Anil Bagri

Slit Proteins Prevent Midline Crossing and Determine the Dorsoventral Position of Major Axonal Pathways in the Mammalian Forebrain

We report that Slit proteins, a family of secreted chemorepellents, are crucial for the proper development of several major forebrain tracts. Mice deficient in Slit2 and, even more so, mice deficient in both Slit1 and Slit2 show significant axon guidance errors in a variety of pathways, including corticofugal, callosal, and thalamocortical tracts. Analysis of multiple pathways suggests several generalizations regarding the functions of Slit proteins in the brain, which appear to contribute to (1) the maintenance of dorsal position by prevention of axonal growth into ventral regions, (2) the prevention of axonal extension toward and across the midline, and (3) the channeling of axons toward particular regions.

Neuropilin-2 Regulates the Development of Select Cranial and Sensory Nerves and Hippocampal Mossy Fiber Projections

Neuropilin-1 and neuropilin-2 bind differentially to different class 3 semaphorins and are thought to provide the ligand-binding moieties in receptor complexes mediating repulsive responses to these semaphorins. Here, we have studied the function of neuropilin-2 through analysis of a neuropilin-2 mutant mouse, which is viable and fertile. Repulsive responses of sympathetic and hippocampal neurons to Sema3F but not to Sema3A are abolished in the mutant. Marked defects are observed in the development of several cranial nerves, in the initial central projections of spinal sensory axons, and in the anterior commissure, habenulo-interpeduncular tract, and the projections of hippocampal mossyfiber axons in the infrapyramidal bundle. Our results show that neuropilin-2 is an essential component of the Sema3F receptor and identify key roles for neuropilin-2 in axon guidance in the PNS and CNS.

Cell migration from the ganglionic eminences is required for the development of hippocampal GABAergic interneurons.

GABAergic interneurons have major roles in hippocampal function and dysfunction. Here we provide evidence that, in mice, virtually all of these cells originate from progenitors in the basal telencephalon. Immature interneurons tangentially migrate from the basal telencephalon through the neocortex to take up their final positions in the hippocampus. Disrupting differentiation in the embryonic basal telencephalon (lateral and medial ganglionic eminences) through loss of Dlx1/2 homeobox function blocks the migration of virtually all GABAergic interneurons to the hippocampus. On the other hand, disrupting specification of the medial ganglionic eminence through loss of Nkx2.1 homeobox function depletes the hippocampus of a distinct subset of hippocampal interneurons. Loss of hippocampal interneurons does not appear to have major effects on the early development of hippocampal projection neurons nor on the pathfinding of afferrent tracts.

Blocking Neuropilin-2 Function Inhibits Tumor Cell Metastasis

Metastasis, which commonly uses lymphatics, accounts for much of the mortality associated with cancer. The vascular endothelial growth factor (VEGF)-C coreceptor, neuropilin-2 (Nrp2), modulates but is not necessary for developmental lymphangiogenesis, and its significance for metastasis is unknown. An antibody to Nrp2 that blocks VEGFC binding disrupts VEGFC-induced lymphatic endothelial cell migration, but not proliferation, in part independently of VEGF receptor activation. It does not affect established lymphatics in normal adult mice but reduces tumoral lymphangiogenesis and, importantly, functional lymphatics associated with tumors. It also reduces metastasis to sentinel lymph nodes and distant organs, apparently by delaying the departure of tumor cells from the primary tumor. Our results demonstrate that Nrp2, which was originally identified as an axon-guidance receptor, is an attractive target for modulating metastasis.

Semaphorin–Neuropilin Interactions Underlying Sympathetic Axon Responses to Class III Semaphorins

Neuropilin-1 and neuropilin-2 show specificity in binding to different class III semaphorins, including Sema III, Sema E, and Sema IV, suggesting that the specificity of action of these semaphorins is dictated by the complement of neuropilins expressed by responsive neurons. In support of this, we show that sympathetic axons coexpress neuropilin-1 and -2, that their responses to Sema III, Sema E, and Sema IV are affected in predicted ways by antibodies to neuropilin-1, and that neuropilin-1 and -2 can form homo- and heterooligomers through an interaction involving at least partly the neuropilin MAM (meprin, A5, mu) domain. These results support the idea that in sympathetic axons, the Sema III signal is mediated predominantly by neuropilin-1 oligomers, the Sema IV signal by neuropilin-2 oligomers, and the Sema E signal by neuropilin-1 and -2, either as homo- or heterooligomers.

Stereotyped Pruning of Long Hippocampal Axon Branches Triggered by Retraction Inducers of the Semaphorin Family

Like naturally occurring neuronal cell death, stereotyped pruning of long axon branches to temporary targets is a widespread regressive phenomenon in the developing mammalian brain that helps sculpt the pattern of neuronal connections. The mechanisms controlling stereotyped pruning are, however, poorly understood. Here, we provide evidence that semaphorins, activating the Plexin-A3 receptor, function as retraction inducers to trigger-stereotyped pruning of specific hippocampal mossy fiber and pyramidal axon branches. Both pruning events are defective in Plexin-A3 mutants, reflecting a cell-autonomous requirement for Plexin-A3. The distribution of mRNAs for Sema3F and Sema3A makes them candidates for triggering the pruning. In vitro, hippocampal neurons respond to semaphorins by retracting axon branches. These results implicate semaphorins as retraction inducers controlling stereotyped pruning in the mammalian brain.

Plexin-A3 Mediates Semaphorin Signaling and Regulates the Development of Hippocampal Axonal Projections

Plexins are receptors implicated in mediating signaling by semaphorins, a family of axonal chemorepellents. The role of specific plexins in mediating semaphorin function in vivo has not, however, yet been examined in vertebrates. Here, we show that plexin-A3 is the most ubiquitously expressed plexin family member within regions of the developing mammalian nervous system known to contain semaphorin-responsive neurons. Using a chimeric receptor construct, we provide evidence that plexin-A3 can transduce a repulsive signal in growth cones in vitro. Analysis of plexin-A3 knockout mice shows that plexin-A3 contributes to Sema3F and Sema3A signaling and that plexin-A3 regulates the development of hippocampal axonal projections in vivo.

Neuropilin-2 mediates VEGF-C-induced lymphatic sprouting together with VEGFR3.

If neuropilin-2 and the growth factor VEGF-C don’t come together, lymphatic vessels don’t branch apart.

Neuropilin-1 Binds to VEGF121 and Regulates Endothelial Cell Migration and Sprouting

Neuropilin-1 (NRP1) was first described as a receptor for the axon guidance molecule, Semaphorin3A, regulating the development of the nervous system. It was later shown that NRP1 is an isoform-specific receptor for vascular endothelial growth factor (VEGF), specifically VEGF165. Much interest has been placed on the role of the various VEGF isoforms in vascular biology. Here we report that blocking NRP1 function, using a recently described antibody that inhibits VEGF165 binding to NRP1, surprisingly reduces VEGF121-induced migration and sprout formation of endothelial cells. Intrigued by this observation, direct binding studies of NRP1 to various VEGF isoforms were performed. We show that VEGF121 binds directly to NRP1; however, unlike VEGF165, VEGF121 is not sufficient to bridge the NRP1·VEGFR2 complex. Additionally, we show that VEGFR2 enhances VEGF165, but not VEGF121 binding to NRP1. We propose a new model for NRP1 interactions with various VEGF isoforms.

Structural studies of neuropilin/antibody complexes provide insights into semaphorin and VEGF binding

Neuropilins (Nrps) are co‐receptors for class 3 semaphorins and vascular endothelial growth factors and important for the development of the nervous system and the vasculature. The extracellular portion of Nrp is composed of two domains that are essential for semaphorin binding (a1a2), two domains necessary for VEGF binding (b1b2), and one domain critical for receptor dimerization (c). We report several crystal structures of Nrp1 and Nrp2 fragments alone and in complex with antibodies that selectively block either semaphorin or vascular endothelial growth factor (VEGF) binding. In these structures, Nrps adopt an unexpected domain arrangement in which the a2, b1, and b2 domains form a tightly packed core that is only loosely connected to the a1 domain. The locations of the antibody epitopes together with in vitro experiments indicate that VEGF and semaphorin do not directly compete for Nrp binding. Based upon our structural and functional data, we propose possible models for ligand binding to neuropilins.