DOI: 10.5061/dryad.6m905qgcj
title: Local nuclear to cytoplasmic ratio regulates H3.3 incorporation via cell cycle state during zygotic genome activation

description: Early embryos often have unique chromatin states prior to zygotic genome
 activation (ZGA). In Drosophila, ZGA occurs after 13 reductive
 nuclear divisions during which the nuclear to cytoplasmic (N/C) ratio
 grows exponentially. Previous work found that histone H3 chromatin
 incorporation decreases while its variant H3.3 increases leading up to
 ZGA. In other cell types, H3.3 is associated with sites of active
 transcription and heterochromatin, suggesting a link between H3.3 and ZGA.
 Here, we test what factors regulate H3.3 incorporation at ZGA. We find
 that H3 nuclear availability falls more rapidly than H3.3 leading up to
 ZGA. We generate H3/H3.3 chimeric proteins at the endogenous H3.3A locus
 and observe that chaperone binding, but not gene structure, regulates H3.3
 behavior. We identify the N/C ratio as a major determinant of H3.3
 incorporation. To isolate how the N/C ratio regulates H3.3 incorporation
 we test the roles of genomic content, zygotic transcription, and cell
 cycle state. We determine that cell cycle regulation, but not H3
 availability or transcription, controls H3.3 incorporation. Overall, we
 propose that local N/C ratios control histone variant usage via cell cycle
 state during ZGA.
description: # Local nuclear to cytoplasmic ratio regulates H3.3 incorporation via cell
 cycle state during zygotic genome activation Dataset DOI:
 [10.5061/dryad.6m905qgcj](10.5061/dryad.6m905qgcj) ## Description of the
 data and file structure This dataset includes all the source data files
 used for the main figures of the biorxiv paper titled "Local nuclear
 to cytoplasmic ratio regulates H3.3 incorporation via cell cycle state
 during zygotic genome activation"
 doi:[https://doi.org/10.1101/2024.07.15.603602](https://doi.org/10.1101/2024.07.15.603602) There are 3 kinds of files: 1) Tables (CSV or XLSX format) 2) Image files (TIF format) 3) R notebook with code (RMD format) and the parameters used to run the code (.R format) ### Files and Description ##### File: EMBOR-2025-61506_Fig1C.csv **Description:** Figure 1C numeric data: Maximum nuclear volume attained by each nucleus reduces as the total number of nuclei doubles with each nuclear cycle (NC). **Variables:** The variables in the dataset column names are listed along with their definitions 1. **NC:** Nuclear cycle identifier. The measured stage of development of the embryo. (NC11 refers to nuclear cycle 11 and so on) 2. **# Total nuclei:** Total count of nuclei in the embryo during the given nuclear cycle. 3. **Maximum Nuclear volume:** The maximum volume achieved by an individual nucleus during the given nuclear cycle measured in cubic micrometers (µm³) **File: EMBOR-2025-61506_Fig1D.csv** **Description:** Figure 1D numeric data: Total amount of histone H3-Dendra2 and its variant H3.3-Dendra2 on mitotic chromatin. **Variables:** The variables in the dataset column names are listed along with their definitions 1. **Nuclear cycle:** The measured stage of development of the embryo. (NC11 refers to nuclear cycle 11 and so on) 2. **Mitotic chromatin Total Intensity (AU):** The total fluorescence intensity of mitotic chromatin measured during imaging, expressed in arbitrary units (AU), reflecting protein abundance. 3. **Dataset:** Identifier referring to the nuclei from different embryos used in the analysis. 4. **Genotype:** The genetic makeup of the embryo from which the data was obtained. H3 or H3.3 refers to the protein that was fluorescently tagged with Dendra2 in the organism imaged. **File: EMBOR-2025-61506_Fig2A_H3_MI.tif** **Description:** Figure 2A Image data: Maximum intensity projections of H3-Dendra2 interphase nuclei 45 seconds before nuclear envelope breakdown (NEB) from NC10-13, along with their timestamps. **File: EMBOR-2025-61506_Fig2A_H3_MI_Montage.tif** **Description:** Figure 2A Image data: Montage of Maximum intensity projections of H3-Dendra2 interphase nuclei 45 seconds before nuclear envelope breakdown (NEB) from NC10-13. Images are pseudo-colored with non-linear look-up tables where purple indicates low and yellow indicates high intensities. Scale bar 20µm **File: EMBOR-2025-61506_Fig2A_H3.3MI.tif** **Description:** Figure 2A Image data: Maximum intensity projections of H3.3-Dendra2 interphase nuclei 45 seconds before nuclear envelope breakdown (NEB) from NC10-13, along with their timestamps. **File: EMBOR-2025-61506_Fig2A_H3.3_MI_Montage.tif** **Description:** Figure 2A Image data: Montage of Maximum intensity projections of H3.3-Dendra2 interphase nuclei 45 seconds before nuclear envelope breakdown (NEB) from NC10-13. Images are pseudo-colored with non-linear look-up tables where purple indicates low and yellow indicates high intensities. Scale bar 20µm **File: EMBOR-2025-61506_Fig2B.csv** **Description:** Figure 2B numeric data: Average interphase nuclear pixel intensities for H3-Dendra2 and H3.3-Dendra2 45 seconds before NEB in NC10-13, normalized to the average individual NC10 values. **Variables:** The variables in the dataset column names are listed along with their definitions 1. **Mean.Intensity:** The average fluorescence intensity measured, indicating the mean level of a protein within the region measured (here nucleus). 2. **Nuclear cycle:** The measured stage of development of the embryo. (NC11 refers to nuclear cycle 11 and so on) 3. **Dataset:** Identifier referring to the nuclei from different embryos used in the analysis. 4. **Nuclear Conc (AU):** The concentration of a nuclear-localized molecule, measured as fluorescence intensity in arbitrary units (AU), reflecting abundance in the nucleus. 5. **Genotype:** The genetic makeup of the embryo from which the data was obtained. H3 or H3.3 refers to the protein that was fluorescently tagged with Dendra2 in the organism imaged. **File: EMBOR-2025-61506_Fig2C_H3_IC.csv** **Description:** Figure 2C numeric data: Summed (total) pixel intensities for each nucleus over time for NC11-13 normalized to the maximum NC11 values for H3-Dendra2. **Variables:** The variables in the dataset column names are listed along with their definitions 1. **Total Intensity:** The overall measured fluorescence intensity summed across a specified region, here individual nuclei, indicating total signal amount. 2. **Time:** The time point or duration at which the measurement was taken, in minutes relative to a defined starting point. 3. **NC:** Nuclear cycle identifier. The measured stage of development of the embryo. (NC11 refers to nuclear cycle 11 and so on) 4. **Dataset:** Identifier referring to the nuclei from different embryos used in the analysis. 5. **Total Intensity (AU):** The total fluorescence intensity measured during imaging, expressed in arbitrary units (AU), here normalized to the maximum NC11 values. **File: EMBOR-2025-61506_Fig2D_H3.3_IC.csv** **Description:** Figure 2D numeric data: Summed (total) pixel intensities for each nucleus over time for NC11-13 normalized to the maximum NC11 values for H3.3-Dendra2. **Variables:** The variables in the dataset column names are listed along with their definitions 1. **Total Intensity:** The overall measured fluorescence intensity summed across a specified region, here individual nuclei, indicating total signal amount. 2. **Time:** The time point or duration at which the measurement was taken, in minutes relative to a defined starting point. 3. **NC:** Nuclear cycle identifier. The measured stage of development of the embryo. (NC11 refers to nuclear cycle 11 and so on) 4. **Dataset:** Identifier referring to the nuclei from different embryos used in the analysis. 5. **Total Intensity (AU):** The total fluorescence intensity measured during imaging, expressed in arbitrary units (AU), here normalized to the maximum NC11 values. **File: EMBOR-2025-61506_Fig2E_Bound.xlsx** **Description:** Figure 2E Numeric data: The fraction of photoconverted unbound H3.3-Dendra2 after nuclear envelope breakdown in NC11-13 **Variables:** The variables in the dataset column names are listed along with their definitions 1. **Label:** Identifier referring to the nuclei from different embryos used in the analysis. 2. **Nuclear cycle:** The measured stage of development of the embryo. (NC11 refers to nuclear cycle 11 and so on) 3. **Total:** The overall amount of photoconverted H3.3-Dendra2, representing the sum of bound and unbound fractions. 4. **Bound:** The fraction or amount of photoconverted H3.3-Dendra2, that are associated with the chromatin. 5. **Unbound:** The fraction or amount of photoconverted H3.3-Dendra2, that are not associated with the chromatin. 6. **%Bound:** The percentage of the total measured molecules that are in the bound state. 7. **%Unbound:** The percentage of the total measured molecules that are in the unbound state. **File: EMBOR-2025-61506_Fig3B.csv** **Description:** Figure 3B numeric data: Total intensities on mitotic chromatin of chimeras during NC10-13 normalized to their NC10 values. **Variables:** The variables in the dataset column names are listed along with their definitions 1. **Nuclear cycle:** The measured stage of development of the embryo. (NC11 refers to nuclear cycle 11 and so on) 2. **Total Intensity (AU):** The total fluorescence intensity of mitotic chromatin measured during imaging, expressed in arbitrary units (AU), reflecting protein abundance. 3. **Dataset:** Identifier referring to the nuclei from different embryos used in the analysis. 4. **Genotype:** The genetic makeup of the embryo from which the data was obtained. Refers to the protein that was fluorescently tagged with Dendra2 in the organism imaged. **File: EMBOR-2025-61506_Fig3C.csv** **Description:** Figure 3C numeric data: Interphase nuclear concentrations of chimeras 45 seconds before nuclear envelope breakdown during NC10-13 normalized to their NC10 values. **Variables:** The variables in the dataset column names are listed along with their definitions 1. **Mean.Intensity:** The average fluorescence intensity measured, indicating the mean level of a protein within the region measured (here nucleus). 2. **Nuclear cycle:** The measured stage of development of the embryo. (NC11 refers to nuclear cycle 11 and so on) 3. **Dataset:** Identifier referring to the nuclei from different embryos used in the analysis. 4. **Nuclear Conc (AU):** The concentration of a nuclear-localized molecule, measured as fluorescence intensity in arbitrary units (AU), reflecting abundance in the nucleus. 5. **Genotype:** The genetic makeup of the embryo from which the data was obtained. Refers to the protein that was fluorescently tagged with Dendra2 in the organism imaged. **File: EMBOR-2025-61506_Fig3E_Chromatin_H3.3ssm.tif** **Description:** Figure 3E Image data: Representative maximum intensity projections of mitotic chromatin for H3.3-Dendra2 over NC10-14 in Hira ssm background. **File: EMBOR-2025-61506_Fig3E_Chromatin_H3.3ssm_Montage.tif** **Description:** Figure 3E Image data: Montage of representative maximum intensity projections of mitotic chromatin for H3.3-Dendra2 over NC10-14 in Hira ssm background. Scale bar 20µm. **File: EMBOR-2025-61506_Fig3E_Max_Import_H3.3ssm.tif** **Description:** Figure 3E Image data: Representative maximum intensity projections of interphase nuclei for H3.3-Dendra2 over NC10-14 in Hira ssm background. **File: EMBOR-2025-61506_Fig3E_Max_Import_H3.3ssm_Montage.tif** **Description:** Figure 3E Image data: Montage of representative maximum intensity projections of interphase nuclei for H3.3-Dendra2 over NC10-14 in Hira ssm background. Scale bar 20µm. **File: EMBOR-2025-61506_Fig3F.csv** **Description:** Figure 3F numeric data: Total intensities of H3-Dendra2 and H3.3ASVM-Dendra2 on mitotic chromatin in Hira ssm embryos between NC10-14 normalized to their average NC10 values. **Variables:** The variables in the dataset column names are listed along with their definitions 1. **Nuclear cycle:** The measured stage of development of the embryo. (NC11 refers to nuclear cycle 11 and so on) 2. **Total Intensity (AU):** The total fluorescence intensity of mitotic chromatin measured during imaging, expressed in arbitrary units (AU), reflecting protein abundance. 3. **Dataset:** Identifier referring to the nuclei from different embryos used in the analysis. 4. **Genotype:** The genetic makeup of the embryo from which the data was obtained. Refers to the protein that was fluorescently tagged with Dendra2 in the organism imaged. **File: EMBOR-2025-61506_Fig3G_Chromatin_H3ssm.tif** **Description:** Figure 3G Image data: Representative maximum intensity projections of mitotic chromatin for H3-Dendra2 over NC10-14 in Hira ssm background. **File: EMBOR-2025-61506_Fig3G_Chromatin_H3ssm_Montage.tif** **Description:** Figure 3G Image data: Montage of representative maximum intensity projections of mitotic chromatin for H3-Dendra2 over NC10-14 in Hira ssm background. Scale bar 20µm. **File: EMBOR-2025-61506_Fig3G_Max_import_H3_ssm.tif** **Description:** Figure 3G Image data: Representative maximum intensity projections of interphase nuclei for H3-Dendra2 over NC10-14 in Hira ssm background. **File: EMBOR-2025-61506_Fig3G_Max_import_H3ssm_Montage.tif** **Description:** Figure 3G Image data: Montage of representative maximum intensity projections of interphase nuclei for H3-Dendra2 over NC10-14 in Hira ssm background. Scale bar 20µm. **File: EMBOR-2025-61506_Fig3H_Chromatin_ssmASVM.tif** **Description:** Figure 3H Image data: Representative maximum intensity projections of mitotic chromatin for H3.3-ASVM-Dendra2 over NC10-14 in Hira ssm background. **File: EMBOR-2025-61506_Fig3H_Chromatin_ssmASVM_Montage.tif** **Description:** Figure 3H Image data: Montage of representative maximum intensity projections of mitotic chromatin for H3.3-ASVM-Dendra2 over NC10-14 in Hira ssm background. Scale bar 20µm. **File: EMBOR-2025-61506_Fig3H_Max_Import_ssmASVM.tif** **Description:** Figure 3H Image data: Representative maximum intensity projections of interphase nuclei for H3.3-ASVM-Dendra2 over NC10-14 in Hira ssm background. **File: EMBOR-2025-61506_Fig3H_Max_Import_ssmASVM_Montage.tif** **Description:** Figure 3H Image data: Montage of representative maximum intensity projections of interphase nuclei for H3.3-ASVM-Dendra2 over NC10-14 in Hira ssm background. Scale bar 20µm. **File: EMBOR-2025-61506_Fig4A_NC11_Control_100um_scalebar-Enlarged.tif** **Description:** Figure 4A Image data: Example NC11 control embryo. Scale bar 100µm. **File: EMBOR-2025-61506_Fig4B_NC11_Shkl_100um_scalebar_1-Enlarged.tif** **Description:** Figure 4B Image data: Example NC11 shkl embryo. Scale bar 100µm. **File: EMBOR-2025-61506_Fig4C.csv** **Description:** Figure 4C numeric data: Total intensities on mitotic chromatin of H3-Dendra2 during NC11-13 in a representative control embryo where each point indicates a single nucleus. **Variables:** The variables in the dataset column names are listed along with their definitions 1. **Total Intensity:** The overall measured fluorescence intensity summed across a specified region, here individual nuclei, indicating total signal amount. 2. **Nuclear cycle:** The measured stage of development of the embryo. (NC11 refers to nuclear cycle 11 and so on) 3. **Region:** The specific area within the embryo from which the nuclei were used in the analysis. 4. **Total Intensity (AU):** The total fluorescence intensity measured during imaging, expressed in arbitrary units (AU), here normalized to the mean NC11 values. **File: EMBOR-2025-61506_Fig4D.csv** **Description:** Figure 4D numeric data: Total intensities on mitotic chromatin of H3.3-Dendra2 during NC11-13 in a representative control embryo where each point indicates a single nucleus. **Variables:** The variables in the dataset column names are listed along with their definitions 1. **Total Intensity:** The overall measured fluorescence intensity summed across a specified region, here individual nuclei, indicating total signal amount. 2. **Nuclear cycle:** The measured stage of development of the embryo. (NC11 refers to nuclear cycle 11 and so on) 3. **Region:** The specific area within the embryo from which the nuclei were used in the analysis. 4. **Total Intensity (AU):** The total fluorescence intensity measured during imaging, expressed in arbitrary units (AU), here normalized to the mean NC11 values. **File: EMBOR-2025-61506_Fig4E.csv** **Description:** Figure 4E numeric data: Total intensities on mitotic chromatin of H3-Dendra2 during NC11-13 in a representative shkl embryo where each point indicates a single nucleus. NC14 represents a partial extra division in the low-density region. **Variables:** The variables in the dataset column names are listed along with their definitions 1. **Total Intensity:** The overall measured fluorescence intensity summed across a specified region, here individual nuclei, indicating total signal amount. 2. **Nuclear cycle:** The measured stage of development of the embryo. (NC11 refers to nuclear cycle 11 and so on) 3. **Density:** Identifier to describe how packed the nuclei within a region were for the given region of the embryo used in the analysis, indicated with high or low. 4. **Total Intensity (AU):** The total fluorescence intensity measured during imaging, expressed in arbitrary units (AU), here normalized to the mean NC11 values. **File: EMBOR-2025-61506_Fig4F.csv** **Description:** Figure 4F numeric data: Total intensities on mitotic chromatin of H3.3-Dendra2 during NC11-13 in a representative shkl embryo where each point indicates a single nucleus. NC14 represents a partial extra division in the low-density region. **Variables:** The variables in the dataset column names are listed along with their definitions 1. **Total Intensity:** The overall measured fluorescence intensity summed across a specified region, here individual nuclei, indicating total signal amount. 2. **Nuclear cycle:** The measured stage of development of the embryo. (NC11 refers to nuclear cycle 11 and so on) 3. **Density:** Identifier to describe how packed the nuclei within a region were for the given region of the embryo used in the analysis, indicated with high or low. 4. **Total Intensity (AU):** The total fluorescence intensity measured during imaging, expressed in arbitrary units (AU), here normalized to the mean NC11 values. **File: EMBOR-2025-61506_Fig5B.csv** **Description:** Figure 5B numeric data: Total intensities over time for nuclei in representative NC13 control embryos binned by the number of neighbors for H3-Dendra2. **Variables:** The variables in the dataset column names are listed along with their definitions 1. **#Neighbours:** The number of neighboring nuclei surrounding a given nucleus within a 20µm radius. 2. **Time (mins):** The time point or duration at which the measurement was taken, in minutes relative to a defined starting point. 3. **Total intensity (AU):** The total fluorescence intensity measured during imaging, expressed in arbitrary units (AU). 4. **#Nuclei:** The total count of nuclei binned to obtain the trace. **File: EMBOR-2025-61506_Fig5B_D_RawData_H3Ctrl-NC13_231127S3.csv** **Description:** Raw data used to generate figures 5B&D using the attached code and parameters file **Variables:** The variables in the dataset column names are listed along with their definitions 1. **frame:** The time point or frame number in a time-lapse image sequence. 2. **labelimageId:** Identifier for the labeled image or segmented object in the dataset. 3. **trackId:** Unique identifier assigned to a tracked object across frames. 4. **lineageId:** Identifier denoting the lineage or family of related tracked objects (e.g., cell lineage through division). 5. **parentTrackId:** The trackId of the parent object from which the current object originated (e.g., previous generation in lineage). 6. **mergerLabelId:** Identifier indicating if objects merged together and associated merged label. 7. **Convexity_0:** Convexity of the object in a specific dimension or channel (dimension 0), indicating shape smoothness (ratio of object area to convex hull area). 8. **Number_of_Defects_0:** The total number of defects or irregularities detected within the object (channel/dimension 0). 9. **Mean_Defect_Displacement_0:** Average displacement of defects within the object (channel/dimension 0), reflecting spatial distribution of defects. 10. **Mean_Defect_Area_0:** Average area of detected defects within the object (channel/dimension 0). 11. **Variance_of_Defect_Area_0:** Variability or spread in the size of defects within the object (channel/dimension 0). 12. **Convex_Hull_Center_0:** X coordinate of the centroid of the object's convex hull. 13. **Convex_Hull_Center_1:** Y coordinate of the centroid of the object's convex hull. 14. **Convex_Hull_Center_2:** Z coordinate of the centroid of the object's convex hull. 15. **Object_Center_0:** X coordinate of the center of mass or centroid of the object. 16. **Object_Center_1:** Y coordinate of the center of mass or centroid of the object. 17. **Object_Center_2:** Z coordinate of the center of mass or centroid of the object. 18. **Object_Area_0:** Area of the object measured in pixels or appropriate spatial units (channel/dimension 0). 19. **Kurtosis_of_Intensity_0:** Measure of the "tailedness" or peakedness of the intensity distribution within the object (channel/dimension 0). 20. **Maximum_intensity_0:** Maximum pixel intensity value within the object (channel/dimension 0). 21. **Mean_Intensity_0:** Average pixel intensity value within the object (channel/dimension 0). 22. **Minimum_intensity_0:** Minimum pixel intensity value within the object (channel/dimension 0). 23. **Principal_components_of_the_object_0:** First principal component value describing object's shape or intensity variation. 24. **Principal_components_of_the_object_1:** Second principal component value describing object's shape or intensity variation. 25. **Principal_components_of_the_object_2:** Third principal component value describing object's shape or intensity variation. 26. **Principal_components_of_the_object_3:** Fourth principal component value describing object's shape or intensity variation. 27. **Principal_components_of_the_object_4:** Fifth principal component value describing object's shape or intensity variation. 28. **Principal_components_of_the_object_5:** Sixth principal component value describing object's shape or intensity variation. 29. **Principal_components_of_the_object_6:** Seventh principal component value describing object's shape or intensity variation. 30. **Principal_components_of_the_object_7:** Eighth principal component value describing object's shape or intensity variation. 31. **Principal_components_of_the_object_8:** Ninth principal component value describing object's shape or intensity variation. 32. **Radii_of_the_object_0:** Radius of the object along the first principal axis. 33. **Radii_of_the_object_1:** Radius of the object along the second principal axis. 34. **Radii_of_the_object_2:** Radius of the object along the third principal axis. 35. **Skewness_of_Intensity_0:** Measure of the asymmetry of the intensity distribution within the object (channel/dimension 0). 36. **Total_Intensity_0:** Sum of all pixel intensities within the object (channel/dimension 0), representing total signal strength. 37. **Variance_of_Intensity_0:** Variability or spread of pixel intensity values within the object (channel/dimension 0). 38. **Bounding_Box_Maximum_0:** Maximum X coordinate defining the bounding box around the object. 39. **Bounding_Box_Maximum_1:** Maximum Y coordinate defining the bounding box around the object. 40. **Bounding_Box_Maximum_2:** Maximum Z coordinate defining the bounding box around the object. 41. **Bounding_Box_Minimum_0:** Minimum X coordinate defining the bounding box around the object. 42. **Bounding_Box_Minimum_1:** Minimum Y coordinate defining the bounding box around the object. 43. **Bounding_Box_Minimum_2:** Minimum Z coordinate defining the bounding box around the object. 44. **Size_in_pixels_0:** The size of the object expressed as the number of pixels contained within it (channel/dimension 0). 45. **Center_of_the_object_0:** X coordinate specifying the geometric center or centroid of the object. 46. **Center_of_the_object_1:** Y coordinate specifying the geometric center or centroid of the object. 47. **Center_of_the_object_2:** Z coordinate specifying the geometric center or centroid of the object. **File: EMBOR-2025-61506_Fig5B_D_parameters_H3_Ctrl.R** **Description:** Code parameters to run Raw data for Figure 5B&D **File: EMBOR-2025-61506_Fig5C.csv** **Description:** Figure 5C numeric data: Total intensities over time for nuclei in representative NC13 control embryos binned by the number of neighbors for H3.3-Dendra2. **Variables:** The variables in the dataset column names are listed along with their definitions 1. **#Neighbours:** The number of neighboring nuclei surrounding a given nucleus within a 20µm radius. 2. **Time (mins):** The time point or duration at which the measurement was taken, in minutes relative to a defined starting point. 3. **Total intensity (AU):** The total fluorescence intensity measured during imaging, expressed in arbitrary units (AU). 4. **#Nuclei:** The total count of nuclei binned to obtain the trace. **File: EMBOR-2025-61506_Fig5C_E_RawData_H3.3_Ctrl-NC13_230719S5.csv** **Description:** Raw data used to generate figures 5C&E using the attached code and parameters file **Variables:** The variables in the dataset column names are listed along with their definitions 1. **frame:** The time point or frame number in a time-lapse image sequence. 2. **labelimageId:** Identifier for the labeled image or segmented object in the dataset. 3. **trackId:** Unique identifier assigned to a tracked object across frames. 4. **lineageId:** Identifier denoting the lineage or family of related tracked objects (e.g., cell lineage through division). 5. **parentTrackId:** The trackId of the parent object from which the current object originated (e.g., previous generation in lineage). 6. **mergerLabelId:** Identifier indicating if objects merged together and associated merged label. 7. **Convexity_0:** Convexity of the object in a specific dimension or channel (dimension 0), indicating shape smoothness (ratio of object area to convex hull area). 8. **Number_of_Defects_0:** The total number of defects or irregularities detected within the object (channel/dimension 0). 9. **Mean_Defect_Displacement_0:** Average displacement of defects within the object (channel/dimension 0), reflecting spatial distribution of defects. 10. **Mean_Defect_Area_0:** Average area of detected defects within the object (channel/dimension 0). 11. **Variance_of_Defect_Area_0:** Variability or spread in the size of defects within the object (channel/dimension 0). 12. **Convex_Hull_Center_0:** X coordinate of the centroid of the object's convex hull. 13. **Convex_Hull_Center_1:** Y coordinate of the centroid of the object's convex hull. 14. **Convex_Hull_Center_2:** Z coordinate of the centroid of the object's convex hull. 15. **Object_Center_0:** X coordinate of the center of mass or centroid of the object. 16. **Object_Center_1:** Y coordinate of the center of mass or centroid of the object. 17. **Object_Center_2:** Z coordinate of the center of mass or centroid of the object. 18. **Object_Area_0:** Area of the object measured in pixels or appropriate spatial units (channel/dimension 0). 19. **Kurtosis_of_Intensity_0:** Measure of the "tailedness" or peakedness of the intensity distribution within the object (channel/dimension 0). 20. **Maximum_intensity_0:** Maximum pixel intensity value within the object (channel/dimension 0). 21. **Mean_Intensity_0:** Average pixel intensity value within the object (channel/dimension 0). 22. **Minimum_intensity_0:** Minimum pixel intensity value within the object (channel/dimension 0). 23. **Principal_components_of_the_object_0:** First principal component value describing object's shape or intensity variation. 24. **Principal_components_of_the_object_1:** Second principal component value describing object's shape or intensity variation. 25. **Principal_components_of_the_object_2:** Third principal component value describing object's shape or intensity variation. 26. **Principal_components_of_the_object_3:** Fourth principal component value describing object's shape or intensity variation. 27. **Principal_components_of_the_object_4:** Fifth principal component value describing object's shape or intensity variation. 28. **Principal_components_of_the_object_5:** Sixth principal component value describing object's shape or intensity variation. 29. **Principal_components_of_the_object_6:** Seventh principal component value describing object's shape or intensity variation. 30. **Principal_components_of_the_object_7:** Eighth principal component value describing object's shape or intensity variation. 31. **Principal_components_of_the_object_8:** Ninth principal component value describing object's shape or intensity variation. 32. **Radii_of_the_object_0:** Radius of the object along the first principal axis. 33. **Radii_of_the_object_1:** Radius of the object along the second principal axis. 34. **Radii_of_the_object_2:** Radius of the object along the third principal axis. 35. **Skewness_of_Intensity_0:** Measure of the asymmetry of the intensity distribution within the object (channel/dimension 0). 36. **Total_Intensity_0:** Sum of all pixel intensities within the object (channel/dimension 0), representing total signal strength. 37. **Variance_of_Intensity_0:** Variability or spread of pixel intensity values within the object (channel/dimension 0). 38. **Bounding_Box_Maximum_0:** Maximum X coordinate defining the bounding box around the object. 39. **Bounding_Box_Maximum_1:** Maximum Y coordinate defining the bounding box around the object. 40. **Bounding_Box_Maximum_2:** Maximum Z coordinate defining the bounding box around the object. 41. **Bounding_Box_Minimum_0:** Minimum X coordinate defining the bounding box around the object. 42. **Bounding_Box_Minimum_1:** Minimum Y coordinate defining the bounding box around the object. 43. **Bounding_Box_Minimum_2:** Minimum Z coordinate defining the bounding box around the object. 44. **Size_in_pixels_0:** The size of the object expressed as the number of pixels contained within it (channel/dimension 0). 45. **Center_of_the_object_0:** X coordinate specifying the geometric center or centroid of the object. 46. **Center_of_the_object_1:** Y coordinate specifying the geometric center or centroid of the object. 47. **Center_of_the_object_2:** Z coordinate specifying the geometric center or centroid of the object. **File: EMBOR-2025-61506_Fig5C_E_parameters_H3.3_Ctrl.R** **Description:** Code parameters to run Raw data for Figure 5C&E **File: EMBOR-2025-61506_Fig5D.csv** **Description:** Figure 5D numeric data: Initial slopes of nuclear import curves (change in total nuclear intensity over time for the first 5 timepoints) from representative NC13 control embryos for H3-Dendra2. **Variables:** The variables in the dataset column names are listed along with their definitions 1. **trackID:** Unique identifier assigned to a tracked nucleus throughout time-lapse imaging. 2. **Initial slope:** The rate of change in total nuclear intensity over time for the first 5 timepoints of nuclear import. 3. **#Neighbours at min volume:** The number of neighboring nuclei surrounding a given nucleus within a 20µm radius at the time it reaches its minimum volume. **File: EMBOR-2025-61506_Fig5E.csv** **Description:** Figure 5E numeric data: Initial slopes of nuclear import curves (change in total nuclear intensity over time for the first 5 timepoints) from representative NC13 control embryos for H3.3-Dendra2. **Variables:** The variables in the dataset column names are listed along with their definitions 1. **trackID:** Unique identifier assigned to a tracked nucleus throughout time-lapse imaging. 2. **Initial slope:** The rate of change in total nuclear intensity over time for the first 5 timepoints of nuclear import. 3. **#Neighbours at min volume:** The number of neighboring nuclei surrounding a given nucleus within a 20µm radius at the time it reaches its minimum volume. **File: EMBOR-2025-61506_Fig5F.csv** **Description:** Figure 5F numeric data: Total intensities over time for nuclei in representative NC13 shkl embryos binned by the number of neighbors for H3-Dendra2. **Variables:** The variables in the dataset column names are listed along with their definitions 1. **#Neighbours:** The number of neighboring nuclei surrounding a given nucleus within a 20µm radius. 2. **Time (mins):** The time point or duration at which the measurement was taken, in minutes relative to a defined starting point. 3. **Total intensity (AU):** The total fluorescence intensity measured during imaging, expressed in arbitrary units (AU). 4. **#Nuclei:** The total count of nuclei binned to obtain the trace. **File: EMBOR-2025-61506_Fig5F_H_RawData_H3_Shkl-NC13_230831S6.csv** **Description:** Raw data used to generate figures 5F&H using the attached code and parameters file **Variables:** The variables in the dataset column names are listed along with their definitions 1. **frame:** The time point or frame number in a time-lapse image sequence. 2. **labelimageId:** Identifier for the labeled image or segmented object in the dataset. 3. **trackId:** Unique identifier assigned to a tracked object across frames. 4. **lineageId:** Identifier denoting the lineage or family of related tracked objects (e.g., cell lineage through division). 5. **parentTrackId:** The trackId of the parent object from which the current object originated (e.g., previous generation in lineage). 6. **mergerLabelId:** Identifier indicating if objects merged together and associated merged label. 7. **Convexity_0:** Convexity of the object in a specific dimension or channel (dimension 0), indicating shape smoothness (ratio of object area to convex hull area). 8. **Number_of_Defects_0:** The total number of defects or irregularities detected within the object (channel/dimension 0). 9. **Mean_Defect_Displacement_0:** Average displacement of defects within the object (channel/dimension 0), reflecting spatial distribution of defects. 10. **Mean_Defect_Area_0:** Average area of detected defects within the object (channel/dimension 0). 11. **Variance_of_Defect_Area_0:** Variability or spread in the size of defects within the object (channel/dimension 0). 12. **Convex_Hull_Center_0:** X coordinate of the centroid of the object's convex hull. 13. **Convex_Hull_Center_1:** Y coordinate of the centroid of the object's convex hull. 14. **Convex_Hull_Center_2:** Z coordinate of the centroid of the object's convex hull. 15. **Object_Center_0:** X coordinate of the center of mass or centroid of the object. 16. **Object_Center_1:** Y coordinate of the center of mass or centroid of the object. 17. **Object_Center_2:** Z coordinate of the center of mass or centroid of the object. 18. **Object_Area_0:** Area of the object measured in pixels or appropriate spatial units (channel/dimension 0). 19. **Kurtosis_of_Intensity_0:** Measure of the "tailedness" or peakedness of the intensity distribution within the object (channel/dimension 0). 20. **Maximum_intensity_0:** Maximum pixel intensity value within the object (channel/dimension 0). 21. **Mean_Intensity_0:** Average pixel intensity value within the object (channel/dimension 0). 22. **Minimum_intensity_0:** Minimum pixel intensity value within the object (channel/dimension 0). 23. **Principal_components_of_the_object_0:** First principal component value describing object's shape or intensity variation. 24. **Principal_components_of_the_object_1:** Second principal component value describing object's shape or intensity variation. 25. **Principal_components_of_the_object_2:** Third principal component value describing object's shape or intensity variation. 26. **Principal_components_of_the_object_3:** Fourth principal component value describing object's shape or intensity variation. 27. **Principal_components_of_the_object_4:** Fifth principal component value describing object's shape or intensity variation. 28. **Principal_components_of_the_object_5:** Sixth principal component value describing object's shape or intensity variation. 29. **Principal_components_of_the_object_6:** Seventh principal component value describing object's shape or intensity variation. 30. **Principal_components_of_the_object_7:** Eighth principal component value describing object's shape or intensity variation. 31. **Principal_components_of_the_object_8:** Ninth principal component value describing object's shape or intensity variation. 32. **Radii_of_the_object_0:** Radius of the object along the first principal axis. 33. **Radii_of_the_object_1:** Radius of the object along the second principal axis. 34. **Radii_of_the_object_2:** Radius of the object along the third principal axis. 35. **Skewness_of_Intensity_0:** Measure of the asymmetry of the intensity distribution within the object (channel/dimension 0). 36. **Total_Intensity_0:** Sum of all pixel intensities within the object (channel/dimension 0), representing total signal strength. 37. **Variance_of_Intensity_0:** Variability or spread of pixel intensity values within the object (channel/dimension 0). 38. **Bounding_Box_Maximum_0:** Maximum X coordinate defining the bounding box around the object. 39. **Bounding_Box_Maximum_1:** Maximum Y coordinate defining the bounding box around the object. 40. **Bounding_Box_Maximum_2:** Maximum Z coordinate defining the bounding box around the object. 41. **Bounding_Box_Minimum_0:** Minimum X coordinate defining the bounding box around the object. 42. **Bounding_Box_Minimum_1:** Minimum Y coordinate defining the bounding box around the object. 43. **Bounding_Box_Minimum_2:** Minimum Z coordinate defining the bounding box around the object. 44. **Size_in_pixels_0:** The size of the object expressed as the number of pixels contained within it (channel/dimension 0). 45. **Center_of_the_object_0:** X coordinate specifying the geometric center or centroid of the object. 46. **Center_of_the_object_1:** Y coordinate specifying the geometric center or centroid of the object. 47. **Center_of_the_object_2:** Z coordinate specifying the geometric center or centroid of the object. 48. **Volume:** Volume of the object in cubic micrometers (µm³) **File: EMBOR-2025-61506_Fig5F_H_parameters_H3_Shkl.R** **Description:** Code parameters to run Raw data for Figure 5F&H **File: EMBOR-2025-61506_Fig5G.csv** **Description:** Figure 5G numeric data: Total intensities over time for nuclei in representative NC13 shkl embryos binned by the number of neighbors for H3.3-Dendra2. **Variables:** The variables in the dataset column names are listed along with their definitions 1. **#Neighbours:** The number of neighboring nuclei surrounding a given nucleus within a 20µm radius. 2. **Time (mins):** The time point or duration at which the measurement was taken, in minutes relative to a defined starting point. 3. **Total intensity (AU):** The total fluorescence intensity measured during imaging, expressed in arbitrary units (AU). 4. **#Nuclei:** The total count of nuclei binned to obtain the trace. **File: EMBOR-2025-61506_Fig5G_I_RawData_H3.3_Shkl-NC13_230617S8.csv** **Description:** Raw data used to generate figures 5G&I using the attached code and parameters file **Variables:** The variables in the dataset column names are listed along with their definitions 1. **frame:** The time point or frame number in a time-lapse image sequence. 2. **labelimageId:** Identifier for the labeled image or segmented object in the dataset. 3. **trackId:** Unique identifier assigned to a tracked object across frames. 4. **lineageId:** Identifier denoting the lineage or family of related tracked objects (e.g., cell lineage through division). 5. **parentTrackId:** The trackId of the parent object from which the current object originated (e.g., previous generation in lineage). 6. **mergerLabelId:** Identifier indicating if objects merged together and associated merged label. 7. **Convexity_0:** Convexity of the object in a specific dimension or channel (dimension 0), indicating shape smoothness (ratio of object area to convex hull area). 8. **Number_of_Defects_0:** The total number of defects or irregularities detected within the object (channel/dimension 0). 9. **Mean_Defect_Displacement_0:** Average displacement of defects within the object (channel/dimension 0), reflecting spatial distribution of defects. 10. **Mean_Defect_Area_0:** Average area of detected defects within the object (channel/dimension 0). 11. **Variance_of_Defect_Area_0:** Variability or spread in the size of defects within the object (channel/dimension 0). 12. **Convex_Hull_Center_0:** X coordinate of the centroid of the object's convex hull. 13. **Convex_Hull_Center_1:** Y coordinate of the centroid of the object's convex hull. 14. **Convex_Hull_Center_2:** Z coordinate of the centroid of the object's convex hull. 15. **Object_Center_0:** X coordinate of the center of mass or centroid of the object. 16. **Object_Center_1:** Y coordinate of the center of mass or centroid of the object. 17. **Object_Center_2:** Z coordinate of the center of mass or centroid of the object. 18. **Object_Area_0:** Area of the object measured in pixels or appropriate spatial units (channel/dimension 0). 19. **Kurtosis_of_Intensity_0:** Measure of the "tailedness" or peakedness of the intensity distribution within the object (channel/dimension 0). 20. **Maximum_intensity_0:** Maximum pixel intensity value within the object (channel/dimension 0). 21. **Mean_Intensity_0:** Average pixel intensity value within the object (channel/dimension 0). 22. **Minimum_intensity_0:** Minimum pixel intensity value within the object (channel/dimension 0). 23. **Principal_components_of_the_object_0:** First principal component value describing object's shape or intensity variation. 24. **Principal_components_of_the_object_1:** Second principal component value describing object's shape or intensity variation. 25. **Principal_components_of_the_object_2:** Third principal component value describing object's shape or intensity variation. 26. **Principal_components_of_the_object_3:** Fourth principal component value describing object's shape or intensity variation. 27. **Principal_components_of_the_object_4:** Fifth principal component value describing object's shape or intensity variation. 28. **Principal_components_of_the_object_5:** Sixth principal component value describing object's shape or intensity variation. 29. **Principal_components_of_the_object_6:** Seventh principal component value describing object's shape or intensity variation. 30. **Principal_components_of_the_object_7:** Eighth principal component value describing object's shape or intensity variation. 31. **Principal_components_of_the_object_8:** Ninth principal component value describing object's shape or intensity variation. 32. **Radii_of_the_object_0:** Radius of the object along the first principal axis. 33. **Radii_of_the_object_1:** Radius of the object along the second principal axis. 34. **Radii_of_the_object_2:** Radius of the object along the third principal axis. 35. **Skewness_of_Intensity_0:** Measure of the asymmetry of the intensity distribution within the object (channel/dimension 0). 36. **Total_Intensity_0:** Sum of all pixel intensities within the object (channel/dimension 0), representing total signal strength. 37. **Variance_of_Intensity_0:** Variability or spread of pixel intensity values within the object (channel/dimension 0). 38. **Bounding_Box_Maximum_0:** Maximum X coordinate defining the bounding box around the object. 39. **Bounding_Box_Maximum_1:** Maximum Y coordinate defining the bounding box around the object. 40. **Bounding_Box_Maximum_2:** Maximum Z coordinate defining the bounding box around the object. 41. **Bounding_Box_Minimum_0:** Minimum X coordinate defining the bounding box around the object. 42. **Bounding_Box_Minimum_1:** Minimum Y coordinate defining the bounding box around the object. 43. **Bounding_Box_Minimum_2:** Minimum Z coordinate defining the bounding box around the object. 44. **Size_in_pixels_0:** The size of the object expressed as the number of pixels contained within it (channel/dimension 0). 45. **Center_of_the_object_0:** X coordinate specifying the geometric center or centroid of the object. 46. **Center_of_the_object_1:** Y coordinate specifying the geometric center or centroid of the object. 47. **Center_of_the_object_2:** Z coordinate specifying the geometric center or centroid of the object. **File: EMBOR-2025-61506_Fig5G_I_parameters_H3.3_Shkl.R** **Description:** Code parameters to run Raw data for Figure 5G&I **File: EMBOR-2025-61506_Fig5H.csv** **Description:** Figure 5H numeric data: Initial slopes of nuclear import curves (change in total nuclear intensity over time for the first 5 timepoints) from representative NC13 shkl embryos for H3-Dendra2. **Variables:** The variables in the dataset column names are listed along with their definitions 1. **trackID:** Unique identifier assigned to a tracked nucleus throughout time-lapse imaging. 2. **Initial slope:** The rate of change in total nuclear intensity over time for the first 5 timepoints of nuclear import. 3. **#Neighbours at min volume:** The number of neighboring nuclei surrounding a given nucleus within a 20µm radius at the time it reaches its minimum volume. **File: EMBOR-2025-61506_Fig5I.csv** **Description:** Figure 5I numeric data: Initial slopes of nuclear import curves (change in total nuclear intensity over time for the first 5 timepoints) from representative NC13 shkl embryos for H3.3-Dendra2. **Variables:** The variables in the dataset column names are listed along with their definitions 1. **trackID:** Unique identifier assigned to a tracked nucleus throughout time-lapse imaging. 2. **Initial slope:** The rate of change in total nuclear intensity over time for the first 5 timepoints of nuclear import. 3. **#Neighbours at min volume:** The number of neighboring nuclei surrounding a given nucleus within a 20µm radius at the time it reaches its minimum volume. **File: EMBOR-2025-61506_Fig6B.csv** **Description:** Figure 6B numeric data: Total intensities of H3.3-Dendra2 on mitotic chromatin in white-RNAi (control) and Slbp-RNAi backgrounds during NC10-13. **Variables:** The variables in the dataset column names are listed along with their definitions 1. **Nuclear cycle:** The measured stage of development of the embryo. (NC11 refers to nuclear cycle 11 and so on) 2. **Total Intensity:** The total fluorescence intensity of mitotic chromatin measured during imaging, reflecting protein abundance. 3. **Genotype:** The genetic background of the embryo from which the data was obtained. 4. **Dataset:** Identifier referring to the nuclei from different embryos used in the analysis. **File: EMBOR-2025-61506_Fig6D.csv** **Description:** Figure 6D numeric data: Total intensities of H3.3-Dendra2 on mitotic chromatin in white-RNAi (control) and zelda-RNAi backgrounds during NC10-13. **Variables:** The variables in the dataset column names are listed along with their definitions 1. **Nuclear cycle:** The measured stage of development of the embryo. (NC11 refers to nuclear cycle 11 and so on) 2. **Total Intensity:** The total fluorescence intensity of mitotic chromatin measured during imaging, reflecting protein abundance. 3. **Genotype:** The genetic background of the embryo from which the data was obtained. 4. **Dataset:** Identifier referring to the nuclei from different embryos used in the analysis. **File: EMBOR-2025-61506_Fig6F.csv** **Description:** Figure 6F Numeric data: Total intensities of H3.3-Dendra2 on mitotic chromatin in control and embryos from chk1-/- mothers during NC10-13. **Variables:** The variables in the dataset column names are listed along with their definitions 1. **Nuclear cycle:** The measured stage of development of the embryo. (NC11 refers to nuclear cycle 11 and so on) 2. **Total Intensity:** The total fluorescence intensity of mitotic chromatin measured during imaging, reflecting protein abundance. 3. **Genotype:** The genetic background of the embryo from which the data was obtained. 4. **Dataset:** Identifier referring to the nuclei from different embryos used in the analysis. **File: Neighborhood_analysis_v2.Rmd** **Description:** R notebook containing the code to run the provided Raw data is included in RMD format. ## Code/software R notebook containing the code to run the provided Raw data is included in RMD format. ## Access information Other publicly accessible locations of the data: [https://github.com/anushadbhatt/AM_paper](https://github.com/anushadbhatt/AM_paper)