DOI: 10.5061/dryad.fttdz090c
title: Single-cell RNA sequencing of sclerotome-derived fibroblasts in zebrafish

description: Despite their importance in tissue maintenance and repair, fibroblast
 diversity and plasticity remain poorly understood. Using single-cell RNA
 sequencing, we uncover distinct sclerotome-derived fibroblast populations
 in zebrafish, including progenitor-like perivascular/interstitial
 fibroblasts, and specialized fibroblasts such as tenocytes. To determine
 fibroblast plasticity in vivo, we develop a laser-induced tendon ablation
 and regeneration model. Lineage tracing reveals that laser-ablated
 tenocytes are quickly regenerated by preexisting fibroblasts. By combining
 single-cell clonal analysis and live imaging, we demonstrate that
 perivascular/interstitial fibroblasts actively migrate to the injury site,
 where they proliferate and give rise to new tenocytes. By contrast,
 perivascular fibroblast-derived pericytes or specialized fibroblasts,
 including tenocytes, exhibit no regenerative plasticity. Interestingly,
 active Hedgehog (Hh) signaling is required for the proliferation of
 activated fibroblasts to ensure efficient tenocyte regeneration. Together,
 our work highlights the functional diversity of fibroblasts and
 establishes perivascular/interstitial fibroblasts as tenocyte progenitors
 that promote tendon regeneration in a Hh signaling-dependent manner.
description: # Single-cell RNA sequencing of sclerotome-derived fibroblasts in
 zebrafish
 [https://doi.org/10.5061/dryad.fttdz090c](https://doi.org/10.5061/dryad.fttdz090c) Data repository containing code used to generate single-cell RNA sequencing (scRNA-seq) data in the manuscript entitled “Single-cell analysis reveals distinct fibroblast plasticity during tenocyte regeneration in zebrafish” available at: https://www.biorxiv.org/content/10.1101/2023.04.18.537403v1 (preprint). ## Description of data and file structure The sclerotome compartment of the somite (labelled by *nkx3-1* expression) is the embryonic source of diverse fibroblast populations. Sclerotome-derived cells were collected by fluorescence-activated cell sorting (FACS) of mCherry-positive cells from trunks of *nkx3-1:Gal4; UAS:NTR-mCherry* transgenic embryos at 52 hours post fertilization (hpf). Isolated cells were sequenced using the 10X Genomics platform and the Illumina Novaseq S2. The raw counts matrix was generated using CellRanger v.5.0.0 and .loom files for trajectory analysis were generated using velocyto 0.17.16. The resulting unprocessed counts matrix was exported into R/Python for further analysis and is provided here as **GSM7181573\_filtered\_feature\_bc\_matrix.h5**. Additionally, the code used for downstream analysis is provided in the following files: **R analysis.R:** contains code used for pre-processing, quality control, normalization and dimension reduction, clustering, and marker exploration. **RNAvelocity.ipynb:** contains code used for RNA velocity inference. ## Sharing/Access information The raw sequencing FASTQ files are available at NCBI GEO [GSE229939](https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE229939). UMI counts matrix is available at NCBI GEO [GSE229939](https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE229939). The code files are also available at [https://github.com/hpeng031/Sclerotome](https://github.com/hpeng031/Sclerotome). ## Code/Software The code provided (stored in Zenodo) utilized the following additional software/packages: **R analysis.R:** This script was executed in R version 4.0.3 and used the following packages - SingleCellExperiment 1.12.0, Seurat 4.0.0, tidyverse 1.3, Matrix 1.2-18, scales, 1.1.1, cowplot 1.1.1, RCurl 1.98-1.3, dplyr 1.0.4, ggplot2 3.3.3, ggprism 1.0.3, AnnotationHub 2.22.0, and tibble 3.0.6. **RNAvelocity.ipynb:** This script was executed using Python and used the following packages - numpy, pandas, matplotlib.pyplot, scanpy, igraph, scvelo, loompy, and anndata.