Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Development
2004 Mar 01;1315:1135-44. doi: 10.1242/dev.01015.
Show Gene links
Show Anatomy links
Cytoplasmic and molecular reconstruction of Xenopus embryos: synergy of dorsalizing and endo-mesodermalizing determinants drives early axial patterning.
Katsumoto K
,
Arikawa T
,
Doi JY
,
Fujii H
,
Nishimatsu S
,
Sakai M
.
???displayArticle.abstract???
Ablation of vegetal cytoplasm from newly fertilized Xenopus eggs results in the development of permanent blastula-type embryos (PBEs). PBEs cleave normally and develop into a very simple tissue consisting only of atypical epidermis. We tried to restore complete embryonic development in PBEs by cytoplasmic transplantation or by mRNA injection. We show a two-step reconstruction of the body plan. In the first step, PBEs injected with either marginal cytoplasm or synthetic VegT RNA restored gastrulation and mesoderm formation, but not axial patterning. Injection of Xwnt8 mRNA (acting upstream of beta-catenin and thus substitutes for the dorsal determinant) did not restore axial development in PBEs. Simultaneous injections of Xwnt8 and VegT into PBEs resulted in dorsal axis development, showing the synergy of these molecules in axial development. These results suggest that the mixing of two cytoplasmic determinants, i.e. the dorsal determinant in the vegetal pole and the endo-mesodermal determinant in the whole vegetal half, triggers the early axial developmental process in Xenopus embryos.
Fig. 1. Permanent blastula-type embryos (PBEs) are simple organisms consisting only of epidermal tissue. All embryos are at control stage 17. (A,B) Midline sections of a PBE (A) and a GNE (B). (C) PBEs did not show Xbra expression. (D) GNEs expressed Xbra in their vegetally shifted marginal zones. (E,F) PBEs (E) and GNEs (F) showed no chordin expression. Scale bars: 250 μm in A,B; 1 mm in C-F.
Fig. 2. VegT mRNA was absent in PBEs but was present in GNEs. Whole-mount in situ hybridization for VegT. Top, PBEs; middle, GNEs; bottom, controls. Scale bar: 1 mm.
Fig. 4. Injection of MC but not AC transfers VegT into PBEs. Whole-mount in situ hybridization for VegT. Top, PBEs injected with 50 nl PBE-animal cytoplasm (AC). Bottom, PBEs injected with 50 nl MC. Scale bar: 1 mm.
Fig. 5. Injection of the MC or VegT into PBEs resulted in gastrulation and zygotic expression of Xbra. All embryos are at control stage 17. (A) A PBE transplanted with 50 nl of MC. (B) Epifluorescent view of A. (C) A midline section of another MC-injected PBE. (D) A VegT (12 pg) injected PBE. (E-G) Whole-mount in situ hybridization of PBEs injected with MC for Xbra (E), chordin (F) and VegT probes (G). (H) VegT-injected PBEs. Left to right: zygotic expression of VegT, Xbra and chordin. (I) In situ hybridization on sections of a PBE injected with MC. (J) Enlarged view of I. Scale bars: 1 mm in A,B,E-H; 250μ m in C,D,I,J.
Fig. 6. Injection of Xwnt8 mRNA into PBEs did not result in dorsal axis formation. (A,B) Stage 17. (C) Stage 11. (D) Stage 38. (A) A PBE-injected with vegetal pole cytoplasm (VPC, 50 nl) into the bottom region formed a proboscis indicating a hyperdorsal phenotype. (B) PBEs (top) and GNEs (bottom) injected with Xwnt8 alone (3 pg into a single cell at the eight-cell stage). (Inset) A control embryo. (C) Whole-mount in situ hybridization for chordin in Xwnt8-injected embryos. (Top) PBEs. Bottom five embryos are GNEs. (Inset) A control embryo (no injection). Note that chordin expression is restricted in the upper blastopore region in both control and Xwnt8-injected GNEs. (D) An Xwnt8 (3 pg)-injected GNE. cg, cement gland; e, eye. Scale bars: 1 mm.
Fig. 7. Double injections of dorsalizing and endo-mesodermalizing determinants restored the dorsal axis. (A,B,H) Stage 38. (C,D) Stage 11. (E-G) Stage 17. (A) A PBE injected with MC (50 nl) and Xwnt8 (3 pg). (B) A PBE injected with VegT and Xwnt8. A single blastomere of four-cell stage PBE was injected with a mixture of 12 pg VegT and 3 pg Xwnt8, thereafter an adjacent cell was injected with 12 pg VegT only. With this protocol, we aimed to make an organizer and a posteriorizing center in the host PBE. (C) Expression of chordin in PBEs. (Top) VegT- and Xwnt8-injected embryos as shown in B. (Middle) These embryos also received 12 pg VegT and 3 pg Xwnt8 at the four-cell stage, but in separate blastomeres. (Bottom) PBEs injected with 12 pg VegT into two adjacent cells of a four-cell stage PBE. (D) Injection of MC and Xwnt8 (top) and MC and VPC (bottom, see text) restored chordin expression in PBEs. (E) NCAM expression in PBEs injected with MC (50 nl) and Xwnt8 (3 pg). (F) Krox20 expression in PBEs injected with MC (50 nl) and Xwnt8 (3 pg). Note the two-band structures in the upper-left sample. (G) Negative controls in which 50 nl animal pole cytoplasm (AC) and Xwnt8 (3 pg) was injected into PBEs. (Top) In situ hybridization for NCAM. (Bottom) In situ hybridization for Krox20. (H) A normal-looking embryo derived from a PBE injected with 50 nl MC and 18 nl VPC. Scale bars: 1 mm.
