Local signaling specifies tissue-resident fibroblasts from multipotent sclerotome progenitors 2 in zebrafish 3 4

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 populations, including tenocytes (tendon fibroblasts), blood vessel associated fibroblasts, and fin mesenchymal cells. High resolution imaging shows that different fibroblast subtypes occupy unique anatomical locations with distinct morphologies. Photoconversion-based cell lineage analysis reveals that sclerotome progenitors at different dorsal-ventral and anterior-posterior positions display distinct differentiation potentials. Single cell clonal analysis suggests that sclerotome progenitors are multipotent, and the fate of their daughter cells is biased by their migration paths and relative positions. Using a small molecule inhibitor, we show that BMP signaling is required for the development of fin mesenchymal cells in the peripheral fin fold. Together, our work demonstrates that the sclerotome contains multipotent progenitors that respond to local signals to generate a diverse population of tissue-resident fibroblasts.Fibroblasts are present in most organs in our body. They not only provide structural support to corresponding tissues, but also play important roles in wound healing and tissue fibrosis. Although the function of fibroblasts has been well appreciated, how different tissue-resident fibroblasts emerge during embryonic development is still poorly understood. Using the zebrafish model, we identify the sclerotome, a sub-compartment of the embryonic somite, as the main source of multiple fibroblast populations. Different fibroblast subtypes display distinct morphologies and locate at unique positions to support different tissues, including the muscles, blood vessels and the fin fold. Using cell tracing in live animals, we find that single sclerotome progenitors are able to generate more than one type of fibroblasts. The differentiation potential of sclerotome progenitors is biased by their initial locations in the trunk as well as their migration directions and relative positions. Local BMP signaling in the fin fold is essential for the proper development of sclerotome derived fin mesenchymal cells. Together, our results show that local microenvironment contributes to the diversification of multipotent sclerotome progenitors into distinct fibroblast subtypes.


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Fibroblasts are connective tissue cells that are present in most organs in animals. They are 56 traditionally viewed as tissue support cells by synthesizing and remodeling extracellular matrix (ECM) 57 components. Recent work has shown that tissue-resident fibroblasts also play important regulatory 58 roles in wound healing, inflammation, tumor microenvironment, and tissue fibrosis (Kalluri, 2016;

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The somite is the embryonic source of many tissue support cells, including fibroblasts. During 73 development, the somite forms three separate domains: the dermatome, the myotome, and the 74 sclerotome. The sclerotome contributes to the axial skeleton and cartilage of the animal. Work in 75 mouse and chick has shown that the sclerotome is the developmental origin of tendon fibroblasts 76 (tenocytes) (Brent et al., 2003;Schweitzer et al., 2001) and vascular smooth muscle cells (Pouget et

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In fish and amphibians, fin mesenchymal cells are a population of fibroblasts present in the larval 82 fin fold. They express many ECM components to provide structural support to the developing fin fold 83 (Durán et al., 2011;Feitosa et al., 2012). However, the developmental origin of fin mesenchymal cells 84 has been controversial. Dye labeling experiments in Xenopus and zebrafish suggest that the neural 85 crest contributes to the fin mesenchyme (Smith and Hall, 1990;Smith et al., 1994). However, later 86 experiments in Xenopus suggest that the mesoderm also contributes to fin mesenchymal cells in both 87 5 the dorsal and ventral fin (Garriock and Krieg, 2007;Tucker and Slack, 2004). Similarly, cell 88 transplantation experiments in axolotls reveal dual origin of the fin mesenchyme from both the neural 89 crest and the somites (Sobkow et al., 2006). More recent work using genetic lineage tracing in fish 90 (zebrafish) and amphibians (Xenopus and axolotl) supports a model where fin mesenchymal cells 91 originate exclusively from the mesoderm with no contribution from the neural crest lineage (Lee et al., 92 2013; Taniguchi et al., 2015). In particular, the dermomyotome compartment of the somite has been 93 suggested to be the source of fin mesenchymal cells in zebrafish (Lee et al., 2013).

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Here, we show that the sclerotome is a major source of multiple distinct fibroblast populations in  To explore the lineage potential of the sclerotome, we developed a sclerotome-specific reporter 105 line, nkx3.1:Gal4; UAS:NTR-mCherry (nkx3.1 NTR-mCherry , similar designations are used for all Gal4/UAS 106 transgenic lines in this paper) . Due to the perdurance of the mCherry protein, we 107 were able to label the initial sclerotome domains as well as all their descendants. We crossed 108 nkx3.1 NTR-mCherry to the endothelial reporter kdrl:EGFP to examine the different populations of 109 sclerotome derived cells at 2 dpf (days post fertilization). Based on their anatomical locations, we 110 broadly defined four main groups of mCherry + cells from the sclerotome lineage (Fig 1A and 1B): 1) 111 cells located in both dorsal and ventral fin folds; 2) cells closely associated with blood vessels in the 112 trunk, including the dorsal longitudinal anastomotic vessel (DLAV), intersegmental vessels (ISV), the 113 dorsal aorta (DA), and the caudal vein plexus (CVP); 3) tenocytes between neighboring somites as 114 we have previously described ; and 4) interstitial cells located in the space between 115 the notochord, the spinal cord and muscles. It is worth noting that the nkx3.1 NTR-mCherry line also drove 116 expression in some muscle cells  and in some cuboidal cells at the edge of the fin 117 fold (Fig 1B), neither of which were derived from the sclerotome lineage. We have previously shown 118 that both tenocytes and ISV-associated perivascular fibroblasts express the pan-fibroblast markers 119 such as col1a2 and col5a1 Rajan et al., 2020). We asked whether other sclerotome 120 derived cells are also fibroblasts. Indeed, all four groups of nkx3.1 NTR-mCherry -expressing cells were 121 positive for col1a2:GFP, a pan-fibroblast reporter, at 2 dpf ( Fig S1C). This result suggests that the 122 sclerotome is the embryonic source of multiple types of fibroblasts in zebrafish.

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At 2 dpf, the fin fold is subdivided into two lobes (Parichy et al., 2009) (Fig S1A). The major lobe 124 arises from the dorsal edge of somite 7, wraps around the trunk and the tail, and ends at the ventral 125 edge at somite 18 where it meets the end of the yolk extension. The minor lobe extends ventrally 126 underneath the yolk extension. In nkx3.1 NTR-mCherry embryos, mCherry + cells can be seen populating 127 both the dorsal and ventral regions in the major lobe of the fin fold (Fig 1B and S1B). Using a mosaic 128 col1a2:Gal4; UAS:Kaede line (referred to as col1a2 Kaede ) (Sharma et al., 2019), we visualized the cell 129 morphology at high resolution ( Fig 1C). These Kaede + fin cells were characterized by extensive "tree-130 like" cellular processes projecting towards the periphery of the fin fold (Fig 1B and 1C). The location, 131 morphology, as well as the marker expression (shown later) suggest that these sclerotome derived fin 132 fold residing cells correspond to fin mesenchymal cells as previously described (Feitosa et al., 2012; 133 Lee et al., 2013). Interestingly, mCherry + fin mesenchymal cells were absent in the fin fold at the tip of 7 the tail and the minor lobe (Fig S1B), suggesting that the sclerotome is not the only lineage that gives 135 rise to fin mesenchymal cells.

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Within the zebrafish trunk, many sclerotome derived fibroblasts were found associated with 137 different vascular beds (Fig 1A and 1B). We have previously described ISV-associated perivascular 138 fibroblasts, characterized by a globular cell body with a few short processes extended around the ISV 139 (Rajan et al., 2020). By contrast, fibroblasts associated with DLAV or DA (referred to as DLAV 140 fibroblasts and DA fibroblasts, respectively) were more elongated along the long axis of the blood 141 vessel (Fig 1B and 1C). The CVP is a transient venous network with a stereotypical "honeycomb-like" 142 structure. Fibroblasts marked by nkx3.1 NTR-mCherry were found occupying the space in the honeycomb 143 pockets of the CVP (Fig 1B). Mosaic labeling with col1a2 Kaede revealed that individual Kaede + cells 144 had a small and elongated cell body with long cellular processes along the underlying endothelium 145 and sometimes across the honeycomb pocket ( Fig 1C). Their location and morphology suggest that

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Finally, tenocytes and interstitial fibroblasts were two fibroblast subtypes that populated in the 149 trunk region but were not closely associated with blood vessels. We have previously shown that 150 tenocytes, marked by scxa and tnmd expression, are located along the myotendinous junction (MTJ), 151 extending long cellular processes into the intersomitic space (Fig 1B and 1C) . By 152 contrast, interstitial fibroblasts were scattered throughout the interstitial space, displaying varied 153 morphology (Fig 1B and 1C). Together, our results show that the sclerotome generates multiple types 154 of fibroblasts in the zebrafish trunk, supporting different tissues, such as muscles, blood vessels and

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presence of some small projections from these Kaede red cells suggests that cell migration may be 172 initiating at this stage ( Fig 2B). By 48 hpf, multiple Kaede red cells can be seen populating the dorsal 173 region of the fish (Fig 2B). Based on their morphology and location relative to the vasculature, we

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Indeed, double staining revealed two juxtaposing cell layers in the fin fold: hmcn2 + fbln1 + fin 265 mesenchymal cells in the proximal layer and hmcn2 + fras1 + apical epidermal cells in the distal layer 266 ( Fig 6B). As expected, double staining in nkx3.1 NTR-mCherry embryos showed that mCherry + cells in the 267 fin fold co-expressed fbln1 but not fras1 (Fig 6C), confirming that the sclerotome contributes to fin 268 mesenchymal cells, a specific cell population in the fin fold.

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We hypothesized that the development of fin mesenchymal cells is mediated by local signals in 270 the fin fold. To test this model, we performed expression analysis to identify signaling ligands enriched 271 in the fin fold. Interestingly, we found that a number of bmp genes, including bmp2b, bmp4, and 272 bmp6, were expressed specifically in fras1 + apical epidermal cells, adjacent to fbln1 + fin mesenchymal 273 cells (Fig 6D and S2). Accordingly, nkx3.1 NTR-mCherry -positive fin mesenchymal cells were positioned 274 along a layer of bmp4-expressing cells (Fig 6C). This result suggests that BMP ligands secreted from  298 expansion in DMH1-treated fish (Fig 7B). By contrast, quantification of a 10-somite region showed 299 that inhibition of BMP signaling lead to 49% reduction of dorsal fin mesenchymal cells (from 54.2 to 300 27.7), and 63% decrease in ventral fin mesenchymal cells (from 39.8 to 14.6) ( Fig 7C). Interestingly, 301 the few remaining fin mesenchymal cells in DMH1-treated fish showed an aberrant cell morphology 302 with more cellular projections compared to controls (Fig 7A). To determine whether BMP signaling 12 regulates the branching morphogenesis of fin mesenchymal cells, we performed late DMH1 304 treatments on kdrl:EGFP; nkx3.1 NTR-mCherry embryos at 25-49 hpf (Fig S4). Similar to early DMH1 305 treatment, fin folds in DMH1-treated embryos were significantly expanded (Fig S4A and S4B).

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Although the number of fin mesenchymal cells was largely unaffected in DMH1-treated fish (Fig S4C), 307 they displayed the similar hyperbranching morphology (Fig S4A). Our results suggest that BMP 308 signaling is required in not only the early migration but also the late morphogenesis of fin    This result suggests that individual sclerotome progenitors are likely multipotent, rather than "lineage-386 restricted" to differentiate into one specific fibroblast subtype. Our studies reveal three key features in 387 cell fate diversification of sclerotome progenitors (Fig 7E). First, sclerotome progenitors display