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

Guidance of trunk neural crest migration requires neuropilin 2/semaphorin 3F signaling RESEARCH ARTICLE 99 Development 133, 99-106 doi:10.1242/dev.02187 Guidance of trunk neural crest migration requires neuropilin 2/semaphorin 3F signaling 1 1 2,3 1, Laura S. Gammill , Constanza Gonzalez , Chenghua Gu and Marianne Bronner-Fraser * In vertebrate embryos, neural crest cells migrate only through the anterior half of each somite while avoiding the posterior half. We demonstrate that neural crest cells express the receptor neuropilin 2 (Npn2), while its repulsive ligand semaphorin 3F (Sema3f) is restricted to the posterior-half somite. In Npn2 and Sema3f mutant mice, neural crest cells lose their segmental migration pattern and instead migrate as a uniform sheet, although somite polarity itself remains unchanged. Furthermore, Npn2 is cell autonomously required for neural crest cells to avoid Sema3f in vitro. These data show that Npn2/Sema3f signaling guides neural crest migration through the somite. Interestingly, neural crest cells still condense into segmentally arranged dorsal root ganglia in Npn2 nulls, suggesting that segmental neural crest migration and segmentation of the peripheral nervous system are separable processes. KEY WORDS: Trunk neural crest migration, Sclerotome, Neuropilin 2, Semaphorin 3F, Mouse, Chick INTRODUCTION Likewise, neuropilin 1 and its ligand semaphorin 3A have been The neural crest is a multipotent population of migratory cells that suggested to play a role (Eickholt et al., 1999), but are not expressed gives rise to a wide variety of different lineages in vertebrates. at the right time (reviewed by Kuan et al., 2004) and are not required During development, neural crest cells arise in the central nervous in the mouse for appropriate trunk neural crest migration (Kawasaki system, but subsequently migrate away and follow defined et al., 2002). It is not clear whether the inability to identify a trunk stereotypic pathways. In the trunk, for example, neural crest cells neural crest mutant phenotype is due to redundancy or whether the invade only the anterior but not the posterior portion of each somitic true regulatory molecules have not been found. sclerotome (Bronner-Fraser, 1986; Rickman et al., 1985; Serbedzija We isolated chick neuropilin 2a1 (Npn2a1) in a screen for genes et al., 1990). This selective migration results in the formation of upregulated as a consequence of neural crest induction (Gammill segmentally arranged streams of migrating neural crest cells. This and Bronner-Fraser, 2002). Npn2 is a receptor for class 3 secreted pattern appears to be imposed by the somites, with the anterior semaphorins (Sema) 3C and 3F as well as vascular endothelial sclerotome being permissive and posterior sclerotome repulsive for growth factor (Bagri and Tessier-Lavigne, 2002; Neufeld et al., neural crest migration (Bronner-Fraser and Stern, 1991; Kalcheim 2002). Npn2 is required for appropriate axon guidance and and Teillet, 1989). Accordingly, surgical or genetic modification of fasciculation in the central and peripheral nervous system (Chen et anteroposterior somite polarity results in loss of segmental neural al., 2000; Cloutier et al., 2002; Giger et al., 2000). However, the crest migration (Kalcheim and Teillet, 1989) and formation of fused importance of Npn2 and its ligands during trunk neural crest neural crest-derived dorsal root ganglia (Bussen et al., 2004; development has not been examined. Kalcheim and Teillet, 1989; Leitges et al., 2000; Mansouri et al., Here, we explore the role of Npn2 signaling during neural crest 2000). Thus, the segmental pattern of neural crest migration is migration. We demonstrate that the Npn2 receptor on neural crest believed to be responsible for the metameric organization of the cells detects a Sema3f repellant cue in the posterior sclerotome that ganglia of the peripheral nervous system (Kuan et al., 2004; guides neural crest migration through the somites. Surprisingly, LeDouarin and Kalcheim, 1999). The segmented arrangement of individualized dorsal root ganglia still form, albeit less well these ganglia relative to the somites, which will form the vertebrae, separated than normal, suggesting that the pattern of neural crest is crucial for proper wiring of the ganglia and peripheral nerves to migration alone does not dictate the arrangement of the peripheral targets in the periphery. ganglia, and that multiple signaling pathways are required to create The identity of the molecular cues that direct neural crest migration a segmented peripheral nervous system. exclusively through the anterior sclerotome is still open to debate. Although previous reports suggested that Eph/ephrin signaling might MATERIALS AND METHODS pattern trunk neural crest migration (Krull et al., 1997; Wang and Embryos Anderson, 1997), the Eph and ephrin mutant mice that have been Fertile chicken eggs were incubated at 37°C to the desired stage (Hamburger examined fail to exhibit trunk neural crest migration defects (Adams and Hamilton, 1992). Embryos were isolated in Ringers Saline and fixed et al., 2001; Davy et al., 2004; Orioli et al., 1996; Wang et al., 1998). overnight at 4°C in 4% paraformaldehyde. Mouse embryos were surgically isolated, with day 0.5 being the day of the plug, into ice cold phosphate- buffered saline (PBS) and fixed for 2 hours at room temperature or overnight Division of Biology 139-74, California Institute of Technology, Pasadena, CA 91125, at 4°C in 4% paraformaldehyde. Embryos were genotyped by polymerase USA. Department of Neuroscience, The Johns Hopkins University School of chain reaction (PCR) (Giger et al., 2000). Medicine, Baltimore, MD 21205, USA. Howard Hughes Medical Institute, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. In situ hybridization Chick in situ hybridization was performed as described previously (Gammill *Author for correspondence (e-mail: [email protected]) and Bronner-Fraser, 2002). Mouse in situ hybridization was performed as described previously (Wilkinson, 1992), except that hybridization was Accepted 27 October 2005 DEVELOPMENT 100 RESEARCH ARTICLE Development 133 (1) performed in 50% formamide, 1.3 SSC (pH 5), 5 mM EDTA, 50 g/ml medium, prepared as described (Stemple and Anderson, 1992) except that yeast RNA, 0.2% Tween 20, 0.5% CHAPS and 50 g/ml heparin at 70°C. DMEM-F12 was used and retinoic acid was omitted. The tubes were Embryos were washed twice in hybridization mix at 70°C, three times in allowed to stick to the coverslip for one hour at 37°C, then 2 ml of additional wash solution I at 65°C, and antibody pre-treatment was performed in 100 complete medium was added slowly down the side of the dish, and explants mM maleic acid, 150 mM NaCl, 0.1% Tween (pH 7.5) with 2% Blocking were cultured for an additional 28-48 hours at 37°C. Reagent (Boehringer Mannheim). Templates for digoxigenin-labeled antisense riboprobes were as follows: chick Npn2 (Gammill and Bronner- RESULTS Fraser, 2002), mouse Npn2 (Giger et al., 2000), Sox10 (Kuhlbrodt et al., Neural crest migrates through anterior and 1998), Sema3f (Giger et al., 2000), ephrinB2 (Wang and Anderson, 1997), posterior sclerotome in Npn2 mutants Tbx18 (Kraus et al., 2001) and Uncx4.1 (Mansouri et al., 1997). Stained embryos were infiltrated with 5% sucrose, 15% sucrose and 7.5% gelatin in We identified chick Npn2 as an early response to neural crest 15% sucrose, frozen in liquid nitrogen, sectioned at 20 M by cryostat induction (Gammill and Bronner-Fraser, 2002). Npn2 is expressed (Microm) and mounted in permafluor (Thermo Electron Corporation). in premigratory neural crest cells in the dorsal neural tube as well as on migratory neural crest in both the chick (Fig. 1A,B) (Gammill Immunohistochemistry and Bronner-Fraser, 2002) and the mouse (Fig. 1C-F). In the trunk, Neural crest cells with were stained with 1:50 anti-HNK-1 (American Type Npn2 was clearly expressed in a segmental pattern in both species. Culture; Tucker et al., 1984) followed by 1:400 anti-mouse-IgM-Rhodamine Longitudinal sections through chick embryos revealed that Npn2 Red X (Jackson Immuno Research) or 1:2000 anti-p75 (Weskamp and was expressed by neural crest cells, identified by HNK-1 Reichardt, 1991) followed by 1:400 anti-rabbit-Rhodamine Red X (Jackson Immuno Research). Sema3f spots were visualized using an anti-mouse IgG immunoreactivity, as they migrate through the anterior half of each Alexa 488 secondary at 1:1000 (Molecular Probes). Unstained embryos were somitic sclerotome (Fig. 1B). Similarly, in transverse sections of infiltrated with 5% sucrose, 15% sucrose and 7.5% gelatin in 15% sucrose, mouse embryos, Npn2 expression colocalized with the neural crest frozen in liquid nitrogen, sectioned at 15 M by cryostat (Microm) and marker p75 (Fig. 1D-F). degelatinized for 20 minutes at 42°C in PBS. Dorsal root ganglia were stained The distribution of Npn2 on neural crest cells made this receptor with 1:500 anti-TUJ1 (neuron specific class III -tubulin; Babco) followed a potential candidate for influencing neural crest formation and by 1:500 anti-mouse-Biotin (Jackson Immuno Research), and developed migration. To address the requirement for Npn2 during neural crest using the ABC-horseradish peroxidase kit (Vector Laboratories) and 0.1 development, we assessed the loss-of-function phenotype by mg/ml 3,3-diaminobenzidine tetrahydrochloride (DAB; Sigma) with 0.009% hydrogen peroxide according to the manufacturer’s instructions. Conditioned medium 293T cells were transfected with 24 g of AP-Sema3f (Giger et al., 2000) or AP empty vector using 45 l Lipofectamine 2000 (Invitrogen) per 10 cm dish according to the manufacturer’s instructions. Media (DMEM + 0.1% BSA + Pen Strep) was collected after 3 days and concentrated using a Centriplus YM-100 filter device (Millipore). Alkaline phosphatase activity was determined using AP Assay Reagent A (GenHunter Corporation) and the molarity of the collected protein calculated according to the manufacturer’s instructions. Sema3f spot preparation Aminopropyltriethoxysilane (2%; APTES; Sigma) was prepared in 95% ethanol and allowed to hydrolyze for 5 minutes in a fume hood. Thermanox (25 mm; Nunc) cover slips in a wafer basket (Fluoroware) were incubated in the 2% APTES for 10 minutes, and washed three times for 5 minutes with 95% ethanol. Coverslips were cured for 15 minutes at 100°C in a vacuum oven, immobilized onto 35 mm tissue culture plates with four spots of silicone vacuum grease, and UV sterilized in a tissue culture hood for 15 minutes. AP-Sema3f (75 nM) was preincubated at room temperature for 1 hour with 50 g/ml mouse anti-human placental alkaline phosphatase (Chemicon), 0.2 l drops were spotted manually in a grid on the coverslip, and the location of the spots was marked on the underside of the dish. After 1 hour at 37°C, the coverslips were washed three times with 4 ml of 1 Hank’s buffered saline solution (HBSS; Invitrogen). After all remaining traces of HBSS had been aspirated, 150-200 l of 125 g/ml fibronectin (BD Biosciences) was laid over the spots and incubated for 1.5 to 2 hours at 37°C. After aspirating the fibronectin, the coverslips were washed once with 4 ml of HBSS and stored overnight at 4°C in 2 ml DMEM-F12 (Invitrogen) + 1 mg/ml BSA. Fig. 1. Npn2 is expressed in migrating neural crest cells. Npn2 expression was revealed by whole-mount in situ hybridization of 31 Mouse neural tube culture somite stage chick (A) and E10.0 (28 somite) mouse (C) embryos. E9.5 embryos (14-24 somites) were isolated into ice cold HBSS. The region Neural crest cells were immunostained with anti-HNK-1 in longitudinal of the trunk containing the last 10 somites was dissected, trimming the sections (B) and anti-p75 in transverse sections (D-F) through the membranes lateral to the somites and removing the gut tube. Trunk pieces embryos shown in A and C. Plane of section is indicated on the whole- were incubated for 8 minutes at 37°C in room temperature 3 g/ml dispase mount view. (E,F) Higher magnification view of the regions boxed in D. made fresh in HBSS and 0.2 m filter sterilized. After rinsing several times Arrowheads indicate migrating neural crest cells. Identical results were with DMEM-F12 + 10% fetal bovine serum (Hyclone), neural tubes were obtained at E9.5. a, anterior; p, posterior; dm, dermomyotome; sc, isolated by trituration with a fire-polished pasteur pipette and plated on the sclerotome; nt, neural tube. Scale bars: 0.1 mm in A-D; 0.01 mm in E,F. spotted region of the coverslips in 1 ml of pre-warmed neural crest complete DEVELOPMENT Npn2/Sema3f in trunk neural crest migration RESEARCH ARTICLE 101 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. characterizing neural crest marker gene expression in Npn2-null mutant mice (Giger et al., 2000). In situ hybridization with probes for the neural crest markers Sox10, Foxd3 and Pax3 at E8-8.5 (4 to 12 somites) showed no obvious effects on the specification or generation of neural crest cells in wild-type, heterozygous or Npn2 mutant mice (data not shown). Profound defects were observed, however, on the pattern of neural crest migration in Npn2-null mice. In the trunk of wild- type (not shown) or Npn2 heterozygous (Fig. 2A) embryos, neural crest migration appeared as segmentally iterated blocks of Sox10 staining cells. In sections through these embryos, Sox10- positive neural crest cells were found only in the anterior-half sclerotome at levels adjacent to the intermediate region of the neural tube (Fig. 2B,C) (Serbedzija et al., 1990). In Npn2 mutant mice, however, neural crest cells migrated in a uniform sheet rather than in streams (Fig. 2D), and Sox10-positive cells were present throughout both anterior and posterior portions of the sclerotome (Fig. 2E,F). More ventrally, approaching the dorsal aorta where the neural crest-derived sympathetic ganglia will form, some segmental neural crest migration was still apparent in Npn2-null mice (Fig. 2D). Interestingly, motor axons, which normally are restricted to the anterior sclerotome, also project into both anterior and posterior sclerotome in Npn2 mutants, although ventral roots still form (data not shown). Together, these data suggest that Npn2 signaling is required for neural crest guidance events through dorsal and intermediate levels of the somite. Npn2 and Sema3f exhibit complementary Fig. 3. Sema3f expression is complementary to Npn2 and expression patterns required to pattern neural crest migration. Npn2 (A) and Sema3f Npn2 can bind to three different ligands: Sema3C, Sema3f and (B) are expressed in reciprocal patterns at E9.5, most notably in the vascular endothelial growth factor (Chen et al., 1997; Gluzman- hindbrain, branchial arches and trunk, where Npn2 is expressed in the anterior-half somite (C) and Sema3f in the posterior-half somite (D). At Poltorak et al., 2000). To determine which ligand was mediating E9.5, the segmental appearance of migrating trunk neural crest (E) that the patterning functions revealed by the Npn2 mutant, we next results from migration exclusively through the anterior-half sclerotome assessed the expression patterns of these molecules by in situ (F) is disrupted in Sema3f mutants (G) because neural crest cells hybridization and immunohistochemistry. Sema3c is first migrate throughout the sclerotome (H). (A-D) In situ hybridization for expressed in the somites around E10, and then only in the Npn2 (A,C) or Sema3f (B,D). (E-H) Neural crest was visualized by in situ dermomyotomal compartment (Adams et al., 1996). hybridization for Sox10. (E,G) Anterior is towards the left. Immunostaining for vascular endothelial growth factor was also (F,H) Longitudinal sections of embryos shown in E and G. i, isthmus; detected at low levels in the dermomyotome but not in the r2, rhombomere 2; r4, rhombomere 4; ov, otic vesicle; a, anterior; sclerotome at E9.5 (data not shown). By contrast, Sema3f was p, posterior; dm, dermomyotome; sc, sclerotome; nt, neural tube. Scale expressed in a pattern complementary to that of Npn2 (Fig. 3A,B), bars: 0.1 mm. DEVELOPMENT 102 RESEARCH ARTICLE Development 133 (1) Somite patterning is normal in Npn2 and Sema3f mutants Two alternate mechanisms could explain the trunk neural crest migration defects observed in the Npn2 (Fig. 2) and Sema3f (Fig. 3) mutant mice. The phenotype could reflect a requirement for signaling between the Sema3f repulsive ligand in the posterior somite and the Npn2 receptor on neural crest cells to guide neural crest migration. Alternatively, Npn2/Sema3f signaling between the anterior and posterior somite could be important for maintenance of anterior and/or posterior sclerotomal identity. Disrupting this signaling could affect somite polarity and thus the environment through which neural crest migrates, secondarily impacting the pattern of neural crest migration. For example, anteroposterior somite polarity is abolished in Delta1 mutant mice (deAngelis et al., 1997), and as a consequence, neural crest cells migrate aberrantly through the posterior sclerotome of these animals (DeBellard et al., 2002). To differentiate between these two possibilities, markers of anterior and posterior sclerotome were examined in wild-type, Npn2 mutant and Sema3f mutant mice to determine whether anterior and posterior somite identity was retained in the mutants. Sema3f was expressed in the posterior sclerotome of both wild-type (Fig. 4B) and Npn2 mutant mice (Fig. 4C). Ephrin B2, a ligand that repels migrating neural crest cells in vitro (Wang and Anderson, 1997), was also equivalently restricted to the posterior sclerotome of wild-type Fig. 4. Somite polarity is normal in Npn2 and Sema3f mutant (Fig. 4D) and Npn2 mutant embryos (Fig. 4E). Thus, two posteriorly mice. In E9.5 embryos, Npn2 is expressed in anterior sclerotome (A), expressed guidance molecules are appropriately localized in Npn2 and Sema3f in posterior sclerotome (B). (C) In Npn2 mutants, Sema3f is mutants. still posteriorly restricted. EphrinB2 is expressed in the posterior Somite polarity is established during segmentation of the somitic sclerotome (D), Tbx18 is expressed in the anterior sclerotome (F) and mesoderm, with anterior and posterior somite identity maintained Uncx4.1 is expressed in the posterior sclerotome (H). Expression of these three genes remains unchanged in Npn2 mutants (E,G,I). and promoted by two different transcription factors, Tbx18 (Bussen (J) Uncx4.1 also remains restricted to the posterior sclerotome of et al., 2004) and Uncx4.1 (Leitges et al., 2000; Mansouri et al., Sema3f mutants. Gene expression was visualized by in situ 2000). Tbx18 was restricted to the anterior sclerotome of wild-type hybridization and embryos sectioned longitudinally. a, anterior; (Fig. 4F) and Npn2 mutant embryos (Fig. 4G). Uncx4.1 was also p, posterior; dm, dermomyotome; sc, sclerotome; nt, neural tube. properly expressed in the posterior sclerotome of wild-type (Fig. 4H), Npn2 mutant (Fig. 4I) and Sema3f mutant mice (Fig. 4J). These results demonstrate that anterior and posterior sclerotomal character with Npn2 expression in the anterior somite (Fig. 3C) mirrored by is maintained in Npn2 and Sema3f mutants, suggesting that the Sema3f expression in the posterior somite (Fig. 3D). In sections, requirement for Npn2/Sema3f signaling is likely to reside in the Npn2 was clearly expressed in the anterior half of each sclerotome neural crest. (Fig. 4A), and Sema3fF in the posterior half (Fig. 4B). This expression pattern made Sema3f an ideal candidate for signaling Npn2 is required in the neural crest for Sema3f- through the Npn2 receptor during trunk neural crest migration. The mediated repulsion complementary distribution of Npn2 and Sema3f expression has To test the requirement for Npn2 in the neural crest directly, we also been observed at later stages of development (Giger et al., explanted wild-type and Npn2 mutant neural tubes, and cultured 2000; Giger et al., 1998). them on fibronectin-coated substrates containing spots of Sema3f. Wild-type neural crest cells avoided Sema3f (Fig. 5A), with the Sema3f mutants phenocopy Npn2-null mice majority of cells remaining at the spot border and only individual, We next assessed the requirement for Sema3f during neural crest rare cells migrating onto the Sema3f substrate, consistent with their development by examining Sox10 expression in Sema3f mutant mice behavior in vivo (Kasemeier-Kulesa et al., 2004). By contrast, Npn2 (Sahay et al., 2003). Strikingly, the Sema3f-null neural crest mutant neural crest cells migrated equally well on fibronectin with migration phenotype was identical to that observed in Npn2 mutants. or without Sema3f protein (Fig. 5B,C). This demonstrates that the Instead of segmentally arranged streams of neural crest cells in the Npn2 receptor on neural crest cells detects a Sema3f repulsive cue trunk (Fig. 3E), Sema3f nulls contained uniform sheets of migrating in the environment, and supports a cell-autonomous requirement for neural crest (Fig. 3G). In sections, alternating Sox10-positive and - Npn2 on the neural crest during trunk neural crest migration. negative regions were observed in the anterior- and posterior-half sclerotome, respectively, of wild-type mice (Fig. 3F), whereas Segmentally arranged dorsal root ganglia form in uniformly distributed Sox10-labeled cells were seen throughout the Npn2 mutant mice sclerotome of Sema3f mutants (Fig. 3H). Together, these results The segmental migration of neural crest through the somite is demonstrate that signaling between the receptor Npn2 and its ligand thought to prefigure the segmented organization of the neural crest- Sema3f is required to restrict neural crest migration to the anterior derived ganglia of the peripheral nervous system. This pattern somite. ensures that the ganglia and the vertebrae, which differentiate from DEVELOPMENT Npn2/Sema3f in trunk neural crest migration RESEARCH ARTICLE 103 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. the somites, will form in register with one another. For example, in each somite, neural crest cells in the anterior sclerotome coalesce to form the dorsal root ganglia. As a result, in Npn2 mutants, one might expect a continuous mass of dorsal root ganglia to form instead of individualized ganglia. This is the case when neural crest migrates non-segmentally through somites that are genetically (deAngelis et al., 1997; DeBellard et al., 2002) or surgically manipulated (Kalcheim and Teillet, 1989) to contain only anterior character. In Fig. 6. Non-segmentally migrating neural crest cells give rise to wild-type embryos at E10.5 and E11.5, the streams of Sox10- segmental dorsal root ganglia in Npn2 mutant mice. In E10.5 (A) expressing neural crest cells in the trunk condensed into ganglia in and E11.5 (C) wild-type embryos, in situ hybridization for Sox10 reveals an anterior to posterior progression (Fig. 6A,C). Strikingly, neural segmental streams of migrating neural crest cells in the posterior trunk, crest cells in Npn2 mutant mice also coalesced into recognizable and condensed dorsal root ganglia anteriorly. (B,D) In Npn2 mutant ganglia. At E10.5, neural crest cells were still distributed throughout embryos, in the absence of segmental neural crest migration the somites, but became excluded from the somite boundaries (Fig. posteriorly, individualized dorsal root ganglia segregate anteriorly. Black arrowheads indicate condensing dorsal root ganglia at the same axial 6B). By E11.5, individualized ganglia appeared to have sorted out level in all panels. Scale bars: 0.5 mm. (E,F) In sections of Npn2 from the sheet of migrating neural crest in the somite (Fig. 6D, heterozygous (E) and mutant (F) E11.5 embryos stained with anti-TUJ1, arrowheads mark the same axial levels in all panels). In longitudinal dorsal root ganglia had a similar appearance but were less well sections at E11.5, TUJ1 immunoreactivity confirmed the apparent separated in the mutant. Scale bar: 0.1 mm. segmentation, with space between each Npn2 mutant dorsal root ganglion (Fig. 6F). Although morphologically similar to those in wild-type embryos (Fig. 6E), the mutant ganglia were not as well separated, suggesting that the process of gangliogenesis occurred but Murine Npn2 is expressed by neural crest cells (Fig. 1D-F) as well was somewhat compromised. The sympathetic ganglia, which form as cells of the anterior sclerotome (Fig. 4A). Therefore, the in a segmental pattern ventral to the somites, are normal in Npn2 migration defects in Npn2 mutants could reflect a requirement for mutants (Giger et al., 2000). Npn2 in one or both cell types. One possibility is that Npn2 receptors on neural crest cells detect a Sema3f repulsive cue in posterior DISCUSSION sclerotome, leading to anterior-only migration. Alternatively, Npn2 We have examined the importance of the receptor Npn2 and its receptors on anterior sclerotomal cells may interact with the Sema3f repulsive ligand Sema3f during neural crest development in the ligand secreted by cells of the adjacent posterior sclerotome to affect trunk. We demonstrate that Npn2 is expressed in migrating neural the environment through which neural crest cells migrate, thus crest cells, and that Npn2/Sema3f signaling is required for segmental influencing neural crest migration in a non-cell-autonomous manner. neural crest migration but not for somite patterning. Surprisingly, we The latter mechanism seems unlikely for several reasons. First and found that segmental migration was not essential for the formation foremost, in contrast to wild-type cells, Npn2 mutant neural crest of individualized dorsal root ganglia. cells do not avoid Sema3f in culture. This indicates a cell autonomous requirement for Npn2 in the neural crest. Second, as Npn2/Sema3f signaling patterns neural crest anteroposterior polarity of the somites is indistinguishable in wild- migration in the trunk type, Npn2 and Sema3f mutant embryos (Fig. 4), Npn2/Sema3f In Npn2 and Sema3f mutant mice, trunk neural crest cells migrate signaling does not appear to be required in this process. In support through both the anterior and posterior sclerotome, rather than of this conclusion, neural crest cells are present all along the exclusively through the anterior-half sclerotome as in wild-type anteroposterior axis in the Npn2 (Fig. 2E,F) and Sema3f mutants mice. This demonstrates that signaling between the receptor Npn2 (Fig. 3H), even at somite borders. This contrasts with the Delta1 and its ligand Sema3f is required to restrict neural crest migration to mutant mouse, in which somite polarity is lost (deAngelis et al., the anterior somite. 1997), where neural crest migration has a pseudo-segmental DEVELOPMENT 104 RESEARCH ARTICLE Development 133 (1) appearance, with migrating neural crest cells present throughout the A second possibility is that dorsal root ganglia form in the absence sclerotome but avoiding somite boundaries (DeBellard et al., 2002). of segmental neural crest migration simply because neurons tend to This comparison suggests that, unlike Delta1, the Npn2 defect is not aggregate (M. Bronner-Fraser, unpublished). In support of this in the somites themselves. Third, as Npn2 is expressed on anterior stochastic mechanism, when normal somites are surgically replaced sclerotomal cells, if Npn2/Sema3f were involved in somite with multiple anterior or posterior somite halves, a giant mass of patterning, loss of signaling through this receptor would most ganglia forms that exhibits a pseudo-segmental appearance, with probably posteriorize the sclerotome. A posteriorized somite alternating thick and thin regions at random intervals within the giant impedes neural crest migration, leading to reduced numbers of ganglion (Kalcheim and Teillet, 1989). That they are fused, however, migratory neural crest cells and their derivatives (Bussen et al., 2004; argues that a combination of these two mechanisms is normally at Kalcheim and Teillet, 1989). However, this is not the case in Npn2- play. null mice. For these reasons, we favor the idea that Npn2 is required Finally, it is also possible that the physical structure of the somite in the neural crest. itself can impose segmentation during gangliogenesis. In addition to What is the purpose, then, of Npn2 expression in both the neural regionally restricted molecular cues, such as Sema3f expression crest and the sclerotome through which it migrates? Interestingly, posteriorly, there are embryological boundaries and differences like Npn2, Npn1 (Eickholt et al., 1999) and Ephb3 receptors (Krull within the sclerotome. These include, most notably, the intersomitic et al., 1997), both of which have been postulated to play a role in space, as well as von Ebner’s fissure between anterior and posterior patterning trunk neural crest migration, are also distributed on both sclerotome, and the various subdomains within the sclerotome neural crest and anterior sclerotomal cells. One intriguing possibility (reviewed by Christ et al., 2004). Anterior sclerotome is less cell is that these receptors do not play a signaling role in the somite, but dense than posterior sclerotome (Christ et al., 2004), is mitogenic rather serve as a sink to bind up any repulsive ligand diffusing from for dorsal root ganglia (Goldstein et al., 1990) and will undergo the posterior sclerotome, thus ensuring a sharp boundary such that apoptosis in the absence of neural crest cells (see Christ et al., 2004). the anterior sclerotome is devoid of the repulsive cue. All of these segmentally restricted differences could have a This is the first report of a single receptor/ligand pair that is morphological impact during gangliogenesis. In the case of the Npn2 absolutely required to pattern trunk neural crest migration. The mutants, the uniform sheet of migrating neural crest cells appears to molecular basis for segmental neural crest migration has segment into individual dorsal root ganglia at the somite border (Fig. preoccupied this field since the phenomenon was first observed. 6). Interestingly, when chick embryos are surgically modified to Many different cell adhesion molecules, extracellular matrix contain only anterior sclerotome, in other words have no somite molecules and receptor/ligand pairs have been identified that are boundary, neural crest migrates non-segmentally and dorsal root expressed in anterior or posterior sclerotome or in the neural crest, ganglia are fused (Kalcheim and Teillet, 1989). However, dorsal root and in some cases they have been shown to be sufficient to direct ganglia are also fused in Uncx4.1 and Tbx18 mutants, where neural crest migration (Kuan et al., 2004). But in no case has a anteroposterior somite polarity is abolished but physical somites still requirement for any molecule been previously demonstrated in the form (Bussen et al., 2004; Leitges et al., 2000; Mansouri et al., embryo. Other signals, such as Eph receptor/ephrin ligand 2000). Together, these data suggest that somite polarity creates interactions, might fine tune neural crest migration, or in the case of positional information at the somite boundary that impacts upon the Npn1/Sema3A, be involved in later steps in the process. However, segmentation of the peripheral nervous system. Migrating neural Npn2/Sema3f signaling is clearly the key determinant patterning crest cells normally maintain filopodial contact across the posterior anterior-only migration through the sclerotome. somite and can even cross over between adjacent streams (Kasemeier-Kulesa et al., 2004), thus the somite boundary could Segmental neural crest migration may not be normally curtail this movement as dorsal root ganglia condense. required for segmental dorsal root ganglion The sympathetic ganglia also are not dependent upon the pattern formation of neural crest migration for their segmented organization. By The requirement for Npn2/Sema3f signaling in neural crest imaging actively migrating neural crest cells, Kasemeier-Kulesa and migration has uncovered an additional, previously unrecognized colleagues (Kasemeier-Kulesa et al., 2004) showed that, once they process that results in dorsal root ganglion segmentation irrespective have passed through the somites, neural crest cells no longer maintain of the neural crest migration pattern. Despite the fact that neural crest their segmental position and can migrate as far as two segments cells migrate through the anterior and posterior sclerotome of Npn2 anteriorly or posteriorly. The mechanisms that ultimately result in the mutant mice, segmentally arranged dorsal root ganglia still form. aggregation of these cells into individualized sympathetic ganglia are This result suggests that segmental neural crest migration and likely to be similar to those we propose for the formation of subsequent sequestration of ganglia are separable events. One metameric dorsal root ganglia in Npn2 mutants. Interestingly, Npn1 explanation is that there may be independent signals restricting and Sema3A are required for localization and condensation of neural crest migration and the pattern of ganglion aggregation. For sympathetic precursors as well (Kawasaki et al., 2002). example, either a cell-adhesive, ‘sorting’ signal or a repulsive cue in The overriding message is that the segmental migration of neural the posterior sclerotome could promote aggregation within the crest cells through the somites itself is not requisite for the creation anterior sclerotome, irrespective of the starting location of the neural of a segmented peripheral nervous system, despite what has been crest cells within the sclerotome. In favor of this possibility, fused assumed for 20 years (reviewed by Kuan et al., 2004). Although the dorsal root ganglia form when anteroposterior somite polarity is dorsal root ganglia eventually segment in the Npn2 mutants, they are abolished by either surgical or genetic manipulation (Bussen et al., more closely spaced than normal. Thus, the pattern of neural crest 2004; Kalcheim and Teillet, 1989; Leitges et al., 2000; Mansouri et migration is important, but not essential for the formation of al., 2000). This indicates that anteroposteriorly patterned signals in segmented dorsal root ganglia. This may not be surprising given the the somite are required for segmental formation of dorsal root regulative nature of vertebrate embryos, which may have ‘back-up’ ganglia. Candidates for such signals include F-spondin (Debby- mechanisms for formation of important organ systems in the event Brafman et al., 1999) and Npn1 (Kitsukawa et al., 1997). that primary mechanisms are perturbed. DEVELOPMENT Npn2/Sema3f in trunk neural crest migration RESEARCH ARTICLE 105 Christ, B., Huang, R. and Scaal, M. (2004). Formation and differentiation of the Functional validation of the neural crest gene avian sclerotome. Anat. Embryol. 208, 333-350. expression profile Cloutier, J.-F., Giger, R., Koentges, G., Dulac, C., Kolodkin, A. and Ginty, D. We originally identified Npn2a1 in a screen for genes upregulated in (2002). Neuropilin-2 mediates axonal fasciculation, zonal segregation, but not response to neural crest induction (Gammill and Bronner-Fraser, axonal convergence, of primary accessory olfactory neurons. Neuron 33, 877- 2002). Our current analysis of Npn2 function has several Coles, E., Gammill, L., Miner, J. and Bronner-Fraser, M. (2005). Abnormalities implications. First of all, the importance of Npn2 for neural crest in neural crest cell migration in laminin alpha5 mutant mice. Dev. Biol. (in press) migration validates our neural crest gene expression profile and Davy, A., Aubin, J. and Soriano, P. (2004). Ephrin-B1 forward and reverse signaling are required during mouse development. Genes Dev. 18, 572-583. demonstrates that our collection of genes contains true regulators of deAngelis, M., McIntyre, J. and Gossler, A. (1997). Maintenance of somite neural crest development. This conclusion is supported by the borders in mice requires the Delta homologue Dll1. Nature 386, 717-721. demonstration that Laminin-5, another gene identified in our Debby-Brafman, A., Burstyn-Cohen, T., Klar, A. and Kalchiem, C. (1999). F- Spondin, expressed in somite regions avoided by neural crest cells, mediates screen, is also important for proper emigration of neural crest cells inhibition of distinct somite domains to neural crest migration. Neuron 22, 475- (Coles et al., 2005). In addition, although we screened for genes expressed in DeBellard, M., Ching, W., Gossler, A. and Bronner-Fraser, M. (2002). Disruption of segmental neural crest migration and ephrin expression in Delta-1 premigratory neural crest (Gammill and Bronner-Fraser, 2002), null mice. Dev. Biol. 249, 121-130. Npn2 is required for neural crest migration and apparently not for Eickholt, B., Mackenzie, S., Graham, A., Walsh, F. and Doherty, P. (1999). specification, as no differences were noted in the expression of early Evidence for collapsin-1 functioning in the control of neural crest migration in neural crest markers Sox10, Pax3 and FoxD3 in the neural folds and both trunk and hindbrain regions. Development 126, 2181-2189. Gammill, L. S. and Bronner-Fraser, M. (2002). Genomic analysis of neural crest dorsal neural tube of the mouse. We cannot, however, rule out the induction. Development 129, 5731-5741. possibility of an early role for chick Npn2 in neural crest Giger, R., Urquhart, E., Gillespie, S., Levengood, D., Ginty, D. and Kolodkin, specification, as it is expressed earlier and at higher levels in this A. (1998). Neuropilin-2 is a receptor for semaphorin IV: insight into the structural basis of receptor function and specificity. Neuron 21, 1079-1092. organism. Regardless, genes crucial for migration are clearly Giger, R., Cloutier, J.-F., Sahay, A., Prinjha, R., Levengood, D., Moore, S., expressed in premigratory neural crest as a consequence of neural Pickering, S., Simmons, D., Rastan, S., Walsh, F. et al. (2000). Neuropilin-2 is crest induction. This supports our model that early neural crest required in vivo for selective axon guidance responses to secreted semaphorins. Neuron 25, 29-41. development entails a sequential activation of migratory potential, Gluzman-Poltorak, Z., Cohen, T., Herzog, Y. and Neufeld, G. (2000). with a signal to migrate activating this potential in a subset of Neuropilin-2 and neuropilin-1 are receptors for the 165-amino acid form of premigratory neural crest cells (Gammill and Bronner-Fraser, 2002). vascular endothelial growht factor (VEGF) and of placenta growth factor-2, but Further analysis of our neural crest gene collection promises to only neuropilin-2 functions as a receptor for the 145-amino acid form of VEGF. J. Biol. Chem. 275, 18040-18045. reveal the roles of additional genes in this process. Goldstein, R., Teillet, M.-A. and Kalcheim, C. (1990). The microenvironment created by grafting rostral half-somites is mitogenics for neural crest cells. PNAS We are indebted to David Ginty for providing the Npn2 knockout mice and 87, 4476-4480. Sema3f mutant embryos, as well as helpful comments throughout the course Hamburger, V. and Hamilton, H. (1992). A series of normal stages in the of this work. Special thanks to Vivian Lee and York Marahrens for comments development of the chick embryo (originally published in 1951). Dev. Dyn. 195, on the manuscript, and to Joaquin Gutierrez for exceptional animal care. We 231-272. are grateful to Drs David Anderson, Peter Gruss, Ahmed Mansouri, Andreas Kalcheim, C. and Teillet, M.-A. (1989). Consequences of somite manipulation Kispert, Patricia Labosky, Andreas Püschel, Kirsten Kuhlbrodt and Michael on the pattern of the dorsal root ganglion development. Development 106, 85- Wegner for kind gifts of plasmids, and to Lou Reichardt for contributing the 93. p75 antibody. Many thanks to Vivian Lee for tips on immunostaining, Kasemeier-Kulesa, J., Kulesa, P. and Lefcort, F. (2004). Imaging neural crest cell dynamics during formation of dorsal root ganglia and sympathetic ganglia. Christian Hochstim for advice on Neural Crest Complete Medium, Pat White Development 132, 235-245. and Isabelle Miletich for help with neural tube cultures, Andy Ewald for the Kawasaki, T., Bekku, Y., Suto, F., Kisukawa, T., Taniguchi, M., Nagatsu, I., APTES protocol, and Chathurani Jayasena for the substratum choice assay Nagatsu, T., Itoh, K., Yagi, T. and Fujisawa, H. (2002). Requirement of protocol. This work was supported by USPHS grants DE15309 and neuropilin-1-mediated Sema3A signals in patterning of the sympathetic nervous NS051051. system. Development 129, 671-680. Kitsukawa, T., Shimizu, M., Sanbo, M., Hirata, T., Taniguchi, M., Bekku, Y., References Yagi, T. and Fujisawa, H. (1997). Neuropilin-semaphorin III/D-mediated Adams, R., Betz, H. and Püschel, A. (1996). A novel class of murine semaphorins chemorepulsive signalis play a crucial rold in peripheral nerve projection in mice. with homology to thrombospondin is differentially expressed during early Neuron 19, 995-1005. embryogenesis. Mech. Dev. 57, 33-45. Kraus, F., Haenig, B. and Kispert, A. (2001). Cloning and expression analysis of Adams, R., Diella, F., Hennig, S., Helmbacher, F., Deutsch, U. and Klein, R. the mouse T-box gene Tbx18. Mech. Dev. 100, 83-86. (2001). The cytoplasmic domain of the ligand EphrinB2 is required for vascular Krull, C., Lansford, R., Gale, N., Collazo, A., Marcelle, C., Yancopoulos, G., morphogenesis but not cranial neural crest migration. Cell 104, 57-69. Fraser, S. and Bronner-Fraser, M. (1997). Interactions of Eph-related receptors Bagri, A. and Tessier-Lavigne, M. (2002). Neuropilins as semaphorin receptors: and ligands confer rostrocaudal pattern to trunk neural crest migration. Curr. in vivo functions in neuronal cell migration and axon guidance. Adv. Exp. Med. Biol. 7, 571-580. Biol. 515, 13-31. Kuan, C.-Y., Tannahill, D., Cook, G. and Keynes, R. (2004). Somite polarity and Bronner-Fraser, M. (1986). Analysis of the early stages of trunk neural crest segmental patterning of the peripheral nervous system. Mech. Dev. 121, 1055- migration in avian embryos using monoclonal antibody HNK-1. Dev. Biol. 115, 44-55. Kuhlbrodt, K., Herbarth, B., Sock, W., Hermans-Bogmeyer, I. and Wegner, Bronner-Fraser, M. and Stern, C. (1991). Effects of mesodermal tissues on avian M. (1998). sox10, a novel transcriptional modulator in glial cells. J. Neurosci. 18, neural crest cell migration. Dev. Biol. 143, 213-217. 237-250. Bussen, M., Petry, M., Schuster-Gossler, K., Leitges, M., Gossler, A. and LeDouarin, N. and Kalcheim, C. (1999). The Neural Crest. Cambridge, UK: Kispert, A. (2004). The T-box transcription factor Tbx18 maintains the Cambridge University Press. separation of anterior and posterior somite compartments. Genes Dev. 18, Leitges, M., Neidhardt, L., Haenig, B., Herrmann, B. and Kispert, A. (2000). 1209-1221. The paired homeobox gene Uncx4.1 specifies pedicles, transverse processes and Chen, H., Chédotal, A., He, Z., Goodman, C. and Tessier-Lavigne, M. proximal ribs of the vertebral column. Development 127, 2259-2267. (1997). Neuropilin-2, a novel member of the neuropilin family, is a high affinity Mansouri, A., Yokota, Y., Wehr, R., Copeland, N., Jenkins, N. and Gruss, P. receptor for the semaphorins sema E and sema IV but not sema III. Neuron 19, (1997). Paired-related murine homeobox gene expressed in the developing 547-559. sclerotome, kidney, and nervous system. Dev. Dyn. 210, 53-65. Chen, H., Bagri, A., Zupicich, J., Zou, Y., Stoeckli, E., Pleasure, S., Mansouri, A., Voss, A., Thomas, T., Yokota, Y. and Gruss, P. (2000). Uncx4.1 is Lowenstein, D., Skarnes, W., Chédotal, A. and Tessier-Lavigne, M. (2000). required for the formation of the pedicles and proximal ribs and acts upstream Neuropilin-2 regulates the development of select cranial and sensory nerves and of Pax9. Development 127, 2251-2258. hippocampal mossy fiber projections. Neuron 25, 43-56. Neufeld, G., Cohen, T., Shraga, N., Lange, T., Kessler, O. and Herzog, Y. DEVELOPMENT 106 RESEARCH ARTICLE Development 133 (1) (2002). The neuropilins: multifunctional semaphorin and VEGF receptors that Tucker, G., Aoyama, H., Lipinski, M., Tursz, T. and Thiery, J. (1984). Identical modulate axon guidance and angiogenesis. Trends Cardiovasc. Med. 12, 13-19. reactivity of monoclonal antibodies HNK-1 and NC-1: conservation in vertebrates Orioli, D., Henkemeyer, M., Lemke, G., Klein, R. and Pawson, T. (1996). Sek4 on cells derived from the neural primordium and on some leukocytes. Cell Differ. and Nuk receptors cooperate in guidance of commissural axons and in palate 14, 223-230. formation. EMBO J. 6035-6049. Wang, H. and Anderson, D. (1997). Eph family transmembrane ligands can Rickman, M., Fawcett, J. and Keynes, R. (1985). The migration of neural acrest mediate repulsive guidance of trunk neural crest migration and motor axon cells and the growth of motor axons through the rostral half of the chick somite. outgrowth. Neuron 18, 383-396. J. Embryol. Exp. Morphol. 90, 437-455. Wang, H., Chen, Z.-F. and Anderson, D. (1998). Molecular distinction and Sahay, A., Molliver, M., Ginty, D. and Kolodkin, A. (2003). Semaphorin 3F is angiogenic interaction between embryonic arteries and veins revealed by ephrin- critical for development of limbic system circuitry and is required in neurons for B2 and its receptor Eph-B4. Cell 93, 741-753. selective CNS axon guidance events. J. Neurosci. 23, 6671-6680. Weskamp, G. and Reichardt, L. (1991). Evidence that biological activity of Serbedzija, G., Fraser, S. and Bronner-Fraser, M. (1990). Pathways of trunk NGF is mediated through a novel sublass of high affinity receptors. Neuron 6, neural crest migration in the mouse embryo as revealed by vital dye labelling. 649-663. Development 108, 605-612. Wilkinson, D. (1992). Whole mount in situ hybridization of vertebrate embryos. In Stemple, D. and Anderson, D. (1992). Isolation of a stem cell for neurons and In Situ Hybridization: A Practical Approach (ed. D. Wilkinson), pp. 75-83. Oxford, glia from the mammalian neural crest. Cell 71, 973-985. UK: Oxford University Press. DEVELOPMENT http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Development The Company of Biologists

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

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0950-1991
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10.1242/dev.02187
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RESEARCH ARTICLE 99 Development 133, 99-106 doi:10.1242/dev.02187 Guidance of trunk neural crest migration requires neuropilin 2/semaphorin 3F signaling 1 1 2,3 1, Laura S. Gammill , Constanza Gonzalez , Chenghua Gu and Marianne Bronner-Fraser * In vertebrate embryos, neural crest cells migrate only through the anterior half of each somite while avoiding the posterior half. We demonstrate that neural crest cells express the receptor neuropilin 2 (Npn2), while its repulsive ligand semaphorin 3F (Sema3f) is restricted to the posterior-half somite. In Npn2 and Sema3f mutant mice, neural crest cells lose their segmental migration pattern and instead migrate as a uniform sheet, although somite polarity itself remains unchanged. Furthermore, Npn2 is cell autonomously required for neural crest cells to avoid Sema3f in vitro. These data show that Npn2/Sema3f signaling guides neural crest migration through the somite. Interestingly, neural crest cells still condense into segmentally arranged dorsal root ganglia in Npn2 nulls, suggesting that segmental neural crest migration and segmentation of the peripheral nervous system are separable processes. KEY WORDS: Trunk neural crest migration, Sclerotome, Neuropilin 2, Semaphorin 3F, Mouse, Chick INTRODUCTION Likewise, neuropilin 1 and its ligand semaphorin 3A have been The neural crest is a multipotent population of migratory cells that suggested to play a role (Eickholt et al., 1999), but are not expressed gives rise to a wide variety of different lineages in vertebrates. at the right time (reviewed by Kuan et al., 2004) and are not required During development, neural crest cells arise in the central nervous in the mouse for appropriate trunk neural crest migration (Kawasaki system, but subsequently migrate away and follow defined et al., 2002). It is not clear whether the inability to identify a trunk stereotypic pathways. In the trunk, for example, neural crest cells neural crest mutant phenotype is due to redundancy or whether the invade only the anterior but not the posterior portion of each somitic true regulatory molecules have not been found. sclerotome (Bronner-Fraser, 1986; Rickman et al., 1985; Serbedzija We isolated chick neuropilin 2a1 (Npn2a1) in a screen for genes et al., 1990). This selective migration results in the formation of upregulated as a consequence of neural crest induction (Gammill segmentally arranged streams of migrating neural crest cells. This and Bronner-Fraser, 2002). Npn2 is a receptor for class 3 secreted pattern appears to be imposed by the somites, with the anterior semaphorins (Sema) 3C and 3F as well as vascular endothelial sclerotome being permissive and posterior sclerotome repulsive for growth factor (Bagri and Tessier-Lavigne, 2002; Neufeld et al., neural crest migration (Bronner-Fraser and Stern, 1991; Kalcheim 2002). Npn2 is required for appropriate axon guidance and and Teillet, 1989). Accordingly, surgical or genetic modification of fasciculation in the central and peripheral nervous system (Chen et anteroposterior somite polarity results in loss of segmental neural al., 2000; Cloutier et al., 2002; Giger et al., 2000). However, the crest migration (Kalcheim and Teillet, 1989) and formation of fused importance of Npn2 and its ligands during trunk neural crest neural crest-derived dorsal root ganglia (Bussen et al., 2004; development has not been examined. Kalcheim and Teillet, 1989; Leitges et al., 2000; Mansouri et al., Here, we explore the role of Npn2 signaling during neural crest 2000). Thus, the segmental pattern of neural crest migration is migration. We demonstrate that the Npn2 receptor on neural crest believed to be responsible for the metameric organization of the cells detects a Sema3f repellant cue in the posterior sclerotome that ganglia of the peripheral nervous system (Kuan et al., 2004; guides neural crest migration through the somites. Surprisingly, LeDouarin and Kalcheim, 1999). The segmented arrangement of individualized dorsal root ganglia still form, albeit less well these ganglia relative to the somites, which will form the vertebrae, separated than normal, suggesting that the pattern of neural crest is crucial for proper wiring of the ganglia and peripheral nerves to migration alone does not dictate the arrangement of the peripheral targets in the periphery. ganglia, and that multiple signaling pathways are required to create The identity of the molecular cues that direct neural crest migration a segmented peripheral nervous system. exclusively through the anterior sclerotome is still open to debate. Although previous reports suggested that Eph/ephrin signaling might MATERIALS AND METHODS pattern trunk neural crest migration (Krull et al., 1997; Wang and Embryos Anderson, 1997), the Eph and ephrin mutant mice that have been Fertile chicken eggs were incubated at 37°C to the desired stage (Hamburger examined fail to exhibit trunk neural crest migration defects (Adams and Hamilton, 1992). Embryos were isolated in Ringers Saline and fixed et al., 2001; Davy et al., 2004; Orioli et al., 1996; Wang et al., 1998). overnight at 4°C in 4% paraformaldehyde. Mouse embryos were surgically isolated, with day 0.5 being the day of the plug, into ice cold phosphate- buffered saline (PBS) and fixed for 2 hours at room temperature or overnight Division of Biology 139-74, California Institute of Technology, Pasadena, CA 91125, at 4°C in 4% paraformaldehyde. Embryos were genotyped by polymerase USA. Department of Neuroscience, The Johns Hopkins University School of chain reaction (PCR) (Giger et al., 2000). Medicine, Baltimore, MD 21205, USA. Howard Hughes Medical Institute, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. In situ hybridization Chick in situ hybridization was performed as described previously (Gammill *Author for correspondence (e-mail: [email protected]) and Bronner-Fraser, 2002). Mouse in situ hybridization was performed as described previously (Wilkinson, 1992), except that hybridization was Accepted 27 October 2005 DEVELOPMENT 100 RESEARCH ARTICLE Development 133 (1) performed in 50% formamide, 1.3 SSC (pH 5), 5 mM EDTA, 50 g/ml medium, prepared as described (Stemple and Anderson, 1992) except that yeast RNA, 0.2% Tween 20, 0.5% CHAPS and 50 g/ml heparin at 70°C. DMEM-F12 was used and retinoic acid was omitted. The tubes were Embryos were washed twice in hybridization mix at 70°C, three times in allowed to stick to the coverslip for one hour at 37°C, then 2 ml of additional wash solution I at 65°C, and antibody pre-treatment was performed in 100 complete medium was added slowly down the side of the dish, and explants mM maleic acid, 150 mM NaCl, 0.1% Tween (pH 7.5) with 2% Blocking were cultured for an additional 28-48 hours at 37°C. Reagent (Boehringer Mannheim). Templates for digoxigenin-labeled antisense riboprobes were as follows: chick Npn2 (Gammill and Bronner- RESULTS Fraser, 2002), mouse Npn2 (Giger et al., 2000), Sox10 (Kuhlbrodt et al., Neural crest migrates through anterior and 1998), Sema3f (Giger et al., 2000), ephrinB2 (Wang and Anderson, 1997), posterior sclerotome in Npn2 mutants Tbx18 (Kraus et al., 2001) and Uncx4.1 (Mansouri et al., 1997). Stained embryos were infiltrated with 5% sucrose, 15% sucrose and 7.5% gelatin in We identified chick Npn2 as an early response to neural crest 15% sucrose, frozen in liquid nitrogen, sectioned at 20 M by cryostat induction (Gammill and Bronner-Fraser, 2002). Npn2 is expressed (Microm) and mounted in permafluor (Thermo Electron Corporation). in premigratory neural crest cells in the dorsal neural tube as well as on migratory neural crest in both the chick (Fig. 1A,B) (Gammill Immunohistochemistry and Bronner-Fraser, 2002) and the mouse (Fig. 1C-F). In the trunk, Neural crest cells with were stained with 1:50 anti-HNK-1 (American Type Npn2 was clearly expressed in a segmental pattern in both species. Culture; Tucker et al., 1984) followed by 1:400 anti-mouse-IgM-Rhodamine Longitudinal sections through chick embryos revealed that Npn2 Red X (Jackson Immuno Research) or 1:2000 anti-p75 (Weskamp and was expressed by neural crest cells, identified by HNK-1 Reichardt, 1991) followed by 1:400 anti-rabbit-Rhodamine Red X (Jackson Immuno Research). Sema3f spots were visualized using an anti-mouse IgG immunoreactivity, as they migrate through the anterior half of each Alexa 488 secondary at 1:1000 (Molecular Probes). Unstained embryos were somitic sclerotome (Fig. 1B). Similarly, in transverse sections of infiltrated with 5% sucrose, 15% sucrose and 7.5% gelatin in 15% sucrose, mouse embryos, Npn2 expression colocalized with the neural crest frozen in liquid nitrogen, sectioned at 15 M by cryostat (Microm) and marker p75 (Fig. 1D-F). degelatinized for 20 minutes at 42°C in PBS. Dorsal root ganglia were stained The distribution of Npn2 on neural crest cells made this receptor with 1:500 anti-TUJ1 (neuron specific class III -tubulin; Babco) followed a potential candidate for influencing neural crest formation and by 1:500 anti-mouse-Biotin (Jackson Immuno Research), and developed migration. To address the requirement for Npn2 during neural crest using the ABC-horseradish peroxidase kit (Vector Laboratories) and 0.1 development, we assessed the loss-of-function phenotype by mg/ml 3,3-diaminobenzidine tetrahydrochloride (DAB; Sigma) with 0.009% hydrogen peroxide according to the manufacturer’s instructions. Conditioned medium 293T cells were transfected with 24 g of AP-Sema3f (Giger et al., 2000) or AP empty vector using 45 l Lipofectamine 2000 (Invitrogen) per 10 cm dish according to the manufacturer’s instructions. Media (DMEM + 0.1% BSA + Pen Strep) was collected after 3 days and concentrated using a Centriplus YM-100 filter device (Millipore). Alkaline phosphatase activity was determined using AP Assay Reagent A (GenHunter Corporation) and the molarity of the collected protein calculated according to the manufacturer’s instructions. Sema3f spot preparation Aminopropyltriethoxysilane (2%; APTES; Sigma) was prepared in 95% ethanol and allowed to hydrolyze for 5 minutes in a fume hood. Thermanox (25 mm; Nunc) cover slips in a wafer basket (Fluoroware) were incubated in the 2% APTES for 10 minutes, and washed three times for 5 minutes with 95% ethanol. Coverslips were cured for 15 minutes at 100°C in a vacuum oven, immobilized onto 35 mm tissue culture plates with four spots of silicone vacuum grease, and UV sterilized in a tissue culture hood for 15 minutes. AP-Sema3f (75 nM) was preincubated at room temperature for 1 hour with 50 g/ml mouse anti-human placental alkaline phosphatase (Chemicon), 0.2 l drops were spotted manually in a grid on the coverslip, and the location of the spots was marked on the underside of the dish. After 1 hour at 37°C, the coverslips were washed three times with 4 ml of 1 Hank’s buffered saline solution (HBSS; Invitrogen). After all remaining traces of HBSS had been aspirated, 150-200 l of 125 g/ml fibronectin (BD Biosciences) was laid over the spots and incubated for 1.5 to 2 hours at 37°C. After aspirating the fibronectin, the coverslips were washed once with 4 ml of HBSS and stored overnight at 4°C in 2 ml DMEM-F12 (Invitrogen) + 1 mg/ml BSA. Fig. 1. Npn2 is expressed in migrating neural crest cells. Npn2 expression was revealed by whole-mount in situ hybridization of 31 Mouse neural tube culture somite stage chick (A) and E10.0 (28 somite) mouse (C) embryos. E9.5 embryos (14-24 somites) were isolated into ice cold HBSS. The region Neural crest cells were immunostained with anti-HNK-1 in longitudinal of the trunk containing the last 10 somites was dissected, trimming the sections (B) and anti-p75 in transverse sections (D-F) through the membranes lateral to the somites and removing the gut tube. Trunk pieces embryos shown in A and C. Plane of section is indicated on the whole- were incubated for 8 minutes at 37°C in room temperature 3 g/ml dispase mount view. (E,F) Higher magnification view of the regions boxed in D. made fresh in HBSS and 0.2 m filter sterilized. After rinsing several times Arrowheads indicate migrating neural crest cells. Identical results were with DMEM-F12 + 10% fetal bovine serum (Hyclone), neural tubes were obtained at E9.5. a, anterior; p, posterior; dm, dermomyotome; sc, isolated by trituration with a fire-polished pasteur pipette and plated on the sclerotome; nt, neural tube. Scale bars: 0.1 mm in A-D; 0.01 mm in E,F. spotted region of the coverslips in 1 ml of pre-warmed neural crest complete DEVELOPMENT Npn2/Sema3f in trunk neural crest migration RESEARCH ARTICLE 101 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. characterizing neural crest marker gene expression in Npn2-null mutant mice (Giger et al., 2000). In situ hybridization with probes for the neural crest markers Sox10, Foxd3 and Pax3 at E8-8.5 (4 to 12 somites) showed no obvious effects on the specification or generation of neural crest cells in wild-type, heterozygous or Npn2 mutant mice (data not shown). Profound defects were observed, however, on the pattern of neural crest migration in Npn2-null mice. In the trunk of wild- type (not shown) or Npn2 heterozygous (Fig. 2A) embryos, neural crest migration appeared as segmentally iterated blocks of Sox10 staining cells. In sections through these embryos, Sox10- positive neural crest cells were found only in the anterior-half sclerotome at levels adjacent to the intermediate region of the neural tube (Fig. 2B,C) (Serbedzija et al., 1990). In Npn2 mutant mice, however, neural crest cells migrated in a uniform sheet rather than in streams (Fig. 2D), and Sox10-positive cells were present throughout both anterior and posterior portions of the sclerotome (Fig. 2E,F). More ventrally, approaching the dorsal aorta where the neural crest-derived sympathetic ganglia will form, some segmental neural crest migration was still apparent in Npn2-null mice (Fig. 2D). Interestingly, motor axons, which normally are restricted to the anterior sclerotome, also project into both anterior and posterior sclerotome in Npn2 mutants, although ventral roots still form (data not shown). Together, these data suggest that Npn2 signaling is required for neural crest guidance events through dorsal and intermediate levels of the somite. Npn2 and Sema3f exhibit complementary Fig. 3. Sema3f expression is complementary to Npn2 and expression patterns required to pattern neural crest migration. Npn2 (A) and Sema3f Npn2 can bind to three different ligands: Sema3C, Sema3f and (B) are expressed in reciprocal patterns at E9.5, most notably in the vascular endothelial growth factor (Chen et al., 1997; Gluzman- hindbrain, branchial arches and trunk, where Npn2 is expressed in the anterior-half somite (C) and Sema3f in the posterior-half somite (D). At Poltorak et al., 2000). To determine which ligand was mediating E9.5, the segmental appearance of migrating trunk neural crest (E) that the patterning functions revealed by the Npn2 mutant, we next results from migration exclusively through the anterior-half sclerotome assessed the expression patterns of these molecules by in situ (F) is disrupted in Sema3f mutants (G) because neural crest cells hybridization and immunohistochemistry. Sema3c is first migrate throughout the sclerotome (H). (A-D) In situ hybridization for expressed in the somites around E10, and then only in the Npn2 (A,C) or Sema3f (B,D). (E-H) Neural crest was visualized by in situ dermomyotomal compartment (Adams et al., 1996). hybridization for Sox10. (E,G) Anterior is towards the left. Immunostaining for vascular endothelial growth factor was also (F,H) Longitudinal sections of embryos shown in E and G. i, isthmus; detected at low levels in the dermomyotome but not in the r2, rhombomere 2; r4, rhombomere 4; ov, otic vesicle; a, anterior; sclerotome at E9.5 (data not shown). By contrast, Sema3f was p, posterior; dm, dermomyotome; sc, sclerotome; nt, neural tube. Scale expressed in a pattern complementary to that of Npn2 (Fig. 3A,B), bars: 0.1 mm. DEVELOPMENT 102 RESEARCH ARTICLE Development 133 (1) Somite patterning is normal in Npn2 and Sema3f mutants Two alternate mechanisms could explain the trunk neural crest migration defects observed in the Npn2 (Fig. 2) and Sema3f (Fig. 3) mutant mice. The phenotype could reflect a requirement for signaling between the Sema3f repulsive ligand in the posterior somite and the Npn2 receptor on neural crest cells to guide neural crest migration. Alternatively, Npn2/Sema3f signaling between the anterior and posterior somite could be important for maintenance of anterior and/or posterior sclerotomal identity. Disrupting this signaling could affect somite polarity and thus the environment through which neural crest migrates, secondarily impacting the pattern of neural crest migration. For example, anteroposterior somite polarity is abolished in Delta1 mutant mice (deAngelis et al., 1997), and as a consequence, neural crest cells migrate aberrantly through the posterior sclerotome of these animals (DeBellard et al., 2002). To differentiate between these two possibilities, markers of anterior and posterior sclerotome were examined in wild-type, Npn2 mutant and Sema3f mutant mice to determine whether anterior and posterior somite identity was retained in the mutants. Sema3f was expressed in the posterior sclerotome of both wild-type (Fig. 4B) and Npn2 mutant mice (Fig. 4C). Ephrin B2, a ligand that repels migrating neural crest cells in vitro (Wang and Anderson, 1997), was also equivalently restricted to the posterior sclerotome of wild-type Fig. 4. Somite polarity is normal in Npn2 and Sema3f mutant (Fig. 4D) and Npn2 mutant embryos (Fig. 4E). Thus, two posteriorly mice. In E9.5 embryos, Npn2 is expressed in anterior sclerotome (A), expressed guidance molecules are appropriately localized in Npn2 and Sema3f in posterior sclerotome (B). (C) In Npn2 mutants, Sema3f is mutants. still posteriorly restricted. EphrinB2 is expressed in the posterior Somite polarity is established during segmentation of the somitic sclerotome (D), Tbx18 is expressed in the anterior sclerotome (F) and mesoderm, with anterior and posterior somite identity maintained Uncx4.1 is expressed in the posterior sclerotome (H). Expression of these three genes remains unchanged in Npn2 mutants (E,G,I). and promoted by two different transcription factors, Tbx18 (Bussen (J) Uncx4.1 also remains restricted to the posterior sclerotome of et al., 2004) and Uncx4.1 (Leitges et al., 2000; Mansouri et al., Sema3f mutants. Gene expression was visualized by in situ 2000). Tbx18 was restricted to the anterior sclerotome of wild-type hybridization and embryos sectioned longitudinally. a, anterior; (Fig. 4F) and Npn2 mutant embryos (Fig. 4G). Uncx4.1 was also p, posterior; dm, dermomyotome; sc, sclerotome; nt, neural tube. properly expressed in the posterior sclerotome of wild-type (Fig. 4H), Npn2 mutant (Fig. 4I) and Sema3f mutant mice (Fig. 4J). These results demonstrate that anterior and posterior sclerotomal character with Npn2 expression in the anterior somite (Fig. 3C) mirrored by is maintained in Npn2 and Sema3f mutants, suggesting that the Sema3f expression in the posterior somite (Fig. 3D). In sections, requirement for Npn2/Sema3f signaling is likely to reside in the Npn2 was clearly expressed in the anterior half of each sclerotome neural crest. (Fig. 4A), and Sema3fF in the posterior half (Fig. 4B). This expression pattern made Sema3f an ideal candidate for signaling Npn2 is required in the neural crest for Sema3f- through the Npn2 receptor during trunk neural crest migration. The mediated repulsion complementary distribution of Npn2 and Sema3f expression has To test the requirement for Npn2 in the neural crest directly, we also been observed at later stages of development (Giger et al., explanted wild-type and Npn2 mutant neural tubes, and cultured 2000; Giger et al., 1998). them on fibronectin-coated substrates containing spots of Sema3f. Wild-type neural crest cells avoided Sema3f (Fig. 5A), with the Sema3f mutants phenocopy Npn2-null mice majority of cells remaining at the spot border and only individual, We next assessed the requirement for Sema3f during neural crest rare cells migrating onto the Sema3f substrate, consistent with their development by examining Sox10 expression in Sema3f mutant mice behavior in vivo (Kasemeier-Kulesa et al., 2004). By contrast, Npn2 (Sahay et al., 2003). Strikingly, the Sema3f-null neural crest mutant neural crest cells migrated equally well on fibronectin with migration phenotype was identical to that observed in Npn2 mutants. or without Sema3f protein (Fig. 5B,C). This demonstrates that the Instead of segmentally arranged streams of neural crest cells in the Npn2 receptor on neural crest cells detects a Sema3f repulsive cue trunk (Fig. 3E), Sema3f nulls contained uniform sheets of migrating in the environment, and supports a cell-autonomous requirement for neural crest (Fig. 3G). In sections, alternating Sox10-positive and - Npn2 on the neural crest during trunk neural crest migration. negative regions were observed in the anterior- and posterior-half sclerotome, respectively, of wild-type mice (Fig. 3F), whereas Segmentally arranged dorsal root ganglia form in uniformly distributed Sox10-labeled cells were seen throughout the Npn2 mutant mice sclerotome of Sema3f mutants (Fig. 3H). Together, these results The segmental migration of neural crest through the somite is demonstrate that signaling between the receptor Npn2 and its ligand thought to prefigure the segmented organization of the neural crest- Sema3f is required to restrict neural crest migration to the anterior derived ganglia of the peripheral nervous system. This pattern somite. ensures that the ganglia and the vertebrae, which differentiate from DEVELOPMENT Npn2/Sema3f in trunk neural crest migration RESEARCH ARTICLE 103 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. the somites, will form in register with one another. For example, in each somite, neural crest cells in the anterior sclerotome coalesce to form the dorsal root ganglia. As a result, in Npn2 mutants, one might expect a continuous mass of dorsal root ganglia to form instead of individualized ganglia. This is the case when neural crest migrates non-segmentally through somites that are genetically (deAngelis et al., 1997; DeBellard et al., 2002) or surgically manipulated (Kalcheim and Teillet, 1989) to contain only anterior character. In Fig. 6. Non-segmentally migrating neural crest cells give rise to wild-type embryos at E10.5 and E11.5, the streams of Sox10- segmental dorsal root ganglia in Npn2 mutant mice. In E10.5 (A) expressing neural crest cells in the trunk condensed into ganglia in and E11.5 (C) wild-type embryos, in situ hybridization for Sox10 reveals an anterior to posterior progression (Fig. 6A,C). Strikingly, neural segmental streams of migrating neural crest cells in the posterior trunk, crest cells in Npn2 mutant mice also coalesced into recognizable and condensed dorsal root ganglia anteriorly. (B,D) In Npn2 mutant ganglia. At E10.5, neural crest cells were still distributed throughout embryos, in the absence of segmental neural crest migration the somites, but became excluded from the somite boundaries (Fig. posteriorly, individualized dorsal root ganglia segregate anteriorly. Black arrowheads indicate condensing dorsal root ganglia at the same axial 6B). By E11.5, individualized ganglia appeared to have sorted out level in all panels. Scale bars: 0.5 mm. (E,F) In sections of Npn2 from the sheet of migrating neural crest in the somite (Fig. 6D, heterozygous (E) and mutant (F) E11.5 embryos stained with anti-TUJ1, arrowheads mark the same axial levels in all panels). In longitudinal dorsal root ganglia had a similar appearance but were less well sections at E11.5, TUJ1 immunoreactivity confirmed the apparent separated in the mutant. Scale bar: 0.1 mm. segmentation, with space between each Npn2 mutant dorsal root ganglion (Fig. 6F). Although morphologically similar to those in wild-type embryos (Fig. 6E), the mutant ganglia were not as well separated, suggesting that the process of gangliogenesis occurred but Murine Npn2 is expressed by neural crest cells (Fig. 1D-F) as well was somewhat compromised. The sympathetic ganglia, which form as cells of the anterior sclerotome (Fig. 4A). Therefore, the in a segmental pattern ventral to the somites, are normal in Npn2 migration defects in Npn2 mutants could reflect a requirement for mutants (Giger et al., 2000). Npn2 in one or both cell types. One possibility is that Npn2 receptors on neural crest cells detect a Sema3f repulsive cue in posterior DISCUSSION sclerotome, leading to anterior-only migration. Alternatively, Npn2 We have examined the importance of the receptor Npn2 and its receptors on anterior sclerotomal cells may interact with the Sema3f repulsive ligand Sema3f during neural crest development in the ligand secreted by cells of the adjacent posterior sclerotome to affect trunk. We demonstrate that Npn2 is expressed in migrating neural the environment through which neural crest cells migrate, thus crest cells, and that Npn2/Sema3f signaling is required for segmental influencing neural crest migration in a non-cell-autonomous manner. neural crest migration but not for somite patterning. Surprisingly, we The latter mechanism seems unlikely for several reasons. First and found that segmental migration was not essential for the formation foremost, in contrast to wild-type cells, Npn2 mutant neural crest of individualized dorsal root ganglia. cells do not avoid Sema3f in culture. This indicates a cell autonomous requirement for Npn2 in the neural crest. Second, as Npn2/Sema3f signaling patterns neural crest anteroposterior polarity of the somites is indistinguishable in wild- migration in the trunk type, Npn2 and Sema3f mutant embryos (Fig. 4), Npn2/Sema3f In Npn2 and Sema3f mutant mice, trunk neural crest cells migrate signaling does not appear to be required in this process. In support through both the anterior and posterior sclerotome, rather than of this conclusion, neural crest cells are present all along the exclusively through the anterior-half sclerotome as in wild-type anteroposterior axis in the Npn2 (Fig. 2E,F) and Sema3f mutants mice. This demonstrates that signaling between the receptor Npn2 (Fig. 3H), even at somite borders. This contrasts with the Delta1 and its ligand Sema3f is required to restrict neural crest migration to mutant mouse, in which somite polarity is lost (deAngelis et al., the anterior somite. 1997), where neural crest migration has a pseudo-segmental DEVELOPMENT 104 RESEARCH ARTICLE Development 133 (1) appearance, with migrating neural crest cells present throughout the A second possibility is that dorsal root ganglia form in the absence sclerotome but avoiding somite boundaries (DeBellard et al., 2002). of segmental neural crest migration simply because neurons tend to This comparison suggests that, unlike Delta1, the Npn2 defect is not aggregate (M. Bronner-Fraser, unpublished). In support of this in the somites themselves. Third, as Npn2 is expressed on anterior stochastic mechanism, when normal somites are surgically replaced sclerotomal cells, if Npn2/Sema3f were involved in somite with multiple anterior or posterior somite halves, a giant mass of patterning, loss of signaling through this receptor would most ganglia forms that exhibits a pseudo-segmental appearance, with probably posteriorize the sclerotome. A posteriorized somite alternating thick and thin regions at random intervals within the giant impedes neural crest migration, leading to reduced numbers of ganglion (Kalcheim and Teillet, 1989). That they are fused, however, migratory neural crest cells and their derivatives (Bussen et al., 2004; argues that a combination of these two mechanisms is normally at Kalcheim and Teillet, 1989). However, this is not the case in Npn2- play. null mice. For these reasons, we favor the idea that Npn2 is required Finally, it is also possible that the physical structure of the somite in the neural crest. itself can impose segmentation during gangliogenesis. In addition to What is the purpose, then, of Npn2 expression in both the neural regionally restricted molecular cues, such as Sema3f expression crest and the sclerotome through which it migrates? Interestingly, posteriorly, there are embryological boundaries and differences like Npn2, Npn1 (Eickholt et al., 1999) and Ephb3 receptors (Krull within the sclerotome. These include, most notably, the intersomitic et al., 1997), both of which have been postulated to play a role in space, as well as von Ebner’s fissure between anterior and posterior patterning trunk neural crest migration, are also distributed on both sclerotome, and the various subdomains within the sclerotome neural crest and anterior sclerotomal cells. One intriguing possibility (reviewed by Christ et al., 2004). Anterior sclerotome is less cell is that these receptors do not play a signaling role in the somite, but dense than posterior sclerotome (Christ et al., 2004), is mitogenic rather serve as a sink to bind up any repulsive ligand diffusing from for dorsal root ganglia (Goldstein et al., 1990) and will undergo the posterior sclerotome, thus ensuring a sharp boundary such that apoptosis in the absence of neural crest cells (see Christ et al., 2004). the anterior sclerotome is devoid of the repulsive cue. All of these segmentally restricted differences could have a This is the first report of a single receptor/ligand pair that is morphological impact during gangliogenesis. In the case of the Npn2 absolutely required to pattern trunk neural crest migration. The mutants, the uniform sheet of migrating neural crest cells appears to molecular basis for segmental neural crest migration has segment into individual dorsal root ganglia at the somite border (Fig. preoccupied this field since the phenomenon was first observed. 6). Interestingly, when chick embryos are surgically modified to Many different cell adhesion molecules, extracellular matrix contain only anterior sclerotome, in other words have no somite molecules and receptor/ligand pairs have been identified that are boundary, neural crest migrates non-segmentally and dorsal root expressed in anterior or posterior sclerotome or in the neural crest, ganglia are fused (Kalcheim and Teillet, 1989). However, dorsal root and in some cases they have been shown to be sufficient to direct ganglia are also fused in Uncx4.1 and Tbx18 mutants, where neural crest migration (Kuan et al., 2004). But in no case has a anteroposterior somite polarity is abolished but physical somites still requirement for any molecule been previously demonstrated in the form (Bussen et al., 2004; Leitges et al., 2000; Mansouri et al., embryo. Other signals, such as Eph receptor/ephrin ligand 2000). Together, these data suggest that somite polarity creates interactions, might fine tune neural crest migration, or in the case of positional information at the somite boundary that impacts upon the Npn1/Sema3A, be involved in later steps in the process. However, segmentation of the peripheral nervous system. Migrating neural Npn2/Sema3f signaling is clearly the key determinant patterning crest cells normally maintain filopodial contact across the posterior anterior-only migration through the sclerotome. somite and can even cross over between adjacent streams (Kasemeier-Kulesa et al., 2004), thus the somite boundary could Segmental neural crest migration may not be normally curtail this movement as dorsal root ganglia condense. required for segmental dorsal root ganglion The sympathetic ganglia also are not dependent upon the pattern formation of neural crest migration for their segmented organization. By The requirement for Npn2/Sema3f signaling in neural crest imaging actively migrating neural crest cells, Kasemeier-Kulesa and migration has uncovered an additional, previously unrecognized colleagues (Kasemeier-Kulesa et al., 2004) showed that, once they process that results in dorsal root ganglion segmentation irrespective have passed through the somites, neural crest cells no longer maintain of the neural crest migration pattern. Despite the fact that neural crest their segmental position and can migrate as far as two segments cells migrate through the anterior and posterior sclerotome of Npn2 anteriorly or posteriorly. The mechanisms that ultimately result in the mutant mice, segmentally arranged dorsal root ganglia still form. aggregation of these cells into individualized sympathetic ganglia are This result suggests that segmental neural crest migration and likely to be similar to those we propose for the formation of subsequent sequestration of ganglia are separable events. One metameric dorsal root ganglia in Npn2 mutants. Interestingly, Npn1 explanation is that there may be independent signals restricting and Sema3A are required for localization and condensation of neural crest migration and the pattern of ganglion aggregation. For sympathetic precursors as well (Kawasaki et al., 2002). example, either a cell-adhesive, ‘sorting’ signal or a repulsive cue in The overriding message is that the segmental migration of neural the posterior sclerotome could promote aggregation within the crest cells through the somites itself is not requisite for the creation anterior sclerotome, irrespective of the starting location of the neural of a segmented peripheral nervous system, despite what has been crest cells within the sclerotome. In favor of this possibility, fused assumed for 20 years (reviewed by Kuan et al., 2004). Although the dorsal root ganglia form when anteroposterior somite polarity is dorsal root ganglia eventually segment in the Npn2 mutants, they are abolished by either surgical or genetic manipulation (Bussen et al., more closely spaced than normal. Thus, the pattern of neural crest 2004; Kalcheim and Teillet, 1989; Leitges et al., 2000; Mansouri et migration is important, but not essential for the formation of al., 2000). This indicates that anteroposteriorly patterned signals in segmented dorsal root ganglia. This may not be surprising given the the somite are required for segmental formation of dorsal root regulative nature of vertebrate embryos, which may have ‘back-up’ ganglia. Candidates for such signals include F-spondin (Debby- mechanisms for formation of important organ systems in the event Brafman et al., 1999) and Npn1 (Kitsukawa et al., 1997). that primary mechanisms are perturbed. DEVELOPMENT Npn2/Sema3f in trunk neural crest migration RESEARCH ARTICLE 105 Christ, B., Huang, R. and Scaal, M. (2004). Formation and differentiation of the Functional validation of the neural crest gene avian sclerotome. Anat. Embryol. 208, 333-350. expression profile Cloutier, J.-F., Giger, R., Koentges, G., Dulac, C., Kolodkin, A. and Ginty, D. We originally identified Npn2a1 in a screen for genes upregulated in (2002). Neuropilin-2 mediates axonal fasciculation, zonal segregation, but not response to neural crest induction (Gammill and Bronner-Fraser, axonal convergence, of primary accessory olfactory neurons. Neuron 33, 877- 2002). Our current analysis of Npn2 function has several Coles, E., Gammill, L., Miner, J. and Bronner-Fraser, M. (2005). Abnormalities implications. First of all, the importance of Npn2 for neural crest in neural crest cell migration in laminin alpha5 mutant mice. Dev. Biol. (in press) migration validates our neural crest gene expression profile and Davy, A., Aubin, J. and Soriano, P. (2004). Ephrin-B1 forward and reverse signaling are required during mouse development. Genes Dev. 18, 572-583. demonstrates that our collection of genes contains true regulators of deAngelis, M., McIntyre, J. and Gossler, A. (1997). Maintenance of somite neural crest development. This conclusion is supported by the borders in mice requires the Delta homologue Dll1. Nature 386, 717-721. demonstration that Laminin-5, another gene identified in our Debby-Brafman, A., Burstyn-Cohen, T., Klar, A. and Kalchiem, C. (1999). F- Spondin, expressed in somite regions avoided by neural crest cells, mediates screen, is also important for proper emigration of neural crest cells inhibition of distinct somite domains to neural crest migration. Neuron 22, 475- (Coles et al., 2005). In addition, although we screened for genes expressed in DeBellard, M., Ching, W., Gossler, A. and Bronner-Fraser, M. (2002). Disruption of segmental neural crest migration and ephrin expression in Delta-1 premigratory neural crest (Gammill and Bronner-Fraser, 2002), null mice. Dev. Biol. 249, 121-130. Npn2 is required for neural crest migration and apparently not for Eickholt, B., Mackenzie, S., Graham, A., Walsh, F. and Doherty, P. (1999). specification, as no differences were noted in the expression of early Evidence for collapsin-1 functioning in the control of neural crest migration in neural crest markers Sox10, Pax3 and FoxD3 in the neural folds and both trunk and hindbrain regions. Development 126, 2181-2189. Gammill, L. S. and Bronner-Fraser, M. (2002). Genomic analysis of neural crest dorsal neural tube of the mouse. We cannot, however, rule out the induction. Development 129, 5731-5741. possibility of an early role for chick Npn2 in neural crest Giger, R., Urquhart, E., Gillespie, S., Levengood, D., Ginty, D. and Kolodkin, specification, as it is expressed earlier and at higher levels in this A. (1998). Neuropilin-2 is a receptor for semaphorin IV: insight into the structural basis of receptor function and specificity. Neuron 21, 1079-1092. organism. Regardless, genes crucial for migration are clearly Giger, R., Cloutier, J.-F., Sahay, A., Prinjha, R., Levengood, D., Moore, S., expressed in premigratory neural crest as a consequence of neural Pickering, S., Simmons, D., Rastan, S., Walsh, F. et al. (2000). Neuropilin-2 is crest induction. This supports our model that early neural crest required in vivo for selective axon guidance responses to secreted semaphorins. Neuron 25, 29-41. development entails a sequential activation of migratory potential, Gluzman-Poltorak, Z., Cohen, T., Herzog, Y. and Neufeld, G. (2000). with a signal to migrate activating this potential in a subset of Neuropilin-2 and neuropilin-1 are receptors for the 165-amino acid form of premigratory neural crest cells (Gammill and Bronner-Fraser, 2002). vascular endothelial growht factor (VEGF) and of placenta growth factor-2, but Further analysis of our neural crest gene collection promises to only neuropilin-2 functions as a receptor for the 145-amino acid form of VEGF. J. Biol. Chem. 275, 18040-18045. reveal the roles of additional genes in this process. Goldstein, R., Teillet, M.-A. and Kalcheim, C. (1990). The microenvironment created by grafting rostral half-somites is mitogenics for neural crest cells. PNAS We are indebted to David Ginty for providing the Npn2 knockout mice and 87, 4476-4480. Sema3f mutant embryos, as well as helpful comments throughout the course Hamburger, V. and Hamilton, H. (1992). A series of normal stages in the of this work. Special thanks to Vivian Lee and York Marahrens for comments development of the chick embryo (originally published in 1951). Dev. Dyn. 195, on the manuscript, and to Joaquin Gutierrez for exceptional animal care. We 231-272. are grateful to Drs David Anderson, Peter Gruss, Ahmed Mansouri, Andreas Kalcheim, C. and Teillet, M.-A. (1989). Consequences of somite manipulation Kispert, Patricia Labosky, Andreas Püschel, Kirsten Kuhlbrodt and Michael on the pattern of the dorsal root ganglion development. Development 106, 85- Wegner for kind gifts of plasmids, and to Lou Reichardt for contributing the 93. p75 antibody. Many thanks to Vivian Lee for tips on immunostaining, Kasemeier-Kulesa, J., Kulesa, P. and Lefcort, F. (2004). Imaging neural crest cell dynamics during formation of dorsal root ganglia and sympathetic ganglia. 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Journal

Development – The Company of Biologists

Published: Jan 1, 2006

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