The development of a patterned vasculature is essential for normal organogenesis. We found that signaling by semaphorin 3E (Sema3E) and its receptor plexin-D1 controls endothelial cell positioning and the patterning of the developing vasculature in the mouse. Sema3E is highly expressed in developing somites, where it acts as a repulsive cue for plexin-D1-expressing endothelial cells of adjacent intersomitic vessels. Sema3E-plexin-D1 signaling did not require neuropilins, which were previously presumed to be obligate Sema3 coreceptors. Moreover, genetic ablation of Sema3E or plexin-D1 but not neuropilin-mediated Sema3 signaling disrupted vascular patterning. These findings reveal an unexpected semaphorin signaling pathway and define a mechanism for controlling vascular patterning.

In animals with binocular vision, retinal ganglion cell (RGC) axons either cross or avoid the midline at the optic chiasm. Here, we show that ephrin-Bs in the chiasm region direct the divergence of retinal axons through the selective repulsion of a subset of RGCs that express EphB1. Ephrin-B2 is expressed at the mouse chiasm midline as the ipsilateral projection is generated and is selectively inhibitory to axons from ventrotemporal (VT) retina, where ipsilaterally projecting RGCs reside. Moreover, blocking ephrin-B2 function in vitro rescues the inhibitory effect of chiasm cells and eliminates the ipsilateral projection in the semiintact mouse visual system. A receptor for ephrin-B2, EphB1, is found exclusively in regions of retina that give rise to the ipsilateral projection. EphB1 null mice exhibit a dramatically reduced ipsilateral projection, suggesting that this receptor contributes to the formation of the ipsilateral retinal projection, most likely through its repulsive interaction with ephrin-B2.

The establishment of functional neural circuits requires the guidance of axons in response to the actions of secreted and cell-surface molecules such as the semaphorins. Semaphorin 3E and its receptor PlexinD1 are expressed in the brain, but their functions are unknown. Here, we show that Sema3E/PlexinD1 signaling plays an important role in initial development of descending axon tracts in the forebrain. Early errors in axonal projections are reflected in behavioral deficits in Sema3E null mutant mice. Two distinct signaling mechanisms can be distinguished downstream of Sema3E. On corticofugal and striatonigral neurons expressing PlexinD1 but not Neuropilin-1, Sema3E acts as a repellent. In contrast, on subiculo-mammillary neurons coexpressing PlexinD1 and Neuropilin-1, Sema3E acts as an attractant. The extracellular domain of Neuropilin-1 is sufficient to convert repulsive signaling by PlexinD1 to attraction. Our data therefore reveal a "gating" function of neuropilins in semaphorin-plexin signaling during the assembly of forebrain neuronal circuits.

During development, the retinal vasculature grows toward hypoxic areas in an organized fashion. By contrast, in ischemic retinopathies, new blood vessels grow out of the retinal surfaces without ameliorating retinal hypoxia. Restoration of proper angiogenic directionality would be of great benefit to reoxygenize the ischemic retina and resolve disease pathogenesis. Here, we show that binding of the semaphorin 3E (Sema3E) ligand to the transmembrane PlexinD1 receptor initiates a signaling pathway that normalizes angiogenic directionality in both developing retinas and ischemic retinopathy. In developing mouse retinas, inhibition of VEGF signaling resulted in downregulation of endothelial PlexinD1 expression, suggesting that astrocyte-derived VEGF normally promotes PlexinD1 expression in growing blood vessels. Neuronderived Sema3E signaled to PlexinD1 and activated the small GTPase RhoJ in ECs, thereby counteracting VEGF-induced filopodia projections and defining the retinal vascular pathfinding. In a mouse model of ischemic retinopathy, enhanced expression of PlexinD1 and RhoJ in extraretinal vessels prevented VEGFinduced disoriented projections of the endothelial filopodia. Remarkably, intravitreal administration of Sema3E protein selectively suppressed extraretinal vascular outgrowth without affecting the desired regeneration of the retinal vasculature. Our study suggests a new paradigm for vascular regeneration therapy that guides angiogenesis precisely toward the ischemic retina.

Neurons, glia, and callosal axons operate as a “ménage à trois” in the development of the corpus callosum.

Ephrin-B and EphB are distributed in matching dorsoventral gradients in the embryonic Xenopus visual system with retinal axons bearing high levels of ligand (dorsal) projecting to tectal regions with high receptor expression (ventral). In vitro stripe assays show that dorsal retinal axons prefer to grow on EphB receptor stripes supporting an attractive guidance mechanism. In vivo disruption of EphB/ephrin-B function by application of exogenous EphB or expression of dominant-negative ephrin-B ligand in dorsal retinal axons causes these axons to shift dorsally in the tectum, while misexpression of wild-type ephrin-B in ventral axons causes them to shift ventrally. These dorsoventral targeting errors are consistent with the hypothesis that an attractive mechanism that requires ephrin-B cytoplasmic domain is critical for retinotectal mapping in this axis.

Summary Precise intrathymic cell migration is important for thymocyte maturation and architecture. The orchestration of thymocyte trafficking, however, is not well understood at a molecular level. Here, we describe highly-regulated plexinD1 expression on CD4+CD8+ double positive (DP) thymocytes which is further affected by T cell receptor/co-receptor engagement. Activation of plexinD1 via one of its ligands, semaphorin3E, represses CCR9/CCL25 signaling in CD69+ thymocytes. In the absence of plexinD1, CD69+ thymocytes remain in the cortex, maturing to form ectopic SP thymocyte clusters in Plxnd1-deficient fetal liver cell-transplanted mice. As a consequence, the boundary between DP and SP thymocytes at corticomedullary junctions is disrupted and medullary structures form under the thymic capsule. These results demonstrate the importance of plexinD1 in directing migration of maturing thymocytes via modulation of biological responses to chemokine gradients.

Common factors are thought to control vascular and neuronal patterning. Here we report an in vivo requirement for the vascular endothelial growth factor receptor type 2 (VEGFR2) in axon tract formation in the mouse brain. We show that VEGFR2 is expressed by neurons of the subiculum and mediates axonal elongation in response to the semaphorin (Sema) family molecule, Sema3E. We further show that VEGFR2 associates with the PlexinD1/Neuropilin-1 (Nrp1) receptor complex for Sema3E and becomes tyrosine-phosphorylated upon Sema3E stimulation. In subicular neurons, Sema3E triggers VEGFR2-dependent activation of the phosphatidylinositol-3 kinase (PI3K)/Akt pathway that is required for the increase in axonal growth. These results implicate VEGFR2 in axonal wiring through a mechanism dependent on Sema3E and independent of vascular endothelial growth factor (VEGF) ligands. This mechanism provides an explanation as to how a semaphorin can activate an axon growth promoting response in developing neurons.