First published online February 18, 2004
doi: 10.1242/10.1242/dev.01015
Development 131, 1135-1144 (2004)
Published by The Company of Biologists 2004
Cytoplasmic and molecular reconstruction of Xenopus embryos: synergy of dorsalizing and endo-mesodermalizing determinants drives early axial patterning
Keiichi Katsumoto1,
Tomohiro Arikawa1,
Jun-ya Doi2,
Hidefumi Fujii1,*,
Shin-ichiro Nishimatsu3 and
Masao Sakai1,
1 Department of Chemistry and Bioscience, Faculty of Science, Kagoshima
University, Kagoshima 890-0065, Japan
2 Department of Bioengineering, Yatsushiro National College of Technology, 2627
Hirayama Shin-Machi, Yatsushiro, 866-8501, Japan
3 Department of Molecular Biology, Kawasaki Medical School, Kurashiki, Okayama
701-0192, Japan
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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.
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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.
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Fig. 3. Injection of marginal cytoplasm (MC, see text) transfers a large yolk
sphere into PBEs. All embryos are at the one-cell stage. (A-D) Injection of MC
resulted in the formation of large yolk-rich sphere in the PBE. (A) Midline
section of a PBE that received MC (50 nl). (B) Midline section of a PBE just
before the first cleavage. Note the absence of large yolk platelets. (C)
Midline section of a GNE at the same stage. (D) A control embryo. (E-H)
Transfer of MC into a host PBE shown by conventional light (E,G) and
epifluorescent micrographs (F,H). (E,F) A recipient PBE transplanted with
neutral-red stained MC. (G,H) A host GNE. Scale bars: 250 µm in A-D; 1 mm
in E-H.
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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.
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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; 250
µm in C,D,I,J.
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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.
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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.
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Fig. 8. A model for the formation of the organizer. Cortical rotation drives the
vegetally localized dorsal determinants (red) to a meridian where the primary
dorsal axis forms. The marginal zone (broken rectangle), which receives the
determinants becomes the Spemann organizer (yellow). The marginal zone is the
margin of VegT containing cytoplasm, which spreads in the whole vegetal half
(green). The two determinants act cell autonomously to form the Spemann
organizer.
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© The Company of Biologists Ltd 2004
