Whole-body single-cell atlas of an adult vertebrate in homeostasis and regeneration

A complete cell-type transcriptome atlas of a vertebrate could promote understanding of animal cell-type composition, organization, and evolution. The miniaturized, transparent, and regenerative teleost Danionella cerebrum brings whole-vertebrate single-cell profiling experiments within reach. We performed region-stratified single-cell RNA sequencing across the adult Danionella body and mapped cell types and gene expression at single-cell resolution using spatial transcriptomics. We delineated spatially-distinct neural cell types based on their regional gene expression signatures. The body-wide atlas elucidated cell types harboring adult positional information, uncovered paedomorphic features, and revealed conserved body-region and appendage-specification programs in adult connective tissue. Comparative analyses revealed conserved neural cell types, and regeneration datasets uncovered expression dynamics during telencephalon regeneration. This whole-vertebrate transcriptome atlas yields a comprehensive resource for myriad questions in biology and neuroscience. bioRxiv preprint: https://www.biorxiv.org/content/10.64898/2026.02.03.703562v1 File Description readme.xlsx This table describing all Zenodo files. merfish.tar.zst Compressed MERFISH data for whole body sections, sagittal brain sections, and coronal brain sections. Includes cell_boundaries.parquet, cell_by_gene.csv, cell_metadata.csv, detected_transcripts.csv. Sagittal brain sections also include explorer and zarr directories from cellpose/baysor. Danionella_Bolton.v2.gff3.zip Compressed GFF3 used for mapping sequencing data and SAMap analyses. DE Markers: pseudobulk_deseq2_markers.xlsx Excel file with filtered markers from the DESeq2 pseudobulk analyses, filtered markers from Seurat FindAllMarkers, and the top 20 markers per cluster for both tests. Includes analyses for clusters from the internal organs subset and the CNS subset. regeneration_timecourse_DE_markers.xlsx Excel file with all DE markers (via Seurat's FindAllMarkers) for regeneration timecourse data. Seurat objects: The following are RDS files of Seurat objects: atlas.rds Seurat object of the entire atlas. cns.rds Seurat object of the CNS subset of the atlas. connective.rds Seurat object of the connective tissue subset of the atlas. cranium_and_body.rds Seurat object of the cranium and body subset of the atlas. epithelial.rds Seurat object of the epithelial subset of the atlas. eyes.rds Seurat object of the eye subset of the atlas. fibroblasts.rds Seurat object of the fibroblast subset of the atlas. fins_atlas.rds Seurat object of the fin subset of the atlas. gabaergic_neurons.rds Seurat object of the GABAergic neuron subset of the atlas. glutamatergic_neurons.rds Seurat object of the glutamatergic neuron subset of the atlas. internal_organs.rds Seurat object of the internal organ subset of the atlas. muscle.rds Seurat object of the muscle subset of the atlas. neural.rds Seurat object of the neural subset of the atlas. opc_oligo_schwann.rds Seurat object of the OPC, Oligodendrocyte, Schwann Cell subset of the atlas. other_neurons.rds Seurat object of the other neurotransmitter (non-glu/GABA) subset of the atlas. rg_ip_nbn.rds Seurat object of the Radial Glia and IP/NB subset of the atlas. scp_ncl.rds Seurat object of the Schwann Cell Precursor / Neural-crest-like subset of the atlas. regeneration_timecourse.rds Seurat object of the regeneration timecourse. fins_supplemental.rds Seurat object of additional fin sections sequenced separately from the atlas. samap_code.zip: Contains the following code for cross species analyses: format_zebrafish_genome.sh SLURM script to format zebrafish genome into an mRNA fasta file for SAMap. makeUniqueGeneNameGtf.py Script used for formatting gene names from zebrafish genome. snakemake_samap_mouse_danionella.zip Snakemake pipeline to run SAMap on mouse and Danionella brains. mouse_cds_agat.sh SLURM script to format mouse genome into a CDS fasta file for SAMap. danionella_cds_agat.sh SLURM script to format Danionella genome into a CDS fasta file for SAMap. snakemake_samap_zebrafish_danionella_telencephalon.zip Snakemake pipeline to run SAMap on zebrafish and Danionella telencephalons. cellbender_code.zip Contains the following code for CellBender: snakemake_cellbender.zip Snakemake pipeline to run CellBender for additional fin sections. python_cellbender_batch_fpr0.3_rerun.py Python script to create SLURM scripts to run CellBender in batch mode for samples used in the atlas and regeneration timecourse datasets. cellbender_parameters_fpr_0.3_corrected.csv CSV used to specify parameters for Python CellBender batch script. r_code.zip Contains the following R code: alluvial_fins.R Create alluvial plots illustrating cells filtered at each step of QC filtering and save formatted table used to generate plots (for supplemental data of additional fin sections). alluvial.R Create alluvial plots illustrating cells filtered at each step of QC filtering and save formatted table used to generate plots (for atlas and regeneration timecourse). clustree_atlas_headless.R Headless script to run on HPC cluster and perform clustering of the atlas via Seurat's FindClusters. clustree_atlas_sbatch_helper.R Helper script to create batch jobs of Seurat's FindClusters at various resolutions for the atlas. Resolution are split into separate jobs to reduce overall run time. Execute via Rscript command on HPC cluster. clustree_combine_atlas_resolutions.R Create a new Seurat object for the atlas that combines separate objects with various clustering resolutions. clustree_headless.R Headless script to run on HPC cluster and perform clustree analyses. clustree_sbatch_helper.R Helper script to create batch jobs of headless clustree analyses. Execute via Rscript command on HPC cluster. colors.R Sourced by other scripts to provide color mappings for the atlas, regeneration timecourse, and supplemental fins datasets. danionella_paedomorphy_analysis.R Danionella paedomorphy three-way developmental comparison. Compares the paedomorphic Danionella atlas to adult zebrafish and ZMAP larval zebrafish atlas. export_deseq2_functions.R Sourced by other scripts to provide functions for running pseudobulk analyses via DESeq2. export_deseq2_headless.R Headless script to run on HPC cluster and perform pseudobulk analyses via DESeq2. export_deseq2_sbatch_helper.R Helper script to create batch jobs of headless DESeq2 runs. Execute via Rscript command on HPC cluster. export_deseq2_table.R Create an Excel file with tables containing per sample QC metrics for datasets. export_qc_tables.R Create an excel file with QC tables for data processing. it_export_dotplot_data.R Read in every dataset and corresponding markers to export CSVs for website's dotplot data. it_export_findAllMarkers.R Run and save results of Seurat's FindAllMarkers on every dataset to use for website's dotplot data. it_export_functions.R Sourced by other scripts to provide functions for exporting plots and data for our website. it_export_umap_headless.R Headless script to run on HPC cluster and create UMAP plots for our website. it_export_umap_sbatch_helper.R Helper script to create batch jobs of headless UMAP plot exports. Execute via Rscript command on HPC cluster. mito_ribo_function.R Sourced by other scripts to provide a function to assign percent.mt and percent.ribo via PercentageFeatureSet to Seurat objects. monocle_pseudotime_bin_markers.R Create an Excel file with tables containing top genes per pseudotime bin for each pseudotime object. monocle_Tel_Dien_Rhomb.R Run Monocle 3 pseudotime seprately on telencephalon, diencephalon, and rhombencephalon. monocle_Tel_Regen.R Run Monocle 3 pseudotime on regenerating telencephalon. monocle.R Source by other scripts to provide functions for running Monocle 3 pseudotime analyses and plotting results. plot_qc_violin.R Plots nFeature_RNA, nCount_RNA, percent.mt, and percent.ribo for datasets. process_atlas.R Process the entire atlas via Seurat. process_functions.R Provides functions for processing single cell sequencing data with Seurat. process_supplemental_fins.R Process the supplemental data for additional fin sections via Seurat. process_timecourse.R Process the regeneration timecourse via Seurat. region_vs_nt_id_code.R Region vs Neurotransmitter identity in Danionella CNS neurons. rename_features.R Provides a function to rename features (genes) for a Seurat object. The Danionella cerebrum genome labeled transcripts as DT#####, per reviewer suggestions we have renamed all features to DC##### to reflect the name of the species. samap_mouse_assortativity.R Calculate assortativity scores based on either neural class or brain region for mouse and Danionella SAMap results. samap_mouse_plots.R Create plots for mouse and Danionella SAMap results. samap_plots_zebrafish.R Create plots for zebrafish and Danionella SAMap results. samap_zebrafish_anneser_e_i_pairings.R Calculate pairings between neural class+cluster labels of all cells (Fig. 2A, Anneser et al., 2024) and excitatory/inhibitory labels of subclustered neuronal cells (Fig. 2C, Anneser et al., 2024) for the Anneser dataset. samap_zebrafish_export_danionella_subset.R To obtain input for SAMap, subset our atlas to just telencephalon samples and subsample each cluster proportionally to the same number of cells present in the zebrafish telencephalon dataset (Anneser et al., 2024). saveTables.R Sourced by other scripts to provide a function to easily create a multi-sheet Excel file. single_cell_sex_prediction_code.R Single-cell sex prediction for whole-body Danionella atlas. timecourse_export_markers_and_venn_diagrams.R Plots venn diagrams for marker genes at each timepoint in our regeneration timecourse dataset and exports an Excel file with all marker tables.