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During neural tube closure, neural ectoderm cells constrict their apical surfaces to bend and fold the tissue into a tube that will become the central nervous system. Recent data from mice and humans with neural tube defects suggest that key genes required for neural tube closure can exert non-cell autonomous effects on cell behavior, but the nature of these effects remains obscure. Here, we coupled tissue-scale, high-resolution time-lapse imaging of the closing neural tube of Xenopus to multivariate regression modeling, and we show that medial actin accumulation drives apical constriction non-autonomously in neighborhoods of cells, rather than solely in individual cells. To further explore this effect, we examined mosaic crispant embryos and identified both autonomous and non-autonomous effects of the apical constriction protein Shroom3.
Fig. 1. Apical actin accumulation is heterogenous in the Xenopus neural ectoderm. A, Diagram of medial and junctional quantification domains on the apical surface of apically constricting cells. B, Lifeact-RFP localization in the anterior neural ectoderm of apically constricting cells. White “X” marks the same cell in each panel. Scale bar = 25 μm. C, mean actin accumulation at the medial domain of neural ectoderm cells is heterogeneous. Actin intensity is measured in arbitrary units. Scale bar = 25 μm. D, mean actin accumulation at individual cell-cell junctions is also heterogeneous. Actin intensity is measured in arbitrary units. Black asterisks mark the same cells in each frame. Scale bar = 25 μm. D′, insets of cells from D. Scale bar = 12.5 μm.
Fig. 2. Neighbor analysis and partial least squares regression reveal multiple modes of non-cell autonomous feedback on apical constriction. A, diagram of neighbor analyses. Neighbor relationships in each frame are determined by sharing of at least one cell vertex, represented by black circles. Average values for the neighbors of each cell were then calculated. B, sample track of a cell and its neighbors. Apical area is shown in black, mean medial actin localization within the cell in magenta, and mean medial actin of neighboring cells shown in green. s.d. = standard deviations. C, partial least squares regression (PLSR) of apical area in control cells in the anterior neural ectoderm indicates non-cell autonomous control of apical constriction. Regression coefficients represent the independent effect of each variable on apical surface area. P-values for each regression component were calculated via jackknife resampling included in the R pls package.
Fig. 3. Loss of Shroom3 disrupts junction behaviors along the mosaic interface. A, control and shroom3 crispant cells meet at the mosaic interface. Magenta = Lifeact-RFP/actin, green = N-cadherin-GFP, cyan = membrane(CAAX)-BFP + Cas9 protein (PNA Bio) + shroom3-targeted sgRNA. Scale bar = 50 μm. A′, cell tracking can automatically identify cell junctions at the mosaic interface. Red cells = control cells, blue cells = shroom3 crispant cells. Red junctions = control × control interaction, green junctions = control x shroom3 crispant interaction (mosaic interface), blue junctions = shroom3 crispant x shroom3 crispant interactions. Scale bar = 50 μm. B, C, D, overall change in junction length, mean junctional actin, and mean junctional N-cadherin in control, mosaic interface, and shroom3 crispant junctions. Both constriction and N-cadherin accumulation are significantly disrupted in interface junctions. Each dot is an individual junction. Horizontal lines within each violin delineate quartiles along each distribution. s.d. = standard deviations. Median value in each group is listed at the top of the panel. P-values were calculated via KS test.
Fig. 4. Loss of Shroom3 causes non-cell autonomous apical constriction defects along the mosaic interface. A, sample embryo showing cells within control and shroom3 crispant clones (left panel) and cells located at the mosaic interface (right panel). Red = control cells, blue = shroom3 crispant cells. B, change in apical area among control and shroom3 crispant cells both within clones (left) and at the mosaic interface (right). shroom3 crispant cells situated next to control cells at the mosaic interface are significantly more constrictive then crispant cells within clones. Red violins = control cells, blue violins = shroom3 crispant cells. Each dot is an individual cell. Horizontal lines within each violin delineate quartiles along each distribution. s.d. = standard deviations. Median value in each group is listed at the top of the panel. P-values were calculated via KS test. C, schematic of calculation of proportion of neighbors at mosaic interface. D, change in apical area versus proportion of cell boundary with unlike neighbors (i.e. at mosaic interface) for both control and shroom3 crispant cells at the mosaic interface. s.d. = standard deviations. R-values calculated by Pearson's correlation.
Fig. 5. Loss of Shroom3 disrupts non-cell autonomous effects of actin localization. A, schematic of neighbor analysis of cells along the mosaic interface. Mean behaviors of both control and shroom3 crispant neighbors can be calculated separately. B, sample cell track showing the behavior of one control cell at the mosaic interface and its neighbors. Black = apical area of cell, magenta = mean medial actin of cell, red = mean medial actin of control neighbors, blue = mean medial actin of shroom3 crispant neighbors. C, partial least squares regression (PLSR) of apical area in both control and shroom3 crispant cells along the mosaic interface. Protein localization variables on the right indicate whether the resulting coefficient was generated based on influence of the variable within a cell, from a cell's control neighbors, or a cell's shroom3 crispant neighbors. Regression coefficients show the influence of each variable on apical area in both control cells (red bars) and shroom3 crispant cells (blue bars). P-values for each regression component were calculated via jackknife resampling included in the R pls package.
figs1. Additional PLSR models. A, partial least squares regression (PLSR) of apical area in control cells in the anterior neural ectoderm, including cell orientation. Regression coefficients represent the independent effect of each variable on apical surface area. P-values for each regression component were calculated via jackknife resampling. B, partial least squares regression (PLSR) of apical area in control cells in the anterior neural ectoderm, excluding N-cadherin localization. Regression coefficients represent the independent effect of each variable on apical surface area. P-values for each regression component were calculated via jackknife resampling.
figs2. Mosaic patterns of shroom3 loss and corresponding apical constriction phenotypes. Red = control cells, blue = shroom3 crispant cells Left panels, tissue geometry of shroom3 crispant cells in each embryo in this study. Vertical white lines represent approximate location of the embryonic midline in each embryo. Right panels, change in apical area among control and shroom3 crispant cells both within clones (left) and at the mosaic interface (right) in each embryo in this study. Red violins = control cells, blue violins = shroom3 crispant cells. Each dot is an individual cell. Horizontal lines within each violin delineate quartiles along each distribution. s.d. = standard deviations.
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