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doi: 10.1242/10.1242/dev.00412

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Xbra functions as a switch between cell migration and convergent extension in the Xenopus gastrula

Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA

View larger version (126K): [in a new window]   Fig. 1. Typical morphology of dissociated cells plated on fibronectin and analyzed in cell migration assays. (A) Uninjected, untreated animal cap. (B) Uninjected, activin-treated animal cap. (C) Head mesoderm, dissected from stage 10 embryo. (D) Dorsal marginal zone (chordamesoderm), dissected from stage 10 embryo.

View larger version (46K): [in a new window]   Fig. 2. VegT is necessary for activin-induced cell migration and convergent extension. Increasing amounts of VegT-EnR RNA (0.5-1 ng) were injected into the animal pole. Sibling animal caps were dissected at stage 9 and processed for each of the assays. (A) Cell migration. Numbers immediately below the graph in parentheses reflect total number of cells scored. (B) Convergent extension.

View larger version (34K): [in a new window]   Fig. 3. Xbra mutants reveal separable pathways for convergent extension and cell migration. Embryos were injected with 300 or 600 pg of RNA encoding either Xbra-EnR, wild type Xbra or XbraDNABD. Sibling animal caps were dissected at stage 9 and processed for each of the assays. (A) Cell migration. Numbers immediately below the graph in parentheses reflect total number of cells scored. (B) Convergent extension.

View larger version (16K): [in a new window]   Fig. 4. The Xbra-mediated inhibition of cell migration can be partially rescued by an inhibitor of convergent extension. Embryos were injected with 300 or 600 pg of RNA encoding wild-type Xbra, plus or minus dominant-negative Wnt11 (2 ng RNA). Animal caps were dissected at stage 9 and processed for the cell migration assay. Numbers immediately below the graph in parentheses reflect total number of cells scored.

View larger version (36K): [in a new window]   Fig. 5. Wnt11 is a downstream target of Xbra that is necessary for convergent extension but not for cell migration. Levels of Wnt11 signal dramatically affect convergent extension, but similarly have no effect on cell migration. Embryos were injected with either dominant-negative Wnt11 (2 ng RNA) or wild-type Wnt11 (100 pg). Sibling animal caps were dissected at stage 9 and processed for each of the assays. (A) Cell migration. Numbers immediately below the graph in parentheses reflect total number of cells scored. (B) Convergent extension.

View larger version (48K): [in a new window]   Fig. 6A. Xbra inhibits cell shape changes induced by V12 Rac or V12 Cdc42, and cell spreading on fibronectin but not poly-L-lysine. (A) V12 Rac and V12 Cdc42 induce cell shape changes similar to those exhibited by cultured cells. Embryos were injected with 150 pg DNA of either V12 Cdc42 or V12 Rac into each blastomere. Animal caps were dissected at stage 10, dissociated and plated onto a fibronectincoated coverslip. Cells were then stained for actin filaments using rhodamine-labeled phalloidin. (a) Uninjected animal cap cells. (b) Animal cap cell expressing V12 Cdc42. (c) Animal cap cell expressing V12 Rac. (B) Xbra inhibits cell shape changes induced by the activated GTPases. Xbra (500 pg RNA) was co-injected with either V12 Rac or V12 Cdc42 (150 pg DNA). Plated animal cap cells were fixed and the number of cells exhibiting a morphology change such as those in Fig. 5A were counted. Numbers immediately below the graph in parentheses reflect total number of cells scored. When normalized for number of cells which respond to V12 Rac or V12 Cdc42, percent inhibition is as follows: for V12 Rac, 77.92% (±6.37%), for V12 Cdc42, 86.06% (±6.11%). (C) Uninjected, dissociated animal cap cells spread upon fibronectin and poly-L-lysine substrates. (a) Uninjected cells plated onto fibronectin. (b) Xbra-injected cells (400 pg RNA) plated onto fibronectin. (c) Uninjected cells plated onto poly-L-lysine. (d,d') Xbra-injected cells (400 pg RNA) plated onto poly-L-lysine. (D) Xbra inhibits cell spreading on fibronectin but not poly-L-lysine. Uninjected or Xbra-injected (400 pg RNA) animal cap cells were plated onto either fibronectin or poly-L-lysine. Plated cells were fixed and stained with rhodamine-phalloidin to visualize actin-rich protrusions. Cells exhibiting a spread morphology, i.e. flattened with irregular borders and often projecting actin-rich protrusions along the substrate, were counted. Numbers immediately below the graph in parentheses reflect total number of cells scored. (E) Dose-response of the effects of Xbra on cell spreading on fibronectin. See text and Materials and Methods for details regarding fibronectin concentrations. At least 170 cells were scored for each data point.

View larger version (15K): [in a new window]   Fig. 6D. Xbra inhibits cell shape changes induced by V12 Rac or V12 Cdc42, and cell spreading on fibronectin but not poly-L-lysine. (A) V12 Rac and V12 Cdc42 induce cell shape changes similar to those exhibited by cultured cells. Embryos were injected with 150 pg DNA of either V12 Cdc42 or V12 Rac into each blastomere. Animal caps were dissected at stage 10, dissociated and plated onto a fibronectincoated coverslip. Cells were then stained for actin filaments using rhodamine-labeled phalloidin. (a) Uninjected animal cap cells. (b) Animal cap cell expressing V12 Cdc42. (c) Animal cap cell expressing V12 Rac. (B) Xbra inhibits cell shape changes induced by the activated GTPases. Xbra (500 pg RNA) was co-injected with either V12 Rac or V12 Cdc42 (150 pg DNA). Plated animal cap cells were fixed and the number of cells exhibiting a morphology change such as those in Fig. 5A were counted. Numbers immediately below the graph in parentheses reflect total number of cells scored. When normalized for number of cells which respond to V12 Rac or V12 Cdc42, percent inhibition is as follows: for V12 Rac, 77.92% (±6.37%), for V12 Cdc42, 86.06% (±6.11%). (C) Uninjected, dissociated animal cap cells spread upon fibronectin and poly-L-lysine substrates. (a) Uninjected cells plated onto fibronectin. (b) Xbra-injected cells (400 pg RNA) plated onto fibronectin. (c) Uninjected cells plated onto poly-L-lysine. (d,d') Xbra-injected cells (400 pg RNA) plated onto poly-L-lysine. (D) Xbra inhibits cell spreading on fibronectin but not poly-L-lysine. Uninjected or Xbra-injected (400 pg RNA) animal cap cells were plated onto either fibronectin or poly-L-lysine. Plated cells were fixed and stained with rhodamine-phalloidin to visualize actin-rich protrusions. Cells exhibiting a spread morphology, i.e. flattened with irregular borders and often projecting actin-rich protrusions along the substrate, were counted. Numbers immediately below the graph in parentheses reflect total number of cells scored. (E) Dose-response of the effects of Xbra on cell spreading on fibronectin. See text and Materials and Methods for details regarding fibronectin concentrations. At least 170 cells were scored for each data point.

View larger version (71K): [in a new window]   Fig. 7. Xbra has effects in the marginal zone consistent with an in vivo role for inhibiting cell migration and promoting convergent extension. Wild-type Xbra inhibits cell migration of the head mesoderm and gives rise to anteriorly truncated embryos. Amounts of RNA listed are per blastomere. (A) Head mesoderm migration. Wild-type Xbra was injected into the dorsal marginal zone (two cells from a four-cell embryo). Prechordal mesoderm was dissected out of resulting stage 10 embryos. Numbers immediately below the graph in parentheses reflect total number of cells scored. (B) Whole embryo phenotype. (C) Mesendoderm extension assay. Selected frames from a timelapse are shown, with time relative to the first frame shown in the bottom right corner. Explants are numbered 1-3 above the first frame. (1) Explant injected with Xbra (100 pg). (2) Uninjected explant. (3) Explant injected with Xbra-EnR (400 pg).

View larger version (45K): [in a new window]   Fig. 8. Xbra-EnR inhibits convergent extension of the dorsal marginal zone, giving rise to tadpoles with fully formed heads, but very short trunks. In addition, inhibiting Xbra function in the dorsal marginal zone increases the number of cells which spread and crawl on fibronectin. (A) DMZ convergent extension. (B) Whole embryo phenotype. (C) DMZ cell migration. Numbers immediately below the graph in parentheses reflect total number of cells scored.

View larger version (46K): [in a new window]   Fig. 9. Xdsh mutants that block the PCP pathway inhibit convergent extension and promote cell migration. (A) DMZ convergent extension. (B) DMZ cell migration. Numbers immediately below the graph in parentheses reflect total number of cells scored.