glands were examined with the electron microscope, no axons or nerve terminals were evident. These observations indicate that the elaboration of the sweat gland plexus is NGF-dependent and suggest that at least one population of cholinergic sympathetic neurons in the rat requires NGF for survival. Our findings are consistent with the idea that during development NGF is a required trophic factor not only for noradrenergic sympathetic but also for cholinergic sympathetic neurons.

Neonatal 6-hydroxydopamine treatment eliminates cholinergic sympathetic innervation and induces sensory sprouting in rat sweat glands.

Previous studies of the development of cholinergic sympathetic innervation of sweat glands in rat footpads suggested that these terminals initially exhibit noradrenergic properties which are lost as the glands and their innervation mature. We have treated neonatal and adult rats with 6-hydroxydopamine (6-OHDA), a toxic congener of norepinephrine, and compared its effects on the cholinergic sympathetic innervation of sweat glands and the noradrenergic sympathetic innervation of the iris, salivary gland, and blood vessels. As reported by others, 6-OHDA treatment of neonates caused the destruction of noradrenergic fibers in the iris and salivary gland but did not affect other fibers projecting to these targets that stain for acetylcholinesterase (AChE). We found that 6-OHDA treatment of neonatal animals also caused the destruction of the sympathetic axons in immature sweat glands that possess catecholamine histofluorescence and tyrosine-hydroxylase-like immunoreactivity. Furthermore, when such animals were examined as adults, we found no AChE staining, vasoactive intestinal peptide (VIP)-like immunoreactivity, or characteristic sympathetic axonal varicosities. However, the denervated glands were invested by a plexus of sensory axons, some of which exhibited substance P-like immunoreactivity (SP-IR). An increase in the number of SP-IR fibers also occurred in the sympathetically denervated irides of these animals. Chronic treatment of neonates with guanethidine, another adrenergic sympathetic neurotoxin, resulted in similar loss of cholinergic sweat gland innervation. Treatment of adults rats with doses of 6-OHDA identical to those used to treat neonates caused the loss of noradrenergic fibers from the iris, salivary gland, and many blood vessels but did not noticeably affect AChE and VIP staining or axonal ultrastructure in the sweat glands. However, treatment with higher doses of 6-OHDA did cause significant axonal degeneration. The response of the sympathetic innervation of developing but not mature sweat glands to 6-OHDA provides evidence for a transition from noradrenergic to cholinergic phenotype during the development of sympathetic neurons in vivo similar to the transition observed in cell culture. The sprouting of sensory axons may be caused by NGF-like trophic influences present in some sympathetically denervated tissues.