Global analysis of cell behavior and protein dynamics reveals region-specific roles for Shroom3 and N-cadherin during neural tube closure
- Department of Molecular Biosciences, University of Texas at Austin, United States
Figures
Figure 1 with 1 supplement
Tissue-level imaging and analysis of contractile protein dynamics during neural tube closure in Xenopus.
(A) Schematic of mRNA injections and subsequent imaged regions of the Xenopus tropicalis embryo. (B) Cell segmentation and tracking workflow. Binary segmentation, cell surface tracking, and cell …
Figure 1—figure supplement 1
Per cell data processing and analysis.
(A) Raw data for apical area (converted to square microns) of an individual cell over time. (B) Apical area averaged/smoothed over 7 frames. (C) Smoothed data after mean-centering and …
Figure 2
Tissue-level analysis of individual cell behaviors reveals dynamic heterogeneity.
(A) Overall change (Δ) in apical surface area (standardized) across anterior (left) and posterior (right) control embryos. (B) Overall change in medial LifeAct/actin localization (standardized) …
Figure 3 with 1 supplement
Cells in the anterior and posterior neural ectoderm both apically constrict but differ in their contractile protein dynamics.
Tissue-level cell size and protein localization dynamics from control embryos in Figure 2. (X) Distribution of overall change (Δ) in displayed parameter (standardized) among cells from control …
Figure 3—figure supplement 1
Endogenous N-cadherin location.
Xenopus tropicalis embryos were fixed in paraformaldehyde then stained with the monoclonal N-cadherin antibody mNCD-2. Increased medial N-cadherin localization is apparent as cells constrict.
Figure 4 with 1 supplement
N-cadherin localizes both at the apical surface and basally as well.
(A) XY (top row) and XZ (bottom row) projections of N-cadherin-GFP and LifeAct-RFP in the anterior neural ectoderm of a Xenopus tropicalis embryo. (B) XY (top panel) and XZ (bottom panel) …
Figure 4—figure supplement 1
Shroom3ΔC-term colocalizes with LifeAct in the neural ectoderm.
Three time points of GFP-Shroom3ΔC-term and LifeAct-RFP in constricting neural ectoderm cells of a Xenopus embryo.
Figure 5 with 3 supplements
Disruption of shroom3 via mosaic F0 CRISPR mosaic causes differential apical constriction phenotypes between regions of the neural ectoderm.
(A) Schematic of mosaic F0 CRISPR/Cas9 injections in Xenopus tropicalis embryos. (B) Workflow of identification and analysis of mosaic F0 crispants. (C) Top row, distribution of initial area (square …
Figure 5—figure supplement 1
shroom3 CRISPR validation.
(A) Gene model for shroom3 in Xenopus tropicalis per Xenbase. The sgRNA was designed to target the 5’ end of the second exon. (B) Fragment analysis to validate efficacy of sgRNA+ Cas9 injections. A …
Figure 5—figure supplement 2
Maps of initial frames and shroom3 crispant calls for each analyzed embryo.
Figure 5—figure supplement 3
Fluorescent aberration in an anterior-imaged embryo.
(A) Smoothed mean of medial LifeAct-RFP/actin fluorescent intensity across all imaged cells across time. (B) Smoothed mean of medial N-cadherin-GFP fluorescent intensity across all imaged cells …
Figure 6
Loss of shroom3 disrupts actin and N-cadherin accumulation and constriction in the anterior neural ectoderm.
(A) Representative images of LifeAct/actin and N-cadherin-GFP (N-cad-GFP) localization in control cells (left) and shroom3 crispant cells (right) from the anterior region of the neural ectoderm. …
Figure 7
Medial N-cadherin accumulation is severely disrupted in anterior shroom3 crispant cells that fail to apically constrict.
(A-D) 2D density plots of all observations of apical area versus medial (A) or junctional (B) LifeAct/actin or medial (C) or junctional (D) N-cadherin for all cells within each group. Percentages in …
Figure 8
Actin and N-cadherin accumulation are uncoupled in anterior shroom3 crispant cells.
(A and B) 2D density plots of all observations of medial (A) or junctional (B) LifeAct/actin versus apical area for all cells within each group. (C and D) 2D density plots of all observations of …
Figure 9
Loss of shroom3 disrupts actin dynamics in the posterior neural ectoderm.
(A) Representative images of LifeAct/actin and N-cadherin-GFP (N-cad-GFP) localization in control cells (left) and shroom3 crispant cells (right) from the posterior region of the neural ectoderm. …
Figure 10
Actin and N-cadherin dynamics are highly heterogenous in the posterior neural ectoderm and poorly correlated with apical constriction.
(A and B) 2D density plots of all observations of apical area versus medial (A) or junctional (B) LifeAct/actin for all cells within each group. (C and D) 2D density plots of all observations of …
Figure 11 with 1 supplement
Individual junction behaviors are polarized in the posterior neural ectoderm.
(A) Junction orientation from posterior control embryo from Figure 2. Scale bars = 100 µm. (B) Distribution of overall change (Δ) in junction length (standardized) from the posterior neural …
Figure 11—figure supplement 1
Individual junction behaviors are mainly anisotropic in the neural ectoderm.
(A) Distribution of overall change (Δ) in junction length (standardized) from the anterior neural ectoderm. Horizontal lines on density plots/violins indicate quartiles of distribution, black …
