A–E, Endothelial-specific expression of plxnD1 mRNA (A, purple; B–C, blue) and PlxnD1 protein (D–E). A, E9.5 mouse embryo (left lateral view). The head is up. Anterior dorsal side, left. plxnD1 is detected in the entire vasculature. Segmental vessel, yellow arrow. B, C, 28 hpf zebrafish embryo. Left lateral views. Anterior, left. Dorsal is up. B, plxnD1 is detected in the entire vasculature. C, Trunk detail. Segmental Arteries (SeAs; black arrowhead). Dorsal Aorta (DA; red arrowhead). Posterior Cardinal Vein (PCV; yellow arrowhead). Floorplate's hypochord, black asterisk. D, E11.5 murine heart cross-section. PlxnD1 (brown). E, Murine dorsal root ganglia (DRG; blue, delimited by yellow dotted line). Vessel with PlxnD1 (green) expression, yellow arrow. F, Murine immune system (paraffin section of the thymus). Cortical thymocytes expressing PlxnD1, dark blue. G–J, Expression of plxnD1/PlxnD1 in the murine nervous system. G, P8 cortex section. Only some cortical commissural neurons (callosal projection neurons; CPN, red) express plxnD1 (green) (rectangle). H, E14.5 murine section of the brain. Forebrain, fb. Trigeminal ganglion, gv. Choroid plexus, cp. plxnD1, red. I–J, Diagrams of coronal sections of E17.5 murine forebrains showing sites were Sema3E (blue) and PlxnD1 (red) are expressed and locales where PlxnD1 and Nrp1 are co-expressed (light orange). Rostral, left. Caudal, right. Dorsal is up. I, PlxnD1 is expressed along the corticofugal and striatonigral projections that originate from the ventrolateral cortex (Ctx) and striatum (St), respectively and which transverse both the internal capsule (ic) and the cerebral peduncle (cp). At the level of the midbrain the striatonigral projections leave the cp and terminate in the substantia nigra (SN), while corticofugal axons continue into the pons (not shown). Robust Sema3E expression is observed in the globus pallidus (GP) and thalamic reticular nucleus (TRN). J, PlxnD1 and Nrp1 are co-expressed in the subiculo-mammillary tract, which projects from the subiculum (Sub) through the fimbria (fim), fornix (f) and postcommissural fornix (pf). Sema3E expression is observed in the hippocampal pyramidal cell layers of the cornus ammonis 1 and 3 (CA1/3) adjacent to the Sub. The anterior commissure (ac), hippocampal commissure (hipc) and septum (Sp) are indicated. K, E18.5 murine vertebral bodies, lateral cross-section. Ossification center, oc. plxnD1, red. Credits for reproduced and/or modified images: A, D and E from (Gitler et al., 2004); B and C from (Torres-Vázquez et al., 2004); F from (Choi et al., 2008); G from (Molyneaux et al., 2009a); H and K from (Zhang et al., 2008); I-J from (Chauvet et al., 2007).

Sema3/PlxnD1 signaling is capable of promoting attraction and repulsion in both the vascular and nervous systems. A, In adenocarcinoma cells the full-length Sema3E (p87-Sema3E) is cleaved by a furin-like endoprotease to yield two products. The longer one contains the Sema domain (p61-Sema3E) while the shorter one contains the dimerization domain (p25-Sema3E). B, As a result, adenocarcinoma cells secrete three combinations of Sema3E dimers (1–3) and one monomer (4) (Christensen et al., 2005). C, In the vascular system (left) full-length Sema3E dimers induce repulsion via PlxnD1. However, the p61-Sema3E monomer containing the Sema domain can function as an attractant in some endothelial lines expressing PlxnD1 (right). However, the requirement for PlxnD1 to mediate this attractive response has not been tested (Christensen et al., 2005). D, In the nervous system, Sema3E acts as a repellant when signaling through PlxnD1 alone (left). However, in neurons expressing also Nrp1 and the VEGF receptor VEGFR2, binding of Sema3E elicits an attractive/growth-promoting axonal response (right). Remarkably, this attractive response requires only the extracellular domains of PlxnD1 and Nrp1 (as shown), with PlxnD1 functioning as the ligand-binding subunit of the complex. In contrast, VEGFR2’s intracellular tail is required in this context as the signal transducing subunit (Bellon et al., 2010). Note however, that the precise Sema3E oligomerization form that mediates these responses is unknown.

Sequence conservation of PlxnD1’s intracellular domains and motifs across vertebrates. A, B, The two RasGAP motifs are one-hundred-percent conserved amongst the species shown, including the arginine residues (outlined in red) essential to functionally antagonize R-Ras family members via GAP activity and/or sequestration. C, The Rho-GTPase binding domains are highly conserved and show a high degree of similarity to the seven amino acid sequence of Drosophila PlxnB (outlined in red) that is required for binding to active, GTP-bound Rac (Hu et al., 2001). D, The PlxnD1-specific T-segment is highly conserved. It includes an invariable PDZ-binding motif SEA-COOH (outlined in red) that mediates the physical interaction between PlxnD1 and the PDZ-containing protein GIPC1. The accession numbers of the PlxnD1 proteins used for sequence analysis are: human/Homo sapiens (AAI50281), chimpanzee/Pan troglodytes (XP_001144444), mouse/Mus musculus (NP_080652), rat/Rattus norvegicus (NP_001101351) and zebrafish/Danio rerio (AAT64905).

A–B, E11.5 murine trunks. Lateral views. Dorsal is up. Endothelium, dark brown. A, Phenotypically wild type sema3E^+/− heterozygote. Segmental vessel, white arrow. Note loss of avascular areas. B, sema3E^−/− homozygous mutant displaying disorganized Segmental vessels. Notably, plxnD1 ^−/− homozygous mutants display identical vascular patterning defects (not shown). C, D, 48 hpf zebrafish embryos. Lateral views. Anterior, left. Dorsal is up. Endothelium, green. Segmental Arteries (SeAs), white arrowheads. Dorsal Aorta (DA), red arrowheads. Posterior Cardinal Vein (PCV), yellow arrowheads. C, Wild type. D, Animal lacking plxnD1 activity. Note aberrant vascular pattern, resembling that of sema3E^−/− homozygous mutant mice (B). E, Model for the evolutionarily conserved role of Sema-PlxnD1 signaling in shaping the Segmental vasculature. Paracrine Sema signals from the somites (pink) are sensed by PlxnD1 receptors expressed in the endothelium (blue) to guide the patterning of nascent Se vessels via a repulsive mechanism. F, Without Sema-PlxnD1 signaling Se vesels grow aberrantly. G–H, plxnD1 activity is required for proper formation of the murine heart’s outflow tract. Images of P0-stage hearts. G, Wild type heart showing two major vessels, the aorta (Ao) and the pulmonary artery (PA). H, plxnD1 mutant displaying persistent truncus arteriosus (TA; a single great vessel), a form of congenital heart disease. In addition, the coronary artery (white arrow) displays an abnormal origin. I–J, P0 murine lumbar skeletons (dorsal views) Anterior, left. Mineralized bone, red. Cartilage, blue. I, Wild type. J, plxnD1^−/− mutant pup showing axial skeletal defects and malformations such as splitting (green arrow) and fusion (green circle) of the vertebral bodies. K, Connectivity patterns between sensory and motor neurons in the triceps (Tri, dark brown) and cutaneous maximus (Cm, light green) reflex arcs in the mouse. Muscles, ovals (Tri; light brown, Cm; dark green). Dotted lines surround synapses between sensory afferents and motor neurons in these reflex arcs. L, Diagram summarizing the role of Sema3E-PlxnD1 repulsive signaling in establishing the fine synaptic specificity between sensory afferents (top) and motor neurons (bottom) in the Cm (green) and Tri (brown) arcs. Left half, wild type wiring pattern. Normally, plxnD1 is expressed in both Cm and Tri sensory neurons and sema3E is expressed only in Cm motor neurons. Therefore only the Tri motor neurons receive direct proprioceptive inputs from Tri sensory neurons (homonymous connectivity). The right half of the figure shows the rewiring of homonymous connectivity resulting from perturbing Sema3E–PlxnD1 signaling. Absence of either plxnD1 activity or sema3E expression induces abnormal homonymous monosynaptic Cm connectivity (left). In contrast, ectopic sema3E expression in the Tri motorneurons (right) prevents the establishment of monosynaptic connections in the Tri reflex arc. Notably, perturbing Sema3E–PlxnD1 signaling does not induce heteronymous sensory–motor connections (Cm to Tri or vice versa). This indicates that additional factors regulate motor pool specificity. Credits for reproduced and/or modified images: A–B from (Gu et al., 2005); C–F from (Torres-Vázquez et al., 2004); G–H from (Gitler et al., 2004); I–J from (Zhang et al., 2008); K adapted from (Maro et al., 2009) L adapted from (Pecho-Vrieseling et al., 2009).

Structure of the Sema receptor PlxnD1. A, PlxnD1 is a type I transmembrane protein. The extracellular N-terminal portion contains a Sema domain (blue), likely involved in mediating ligand binding. Following the Sema domain there are three MRS (MET-Related Sequence) repeats (green), four IPT (Immunoglobin-like fold shared by Plexins and Transcription factors) domains (orange) and the transmembrane domain (brown). The cytosolic tail of PlxnD1 is also known as the Sex and Plexins (SP) domain. It contains a split GAP (GTPase Activating Protein) domain with two highly conserved C regions (C1 and C2; red). Each C region contains a Ras GAP motif (RasGAP1 and RasGAP2; black), each of which includes conserved arginine residues required to inhibit the activity of R-Ras proteins. A Rho GTPase-binding domain (RBD, beige) is located between the C1 and C2 regions. Finally, the GAP domain is followed by a short C-terminal region that lacks any resemblance to known protein domains and which is highly conserved between members of the same Plxn subfamily. Here we designate this region as the terminal (T) segment (pink). The T segment of PlxnD1 ends in a short PDZ-binding motif (D1-PBM; aqua) that physically associates with GIPC1. B, Activation model of PlxnD1. In the absence of its Sema ligands, PlxnD1 is in a conformationally inactive folded state, in which the Sema domain contacts the rest of the extracellular portion and the GAP domain is non-functional. Upon Sema binding PlexinD1 undergoes conformational changes that activate its GAP domain and likely enable additional protein-protein interactions.

A general model of Sema3/PlxnD1-mediated repulsion. A, In the absence of ligand-mediated activation PlxnD1 is in a conformation that enables its association with GTP-bound Rnd2 but prevents its interaction with active, GTP-bound Rac and R-Ras. Thus, GTP-bound Rac is able to bind to PAK (p21-activated kinase) to stimulate the assembly of actin filaments to support cell migration while active GTP-bound Ras promotes integrin-mediated adhesion to the extracellular matrix (ECM) and mediates additional downstream signaling events. B, Upon binding of its Sema3 ligand, PlxnD1 undergoes a conformational change and binds the active forms of both Rac and R-Ras GTPases. By sequestering Rac, PlxnD1 leads to the inactivation of PAK and the collapse of the actin-based cytoskeleton leading to retraction and/or turning responses. PlxnD1 inactivates R-Ras GTPases by either enhancing GTP hydrolysis (as shown) or by sequestering them resulting in the loss of integrin-based adhesion to the ECM (Ito et al., 2006; Oinuma et al., 2004a; Oinuma et al., 2006; Rohm et al., 2000; Sakurai et al., 2010; Uesugi et al., 2008) and likely reducing as well other R-Ras mediated signaling events.