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Spatial and temporal analysis of PCP protein dynamics during neural tube closure

  1. Mitchell T Butler
  2. John B Wallingford  Is a corresponding author
  1. University of Texas at Austin, United States
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Cite this article as: eLife 2018;7:e36456 doi: 10.7554/eLife.36456

Abstract

Planar cell polarity (PCP) controls convergent extension and axis elongation in all vertebrates. Although asymmetric localization of PCP proteins is central to their function, we understand little about PCP protein localization during convergent extension. Here, we use quantitative live imaging to simultaneously monitor cell intercalation behaviors and PCP protein dynamics in the Xenopus laevis neural plate epithelium. We observed asymmetric enrichment of PCP proteins, but more interestingly, we observed tight correlation of PCP protein enrichment with actomyosin-driven contractile behavior of cell-cell junctions. Moreover, we found that the turnover rates of junctional PCP proteins also correlated with the contractile behavior of individual junctions. All these dynamic relationships were disrupted when PCP signaling was manipulated. Together, these results provide a dynamic and quantitative view of PCP protein localization during convergent extension and suggest a complex and intimate link between the dynamic localization of core PCP proteins, actomyosin assembly, and polarized junction shrinking during cell intercalation in the closing vertebrate neural tube.

https://doi.org/10.7554/eLife.36456.001

Introduction

Convergent extension (CE) is the evolutionarily conserved morphogenetic engine that drives elongation of the body axis in animals ranging from insects to mammals (Tada and Heisenberg, 2012). Multiple cell behaviors can contribute to CE, but by far the most well-understood process is cell intercalation, by which cells rearrange in a polarized manner (Walck-Shannon and Hardin, 2014). Cell intercalation, in turn, is thought to be driven by multiple subcellular behaviors, including extension of mediolaterally directed cellular protrusions (e.g. [Keller and Hardin, 1987Shih and Keller, 1992]) and active shrinkage of mediolaterally oriented cell-cell junctions (e.g. [Bertet et al., 2004; Blankenship et al., 2006]). More recent data suggest that these two subcellular behaviors likely work in concert (Sun et al., 2017; Williams et al., 2014). Understanding the molecular mechanisms governing protrusive activity and junction shrinking during cell intercalation will be essential to understanding convergent extension.

The junction shrinking mechanism for cell intercalation was initially described in Drosophila (Bertet et al., 2004; Blankenship et al., 2006) and was subsequently identified in both epithelial and mesenchymal cells in vertebrates (Lienkamp et al., 2012; Nishimura et al., 2012; Shindo and Wallingford, 2014; Trichas et al., 2012; Williams et al., 2014). In all tissues examined by live imaging, junction shrinkage is accompanied by pulsed actomyosin contractions that are restricted to or enriched at mediolaterally oriented cell-cell junctions and absent from or less common at the junctions perpendicular to the anterior-posterior axis (Bertet et al., 2004; Blankenship et al., 2006; Shindo and Wallingford, 2014; Williams et al., 2014). A major unresolved question concerns the molecular mechanism by which actomyosin activity is restricted to specific cell-cell junctions during intercalation.

In Drosophila, pair-rule genes and Toll receptors are crucial regulators of polarized actomyosin (Paré et al., 2014; Zallen and Wieschaus, 2004), but homologous genes do not appear to be involved in vertebrates. Instead, the most well-characterized regulator of mediolateral cell intercalation in vertebrates is the Planar Cell Polarity (PCP) signaling system (Butler and Wallingford, 2017). Indeed, PCP signaling controls cell intercalation during gastrulation and neural tube closure in frogs, fish, and mice, and mutations in PCP genes are now a well-defined genetic risk factor for human neural tube birth defects (NTDs) (De Marco et al., 2014; Juriloff and Harris, 2012; Wallingford et al., 2013). Understanding PCP signaling is therefore a critical challenge in developmental cell biology.

One fundamental principle of PCP signaling is that cell polarity is imparted by asymmetric enrichment of the core PCP proteins (Butler and Wallingford, 2017; Strutt and Strutt, 2009). As shown first in Drosophila, the Prickle (Pk) and Van Gogh (Vangl) proteins act in concert on one side of the cell, and Dishevelled and Frizzled act on the complementary side (Axelrod, 2001; Bastock et al., 2003; Strutt, 2001; Tree et al., 2002). Interestingly, FRAP studies in Drosophila have shown that these patterns of enrichment are driven by planar polarization of the junctional turnover kinetics of PCP proteins, underscoring the dynamic nature of the PCP signaling system (Strutt et al., 2011). Similar patterns of enrichment and turnover have been reported in vertebrate epithelia (Butler and Wallingford, 2015; Chien et al., 2015; Shi et al., 2016), but less is known about PCP protein localization dynamics during cell intercalation.

For example, complementary, asymmetric domains of PCP protein enrichment have been described during vertebrate CE (Ciruna et al., 2006; Jiang et al., 2005; McGreevy et al., 2015; Ossipova et al., 2015; Roszko et al., 2015; Yin et al., 2008), but how PCP protein enrichment is coordinated in space and time with the subcellular behaviors that drive intercalation remains essentially unexplored. This gap in our knowledge is critical, because recent work demonstrates that PCP proteins are required for the junction shrinking behaviors that contribute critically to cell intercalation (Lienkamp et al., 2012; Nishimura et al., 2012; Shindo and Wallingford, 2014). Thus, there is a pressing need for a quantitative, dynamic picture of PCP protein localization as it relates both to subcellular behaviors involved in cell intercalation and to the actomyosin machinery that drives them.

To this end, we established methods for robust quantification of PCP protein localization in a living vertebrate neural plate as well as methods for correlating PCP protein dynamics with the subcellular behaviors that drive epithelial cell intercalation. Strikingly, we find that in addition to expected patterns of spatial asymmetry, PCP protein enrichment is tightly linked to cell-cell junction behavior: Prickle2 (Pk2) and Vangl2 were dynamically enriched specifically at shrinking cell-cell junctions and depleted from elongating junctions during cell intercalation. FRAP analysis revealed that these patterns of enrichment reflected differences in the kinetics of protein turnover at these sites. Moreover, Pk2 enrichment was temporally and spatially correlated with planar polarized oscillations of junctional actomyosin enrichment. Importantly, all these dynamic relationships were disrupted when PCP signaling was manipulated. Thus, our studies reveal an intimate link between the dynamic localization of core PCP proteins, actomyosin assembly, and polarized junction shrinking during cell intercalation of the closing vertebrate neural tube.

Results

We characterized PCP protein dynamics in the neural plate of Xenopus, as studies in this animal consistently prefigure similar results in mammalian systems yet provide exceptional views of dynamic cell biological processes. Because Dishevelled and Frizzled function in both PCP and canonical Wnt signaling, their roles are more difficult to interrogate, so we focused instead on Prickle and Vangl, which act solely in PCP signaling and are required for CE and neural tube closure in vertebrates, including Xenopus (Darken et al., 2002; Goto et al., 2005; Goto and Keller, 2002; Kibar et al., 2001; Takeuchi et al., 2003). Previous work suggests that Prickle and Vangl localize to the anterior face of cells in the Xenopus neural plate (Ossipova et al., 2015), but we sought to establish a more robust quantification of this pattern as a foundation for the time-lapse studies described below.

We used mRNA injection to express fluorescent protein (FP) fusions to PCP proteins in Xenopus. Because overexpression of PCP proteins has a well-documented effect on PCP signaling, we used titration experiments to empirically determine the lowest possible dose of mRNA that allowed imaging in the Xenopus neural plate. Under these conditions, GFP-Vangl2 displayed a strong asymmetric bias to anterior cell faces (Figure 1A), while at slightly higher doses of injected mRNA, low levels of Vangl2 could be observed at the posterior faces as well (not shown), consistent with previous reports (Ossipova et al., 2015; Roszko et al., 2015). Core PCP proteins are encoded by multi-gene families, and despite the reported role of Prickle1 in convergent extension, GFP-Pk1 did not display asymmetric enrichment in our hands (not shown). However, GFP-Prickle2 (Butler and Wallingford, 2015) was strongly enriched anteriorly (Figure 1A). GFP-Pk2 was also restricted to the apicolateral cell junctional regions, as expected (Figure 1—figure supplement 1). Knockdown experiments confirmed that Pk2 was physiologically relevant for convergent extension and neural tube closure in Xenopus (Figure 1—figure supplement 2). In double-labeling experiments, GFP-Vangl2 strongly co-localized with RFP-Pk2 (Figure 1A, Video 1).

Figure 1 with 3 supplements see all
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 GFP-Pk2 intensity along V-junctions (0–45° relative to mediolateral axis) and T-junctions (46–90° relative to mediolateral axis) normalized as a ratio to the mean cytoplasmic intensity in control cells and cells expressing Xdd1 and Pk2-ΔPΔL. Error bars represent standard deviation. Ctrl V vs. T, p<0.0001***; Xdd1 V vs. T, p=0.5799; Pk2-ΔPΔL V vs. T, p=0.173; Ctrl V vs. Xdd1 T, p=0.5770; Ctrl T vs. Pk2-ΔPΔL V, p=0.0268 (Mann Whitney test for significance). n = 101 V and 71 T from three experiments, seven embryos (Ctrl); n = n = 171 V and 199 T from four experiments, five embryos (Xdd1); n = 128 V and 142 T from three experiments, seven embryos (Pk2-ΔPΔL). (C) Distributions of data shown in (B) plotted against the angle of the junction at which the intensity was measured. Correlation coefficients for Xdd1 and Pk2-ΔPΔL were significantly different from controls using the Fischer R-to-Z transformation. n = 172 junctions (Control), n = 263 junctions (Xdd1) and n = 245 junctions (Pk2-ΔPΔL) (D-E) Confocal images of Xenopus neural epithelia labeled evenly with GFP-Pk2 and membraneBFP and mosaically with H2B-RFP, serving as a tracer for either Xdd1 (C) or Pk2-ΔPΔL (D) expression. Scale = 10 µm.

https://doi.org/10.7554/eLife.36456.002
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.

https://doi.org/10.7554/eLife.36456.006

We quantified these localization patterns at the population level by binning cell-cell junctions into two groups based on their orientation. To maintain a consistent nomenclature with previous work on cell intercalation, we refer to junctions aligned within 45 degrees of the mediolateral axis as ‘V-junctions’ and the perpendicular junctions (between 45 and 90 degrees off the mediolateral axis) as ‘T-junctions.’ An important subtlety to note here is that the V-junctions are mediolaterally aligned, but actually separate cells that are anteroposterior neighbors. We found that GFP-Pk2 was significantly enriched at V-junctions as compared to T-junctions (Figure 1B). In a more granular view of the data, we observed a significant correlation between GFP-Pk2 pixel intensity and junction angle, with higher enrichment along more mediolaterally oriented junctions (Figure 1C).

To test these quantification schemes, we took advantage of the fact that in many other systems, disruption of any one core PCP protein leads to loss of polarized enrichment of the others. We used targeted injection to co-express reagents for disrupting PCP signaling together with a nuclear RFP lineage tracer, allowing us to compare normal and experimental cells in the same embryo (Figure 1D,E). Xdd1 is a well-defined, PCP-specific dominant negative of Dvl2 (Sokol, 1996; Wallingford et al., 2000) that disrupts convergent extension of the neural tube in Xenopus (Wallingford and Harland, 2001; Wallingford and Harland, 2002); we found that Xdd1 expression severely disrupted Prickle2 asymmetries in the neural plate using both ensemble and individual metrics (Figure 1B,C).

To extend this analysis, we explored Pk2 signaling by expressing a deletion construct lacking the PET and LIM domains (Pk2-ΔPΔL), as this construct disrupted PCP in Xenopus multiciliated cells and an equivalent deletion of Pk1 disrupts CE (Butler and Wallingford, 2015; Takeuchi et al., 2003). We found that Pk2-ΔPΔL strongly disrupted axis elongation in Xenopus (Figure 1—figure supplement 3) and also severely disrupted the planar asymmetry of co-expressed wild-type Pk2-GFP in the neural plate (Figure 1B,C). These results establish the Xenopus neural plate as an effective, quantitative platform for studies of PCP protein localization.

Epithelial convergent extension in the closing Xenopus neural tube involves PCP-dependent polarized junction shrinking

Convergent extension is an inherently dynamic process, as cells constantly exchange neighbors. The dynamic nature of tissues engaged in convergent extension differs markedly from the settings in which PCP protein localization is most commonly studied. We therefore sought to exploit the strengths of Xenopus embryos to image PCP protein dynamics together with the subcellular behaviors that drive convergent extension in the closing neural tube. Curiously, the neural plate of Xenopus consists of two cell layers, an outer epithelial layer and a deeper mesenchymal layer (Schroeder, 1970). While cell intercalation of the deep mesenchymal cells has been characterized (Elul et al., 1997; Keller et al., 1992), the behaviors of the overlying epithelial cells have not. This distinction is not trivial, because it is the outer epithelial cells that display the robust patterns of PCP protein localization described above. To understand how PCP protein localization relates to convergent extension cell behaviors, we had first to characterize cell behaviors in this epithelium.

Neural tube closure spans roughly 6 hr in Xenopus, starting with the shaping of the neural tube at last gastrula stages, followed by the progressive elevation and apposition of the neural folds (Figure 2A). We immobilized embryos on a confocal microscope stage (Kieserman et al., 2010) and used image tiling to collect high-magnification images of the superficial neural plate across the entirety of neural tube closure (Video 2). This approach allowed assessment of tissue-level morphogenetic changes in the neural plate, as well as individual cell trajectories, which closely resembled those of previous studies ((Figure 2B; Figure 2—figure supplement 1) and see [Keller et al., 1992]). Moreover, our tiling approach provided sufficient magnification to quantify the discrete behaviors of individual cells, and tracking of cell clusters revealed extensive intercalations that were mediolaterally biased, resulting in convergence and extension (Figure 2C, Video 3).

Figure 2 with 1 supplement see all
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 to 16. Cells labeled with membraneGFP and nuclear H2B-RFP are merged with the DIC image of the same embryo. Images are shown ~2 hr. apart (128 min. interval). Scale = 200 µm. (C) Higher magnification images of cell rearrangements in neural ectoderm from the time-lapse shown in (B). Labeling of individual cells with colored dots across time points demonstrates the cellular rearrangements contributing to the narrowing and lengthening of tissue and T transitions labeled with magenta for shrinking (T1-T2) and cyan for growing (T2-T3) junctions. Scale = approx. 20 µm.

https://doi.org/10.7554/eLife.36456.007
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.

https://doi.org/10.7554/eLife.36456.009
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.

https://doi.org/10.7554/eLife.36456.010

Cell intercalations in the neural plate were associated with so-called T transitions, which are characterized by preferential contraction of junctions aligned in the mediolateral axis (T1-T2 transition) (Figure 3A,a’), followed by elongation of new junctions perpendicularly along the anteroposterior axis (T2-T3 transition)(Figure 3a’, a’’)(Bertet et al., 2004). As above, we first quantified these behaviors at the ensemble level and found that mediolaterally oriented V-junctions preferentially shrank, while the perpendicular T-junctions elongated (Figure 3B). Moreover, when we plotted the change in the length of cell-cell junctions against the average angle of that junction for all cells examined (n > 250), we observed a significantly positive correlation; V-junctions preferentially shrank along the mediolateral axis and T-junctions elongated perpendicularly (Figure 3C). Importantly, the orientation of shrinking and growing junctions in this analysis remained fairly constant, changing on average only 5 (±4) degrees over the course of measurement, and no junctions shifted by more than 20 degrees. Thus, in general, V-junctions separate anteroposterior (AP) neighbors and T-junctions separate mediolateral (ML neighbors). Finally, we also observed planar polarized formation and resolution of multicellular rosettes (Figure 3D–F), as have been described in other epithelia (Blankenship et al., 2006; Lienkamp et al., 2012; Trichas et al., 2012).

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 shrinkage mediolaterally (T1-T2) (a’), a new junction (cyan) elongates perpendicularly along the AP axis (T2-T3) (a’’). Scale = approx. 20 µm. (B) Graph showing the mean change (±s.d.) in junction length for V- and T-junctions. (C) Plot of the average angles of junctions over 1800 s against the change in junction length. Each dot represents on cell-cell junction. n = 267 junctions from three embryos across three different experiments. (D) The simultaneous mediolateral shrinking of two neighboring v-junctions (magenta) leads to formation of a multicellular rosette (d’), and new junctions (cyan) that emerge from the resolving rosette are oriented along the AP axis (d’’). Scale = approx. 20 µm. (E) Rose diagram plotting the orientation of shrinking junctions that lead to the formation of multicellular rosettes and the mean resultant vector (arrow). n = 42 junctions from four embryos across three separate experiments. (F) Rose diagram plotting the orientation of new junctions emerging from resolving rosettes and mean resultant vector (arrow). n = 36 new junctions from 3 embryos across three separate experiments.

https://doi.org/10.7554/eLife.36456.011

Because PCP signaling is essential for neural convergent extension, we next assessed the effect of PCP disruption specifically on junction shrinking behaviors in the neural epithelium. Using the mosaic approach described above, we found that expression of Xdd1 elicited the expected tissue level defect in the medially directed movement of the neural folds (Figure 4A, magenta nuclei indicate Xdd1 expressing cells).

Figure 4 with 1 supplement see all
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 side (right). The lateral boundaries of the neural plate are marked in each frame by yellow lines, demonstrating that Xdd1 expression disrupts the medial movement of the right neural fold in comparison to the control fold on the left. Scale = 50 µm (B) Graph of average angle of junctions versus junction length change for cells expressing Xdd1 to compare with control plot in Figure 3C. n = 187 junctions from four embryos across three experiments. (C) Graph with the total number of T transition events expressed as transitions per hour per the number of cells examined, with each point representing a single embryo. Error bars represent standard deviation. For statistical analysis, Control vs. Xdd1, and Control vs. Pk2-ΔPΔL ML, p=0.0025** for both classes of transitions (Mann-Whitney Test for significance). n = 4 experiments, seven embryos, 1167 cells (Control); three experiments, five embryos, 560 cells (Xdd1); three experiments, five embryos, 841 cells (Pk2-ΔPΔL). (D) The calculated rate of junction contraction for completed Type one to Type two transition (T1-T2) (complete contraction of a V-junction, see Figure 3A–a’). Error bars represent standard deviation. Ctrl vs. Xdd1, p<0.0001****, Ctrl vs. Pk2-ΔPΔL, p<0.0001****, Xdd1 vs. Pk2-ΔPΔL, p=0.2051. (Mann-Whitney statistical test). n = 24 junctions from four embryos across three experiments (Ctrl), n = 19, 2, 2 (Xdd1), and n = 9, 3, 3 (Pk2-ΔPΔL). (E) Plot of the angle of the long axis of control and Xdd1-expressing cells at Stages 12–12.5 (early) and Stages 13–14 (late), with lines connecting angles of the same cell at the two different time points. All measurements are from different regions of embryos that mosaically express Xdd1, similar to as shown in (A). n = 136 control cells and 114 Xdd1-expressing cells from four embryos across three experiments.

https://doi.org/10.7554/eLife.36456.012

Analysis of individual cell behaviors in these embryos revealed that Xdd1 expression significantly disrupted planar polarized junction shrinking, eliminating the strong correlation between junction angle and junction shrinkage or growth (Figure 4B, compare with Figure 3C). This uncoupling of junction behavior from orientation was associated with reduced junction shrinkage generally, and a reduction in the number of productive T transitions (Figure 4C). In fact, even the few productive T transitions that were observed were significantly slowed (Figure 4D).

Finally, these defects in cell intercalation behavior had a profound impact on cell shape. During neural plate shaping stages (12-14), the majority of neural epithelial cells in control embryos exhibited a shift from an anteroposterior orientation to a mediolateral orientation (Figure 4E, green), while Xdd1 expressing cells maintain their alignment in the anteroposterior axis (Figure 4E, blue). In fact, these cells actually increased their length-to-width ratios along the AP axis (not shown), likely as a result of forces generated by other cell behaviors that shape the neural plate at these stages (e.g. apical constriction, elongation of underlying mesoderm, etc.). Notably, genetic mutation of PCP genes in zebrafish also elicits similar spectrum of cell shape and orientation defects (Roszko et al., 2015). Finally, disruption of Prickle function by expression of the dominant-negative Pk2-ΔPΔL elicited the same spectrum of defects (Figure 4C,D, pink; Figure 4—figure supplement 1). Together with our data on PCP protein localization (Figure 1, above), these results establish the Xenopus neural plate as an effective platform with which to probe the relationship between epithelial cell intercalation behaviors and core PCP protein dynamics.

Prickle2 and Vangl2 are dynamically enriched specifically at shrinking cell-cell junctions

With these imaging and analysis systems in place, we performed time-lapse imaging with an eye toward understanding the dynamic relationship between PCP protein localization and epithelial cell behaviors. This analysis revealed several novel insights. First, we noted that the accumulation of Pk2 was junction-specific, displaying overt changes in intensity precisely at tricellular junctions (Figure 5A). Moreover, we found that GFP-Pk2 was dynamically enriched at shrinking junctions but depleted from elongating junctions (Figure 5A), suggesting that dynamic enrichment of Pk2 might not simply reflect the junction’s spatial orientation (e.g. to V- vs. T-junctions). This notion was support by the observation that even when adjacent junctions share a similar orientation, the GFP-Pk2 intensity at these junctions frequently differed substantially (Videos 4 and 5). For example, the still images in Figure 5B show three adjacent junctions sharing a roughly similar mediolateral alignment; none deviates by more than 20 degrees from the mediolateral at any point in the movie. Nonetheless, two of the junctions shrink during the movie (red brackets) while the third one grows (yellow brackets). Even among these similarly oriented junctions, shrinking is associated with increasing levels of Pk2-GFP intensity, and elongation with decreasing Pk2-GFP (Figure 5C). Similar results were obtained for Vangl2 (Figure 5—figure supplement 1).

Figure 5 with 1 supplement see all
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 junctions (outward facing arrows) along with the corresponding change in GFP-Pk2 intensity at two different time points. Scale = 10 µm (B) Higher magnification view of the horizontal junctions shown in A, shown at 5-min intervals. Red brackets indicated shrinking junctions; yellow brackets indicate growing junctions. Scale = 10 µm (C) Plots of intensity (red/yellow traces) and length (black dashed traces) for each of the indicated junctions in Panel B. Plots show 30 min of junction dynamics ending in either junction resolution or junction formation and expansion; gray area behind select data points indicate the interval of the four frames shown in (B), which are offset due to different junctions appearing and resolving at different times.

https://doi.org/10.7554/eLife.36456.014
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 Figure 5B,C.

https://doi.org/10.7554/eLife.36456.016
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.

https://doi.org/10.7554/eLife.36456.017

To quantify these observations, we plotted the changes in GFP-Pk2 fluorescence intensity against corresponding changes in junction length, and we observed a strong and highly significant correlation (Figure 6A). Similar results were observed with Vangl2 (Figure 6B). We considered the possibility that these changes in intensity could reflect density, increasing simply if the amount of protein on a junction remains constant as that junction shrinks. To explore this idea, we examined a generic membrane marker (FP-caax) and found that while it did show a tendency to increase as junctions shrink, this increase was modest and the correlation was far weaker than that observed for PCP proteins (Figure 6C). Importantly, even when normalized against the membrane marker to account for changes in membrane-FP in the same junctions, the intensities of GFP-Vangl2 and GFP-Prickle2 still displayed strong and significant correlations with junction shrinkage (Figure 6—figure supplement 1).

Figure 6 with 2 supplements see all
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) Raw GFP-Vangl2 pixel intensities strongly correlate with junction length changes. n = 37 junctions from 2 embryos from two different experiments. (C) GFP-caax displays only a weak correlation with junction length changes. Inset shows a magnified view of the core of the plot. n = 108 junctions from seven embryos across six experiments. Even when normalized against GFP-caax, Pk2 and Vangl2 levels show a strong correlation to junction length changes, as shown in Figure 6figure supplement 1 Figure 6

https://doi.org/10.7554/eLife.36456.018

These findings suggest that the strength of asymmetric Pk2 and Vangl2 enrichment at a particular junction is at least as strongly tied to the dynamic behavior of that junction as it is to the junction’s orientation. As a final test of this idea, we selected a subset of V-junctions which remained aligned within 30 degrees of the ML axis for the entire movie and then plotted the average velocity of shrinking or growth against the average intensity of GFP-Pk2 at that junction. Again, we found a highly significant correlation (Figure 6—figure supplement 2). Together, these data suggest that the enrichment of Pk2 and Vangl2 is governed both by the reciprocal interaction of each individual pair of neighboring cells and is intricately linked to the behavior of the shared junction between them.

Turnover of Prickle2 and Vangl2 at cell-cell junctions is planar polarized during cell intercalation

Of the PCP protein dynamics we observed, we felt that the enrichment specifically at shrinking V-junctions was the most significant. Our data with Caax-GFP (Figure 6C) suggest that simply reducing the junction length is not sufficient to increase density to the full extent observed for Pk2 and Vangl2 at shrinking junctions. We reasoned, therefore, that PCP enrichment could also involve an active process, for example in which regulated turnover kinetics are more dynamic at some junctions and less so at others. FRAP studies have demonstrated that polarized PCP protein turnover is planar polarized in other cell types (Butler and Wallingford, 2015; Chien et al., 2015; Shi et al., 2016; Strutt et al., 2011), so we used this method to assess localization dynamics during cell intercalation in the closing neural tube (Figure 7A).

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 applied for warmer colors representing higher fluorescence intensities. Dashed red box marks the bleached region of interest. Note that the cell on the anterior side of the junctions is unlabeled. Scale = 5 µm. (B, C) Graphs showing mean fluorescence recovery after photobleaching at shrinking and nonshrinking junctions for GFP-Pk2 (n = 34 nonshrinking, n = 15 shrinking) and GFP-Vangl2 (n = 17 nonshriking, n = 16 shrinking). Shrinking junctions were defined as those that were reduced by 0.5 μm or more in length over the course of bleaching and fluorescence intensity recovery. Error bars represent SEM. (DE) Graphs plotting the change in junction length during photobleaching and recovery against the calculated nonmobile fraction for the individual junctions analyzed in (B) and (C) with associated linear regression model and correlation analysis statistics included. n = 49 (GFP-Prickle2); n = 33 (GFP-Vangl2).

https://doi.org/10.7554/eLife.36456.021

Both Pk2 and Vangl2 displayed striking differences in turnover kinetics at shrinking versus non-shrinking junctions, with significantly less recovery (i.e. higher stable fraction) for both Vangl2 and Prickle2 at shrinking junctions compared to non-shrinking junctions (Figure 7B,C). Moreover, when we integrated these FRAP data with time-lapse analysis of cell behaviors, we found that the stable fraction of both proteins correlated significantly with changes in junction length: more rapidly shrinking junctions displayed higher stable fractions of junctional PCP proteins (Figure 7D,E). Thus, the overall accumulation of Prickle2 and Vangl2 at shrinking junctions parallels the increased stability of these proteins at these sites, and together, these data suggest a key role for PCP protein trafficking in the coordination of cell-cell junction shrinkage.

Planar polarization of actomyosin during junction shrinking in the neural epithelium is PCP-dependent

We next sought to understand the link between PCP protein localization and the actomyosin machinery known to drive cell-cell junction shrinkage. Time-lapse imaging in Drosophila first demonstrated that cell intercalation by junction shrinkage is accompanied by pulsed accumulations of actomyosin at V-junctions (Bertet et al., 2004; Fernandez-Gonzalez et al., 2009; Rauzi et al., 2008). While this process is independent of PCP signaling in Drosophila, similar actomyosin pulses have been observed during PCP-dependent junction shrinking in mesenchymal cells of the Xenopus gastrula mesoderm (Shindo and Wallingford, 2014). Static analyses of Xenopus, chicks and mice also indicate that actomyosin is enriched at mediolaterally oriented junctions (McGreevy et al., 2015; Nishimura et al., 2012; Williams et al., 2014). However, the spatiotemporal relationship between actomyosin dynamics and subcellular behaviors during cell intercalation in the vertebrate neural tube remains poorly defined.

Using a GFP-fusion to the myosin regulatory light chain Myl9 (Shindo and Wallingford, 2014), we observed a strong enrichment at V-junctions as compared to T-junctions. This enrichment was apparent at both the population level (Figure 8A–C, green) and in the significant correlation between Myl9 intensity and junction angle for individual junctions (Figure 8D, green, n > 450). Consistent with previous reports in other systems (Bertet et al., 2004; Shindo and Wallingford, 2014), Myl9 intensity was elevated in shrinking junctions (Figure 8—figure supplement 1A). These correlations are likely to be functionally relevant, because changes in myosin enrichment strongly correlated with decreases in junction length. Similar results were obtained using the actin biosensor Utrophin-RFP (Burkel et al., 2007), and the changes in myosin intensity were also strongly correlated to changes in actin on individual junctions (Figure 8—figure supplement 1).

Figure 8 with 1 supplement see all
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 = 10 µm (C) Graph plotting Myl9-GFP intensity along V-junctions (0–45° relative to mediolateral axis) and T-junctions (46–90° relative to mediolateral axis) normalized as a ratio to the mean cytoplasmic intensity of the cells sharing the junction. Control cells (n = 91 V, 91T) and cells expressing Xdd1 (n = 44 V, 53 T) and Pk2-ΔPΔL (n = 45 V, 45 T). Ctrl V vs. T, p<0.0001****; Pk2-ΔPΔL V vs. T, p=0.2304; Xdd1 V vs. T, p=0.0022**; Control V vs. Xdd1 V, p=0.5826; Control T vs. Xdd1 T, p<0.0001****; Control T vs. Pk2-ΔPΔL T, p<0.0001**** (Mann-Whitney Test for significance). Error bars represent standard deviation. (D) Distributions of normalized Myl9-GFP intensity plotted against the angle of the junction at which intensity was measured in control cells and cells expressing Xdd1 or Pk2-ΔPΔL. Correlation coefficients for Xdd1 and Pk2-ΔPΔL were shown to be significantly different from controls using the Fischer R-to-Z transformation. n = 498 junctions (Control), n = 263 junctions (Xdd1) and n = 245 junctions (Pk2-ΔPΔL) from four experiments, five embryos (Xdd1); three experiments, seven embryos (Pk2-ΔPΔL).

https://doi.org/10.7554/eLife.36456.022

Expression of Xdd1 or Pk2-ΔPΔL significantly disrupted the planar polarization of myosin enrichment (Figure 8A–D). Interestingly, these reagents appear to act via converse mechanisms: Expression of Xdd1 elicited an elevation of myosin levels at T-junctions, while expression of Pk2-ΔPΔL elicited a reduction of myosin enrichment at V-junctions (Figure 8C). This result was strikingly similar to the trend observed above for GFP-Pk2 intensities in these conditions (Figure 1B), further suggesting that PCP and Myl9 enrichments are functionally related. Thus, disruption of PCP signaling disrupts both asymmetric PCP protein localization and the planar polarization of actomyosin contraction in the closing neural tube.

PCP proteins and actomyosin dynamics are spatiotemporally coordinated with junction shrinkage

In light of observed spatiotemporal patterns of PCP protein localization (Figure 8), we more closely examined Myl9 and Pk2 localization during time-lapse movies. As expected from results in other systems, Myl9-GFP displayed a pulsatile behavior at shrinking V-junctions (Figure 9A–B, Video 6). Myl9 also pulsed at T-junctions, although such pulses tended to involve larger fluctuations and failed to persistently shrink the junction (Figure 9C). Strikingly, Pk2 intensity also displayed pulsatile enrichment, and moreover, changes in Pk2 were strongly correlated with similar changes in Myl9 intensity at cell-cell junctions (Figure 9A–D). Finally, the pulses of Pk2 and Myl9 were also strongly cross-correlated in time (Figure 9E). Together, these data demonstrate shared dynamic patterns of core PCP protein and actomyosin localization in space and time during cell intercalation and suggest that these systems work in close concert to drive junction shrinking in the Xenopus neural epithelium.

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 fluorophore across the length of the junction. Scale = 10 µm. (B) Plot of mean intensities over time for the V-junction in (A) which shows the pulsed co-accumulation of Pk2 (pink) and Myl9 (green) as junction length shrinks (black dashed line). The orange box marks the time points portrayed in (A) (C) Plot of mean intensities over time for a mosaically labeled T-junction showing the pulsed co-accumulation of Pk2 (pink) and Myl9 (green) as junction fluctuates between shrinking and growing (black dashed line). (D) Scatter plot of the change in RFP-Pk2 intensity against the change in GFP-Myl9 intensity and associated correlation; a magnified view of the core of this plot is shown in Panel c’. n = 96 junctions from three embryos across two experiments. (E) Cross correlation analysis of changes in RFP-Pk2 and GFP-Myl9 intensities over time, with a mean cross correlation coefficient of 0.7 at 0 s time lag demonstrating synchronous accumulation dynamics. n = 22 junctions from three embryos; error bars represent SEM.

https://doi.org/10.7554/eLife.36456.024
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.

https://doi.org/10.7554/eLife.36456.025

Discussion

Here, we have used image tiling and time-lapse microscopy in Xenopus embryos to generate high magnification movies of the closing vertebrate neural tube. These movies allowed us to quantify core PCP protein localization and dynamics as they relate to the cell behaviors associated with convergent extension. We focus here on junction shrinkage, which together with mediolateral protrusions is an essential sub-cellular behavior contributing to cell intercalation. We find that the Prickle2 and Vangl2 display a consistent pattern of localization and turnover in space and time during cell intercalation that is strongly linked to actomyosin assembly at cell-cell junctions. Directed, mechanistic studies will be required to fully understand the relationships reported here, but our data are nonetheless significant for providing a comprehensive and quantitative view of the spatial and temporal patterns of PCP protein localization during vertebrate collective cell movements.

PCP protein localization in time and space during convergent extension

The spatial asymmetry of core PCP proteins is fundamental to their function. In a wide array of cell types, planar polarization is defined by Dvl and Frizzled enrichment to one region of the cell and Vangl and Prickle in a reciprocal pattern. Feedback across cell membranes is thought to reinforce initially weak asymmetry, leading to the robust asymmetry at later stages (Butler and Wallingford, 2017; Strutt and Strutt, 2009).

Interestingly, while cell intercalation was the first setting in which vertebrate PCP was defined, the first reports of asymmetric protein localization in vertebrates came instead from studies of cochlear hair cells and later from ciliated cells of the node and airway (Rida and Chen, 2009; Wallingford, 2010). Indeed, even now we know little about the spatial patterns and almost nothing about the temporal patterns of PCP protein localization in the context of convergent extension.

Currently, a consensus has emerged that PCP proteins spatially delineate an anterior-to-posterior axis in cells undergoing cell intercalation. This consensus has support from studies of various PCP proteins in diverse tissues, first in zebrafish and later in other animals (Ciruna et al., 2006; McGreevy et al., 2015; Nishimura et al., 2012; Ossipova et al., 2015; Roszko et al., 2015; Yin et al., 2008). This model is consistent with the similar pattern of anteroposterior localization for PCP proteins in the orientation of directional ciliary beating in the embryonic node and spinal cord (Antic et al., 2010; Borovina et al., 2010; Hashimoto et al., 2010). Moreover, this axis of polarization is consistent with embryological data suggesting that anteroposterior patterning is crucial to convergent extension in Xenopus (Ninomiya et al., 2004). However, it should be noted that several studies suggest additional regions of localization in the mediolateral ends of cells during cell intercalation (e.g. [Jiang et al., 2005; Kinoshita et al., 2003; Panousopoulou et al., 2013]).

Our data here are consistent with the idea that anteroposterior localization of PCP proteins is critical for cell intercalation, although they do not exclude additional roles in mediolateral protrusions, and disruption of PCP does disrupt the polarity and stability of mediolateral protrusions (Wallingford et al., 2000). Significantly, recent work suggests that both mediolateral protrusions and junction shrinking act together during convergent extension (Sun et al., 2017; Williams et al., 2014), so it is clear that additional studies will be required.

In our view, the more important findings here relate to the temporal aspects of PCP protein localization, as previous studies provided only static snapshots of what is a highly dynamic process. Our time-lapse studies not only reveal that Prickle2 and Vangl2 are dynamically enriched at shrinking junctions, but also suggest that the shrinking/growing status of a junction may be as important a determinant of PCP protein enrichment as is its orientation along the mediolateral/anteroposterior axes. In addition, our FRAP data reveal that turnover kinetics also differ with the dynamic behavior of the junctions, with higher stable fractions associated with shrinking junctions. Thus, our study reveals a new complexity to the pattern of PCP protein localization that likely reflects the dynamic nature of the cells involved, as they are constantly exchanging neighbors as they intercalate.

Finally, our data provide an interesting complement to previous studies linking turnover at cell junctions to planar polarized PCP protein localization. In Drosophila, the enriched regions of PCP protein localization display higher stable fractions than do non-enriched regions, and the loss of asymmetric protein localization after disrupting PCP signaling is accompanied by a loss of this asymmetric turnover (Strutt et al., 2011). Similar results have been obtained in cells of the Xenopus epidermis and in the mouse oviduct (Butler and Wallingford, 2015; Chien et al., 2015; Shi et al., 2016). However, cell-cell neighbor exchange is minimal in those contexts, in contrast to CE, where such neighbor exchanges are constant. We therefore find it interesting that we observed a similar trend during cell intercalation, where enriched regions of PCP protein localization also display higher stable fractions of PCP protein even as these junctions shrink dramatically. Interestingly, when we calculated the mean stable fraction for PCP proteins (i.e. averaging all junctions), this value was substantially lower than what we previously observed using similar methods in Xenopus multiciliated cells (Butler and Wallingford, 2015); this difference may represent an adaptation to the dynamic nature of the junctions involved. Together with the previous studies, our data demonstrate that planar polarized junction turnover kinetics are a general feature of PCP protein localization, spanning a broad spectrum of organisms and cell types.

PCP protein and myosin interplay during convergent extension

Another interesting result in this study is the tight spatiotemporal relationship between PCP proteins and myosin at shrinking junctions. PCP proteins have been shown to be required for the phosphorylation of Myosin during cell intercalation in Xenopus, chicks, and mice (Lienkamp et al., 2012; McGreevy et al., 2015; Nishimura et al., 2012; Shindo and Wallingford, 2014; Williams et al., 2014), as well as in the cochlea of mice (Lee et al., 2012). The mechanism by which PCP proteins act on Myosin remains unclear, but of the PCP proteins, it is Dvl that is most directly implicated. Dvl acts via the formin Daam1, the PDZ-RhoGEF, and RhoA to activate Rho Kinase (Habas et al., 2001; Nishimura et al., 2012), which in turn is essential for cell intercalation (Marlow et al., 2002; Ybot-Gonzalez et al., 2007). Thus, our focus here on Prickle and Vangl (necessitated by the complexity of Dvl/Fzd function) is a limitation, because the mechanisms by which Pk2 and Vangl2 impact myosin activation are unknown.

Because mechanical feedback contributes to the oscillations of actomyosin at shrinking junctions during PCP-independent cell intercalation in Drosophila (e.g. [Fernandez-Gonzalez et al., 2009]), one attractive hypothesis involves Dvl/RhoA-mediated myosin accumulation on one cell face resulting in mechanically induced myosin accumulation on the opposing cell face. In this case, Pk2 and Vangl2 function only to ensure Dvl/Fzd localization on the other side of the junction. Alternatively, Pk2 and Vangl2 may also interact directly with actomyosin machinery by a yet to be described mechanism.

Regardless of precise mechanism, it is clear that a functional PCP system is required to drive myosin contraction, so it is interesting that two recent studies suggest that, conversely, myosin is required for normal PCP protein localization. Disruption of myosin action disrupts the polarized localization of Vangl2 in the Xenopus neural plate and of Pk in the ascidian notochord (Newman-Smith et al., 2015; Ossipova et al., 2015). These data may suggest a ‘reciprocal’ relationship between PCP proteins and myosin, an idea supported by our observations here of very tight temporal and spatial correlation between Myosin levels and Pk2 levels at cell-cell junctions. However, because there is evidence that mechanical cues can impact PCP signaling in Drosophila, Xenopus, and mice (Aigouy et al., 2010; Bosveld et al., 2012; Chien et al., 2015; Luxenburg et al., 2015), it is important to consider that both the neural plate and the notochord are engaged in large-scale collective cell movement. The broad application of myosin inhibitors would clearly disrupt global patterns of cell movement and even the orientation and magnitude of tension exerted on cell-cell junctions, so myosin inhibition might disrupt PCP protein localization secondarily. Future experiments using acute, spatially resolved myosin inhibition will be required to adequately address this issue.

Conclusions

In sum, the data here provide a quantitative, dynamic view of PCP protein localization as it relates to a subcellular behavior that drives cell intercalation. This work provides new insights into the general problem of how developmental signaling systems such as PCP interface with fundamental cellular machines such as actomyosin. Finally, because defects in PCP signaling are strongly linked to human neural tube defects, this work also lays a foundation for understanding the growing spectrum of human disease-associated mutations in PCP genes.

Materials and methods

Xenopus manipulations

Eggs were and externally fertilized according to standard protocols (Sive et al., 2000). The jelly coat was removed from embryos at the two-cell stage by bathing in a solution of 2% cysteine (pH 7.9). The embryos were then washed in 1/3x Marc’s Modified Ringer’s (MMR) solution and microinjected in a solution of 1/3x MMR with 2% Ficoll using an Oxford manipulator. The mRNAs coding for fluorescent protein fusions were synthesized using mMessage mMachine kits (Ambion) and injected into one of eight dorsal blastomeres for even labeling of the neural plate at the following concentrations: 50 pg membraneGFP, 60 pg of membraneRFP, 80 pg of membraneBFP, 200 pg for GFP- or RFP-Pk2, 60 pg for GFP-Vangl2, 50 pg for H2B-RFP, and 30 pg for Myl9-GFP. For mosaically labeled tissues, mRNAs were injected at the 16- or 32 cell stages with approximately 70% of the totals amounts listed above. Dominant-negative Pk2 (Pk2-ΔPETΔLIM) was injected at 700–800 pg for overexpression, at the eight-cell stage, as was the dominant-negative Dvl (Xdd1), and both were similarly reduced by 70% for later stage injections. For Pk2 morpholino treatments, 20–25 ng was injected into one cell at the eight-cell stage, and 400 pg of GFP-Pk2 were used to rescue the morpholino phenotypes. Developmental stages were determined according to (Nieuwkoop and Faber, 1994).

Live imaging and image quantification

Confocal imaging was done using live embryos submerged in 1/3x MMR in AttoFluor Cell Chambers (Life Technologies A7816) and between coverglasses, using silicon grease as an adhesive spacer, and carried out with a Zeiss LSM700 confocal microscope. Images were processed with the Fiji distribution of ImageJ, Imaris (Bitplane) and Photoshop (Adobe) software suites, and figures were assembled in Illustrator (Adobe). For junction length and protein enrichment measures, lines (3–6 pixels wide, depending on image scale/zoom) were drawn over cell junctional regions (excluding the tricellular junctional vertices), while cytoplasmic measures were taken using the freehand shapes tools within the apicolateral cortical regions. Mean PCP and Myl9 fluorescence intensities along a cell-cell junction were normalized to against the membrane label in analysis of changes in PCP vs. during junction length changes (Supp. Figure 6) and against the average of cytoplasmic intensities of the two cells sharing a junction in the analyses when cells are labeled.(Figures 1B and 8C). Statistical analyses were carried out using Prism (Graphpad) software with Mann Whitney tests for significance and Spearman non-parametric correlations. Extreme outliers with fluorescent intensities more than three standard deviations away from the mean were removed from the analysis; in all datasets such outliers were very rare (<6). These outliers likely represent non-specific contraction waves sometimes observed in the Xenopus neural plate. For FRAP analysis, time-lapse movies were acquired after photobleaching discrete domains of core PCP GFP fusions localization. Intensity measurements were taken in Fiji, with recordings for each time point taken individually from each frame captured at bleached regions and normalized as detailed in Goldman and Spector (2005). Statistical analysis was performed in Prism (Graphpad) software with exponential decay functions. Angles of junctions shrinking to form and emerging from rosettes were measured manually in Fiji, and rose diagrams were plotted with Oriana software (Kovach Computing Services). Cross-correlation analysis was performed using a free web-based statistics calculator at www.wessa.net. Stereo time-lapse imaging was performed using a Zeiss AXIO Zoom.V16 Stereomicroscope and associated Zen software. Movies were exported from Fiji and processed in Adobe Photoshop, and cell tracking was performed with the Fiji manual tracking plug-in.

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Decision letter

  1. Lilianna Solnica-Krezel
    Reviewing Editor; Washington University School of Medicine, United States
  2. Didier YR Stainier
    Senior Editor; Max Planck Institute for Heart and Lung Research, Germany

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for sending your article entitled "Spatial and temporal PCP protein dynamics coordinate cell intercalation during neural tube closure" for peer review at eLife. Your article is being evaluated by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation is being overseen by Didier Stainier as the Senior Editor.

Given the list of essential revisions, including new experiments, the editors and reviewers invite you to respond within the next two weeks with an action plan and timetable for the completion of the additional work. We plan to share your responses with the reviewers and then issue a binding recommendation.

Summary:

Butler and Wallingford report their studies of the dynamic localization of Vangl2-GFP and Prickle2-GFP core components of the Planar Cell Polarity (PCP) pathway during convergent extension movements of neuroectodermal tissue in Xenopus. They confirm and extend earlier studies in zebrafish, frog and mouse providing snapshots of PCP protein localization that revealed enrichment of Vangl2 and Pk at the anterior edges of mediolaterally (ML) elongated cells, consistent with asymmetric localization of PCP components shown by pioneering studies in Drosophila. The value of this work is in careful temporal assessment of protein localization in relation to cell behavior and in particular to junction shrinking – one of the drivers of the ML intercalation process. A major conclusion of this study is that "the strength of asymmetric Pk2 and Vangl2 enrichment at a particular junction is more strongly tied to the dynamic behavior of that junction than it is to the junction's orientation or to positional information across the tissue." In addition, they show that Pk and Vangl2 are more stable at shrinking rather than growing junctions, and PCP enrichment correlates with myosin junctional localization.

This is an important study because it provides direct evidence in a live, dynamic system what the PCP field has generally assumed to be true – that PCP asymmetry accompanies myosin recruitment to promote junction shrinking during epithelial CE. The study adds further insight by showing how PCP enrichment and stability are better correlated with junction behavior than its orientation. These findings in dynamic cellular context are consistent with those of Strutt and Strutt in Drosophila planar polarized and more static epithelia that junctions enriched in PCP components exhibit higher stable fractions of PCP proteins. Together, these findings underscore the conservation of the molecular mechanisms of the core PCP pathway signaling between the dynamic vertebrate tissue during gastrulation/neurulation and Drosophila epithelia. Whereas the manuscript is largely correlative, these are very demanding experiments and the observed correlations are significant and should be of interest to the broad scientific readership given the growing list of developmental processes in which PCP pathway is being implicated.

However, some conclusions require further support and additional experimental details are needed for the reviewers to properly interpret some of the results. Moreover, it will be important to move beyond correlations.

The full reviews are also included for your reference, as they contain detailed and useful suggestions.

Essential revisions:

1) While there is good evidence that Pk and Vangl2 enrichment is better correlated with junction shrinkage than orientation, the authors do not address whether it is tied to "positional information across the tissue", i.e. whether a junction separates AP or ML neighbors. V vs T junctions - Considering the analysis is performed on a dynamic tissue where junction angles are not constant, a method that distinguishes junctions based on whether they separate AP or ML cells would be more appropriate.

2) The temporal relationship between PCP enrichment and myosin oscillations needs better documentation.

3) Please include movies for time-lapse images.

4) The data on the dynamic behavior of Vangl and Prickle fusion proteins is compelling as there are correlations with junction shrinking, actomyosin assembly, however they are just correlations. In the text the authors in general acknowledge the limitation of this analysis as for example in the summary "suggest a complex and intimate link between the dynamic localization of core PCP proteins, actomyosin assembly, and polarized junction shrinking". However, the title "Spatial and temporal PCP protein dynamics coordinate cell intercalation during neural tube closure", indicates causal relationships that are not experimentally supported and is an overstatement of the finding in this manuscript. The Title needs to be revised.

5) Moreover, it would be important to move beyond correlations. To do this, a more mechanistic understanding of relationships between PCP protein localization, myosin localization, and junction shrinking is needed. As the authors mentioned in the discussion, PCP signaling is known to regulate Myosin activity via Daam1 and Rho Kinase. One key question is whether PCP components drive myosin contractility, contractility drives PCP localization, or both. The key experiment that would take this paper beyond correlations is to perform FRAP assays under myosin inhibition. This would test whether myosin/junction shrinkage underlies the increase in Pk and Vang stability at V-junctions, or whether their stability is upstream of myosin (and perhaps due to AP position).

6) The observed enrichment of PCP components at shrinking V-junctions is a striking observation here. The authors acknowledge that "intensity could reflect increased density due to junction shrinking" as observed for generic membrane markers. But they discard this possibility because "even when normalized against such a membrane label, intensities of Vangl2 and Prickle2 still displayed a significant correlation with junction shrinkage". So, they put forward "the alternative hypothesis" whereby the observed enrichment could be an active process, the hypothesis they pursue and find experimental support for. However, these hypotheses are not mutually exclusive. It would be important to present data for the changes in the density of the PCP and general membrane markers, to more fully understand the relative contributions of these two mechanisms. For example, it would be valuable to see a line for the intensity of the generic membrane marker on the graph in Figure 8B. And although PCP fusion protein localization is normalized to a generic membrane marker in Figure 6—figure supplement 1, the legend of Figure 1 states that fluorescence intensity was normalized to the cytoplasm. Additional figure legends should specify to what fluorescence intensity is normalized.

7) Figure 4 presents defective CE in neuroectoderm expressing Xdd1. Junction shrinking is analyzed. However, visually abnormal cell alignment of cells is evident and should be quantified. It seems that instead of ML alignment, cell bodies are aligned with the AP embryonic axis, as has been seen in some zebrafish PCP mutants (e.g. Roszko et al., 2015). If this is the case, how V junction should be defined? Simply as ML aligned or should be longer junction (as V junctions in ML-elongated cells are). This should be analyzed and considered.

8) Reduced number of productive T1 transitions is perceived as the main phenotype. However, it would be important to analyze the total frequency of any transitions and intercalations. Does Xdd1 overexpression lead to loss of polarized transitions and intercalations or does it generally impair junction shrinking and intercalations. This is very important to assess whether Xdd1 overexpression impairs CE in the same way as loss of individual PCP components in mouse or zebrafish mutants.

9) The paper could be improved by a restructuring of the discussion to present a working model of PCP and myosin regulation during cell intercalation in CE, including how the present study supports or challenges prevailing models.

Reviewer #1:

Butler and Wallingford report their studies of the dynamic localization of Vangl2-GFP and Prickle2-GFP core components of the Planar Cell Polarity (PCP) pathway during convergent extension movements of neuroectodermal tissue in Xenopus. They confirm and extend earlier studies in zebrafish, frog and mouse providing snapshots of PCP protein localization that revealed enrichment of Vangl2 and Pk at the anterior edges of mediolaterally (ML) elongated cells, consistent with asymmetric localization of PCP components shown by pioneering studies in Drosophila. The value of this work is in careful temporal assessment of protein localization in relation to cell behavior and in particular to junction shrinking – one of the drivers of the ML intercalation process. The authors propose that Vangl and Prickle are dynamically enriched at shrinking junctions and suggest "the shrinking/growing status of a junction is a better indicator of PCP protein enrichment than its orientation in the ML/AP axis. Another important insight from FRAP experiments reported here is that turnover kinetics also vary with the dynamic junction behavior such that more stable fractions are associated with shrinking junctions and this is correlated with behavior of Myosin at junctions. These findings in dynamic cellular context are consistent with those of Strutt and Strutt in Drosophila planar polarized and more static epithelia that junctions enriched in PCP components exhibit higher stable fractions of PCP proteins. Together, these findings underscore the conservation of the molecular mechanisms of the core PCP pathway signaling between the dynamic vertebrate tissue during gastrulation/neurulation and Drosophila epithelia. Whereas the manuscript is largely correlative, these are very demanding experiments and the observed correlations are significant and should be of interest to the broad scientific readership given the growing list of developmental processes in which PCP pathway is being implicated.

However, there are several conclusions that require additional experimental and several conclusions need further discussion. Considering the following points will significantly improve the manuscript.

- The data on the dynamic behavior of Vangl and Prickle fusion proteins is compelling as there are correlations with junction shrinking, actomyosin assembly, however they are just correlations. In the text the authors in general acknowledge the limitation of this analysis as for example in the summary "suggest a complex and intimate link between the dynamic localization of core PCP proteins, actomyosin assembly, and polarized junction shrinking". However, the title "Spatial and temporal PCP protein dynamics coordinate cell intercalation during neural tube closure", indicates causal relationships that are not experimentally supported and is an overstatement of the finding in this manuscript. The title needs to be revised.

- Moreover, it would be important to move beyond correlations. To do this, a more mechanistic understanding of relationships between PCP protein localization, myosin localization, and junction shrinking is needed. As the authors mentioned in the discussion, PCP signaling is known to regulate Myosin activity via Daam1 and Rho Kinase. It would be important to know whether loss of function of either Rho or Daam1 leads to an uncoupling of Myl9 and Pk2 at V junctions. Another important question is whether PCP components drive myosin contractility, contractility drives PCP localization, or both. They show that function of Pk and Dvl is necessary for polarized Myosin localization, but is it sufficient? I.e. Does ectopic expression of PCP components drive asymmetric Myosin accumulation? And would pharmacologically increasing or decreasing myosin contractility lead to increased or decreased Pk2 localization?

- The observed enrichment of PCP components at shrinking V-junctions is a striking observation here. The authors acknowledge that "intensity could reflect increased density due to junction shrinking" as observed for generic membrane markers. But they discard this possibility because "even when normalized against such a membrane label, intensities of Vangl2 and Prickle2 still displayed a significant correlation with junction shrinkage". So, they put forward "the alternative hypothesis" whereby the observed enrichment could be an active process, the hypothesis they pursue and find experimental support for. However, these hypotheses are not mutually exclusive. It would be important to present data for the changes in the density of the PCP and general membrane markers, to more fully understand the relative contributions of these two mechanisms. For example, it would be valuable to see a line for the intensity of the generic membrane marker on the graph in Figure 8B. And although PCP fusion protein localization is normalized to a generic membrane marker in Figure 6—figure supplement 1, the legend of Figure 1 states that fluorescence intensity was normalized to the cytoplasm. Additional figure legends should specify to what fluorescence intensity is normalized.

- Expression of Xdd1 and Pk2-∆P∆L is used to disrupt PCP signaling, and Xdd1 has been used in several previous studies as a model for loss of PCP signaling. However, dominant negative approaches can have additional effects. Indeed, as the authors acknowledge these two reagents appear to act via converse mechanisms on the planar polarization of myosin enrichment. This hints that it is not simply a loss of PCP signaling that disrupts junction polarity and contractility, but rather that each of these constructs influences cell polarity in distinct ways. A closer examination of the interplay between Pk2/Vangl2, Dvl, and Myosin would distinguish between unintended effects on dominant-negative constructs and biologically relevant consequences of PCP signaling.

- It is important to note possible caveats associated with studying the behavior of misexpressed fusion proteins. This has been a standard in the field, but it is puzzling that in this work Vangl-GFP appears to be absent on posterior membranes, whereas Roszko et al., 2015) using antibodies detected endogenous Vangl2 in zebrafish mesodermal and neuroectodermal cells along the entire cell membrane and using Vangl2-GFP fusion protein along the entire cell membrane but anteriorly enriched. Do longer exposures show lower level of Vangl-GFP at other membranes as well?

- Similarly, arguable is the conclusion in the Discussion section "More important than the spatial localization of PCP proteins during cell intercalation is the temporal aspect reported here." First, as the authors note the asymmetric distribution of core PCP components and that junctions enriched in PCP components exhibit higher stable fractions of PCP proteins has been observed in more static epithelia in Drosophila. The more dynamic aspects reported here for shrinking junctions may represent a phase in the course of PCP signaling. Indeed, this phase likely depends on the earlier phases of PCP signaling where both in Drosophila and zebrafish endogenous Vangl accumulates uniformly at cell membrane and then its distribution becomes asymmetric. As disruption of PCP signaling disrupts all asymmetries including the dynamic ones reported here, the "more important" statement is not justified.

- Figure 4 presents defective CE in neuroectoderm expressing Xdd1. Junction shrinking is analyzed. However, visually abnormal cell alignment of cells is evident and should be quantified. It seems that instead of ML alignment, cell bodies are aligned with the AP embryonic axis, as has been seen in some zebrafish PCP mutants (e.g. Roszko et al., 2015). If this is the case, how V junction should be defined? Simply as ML aligned or should be longer junction (as V junctions in ML-elongated cells are). This should be analyzed and considered.

- Reduced number of productive T1 transitions is perceived as the main phenotype. However, it would be important to analyze the total frequency of any transitions and intercalations. Does Xdd1 overexpression lead to loss of polarized transitions and intercalations or does it generally impair junction shrinking and intercalations. This is very important to assess whether Xdd1 overexpression impairs CE in the same way as loss of individual PCP components in mouse or zebrafish mutants.

Reviewer #2:

Butler and Wallingford investigate PCP protein localization during convergent extension (CE) movements that accompany neural tube closure in Xenopus embryos. They find that Pk-GFP and Vangl2-GFP are asymmetrically distributed at intercellular junctions and that the proteins are enriched most strongly at actively shrinking junctions. A major conclusion of this study is that "the strength of asymmetric Pk2 and Vangl2 enrichment at a particular junction is more strongly tied to the dynamic behavior of that junction than it is to the junction's orientation or to positional information across the tissue." In addition, they show that Pk and Vangl2 are more stable at shrinking rather than growing junctions, and PCP enrichment correlates with myosin junctional localization.

This is an important study because it provides direct evidence in a live, dynamic system what the PCP field has generally assumed to be true – that PCP asymmetry accompanies myosin recruitment to promote junction shrinking during epithelial CE. The study adds further insight by showing how PCP enrichment and stability are better correlated with junction behavior than its orientation.

However, some conclusions require further support and additional experimental details are needed for the reviewer to properly interpret some of the results. Specifically, while the there is good evidence that Pk and Vangl2 enrichment is better correlated with junction shrinkage than orientation, they do not address whether it is tied to "positional information across the tissue", i.e. whether a junction separates AP or ML neighbors. Second, the temporal relationship between PCP enrichment and myosin oscillations needs better documentation. Third, the paper does not address whether myosin activity or AP position accounts for the observed increased Pk and Vangl2 stability. Most of these points could be addressed with existing data sets. Finally, I feel that the paper could be improved by a restructuring of the discussion to present a working model of PCP and myosin regulation during cell intercalation in CE, including how the present study supports or challenges prevailing models.

Revisions needed to strengthen the major conclusions:a) Please include movies for time-lapse images.

b) All experiments use exogenously expressed GFP constructs. Do Vangl2, Pk2 and MyI9 endogenous proteins show the same degree of polarization?

c) V vs T junctions- Considering the analysis is performed on a dynamic tissue where junction angles are not constant, a method that distinguishes junctions based on whether they separate AP or ML cells would be more appropriate. For example, in Figure 5A, an expanding junction is shown oriented roughly perpendicular to a neighboring shrinking junction. The junction changes its orientation ~30 degrees as it lengthens. When during this process would the angle of the junction be measured? Since junction angles fluctuate more rapidly than cells exchange neighbors, a more meaningful categorization of V vs T could be whether the junction separates AP or ML neighbors. Since the AP relationship between cells is thought to be the primary determinant of PCP asymmetry in other systems, I feel this is important to consider.

d) Pk and Vangl stability – The data shown that Pk and Vangl2 stability correlates with shrinking junctions, but does it also correlate with junction angle or AP neighbor relationships? Perhaps stability is dependent on whether Pk and Vangl are incorporated in an intercellular PCP complex, which would be expected to occur between AP neighbors, but less so at ML neighbors (Strutt et al., 2011).

e) Temporal correlation between Pk and myosin – Figure 8A,B. A major conclusion of the paper is that "recruitment of Prickle2 and Vangl2 to cell-cell junctions was temporally and spatially coordinated with planar polarized oscillations of actomyosin enrichment", but the temporal correlations and oscillatory behaviors are not clear from the image shown in Figure 8B or intensity plot over the corresponding junction in Figure 8B. Only one example is given, and the behavior is not obviously oscillatory. Accompanying movies and analysis of additional shrinkage events would strengthen the data and potentially provide insights into the order of events, i.e. whether Pk enrichment precedes myosin accumulation.

Clarifications to the experimental procedures and data analysis:a) Figure 1B. – A more complete description of how GFP intensities were measured is needed. Is the average GFP intensity across the junction plotted? Are junctions between two cells that both express Pk-GFP or Vangl2-GFP included in the analysis? Or are only individual GFP+ cells that are surrounded by GFP- neighbors included?

b) Figure 5A-B. Pk-GFP intensity is particularly strong at vertices, which raises the question of how the boundaries of a junction are defined. Are the intensities at vertices included or excluded as part of the junction? In both growing and shrinking junctions? These bright points could really skew the data.

c) Figure 5B. Could the authors provide more information about how the changes in GFP intensity are determined? Are they choosing the same time points used to calculate the change in junction length (Intensity at t=end minus intensity at t=0)? Or is this the net change (max minus min) over the course of junction length changes? How are the max and min junction lengths chosen?

d) Figure 5B. There are many junctions that display a substantial change in length but no change in Pk intensity. I'm not sure how to think about these data points. Can the authors comment? If Pk2/PCP localization instructs junction shrinkage, then these results are unexpected. Perhaps these data points represent unproductive shrinkage events that never result in complete junction loss and neighbor exchange? If the data were separated into productive shrinkage events that lead to junction loss vs fluctuating junctions would the correlation between Pk intensity and length changes be even stronger?

e) Figure 6A-E. In the FRAP analysis, were both V and T-junctions analyzed (oriented along the full range of 0-90 degrees)? Does Pk-GFP stability correlate with the junction angle (see point 1d above)?

Reviewer #3:

I enjoyed reading this manuscript and believe it will be a valuable addition to the literature. It is essentially 'descriptive', in the positive sense of providing a careful quantitative description of events that will be of great value in underpinning further work in the field. The findings are correlative rather than mechanistic. Looking over my brief notes I made as I read through, I also find they are largely confirmatory:

- establish/confirm Pk2 and Vangl2 colocalize preferentially on A/P cell boundaries (but asymmetry weak).

- confirm that convergent extension occurs in epithelium of neural plate via T1 transitions and rosette formation and is dependent on PCP pathway function.

- GFP-Pk2/GFP-Vangl2 are enriched on shrinking junctions (this of course follows from the known localizations on A/P cell boundaries and that the tissue undergoes convergent extension on the AP axis). Interestingly, Pk2/Vangl2 are preferentially enriched on shrinking junctions: possibly they are actively recruited to shrinking junctions, or actively driving shrinkage? Or maybe turnover is slow, so get concentrated on shrinking junctions by virtue of the shrinkage?

- find by FRAP that Pk2/Vangl2 show higher stable fraction on shrinking junctions, where their concentrations are higher. This is consistent with Drosophila data (e.g. Strutt et al., 2011?) suggesting at higher junctional concentrations, PCP proteins show higher stable fractions?

- see expected pulsatile actomyosin behavior on shrinking junctions which is PCP pathway dependent. Pk2 seems to pulse with myosin, due to shared membrane enrichment?

There is an interesting hint that there may be an active relationship between PCP protein behavior and actomyosin dynamics but cause and effect is not investigated.

This would sit best in a good developmental biology journal?

https://doi.org/10.7554/eLife.36456.028

Author response

[Editors’ note: formal revisions were requested, following approval of the authors’ plan of action.]

Essential revisions:

1) While there is good evidence that Pk and Vangl2 enrichment is better correlated with junction shrinkage than orientation, the authors do not address whether it is tied to "positional information across the tissue", i.e. whether a junction separates AP or ML neighbors. V vs T junctions- Considering the analysis is performed on a dynamic tissue where junction angles are not constant, a method that distinguishes junctions based on whether they separate AP or ML cells would be more appropriate.

We agree that this is an important point and we addressed it in three ways.

First, because "junction angles are not constant," we re-examined our data from Figure 3B and C. We found that the average change in junction angle during the period of analysis for this dataset was only 5 degrees (+/- 4 degrees), with no changes greater than 20 degrees observed. This result is now reported (subsection “Epithelial convergent extension in the closing Xenopus neural tube involves PCP-194 dependent polarized junction shrinking”), as it will help the reader to evaluate the dataset. This result argues that our use of junction orientation is an effective proxy for junctions that separate A/P neighbors.

Second, even with these data, we feel the reviewer's point is valid, so we added a new analysis using more stringent criteria. Because we feel it is essential to continue identifying junctions by their orientation (this is an unambiguous metric and is the standard in the field for many labs), we selected junctions for this analysis that remain within 30 degrees of mediolateral for the entire duration of analysis, and for these junctions, we plotted the average velocity of shrinking or growth against the average Pk2 intensity for every time point in the movie (subsection “Prickle2 and Vangl2 are dynamically enriched specifically at shrinking cell-cell 266 junctions”, new Figure 6—figure supplement 2). Again, we observed a very strong correlation.

Finally, we softened our language. We no longer claim that behavior is "better" correlated than orientation, but instead suggest that it is "at least as" correlated.

We hope these changes address the reviewers’ intended question; if not, we apologize and ask for clarification.

2) The temporal relationship between PCP enrichment and myosin oscillations needs better documentation.

We agree, and we have made three changes:

1) We have added better, higher magnification images supporting this claim, as well as intensity plots for the frames shown. These are provided in Figure 9A of the revision.

2) We provide a representative time-lapse movie.

3) We provide data showing a strong cross-correlation between Pk2 and Myl9 levels (new Figure 9E), as detailed below in our response to comment 5.

3) Please include movies for time-lapse images.

We regret this oversight. We have supplied several representative time-lapse movies to accompany the figures.

4) The data on the dynamic behavior of Vangl and Prickle fusion proteins is compelling as there are correlations with junction shrinking, actomyosin assembly, however they are just correlations. In the text the authors in general acknowledge the limitation of this analysis as for example in the summary "suggest a complex and intimate link between the dynamic localization of core PCP proteins, actomyosin assembly, and polarized junction shrinking". However, the title "Spatial and temporal PCP protein dynamics coordinate cell intercalation during neural tube closure", indicates causal relationships that are not experimentally supported and is an overstatement of the finding in this manuscript. The Title needs to be revised.

We have re-titled the manuscript: "Spatial and temporal analysis of PCP protein dynamics during neural tube closure".

5) Moreover, it would be important to move beyond correlations. To do this, a more mechanistic understanding of relationships between PCP protein localization, myosin localization, and junction shrinking is needed. As the authors mentioned in the discussion, PCP signaling is known to regulate Myosin activity via Daam1 and Rho Kinase. One key question is whether PCP components drive myosin contractility, contractility drives PCP localization, or both. The key experiment that would take this paper beyond correlations is to perform FRAP assays under myosin inhibition. This would test whether myosin/junction shrinkage underlies the increase in Pk and Vang stability at V-junctions, or whether their stability is upstream of myosin (and perhaps due to AP position).

In the requested "action plan," we proposed addressing this concern with a cross-correlation analysis of PCP protein and Myosin dynamics to ask if these pulses are truly coincident or if in fact one pulse precedes the other. We now report a strong cross-correlation between Myl9 and Pk2 intensities - which we feel adds value to the paper (New Figure 9E). However, we find that neither one clearly preceded the other in this analysis. This may reflect true simultaneous enrichment, or it may indicate that the time-resolution of our movies is insufficient to detect a difference.

We regret, then, that without substantial additional experimentation, we are unable to make more mechanistic conclusions. That being said, we respectfully point out that the data here nonetheless represent both a quantum leap forward in analysis of PCP protein localization during CE and a very thorough body of work.

We will revise the manuscript to (a) explicitly acknowledge the limitations of our descriptive approach (Discussion section) and (b) provide a careful synthesis of our current understanding of the relationships between PCP proteins and myosin in the Discussion section.

We very much hope the reviewers find this proposal acceptable.

6) The observed enrichment of PCP components at shrinking V-junctions is a striking observation here. The authors acknowledge that "intensity could reflect increased density due to junction shrinking" as observed for generic membrane markers. But they discard this possibility because "even when normalized against such a membrane label, intensities of Vangl2 and Prickle2 still displayed a significant correlation with junction shrinkage". So, they put forward "the alternative hypothesis" whereby the observed enrichment could be an active process, the hypothesis they pursue and find experimental support for. However, these hypotheses are not mutually exclusive. It would be important to present data for the changes in the density of the PCP and general membrane markers, to more fully understand the relative contributions of these two mechanisms. For example, it would be valuable to see a line for the intensity of the generic membrane marker on the graph in Figure 8B.

This is another important point, so we have moved our raw correlation data for Pk2 and Vangl2 to a new Figure 6 that also includes the correlation data for the general membrane marker, Caax-GFP. We present these raw data first and then discuss the Pk2/Vangl2 data normalized against Caax-GFP, which is now shown in Figure 6—figure supplement 1 of the revision. We also reworded how we interpret these data to reflect that reduced turnover could in fact help produce the increased enrichment we see for the PCP proteins over the membrane label rather than use language that suggests they are mutually exclusive

And although PCP fusion protein localization is normalized to a generic membrane marker in Figure 6—figure supplement 1, the legend of Figure 1 states that fluorescence intensity was normalized to the cytoplasm. Additional figure legends should specify to what fluorescence intensity is normalized.

All legends now accurately reflect if and how normalization was used for each particular dataset. We also included additional language in the methods to point out the circumstances and details for the two alternative normalization methods.

7) Figure 4 presents defective CE in neuroectoderm expressing Xdd1. Junction shrinking is analyzed. However, visually abnormal cell alignment of cells is evident and should be quantified. It seems that instead of ML alignment, cell bodies are aligned with the AP embryonic axis, as has been seen in some zebrafish PCP mutants (e.g. Roszko et al., 2015). If this is the case, how V junction should be defined? Simply as ML aligned or should be longer junction (as V junctions in ML-elongated cells are). This should be analyzed and considered.

We agree that these are important points. We have extracted such data from our movies and now provide them in the new Figure 4 and Figure 4—figure supplement 1 of the revision. This analysis revealed that, as the reviewer intuited, there is a defect in the orientation of the long axes of cells with Xdd1 or dom-neg Pk2 expression. Moreover, we find that the planar polarization of junction shrinkage is entirely randomized by Xdd1 expression, and consistent with the reduction of T1 transitions, junction shrinkage is reduced overall.

Regarding the question of how V junctions should be defined if cell elongation is changed is an interesting one. We strongly feel that it remains critical for junctions to have unambiguous definitions, so we prefer to continue using orientation to define junctions, as outlined in comment 1, above.

Importantly, our analysis suggests that junction-shrinking events are not re-oriented but rather are abrogated and randomized.

8) Reduced number of productive T1 transitions is perceived as the main phenotype. However, it would be important to analyze the total frequency of any transitions and intercalations.

We regret being unclear: The analysis shown in what is now Figure 4C and D is for all junction shrinkage events, regardless of orientation. Those data, combined with the analysis provided in comment 7, should address this concern.

Does Xdd1 overexpression lead to loss of polarized transitions and intercalations or does it generally impair junction shrinking and intercalations. This is very important to assess whether Xdd1 overexpression impairs CE in the same way as loss of individual PCP components in mouse or zebrafish mutants.

If we understand this correctly, the analysis we provide in comments 1 and 7 should address this concern.

9) The paper could be improved by a restructuring of the discussion to present a working model of PCP and myosin regulation during cell intercalation in CE, including how the present study supports or challenges prevailing models.

We have added extensively to the Discussion section, but we preferred stop short of presenting a "working model," which we feel would be premature. As mentioned above, our data do not provide mechanistic insights into this question, though we feel they do add important dynamic information about the relationship between PCP and myosin. Moreover, as one of the reviewers points out in the last minor comment below, this relationship must be handled with great care. We hope the reviewers feel our additional verbiage is sufficient to render the discussion useful.

https://doi.org/10.7554/eLife.36456.029

Article and author information

Author details

  1. Mitchell T Butler

    Department of Molecular Biosciences, University of Texas at Austin, Austin, United States
    Present address
    UNC Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, United States
    Contribution
    Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3130-1186
  2. John B Wallingford

    Department of Molecular Biosciences, University of Texas at Austin, Austin, United States
    Contribution
    Conceptualization, Funding acquisition, Visualization, Project administration, Writing—review and editing
    For correspondence
    wallingford@austin.utexas.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6280-8625

Funding

National Institute of General Medical Sciences (R01GM104853)

  • John B Wallingford

Eunice Kennedy Shriver National Institute of Child Health and Human Development (R21HD084072)

  • John B Wallingford

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocols (AUP-2015-00160) of the University of Texas at Austin.

Senior Editor

  1. Didier YR Stainier, Max Planck Institute for Heart and Lung Research, Germany

Reviewing Editor

  1. Lilianna Solnica-Krezel, Washington University School of Medicine, United States

Publication history

  1. Received: March 7, 2018
  2. Accepted: July 25, 2018
  3. Accepted Manuscript published: August 6, 2018 (version 1)
  4. Version of Record published: August 29, 2018 (version 2)

Copyright

© 2018, Butler et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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