DOI: 10.5061/dryad.5dv41nsk3
title: Data from: Cohesin protein Smc3 influences kinocilial structure and function

description: Cohesinopathies and ciliopathies are congenital disorders affecting
 overlapping body systems. The extent to which these syndromes may be
 linked remains largely untested. Recently, reduced expression of a cohesin
 core subunit, Smc3, was found to result in abnormal otolith development in
 zebrafish embryos. This finding suggests that Smc3 may contribute to
 kinociliary development and function, which would represent a novel role
 for Smc3. Using hair cells found in neuromasts of the posterior lateral
 line, we found that Smc3 knockdown resulted in reduced kinociliary length.
 To address the role of Smc3 in kinocilial function, we monitored neomycin
 resistance of neuromasts (associated with several cilial gene mutants) and
 FM1-43X uptake in hair cells (associated with mechanotransduction). We
 found that Smc3 knockdown indeed led to neomycin resistance of the
 posterior lateral line neuromasts, suggesting impaired kinocilium
 function. However, neuromast hair cells did not have defects in FM1-43X
 uptake. We further demonstrated that hair cell number is reduced within
 neuromasts. This study suggests a significant influence of cohesin subunit
 Smc3 in ciliary structure and function and provides a preliminary link
 between cohesinopathy and ciliopathy etiologies.
description: # Data from: Cohesin protein Smc3 influences kinocilial structure and
 function Dataset DOI:
 [10.5061/dryad.5dv41nsk3](https://doi.org/10.5061/dryad.5dv41nsk3) ##
 Description of the data and file structure This dataset includes image
 files captured and quantitative analysis performed to discern the
 influence of cohesin protein Smc3 on kinociliary structure and/or
 development in zebrafish larvae. Datasets are organized in separate .zip
 files for separate experimental efforts corresponding to each figure in
 the manuscript. Each .zip file contains additional README files with
 details to provide necessary information. ### Files and variables ####
 File: Figure_1_Data.zip **Description:** This .zip file contains .tiff
 files of each imaged and evaluated neuromast for kinociliary length
 analysis. Folder names indicate the injection type. The dataset includes
 .tiff files of observed neuromasts used for kinociliary length analysis.
 Each file name corresponds directly to the identifiers used in the .csv
 file for that injection type. The .csv file contains measurements of a
 representative kinocilium from the indicated neuromast and calculations to
 estimate the length of the kinocilium based on the measurements. Variables
 within each .csv file are defined as described below:  Name: corresponds
 to the name of the file from the dataset analyzed in that row. First z
 slice: indicates the image within the z-stack where the longest kinocilium
 first appears. Last z slice: indicates the image within the z-stack where
 the longest kinocilium last appears. z-thickness: indicates the thickness,
 microns, of each image within the stack. z-displacement: calculation of
 the displacement, in microns, of the longest kinocilium in the
 z-dimension. First x: indicates the x-coordinate where signal from the
 kinocilium in the first z slice occurs. Last x: indicates the x-coordinate
 where signal from the kinocilium in the last z slice occurs.
 x-displacement: calculation of the displacement, in microns, of the
 longest kinocilium in the x-dimension. First y: indicates the y-coordinate
 where signal from the kinocilium in the first z slice occurs. Last y:
 indicates the y-coordinate where signal from the kinocilium in the last z
 slice occurs. y-displacement: calculation of the displacement, in microns,
 of the longest kinocilium in the y-dimension. 2D displacement: calculation
 of the displacement, in microns, of the longest kinocilium in x and y
 dimensions. Ciliary length: calculation of the displacement, in microns,
 of the longest kinocilium in x, y and z dimensions. #### File:
 Figure_2_data.zip **Description:** ## Overview This dataset contains image
 and analysis files related to neuromast imaging and hair cell
 quantification. \## File Types \- **.czi files:** These are the **raw
 image files** acquired directly from the microscope. \- **.tiff / .mp4
 files:** These are **converted versions** of the raw data, exported for
 ease of viewing and compatibility with analysis software. \## Required
 Software To open and view the proprietary **.czi** files, use **ImageJ**
 or **Fiji** (which includes the Bio-Formats plugin). Both programs are
 freely available and widely used for microscopy data visualization and
 analysis. \## Data Conversion Raw **.czi** files were exported either as
 **TIFF** or **MP4** formats using standard export functions within the
 imaging software. No additional processing or filtering was applied during
 conversion. \## Imaging Notes Not all neuromasts were imaged for every
 embryo due to time constraints during acquisition. However, all imaged
 neuromasts are **representative of the overall population** and were
 selected to ensure consistent sampling across embryos. \## Quantification
 Data The accompanying Excel file includes **raw hair cell counts**,
 recorded manually while observing each embryo under the microscope. These
 values serve as the primary quantitative dataset corresponding to the
 imaging files. \## Definition of Excel Column Header Variables Name:
 larvae were identified by SC KD or Smc3 KD, indicating whether they had
 received a standard control (SC) or Smc3 (Smc3) morpholino injection at
 the one cell stage and a number to differentiate the sample from others of
 the same injection type. MI1: number of hair cells observed in the middle
 1 neuromast. MI2: number of hair cells observed in the middle 2 neuromast.
 O2: number of hair cells observed in the otic 2 neuromast. M2: number of
 hair cells observed in the mandibular 2 neuromast. IO4: number of hair
 cells observed in the infraorbital 4 neuromast. Average: a calculation of
 the average number of hair cells observed across the 5 evaluated
 neuromasts for each larva. #### File: Figure_3_Data.zip
 **Description:** The dataset includes .tiff images of 3dpf embryos from
 which neuromasts were counted following control treatment or neomycin
 treatment. For some embryos, a series of images at different microscope
 focuses were captured in an effort to visualize all observe neuromasts,
 indicated by the decimal series in the file name (5 dpf scmo control
 15.1.tif and 5 dpf scmo control 15.2.tif are unique images of the same
 embryo). The .csv files indicate the number of observed neuromasts in each
 embryo. The embryo name in the .csv file corresponds to the number of the
 embryo indicated by the .tif file names. Variables within each .csv file
 are defined as described below:  Name: corresponds to the name of the file
 from the dataset analyzed in that row. Observed neuromasts: corresponds to
 the number of neuromasts observed in that sample number. #### File:
 Figure_4_Data.zip **Description:** The dataset includes maximum projection
 .tif files of neuromasts used for quantitative analysis, as indicated in
 the accompanying .csv files. Each .tif file name corresponds directly to
 the identifiers used in the .csv files. The .csv files contain
 measurements of the mean intensity of an ROI drawn around the neuromasts,
 the mean intensity of the background of each image, and the ratio of ROI
 to background mean intensity. Outlier ROI/background ratios were excluded
 from the analysis if they fell above or below the interquartile range.
 Inclusion or exclusion of each ROI/background value from analysis is
 indicated in the .csv files. Excluding outliers did not change the results
 of statistical analysis. Variables within each .csv file are defined as
 described below:  Name: corresponds to the name of the file from the
 dataset analyzed in that row. ROI mean intensity: average intensity of all
 pixels falling inside the region of interest drawn in ImageJ around the
 cells comprising the neuromast. Background mean intensity: average
 intensity of all pixels falling inside the background region drawn in
 imageJ in the background of each image. ROI/background: ratio of the “ROI
 mean intensity” value divided by the “Background mean intensity” value.
 Excluded from analysis due to falling outside of the IQR?: indicates
 whether the value was included in final analysis for the construction of
 Figure 4 in the publication based on the value falling outside of the
 interquartile range. Exclusion of these values did not change the results
 of analysis. #### File: Figure_S1_data.zip **Description:** The dataset
 includes lanes that were used for quantitative analysis, as indicated in
 the accompanying Excel file. Each lane label corresponds directly to the
 identifiers used in the Excel sheet. The Excel file contains measurements
 of pigment intensity, along with normalized values relative to the
 designated right-hand control sample. Certain lanes were excluded from the
 analysis due to quality control considerations. Common reasons for
 exclusion include insufficient initial protein quantity, evidence of
 protein degradation, or other technical issues affecting data reliability.
 \## Definition of Excel Column Header Variables Dpf: days post
 fertilization Name: indicates the identifier used for each sample,
 identified by SC KD or Smc3 KD, indicating whether they had received a
 standard control (SC) or Smc3 (Smc3) morpholino injection at the one cell
 stage and a number to differentiate the sample from others of the same
 injection type. Smc3: intensity value for the Smc3 protein band measured
 using ImageJ normalized to the background Tubulin: intensity value for the
 Tubulin protein band measured using ImageJ normalized to the background
 Normalized value: Normalized value of Smc3 intensity to tubulin intensity
 within the same sample Average % reduction: Normalized Smc3 value of
 standard control​​ − Normalized Smc3 value of in Smc3 KD/Normalized Smc3
 value of standard control×100 Normalized to SC: Value of Smc3 intensity of
 all samples normalized to SC samples #### File: Figure_S3_Data.zip
 **Description:** The dataset includes .tiff images of 3dpf embryos from
 which somites were counted. A series of images at different microscope
 focuses were captured of each embryo, indicated by the lettering series in
 the file name (ex. 72hpf_SCKD_1-A.tif and 72hpf_SCKD_1-B.tif are unique
 images of the same embryo). The .csv files indicate the number of somites
 observed in each embryo. The embryo name corresponds to the number of the
 embryo indicated by the .tif file names. Variables within each .csv file
 are defined as described below:  Name: corresponds to the name of the file
 from the dataset analyzed in that row. Somites: number of somites observed
 for that sample. ## Code/software To open and view the proprietary
 **.czi** files, use **ImageJ** or **Fiji** (which includes the Bio-Formats
 plugin). Both programs are freely available and widely used for microscopy
 data visualization and analysis.