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.

**Updated 7-25-2022 to correct some errors in cell tracking and shroom3 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

Xenopus tropicalis 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 tidyverse 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 shroom3 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 shroom3 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, shroom3 crispant junctions are situated between two shroom3 crispant cells, and mosaic interface junctions are situated between a wild-type and a shroom3 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).

Cell Data Parameters

cell_surfaces – frame-by-frame tracked data for cells

region: relative region of the neural ectoderm, i.e. “anterior” or “posterior”

movie: label for each individual embryo analyzed

track_id_cells: cell tracking label that is unique to cells within an embryo but may be repeated between different embryos

minute: time per each individual embryo/movie in minutes

center_x_cells: pixel X coordinate of centroid of each cell in each frame

center_y_cells: pixel Y coordinate of centroid of each cell in each frame

vx_coords_cells: pixel XY coordinates of the vertices of each cell in each frame in X:Y#X:Y format

CRISPR: CRISPR status of each cell, i.e. “control” or “shroom3 crispant”, called based on membrane-BFP localization.

control_neighbors: number of cell neighbors that are “control” in each frame

crispant_neighbors: number of cell neighbors that are “shroom3 crispant” in each frame

apical_area_pixels: cell apical area in pixels (measured within a 1 pixel constriction of the segmented cell junctions)

apical_area_pixels_smoothed: apical_area_pixels averaged over 7 frames, -3 and +3 frame frame in question

apical_area_micron_smoothed: apical_area_pixels_smoothed converted to square microns

apical_area_standardized: apical_area_pixels_smoothed mean-centered and scaled per cell track (via R “scale” function), measured in standard deviations

stretch: 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 et al. 2010 for more information

stretch_smoothed: stretch averaged over 7 frames, -3 and +3 frame frame in question

stretch_standardized: stretch_smoothed mean-centered and scaled per cell track (via R “scale” function), measured in standard deviations

orientation: 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.

 

orientation_standardized: orientation_smoothed mean-centered and scaled per cell track (via R “scale” function), measured in standard deviations (for use in Partial Least Squares regression)

medial_actin: 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)

medial_actin_smoothed: medial_actin averaged over 7 frames, -3 and +3 frame frame in question

medial_actin_standarized: medial_actin_smoothed mean-centered and scaled per cell track (via R “scale” function), measured in standard deviations

junctional_actin: mean LifeAct-RFP fluorescent intensity at the junctional domain of each cell in arbitrary units (measured at segmented cell junctions)

junctional_actin_smoothed: junctional_actin averaged over 7 frames, -3 and +3 frame frame in question

junctional_actin_standarized: junctional_actin_smoothed mean-centered and scaled per cell track (via R “scale” function), measured in standard deviations

medial_Ncadherin: 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)

medial_Ncadherin_smoothed: medial_Ncadherin averaged over 7 frames, -3 and +3 frame frame in question

medial_Ncadherin_standarized: medial_Ncadherin_smoothed mean-centered and scaled per cell track (via R “scale” function), measured in standard deviations

junctional_Ncadherin: mean N-cadherin-GFP fluorescent intensity at the junctional domain of each cell in arbitrary units (measured at segmented cell junctions)

junctional_Ncadherin_smoothed: junctional_Ncadherin averaged over 7 frames, -3 and +3 frame frame in question

junctional_Ncadherin_standarized: junctional_Ncadherin_smoothed mean-centered and scaled per cell track (via R “scale” function), measured in standard deviations

medial_memBFP: 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)

medial_memBFP_smoothed: medial_memBFP averaged over 7 frames, -3 and +3 frame frame in question

junctional_memBFP: mean membrane(CAAX)-BFP fluorescent intensity at the junctional domain of each cell in arbitrary units (measured at segmented cell junctions)

junctional_ memBFP_smoothed: junctional_ memBFP  averaged over 7 frames, -3 and +3 frame frame in question

 

cell_surface_stats – summary statistics for cells

region: relative region of the neural ectoderm, i.e. “anterior” or “posterior”

movie: label for each individual embryo analyzed

track_id_cells: cell tracking label that is unique to cells within an embryo but may be repeated between different embryos

CRISPR: CRISPR status of each cell, i.e. “control” or “shroom3 crispant”, called based on membrane-BFP localization.

at_mosaic_interface: TRUE denotes that cell was next to another cell of the other CRISPR type at some point during tracking, i.e. control cells next to shroom3 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.

start_area_micron: initial apical area of a cell in square microns (calculated from apical_area_micron_smoothed)

end_area_micron: final apical area of a cell in square microns (calculated from apical_area_micron_smoothed)

delta_apical_area: final value of apical_area_standardized minus initial value of apical_area_standardized (measured in standard deviations/s.d.)

delta_medial_actin: final value of medial_actin_standarized minus initial value of medial_actin_standarized (measured in standard deviations/s.d.)

delta_junctional_actin: final value of junctional_actin_standarized minus initial value of junctional_actin_standarized (measured in standard deviations/s.d.)

delta_medial_Ncadherin: final value of medial_Ncadherin_standarized minus initial value of medial_Ncadherin_standarized (measured in standard deviations/s.d.)

delta_junctional_Ncadherin: final value of junctional_Ncadherin_standarized minus initial value of junctional_Ncadherin_standarized (measured in standard deviations/s.d.)

 

junctions – per minute/junction measurements of junctions

region: relative region of the neural ectoderm, i.e. “anterior” or “posterior”

movie: label for each individual embryo analyzed

track_id_junctions: 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.

CRISPR: CRISPR status of each junction based on the cells the junction is between. i.e. “control” is between two control cells, “shroom3 crispant” is between two shroom3 crispant cells, and “at mosaic interface” is between a control cell and a shroom3 crispant cell. Junctions “at mosaic interface” were not included in quantitative analyses in this paper.

minute: time per each individual embryo/movie in minutes

vx_1_x, vx_1_y: pixel XY coordinates of first vertex of junction

vx_2_x, vx_2_y: pixel XY coordinates of second vertex of junction

actin: mean LifeAct-RFP fluorescent intensity across the junction, measured in arbitrary units

actin_smooth: actin averaged over 7 frames, -3 and +3 frame frame in question

actin_standardized: actin_smooth mean-centered and scaled per cell track (via R “scale” function), measured in standard deviations

Ncadherin: N-cadherin-GFP fluorescent intensity across the junction, measured in arbitrary units

Ncadherin_smooth: Ncadherin averaged over 7 frames, -3 and +3 frame frame in question

Ncadherin_standardized: Ncadherin_smooth mean-centered and scaled per cell track (via R “scale” function), measured in standard deviations

length_px: length of junction in maximum intensity projection measured in pixels

length_micron: length_px converted to microns

delta_z_micron: 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.

length_corrected: length_micron corrected for z-distance between vertices using delta_z_micron, measured in microns.

length_smooth: length_corrected averaged over 7 frames, -3 and +3 frame frame in question

length_standardized: length_smooth mean-centered and scaled per cell track (via R “scale” function), measured in standard deviations

orientation: orientation of junction relative to anteroposterior axis in degrees, where 0 is aligned with mediolateral axis and 90 is aligned with anteroposterior axis.

 

junction_stats - summary statistics for junctions

region: relative region of the neural ectoderm, i.e. “anterior” or “posterior”

movie: label for each individual embryo analyzed

track_id_junctions: 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.

CRISPR: CRISPR status of each junction based on the cells the junction is between. i.e. “control” is between two control cells, “shroom3 crispant” is between two shroom3 crispant cells, and “at mosaic interface” is between a control cell and a shroom3 crispant cell. Junctions “at mosaic interface” were not included in quantitative analyses in this paper.

delta_length: final value of length_standarized minus initial value of length_standarized (measured in standard deviations/s.d.)

delta_actin: final value of actin_standarized minus initial value of actin_standarized (measured in standard deviations/s.d.)

delta_Ncadherin: final value of Ncadherin_standarized minus initial value of Ncadherin_standarized (measured in standard deviations/s.d.)

mean_orientation: average of orientation over junction track