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First published online 30 November 2005
doi: 10.1242/dev.02187

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Guidance of trunk neural crest migration requires neuropilin 2/semaphorin 3F signaling

¹ Division of Biology 139-74, California Institute of Technology, Pasadena, CA 91125, USA.
² Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
³ Howard Hughes Medical Institute, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

View larger version (115K): [in a new window]   Fig. 1. Npn2 is expressed in migrating neural crest cells. Npn2 expression was revealed by whole-mount in situ hybridization of 31 somite stage chick (A) and E10.0 (28 somite) mouse (C) embryos. Neural crest cells were immunostained with anti-HNK-1 in longitudinal sections (B) and anti-p75 in transverse sections (D-F) through the embryos shown in A and C. Plane of section is indicated on the whole-mount view. (E,F) Higher magnification view of the regions boxed in D. Arrowheads indicate migrating neural crest cells. Identical results were obtained at E9.5. a, anterior; p, posterior; dm, dermomyotome; sc, sclerotome; nt, neural tube. Scale bars: 0.1 mm in A-D; 0.01 mm in E,F.

View larger version (69K): [in a new window]   Fig. 2. Npn2 is required to pattern segmental trunk neural crest migration. Trunk neural crest normally migrates in streams (A) restricted to the anterior-half sclerotome (B,C). In the absence of Npn2, segmental migration is lost (D) and neural crest cells migrate throughout both anterior- and posterior-half sclerotomes (E,F). Neural crest was visualized at E9.5 by in situ hybridization for Sox10. In A,D, anterior is towards the left, dorsal is upwards. (B,C,E,F) Longitudinal sections; B and E are sections through the embryos shown in A and D at the levels indicated. a, anterior; p, posterior; dm, dermomyotome; sc, sclerotome; nt, neural tube. Scale bars: 0.1 mm.

View larger version (97K): [in a new window]   Fig. 3. Sema3f expression is complementary to Npn2 and required to pattern neural crest migration. Npn2 (A) and Sema3f (B) are expressed in reciprocal patterns at E9.5, most notably in the hindbrain, branchial arches and trunk, where Npn2 is expressed in the anterior-half somite (C) and Sema3f in the posterior-half somite (D). At E9.5, the segmental appearance of migrating trunk neural crest (E) that results from migration exclusively through the anterior-half sclerotome (F) is disrupted in Sema3f mutants (G) because neural crest cells migrate throughout the sclerotome (H). (A-D) In situ hybridization for Npn2 (A,C) or Sema3f (B,D). (E-H) Neural crest was visualized by in situ hybridization for Sox10. (E,G) Anterior is towards the left. (F,H) Longitudinal sections of embryos shown in E and G. i, isthmus; r2, rhombomere 2; r4, rhombomere 4; ov, otic vesicle; a, anterior; p, posterior; dm, dermomyotome; sc, sclerotome; nt, neural tube. Scale bars: 0.1 mm.

View larger version (164K): [in a new window]   Fig. 4. Somite polarity is normal in Npn2 and Sema3f mutant mice. In E9.5 embryos, Npn2 is expressed in anterior sclerotome (A), and Sema3f in posterior sclerotome (B). (C) In Npn2 mutants, Sema3f is still posteriorly restricted. EphrinB2 is expressed in the posterior sclerotome (D), Tbx18 is expressed in the anterior sclerotome (F) and Uncx4.1 is expressed in the posterior sclerotome (H). Expression of these three genes remains unchanged in Npn2 mutants (E,G,I). (J) Uncx4.1 also remains restricted to the posterior sclerotome of Sema3f mutants. Gene expression was visualized by in situ hybridization and embryos sectioned longitudinally. a, anterior; p, posterior; dm, dermomyotome; sc, sclerotome; nt, neural tube.

View larger version (50K): [in a new window]   Fig. 5. Npn2 is cell autonomously required for neural crest cells to avoid Sema3f in vitro. Neural tubes from 14 to 24 somite mouse embryos were cultured on Thermanox coverslips coated with fibronectin and spotted with AP-Sema-3F conditioned medium conjugated with anti-placental alkaline phosphatase. Neural crest cells normally avoid immobilized Sema3f (A), while neural crest cells lacking the Npn2 receptor migrate equally well on fibronectin and Sema3f substrates (B,C; n=5 spots in three separate experiments). Neural crest cells were labeled with anti-p75, while spots (outlined in white) were visualized using anti-mouse IgG-Alexa 488.

View larger version (78K): [in a new window]   Fig. 6. Non-segmentally migrating neural crest cells give rise to segmental dorsal root ganglia in Npn2 mutant mice. In E10.5 (A) and E11.5 (C) wild-type embryos, in situ hybridization for Sox10 reveals segmental streams of migrating neural crest cells in the posterior trunk, and condensed dorsal root ganglia anteriorly. (B,D) In Npn2 mutant embryos, in the absence of segmental neural crest migration posteriorly, individualized dorsal root ganglia segregate anteriorly. Black arrowheads indicate condensing dorsal root ganglia at the same axial level in all panels. Scale bars: 0.5 mm. (E,F) In sections of Npn2 heterozygous (E) and mutant (F) E11.5 embryos stained with anti-TUJ1, dorsal root ganglia had a similar appearance but were less well separated in the mutant. Scale bar: 0.1 mm.