Ectopic NCC migration perturbs DRG segmentation. (A and B) Lateral wholemount view and transverse sections (C and D) of 11.5 dpc wild-types and Nrp1-null mutants labeled with the sensory marker Isl1. (E and F) Dorsal wholemount view of 11.5 dpc wild-types and Nrp1-null mutants labeled with the sensory marker Brn3a; higher magnifications of the boxed areas in E and F are shown in G and H. Black and white arrows indicate ectopic neuron clusters in dorsolateral positions and between neighboring DRGs, respectively; note that the merging of neighboring DRGs is accompanied by abnormal curving of the neural tube in F. (Scale bars, 100 μm.)

Relationship of ectopic NCCs to blood vessels. Transverse (A–D) and longitudinal sections (E–G) stained for p75 (green) and endomucin (red) illustrate the relationship of NCCs to blood vessels at 9.5 dpc. (A and B) At the level of the intersomitic furrow, NCCs in wild-types accumulated in the MSA (bracket in A), whereas NCCs in Sema3a-null mutants formed a ventromedial (double arrowhead) and dorsolateral (arrow) stream that followed the course of intersomitic blood vessels (C and D). (E) Longitudinal sections demonstrated that most NCCs normally migrated into the anterior sclerotome (white arrowhead), but not into the posterior sclerotome (indicated with a bracket). (F and G) In Sema3a- and Nrp1^Sema-null mutants, only few NCCs entered the anterior sclerotome (white arrowhead), while most NCCs (white arrows) migrated alongside intersomitic and perisomitic vessels (clear arrows). In addition, ectopic NCCs were occasionally seen in the posterior sclerotome of mutants (clear arrowheads). Abbreviations: da, dorsal aorta; isv, intersomitic vessel; nt, neural tube; sa, sympathetic anlage. (Scale bar, 100 μm.)

Nrp1 and Sema3a expression during trunk NCC migration. Whole-mount in situ hybridization (ISH) for Sox10 (A and B), Nrp1 (C and D), and Sema3a (E and F) of 9.25 dpc (20–21 somite stage) mouse embryos. (A and C) Trunk NCCs express Sox10 and Nrp1 and migrate into the anterior sclerotome (black arrowheads), while the posterior sclerotome remains NCC-free (brackets); the arrows indicate the region where the sympathetic chain begins to assemble. (E) Sema3a was expressed in pairs of vertical stripes, 1 in the anterior (empty arrow) and 1 in the posterior somite half (empty arrowhead). (B, D, and F) Transverse sections through the wholemount samples shown in A, C, and E at the level of the anterior somite suggested that Sox10 (B) and Nrp1 (D) were co-expressed in NCC in the migration staging area (MSA), within the sclerotome (black arrowheads) and in NCC clusters (black arrows) adjacent to the dorsal aorta (da). Nrp1 expression in Sox10-negative mesenchymal cells (wavy arrow; compare B with D). (F) Sema3a expression in the forelimb (fl) and the dermomyotome (dm). (Scale bars: A, C, and E, 250 μm; B, D, and F, 100 μm.)

SEMA3A/NRP1 signaling promotes NCC migration into the sclerotome. (A–F) In situ hybridization (ISH) for Sox10 at 9.5 dpc (22–25 somite stage); (D–F) are higher magnifications of the boxed areas in (A–C). NCCs preferentially migrated into the anterior sclerotome in wild-types (A), but into the intersomitic furrows in full Nrp1-null (B) or NCC-specific Nrp1-null mutants (C); as an example, 1 NCC stream in the anterior sclerotome is indicated with an arrowhead and 1 of the intersomitic streams with an arrow in (D–F). Whereas wild-type NCCs avoided the posterior sclerotome (indicated with brackets in D), mutant NCCs occasionally invaded the posterior sclerotome (clear arrowheads in E and F). (G–J) Transverse sections through the intersomitic furrow of a wild-type (G), NCC-specific Nrp1-null mutant (H), and mutants deficient in semaphorin signaling through NRP1 (I) or in SEMA3A (J); half of each section is shown. At the level of the intersomitic furrow, wild-type NCCs were only seen in the migration staging area (bracket) adjacent to the dorsal neural tube and in the sympathetic anlagen (sa); in contrast, many mutant NCCs migrated into the intersomitic furrow, where they segregated into a ventromedial (double arrowheads) and dorsolateral (arrow) stream. Consequently, NCCs were seen scattered over a wide area near the dorsal aorta. Abbreviations: da, dorsal aorta; nt, neural tube; sa, sympathetic anlage. (Scale bars, 250 μm.)

Working model: SEMA3A and NRP1 control trunk NCC migration to organize PNS neurons. (A and B) NCC migration pathways at 9.5 dpc. (A) In wild-types, only a few intermediate wave NCCs are NRP1-negative (green) and travel alongside intersomitic and perisomitic vessels (red). Rather, most NCCs are NRP1-positive (yellow) and are channeled into the anterior sclerotome by repulsive SEMA3A signals. Accordingly, Sema3A (blue) is expressed in 2 domains, a narrow stripe in the dermomyotome adjacent to the preceding intersomitic furrow, and a broader domain in the posterior dermomyotome that extends into the posterior sclerotome. (B) In the absence of NRP1 signaling, intermediate wave NCCs are blind to SEMA3A (now shown in gray) and preferentially migrate alongside intersomitic blood vessels (red), similar to early wave NCCs. (C and D) Peripheral neuron position in wild-types and Nrp1-null mutants at 11.5 dpc reflects the migratory patterns of their NCC precursors. (C) In wild-types, sensory neurons condense into segmentally arranged dorsal root ganglia in the anterior sclerotome of the somites, while sympathetic neurons form paired, but nonsegmented primary sympathetic cords next to the dorsal aorta. (D) In the absence of SEMA3A/NRP1 signaling, sensory and sympathetic neurons differentiate in ectopic positions corresponding to the earlier position of their NCC precursors. Consequently, both the segmentation of the sensory system and the assembly of the sympathetic cords are disrupted. Abbreviations: da, dorsal aorta; dm, dermomyotome; isv, intersomitic vessel; psv, perisomitic vessel; pcv, posterior cardinal vein; scl, sclerotome.

Ectopic NCC migration impacts on PNS organization. (A and B) Wholemount in situ hybridization of 9.75 dpc wild-type and Nrp1-null embryos for Sox10 revealed ectopic NCC near the dorsal neural tube and in the intersomitic furrows (arrows). (C–E) Transverse sections of 10.5 dpc wild-type and Nrp1-null embryos labeled for Sox10 (red) and endomucin (green); half of each section is shown. (C) In wild-types, primary sympathetic ganglia (psg) formed between the dorsal aorta (da) and the posterior cardinal veins (pcv), while dorsal root ganglia (drg) form adjacent to the neural tube (nt) and extended 1 spinal nerve each (spn). (D and E) In mutants, dorsal root ganglia and primary sympathetic ganglia formed, but they were smaller than normal; at some axial levels, the sympathetic ganglia were missing (square bracket in E). The insets in D and E are higher magnifications of the squared areas and illustrate the proximity of ectopic cell clusters to superficial blood vessels. (F and G) Transverse sections of 10.5 dpc wild-type and Nrp1-null embryos labeled for neuronal microtubules (Tuj1; blue), endomucin (green), and the sympathetic marker tyrosine hydroxylase (TH; red). The inset in G is a higher magnification of the squared area and illustrates the presence of ectopic neuronal microtubule-positive, TH-negative neurons adjacent to the dorsal root ganglia. In mutants, TH-positive neuronal progenitors were scattered distally to the dorsal aorta. Ventral regions marked with squares in F and G are shown as separate color channels at higher magnification in H and I and J and K, respectively. (Scale bars, 100 μm.)

Nrp1 and Sema3a expression patterns during trunk NCC migration. (A–E) Longitudinal sections through a 9.5 dpc (25-somite) mouse embryo; only one-half of the section is shown; the neural tube is at the top of each panel; somites are counted backwards from the most recent pair formed. (A–C) Double labeling for the NCC marker p75 (green) and NRP1 (red). Early wave NCCs appeared to migrate alongside intersomitic blood vessels (isv) and expressed p75, but not NRP1 (arrow in A). In addition, some early NCCs migrated along the boundary between anterior and posterior sclerotome (curved arrow). (B) NCCs in the nonsegmented migration staging area (MSA) in the dorsal rostral trunk co-expressed p75 and NRP1 (yellow). (C) A section taken at the same rostral level, but further ventrally through the somites, showed that NCCs in the anterior sclerotome (arrowheads) co-expressed p75 and NRP1 (yellow). (A and C) Mesenchymal cells (wavy arrows) and neural progenitors in the neural tube also expressed NRP1, but at lower levels. (D and E) Triple labeling for Sema3a (blue), p75 (NCCs, green), and endomucin (blood vessels, red). (D) Sema3a was expressed in the posterior sclerotome and the dermomyotome, with highest levels in the anterior and posterior part of the dermomyotome, adjacent to the neighboring intersomitic furrows (clear arrow and arrowhead, respectively). The dimensions of the dermomyotome versus sclerotome and the anterior (a) versus posterior (p) sclerotome are indicated with brackets. (E) In wild-types, most intermediate wave NCCs migrated through the avascular anterior sclerotome (arrowhead), and only a few NCCs migrated alongside intersomitic vessels (arrow) or around the dermomyotome (open arrowhead). (F) Schematic representation of NRP1 and SEMA3A expression during trunk NCC migration in the 9.5 dpc mouse embryo. The firstborn trunk NCCs do not express NRP1 (green) and migrate through the intersomitic furrow or along the anteroposterior boundary within the sclerotome. Most NCCs in the intermediate wave are NRP1-positive (yellow) and migrate through the anterior sclerotome; they settle in the sclerotome or travel ventrally to meet NCCs from the early wave at the dorsal aorta; there, NCCs move rostrocaudally along the dorsal aorta to form the sympathetic anlagen. Abbreviations: da, dorsal aorta; dm, dermomyotome; isf, intersomitic furrow; isv, intersomitic vessel; psv, perisomitic vessel; pvp, perineural vascular plexus; sa, sympathetic anlage; scl, sclerotome. (Scale bars: A–C, 50 μm; D and E, 100 μm.)