Neuropilin-1 functions as a VEGFR2 co-receptor to guide developmental angiogenesis independent of ligand binding.

During development, tissue repair, and tumor growth, most blood vessel networks are generated through angiogenesis. Vascular endothelial growth factor (VEGF) is a key regulator of this process and currently both VEGF and its receptors, VEGFR1, VEGFR2, and Neuropilin1 (NRP1), are targeted in therapeutic strategies for vascular disease and cancer. NRP1 is essential for vascular morphogenesis, but how NRP1 functions to guide vascular development has not been completely elucidated. In this study, we generated a mouse line harboring a point mutation in the endogenous Nrp1 locus that selectively abolishes VEGF-NRP1 binding (Nrp1(VEGF-)). Nrp1(VEGF-) mutants survive to adulthood with normal vasculature revealing that NRP1 functions independent of VEGF-NRP1 binding during developmental angiogenesis. Moreover, we found that Nrp1-deficient vessels have reduced VEGFR2 surface expression in vivo demonstrating that NRP1 regulates its co-receptor, VEGFR2. Given the resources invested in NRP1-targeted anti-angiogenesis therapies, our results will be integral for developing strategies to re-build vasculature in disease.

Guidance from above: common cues direct distinct signaling outcomes in vascular and neural patterning.

The nervous and vascular systems are both exquisitely branched and complicated systems and their proper development requires careful guidance of nerves and vessels. The recent realization that common ligand-receptor pairs are used in guiding the patterning of both systems has prompted the question of whether similar signaling pathways are used in both systems. This review highlights recent progress in our understanding of the similarities and differences in the intracellular signaling mechanisms downstream of semaphorins, ephrins and vascular endothelial growth factor in neurons and endothelial cells during neural and vascular development. We present evidence that similar intracellular signaling principles underlying cytoskeletal regulation are used to control neural and vascular guidance, although the specific molecules used in neurons and endothelial cells are often different.

Visualizing glutamatergic cell bodies and synapses in Drosophila larval and adult CNS.

Glutamate is the major excitatory neurotransmitter in the vertebrate central nervous system (CNS) and at Drosophila neuromuscular junctions (NMJs). Although glutamate is also used as a transmitter in the Drosophila CNS, there has been no systematic description of the central glutamatergic signaling system in the fly. With the recent cloning of the Drosophila vesicular glutamate transporter (DVGLUT), it is now possible to mark many, if not all, central glutamatergic neurons and synapses. Here we present the pattern of glutamatergic synapses and cell bodies in the late larval CNS and in the adult fly brain by using an anti-DVGLUT antibody. We also introduce two new tools for studying the Drosophila glutamatergic system: a dvglut promoter fragment fused to Gal4 whose expression labels glutamatergic neurons and a green fluorescent protein (GFP)-tagged DVGLUT transgene that localizes to synapses. In the larval CNS, we find synaptic DVGLUT immunoreactivity prominent in all brain lobe neuropil compartments except for the mushroom body. Likewise in the adult CNS, glutamatergic synapses are abundant throughout all major brain structures except the mushroom body. We also find that the larval ventral nerve cord neuropil is rich in glutamatergic synapses, which are primarily located near the dorsal surface of the neuropil, segregated from the ventrally positioned cholinergic processes. This description of the glutamatergic system in Drosophila highlights the prevalence of glutamatergic neurons in the CNS and presents tools for future study and manipulation of glutamatergic transmission.

A single vesicular glutamate transporter is sufficient to fill a synaptic vesicle.

Quantal size is the postsynaptic response to the release of a single synaptic vesicle and is determined in part by the amount of transmitter within that vesicle. At glutamatergic synapses, the vesicular glutamate transporter (VGLUT) fills vesicles with glutamate. While elevated VGLUT expression increases quantal size, the minimum number of transporters required to fill a vesicle is unknown. In Drosophila DVGLUT mutants, reduced transporter levels lead to a dose-dependent reduction in the frequency of spontaneous quantal release with no change in quantal size. Quantal frequency is not limited by vesicle number or impaired exocytosis. This suggests that a single functional unit of transporter is both necessary and sufficient to fill a vesicle to completion and that vesicles without DVGLUT are empty. Consistent with the presence of empty vesicles, at dvglut mutant synapses synaptic vesicles are smaller, suggesting that vesicle filling and/or transporter level is an important determinant of vesicle size.

Increased expression of the Drosophila vesicular glutamate transporter leads to excess glutamate release and a compensatory decrease in quantal content.

Quantal size is a fundamental parameter controlling the strength of synaptic transmission. The transmitter content of synaptic vesicles is one mechanism that can affect the physiological response to the release of a single vesicle. At glutamatergic synapses, vesicular glutamate transporters (VGLUTs) are responsible for filling synaptic vesicles with glutamate. To investigate how VGLUT expression can regulate synaptic strength in vivo, we have identified the Drosophila vesicular glutamate transporter, which we name DVGLUT. DVGLUT mRNA is expressed in glutamatergic motoneurons and a large number of interneurons in the Drosophila CNS. DVGLUT protein resides on synaptic vesicles and localizes to the presynaptic terminals of all known glutamatergic neuromuscular junctions as well as to synapses throughout the CNS neuropil. Increasing the expression of DVGLUT in motoneurons leads to an increase in quantal size that is accompanied by an increase in synaptic vesicle volume. At synapses confronted with increased glutamate release from each vesicle, there is a compensatory decrease in the number of synaptic vesicles released that maintains normal levels of synaptic excitation. These results demonstrate that (1) expression of DVGLUT determines the size and glutamate content of synaptic vesicles and (2) homeostatic mechanisms exist to attenuate the excitatory effects of excess glutamate release.