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Fig. 1. (A) Cytoplasmic nucleoplasmin indicates mitotic cells in early Xenopus embryos. Immunostaining with anti-nucleoplasmin (Nuc, green) and anti-bcatenin
(b-cat, red). (A) Nucleoplasmin is localized in interphase nuclei and diffuses into the cytoplasm during cell division. (B) b-Catenin indicates cell borders. (C)
DNA-staining (DAPI). (D) Overlay of the three fluorescent signals. Arrows indicate mitotic cells, the bar indicates 50 mm. (E) Cell division in the early gastrula
(st. 10C). Central sections through plastic-embedded embryos were scored for dividing cells in different regions as indicated by the schematic drawing in (E).
Representative micrographs of the corresponding regions are shown in (E) and the mitotic patterns are schematically illustrated in (E00). (E,E0), animal cap; (F,
F0), dorsal marginal zone; nuclei are counterstained with DAPI; (G,G0), bottle cells at the dorsal lip. Note that the bottle cells are non-mitotic. An, animal; D, dorsal;
V, ventral, Veg, vegetal.
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Fig. 2. Inhibition of cell proliferation in activin induced bottle cell fields. (A) Experimental procedure: ectopic bottle cell fields were induced by injection of mRNA
coding for activin into the animal hemisphere of 8-cell embryos. Embryos were analysed at gastrula stages. (B) Activin-induced bottle cell field after preparation for
scanning electron microscopy (SEM). The constricted apices of the bottle cells are visible in the centre of the micrograph and are surrounded by wild type (wt)
unconstricted animal cap cell apices. (C) Nucleoplasmin localization patterns in ectopic bottle cell fields. After injection of activin mRNA at the 8-cell stage the
embryos were fixed at stage 10.5 and stained for nucleoplasmin (green) and b-catenin (red). (C,E) Schematic drawings of activin-injected embryos. The squares
indicate the sectioning planes and the regions shown in (D) and (F). (D) Cross-section through a bottle cell field. Bottle cells appear as slender, elongated cells
(arrows). Nucleoplasmin is localized in the nuclei of these cells indicating that they are non-mitotic. (F) Tangential section through a bottle cell field. The apically
constricted necks of the bottle cells can be detected in the centre of the micrograph (small arrows). A single mitotic cell with cytoplasmic nucleoplasmin staining is
conspicuous at the periphery of the bottle cell field (double arrow). Bars correspond to 100 mm in D and F.
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Fig. 3. Uncontrolled cell proliferation interferes with gastrulation movements without affecting the expression of gsc and Xbra. (A) Experimental procedure, vegetal
injection. Embryos were scored for b-catenin and nucleoplasmin. (B) Control embryo. Note the bottle cells (boc, arrowhead) at the dorsal lip, the tissue separation in
the dorsal mesoderm (mes) and the migration of endomesodermal cells along the blastocoel roof (arrow), which is at least in part a result of the vegetal rotation
movement. ec, ectoderm; en, endoderm; blc, blastocoel; Do, dorsal; Ve, ventral. The squares indicate the regions shown in (D) and (F), respectively. (D) Dorsal
marginal zone. Prospective mesoderm involutes around an inner lip (curved arrow) and the tissue layers are separated by the cleft of Brachet (arrowheads). (F) The
vegetal cell mass is characterized by large yolk loaden cells and a low mitotic rate; a single mitotic cell is indicated by an arrow. (C) Early gastrula after vegetal
injection of cdc25C-mRNA. Gastrulation movements are severely disturbed, bottle cells are missing and the prospective tissue layers are not discernible. The regions
indicated by the squares in (C) are shown in (E) and (G), respectively. (E) In the marginal zone of an cdc25C injected embryo involution movements or Brachets cleft
are not detectable. (G) The vegetal half of such embryos consists of small cells and mitoses are frequent. For reasons of clarity the mitotic cells detected in (D) are
displayed by additional cartoons (DG. (H,I) Overexpression of cdc25C in animal cells interferes with epiboly. (H) Experimental procedure, animal injection. (I)
Section through an stage 10.5 embryo overexpressing cdc25C in the animal hemisphere (b-catenin (red), nucleoplasmin (green)). A part of the animal cap is shown
(square in H). Epiboly is clearly disturbed, the cap remains multilayered and displays folds/invagination sites (arrows). (I0) Schematic drawing illustrating cell shapes
and the proliferating cells (green). Bars correspond to 300 mm in (B), (C) and to 100 mm in (D). (K) Overexpression of cdc25C does not affect the basic
expression patterns of Xbra and gsc. (K) Experimental procedure, vegetal injection. (L) In situ hybridisation with riboprobes against Xbra (L,M) and gsc (N,O) in
uninjected embryos (L,N) and in embryos overexpressing cdc25C (M,O). In embryos injected with cdc25C-mRNA the two genes are expressed at normal levels but
the expression patterns are slightly different to the controls, probably due to the disturbed early gastrulation movements.
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Fig. 4. Ccd25C interferes with BVg1-induced bottle cell formation. Cdc25C-mRNA was injected into the four animal blastomeres of the 8-cell embryo followed by
injection of BVg-1-mRNA into an animal blastomere at the 86-cell-stage. At stage 10.5, embryos were scored for ectopic bottle cell formation. (A,B) BVg1-
induced bottle cell fields (arrowheads in B). (C,D) Ectopic bottle cell formation is not as pronounced after coinjection of cdc25C-mRNA as deduced from pigment
accumulation in the animal cap. In part of the embryos some pigment accumulation is still visible (arrowheads in D), in others no accumulated pigment can be
observed (I). The arrow in C indicates the endogenous lip of an injected embryo (vegetal view). (E) Histology of the embryos displayed in B and D. Bottle cells
are formed after overexpression of BVg1 (E,F) but not after coexpression of BVg1/cdc25C (G,H). (I,K) Coexpression of BVg1 together with wild-type cdc25C (I)
but not with phosphatase mutant cdc25C(C457A) (K) inhibits ectopic bottle cell formation. (L) Percentage of embryos displaying ectopic bottle cell formation after
overexpression of BVg1 (nZ98), BVg1Ccdc25C (nZ82), and BVg1Ccdc25C(C457A) (nZ57).
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Fig. 5. Interference of cdc25C with ectopic bottle cell formation correlates with increased cell proliferation. Embryos injected with the indicated mRNAs were fixed
at early gastrulation and stained for phospho-Histone H3. Representative embryos overexpressing BVg1 (A, nZ15), BVg1Ccdc25C (B, nZ17), and BVg1C
cdc25C(C457A) (C, nZ12) are shown. (D,E) Nucleoplasmin localization in animal caps of embryos overexpressing either BVg1 (D) or BVg1Ccdc25C (E). Apical
constriction is stronger in (D) and cell divisions are more frequent in (E). (D0,E0) Schematic drawings illustrating cell shapes and mitotic rates in (D) and (E),
respectively.
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Fig. 6. (A,B) Increase of the mitotic rate at some distance to ectopic bottle cells. Embryos overexpressing activin were fixed at gastrulation and stained for b-catenin
(red) and nucleoplasmin (green), animalegetal sections through the centre (A,A0) and the periphery (B,B0) of the ectopic bottle cell field, as indicated by the
drawings in (A) an (B). Mitotic cells are schematically illustrated in (A0,B0). (C) Spatial relationship of mitoses and the expression domains of gsc and Xbra. (C)
Double in situ hybridisation revealing a target board like pattern of bottle cells at the centre (asterisk indicates the injection site), gsc expression (blue) directly
adjacent to the bottle cells (and underneath, not shown), and Xbra (magenta) at the periphery. (D) Albino embryo at stage 10.25 after injection of BVg1 mRNA and
in situ hybridisation with riboprobes against gsc, animal view. Ectopic gsc transcripts at the injection site (asterisk). (E,F) Same embryo as in (D) after
counterstaining with anti-phospho-Histone H3 (p-H3) to visualize mitotic nuclei. (G) Albino embryo overexpressing BVg1 after in situ hybridisation with riboprobes
against Xbra, marginal view. Ectopic Xbra forms a ring around the injections site (asterisk), endogenous Xbra in the marginal zone is indicated by arrows. (H,I) Same
embryo as in (G) after counterstaining with anti-p-H3. Mitoses are frequent in the animal cap including the Xbra region but they are absent from the bottle cell region
and rare in the gsc domains. Arrows in (F) and (I) indicate mitotic nuclei in the periphery of the gsc domain. (K) Summary of the spatial relationships of mitotic
nuclei, the ectopic bottle cell field, and gsc and Xbra expression domains.
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Fig. 7. goosecoid expression and the occurrence of mitoses in the early gastrula. (A) gsc-mRNA in the organizer at stage 10 (vegetal view). (B) Phospho-Histone H3
staining at stage 10C(vegetal view, dorsal side). Arrows in (A) and (B) indicate dorsal bottle cells. (C) Double staining of gsc (gsc, blue) and phospho-Histone H3
(p-H3, brown dots) in a stage 10 albino. The dashed line indicates the bottle cell region. (C0) Schematic drawing of the pattern in (C). Arrows in (C) indicate mitotic
nuclei at the distal border of the gsc domain. an, animal; marg, marginal; veg, vegetal.
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Fig. 8. Influence of the mesodermal signal eFGF on TGF-b induced bottle cell formation. mRNAs coding for activin, BVg1, eFGF or cdc25C were injected either
alone or in the indicated combinations into 8-cell stage embryos. These were subsequently scored for ectopic lip morphology (A, I, N), Xbra-expression (E
H) and the occurrence of mitotic nuclei (anti-p-H3 staining, Q). (A,E) Uninjected control embryos, animal view, endogenous Xbra signal is not visible from this
perspective (E). Ectopic bottle cells do form in response to activin or BVg1 alone (B,K,N) but not after overexpression of eFGF (C,L), activinCeFGF (D,M),
BVg1CeFGF (O) or BVg1Ccdc25C (P). Activin induces a ring of Xbra-expressing cells around the ectopic bottle cells (F), eFGF alone strongly induces Xbra (G),
and the inhibition of activin-induced bottle cell formation by eFGF is also accompanied by induction of Xbra (H). The arrowhead in (F) indicates endogenous Xbra
transcripts. (Q) p-H3 staining in representative embryos injected with BVg1 (Q, nZ15), BVg1CeFGF (R, nZ11), and BVg1Ccdc25C (S, nZ17) mRNAs.
Positive nuclei are depicted in the additional cartoons (Q00). The line in Q0 indicates the endogenous lip. Coexpression of eFGF or cdc25C together with BVg1
causes increased mitotic rates (R,S) as compared to embryos overexpressing BVg1 alone (Q). Asterisks in (F,N,Q) indicate injection sites as indicated by the
accumulated pigment of bottle cells.
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Fig. 9. Schematic illustration summarizing the correlation of ectopic lip
morphogenesis to the gene expression patterns of goosecoid and Xbra as well as
to the cell division rate. High levels of activin-like TGF-b signals induce apical
constriction and goosecoid expression, but inhibit cell proliferation. Moderate
to low levels of TGF-bs induce Xbra/eFGF and are accompanied by a higher
mitotic rate.
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