Dataset DOI: 10.5061/dryad.kprr4xhmc

Description of the data and file structure

Dataset is uploaded as a zip file “Archive.zip”.

This dataset includes data collected to assemble the figures and figure supplements in Shen et al. "Zebrafish knock-in lines enabling live visualization of extracellular matrix dynamics during development and regeneration". Data include raw and processed images from confocal microscopy, and stereofluorescence microscopy; Excel and CSV spreadsheets with measurements; prism files for statistical tests and graphs; and genome sequencing data files. 

A Graphpad prism file “Shen et al. Prism analyses” is included that includes all statistical analysis for this study. Analyses are individually labeled within the Prism file.

README for Figure 1

Figure 1 presents a C-terminal tagging strategy for generating fluorescent knock-in alleles of extracellular matrix (ECM) genes in zebrafish. Guide RNAs (gRNAs) were designed to target intronic regions near the terminal coding exons of lamc1 and col1a2, while the tgfbi gRNA target site was located in the fifth intron. A flexible linker sequence (GGSGGTGGSGGT) was inserted between the endogenous protein and a fluorescent reporter to create fusion proteins, with donor DNA constructs indicated by pink dashed boxes and stop codons represented by red hexagons. Representative images show heterozygous knock-in larvae expressing lamc1-eGFP (enhanced green fluorescent protein), col1a2-mScarlet, and tgfbi-mScarlet fluorescent reporters. These larvae were imaged at 5 dpf (days post-fertilization), demonstrating successful expression and localization of the tagged ECM proteins. Additional images from lateral, dorsal, and ventral perspectives provide a comprehensive view of reporter distribution throughout the organism. All images include scale bars of 500 µm (micrometers), highlighting tissue-level patterns of ECM protein expression in developing larvae.

Description: Folders are subdivided into Figure panels. Image files in folders Fig1B and 1C include raw and processed images of larvae, labeled by strain and magnification.

Fig1A folder contains a schematic in PPT and PDF formats.

Fig1B folder contains 3 processed JPEG images chosen for the figure panel. It also contains a subfolder “Raw images” with images containing a strain name, animal number, magnification, animal view, and image number. These images are in .czi format and can be opened using Zeiss imaging software. A subfolder “Processed images” with subfolders containing a strain name, animal number, magnification, animal view, and image number. These images are JPEGs and can be opened by freeware.

Fig1C contains 8 processed JPEG images chosen for the figure panel. It also contains a subfolder “Raw images” with images containing a strain name, animal number, magnification, animal view, and image number. These images are czi format and can be opened using Zeiss imaging software. A subfolder “Processed images” with subfolders containing a strain name, animal number, magnification, animal view, and image number. These images are JPEGs and can be opened by freeware.

README for Figure 2

Figure 2 investigates the dynamic behavior of extracellular matrix (ECM) proteins during zebrafish embryogenesis using FRAP (fluorescence recovery after photobleaching) analysis. Confocal images of Lamc1-eGFP (enhanced green fluorescent protein), Col1a2-mScarlet, and Tgfbi-mScarlet were collected in larvae at 5 dpf (days post-fertilization), and recovery half-lives were measured in the fin fold and myoseptum, although Tgfbi-mScarlet was not detectable in the fin fold. FRAP experiments quantified fluorescence recovery after photobleaching, with bleached regions marked by dashed boxes and recovery tracked over time in living animals. The results demonstrated differences in matrix protein turnover and mobility between tissues and among ECM components, with quantified recovery curves shown for each reporter. To test the role of laminin during matrix maintenance, lamb1a^pd110 mutant embryos carrying the lamc1-eGFP reporter were raised at a permissive temperature of 28.5°C until 5 dpf and then shifted to a restrictive temperature of 33°C before analysis. Representative images and body-length measurements were obtained at 6 dpf, revealing phenotypic differences between heterozygous and mutant larvae. Additional FRAP experiments and fluorescence quantification of myosepta assessed how loss of laminin affected ECM stability and remodeling, with statistical comparisons performed using Student’s t-test. Scale bars represent 50 µm (micrometers) in panel A, 100 µm in panels C, E, G, and H, and 500 µm in panel J.

Description: Folders are subdivided into Figure panels. Files include raw and processed images, organized by timepoints and animal identifier; spreadsheets with measurements; and statistical tests.

Fig2A folder contains processed JPEG images used for figure. A subfolder “More images” contains a number of raw image files (czi format and can be opened using Zeiss imaging software), as well as processed JPEG image files for all of these raw files.

Fig2B folder contains a spreadsheet with FRAP half-life data in Excel format, with 5 tabs labeled by knock-in genotype and tissue; Excel sheets are also provided in CSV format. Values in the files represent arbitrary fluorescence units and percentage fluorescence recovered, as well as calculated half life data in hours.

Fig2C folder contains a folder “Raw images” with subfolders separated by Animal numbers. These subfolders contain raw .czi files and subfolders with processed JPEG images for these raw files. There are also 4 processed images used in the figure (3 images in PNG format, one in JPEG format).

Fig2D folder contains fluorescence measurements expressed as a relative fluorescence ratio with respect to pre-bleaching values in Excel and CSV formats.

Fig2E folder contains a folder “Raw images” with subfolders separated by Animal numbers. These subfolders contain raw .czi files and subfolders with processed JPEG images for these raw files. There are also 4 processed images used in the figure (all PNG format).

Fig2F folder contains fluorescence measurements expressed as a relative fluorescence ratio with respect to pre-bleaching values in Excel and CSV formats.

Fig2G folder contains a folder “Raw images” with subfolders separated by Animal numbers. These subfolders contain raw .czi files to be opened using Zeiss software. A folder “Processed images” contains processed JPEG files corresponding to these raw images. These are also 3 JPEG images used for the figure, and an Excel and CSV spreadsheet (contents are identical) with fluorescence measurements.

Fig2H folder contains a folder “Raw images” with subfolders separated by Animal numbers. These subfolders contain raw .czi files to be opened using Zeiss software. A folder “Processed images” contains processed JPEG files corresponding to these raw images. These are also 3 JPEG images used for the figure, and fluorescence measurements in Excel and CSV format.

Fig2I folder contains a schematic in PPT and PDF formats.

Fig2J folder contains a folder “Raw images” containing subfolders representing either heterozygous or homozygous animals. These subfolders have raw .czi images containing a strain name, temperature of incubation, age, animal number, and magnification. A folder “Processed images” contained processed JPEG versions of the raw images in subfolders. These are also 2 JPEG images used for the figure.

Fig2K folder contains a folder “Raw images” containing subfolders representing either heterozygous or homozygous animals. These subfolders have raw .czi images containing a strain name, temperature of incubation, age, animal number, magnification. A folder “Processed images” contains processed JPEG versions of the raw images in subfolders. There are also Excel and CSV versions of a spreadsheet with length measurements in microns.

Fig2L folder contains a folder “Raw and processed images” containing subfolders labeled by date and genotype. Each subfolder contains raw .czi images labeled by genotype and animal numbers, with subfolders containing processed images of these raw files. There is also a spreadsheet with fluorescence measurements in Excel format, with 3 labeled tabs also separated as individual CSV files. “lamc1-eGFP lamb1a control” and “lamc1-eGFP lamb1a mutant” CSV files contain fluorescence measurements expressed as a relative fluorescence ratio with respect to pre-bleaching values. The file “Relative recovery rate” (also present as a tab of the Excel file) expresses values from these 2 tabs as ratios over the pre-bleach values.

Fig2M folder contains a folder “Raw images” containing subfolders representing either heterozygous or homozygous animals. These subfolders have raw .czi images containing a strain name, temperature of incubation, age, animal number, magnification. A folder “Processed images” contained processed JPEG versions of the raw images in subfolders. There are also Excel and CSV versions of a spreadsheet with fluorescence measurements expressed as arbitrary fluorescence units.

README for Figure 3

Figure 3 examines the localization and regenerative responses of several extracellular matrix (ECM) components in adult zebrafish using fluorescent knock-in reporters. Confocal imaging of lamc1-eGFP (enhanced green fluorescent protein) and col1a2-mScarlet hearts showed differences between uninjured ventricles and regenerating ventricles at 14 dpa (days post-amputation), with accumulation of Col1a2-mScarlet observed near the injury site. Lamc1-eGFP was also detected in both uninjured and regenerating spinal cords at 7 dpi (days post-injury), indicating changes in ECM distribution during tissue repair. Imaging of live col1a2mScarlet/+; fgf20aEGFP scales revealed Col1a2-mScarlet localization within anteroposteriorly oriented fibrils. In adult kidneys, lamc1-eGFP, col1a2-mScarlet, and tgfbi-mScarletreporters were visualized alongside Phalloidin, which labeled the apical surfaces of tubules, and DAPI (4′,6-diamidino-2-phenylindole), which labeled cell nuclei; glomeruli were identified by white arrowheads. Representative images of adult caudal fins demonstrated distinct localization patterns of Lamc1-eGFP, Col1a2-mScarlet, and Tgfbi-mScarlet around fin joints and intraray vasculature. Sample sizes ranged from n = 2–18 animals, depending on the tissue and experimental condition. Scale bars measured 100 µm (micrometers) in panels A–D, 50 µm in panel E, and 1 mm (millimeter) in panel F.

Description: Folders are subdivided into Figure panels 3A-3F. Each folder contains files with raw and processed images, organized by timepoints and sample/animal/image identifier.

Fig3A folder contains a folder “Raw images” with 2 subfolders labeled by injury context and genotype. The subfolders contain raw .lsm images which can be opened using Leica imaging software, labeled by timepoint, genotype, channels, magnification, and image number. It also contains a folder “Processed images” with similar subfolders, these contain JPEG images processed from the raw files and readable by freeware. There are also 2 JPEG images used for the figure panel.

Fig3B folder – please see the description for Fig3A.

Fig3C folder – please see the description for Fig3A. This folder contains 8 JPEG images used for the figure panel.

Fig3D folder contains a folder “col1a2-scale” with a subfolder “raw images” containing subfolders with .lif images for 3 individual animals, which can be read using Leica imaging software. It contains 3 processed .tif images as well readable by freeware. The folder “fgf20a-scale” is structured similarly.

Fig3E folder contains folders “col1a2”, “lamc1”, and “tgfbi” corresponding to 3 genotypes. Each of these folders has several processed JPEG image files labeled by genotype, channel, magnification, and image number. Subfolders in each labeled “Metadata” contain .xml files with Leica confocal imaging information; these can be opened with any web browser.

Fig3F folder contains a folder “Raw images” with 3 subfolders labeled by genotype. The subfolders contain raw .czi images which can be opened using Zeica imaging software, labeled by genotype and image number. It also contains a folder “Processed images” with similar subfolders, these contain JPEG images processed from the raw files and readable by freeware. There are also 3 JPEG images used for the figure panel.

README for Figure 4

Figure 4 examines the spatiotemporal deposition of extracellular matrix (ECM) components during adult zebrafish fin regeneration and suggests that Col1a2-mScarlet forms an early structural foundation for regenerating tissue. Longitudinal imaging of lamc1-eGFP (enhanced green fluorescent protein) and col1a2-mScarlet transgenic fish was performed at 4, 7, and 14 dpa (days post-amputation), with white arrowheads marking the tips of regenerates and cyan arrows indicating the amputation plane. Regenerating col1a2-mScarlet/+ fin rays were also imaged at 2, 4, and 7 dpa, allowing the same individuals to be followed over time and revealing progressive matrix deposition during regeneration. To relate ECM organization to cellular proliferation, regenerating fins expressing sp7:FUCCI (fluorescent ubiquitination-based cell cycle indicator) were analyzed at 4 and 7 dpa. This reporter distinguished cycling osteoblasts expressing Geminin from non-cycling osteoblasts expressing zCdt1, helping visualize the spatial relationship between bone-forming cells and regenerating matrix. Signals associated with fin joints were identified by white arrowheads, highlighting sites of tissue organization during regeneration. Sample sizes ranged from n = 3 for uninjured controls to n = 5 for regenerating fin analyses. Scale bars correspond to 500 µm (micrometers) in panels A and B and 100 µm in panels C and D.

Description: Folders are subdivided into Figure panels 4A-4D. Each folder contains files with raw and processed images, organized by timepoints and sample/animal/image identifier.

Fig4A contains a folder with 3 processed JPEG images chosen for the figure panel. It also contains a folder “Raw images” with images containing a strain name, animal number, magnification, animal view, and image number. These images are .czi format and can be opened using Zeiss imaging software. It also contains a folder “Processed images” with subfolders containing a strain name, animal number, magnification, animal view, and image number. These images are JPEGs and can be opened by freeware.

Fig4B folder – please see the description for Fig4A.

Fig4C folder contains a folder “Raw images” with subfolders representing individual animals. These subfolders contain .czi files readable by Zeiss imaging software and labeled by magnification, amputation timepoint, and ray number. The folder “Processed images” contains these “individual” subfolders each with processed .tif image files generated from the .czi files. The folder also contains images from the experiments in PPT and PDF formats.

Fig4D folder contains 2 folders labeled “4 dpa” and “7 dpa”. Each has similar content: a subfolder “Raw images” with .czi raw image files labeled by timepoint, genotype, magnification, and animal number; a subfolder “Processed images” contained processed JPEG images for these raw files; 4 JPEG image files used for the figure panel.

README for Figure S1

Figure S1 analyzes the expression of the extracellular matrix (ECM) genes lamc1, col1a2, and tgfbi during zebrafish tissue regeneration and development. XY scatter plots identify genes that are differentially regulated during heart, fin, and spinal cord regeneration, using significance thresholds of log₂ fold change > 1 for increased expression and log₂fold change < –1 for decreased expression. Messenger RNA (mRNA) levels were quantified using RPKM (Reads Per Kilobase per Million mapped reads) in uninjured and regenerating caudal fins (4 dpa; days post-amputation), cardiac ventricles (7 days post-ablation), and spinal cords (7 days post-transection), with n = 3 samples per group and statistical analysis performed using Student’s t-test. To examine developmental expression patterns, in situ hybridization was performed for lamc1, col1a2, and tgfbi in larvae at 3 dpf (days post-fertilization), using clutches of 30 embryos per probe and imaging n = 8 representative embryos. The results demonstrate spatial expression of all three genes during early development. In addition, laminin protein localization was assessed by antibody staining in lamc1-eGFP (enhanced green fluorescent protein) larvae at 3 dpf, with 50 embryos stained and n = 5 representative embryos imaged. Together, these analyses provide both transcriptional and protein-level evidence for the expression of ECM components during regeneration and embryonic development. Scale bars in panels E and F correspond to 500 µm (micrometers).

Description: Folders are subdivided into Figure panels. Files include raw and processed images for IVIS imaging, organized by timepoints and animal identifier; spreadsheets with measurements.

FigS1A folder contains .png images of graphs generated from genome sequencing datasets.

FigS1B-D folders contain Excel spreadsheets. Each Excel file has also been saved as CSV file (one file per Excel tab). Excel tabs/CSV spreadsheets contain data on the expression of that gene (lamc1, S1B; col1a2, S1C; tgfbi, S1D) in an individual tissue (Heart, Caudal fin, or Spinal cord) and are named according to tissue type. These files contain gene expression sequencing data values expressed as RPKM (Reads Per Kilobase per Million mapped reads).

FigS1E folder contains a folder “WISH” with subfolders “April 17, 2026” and “March 10, 2026” containing subfolders labeled by a probe name. Each subfolder with a probe name contains raw .czi image files labeled by microscope, magnification, and animal number; processed .tif images for these raw files; and .xml imaging metadata openable by a web browser.  The folder also contains Excel and CSV formats of a spreadsheet with PCR primer nucleotide sequences.

FigS1F folder contains 3 folders representing 3 independent tests of antibodies. Each folder contains subfolders with a gene name. Each of the subfolders has raw .czi image files and a subfolder with processed .tif images corresponding to the raw files.

README for Figure S2

Figure S2 evaluates the viability, growth, and inheritance of homozygous fluorescent knock-in zebrafish carrying lamc1-eGFP (enhanced green fluorescent protein), col1a2-mScarlet, and tgfbi-mScarlet alleles. Representative images compare WT (wild-type) larvae with heterozygous (+/−) and homozygous (+/+) knock-in siblings at 3 dpf (days post-fertilization) for col1a2 and tgfbi, and 4 dpf for lamc1. Body length measurements were performed for each genotype using n = 10 larvae per group, and differences were assessed using Student’s t-test. Genotype ratios were also quantified to determine whether the knock-in alleles were inherited at expected frequencies and whether homozygous animals survived through early development. The analyses showed the morphology and growth characteristics of homozygous knock-in larvae relative to wild-type and heterozygous siblings for all three ECM (extracellular matrix) genes. Comparison of body lengths and genotype distributions provided an assessment of whether insertion of the fluorescent reporters affected larval development or survival. Representative imaging documented normal or altered phenotypes associated with each knock-in line during embryonic development. Scale bars in panels A, D, and G correspond to 500 µm (micrometers).

Description: Folders are subdivided into Figure panels. Files include raw and processed images, organized by timepoints and animal identifier; spreadsheets with measurements.

FigS2A contains a folder with 3 processed JPEG images chosen for the figure panel. It also contains a folder “Raw images” with images containing a strain name, magnification, and animal number. These images are .czi format and can be opened using Zeiss imaging software. It also contains a folder “Processed images” with subfolders containing a strain name, animal number, magnification, and image number. These images are JPEGs and can be opened by freeware.

FigS2B, E, H folders contain Excel (and CSV versions of individual tabs) spreadsheets with measurements expressed as microns.

FigS2C contains numbers of embryos for each genotype from an incross, and corresponding percentages, in Excel and CSV formats.

FigS2D contains a folder “col1a2 processed images” with several processed JPEG image files labeled by age, genotype, magnification, embryo number, and projection type. Other.xml metadata files contain Leica confocal imaging information; these can be opened with any web browser.

FigS2F, I folder contains Excel and CSV spreadsheets. One file “KI_genotyping primers" contains nucleotide sequences of primers. The two files “incross…” contain data with numbers of embryos for each genotype from an incross for each knock-in genotype mentioned, and corresponding percentages/ratios for each genotype. It also contains subfolders with processed images of genotyping gels.

FigS2G contains a folder “tgfbi processed images” with several processed JPEG image files labeled by age, genotype, magnification, embryo number, and projection type. Other.xml metadata files contain Leica confocal imaging information; these can be opened with any web browser.

README for Figure S3

Figure S3 characterizes the distribution of extracellular matrix (ECM) components in adult zebrafish tissues using fluorescent knock-in reporters and confocal microscopy. In uninjured adult ventricles, Lamc1-eGFP (enhanced green fluorescent protein) localization was examined in sections co-stained for tcf21:DsRed2-positive epicardial and epicardial-derived cells, with nuclei labeled by DAPI (4′,6-diamidino-2-phenylindole). In regenerating hearts at 14 dpa (days post-amputation), Col1a2-mScarlet expression was analyzed together with Fibronectin staining, revealing ECM accumulation within the regenerating ventricular apex. Imaging of plucked scales demonstrated distinct localization patterns for Lamc1-eGFP and Col1a2-mScarlet, including Lamc1-associated blood vessels and clustered Col1a2-rich structures identified by arrowheads. The distribution of Col1a2-mScarlet and Tgfbi-mScarlet was also examined in adult kidneys, where both proteins were associated with renal architecture and renal tubules marked by white arrowheads. These analyses highlight tissue-specific ECM organization across the heart, scales, and kidney in adult zebrafish. Sample sizes ranged from n = 3 animals for scale and kidney imaging to n = 10 for regenerating heart analyses. Scale bars correspond to 100 µm (micrometers) in panels A and B and 1 mm (millimeter) in panels C and D.

Description: Folders are subdivided into Figure panels FigS3A-3D. Each folder contains files with raw and processed images, organized by timepoints and sample/animal/image identifier.

FigS3A contains a folder with 6 processed JPEG images chosen for the figure panel. It also contains a folder “Raw images” with images containing a strain name, magnification, and animal number. These images are .lsm format and can be opened using Leica imaging software. It also contains a folder “Processed images” with subfolders containing a strain name, animal number, magnification, and image number. These images are JPEGs and can be opened by freeware.

FigS3B contains a folder with 3 processed JPEG images chosen for the figure panel. It also contains a folder “Raw images” with images containing a strain name, magnification, and animal number. These images are .lsm format and can be opened using Leica imaging software. It also contains a folder “Processed images” with subfolders containing a strain name, animal number, magnification, and image number. These images are JPEGs and can be opened by freeware.

FigS3C contains a folder with 2 processed (JPEG, PNG) images chosen for the figure panel. It also contains a folder “Raw images” with 2 subfolders labeled by genotype and with images containing a strain name, magnification, and animal number. These images are .czi format and can be opened using Zeiss imaging software. It also contains a folder “Processed images” with 2 subfolders labeled by genotype and with images containing a strain name, animal number, magnification, and image number. These images are JPEGs and can be opened by freeware.

FigS3D contains a folder with 2 processed JPEG images chosen for the figure panel. It also contains a folder “Raw images” with 2 subfolders labeled by genotype and with images containing a sex, tissue region and magnification. These images are .czi format and can be opened using Zeiss imaging software. It also contains a folder “Processed images” with 2 subfolders labeled by genotype and with images containing a sex, strain name, animal number, magnification, and image number. These images are JPEGs and can be opened by freeware.

README for Figure S4

Figure S4 investigates the distribution and persistence of extracellular matrix (ECM) components during zebrafish fin regeneration using fluorescent knock-in reporters. Tissue sections from regenerating lamc1-eGFP (enhanced green fluorescent protein) fins at 1 dpa (day post-amputation) and col1a2-mScarlet fins at 4 dpa illustrate the spatial localization of these ECM proteins during early regenerative stages. Quantitative analysis of fluorescence intensity at 7 dpa measured Lamc1-eGFP and Col1a2-mScarlet levels from the amputation plane to the distal tip of regenerating fin rays, with data reported as mean ± S.D. (standard deviation) from n = 5 animals and 20 fin rays. Long-term imaging at 60 dpa demonstrated persistent Col1a2-mScarlet accumulation at the original amputation site, suggesting stable matrix deposition within regenerated tissue. The distribution of Tgfbi-mScarlet was also quantified along regenerating fins at 7 dpa, with measurements taken from the amputation plane (Amp) to the distal tip using n = 5 fish and four fin rays per individual. Representative images of regenerating tgfbi-mScarlet/+ fins at 4 and 7 dpa further documented the temporal pattern of Tgfbi deposition during regeneration. In addition, vascular organization at the amputation plane was examined in fli1:EGFP (enhanced green fluorescent protein) transgenic fins, revealing blood vessel patterning associated with regenerative growth. Scale bars correspond to 100 µm (micrometers) in panels A and B, 250 µm in panel G, and 1 mm (millimeter) in panels D and F.

Description: Folders are subdivided into Figure panels. Files include raw and processed images, organized by timepoints and animal identifier; spreadsheets with measurements.

FigS4A contains a folder with 3 processed JPEG images chosen for the figure panel. It also contains a folder “Raw images” with images containing a timepoint, strain name, channel, magnification, and image number. These images are .lsm format and can be opened using Leica imaging software. It also contains a folder “Processed images” with JPEGs processed from the raw files.

FigS4B contains a folder with 3 processed JPEG images chosen for the figure panel. It also contains a folder “Raw images” with images containing a timepoint, strain name, channel, magnification, and image number. These images are .lsm format and can be opened using Leica imaging software. It also contains a folder “Processed images” with JPEGs processed from the raw files.

FigS4C, E folders contain fluorescence measurements expressed in arbitrary units, in Excel and CSV formats.

FigS4D contains a folder with a processed image chosen for the figure panel. It also contains a folder “Raw images” with images containing a strain name, magnification, and animal number. These images are .czi format and can be opened using Zeiss imaging software. It also contains a folder “Processed images” with JPEGs processed from the raw files.

FigS4F contains a folder with 2 processed JPEG images chosen for the figure panel. It also contains a folder “Raw images” with images containing a timepoint, strain, magnification, and animal number. These images are .czi format and can be opened using Zeiss imaging software. It also contains a folder “Processed images” with subfolders containing JPEGs representing the raw files that can be opened by freeware.

FigS4G contains a folder “Raw images” with subfolders representing individual animals. These subfolders contain .czi files readable by Zeiss imaging software and labeled by date, magnification, and amputation timepoint. The folder “Processed images” contains these “individual” subfolders each with processed .tif image files generated from the .czi files. The folder also contains images from the experiments in PPT and PDF formats.

README for Figure S5

Figure S5 presents a transcriptomic analysis demonstrating that the fin regeneration “foundation” region has a gene expression profile distinct from both uninjured tissue and early regenerating tissue. For the RNA sequencing experiment, fin tissue segments located above and below the amputation plane were collected at 0, 3.5, 7, 14, and 21 dpa (days post-amputation), and total RNA (ribonucleic acid) was isolated for library preparation and sequencing. A volcano plot comparing uninjured fins (0 dpa) with tissue at the 7 dpa amputation site identified genes with altered expression using log₂ fold-change thresholds of > 1 for upregulated genes and < −1 for downregulated genes. Functional analysis using GO (Gene Ontology) enrichment highlighted biological processes associated with the 7 dpa amputation site. Additional GSEA (Gene Set Enrichment Analysis) compared the 7 dpa foundation region with the early regenerate at 1 dpa, revealing differences in pathways related to DNA replication as well as collagen metabolism and catabolism. In the GSEA plots, the ES (enrichment score) on the y-axis quantified the degree of gene-set enrichment, while black vertical lines marked the positions of genes from a given set within the ranked expression list. Color gradients indicated relative expression between conditions, with red denoting higher expression in the first condition and blue indicating higher expression in the comparison condition, and red arrowheads highlighting differences in enrichment-score magnitudes. No spatial scale bars or other physical units of measurement are reported in this figure legend.

Description: The folder contains the results of genome sequencing data analyses, including PDF reports, CSV files of differential analysis results, gene ontology (GO) enrichment analyses of differentially expressed (DE) genes, and gene set enrichment analyses (GSEA).

Figure S5B contains the output from DESeq2 (v1.34.0) for the differential expression analysis comparing 7 dpa versus 0 dpa amputation-site samples. These results were used to generate the volcano plot shown in Figure S5B. The differential expression results are provided in "DESeq2.Salmon.diff.xlsx".

The columns in DESeq2.Salmon.diff.xlsx are defined as follows:

• symbol: Gene symbol.
• baseMean: Mean of normalized counts across all samples.
• log2FoldChange: Estimated log2 fold change between 7 dpa and 0 dpa.
• lfcSE: Standard error of the log2 fold change estimate.
• stat: Wald test statistic.
• pvalue: Raw p-value from the Wald test.
• padj: Benjamini–Hochberg adjusted p-value (false discovery rate, FDR).
• baseMean.shrinked: Mean normalized count reported by DESeq2 after lfcShrink(type = "ashr").
• log2FoldChange.shrinked: Shrunk log2 fold change estimated using lfcShrink(type = "ashr").
• lfcSE.shrinked: Standard error associated with the shrunk log2 fold change estimate.
• pvalue.shrinked: P-value reported in the output of lfcShrink(type = "ashr").
• padj.shrinked: Benjamini–Hochberg adjusted p-value reported in the output of lfcShrink(type = "ashr").

Figure S5C contains the raw data used to generate Figure S5C. The subset of Gene Ontology (GO) Biological Process enrichment results displayed in the figure is provided in "GO.BP.enrichment.sel.golist.csv".

The complete GO Biological Process enrichment results generated by clusterProfiler (v4.14.4) are available in the subdirectory:

"fish_amputation_site/dpa7-dpa0/enrichment.pval0.05.lfc1.up.regulated/"

as GO.BP.enrichment.csv.

The columns in GO.BP.enrichment.csv are defined as follows:

• ID: Gene Ontology (GO) accession identifier.
• Description: GO term description.
• GeneRatio: Ratio of input genes annotated to the GO term (reported as Count/total input genes).
• BgRatio: Ratio of background genes annotated to the GO term (reported as background gene count/total background genes).
• RichFactor: Ratio of the number of enriched genes (Count) to the total number of genes annotated to the GO term.
• FoldEnrichment: Fold enrichment of the GO term relative to the background, calculated as GeneRatio/BgRatio.
• zScore: Enrichment z-score computed by clusterProfiler, indicating the direction and magnitude of enrichment.
• pvalue: Raw p-value from the enrichment test.
• p.adjust: Benjamini-Hochberg adjusted p-value.
• qvalue: Estimated q-value for the enrichment test.
• geneID: Slash-delimited list of input genes annotated to the GO term.
• Count: Number of input genes annotated to the GO term.

 Figure S5D contains the complete output from GSEA (v4.4.0) using the Preranked workflow. The ranked gene list was generated from the DESeq2 Wald test statistic (stat) for the comparison of 7 dpa versus 0 dpa amputation-site samples. Gene identifiers were converted to human homologs before performing GSEA.

 Figure S5E contains the complete output from GSEA using the Preranked workflow. The ranked gene list was generated from the DESeq2 Wald test statistic ('stat') for the differential expression analysis comparing 1 dpa and 0 dpa during fin regeneration. Gene identifiers were mapped to their human homologs prior to GSEA.