Fujii H et al. (2008), VegT, eFGF and Xbra cause overall posteriorizat...

XB-ART-37348

Dev Growth Differ 2008 Mar 01;503:169-80. doi: 10.1111/j.1440-169X.2008.01014.x.

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VegT, eFGF and Xbra cause overall posteriorization while Xwnt8 causes eye-level restricted posteriorization in synergy with chordin in early Xenopus development.

Fujii H , Sakai M , Nishimatsu S , Nohno T , Mochii M , Orii H , Watanabe K .

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We examined several candidate posterior/mesodermal inducing molecules using permanent blastula-type embryos (PBEs) as an assay system. Candidate molecules were injected individually or in combination with the organizer factor chordin mRNA. Injection of chordin alone resulted in a white hemispherical neural tissue surrounded by a large circular cement gland, together with anterior neural gene expression and thus the development of the anterior-most parts of the embryo, without mesodermal tissues. When VegT, eFGF or Xbra mRNAs were injected into a different blastomere of the chordin-injected PBEs, the embryos elongated and formed eye, muscle and pigment cells, and expressed mesodermal and posterior neural genes. These embryos formed the full spectrum of the anteroposterior embryonic axis. In contrast, injection of CSKA-Xwnt8 DNA into PBEs injected with chordin resulted in eye formation and expression of En2, a midbrain/hindbrain marker, and Xnot, a notochord marker, but neither elongation, muscle formation nor more posterior gene expression. Injection of chordin and posteriorizing molecules into the same cell did not result in elongation of the embryo. Thus, by using PBEs as the host test system we show that (i) overall anteroposterior neural development, mesoderm (muscle) formation, together with embryo elongation can occur through the synergistic effect(s) of the organizer molecule chordin, and each of the 'verall posteriorizing molecules'eFGF, VegT and Xbra; (ii) Xwnt8-mediated posteriorization is restricted to the eye level and is independent of mesoderm formation; and (iii) proper anteroposterior patterning requires a separation of the dorsalizing and posteriorizing gene expression domains.

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Species referenced: Xenopus laevis
Genes referenced: chrd egr2 en2 fgf4 hoxb9 ncam1 not tbxt vegt wnt8a

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	Fig. 1. Schematic representation of the experimental procedure. (Left) Permanent blastula-type embryos (PBEs) were made by ablating vegetal cytoplasm and egg surface so that more than 60% of the egg surface was deleted. (Middle) At the 4-cell stage, one of the candidate posteriorizing molecules (VegT, eFGF, or Xbra mRNAs or CSKA-Xwnt8 DNA construct) was injected into the vegetal region of a single blastomere. (Right) At the 8-cell stage, chordin mRNA was injected into a vegetal blastomere opposite the injection site of the candidate posteriorizing molecules.
	Fig. 2. Reverse transcription–polymerase chain reaction (RT– PCR) analysis of experimental embryos. The PCR products from four permanent blastula-type embryos (PBEs) or one control embryo (stage 24) were loaded in each lane. PCR primer pairs and cycling conditions are shown in Table 1. See also Materials and methods. Lane 1, a normal embryo at stage 24. Lane 2, naive PBEs without injection were expressed Krox20, but not dorsal/ neural and endomesodermal genes. Lane 3, PBEs injected with 100 pg chordin mRNA. Lane 4, PBEs injected with 1 pg eFGF mRNA. Lane 5, PBEs injected with 100 pg chordin and 1 pg eFGF (separate injection, see Fig. 1). Lane 6, PBEs injected with 50 pg CSKA-Xwnt8 DNA. Lane 7, PBEs injected with 100 pg chordin and 50 pg CSKA-Xwnt8 (separate injection).
	Fig. 3. Morphology of permanent blastula-type embryos (PBEs) injected with chordin or posteriorizing candidate molecules. (A) Naive PBEs without injection. (B) PBEs injected with 100 pg chordin. A white neural area is surrounded by a circular cement gland. (C) PBEs injected with 1 pg eFGF. Note the accumulation of cells (white arrowheads). (D) PBEs injected with 50 pg CSKA-Xwnt8 DNA. Note the cell accumulation on the right side of the embryos. (E) Fluorescent image showing that chordin-injected blastomeres developed into white neural cells. FDA (green) injected with chordin mRNA at the 8-cell stage was localized in neural cells. (F) eFGF mRNA expressing domain as shown by coinjection of RDA (magenta). All embryos are at stage 24. Bar in A for A–D, 1 mm. Bar in E for E and F, 0.5 mm. Broken lines in E and F indicate the outline of the embryo.
	Fig. 4. Injection of eFGF, VegT and Xbra triggered endogenous expression of these molecules and Xwnt8 expression, whereas CSKA-Xwnt8 did not induce eFGF, VegT or Xbra. Permanent blastula-type embryos (PBEs) were injected into one blastomere with VegT, eFGF, Xbra mRNA or CSKA-Xwnt8 DNA at the 4-cell stage and assayed at stage 11. The PCR products from four PBEs or one control embryo were loaded in each lane. See also Materials and methods. Lane 1, a normal embryo. Lane 2, naive PBEs without injection. Lane 3, PBEs injected with 6 pg VegT mRNA. Lane 4, PBEs injected with 50 pg CSKAXwnt8 DNA. Lane 5, PBEs injected with 1 pg eFGF mRNA. Lane 6, PBEs injected with 400 pg Xbra mRNA. Lane 7, PBEs injected with 100 pg chordin mRNA.
	Fig. 5. Overall posteriorization by VegT, eFGF, Xbra mRNA and eye-level restricted posteriorization by CSKA-Xwnt8 DNA. (A) PBE/chd/eFGFs at stage 24 (B) A PBE/chd/eFGF at stage 40. cg, cement gland; ey, eye; me, melanocytes; tf, tail fin. (C, D) A PBE/chd/eFGFs at stage 24. Chordin mRNA was injected together with a lineage marker, FDA (green) while eFGF was injected together with RDA (magenta). (E) A PBE/chd/VegT at stage 40. (F) A PBE/chd/Xbra at stage 40. Embryos shown in B, E and F twitched, suggesting the presence of motor neurons and skeletal muscle. (G) PBE/chd/CSKA-Xwnt8s at stage 24. These embryos formed smaller cement glands compared with PBE/chds (Fig. 3B). (H) A PBE/chd/CSKA-Xwnt8 at stage 40. This embryo did not elongate, but formed a large eye (ey) and a cement gland (cg). Bars in A, B, and E–H, 1 mm; bars in C and D, 0.5 mm.
	Fig. 6. PBE/chd/eFGF formed eye and notochord while PBE/ chd/CSKA-Xwnt8 formed eye but not notochord. (A, B) Sections of a PBE/chd/eFGF shown in Figure 5(B). (A) A transverse section at the eye level. (B) Posterior region forming a notochord. (C) A section of PBE/chd/CSKA-Xwnt8 shown in Figure 5(H). (D) An eye of a control embryo. le, lens; ne, neuroepithelial cells; no, notochord; re, retinal-pigmented epithelium. Bar in A for A, C, and E, 0.1 mm. Bar in B, 0.2 mm.
	Fig. 7. Anteroposteriorly organized gene expression in PBE/chd/eFGFs. (A–C) In situ hybridization of PBE/chd/eFGFs. Top: normal embryo at stage 24. Bottom: PBE/chd/eFGFs at stage 24. Anterior to the left. (A) NCAM (B) Krox20 (C) HoxB9. (D and E) Immunohistochemical staining using 12/101 (muscle marker, D) and MZ15 (notochord marker, E) antibody. Top: normal embryo at stage 40. Bottom: PBE/chd/eFGFs at stage 40. White arrowheads mark the anterior end of the stain while black arrowheads mark the posterior end. Bars: 1 mm.
	Fig. 8. Co-injection of chordin and posteriorizing molecules into the same blastomere did not result in elongation of the embryos and proper anteroposterior patterning. (A) PBE/ chd + eFGFs at stage 24. These embryos did not elongate and had semicircular cement glands. (B) A PBE/chd + eFGF at stage 40. A small eye remnant was seen. (C) PBE/chd + CSKAXwnt8s at stage 24. These embryos resembled PBE/ chd + eFGFs. (D) A PBE/chd + CSKA-Xwnt8 at stage 40. Eye structure was absent. cg, cement gland; ey, eye remnant. (E) Reverse transcription–polymerase chain reaction (RT–PCR) analysis for coinjection experiments. The PCR products from four permanent blastula-type embryos (PBEs) or one control embryo were loaded in each lane. See also Materials and methods. Lane 1, a normal embryo at stage 24. Lane 2, PBEs injected with the mixture of 100 pg chordin and 1 pg eFGF into one blastomere at the 8-cell stage. Lane 3, PBEs injected with the mixture of 100 pg chordin and 50 pg CSKA-Xwnt8 into one blastomere at the 8-cell stage. In spite of their non-elongated morphology, PBE/chd + eFGFs expressed notochord and muscle markers. Bars in A–D, 1 mm.
	Fig. 9. Proposed model of anteroposterior patterning in Xenopus embryos. (A) After gene expression commences (after mid-blastula transition [MBT]), the Xenopus embryo is divided into three domains, as shown by previous tissue transplantation experiments; competent animal domain (green), dorsalizing organizer domain (the Spemann-Mangold organizer) (magenta) and posteriorizing vegetal domain (cyan–light-blue gradient). Tissue transplantation experiments revealed whole vegetal hemisphere (stage 10) Xenopus embryos have overall posteriorizing activity; however, this activity could not be attributed to zygotic Xwnt8, because Xwnt8 did not exhibit overall posteriorizing activity. The molecule responsible for the overall posteriorizing activity in the non-dorsal marginal zone may be VegT, eFGF and Xbra; however, these molecules are absent in the vegetal pole. Unknown molecules in the gastrula vegetal pole are most likely responsible for the overall posteriorizing activity. (B) Posteriorization at the neurula stage. At this stage, Xwnt8 is expressed in the mid-anterior neural fold, and may be involved in the eye and midbrain level posteriorization. The expression domain of eFGF, VegT and Xbra suggests that these molecules are responsible for overall posteriorization.