DOI: 10.5061/dryad.zw3r2289b
title: Global analysis of cell behavior and protein localization dynamics reveals region-specific functions for Shroom3 and N-cadherin during neural tube closure

description: Failures of neural tube closure are common and serious birth defects, yet
 we have a poor understanding of the interaction of genetics and cell
 biology during neural tube closure. Additionally, mutations that cause
 neural tube defects (NTDs) tend to affect anterior or posterior regions of
 the neural tube but rarely both, indicating a regional specificity to NTD
 genetics. To better understand the regional specificity of cell behaviors
 during neural tube closure, we analyzed the dynamic localization of actin
 and N-cadherin via high-resolution tissue-level time-lapse microscopy
 during Xenopus neural tube closure. To investigate the regionality of gene
 function, we generated mosaic mutations in shroom3, a key regulator or
 neural tube closure This approach elucidates new differences between cell
 behaviors during cranial/anterior and spinal/posterior neural tube
 closure, provides mechanistic insight into the function of shroom3 and
 demonstrates the ability of tissue-level imaging and analysis to generate
 cell-biological mechanistic insights into neural tube closure.
description: **Updated 7-25-2022 to correct some errors in cell tracking
 and <em>shroom3</em> crispant identification, as well as add
 stretch and orientation values** All figure references
 are to the corresponding publication. Full citations
 are also in the corresponding publication. Imaging <em>Xenopus tropicalis</em> embryos injected with mRNAs encoding LifeAct-RFP and N-cadherin-GFP were held at 25C until they reached Nieuwkoop and Faber (NF) stage 12.5.  At NF stage 12.5, vitelline envelopes were removed from embryos and embryos were allowed to “relax” for 30 minutes.  Embryos were then mounted in imaging chambers and positioned for imaging of either the anterior or posterior neural plate. Embryos were imaged on a Nikon A1R confocal microscope using the resonant scanner.  Image quality, Z-stacking, and XY tiling were optimized to generate optimal 3D images of the neural plate at a rate of 1 frame per minute. Ultimately, movies of 9 embryos were of sufficient length and quality for analysis, tissue geometry of the initial frame of each of these embryos is presented in Figure 5 figure supplement 2. Image Analysis Raw 3D images were projected to 2D via maximum intensity and underwent initial segmentation of cell boundaries using the FIJI plugin Tissue Analyzer (Aigouy et al., 2010; Aigouy et al., 2016).  The segmentation of an initial frame was hand-corrected, and this hand-corrected segmentation was used to train a classifier using the programs CSML and EPySEG (Aigouy et al., 2020; Ota et al., 2018). CSML and EPySEG were used to generate segmentation for subsequent frames, which were then further hand-corrected in Tissue Analyzer. After hand-correction, Tissue Analyzer was used to track both cell surfaces and cell junctions, then generate a database of measurements of size and fluorescent intensities for each cell and junction over time. Values for medial and junctional localization of imaged markers in cells were calculated as average pixel fluorescence intensity across the entirety of each respective domain (i.e., total fluorescence of a region divided by the area of the region). Similarly, localization of imaged markers to individual junctions was calculated as an average across the entire junction. For individual junctions, errors in junction length caused by Z-displacement and projection were corrected in Matlab. Tissue Analyzer databases were imported to R and further analyzed and manipulated primarily using the <em>tidyverse </em>package (Wickham et al., 2019). Data Analysis Cell tracks shorter than 30 frames and junction tracks shorter than 15 frames were discarded. Individual cell and junction tracks were smoothed by averaging over a 7 frame/minute window (Figure 1 figure supplement 1A,B). Individual cell tracks were further mean-centered and standardized so that variables are measured in standard deviations rather than fluorescence or size units (Figure 1 figure supplement 1C).  This standardization allows us to analyze dynamics of cell size and protein localization across a population of cells while controlling for initial size and fluorescence of cells. In an example embryo, cells begin and end tracking with a variety of apical surface areas (Figure 1 figure supplement 1D’), but once the cell tracks are mean-centered and standardized it becomes clear that the cells are behaving similarly at a population level (Figure 1 figure supplement 1D”). Embryo “b” had a fluorescence anomaly during imaging that resulted in a reduction in overall observed fluorescence followed by a recovery (Figure 5 figure supplement 3A,B).  Cells were tracked through the anomaly (Figure 5 figure supplement 3C), but fluorescent values for the frames 23 to 45 were discarded (Figure 5 figure supplement 3, red dashed box). Cells were determined to be wild-type versus <em>shroom3 </em>crispant by a membrane-BFP localization threshold specific to each embryo (Figure 5B, middle panel).  Crispant calls were then manually annotated in cases along the mosaic interface where thresholding produced crispant calls deemed incorrect. Individual junctions were determined to be wild-type versus <em>shroom3 </em>crispant versus mosaic interface based on the status of the cells the junction was situated between.  Wild-type junctions are situated between two wild-type cells, <em>shroom3 </em>crispant junctions are situated between two shroom3 crispant cells, and mosaic interface junctions are situated between a wild-type and a <em>shroom3</em> crispant cell (Figure 5B, lower panel). Junction orientations were corrected so that the mediolateral axis of the embryo was set at 0° and the anteroposterior axis of the embryo was set at 90° (Figure 11B).
description: Cell Data
 Parameters <strong>cell_surfaces </strong>–
 frame-by-frame tracked data for cells <em>region</em>:
 relative region of the neural ectoderm, i.e. “anterior” or
 “posterior” <em>movie:
 </em>label for each individual embryo
 analyzed <em>track_id_cells</em>: cell
 tracking label that is unique to cells within an embryo but may be
 repeated between different embryos <em>minute</em>: time
 per each individual embryo/movie in minutes <em>center_x_cells</em>: pixel X
 coordinate of centroid of each cell in each frame
 <em>center_y_cells</em>: pixel Y
 coordinate of centroid of each cell in each frame
 <em>vx_coords_cells</em>: pixel
 XY coordinates of the vertices of each cell in each frame in X:Y#X:Y
 format <em>CRISPR</em>: CRISPR
 status of each cell, i.e. “control” or “<em>shroom3
 </em>crispant”, called based on membrane-BFP
 localization. <em>control_neighbors</em>: number
 of cell neighbors that are “control” in each frame
 <em>crispant_neighbors</em>: number
 of cell neighbors that are “<em>shroom3 </em>crispant” in each
 frame <em>apical_area_pixels</em>: cell
 apical area in pixels (measured within a 1 pixel constriction of the
 segmented cell junctions) <em>apical_area_pixels_smoothed:
 apical_area_pixels</em><em> </em>averaged
 over 7 frames, -3 and +3 frame frame in question
 <em>apical_area_micron_smoothed</em>: <em>apical_area_pixels_smoothed </em>converted to square microns <em>apical_area_standardized: apical_area_pixels_smoothed</em> mean-centered and scaled per cell track (via R “scale” function), measured in standard deviations <em>stretch</em>: a normalized value ranging from 0-1 that describes cell elongation, calculated by Tissue Analyzer. Cells of the same shape but different size will have the same “stretch”. See Aigouy <em>et al. </em>2010 for more information <em>stretch_smoothed</em>: <em>stretch </em>averaged over 7 frames, -3 and +3 frame frame in question <em>stretch_standardized: stretch_smoothed</em> mean-centered and scaled per cell track (via R “scale” function), measured in standard deviations <em>orientation</em>: angle of the major axis of the fit ellipsis of the cell ranging from 0 to 90 (degrees), where 0 is aligned with the mediolateral axis and 90 is aligned with the anteroposterior axis.   <em>orientation_standardized: orientation_smoothed</em> mean-centered and scaled per cell track (via R “scale” function), measured in standard deviations (for use in Partial Least Squares regression) <em>medial_actin</em>: mean LifeAct-RFP fluorescent intensity at the medial apical domain of each cell in arbitrary units (measured within a 1 pixel constriction of the segmented cell junctions) <em>medial_actin_smoothed</em>: <em>medial_actin</em> averaged over 7 frames, -3 and +3 frame frame in question <em>medial_actin_standarized</em>: <em>medial_actin_smoothed </em>mean-centered and scaled per cell track (via R “scale” function), measured in standard deviations <em>junctional_actin</em>: mean LifeAct-RFP fluorescent intensity at the junctional domain of each cell in arbitrary units (measured at segmented cell junctions) <em>junctional_actin_smoothed</em>: <em>junctional_actin</em> averaged over 7 frames, -3 and +3 frame frame in question <em>junctional_actin_standarized</em>: <em>junctional_actin_smoothed </em>mean-centered and scaled per cell track (via R “scale” function), measured in standard deviations <em>medial_Ncadherin</em>: mean N-cadherin-GFP fluorescent intensity at the medial apical domain of each cell in arbitrary units (measured within a 1 pixel constriction of the segmented cell junctions) <em>medial_Ncadherin_smoothed</em>: <em>medial_Ncadherin</em> averaged over 7 frames, -3 and +3 frame frame in question <em>medial_Ncadherin_standarized</em>: <em>medial_Ncadherin_smoothed </em>mean-centered and scaled per cell track (via R “scale” function), measured in standard deviations <em>junctional_Ncadherin</em>: mean N-cadherin-GFP fluorescent intensity at the junctional domain of each cell in arbitrary units (measured at segmented cell junctions) <em>junctional_Ncadherin_smoothed</em>: <em>junctional_Ncadherin</em> averaged over 7 frames, -3 and +3 frame frame in question <em>junctional_Ncadherin_standarized</em>: <em>junctional_Ncadherin_smoothed </em>mean-centered and scaled per cell track (via R “scale” function), measured in standard deviations <em>medial_memBFP</em>: mean membrane(CAAX)-BFP fluorescent intensity at the medial apical domain of each cell in arbitrary units (measured within a 1 pixel constriction of the segmented cell junctions) <em>medial_memBFP_smoothed</em>: <em>medial_memBFP</em> averaged over 7 frames, -3 and +3 frame frame in question <em>junctional_memBFP</em>: mean membrane(CAAX)-BFP fluorescent intensity at the junctional domain of each cell in arbitrary units (measured at segmented cell junctions) <em>junctional_ memBFP_smoothed</em>: <em>junctional_ memBFP</em>  averaged over 7 frames, -3 and +3 frame frame in question   <strong>cell_surface_stats </strong>– summary statistics for cells <em>region</em>: relative region of the neural ectoderm, i.e. “anterior” or “posterior” <em>movie: </em>label for each individual embryo analyzed <em>track_id_cells</em>: cell tracking label that is unique to cells within an embryo but may be repeated between different embryos <em>CRISPR</em>: CRISPR status of each cell, i.e. “control” or “<em>shroom3 </em>crispant”, called based on membrane-BFP localization. <em>at_mosaic_interface</em>: TRUE denotes that cell was next to another cell of the other <em>CRISPR </em>type at some point during tracking, i.e. control cells next to <em>shroom3 </em>crispant cells and vice versa. FALSE indicates that a cell was only next to cells of the same CRISPR type, i.e. control cells next to only other control cells. Cells labeled TRUE were not used in quantitative analyses in this paper. <em>start_area_micron</em>: initial apical area of a cell in square microns (calculated from <em>apical_area_micron_smoothed)</em> <em>end_area_micron</em>: final apical area of a cell in square microns (calculated from <em>apical_area_micron_smoothed)</em> <em>delta_apical_area</em>: final value of <em>apical_area_standardized </em>minus initial value of <em>apical_area_standardized </em>(measured in standard deviations/s.d.) <em>delta_medial_actin</em>: final value of <em>medial_actin_standarized</em> minus initial value of <em>medial_actin_standarized</em> (measured in standard deviations/s.d.) <em>delta_junctional_actin</em>: final value of <em>junctional_actin_standarized</em> minus initial value of <em>junctional_actin_standarized</em> (measured in standard deviations/s.d.) <em>delta_medial_Ncadherin</em>: final value of <em>medial_Ncadherin_standarized</em> minus initial value of <em>medial_Ncadherin_standarized</em> (measured in standard deviations/s.d.) <em>delta_junctional_Ncadherin</em>: final value of <em>junctional_Ncadherin_standarized</em> minus initial value of <em>junctional_Ncadherin_standarized</em> (measured in standard deviations/s.d.) <em> </em> <strong>junctions </strong>– per minute/junction measurements of junctions <em>region</em>: relative region of the neural ectoderm, i.e. “anterior” or “posterior” <em>movie: </em>label for each individual embryo analyzed <em>track_id_junctions</em>: junction tracking label that is unique to junctions within an embryo but may be repeated between different embryos. Junctions are determined as interactions between cells. <em>CRISPR</em>: CRISPR status of each junction based on the cells the junction is between. i.e. “control” is between two control cells, “<em>shroom3 </em>crispant” is between two <em>shroom3 </em>crispant cells, and “at mosaic interface” is between a control cell and a <em>shroom3 </em>crispant cell. Junctions “at mosaic interface” were not included in quantitative analyses in this paper. <em>minute</em>: time per each individual embryo/movie in minutes <em>vx_1_x, vx_1_y: </em>pixel XY coordinates of first vertex of junction <em>vx_2_x, vx_2_y: </em>pixel XY coordinates of second vertex of junction <em>actin</em>: mean LifeAct-RFP fluorescent intensity across the junction, measured in arbitrary units <em>actin_smooth</em>: <em>actin</em> averaged over 7 frames, -3 and +3 frame frame in question <em>actin_standardized: actin_smooth</em> mean-centered and scaled per cell track (via R “scale” function), measured in standard deviations <em>Ncadherin</em>: N-cadherin-GFP fluorescent intensity across the junction, measured in arbitrary units <em>Ncadherin_smooth</em>: <em>Ncadherin</em> averaged over 7 frames, -3 and +3 frame frame in question <em>Ncadherin_standardized</em>: <em>Ncadherin_smooth</em> mean-centered and scaled per cell track (via R “scale” function), measured in standard deviations <em>length_px</em>: length of junction in maximum intensity projection measured in pixels <em>length_micron</em>:<em> length_px </em>converted to microns <em>delta_z_micron</em>: Z-distance between first and second vertex of junction, in microns. Calculated by determining where the maximum intensity projection sampled the Z-stack for each vertex in the LifeAct-RFP channel. <em>length_corrected</em>: <em>length_micron </em>corrected for z-distance between vertices using <em>delta_z_micron, </em>measured in microns. <em>length_smooth</em>: <em>length_corrected</em> averaged over 7 frames, -3 and +3 frame frame in question <em>length_standardized</em>: <em>length_smooth</em> mean-centered and scaled per cell track (via R “scale” function), measured in standard deviations <em>orientation</em>: orientation of junction relative to anteroposterior axis in degrees, where 0 is aligned with mediolateral axis and 90 is aligned with anteroposterior axis.   <strong>junction_stats </strong>- summary statistics for junctions <em>region</em>: relative region of the neural ectoderm, i.e. “anterior” or “posterior” <em>movie: </em>label for each individual embryo analyzed <em>track_id_junctions</em>: junction tracking label that is unique to junctions within an embryo but may be repeated between different embryos. Junctions are determined as interactions between cells. <em>CRISPR</em>: CRISPR status of each junction based on the cells the junction is between. i.e. “control” is between two control cells, “<em>shroom3 </em>crispant” is between two <em>shroom3 </em>crispant cells, and “at mosaic interface” is between a control cell and a <em>shroom3 </em>crispant cell. Junctions “at mosaic interface” were not included in quantitative analyses in this paper. <em>delta_length</em>: final value of <em>length_standarized</em> minus initial value of <em>length_standarized</em> (measured in standard deviations/s.d.) <em>delta_actin</em>: final value of <em>actin_standarized</em> minus initial value of <em>actin_standarized</em> (measured in standard deviations/s.d.) <em>delta_Ncadherin</em>: final value of <em>Ncadherin_standarized</em> minus initial value of <em>Ncadherin_standarized</em> (measured in standard deviations/s.d.) <em>mean_orientation</em>: average of <em>orientation</em> over junction track