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???displayArticle.abstract??? Rho GTPases are molecular switches that regulate many essential cellular processes, including actin dynamics, cell adhesion, cell-cycle progression, and transcription. We have isolated the Xenopus homolog of Rho GTPase Cdc42 and examined its potential role during gastrulation movements in early Xenopus embryos. XCdc42 is expressed in tissues undergoing extensive morphogenetic changes, such as the deep layers of involuting mesoderm and posteriorneuroectoderm during gastrulation, and somitic mesoderm at neurula stages. Overexpression of either wild-type (WT) or dominant-negative (DN) XCdc42 interferes with convergent extension movements in intact embryos, activin-stimulated animal caps, and dorsal marginal zone explants. These effects occur without affecting mesodermal specification. Overexpression of WT or DN XCdc42 leads to the decrease and increase of cell adhesiveness of blastomeres, respectively, as demonstrated by the cell adhesion assay. In addition, when overexpressed, PKC-alpha, XWnt-5a, and Mfz-3 inhibit activin-induced convergent extension in animal cap explants. This inhibition can be rescued by coexpression of DN XCdc42, implying that XCdc42 acts downstream of the Wnt/Ca2+ signaling pathway involving PKC activation. XCdc42 also lies downstream of XWnt-5a in the regulation of Ca2+-dependent cell adhesion. Taken together, our results suggest that XCdc42 plays a role in the regulation of convergent extension movements during gastrulation through the protein kinase C-mediated Wnt/Ca2+ pathway.
FIG. 2. Expression pattern of XCdc42 in early development. (A) Temporal expression pattern of XCdc42. Developmental stages are shown above each lane. Ornithine decarboxylase (ODC) was used as a loading control. (B) Spatial expression pattern of XCdc42. (a) Sagittal section of a stage 11 gastrula. An arrow indicates dorsal lip of blastopore. (b) Higher magnified view of (a). XCdc42 transcripts are detectable in the deep cells of involuting mesoderm (arrowheads) and in the presumptive neuroectoderm (arrows). (c) Sagittal section of a stage 12 gastrula. Arrowheads indicate involuting mesoderm. (d) Higher magnified view of (c). Relatively low levels of XCdc42 expression are present in the deep layers of dorsal mesoderm (arrowheads), with its strong expression in the overlying ectoderm (arrows). (e) Transverse section of an early neurula. Strong expression of XCdc42 is present in the neural plate (NP), somitic mesoderm (S), notochord (No), and ectoderm. (f) Cross section of a tailbud stage embryo. XCdc42 is expressed in the pronephric anlage (PA), neural tube, and notochord, but not in the segmented somites (S).
FIG. 3. Gain and loss of XCdc42 function have similar effects on the gastrulation movement. Embryos were injected into the DMZ at the four-cell stage with 200 pg of either WT or DN XCdc42 mRNA and allowed to develop to stage 32. Prolactin-injected control embryos (A) were normal, whereas embryos expressing WT XCdc42 (B) or DN XCdc42 (C) displayed similarly two distinct phenotypes; one is dorsally kinked and has an open neural tube. The other is stout and fails to straighten the A/P axis. Lateral view is shown; anterior is to the left.
FIG. 4. XCdc42 overexpression affects the localization of mesodermal and neural markers. Four-cell stage embryos were injected in the DMZ with 200 pg of WT XCdc42 mRNA. (A) (a) Expression of chordin at stage 10 in uninjected control (upper) and XCdc42-injected (lower) embryos (vegetal view is shown; dorsal at top). (b) Expression of chordin in uninjected (upper, dorsovegetal view; anterior at top) and XCdc42-injected (lower, vegetal view; dorsal at top) embryos at stage 12. In XCdc42-injected embryos, chordin-expressing cells did not converge toward the dorsal midline and were still localized around the blastopore. (c) The pattern of Xbra expression in uninjected (upper) and XCdc42-injected (lower) embryos at stage 10 (vegetal view; dorsal at top). (d) Expression of Xbra in stage 12 control (upper, dorsovegetal view) and XCdc42-injected (lower, vegetal view; dorsal at top) embryos. Xbra transcripts, which were detectable in the notochordal mesoderm in uninjected control embryos, were absent in XCdc42-injected embryos, indicating the failure of dorsal mesoderm to involute during gastrulation. (e, f) The expression of XmyoD in uninjected (dorsovegetal view) and XCdc42-injected (vegetal view; dorsal at top) embryos at stage 11.5, respectively. In XCdc42-injected embryos, XmyoD remained expressed around the blastopore, but not in the lateralmesoderm. (g) At stage 13, uninjected control embryos had the anterior expression of Otx-2 (dorsal view is shown; anterior at top). (h) In XCdc42-injected embryos at stage 13, the expression of Otx-2 occurred in the dorsal ectoderm (dorsovegetal).
(B) (a, b) In situ hybridization against XmyoD was performed on both uninjected (a) and XCdc42-injected (b) embryos at stage 25. XCdc42-injected embryos failed to close the neural plate, yet retained muscle differentiation. (c, d) As in the uninjected embryo at stage 30 (c), notochord differentiation normally occurred in the XCdc42-injected embryo (d) as assessed by expression of Xnot.
FIG. 5. XCdc42 affects convergent extension, but not mesoderm induction. (A) Embryos were injected as indicated into the animal pole of four blastomeres at the four-cell stage. Animal cap explants were excised at stage 8.5, treated with 10 ng/ml recombinant activin, and cultured to the equivalent of stage 18. (a) Animal cap explants from uninjected control embryos elongated significantly in response to activin. (b) WT XCdc42 mRNA (400 pg) injection interfered with activin-induced explant elongation. (c) Animal cap explants injected with DN XCdc42 mRNA (2 ng) failed to elongate. (d) Coinjection of WT XCdc42 mRNA (400 pg) together with DN XCdc42 mRNA (400 pg) rescued the elongation of animal caps. (B) Injections were targeted to the DMZ at the four-cell stage. DMZ explants were excised at stage 10 and cultured to stage 20 before being measured for elongation. (a) Uninjected control DMZ explants elongated significantly. Injection of either (b) WT XCdc42 (400 pg) or (c) DN XCdc42 (2 ng) suppressed convergent extension of DMZ explants. (C) Quantitation of convergent extension of animal cap (AC) and DMZ explants. RNAs expressed are shown below graph. n, total number of explants. (D) RT-PCR analysis detecting expression of mesodermal markers in animal caps derived from embryos injected with XCdc42 constructs. Animal caps from embryos injected with either WT XCdc42 (400 pg) alone or WT and DN XCdc42 (400 pg each) were treated with activin at stage 8.5 and cultured until stage 10.5 for analysis of goosecoid (gsc), Xbra, and chordin (Chd) expression and until stage 23 for analysis of muscle actin (M. actin) expression. ODC and EF-1alpha are loading controls.
FIG. 6. XCdc42 functions downstream of the Wnt/Ca2ï°„ signaling pathway involving PKC activation. (A) (a) Uninjected animal caps treated with activin (10 ng/ml) showed extensive elongation. (b) Human PKC (hPKC)-ï° mRNA (0.5 ng per blastomere at the 4-cell stage) injection inhibited explant elongation. (c) Coinjection of DN XCdc42 mRNA (200 pg) with hPKC-ï° mRNA (2 ng) efficiently rescued the elongation of animal caps. (B) (a) Animal cap explants derived from uninjected control embryo elongated significantly in response to activin treatment. (b) Animal cap explants expressing XWnt-5a mRNA (800 pg) failed to elongate. (c) Rescue of XWnt-5a-inhibited explant elongation by coexpression of DN XCdc42 mRNA (300 pg). (d) Injection of mouse frizzled-3 (Mfz-3) mRNA (200 pg) inhibited activin-induced elongation. (e) Coexpression of DN XCdc42 mRNA (200 pg) reversed the inhibition of explant elongation caused by Mfz-3. (f) Control animal cap explants with no activin treatment remained rounded. (C) Quantitation of elongation of animal cap explants. RNAs expressed are indicated below graph. Control-1 and control-2 serve for hPKC-ï° and XWnt-5a and Mfz-3, respectively. n, total number of explants.
cdc42 (cell division cycle 42) gene expression in bisected Xenopus laevisembryo, mid-sagittal section, via in situ hybridization, NF stage 12, dorsal right, blastopore down.
cdc42 (cell division cycle 42) gene expression in bisected Xenopus laevis embryo, mid-sagittal section, via in situ hybridization, NF stage 13-15, dorsal up.
cdc42 (cell division cycle 42) gene expression in bisected Xenopus laevisembryo, mid-sagittal section, via in situ hybridization, NF stage 28, mid trunk section, dorsal right.
