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First published online 4 October 2006
doi: 10.1242/dev.02590

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Profilin is an effector for Daam1 in non-canonical Wnt signaling and is required for vertebrate gastrulation

¹ Department of Biochemistry, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson School of Medicine, Piscataway, NJ 08854, USA.
² Department of Pharmacology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson School of Medicine, Piscataway, NJ 08854, USA.
³ Laboratory of Molecular Genetics, National Institutes of Child Health and Human Development, Bethesda, MD 20892-2790, USA.
⁴ The Cancer Institute of New Jersey, 195 Little Albany Street, New Brunswick, NJ 08903-2681, USA.

View larger version (27K): [in a new window]   Fig. 1. Profilin binds to the FH1 domain of Daam1. (A) Schematic diagram of the Daam1 and Profilin1 constructs showing domains, with numbers indicating amino acid positions. (B,C) Coimmunoprecipitation experiments. Plasmids encoding tagged-Daam1 or Daam1 fragments and Profilin1 were cotransfected into HEK293T cells, and lysates were immunoprecipitated (IP) with indicated Abs. Precipitates were immunoblotted with indicated Abs. (B) Profilin1 interacts with Daam1, C-Daam1 and the FH1 domain of Daam1 but not N-Daam1 or the FH2 domain of Daam1. Note the tagged FH1 domain migrates overlapping the IgG band and is not resolved and a nonspecific band comigrates at the same position as HA-Daam1 (arrowhead). (C) Endogenous Profilin1 is precipitated with epitope tagged-Daam1, C-Daam1 or the FH1 domain of Daam1 but not N-Daam1 or the FH2 domain of Daam1. (D) Profilin1 does not induce Rho activation or interfere with Rho activation induced by C-Daam1. Immunoprecipitation and Rho activation assays were carried out as described (Habas et al., 2003; Habas et al., 2001).

View larger version (69K): [in a new window]   Fig. 2. Subcellular localization of Profilin1 and Daam1 in response to Wnt stimulation. (A) NIH3T3 cells respond to Wnt3a and Wnt5a but not control conditioned media (CM). After 3 hours of exposure of cells to Wnt3a CM or Wnt3a protein, endogenous ß-catenin (red) is localized in the nucleus and stress fiber induction is indicated by Phalloidin staining (green). After 3 hours of exposure to Wnt5a CM, no nuclear localization of ß-catenin is seen but stress fiber induction is observed. Position of the nucleus is indicated by arrow. (B) Expression of Flag-DIX-Dishevelled (red) induces stress fiber formation (green) in NIH3T3 cells. (C) Subcellular localization of GFP-Profilin1 in NIH3T3 cells. GFP-Profilin1 is localized diffusely in the cytoplasm of NIH3T3 cells in the presence of control CM but associates with actin stress fibers (arrow) (red) after 3 hours Wnt-3a CM stimulation. (D) Endogenous Daam1 (red) is localized diffusely in the cytoplasm of NIH3T3 cells but is localized to actin stress fibers (arrow) (green) after 3 hours Wnt3a CM stimulation. (E) Quantification of the studies of C and D. Number of cells evaluated is shown at the top of each bar. Note in B,C,D magnifications of boxed areas (white) are shown below each respective panel.

View larger version (51K): [in a new window]   Fig. 3. Wnt stimulation induces a colocalization of Daam1 and Profilin1. (A) Epitope-tagged Daam1 (HA) or (B) endogenous Daam1 (red) colocalizes with GFP-Profilin1 to structures resembling actin stress fibers (arrow) in response to 3 hours Wnt3a but not control CM stimulation. Note in A and B, magnifications of the boxed areas (white) are shown below each respective panel. (C) Transfection of Profilin1 siRNA but not a control GFP siRNA reduces the level of endogenous Profilin1 in NIH3T3 cells. ß-catenin and Daam1 are used as loading controls. (D) Top-Flash assay reveals Wnt3a CM and Dishevelled but not control CM or DIX-Dishevelled induces reporter activation in NIH3T3 cells. Transfection of control or Profilin1 siRNA does not interfere with reporter activation by either Wnt stimulation or Dishevelled expression.

View larger version (59K): [in a new window]   Fig. 4. Profilin1 is required for Wnt3a and Dishevelled-mediated stress fiber induction. (A) Transfection of Profilin1 siRNA but not GFP siRNA into NIH3T3 cells inhibits stress fiber induction (green) by 3 hours Wnt3a CM or Wnt3a protein stimulation. Note the Profilin1 siRNA does not inhibit stress fiber induction induced by serum stimulation. Nuclear translocation of endogenous ß-catenin (red) induced by Wnt3a CM or purified Wnt3a protein stimulation is not affected by Profilin1 siRNA. (B) Transfection of Profilin1 siRNA but not GFP siRNA into NIH3T3 cells inhibits stress fiber induction (green) induced by DIX-Dishevelled expression (red), magnifications of the boxed areas (white) are shown at the side of each respective panel. (C) Quantification of the results of A and B. Number of cells evaluated is shown at the top of each bar.

View larger version (38K): [in a new window]   Fig. 5. Profilin1 is required for Daam1-mediated stress fiber induction. (A) Expression of Myc-tagged C-Daam1 (red) in NIH3T3 cells induces stress fibers (green), and this effect is abrogated by coexpression of Profilin1 siRNA but not GFP siRNA, magnifications of the boxed areas (white) are shown at the side of each respective panel. (B) Quantification of the effects on Profilin1 and GFP siRNA on stress fiber formation induced by C-Daam1 expression in NIH3T3 cells. Number of cells evaluated is shown at the top of each bar.

View larger version (49K): [in a new window]   Fig. 6. XProfilin1 is required for gastrulation. (A) Injection of Profilin1 RNA dorsally but not ventrally inhibits gastrulation with the resulting embryos having open neural folds and reduced anterior structures. (B) XProfilin1 Morpholino (MO) sequence and diagram of the Profilin1 rescue construct (N-Profilin1). (C) Injection of the XProfilin1 MO inhibits translation of Myc-tagged Profilin1 in a dose dependent way but does not affect the endogenous levels of Rho and Rac proteins. (D) Dorsal injection of XProfilin1 MO inhibits gastrulation and results in a similar gastrulation defect phenotype as overexpression of Profilin1 RNA (see A). This phenotype is reversed by injection of N-Profilin1 with the XProfilin1 MO. (E) Quantitation of the phenotypic results from overexpression or depletion of XProfilin1. Injections were performed into the dorsal or ventral marginal zone of the 4-cell embryo, and phenotypes were scored at the tadpole stage.

View larger version (94K): [in a new window]   Fig. 7. Profilin1 does not interfere with mesoderm induction. Embryos injected dorsally with Profilin1 (2 ng) or XProfilin1 MO (50 ng) show normal mesoendoderm and neural induction, but abnormal tissue localization due to gastrulation defects. No effects were observed with injections of the control MO (50 ng) after gastrulation defects. The phenotype induced by the XProfilin1 MO can be rescued by co-expression of N-Profilin1 (50 pg). Injected embryos show normal expression of mesodermal Xbra, and dorsal Gsc and Otx2 at st 10.5, but exhibit an Xbra expression surrounding a large blastopore that does not close at st 12. Gsc expression in control embryos at st 13 is observed in anterior mesoendoderm far from the closed blastopore (arrowheads), but in Profilin or XProfilin1 MO-injected embryo remains trapped near the open blastopore. Otx-2 is expressed anteriorly in both mesodermal and overlying neural tissues in control embryos at st 13 but in Profilin or XProfilin1 MO-injected embryos, two separate Otx-2 expression domains are obvious. Sox-2 is expressed in the neural plate at st 14 (arrowheads), but in Profilin or XProfilin1 MO-injected embryos, Sox-2 expression is seen surrounding the open blastopore.

View larger version (71K): [in a new window]   Fig. 8. Profilin1 is not required for tissue separation or convergent extension. (A) Overexpression (2 ng RNA) or depletion of Profilin1 (50 ng XProfilin1 MO) does not inhibit convergent extension in animal explants treated with activin or (C) in Keller explants whereas dominant negative Dishevelled (Xdd1, 2 ng) inhibits these movements. (E) Overexpression (2 ng RNA) or depletion of Profilin1 (50 ng XProfilin1 MO) does not inhibit tissue separation by 1 hr whereas injection of Xfz7 (1 ng) does. Quantitation of the convergent extension assays in (B) animal explants, (D) Keller explants and (F) separation assays. For animal explant assays, a length/width ratio of 2 or higher was scored as elongated. For Keller explants, a length/width ratio below 2 was scored as a short elongation and a length/width ratio of 2 or higher was scored as a long elongation.

View larger version (60K): [in a new window]   Fig. 9. Profilin1 is specifically required for blastopore closure. (A) Schema of the injection approach to overexpress or deplete Profilin1 in or lateral to the neural folds, adapted from Wallingford and Harland (Wallingford and Harland, 2002). RNA (50 pg) encoding membrane-localized GFP injected into the lateral blastomeres at the 16-cell stage targets expression lateral of the neural folds whereas injection into the medial blastomeres targets expression to the neural folds. (B) Targeted overexpression of Profilin1 RNA (2 ng) or depletion of Profilin1 (50 ng MO) in the neural folds does not inhibit anterior neural fold closure but leads to open neural folds in the posterior, reflecting a failure of blastopore closure. This phenotype can be rescued by coexpression of N-Profilin1 (50 pg). Lateral overexpression or depletion of Profilin1 has no effect on neural fold closure. (C) Time lapse images of embryos injected with Profilin1 RNA (2 ng) or XProfilin1 MO (50 ng) showing a delay and eventual failure of blastopore closure. The XProfilin1 MO phenotype is rescued by coinjection of 50 pg N-Profilin1 RNA. (D) Depletion of XProfilin1 and XDaam1 synergistically inhibit gastrulation. Quantitation of the phenotypes observed with separate or co-injections of XProfilin1 MO and/or XDaam1 MO, Embryos with an open blastopore and significantly reduced anterior posterior (AP) axis were scored as severe embryos. Embryos with a small open blastopore or delayed blastopore closure and a shortened AP axis or bent body axis were scored as intermediate to mild. (E) A model for how Profilin1 functions in the non-canonical Wnt signaling pathway (see Discussion).

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