DOI: 10.5061/dryad.j3tx95xtz
title: Data and code from: Evidence for chronological diversification of spinal neuron subtypes by a shared sequence of transcription factors

description: The mechanisms underlying the generation of the immense diversity of
 neuronal cell types remain a fundamental question of developmental
 biology. In the spinal cord, different “cardinal classes” of neurons that
 share a common molecular identity are produced from spatially segregated
 progenitor domains. Within many such classes, a stereotyped sequence of
 divergent neuronal types of related function is generated over time,
 raising the question of the molecular mechanisms that control this
 process. Here, we show that the successive expression of mouse
 transcription factors Onecut2, Pou2f2, and Pou3f1 within the cardinal
 classes giving rise to motor and sensory circuits, correlates with the
 emergence of sequentially generated subpopulations of neurons within those
 domains. We demonstrate that the genetic loss of Pou2f2 results in
 impaired development of two early-born motor neuron columns and
 re-specification of anterolateral system projection neurons as a
 later-born subset. Similarly, we show that Pou3f1 expression is required
 for the normal development of later-born subsets of motor neurons and
 anterolateral system projection neurons. Together, our observations
 provide functional evidence that horologic diversification of output
 neurons of spinal motor and sensory circuits are driven by a conserved
 sequential order of expression of transcription factors.
description: # Data and code from: Evidence for chronological diversification of spinal
 neuron subtypes by a shared sequence of transcription factors Dataset DOI:
 [10.5061/dryad.j3tx95xtz](https://doi.org/10.5061/dryad.j3tx95xtz) ##
 Description of the data and file structure This repository contains all
 data and the R code to reproduce the plots in Figures 1-7 and S1-S9 + S15
 from Caudet Segarra et al. 2025. ### Files and variables #### Data files
 hosted by Dryad * Input_data_Caudet_Segarra_et_al.zip #### Software files
 hosted by [Zenodo](https://zenodo.org) *
 Supplemental_Experimental_Procedures_-_Imaging_Revision.Rmd *
 Supplemental_Experimental_Procedures___Revision_scRNAseq.Rmd ###
 Supplemental files hosted by [Zenodo](https://zenodo.org) *
 Output_Caudet_Segarra_et_al.zip **Description:** Please unpack
 Input_data_Caudet_Segarra_et_al.zip file and download the R scripts
 Supplemental_Experimental_Procedures_-_Imaging_Revision.Rmd and
 Supplemental_Experimental_Procedures___Revision_scRNAseq.Rmd from Zenodo
 and put them in the same directory as the input data. There should be 2 R
 Markdown files (called Supplemental Experimental Procdures -
 Imaging_Revision.Rmd and Supplemental Experimental Procedures - Revision
 scRNAseq.Rmd), 2 Input folders (Input and Input_scRNAseq) and 2 Output
 folders (Output and Output_scRNAseq). Running the scripts in the R
 Markdown files should generate all the data shown in the manuscript.
 Output data was also uploaded as Supplemental files to Zenodo. #####
 Quantitative image analysis The Input folder contains all data files
 underlying quantitative image analysis performed in Figures 1-7 and S1-S9
 and S15 organized by figures. The code provided in Supplemental
 Experimental Procdures - Imaging_Revision.Rmd should load these files and
 generate all plots provided in the main text and supplemental figures and
 save them to the output folders. Data in the input folder is organized by
 figures. Subfolders in each figure folder are organized by stainings.
 Subfolder names indicate the staining date, genotype + stage, and the
 order of channels during image acquisition. Identity of the fluorophores
 used for fluorescent detection is also indicated in most cases (e.g.
 647Foxp1 indicates that Foxp1 was detected with an Alexa647-conjugated
 secondary antibody). Staining folders in figure folders
 'Figure1+S1', 'Figure2', 'Figure3+4',
 'Figure5,6,7', 'Figure7', 'FigureS2',
 'FigureS4', 'FigureS8' and 'FigureS15'
 contain .csv files containing the data underlying the quantititive image
 analysis. Unless specified otherwise, .csv files contain the following
 columns: * Area: Area of the segmented nucleus in Pixels * Mean: Mean
 fluorescence intensity of the nucleus * XM: X coordinate for centre of
 mass of the nucleus * YM: Y coordinate for centre of mass of the nucleus *
 Ch: Imaging Channel Some .csv files contain additionally the columns: *
 Min: Minimal pixel intensity detected per nucleus (not used for further
 analysis) * Max: Maximal pixel intensity detected per nucleus (not used
 for further analysis) Intensity profile plots in Figures 5E and S8E were
 generated based on .csv files provided in folders
 'Figures5,6,S7/240612_SC_e10.5_405Pou2f2_488Lhx3_568Isl1-2_647Foxp1_slide1401' and 'FigureS8/250717_SC_Nes-Cre_R26-YFP_e10.5_450Pou2f2_488GFP_568Isl1-2_647OC2_slide2429B' and 'FigureS8/250717_SC_Nes-Cre_R26-YFP_e10.5_450Pou2f2_488GFP_568Isl1-2_647Cre_slide2429A'. On each section, independent line profiles for the left and right hemisection were generated using Fiji's 'PlotProfile' function of a 200 µm wide line. .csv files containing data underlying intensity profile plots contain the following columns: * Distance: Distance along the profiles, starting at the ventricular zone (= data in columns X1, X2, X3, X4, X5, X6, X7) * Value: Intensity value of Channel1 along profile 1 * Y1: Intensity value of Channel2 along profile 1 * Y2: Intensity value of Channel3 along profile 1 * Y3: Intensity value of Channel4 along profile 1 * Y4: Intensity value of Channel1 along profile 2 * Y5: Intensity value of Channel2 along profile 2 * Y6: Intensity value of Channel3 along profile 2 * Y7: Intensity value of Channel4 along profile 2 Dynamics of Foxp1 and Mecom positive MNs are provided as Excel sheet in 'Figures5,6,S7/S7F_Foxp1_Mecom_timecourse.xlsx'. Sheets within the Excel file contain data from the different developmental stages (e9.5, e10.5, e11.5, e12.5). The Excel file contains the following columns: * % Foxp1: Percentage of motor neurons expressing Foxp1 * % Mecom: Percentage of motor neurons expressing Mecom Cell counts of Isl1 Mecom+ MMC neurons expressing Pou2f2 and Pou3f1 (Figure 5K) are provided in the folder 'Figures5,6,S7/230510_SC_e11.5_405Pou2f2_488Pou3f1_568Mecom_647Isl1_slide734B' as .csv file. The .csv file contains the following columns: * embryo : ID of the quantified embryo * Isl1 Mecom+: Total number of Isl1 Mecom+ MMC neurons * Pou3f1+: Number of Pou3f1-positive MMC neurons * Pou2f2+: Number of Pou2f2-positive MMC neurons Fraction of motor neurons in thoracic motor columns expressing Pou2f2 and Pou3f1 is provided as .csv file in 'Figure5,6,S7/221012_SC_e11.5_405Pou2f2_488Isl1-2_568Pou3f1_647Foxp1/Quantifications_Pou2f2_Pou3f1_thoracic_MNs.csv' The .csv file contains the following columns: * PGC_Pou2f2: Fraction of PGC neurons expressing Pou2f2 * PGC_Pou3f1: Fraction of PGC neurons expressing Pou3f1 * HMC_Pou2f2: Fraction of HMC neurons expressing Pou2f2 * HMC_Pou3f1: Fraction of HMC neurons expressing Pou3f1 * MMC_Pou2f2: Fraction of MMC neurons expressing Pou2f2 * MMC_Pou3f1: Fraction of MMC neurons expressing Pou3f1 Data underlying the quantification of fraction of nuclei labelled by EdU administration at e9.5 or e10.5 expressing Onecut2, Pou2f2 and Pou3f1 are provided in Excel files in 'FigureS4/IF168_25013_e13.5_SC_488Zfhx3_Cy3Pou2f2_647EdU'. Each sheets in the Excel file correspond to data from one embryo section. The sheets contain the following columns: * Area: Area of the segmented nucleus in Pixels * Mean: Mean fluorescence intensity of the nucleus * Min: Minimal pixel intensity detected per nucleus (not used for further analysis) * Max: Maximal pixel intensity detected per nucleus (not used for further analysis) * Ch: Imaging Channel Some sheets contain additionally the columns: * XM: X coordinate for centre of mass of the nucleus (not used for further analysis) * YM: Y coordinate for centre of mass of the nucleus (not used for further analysis) Outlines of spinal cord sections used to quantify the mediolateral distribution of Onecut2+, Pou2f2+ and double-negative Phox2a neurons in Figure 7 are provided as .tif in the folder 'Figure7/IF115_e12.5_Pou2f2_Lmx1b_Phox2a_OC2'. Each .tif is in a subfolder, whose name matches the .csv files provided in 'Figure7/IF115_e12.5_Pou2f2_Lmx1b_Phox2a_OC2'. ##### Single-cell RNA sequencing To reproduce the scRNAseq analysis, first the object m_neural.rds needs to be generated as specified in Delile et al. 2019, Development, and [https://github.com/juliendelile/MouseSpinalCordAtlas](https://github.com/juliendelile/MouseSpinalCordAtlas). For analysis of scRNAseq from Wang et al. 2022, Nat. Comm., please download the data from the GEO repository (GSM4734731) and put it into the Input_scRNAseq folder. For data from Bell et al. 2024, PNAS, please download GSE240528FinalObject.Robj, provided via the GEO repository (GSE240528) and put this object into the the Input_scRNAseq folder. ## Code/software Data was analyzed using R 4.1.2 using R Studio version 2025.09.0+387 (2025.09.0+387). For some parts of the code Python version 3.8.15 was loaded via the R package reticulate. A complete list of the utilized R packages/libraries is provided at the end of the PDFs generated from the provided R Markdown documents. ## Access information Other publicly accessible locations of the data: * Input data for the scRNAseq analysis is partially available from the GEO repository (GSM4734731, GSE240528) Data was derived from the following sources: * The object m_neural.rds needs to be generated as specified in Delile et al. 2019, Development, and [https://github.com/juliendelile/MouseSpinalCordAtlas](https://github.com/juliendelile/MouseSpinalCordAtlas).