Spatial and temporal analysis of PCP protein dynamics during neural tube closure
- University of Texas at Austin, United States
Figures
Figure 1 with 3 supplements
Planar polarized localization of Prickle2 and Vangl2 in the neural plate.
(A) Neural epithelium mosaically labeled with GFP-Vangl2, RFP-Pk2, and membraneBFP showing the overlapping localizations of Pk2 and Vangl2. Anterior is up, and scale = 10 µm. (B) Graph plotting …
Figure 1—figure supplement 1
GFP-Pk2 localizes to anterior apicolateral regions of cells in the Xenopus neural plate.
Pseudo 3D render of neural epithelium mosaically labeled with GFP-Pk2 and membraneRFP showing Pk2 restricted to the apicolateral anterior regions.
Figure 1—figure supplement 2
Pk2 knockdown results in embryonic convergent extension phenotypes.
(A) Dorsal view: stereoscope images of a representative Stage 20 control embryo, an embryo that has been injected dorsally with 25 ng Pk2 morpholino, an embryo that received morpholino plus a 400 pg …
Figure 1—figure supplement 3
A dominant negative Pk2 disrupts convergent extension.
(A–B) Stereo images of a stage 30 control embryo (A) and clutchmate dorsally expressing Pk2-ΔPΔL (B) which failed to properly extend the body axis.
Figure 2 with 1 supplement
High-magnification time-lapse imaging of convergent extension in the closing Xenopus neural tube.
(A) Stereo image stills from a time-lapse movie of Xenopus neural tube morphogenesis from stages 12 to 19. (B) Stills from time-lapse confocal imaging of the dorsal side of an embryo from stages 12 …
Figure 2—figure supplement 1
In vivo imaging of cell trajectories in the closing neural tube.
Three successive still images from the time-lapse movie shown in Figure 2 and Video 1 with a subset of cells labeled with membraneGFP and H2B-RFP being tracked. Scale = 200 µm. The far right panel …
Figure 3
Polarized apical junction dynamics facilitate mediolateral cell intercalations.
(A) Confocal images of junction dynamics in the Xenopus neural plate epithelium labeled with membraneGFP. Magenta lines mark the shrinking of a V-junction during a T transition; after complete …
Figure 4 with 1 supplement
PCP function is required for polarized junction shrinking in the neural plate.
(A) Confocal images from a Xenopus neural plate evenly labeled with membraneBFP and Utrophin-RFP (actin biosensor), but mosaically co-expressing H2B-RFP (magenta nuclei) together with Xdd1 on one …
Figure 4—figure supplement 1
A dominant negative Pk2 disrupts polarized cell rearrangements.
(A) Confocal images from a Xenopus neural plate at stage 12.5 (top) and stage 14 (bottom) evenly labeled with Myl9-GFP and Utrophin-RFP (actin biosensor), but mosaically co-expressing H2B-RFP …
Figure 5 with 1 supplement
Pk2 is dynamically enriched at shrinking V-junctions.
(A) Confocal images of neural epithelial cells labeled with membraneRFP (pseudocolored blue) and GFP-Pk2 showing the change in length of shrinking junctions (inward facing arrowheads) and growing …
Figure 5—figure supplement 1
Distinct Vangl2 dynamics at adjacent growing and shrinking junctions.
(A) Confocal stills of a neural epithelium mosaically labeled with GFP-Vangl2 (left) and co-labeled with H2B-RFP and membraneBFP merged with the DIC image (right). White lines indicate junctions of …
Figure 6 with 2 supplements
Pk2 and Vangl2 dynamics at shrinking and growing junctions.
(A) Raw GFP-Pk2 pixel intensities strongly correlate with junction length changes. Inset shows a magnified view of the core of the plot. n = 71 junctions from five embryos across four experiments. (B…
Figure 6—figure supplement 1
PCP protein localization is correlated with junctional behavior.
(A) Plot of normalized GFP-Pk2 intensities versus change in junction length (r = −0.49; p<0.001; n = 71). Normalization was performed by subtracting background and expressing the intensity of …
Figure 6—figure supplement 2
Pk2 is more highly enriched at shrinking mediolateral junctions.
(A) Plot of average Pk2 intensities normalized to the membrane label intensities at two time points against the change in the length of the junction divided by the time in seconds between the two …
Figure 7
Turnover of Pk2 and Vangl2 correlates with junction behavior.
(A) Still images from time-lapse movies captured before and after photobleaching a nonshrinking and shrinking junction of cells mosaically labeled with GFP-Pk2 in the neural plate, with a LUT …
Figure 8 with 1 supplement
PCP function is required for polarization of actomyosin contractility during junction shrinking.
(A–B). Confocal images of Xenopus neural epithelia labeled evenly with Myl9-GFP and membraneBFP and mosaically with H2B-RFP serving as a tracer for either Xdd1 (A) or Pk2-ΔPΔL (B) expression. Scale =…
Figure 8—figure supplement 1
(A) Plot showing the correlation between changes in junction length and Utrophin-RFP (actin biosensor) intensities. (B) Plot showing the correlation between changes in junction length and Myl9-GFP …
Figure 9
Spatiotemporal coordination of Prickle2 and actomyosin accumulation at shrinking junctions.
(A) Confocal images of a shrinking V-junction mosaically labeled with Myl9-GFP, RFP-Pk2, and membraneBFP over the course of a 1600 s time lapse and associated intensity plot profile for each …
Videos
Video 1
Time-lapse confocal images of a Xenopus laevis neural plate mosaically labeled with GFP-Vangl2 (left), RFP-Pk2 (middle), and memBFP (right, merged with GFP-Vangl2, RFP-Pk2, and DIC channels).
Still images and scale are shown in Figure 1A.
Video 2
Time-lapse confocal images of the dorsal side of a Xenopus laevis embryo from stages 12 to 16.
Cells labeled with membraneGFP and nuclear H2B-RFP are merged with the DIC image of the same embryo. Still images and scale are shown in Figure 2B.
Video 3
Time-lapse confocal images of the dorsal side of a Xenopus laevis embryo from stages 12 to 16.
Cells labeled with membraneGFP and pseudo-colored to help track cell rearrangements during neural convergent extension. Still images and scale are shown in Figure 2C.
Video 4
Time-lapse confocal images of a Xenopus laevis neural plate mosaically labeled with GFP-Pk2 (left) and memRFP (right, pseudo-colored blue and merged with GFP-Pk2 and DIC channels).
Still images and scale are shown in Figure 5A and a magnified view is shown in Video 5 and analysis of the mediolaterally aligned junctions annotated in the beginning of the movie are provided in Fig…
Video 5
Time-lapse confocal images of a Xenopus laevis neural plate mosaically labeled with GFP-Pk2 and memRFP (pseudo-colored blue) merged with DIC.
Shrinking and growing junctions are annotated with red and yellow lines, respectively, and analysis of these junctions is provided in Figure 5C.
Video 6
Time-lapse confocal images of a Xenopus laevis neural plate mosaically labeled with Myl9-GFP (top left), RFP-Pk2 (bottom left), and memBFP (top right).
The bottom right panel is a merge of the Myl9-GFP and RFP-Pk2 channels. Still images and analysis of these movies is provided in Figure 9A,B.
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.36456.026
