Origin and diversification of fibroblasts from the sclerotome in zebrafish

Fibroblasts play an important role in maintaining tissue integrity by secreting components of the extracellular matrix and initiating response to injury. Although the function of fibroblasts has been extensively studied in adults, the embryonic origin and diversification of different fibroblast subtypes during development remain largely unexplored. Using zebrafish as a model, we show that the sclerotome, a sub-compartment of the somite, is the embryonic source of multiple fibroblast subtypes including tenocytes (tendon fibroblasts), blood vessel associated fibroblasts, fin mesenchymal cells, and interstitial fibroblasts. High-resolution imaging shows that different fibroblast subtypes occupy unique anatomical locations with distinct morphologies. Long-term Cre-mediated lineage tracing reveals that the sclerotome also contributes to cells closely associated with the axial skeleton. Ablation of sclerotome progenitors results in extensive skeletal defects. Using photoconversion-based cell lineage analysis, we find that sclerotome progenitors at different dorsal-ventral and anterior-posterior positions display distinct differentiation potentials. Single-cell clonal analysis combined with in vivo imaging suggests that the sclerotome mostly contains unipotent and bipotent progenitors prior to cell migration, and the fate of their daughter cells is biased by their migration paths and relative positions. Together, our work demonstrates that the sclerotome is the embryonic source of trunk fibroblasts as well as the axial skeleton, and local signals likely contribute to the diversification of distinct fibroblast subtypes.


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The sclerotome generates different types of trunk fibroblasts 123 6 We performed mosaic labeling and marker analysis to characterize fibroblasts derived from the 124 nkx3.1 lineage (Fig 2A-C). nkx3.1 NTR-mCherry -expressing fibroblasts in the trunk, including tenocytes, 125 blood vessel associated fibroblasts, and interstitial fibroblasts, were marked by pan-fibroblast markers 126 such as tgfbi and col5a1 (Metikala et al., 2021;Muhl et al., 2020) (Fig 2B). A subset of these 127 sclerotome-derived fibroblasts was associated with different vascular beds (Fig 1C and 2A). We associated with DLAV or DA (DLAV and DA fibroblasts, respectively) were more elongated along the 132 long axis of the blood vessel (Fig 1C and 2A). CVP is a transient venous network with a stereotypical 133 "honeycomb-like" structure. Fibroblasts marked by nkx3.1 NTR-mCherry occupied the space in the 134 honeycomb pockets of the CVP (Fig 1C and 2A). Mosaic labeling showed that individual Kaede + cells 135 had a small and elongated cell body with long cellular processes along the underlying endothelium 136 and sometimes across the honeycomb pocket (Fig 2A). The location and morphology of these CVP-137 associated fibroblasts suggest that they are stromal reticular cells (SRCs) (Murayama et al., 2015).

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Tenocytes and interstitial fibroblasts were the two fibroblast subtypes that populated the trunk 139 region but were not closely associated with blood vessels (Fig 1C). We and others have previously

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The sclerotome contributes to fin mesenchymal cells

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The nkx3.1 NTR-mCherry line labeled cells in the fin fold (Fig 1B-C). At 2 dpf, the fin fold is subdivided 149 into two parts: the major lobe wrapping around the trunk and tail, and the minor lobe beneath the yolk 150 extension (Parichy et al., 2009) (Fig S2). Cells from the nkx3.1 lineage populated both the dorsal and 151 ventral regions in the major lobe (Fig 1C and S2). Using the mosaic col1a2 Kaede line, we found that 152 Kaede + fin cells displayed extensive "tree-like" cellular processes projecting towards the periphery of 153 the fin fold (Fig 2A). The location and morphology suggest that these sclerotome-derived fin fold revealed two juxtaposing cell layers in the fin fold: hmcn2 + fbln1 + fin mesenchymal cells in the proximal 159 layer, and hmcn2 + fras1 + apical epidermal cells in the distal layer ( Fig 3B). As expected, double 160 staining in nkx3.1 NTR-mCherry embryos showed that mCherry + cells in the fin fold co-expressed fbln1 but 161 not fras1 (Fig 3C), confirming that the sclerotome contributes to fin mesenchymal cells, a specific cell 162 population in the fin fold.

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Interestingly, nkx3.1 Kaede -expressing fin mesenchymal cells were absent in the caudal fin fold and 164 minor lobe (Fig S2). Cre-mediated lineage tracing in nkx3.1:Gal4; UAS:Cre-ERT2; ubi:Switch fish 165 showed similar results (Fig 3D). Switched mCherry + cells derived from the nkx3.1 lineage gave rise to 166 fin mesenchymal cells in both the dorsal and ventral regions of the major lobe at 4 dpf, but cells in the 167 minor lobe and caudal fin remained GFP + mCherry -(un-switched) (Fig 3D). Our results suggest that 168 sclerotome is not the only lineage that gives rise to fin mesenchymal cells. To determine whether the 169 dermomyotome (also known as the external cell layer) contributes to fin mesenchymal cells, we 170 imaged pax7b:Gal4FF; UAS:EGFP (pax7b EGFP ) fish at 50 hpf. Interestingly, despite extensive labeling 171 of dermomyotome cells in all somites, most fin mesenchymal cells were not marked by pax7b EGFP (Fig   172   3E). EGFP + fin cells were not observed in the minor lobe or caudal fin, while on average, only 4.6% 173 and 0.2% of fin mesenchymal cells were pax7b EGFP -positive in the dorsal and ventral regions of the 174 major lobe, respectively ( Fig 3F). This is in striking contrast to 95% (dorsal) and 94% (ventral) 175 contributions from the nkx3.1 NTR-mCherry reporter ( Fig 3F). Therefore, our results suggest that the 176 sclerotome, but not the dermomyotome, is the main contributor of fin mesenchymal cells in the dorsal 177 and ventral fin folds.

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Together, our data show that the sclerotome generates multiple types of fibroblasts in the 179 zebrafish trunk, supporting different tissues such as muscles, blood vessels, and the fin fold. These            fibroblasts. Type II cells, such as tenocytes, ISV-associated perivascular fibroblasts, and interstitial 261 fibroblasts, are located medial to the somite. Interestingly, progenitors with initial dorsal projections 262 predominantly generated type I cells (13/14, 93%), whereas cells projecting ventrally were more likely 263 to give rise to type II cells (11/19, 58%) than type I cells (3/19, 16%) and sometimes generated cells of 264 both types (5/19, 26%) (Fig 6E-F). It is important to note that ventral projecting progenitors at 24 hpf 265 never generated dorsal fin mesenchymal cells and all type I cells generated by this group were DLAV 266 fibroblasts found along the ventral side of the DLAV (Fig 6F). Together, our results suggest that the

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In summary, we demonstrate that the sclerotome is the embryonic source of multiple fibroblast 450 subtypes and contributes to axial skeletal development in zebrafish. Our work shows that the 451 sclerotome is an excellent model for studying cell type diversification during embryonic development.

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Bones can be fluorescently labelled in live larvae using the vital dye, calcein. Larvae at 17 dpf 520 were placed in a 100 mm petri dish in a 0.2% calcein solution for 15 -30 minutes at room 521 temperature. They were then washed at least six times with water from zebrafish vivarium. The larvae 522 were left for 2 -3 hours prior to imaging to allow excess calcein to pass through their digestive tract.

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Fish were anesthetized using tricaine and mounted in 0.8% low-melting-point agarose for live 524 imaging. To obtain transverse views, fish were fixed in 4% formaldehyde and sectioned for imaging.

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All graphs were generated using the GraphPad Prism software. Data were plotted as mean±SEM.