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Furue M
,
Myoishi Y
,
Fukui Y
,
Ariizumi T
,
Okamoto T
,
Asashima M
.
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Activin A has potent mesoderm-inducing activity in amphibian embryos and induces various mesodermal tissues in vitro from the isolated presumptive ectoderm. By using a sandwich culture method established to examine activin A activity, we previously demonstrated that activin-treated ectoderm can function as both a head and trunk-tailorganizer, depending on the concentration of activin A. By using activin A and undifferentiated presumptive ectoderm, it is theoretically possible to reproduce embryonic induction. Here, we test this hypothesis by studying the induction of cartilagetissue by using the sandwich-culture method. In the sandwiched explants, the mesenchymal cell condensation expressed type II collagen and cartilage homeoprotein-1 mRNA, and subsequently, cartilage were induced as they are in vivo. goosecoid (gsc) mRNA was prominently expressed in the cartilage in the explants. Xenopus distal-less 4 (X-dll4) mRNA was expressed throughout the explants. In Xenopus embryos, gsc expression is restricted to the cartilage of the lower jaw, and X-dll4 is widely expressed in the ventralhead region, including craniofacial cartilage. These finding suggest that the craniofacial cartilage, especially lower jaw cartilage, was induced in the activin-treated sandwiched explants. In addition, a normal developmental pattern was recapitulated at the histological and genetic level. This work also suggests that the craniofacial cartilage-induction pathway is downstream of activin A. This study presents a model system suitable for the in vitro analysis of craniofacial cartilage induction in vertebrates.
Fig 3. Type II Collagen protein and mRNA expression in the explants and general embryos. The serial sections of the histologically analyzed explants and the sections of general embryos were immunostained with anti-Col2 antibody which was visualized with AEC and counterstained with hematoxylin. The sections of explants and general embryos were hybridized with DIG-labeled Col2 RNA probe, and the localization was visualized with NBT/BCIP. (A) Immunolocalization of Col2 in the serial section of Fig. 2 B, the 4 day-cultured explant. (B) Immunolocalization of Col2 in the serial section of Fig. 2 C, 7 day-cultured explant. (C) Immunolocalization of Col2 in the serial section of Fig. 2 D, 10 day-cultured explant. (D) Immunolocalization of Col2 in the serial section of Fig. 2 F, 14 day-cultured explant. (E) Col2 mRNA expression in the section of 4 day-cultured explant. (F) Col2 mRNA expression in the section of 7 day-cultured explant. (G) Immunolocalization of Col2 in the section of Stage 35 embryo. (H) Higher magnification of the arrowhead indicating area in G. (I) Immunolocalization of Col2 in the section of Stage 40 embryo. (J) Higher magnification of the arrowhead indicating area in I. (K) Immunolocalization of Col2 in the section of Stage 42 embryo. (L) Higher magnification of the arrowhead indicating area in K. (M) Immunolocalization of Col2 in the section of Stage 44 embryo. (N) Higher magnification of the arrowhead indicating area in M. (O) Col2 mRNA expression in the section of Stage 35 embryo. (P) Higher magnification of indicating area in O. (Q) Col2 mRNA expression in the section of Stage 44 embryo. (R) Higher magnification of indicating area in Q. et, ethmoid trabecular cartilage; ir, infrarostralcartilage; m, Meckel's cartilage; nt, notochord; pq, palatoquadratecartilage. (Bars = 50 μm.)
Fig 5. Expression of Cart-1, X-dll4, and gsc mRNA in the cartilage of the explants and general embryos by in situ hybridization. The Alcian blue-stained section of a 7 day-cultured explant was double-stained with DIG-labeled Cart-1 probe and fluorescein-labeled X-dll4 RNA probes, visualized with Cyanine 3 and fluorescein, respectively. The serial section of above section was stained with DIG-labeled gsc RNA probe and visualized with Cyanine 3. The Alcian blue-stained sections of stage 41 general embryos were stained with DIG-labeled RNA probes and visualized with NBT/BCIP (purple). (A) Cart-1 mRNA (red) and X-dll4 mRNA (green) expression in a section of 7 day-cultured explant. (B) The Alcian blue staining of A. (C) gsc mRNA (red) in the serial section of A. (D) The Alcian blue-stained staining of C. (E) Cart-1 mRNA expression in embryo. (F) X-dll4 mRNA expression in embryos. (G) gsc mRNA expression in embryo. cg, cement gland; et, ethmoid trabecular cartilage; ir, infrarostralcartilage; m, Meckel's cartilage; nt, notochord; oc, oral cavity; ol, olfactory organ; pq, palatoquadratecartilage. (Bars = 10 μm.)
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