Colocalization of neuropilin-1 and Flk-1 in retinal neovascularization in a mouse model of retinopathy.

PURPOSE: To investigate the mechanisms of the development of retinal neovascularization, the localizations of vascular endothelial (VEGF) receptors Flk-1 and neuropilin (NP)-1 mRNAs were examined. METHODS: The model of retinopathy of prematurity (ROP) was produced by ischemia-induced ocular neovascularization, by exposing postnatal day-7 mice to 75% oxygen for 5 days and then returning them to room air for 5 days. Retinal neovascularization was visualized by injection of fluorescein-dextran. Expression of Flk-1 and NP-1 mRNAs were examined by in situ hybridization with flatmount and serial sections of the retina. The localization of NP-1 was also confirmed by immunohistochemistry. Blood vessel patterns were characterized by immunohistochemical localization of von Willebrand factor (vWF). RESULTS: Flatmount in situ hybridization showed intense expression of NP-1 and Flk-1 mRNAs colocalized in the area of neovascularization. In situ hybridization of serial sections of the retina revealed that expression of Flk-1 and NP-1 was restricted to neovascularized vessels of the retina from ROP mice. CONCLUSIONS: The restricted expression of Flk-1 and NP-1 on neovascularized vessels suggests that these molecules may play important roles in retinal neovascularization. This is the first report of the colocalization of NP-1 and Flk-1 on neovascularized vessels of the retina from ROP mice.

Repulsive axon guidance molecule Sema3A inhibits branching morphogenesis of fetal mouse lung.

Semaphorin III/collapsin-1 (Sema3A) guides a specific subset of neuronal growth cones as a repulsive molecule. In this study, we have investigated a possible role of non-neuronal Sema3A in lung morphogenesis. Expression of mRNAs of Sema3A and neuropilin-1 (NP-1), a Sema3A receptor, was detected in fetal and adult lungs. Sema3A-immunoreactive cells were found in airway and alveolar epithelial cells of the fetal and adult lungs. Immunoreactivity for NP-1 was seen in fetal and adult alveolar epithelial cells as well as endothelial cells. Immunoreactivity of collapsin response mediator protein CRMP (CRMP-2), an intracellular protein mediating Sema3A signaling, was localized in alveolar epithelial cells, nerve tissue and airway neuroendocrine cells. The expression of CRMP-2 increased during the fetal, neonate and adult periods, and this pattern paralleled that of NP-1. In a two-day culture of lung explants from fetal mouse lung (E11.5), with exogenous Sema3A at a dose comparable to that which induces growth cone collapse of dorsal root ganglia neurons, the number of terminal buds was reduced in a dose-dependent manner when compared with control or untreated lung explants. This decrease was not accompanied with any alteration of the bromodeoxyuridine-positive DNA-synthesizing fraction. A soluble NP-1 lacking the transmembrane and intracellular region, neutralized the inhibitory effect of Sema3A. The fetal lung explants from neuropilin-1 homozygous null mice grew normally in vitro regardless of Sema3A treatment. These results provide evidence that Sema3A inhibits branching morphogenesis in lung bud organ cultures via NP-1 as a receptor or a component of a possible multimeric Sema3A receptor complex.

A requirement for neuropilin-1 in embryonic vessel formation.

Neuropilin-1 is a membrane protein that is expressed in developing neurons and functions as a receptor or a component of the receptor complex for the class 3 semaphorins, which are inhibitory axon guidance signals. Targeted inactivation of the neuropilin-1 gene in mice induced disorganization of the pathway and projection of nerve fibers, suggesting that neuropilin-1 mediates semaphorin-elicited signals and regulates nerve fiber guidance in embryogenesis. Neuropilin-1 is also expressed in endothelial cells and shown to bind vascular endothelial growth factor (VEGF), a potent regulator for vasculogenesis and angiogenesis. However, the roles of neuropilin-1 in vascular formation have been unclear. This paper reported that the neuropilin-1 mutant mouse embryos exhibited various types of vascular defects, including impairment in neural vascularization, agenesis and transposition of great vessels, insufficient aorticopulmonary truncus (persistent truncus arteriosus), and disorganized and insufficient development of vascular networks in the yolk sac. The vascular defects induced by neuropilin-1 deficiency in mouse embryos suggest that neuropilin-1 plays roles in embryonic vessel formation, as well as nerve fiber guidance.

Receptors for collapsin/semaphorins.

Chemorepulsive signals that repel or paralyze neuronal growth cones have been found to play important roles in axon guidance in a stereotyped manner. Recent progress in the identification of neuropilins as the receptors for class III secreted collapsin/semaphorin subfamily members, which are neuronal repellents, and in the analysis of mutant mice lacking neuropilin function has confirmed the importance of these chemorepellents in axon guidance. In addition, characterization of the neuropilin protein has yielded new insights into the functions of this molecule in vascular formation and in axon guidance.

Neuropilin-semaphorin III/D-mediated chemorepulsive signals play a crucial role in peripheral nerve projection in mice.

Neuropilin is a neuronal cell surface protein and has been shown to function as a receptor for a secreted protein, semaphorin III/D, that can induce neuronal growth cone collapse and repulsion of neurites in vitro. The roles of neuropilin in vivo, however, are unknown. Here, we report that neuropilin-deficient mutant mice produced by targeted disruption of the neuropilin gene show severe abnormalities in the trajectory of efferent fibers of the PNS. We also describe that neuropilin-deprived dorsal root ganglion neurons are perfectly protected from growth cone collapse elicited by semaphorin III/D. Our results indicate that neuropilin-semaphorin III/D-mediated chemorepulsive signals play a major role in guidance of PNS efferents.

Roles of a neuronal cell-surface molecule, neuropilin, in nerve fiber fasciculation and guidance.

Neuropilin is a cell-surface glycoprotein that was first identified in Xenopus tadpole nervous tissues and then in chicken and mouse. The primary structure of neuropilin is highly conserved among these vertebrate species. The extracellular part of the molecule is composed of three domains referred to as a1/a2, b1/b2, and c, each of which is expected to be involved in molecular and/or cellular interactions. Neuropilin can mediate cell adhesion by heterophilic molecular interaction. In all vertebrate species examined, the neuropilin protein is restricted to axons of particular neuron classes, and at stages when axon growth is active. The gain and loss of function of neuropilin in developing mouse embryos causes defasciculation and incorrect sprouting of nerve fibers. These findings suggest that neuropilin serves in a variety of neuronal cell interactions by binding to a variety of molecules, and that it plays essential roles in nerve fiber fasciculation and guidance.

Positional cues that are strictly localized in the telencephalon induce preferential growth of mitral cell axons.

In mice, mitral cells are the major efferent neurons of the main olfactory bulb and elongate axons into a very narrow part of the telencephalon to form a fiber bundle referred to as the lateral olfactory tract (LOT). To clarify the mechanisms responsible for guidance of mitral cell axons along this particular pathway, we co-cultured mouse embryo main olfactory bulbs with the telencephalons, and analyzed the pathways taken by mitral cell axons. Ingrowth of mitral cell axons into the telencephalon was observed in those co-cultures in which the olfactory bulbs had been exactly combined to their normal pathway (the LOT position) of the telencephalon. The axons grew preferentially along the LOT position, and formed a LOT-like fiber bundle. When the olfactory bulbs were grafted at positions apart from their normal pathway, however, no mitral cell axons grew into the telencephalon. Neocortical fragments combined with the telencephalon projected fibers into the telencephalon in random directions. These results suggest that the LOT position of the telencephalon offers a guiding pathway for mitral cell axons and that guiding cues for mitral cell axons are extremely localized.

Developmentally regulated expression of a cell surface protein, neuropilin, in the mouse nervous system.

Neuropilin (previously A5) is a cell surface glycoprotein that was originally identified in Xenopus tadpole nervous tissues. In Xenopus, neuropilin is expressed on both the presynaptic and postsynaptic elements in the visual and general somatic sensory systems, suggesting a role in neuronal cell recognition. In this study, we identified a mouse homologue of neuropilin and examined its expression in developing mouse nervous tissues. cDNA cloning and sequencing revealed that the primary structure of the mouse neuropilin was highly similar to that of Xenopus and that the extracellular segment of the molecule possessed several motifs that were expected to be involved in cell-cell interaction. Immunohistochemistry and in situ hybridization analyses in mice indicated that the expression of neuropilin was restricted to particular neuron circuits. Neuropilin protein was localized on axons but not on the somata of neurons. The expression of neuropilin persisted through the time when axons were actively growing to form neuronal connections. These observations suggest that neuropilin is involved in growth, fasciculation, and targeting for a particular groups of axons.

Overexpression of a membrane protein, neuropilin, in chimeric mice causes anomalies in the cardiovascular system, nervous system and limbs.

Neuropilin is a type 1 membrane protein, which is highly conserved among Xenopus frog, chicken and mouse. The extracellular part of the neuropilin protein is composed of three unique domains, each of which is thought to be involved in molecular and/or cellular interactions. In mice, neuropilin is expressed in the cardiovascular system, nervous system and limbs at particular developmental stages. To clarify the roles of neuropilin in morphogenesis in vivo, we generated mouse embryonic stem (ES) cell clones that constitutively expressed exogenous neuropilin, then produced chimeras using these ES cell clones. The chimeras overexpressed neuropilin and were embryonic lethal. The chimeric embryos exhibited several morphological abnormalities; excess capillaries and blood vessels, dilation of blood vessels, malformed hearts, ectopic sprouting and defasciculation of nerve fibers, and extra digits. All of these abnormalities occurred in the organs in which neuropilin is expressed in normal development. The variety of abnormalities occurring in these chimeric embryos suggested diverse functions of neuropilin in embryonic morphogenesis, which may be ascribed to multiple interaction domains identified in the molecule. Correct spatiotemporal expression of neuropilin seems to be essential for normal development of the cardiovascular system, nervous system and limbs.