August 15, 2009;
Non-redundant roles for Profilin2 and Profilin1 during vertebrate gastrulation.
Gastrulation is a critical morphogenetic event during vertebrate embryogenesis, and it is comprised of directional cell movement resulting from the polarization and reorganization of the actin cytoskeleton. The non-canonical Wnt signaling pathway has emerged as a key regulator of gastrulation. However, the molecular mechanisms by which the Wnt pathway mediates changes to the cellular actin cytoskeleton remains poorly defined. We had previously identified the Formin
and an effector molecule XProfilin1
as links for Wnt-mediated cytoskeletal changes during gastrulation. We report here the identification of XProfilin2
as a non-redundant and distinct effector of Daam1
for gastrulation. XProfilin2
interacts with FH1 domain of Daam1
and temporally interacts with Daam1
during gastrulation. In the Xenopus embryo
is temporally expressed throughout embryogenesis and it is spatially expressed in cells undergoing morphogenetic movement during gastrulation. While we have previously shown XProfilin1
closure, overexpression or depletion of XProfilin2
specifically affects convergent extension movement independent of mesodermal specification. Specifically, we show that XProfilin2
modulates cell polarization and axial alignment of mesodermal cells undergoing gastrulation independent of XProfilin1
. Together, our studies demonstrate that XProfilin2
are non-redundant effectors for Daam1
for non-canonical Wnt signaling and that they regulate distinct functions during vertebrate gastrulation.
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Fig. 2. Temporal and spatial expression pattern of XProfilin2. (A) XProfilin2 is expressed throughout Xenopus development as monitored by RT-PCR analysis; total extracted mRNA is shown as a loading control, −RT without reverse transcriptase. Quantitation of the relative RNA expression levels is shown below (B). The spatial expression pattern of XProfilin2 is dynamic with the highest level of expression refined to the neural fold and nervous system during neurula-stage embryo. Stages and views of the embryos are shown in each panel; AP = animal pole, DL = dorsal lip, NP = neural plate, NF = neural fold, BR = brain, EY = eye, and SC = spinal cord. No signal is detected using an XProfilin2 sense probe.
Fig. 3. XProfilin2 is required for gastrulation. (A) Injection of XProfilin2 RNA dorsally but not ventrally inhibits gastrulation with the resulting embryos having open neural folds and reduced anterior structures (Severe) or delayed blastopore closure and a curved/bent axis (Mild). (B) Quantitation of the phenotypic results from overexpression studies of XProfilin2. Number of embryos scored (n) is shown at the top of each bar. (C) Schematic representation of the XProfilin2 constructs and targeted-Morpholino site. (D) Injection of the XProfilin2 MO but not XProfilin1 MO or a control MO inhibits translation of Myc-tagged 5′UTR-XProfilin2. The δN-XProfilin2 cDNA lacking the XProfilin2 MO recognition sequence is insensitive to the effects of the XProfilin2 MO. (E) Injection of XProfilin2 MO inhibits gastrulation and results in a similar gastrulation-defect phenotype as overexpression of XProfilin2 RNA (see A). This phenotype is reversed by injection of δN-XProfilin2 but not δN-XProfilin1 (F). Quantitation of phenotypic results of XProfilin2-depletion studies. Number of embryos scored (n) is shown at the top of each bar.
Fig. 4. XProfilin2 does not interfere with mesoderm induction. Embryos injected dorsally with Profilin2 (2 ng), XProfilin2 MO (70 ng), XProfilin2 MO + δN-XProfilin2 (70 ng + 100 pg) have no defects in expression of mesodermal marker genes Xbra, Gsc and XWnt8 as monitored by RT-PCR analysis; Ornithine decarboxylase (ODC) is used as a loading control, −RT without reverse transcriptase. (B) Embryos injected dorsally with XProfilin2 (2 ng), XProfilin2 MO (70 ng), XProfilin2 MO + δN-XProfilin2 (70 ng + 100 pg) show abnormal tissue localization due to gastrulation defects. Injected embryos show normal expression of Xbra at st 10.5 but Xbra is observed trapped around the blastopore that does not close at st 14. Sox-2 is expressed in the neural plate at st 14 in uninjected embryos, but in the injected embryos, Sox-2 expression surrounds the open blastopore. Gsc expression is observed in anterior mesendoderm away from the closed blastopore in control embryos at st 14, but in the injected embryos remains trapped near the open blastopore. Otx-2 is expressed anteriorly in both mesodermal and overlying neural tissues in control embryos at st 14, but in the injected embryos, the Otx-2 expression domain remains closer to the blastopore. Number of embryos scored is shown on each panel and yellow arrow indicates position of the blastopore.
Fig. 5. XProfilin2 regulates cell behavior responsible for convergent extension movement. (A) Overexpression (2 ng RNA) or depletion of XProfilin2 (70 ng MO) inhibit convergent extension in Keller explants and dominant negative Dishevelled (Xdd1, 2 ng) is used as a positive control. The effects of the XProfilin2 MO are rescued by co-expression of δN-XProfilin2. (B) Quantitation of the Keller explants. The elongation of explants was measured using ImageJ and the values were expressed relative to that of the uninjected control sample (C). Overexpression (2 ng RNA) or depletion of XProfilin2 (70 ng MO) impairs polarization, elongation and mediolateral alignment of dorsal mesodermal cells undergoing convergent extension movement similar to expression of Xdd1 (2 ng). The induced defects in cell behaviors by XProfilin2 MO are rescued by co-expression of δN-XProfilin2 but not δN-XProfilin1 and the control MO has no effects. The orientation of the explants is shown in the upper right; anterior, posterior and left and right lateral. (D and E) Quantification of the effects of XProfilin2 on cell polarization, elongation and mediolateral alignment. Numbers of cells examined are shown at the top of each bar. Average angular deviation is the average deviation of the angular orientation of the length of cells along the midline.
Fig. 6. Profilin2 modulates actin fibers. (A) Overexpression of XProfilin2 (2 ng RNA) increases the actin fibers in dorsal mesodermal cells of Xenopus embryos at neurula stage, while the depletion of XProfilin2 (70 ng MO) depletes the actin fibers. The MO mediated depletion of the actin fibers is rescued by co-expression of δN-XProfilin2. (B) Quantitation of the effects of Profilin2 on the abundance of actin fibers. Numbers of the cells counted are shown at the top of each bar.