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Bimodal functions of Notch-mediated signaling are involved in neural crest formation during avian ectoderm development

¹ Department of Developmental Neurobiology, Graduate School of Medicine, Tohoku University, Sendai, Miyagi 980-8575, Japan
² PRESTO, Japan Science and Technology Corporation, Japan

View larger version (41K): [in a new window]   Fig. 1. Electroporation of expression plasmids into ectoderm of cultured quail embryos. (A) Electroporation and whole embryo culture. Plasmid DNA is placed on the dorsal surface of the stage 5-6 quail embryo adhering to the collagen-coated membrane (1). Electroporation is performed in the chamber filled with Hanks BSS (2). The embryo is cultured on vitelline membrane in culture medium (3). (B) A demonstrative picture showing how electroporation is performed. As a negative electrode, L-shaped tungsten needle is used. The tip of the electrode is placed over the right side of the ectoderm. The square-shaped positive electrode is fixed underneath the embryo. (C,D) An example of cultured embryo. Electroporation was performed at stage 6, and cultured until stage 10 (approximately 12 hours). Expression of GFP is visible in the right side of the embryo. BF, bright field. (E) Correlation of schedule of electroporation (EP), culture length, Hamburger and Hamilton stages and somite number.

View larger version (75K): [in a new window]   Fig. 2. Notch1 and Delta1 are expressed in the neural fold of quail embryos during neural crest formation. (A-C) Slug expression in the premigratory neural crest cells (arrowheads). At the one-somite stage, Slug expression is barely detectable in whole-mount preparation (but see Fig. 3E). (D-F) Pdgfr expression in the premigratory cranial crest cells (arrowheads). An arrow in E indicates somite expression. (G-I) Delta1 expression in the epidermal ectoderm (arrowheads). Arrow in G indicates strong expression in presomitic mesoderm. Asterisk in I indicates expression in the neural plate. (J-L) Broad expression of Notch1. Arrowheads in J indicate the boundary of the neural plate and the epidermal ectoderm.

View larger version (76K): [in a new window]   Fig. 3. Delta1 expression is restricted to the epidermal side of the ectoderm. Neighboring sections of stage 7 (one-somite stage) and stage 9 (five-somite stage) were hybridized with Notch1, Delta1, Slug and Sox2 probes. Notch1 is expressed in broadly in the ectoderm (A,B). Delta1 mRNA expression (C,D) is highly localized in the epidermal ectoderm, and shows a sharp boundary juxtaposed to the neural crest domain, marked by Slug expression (E,F). Sox2 is expressed in the neural plate, and downregulated in the neural crest domain (G,H).

View larger version (104K): [in a new window]   Fig. 4. Forced activation of Notch signaling in the ectoderm represses expression of head-fold markers. Electroporation of GFP (A,B,G,H), GFP+CNIC (C,D) and GFP+CNICC89 (E,F,I,J) was performed on stage 6 embryos, and the embryos were cultured for 7 hours until stage 9. Transfected areas are visualized by the fluorescence of GFP overlaid on bright field image of electroporated live embryos (A,C,E,G,I). Slug and Pdgfr expression in the head fold (B,D,F,H,J) are shown at higher magnification, corresponding to the boxed areas of A,C,E,G,I, respectively. Forced activation of Notch signaling by CNIC and CNICC89 (cytoplasmic domain of chicken Notch1) reduced the expression of Slug and Pdgfr in the head fold (arrowheads in D,F,H).

View larger version (70K): [in a new window]   Fig. 5. CNICC89-transfected ectoderm cells fail to express Slug and HNK-1, and do not delaminate from the ectoderm. Electroporation of GFP (I-L) or CNICC89 (A-H and M-P) was performed on stage 6 embryo, and the embryos were cultured for 7 hours (A-F), or 24 hours (G-P). (A-F) A CNICC89-transfected neural fold (ipsilateral, D-F) and an untransfected neural fold (contralateral, A-C) of the same embryo, stained with anti-Flag (FITC, green), anti-Slug (cy3, red) and DAPI (blue). In the contralateral neural fold, many Slug-positive premigratory neural crest nuclei are observed (A,C). By contrast, in the ipsilateral side, the number of Slug-positive nuclei is reduced (D). Many Flag-positive nuclei of CNICC89-transfected cells can be identified (E), and most of the Flag-positive nuclei do not possess Slug-immunoreactivity (D-F, arrowheads). (G,H) Whole-mount HNK-1 staining of a CNICC89-transfected embryo. Compared with the contralateral side (G), migrating neural crest cells are severely reduced in the CNICC89-transfected side (H, black arrows in midbrain-forebrain level and black arrowheads in branchial arches). Strong HNK-1 staining indicated by white asterisks is in the axial mesoderm. ba2, second branchial arch; ba3, third branchial arch; e, eye; op, otic placode. (I-L) GFP-transfected HNK-1-positive neural crest cells migrate laterally underneath the epidermis (I,J, arrowheads), and colonize the branchial arch (K,L, arrowheads). (M,N) CNICC89-transfected cells failed to delaminate from the ectoderm. Note that CNICC89-transfected neural fold sometimes fails to close. (O,P) In the contralateral side of the CNICC89-transfected embryo, HNK-1-positive neural crest cells normally colonize the branchial arch (P).

View larger version (95K): [in a new window]   Fig. 6. Reduced Notch signaling results in the decrease of Slug expression. Electroporation of GFP (A,B), GFP+Delta1stu (dominant-negative Delta1, C,D), GFP+chick Numb (E,F) was performed on stage 5 embryo, and the embryos were cultured for 9 hours until stage 9. Transfected areas are visualized by the fluorescence of GFP overlaid on bright field image of electroporated live embryos (A,C,E). Slug expression in the head fold (B,D,F) is shown at higher magnification, corresponding to the boxed areas of A,C,E, respectively. Reduction of Notch signaling results in the decrease of the Slug expression in the head fold (arrowheads in D,F).

View larger version (94K): [in a new window]   Fig. 7. Manipulation of Notch signaling does not affect the differentiation of the neural plate or the epidermis. Electroporation of GFP (A-C,J-L), GFP+CNICC89 (D-F,M-O), GFP+Delta1stu (G-I,P-R), was performed on stage 5 embryo, and the embryos were cultured for 4 hours until stage 7. Transfected areas are visualized by the fluorescence of GFP overlaid on bright field image of electroporated live embryos (A,D,G,J,M,P). Expression of an epidermis marker Keratin19 and a neural plate marker Sox2 in these embryos are shown as whole-mount preparations (B,E,H,K,N,Q) and as sections (C,F,I,L,O,R).

View larger version (107K): [in a new window]   Fig. 8. Manipulation of Notch signaling in the ectoderm downregulates the epidermal expression of Bmp4. Electroporation of GFP (A,B), GFP+CNICC89 (C,D) and GFP+Delta1stu (E,F) was performed on stage 5 embryos, and the embryos were cultured for 4 hours until stage 7. Transfected areas are visualized by the fluorescence of GFP overlaid on bright field image of electroporated live embryos (A,C,E). Both forced activation and inactivation of Notch signaling reduce Bmp4 expression in the epidermal ectoderm (arrowheads in D,F).

View larger version (100K): [in a new window]   Fig. 9. Exogenous Bmp4 rescues the reduction of Slug expression by Delta1stu, but not the reduction by CNICC89. Electroporation of GFP (A,B), GFP+Noggin (C,D), GFP+Bmp4 (E,F), GFP+Bmp4 +CNICC89 (G,H), GFP+Bmp4+Delta1stu (I,J), was performed on stage 5 embryo, and the embryos were cultured for 9 hours until stage 9. Transfected areas are visualized by the fluorescence of GFP overlaid on bright field image of electroporated live embryos (A,C,E,G,I). Misexpression of Noggin reduces Slug expression (D, see also the section in the inset, arrowhead), while overexpression of Bmp4 results in the expansion of Slug domain (F, see also the section in the inset, arrowheads). The loss of Slug expression by CNICC89 cannot be rescued by the co-expression of Bmp4 (H, see also the section in the inset, arrowhead). The decrease of Slug expression by Delta1stu (see Fig. 6D) is rescued by the co-expression of Bmp4 (J, see also the section in the inset).

View larger version (96K): [in a new window]   Fig. 10. Notch activation is necessary and sufficient for Hairy2 expression in the ectoderm, but Su(H)/RBP-J activity is not required. Electroporation of GFP + Su(H)DBM was performed on stage 5 embryos, and the embryos were cultured for 9 hours until stage 9 (A,B). Slug expression is not affected (B). Electroporation of GFP+CNICC89 (C,D) or GFP+Delta1stu (E,F) was performed on stage 5 embryo, and the embryos were cultured for 4 hours until stage 7. Transfection of CNICC89 has no effect on Hairy2 expression in the epidermis (D), while ectopic Hairy2 expression in the neural plate is observed (D, arrows in inset). Transfection of Delta1stu reduced the level of Hairy2 expression in the epidermis (F, arrowheads)

View larger version (35K): [in a new window]   Fig. 11. Alternative models of regulatory relationship of BMP signaling, Notch signaling and Slug expression.

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