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First published online 13 September 2006
doi: 10.1242/dev.02583

Development 133, 3983-3992 (2006)
Published by The Company of Biologists 2006
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Semaphorin 3d promotes cell proliferation and neural crest cell development downstream of TCF in the zebrafish hindbrain

Jason D. Berndt and Mary C. Halloran*

Departments of Zoology and Anatomy and Neuroscience Training Program, University of Wisconsin, Madison, WI 53706, USA.


Figure 1
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  		Fig. 1. Sema3d mRNA is expressed in NCCs and morpholino knockdown of Sema3d disrupts NCC derivatives without affecting NCC induction. (A-C) In situ hybridization for sema3d at 15 hpf (A,B) and 22 hpf (C). (D-F) Alcian Blue labeling of pharyngeal cartilage at 5 dpf; (G-I) DIC imaging of melanophores in 2 dpf in CONMO (D,G), 3DMO (E,H) and 3DI4E5MO (F,I) injected embryos. Arrow in G indicates ventral horn of melanophores. (J) RT-PCR on mRNA extracted from embryos injected with Sema3d splice blocking morpholinos. (K-N) In situ hybridization for crestin at 13 hpf (K,L) and hoxb2a at 18 hpf (M,N) in CONMO (K,M) and 3DMO (L,N) injected embryos. (A,K-N) Dorsal views, anterior is leftwards. (B) Transverse section through rhombomere 4. (C,G-I) Lateral views, anterior is leftwards. (D-F) Ventral views, anterior is leftwards. r2-r5, rhombomeres 2-5; oto, otocyst; m, hs, ch and gill, Meckel's, hyosymplectic, ceratohyal and gill cartilages, respectively; bp, base pairs. Scale bars: 40 µm for A,C,M,N; 20 µm for B; 100 µm for D-I; 80 µm for K,L.

 

Figure 2
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  		Fig. 2. Sema3d knockdown reduces migratory NCC markers without affecting EMT. (A,B) Uncaged FITC-dextran (green) and TOPRO3 labeling of nuclei (blue) in the hindbrain at 17 hpf in CONMO (A) and 3DMO (B) injected embryos. (C-E) Double in situ hybridization for crestin (green) and dlx2 (red); expression is reduced in 3DMO (D) and 3DI4E5MO (E) injected embryos compared with CONMO (C). (F) Quantification of the area of labeling of crestin and dlx2 in the r4 migratory stream, indicated by white outline in C. *P<10-6, t-test. (A-E) Dorsal views, anterior is leftwards. oto, otocyst. Scale bars: 40 µm.

 

Figure 3
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  		Fig. 3. Sema3d knockdown inhibits cell cycle in the hindbrain. (A-D) Immunodetection in the hindbrain at 17 hpf of BrdU (A,B) and PH3 (C,D) in CONMO (A,C) and 3DMO (B,D) injected embryos. (E,F) Quantification of the number of BrdU (E) and PH3 (F) labeled nuclei in the neuroepithelium of rhombomeres 3-5. Numbers in parentheses indicate n. *P<10-4 compared with CONMO, t-test. (A-D) Dorsal views, anterior is leftwards. Scale bar: 40 µm for A-D.

 

Figure 4
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  		Fig. 4. Sema3d knockdown affects the expression of cell cycle genes. (A-I) In situ hybridization in the hindbrain at 17hpf for cyclin D1 (A-C), cyclin A2 (D-F) and p57kip2 (G-I) in CONMO (A,D,G), 3DMO (B,E,H), and 3DI4E5MO (C,F,I) injected embryos. oto, otocyst. (A-I) Dorsal views, anterior is leftwards. Scale bars: 40 µm for A-I.

 

Figure 5
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  		Fig. 5. {Delta}TCFgfp disrupts cell cycle and crestin expression and eliminates cyclin D1 and sema3d expression. (A-I) Immunodetection of BrdU (A,B,I) and in situ hybridization for crestin (C,D), cyclinD1 (E,F), sema3d (G,H) in the hindbrain at 17 hpf of heat-shocked wild-type siblings (A,C,E,G), {Delta}TCFgfp transgenic (B,D,F,H), and {Delta}TCFgfp + Sema3dmyc double-transgenic (I) embryos. (J) Quantification of the number of BrdU-labeled nuclei in the neuroepithelium of rhombomeres 3-5. Numbers in parentheses indicate n. *P<10-13 versus wild-type sibling, #P<10-4 versus {Delta}TCFgfp, t-test. (A-I) Dorsal views, anterior is leftwards. oto, otocyst. Scale bar: 40 µm for A-I.

 

Figure 6
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  		Fig. 6. Schematic models of potential interactions between Wnt/TCF and Sema3d. (A) A linear model with Sema3d signaling downstream of Wnt/TCF. (B) An alternative model showing the interaction of parallel pathways by means of an unidentified inhibitor, X.

 

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© The Company of Biologists Ltd 2006