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First published online 15 April 2009
doi: 10.1242/dev.028373

Development 136, 1717-1726 (2009)
Published by The Company of Biologists 2009
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Role of noggin as an upstream signal in the lack of neuronal derivatives found in the avian caudal-most neural crest

Liliana Osório1,2,3,*,{dagger}, Marie-Aimée Teillet1,2 and Martin Catala1,2

1 UPMC Univ Paris 06, UMR 7622, Laboratoire de Biologie du Développement, F-75005, Paris, France.
2 CNRS, UMR 7622, Laboratoire de Biologie du Développement, F-75005, Paris, France.
3 Life and Health Sciences Research Institute, School of Health Sciences, University of Minho, 4710-057 Braga, Portugal.


Figure 1
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  		Fig. 1. Generation of caudal-most NCCs in the chick embryo. (A-E) Whole-mount detection of Cad7 (A), Sox10 (C) and HNK1 (E) in E4/HH24 embryos, showing the absence of NCCs in the caudal-most region posterior to somite 47 (s47). At E5/HH26, whole-mount preparations and cross-sections (at positions indicated) were hybridized for Cad7 (B,B') and Sox10 (D,D'), revealing the presence of a few NCCs. (F) Cross-section immunolabeled with HNK1, showing NCCs located in the MSA (arrowhead). dm, dermomyotome; scl, sclerotome. Scale bars: 50 µm.

 

Figure 2
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  		Fig. 2. NCC specification in the caudal-most NT in E4/HH24 chick embryos. Whole-mount ISH to detect Pax3 in dorsal (A) and lateral (A') views and cross-section (A''). Pax7 immunodetection in whole mount (B,B') and cross-section (B''). Whole-mount ISH for Msx1 (C,C'), Msx2 (D,D'), FoxD3 (E,E'), Snail2 (F,F') and Sox9 (G,G'), and cross-sections caudally to the level of somite 47 (s47) (C'',D'',E'',F'',G''). All transcription factors, except for Msx1, are expressed in the dorsal region of the caudal-most NT at E4/HH24. Scale bars: 50 µm.

 

Figure 3
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  		Fig. 3. Bmp4 and Wnt1 signaling in the caudal-most NT of E4/HH24 embryos. Whole-mount ISH to detect Bmp4 (A), Nog (B), Wnt1 (C) and Wnt3a (D) and cross-sections (A',B',C',D'). Bmp4 and its inhibitor Nog are both detected all along the dorsal caudal-most NT, facing somites posterior to somite 47 (s47) that are already differentiating (A-B'). (C,C') Wnt1 is not expressed, in contrast to Wnt3a (D,D'). Scale bars: 50 µm.

 

Figure 4
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  		Fig. 4. Cell apoptosis in the caudal-most NT. (A-F) NBS staining and TUNEL assay. (A,C,E,F) NBS staining in whole embryos at E3/HH18 (A), E4/HH24 (C) and E5/HH26 (E,F). Note the intense level of apoptosis detected caudally to somite 47 (s47) at E4/HH24. (B,D) TUNEL assay on cryosections at E3/HH18 (B) and E4/HH24 (D) at equivalent regions. Apoptosis affects the dorsal moiety of the caudal-most NT (nt) and the overlaying ectoderm (ect) at E4/HH24. Scale bars: 50 µm.

 

Figure 5
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  		Fig. 5. Rostral transplantation of quail caudal-most somites or NT into E2/HH11-12 chick embryos. (A) Schematic representation of the heterotopic transplantations of caudal-most somites (A') and caudal-most NT (A'') and respective results (B-H' and I-L'). (B,C,E) Sox10 and QCPN in whole chimeras 16 (B), 24 (C) and 48 (E) hpt. Sox10+ cells are located dorsal to and close to the NT at the level of the graft (B',C',E'). (D) Chimera labeled for Wnt1 and QCPN 24 hpt and cross-section in the graft region (D'). (F) Immunohistochemistry for HNK1 and QCPN, showing sensory ganglia facing the transplanted caudal-most somites at 48 hpt. (G) These ganglia present Isl1/2+ postmitotic neurons. (H) Whole-mount ISH to detect Uncx4.1 and parasagittal section (H') revealing two distinct compartments in the caudal-most somites. (I) Sox10 and QCPN in whole chimera 24 hpt of quail caudal-most NT. No quail NCCs are detected at the level of the graft (I'). Forty-eight hpt, double labeled HNK1-QCPN quail NCCs are detected dorsal to the grafted NT (J,J'). (K) Some quail NCCs are WRS+ (melanocyte progenitor cells; arrowheads). (L) Whole-mount ISH to detect Wnt1 and QCPN 24 hpt and cross-section at graft level (L'); Wnt1 is not restored. Scale bars: 50 µm.

 

Figure 6
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  		Fig. 6. Forced Nog expression in the trunk NT of E2/HH11-12 chick embryos. Whole-mount GFP expression (A) and ISH for Sox10 (B) 8 hpe. Cross-sections showing NCCs located dorsally to the NT at dissociating (B') and epithelial (B'') somite levels. Cross-section immunolabeled with HNK1 (C) and ISH for mNog (C') 8 hpe. Cryosection labeled with HNK1 and GFP 24 hpe (D). Whole mount and cross-section labeled with HNK1 48 hpe (E,E'), with control cross-section (F). (G) Whole-mount Sox10 expression. ISH to detect Cad6B 12 hpe (H) and control (I) cross-sections. Immunodetection of N-Cad 24 hpe (J) and control (K) cross-sections. ISH to detect Cad7 in whole mount (L) and cross-section (L') 48 hpe and control cross-section (M). Msx1 (N), Wnt1 (O) and FoxD3 (P) 24 hpe, all absent in the electroporated NT. Ngn1 in whole mount (Q) and cross-section (Q') 24 hpe and control cross-section (R). (S,T) Immunodetection of TuJ1 and Isl1/2 in adjacent cross-sections 48 hpe. (U) TUNEL assay on cryosection shows massive apoptosis in the electroporated NT and NCCs 24 hpe. Scale bars: 50 µm.

 

Figure 7
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  		Fig. 7. Increased Bmp4 and Wnt1 signaling in the dorsal caudal-most NT of E4/HH24 chick embryos. Twenty-four hpe of Bmp4 (A-D'), miRNA-Nog (E-I') and Wnt1 (J-N'). (A,B,E,F,J,K) Cryosections labeled with HNK1 and GFP, showing numerous NCCs close to the dorsal caudal-most NT. Whole-mount ISH for Sox10 (C,G,L) and cross-sections (C',G',L'). Wnt1 expression is restored in the dorsal aspect of the caudal-most NT after Bmp4 (D,D') or miRNA-Nog (H,H') electroporation. Nog is not detected in the caudal-most NT electroporated with miRNA-Nog (I,I'). Electroporation of Wnt1 in the dorsal caudal-most NT partially restores Msx1 expression (M,M') and increases FoxD3 expression (N,N'). Forty-eight hpe of miRNA-Nog (O-Q'). ISH to detect Sox10 (O,O'), Ngn1 (P,P') and Ngn2 (Q,Q'). s47, somite 47. Scale bars: 50 µm.

 

Figure 8
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  		Fig. 8. A model for the lack of neuronal NCC derivatives in the caudal-most region of the chick embryo. Caudal-most NCCs are characterized by their scarcity and delayed migration. Maintenance of Nog expression impairs Bmp4 signaling, thus leading to the absence of its targets Msx1 and Wnt1. This impairment is concomitant with a massive dorsal NT apoptosis at E4/HH24 that culminates in the elimination of the early migrating NCC population that would contribute to neuronal derivatives. By contrast, later migrating NCCs mainly constituting melanocytic progenitors specifically require Wnt3a. Delamination of these cells involves a mechanism other than one that depends on Bmp4-Wnt1 signaling.

 

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© The Company of Biologists Ltd 2009