DOI: 10.5061/dryad.b2rbnzsqm
title: Data from: Marcks and Marcks-like 1 proteins promote spinal cord development and regeneration in Xenopus

description: Marcks and Marcksl1 are abundant proteins that shuttle between the
 cytoplasm and membrane to modulate multiple cellular processes, including
 cytoskeletal dynamics, proliferation, and secretion. Here, we performed
 loss- and gain-of-function experiments in Xenopus laevis to reveal the
 novel roles of these proteins in spinal cord development and regeneration.
 We show that Marcks and Marcksl1 have partly redundant functions and are
 required for normal neurite formation and proliferation of neuro-glial
 progenitors during embryonic spinal cord development and for its
 regeneration during tadpole stages. Rescue experiments in Marcks and
 Marcksl1 loss of function animals further suggested that some of the
 functions of Marcks and Marcksl1 in the spinal cord are mediated by
 phospholipid signaling. Taken together, these findings identify Marcks and
 Marcksl1 as critical new players in spinal cord development and
 regeneration and suggest new pathways to be targeted for therapeutic
 stimulation of spinal cord regeneration in human patients.
description: Images have been collected using fluorescent and confocal
 microscopy. Fluorescence intensity was measured using Image J and
 statistically analysed in GraphPad Prism. 
description: # Marcks and Marcks-like 1 proteins promote spinal cord development and
 regeneration in Xenopus
 [https://doi.org/10.5061/dryad.b2rbnzsqm](https://doi.org/10.5061/dryad.b2rbnzsqm) ## Description of the data and file structure In this dataset you will find folders for each **Main Figure** and corresponding **Supplementary Figure** in El Amri, M., Pandit, A., & Schlosser, G. (2024). This repository contains raw images, corresponding data points used for quantification and statistical analysis, Sanger sequencing data, and swimming distance values used to create the figures of the associated publication, with each file representing the data for one figure panel named accordingly with the publication. Unmerged immunohistochemical and in-situ hybridisation images were obtained by Fluorescent or Confocal microscopy (.jpg or .tiff). Cell counts or fluorescence intensity were quantified using Image J, averaged and plugged in GraphPad Prism for statistical analysis (.pzfx). Sequencing results (.abi) were analysed using Synthego ICE. Swimming distance values were obtained from EthoVision XT video tracking software (Noldus).  All data collection and analyses are fully described in the associated publication Materials and Methods section. All files in this Data Repository contain the underlying data used to generate the Figures in the associated publication and are named accordingly. ### Files and variables #### File: Fig\_2\_Panel\_C\_Statistical\_Analysis.pzfx **Description:** This data file contains the fluorescence values used to compare and assess neurite outgrowth from the spinal cord section images represented in Figure 2, Panels A and B (raw representative images are uploaded separately in this repository and are named accordingly). This file contains statistical analysis of the average Raw Integrated Density ratio for injected : uninjected spinal cord sides, measured using Image J and analysed in GraphPad Prism, as described in the associated publication Figure Legend and Materials and Methods section.  #### File: Fig\_2\_Panel\_G\_Statistical\_Analysis.pzfx **Description:** This data file contains the fluorescence values used to compare and assess neurite outgrowth from the spinal cord section images represented in Figure 2, Panels D, E, and F (raw representative images are uploaded separately in this repository and are named accordingly)**.** This file contains statistical analysis of the average Raw Integrated Density ratio for injected : uninjected spinal cord sides, measured using Image J and analysed in GraphPad Prism, as described in the associated publication Figure Legend and Materials and Methods section.  #### File: Fig\_2\_Panel\_K\_Statistical\_Analysis.pzfx **Description:** This data file contains the fluorescence values used to compare and assess neurite outgrowth from the spinal cord section images represented in Figure 2, Panels H, I, and J (raw representative images are uploaded separately in this repository and are named accordingly). This file contains statistical analysis of the average Raw Integrated Density ratio for injected : uninjected spinal cord sides, measured using Image J and analysed in GraphPad Prism, as described in the associated publication Figure Legend and Materials and Methods section.  #### File: Fig\_2\_Supp\_Fig\_7\_Panel\_D\_Statistical\_Analysis.pzfx **Description:** This data file contains the fluorescence values used to compare and assess neurite outgrowth from the spinal cord section images represented in Figure 2 - Supplementary Figure 7, Panels A, B, and C (raw representative images are uploaded separately in this repository and are named accordingly). This file contains statistical analysis of the average Raw Integrated Density ratio for injected : uninjected spinal cord sides, measured using Image J and analysed in GraphPad Prism, as described in the associated publication Figure Legend and Materials and Methods section.  #### File: Fig\_3\_Panel\_D\_Statistical\_Analysis.pzfx **Description:** This data file contains the % of PH3+ cells used to compare and assess mitotic activity from the spinal cord section images represented in Figure 3, Panels A, B, and C (raw representative images are uploaded separately in this repository and are named accordingly). This file contains statistical analysis of the % of PH3+ cells in total DAPI nuclei for injected : uninjected spinal cord sides, measured using Image J and analysed in GraphPad Prism, as described in the associated publication Figure Legend and Materials and Methods section.  #### File: Fig\_3\_Panel\_H\_Statistical\_Analysis.pzfx **Description:** This data file contains the % of PH3+ cells used to compare and assess mitotic activity from the spinal cord section images represented in Figure 3, Panels E, F, and G (raw representative images are uploaded separately in this repository and are named accordingly). This file contains statistical analysis of the % of PH3+ cells in total DAPI nuclei for injected : uninjected spinal cord sides, measured using Image J and analysed in GraphPad Prism, as described in the associated publication Figure Legend and Materials and Methods section.  #### File: Fig\_3\_Panel\_L\_Statistical\_Analysis.pzfx **Description:** This data file contains the % of Sox2+ cells used to compare and assess neuro-glial progenitor activity from the spinal cord section images represented in Figure 3, Panels I, J, and K (raw representative images are uploaded separately in this repository and are named accordingly). This file contains statistical analysis of the average number of Sox2+ cell ratio for injected : uninjected spinal cord sides, measured using Image J and analysed in GraphPad Prism, as described in the associated publication Figure Legend and Materials and Methods section.  #### File: Fig\_3\_Panel\_M\_Statistical\_Analysis.pzfx **Description:** This data file contains the % of Sox3+ cells used to compare and assess neuro-glial progenitor activity from the spinal cord section images obtained. Raw representative images used to generate this data are uploaded separately in this repository and are named accordingly. This file contains statistical analysis of the average number of Sox3+ cell ratio for injected : uninjected spinal cord sides, measured using Image J and analysed in GraphPad Prism, as described in the associated publication Figure Legend and Materials and Methods section.  #### File: Fig\_3\_Panel\_N\_O\_Statistical\_Analysis.pzfx **Description:** This data file contains the ratio of total number of DAPI cells in injected : uninjected sides to compare the total number of cells in each side. These datasets represent statistical inputs and analysis of the total DAPI+ cell counts under different conditions (e.g. Morpholino, CRISPR treatment), measured using Image J and analysed in GraphPad Prism as described in the associated publication Figure Legend and Materials and Methods section.  #### File: Fig\_3\_Supp\_Fig\_1\_Panel\_D\_Statistical\_Analysis.pzfx **Description:** This data file contains the % of PH3+ cells used to compare and assess mitotic activity from the spinal cord section images represented in Figure 3 - Supplementary Figure 1, Panels A, B, and C (raw representative images are uploaded separately in this repository and are named accordingly). This file contains statistical analysis of the % of PH3+ cells in total DAPI nuclei for injected : uninjected spinal cord sides, which were measured using Image J and analysed in GraphPad Prism are described in the associated publication Figure Legend and Materials and Methods section. #### File: Fig\_4\_Acetylated\_Tubulin\_Statistical\_Analysis.pzfx **Description:**  This data file contains the fluorescence values used to compare and assess neurite outgrowth from the spinal cord sections after CRISPR and pharmacological treatment, with the chemical names listed accordingly as per Figure 4 - Graphs B, D, F, and H. Raw representative images used for quantification are uploaded separately in this repository and are named accordingly. This file contains the statistical analysis of the average Raw Integrated Density ratio for injected : uninjected spinal cord sides, measured using Image J and analysed in GraphPad Prism, as described in the associated publication Figure Legend and Materials and Methods section. #### File: Fig\_4\_PH3\_Statistical\_Analysis.pzfx **Description**: This data file contains the % of PH3+ cells used to compare and assess mitotic activity from spinal cord sections after CRISPR and pharmacological treatment, with the chemical names listed accordingly as per Figure 4 - Graphs C, E, G, and I. Raw representative images used for quantification are uploaded separately in this repository and are named accordingly. This file contains the statistical analysis of the % of PH3+ cells in total DAPI nuclei for injected : uninjected spinal cord sides, measured using Image J and analysed in GraphPad Prism, as described in the associated publication Figure Legend and Materials and Methods section. #### File: Fig\_4\_Supp\_Fig\_1\_Acetylated\_Tubulin\_Statistical\_Analysis.pzfx **Description:** This data file contains the fluorescence values used to compare and assess neurite outgrowth from the spinal cord sections after CRISPR and pharmacological treatment, with the chemical names listed accordingly as per Figure 4 - Supplementary Figure 1, Graphs A, B, E, F, I, J, M, and N. Raw representative images used for quantification are uploaded separately in this repository and are named accordingly. This file contains the statistical analysis of the average Raw Integrated Density for injected *and* uninjected spinal cord sides listed separately. Values were measured using Image J and analysed in GraphPad Prism as described in the associated publication Figure Legend and Materials and Methods section. #### File: Fig\_4\_Supp\_Fig\_1\_PH3\_Statistical\_Analysis.pzfx **Description:** This data file contains the % of PH3+ cells used to compare and assess mitotic activity from spinal cord sections after CRISPR and pharmacological treatment, with the chemical names listed accordingly as per Figure 4 - Supplementary Figure 1, Graphs C, D, G, H, K, L, O, and P. Raw representative images used for quantification are uploaded separately in this repository and are named accordingly. This file contains the statistical analysis of the % of PH3+ cells in total DAPI nuclei for injected *and* uninjected spinal cord sides listed separately. Values were measured using Image J and analysed in GraphPad Prism as described in the associated publication Figure Legend and Materials and Methods section. #### File: Fig\_5\_Panel\_V\_Statistical\_Analysis.pzfx **Description:** This data file contains the fluorescence values of MARCKS and MARCKSL1 antibody signals in CRISPR-injected tadpoles at different timepoints after spinal cord injury. Data values for the average Raw Integrated Density of MARCKS/MARCKSL1 antibody signal in spinal cord tissues were obtained using Image J and statistically analysed in GraphPad Prism, as described in the associated publication Figure Legend and Materials and Methods section. Raw representative images used for quantification are uploaded separately in this repository and are named accordingly. #### File: Fig\_6\_Swimming\_Distance.pzfx **Description:** This data file contains the total swimming distance of MARCKS/MARCKSL1 CRISPR-injected and uninjected tadpoles before spinal cord injury and at different recovery timepoints, represented in Figure 6 - Graph A. Data values for the total swimming distance were obtained from EthoVision XT video tracking software (Noldus) and statistically analysed in GraphPad Prism, as described in the associated publication Figure Legend and Materials and Methods section. #### File: Fig\_6\_Panel\_J\_Gap\_Length.pzfx **Description:** This data file contains the spinal cord injury gap length of MARCKS/MARCKSL1 CRISPR-injected and uninjected tadpoles at different recovery timepoints, represented in Figure 6 - Graph J. Data values for the injury gap length were measured using Image J and statistically analysed in GraphPad Prism, as described in the associated publication Figure Legend and Materials and Methods section. Raw representative images used for quantification are uploaded separately in this repository and are named accordingly. #### File: Fig\_7\_Panel\_I\_EdU\_Cell\_Counts\_Statistical\_Analysis.pzfx **Description:** This data file contains the % of EdU+ cells used to compare and assess cell proliferation in CRISPR-injected and uninjected tadpoles *during the same timepoints*, represented in Figure 7, Graph I.  This file contains the statistical analysis of the % of EdU+ cells in total DAPI nuclei in spinal cord tissues measured using Image J and analysed in GraphPad Prism, as described in the associated publication Figure Legend and Materials and Methods section. Raw representative images used for quantification are uploaded separately in this repository and are named accordingly. #### File: Fig\_7\_Supp\_Fig\_1\_Panel\_I\_EdU\_Cell\_Counts\_Statistical\_Analysis.pzfx **Description:** Similar to the previous file, this data file contains the % of EdU+ cells used to compare and assess cell proliferation in CRISPR-injected and uninjected tadpole timepoints *separately*, represented in Figure 7 - Supplementary Figure 1. This file contains the statistical analysis of the % of EdU+ cells in total DAPI nuclei in spinal cord tissues measured using Image J and analysed in GraphPad Prism, as described in the associated publication Figure Legend and Materials and Methods section. Raw representative images used for quantification are uploaded separately in this repository and are named accordingly. #### File: Fig\_8\_Panel\_I\_Sox2\_Cell\_Counts\_Statistical\_Analysis.pzfx **Description:** This data file contains the % of Sox2+ cells used to compare and assess neuro-glial progenitor activity in CRISPR-injected and uninjected tadpoles during the same timepoints, represented in Figure 8, Graph I.  This file contains the statistical analysis of the % of Sox2+ cells in total DAPI nuclei in spinal cord tissues measured using Image J and analysed in GraphPad Prism, as described in the associated publication Figure Legend and Materials and Methods section. Raw representative images used for quantification are uploaded separately in this repository and are named accordingly. #### File: Fig\_8\_Supp\_Fig\_1\_\_Sox2\_Cell\_Counts\_Statistical\_Analysis.pzfx **Description:** Similar to the previous file, this data file contains the % of Sox2+ cells used to compare and assess cell proliferation in CRISPR-injected and uninjected tadpole timepoints *separately*, represented in Figure 8 - Supplementary Figure 1.  This file contains the statistical analysis of the % of Sox2+ cells in total DAPI nuclei in spinal cord tissues measured using Image J and analysed in GraphPad Prism, as described in the associated publication Figure Legend and Materials and Methods section. Raw representative images used for quantification are uploaded separately in this repository and are named accordingly. #### File: Figures\_7\_and\_8\_Supplementary\_Figures\_EdU\_Sox2\_Sham\_Surgery.pzfx **Description:** This data file contains the % of EdU+ and Sox2+ cells used to compare and assess cell proliferation and neuro-glial progenitor cell activity, respectively, in sham-operated tadpoles at different timepoints after injury, represented in Figures 7 & 8 - Supplementary Figure 2. This file contains the statistical analysis of the % of EdU+ and Sox2+ cells in total DAPI nuclei in spinal cord tissues measured using Image J and analysed in GraphPad Prism, as described in the associated publication Figure Legend and Materials and Methods section. Raw representative images used for quantification are uploaded separately in this repository and are named accordingly. #### File: Fig\_9\_Panels\_P\_to\_S\_Statistical\_Analysis.pzfx **Description:** This data file contains statistical analysis of total swimming distance, injured spinal cord gap length, % of Sox2+ cells and % of EdU+ cells in spinal cord sections from CRISPR-injected tadpoles treated with phospholipid-mediating pharmacological agents. Swimming data points were measured using EthoVision XT video tracking software (Noldus) and analysed using GraphPad. Gap length, DAPI, Sox2, and EdU signal were measured using Image J and analysed in GraphPad, as described in the associated publication Figure Legend and Materials and Methods section. Raw representative images used for quantification are uploaded separately in this repository and are named accordingly. ## Code/software Synthego Inference for CRISPR Edits (ICE) is a free online tool that provides a quantitative assessment of genome editing.  *EthoVision* XT is a video tracking software that can be used for rodents, fish, insects and other animals that can be used for behavioural assessment.