Blood vessels are vital to sustain life in all vertebrates. While it is known that mural cells (pericytes and smooth muscle cells) regulate vascular integrity, the contribution of other cell types to vascular stabilization has been largely unexplored. Using zebrafish, we identified sclerotome-derived perivascular fibroblasts as a novel population of blood vessel associated cells. In contrast to pericytes, perivascular fibroblasts emerge early during development, express the extracellular matrix (ECM) genes col1a2 and col5a1, and display distinct morphology and distribution. Time-lapse imaging reveals that perivascular fibroblasts serve as pericyte precursors. Genetic ablation of perivascular fibroblasts markedly reduces collagen deposition around endothelial cells, resulting in dysmorphic blood vessels with variable diameters. Strikingly, col5a1 mutants show spontaneous hemorrhage, and the penetrance of the phenotype is strongly enhanced by the additional loss of col1a2. Together, our work reveals dual roles of perivascular fibroblasts in vascular stabilization where they establish the ECM around nascent vessels and function as pericyte progenitors.

24Blood vessels are vital to sustain life in all vertebrates. While it is known that mural cells (pericytes 25 and smooth muscle cells) regulate vascular integrity, the contribution of other cell types to vascular 26 stabilization has been largely unexplored. Using zebrafish, we identified sclerotome-derived 27 perivascular fibroblasts as a novel population of blood vessel associated cells. In contrast to pericytes, 28 perivascular fibroblasts emerge early during development, express the extracellular matrix (ECM) 29 genes col1a2 and col5a1, and display distinct morphology and distribution. Time-lapse imaging 30 reveals that perivascular fibroblasts serve as pericyte precursors. Genetic ablation of perivascular 31 fibroblasts results in dysmorphic blood vessels with variable diameters. Strikingly, col5a1 mutants 32show spontaneous hemorrhage, and the penetrance of the phenotype is strongly enhanced by the 33 additional loss of col1a2. Together, our work reveals dual roles of perivascular fibroblasts in vascular 34 stabilization where they establish the ECM around nascent vessels and function as pericyte 35 progenitors. 36 3 AUTHOR SUMMARY 37 38 Blood vessels are essential to sustain life in humans. Defects in blood vessels can lead to serious 39 diseases, such as hemorrhage, tissue ischemia, and stroke. However, how blood vessel stability is 40 maintained by surrounding support cells is still poorly understood. Using the zebrafish model, we 41 identify a new population of blood vessel associated cells termed perivascular fibroblasts, which 42 originate from the sclerotome, an embryonic structure that is previously known to generate the 43 skeleton of the animal. Perivascular fibroblasts are distinct from pericytes, a known population of 44 blood vessel support cells. They become associated with blood vessels much earlier than pericytes 45 and express several collagen genes, encoding main components of the extracellular matrix. Loss of 46 perivascular fibroblasts or mutations in collagen genes result in fragile blood vessels prone to 47 53 54The vascular system is crucial to the survival of vertebrates. Blood vessels must rapidly expand 55 and contract in response to systemic cues, but also maintain the stability to withstand the stress of 56 blood flow. To maintain their integrity, blood vessels are supported by a highly specialized 57 perivascular architecture comprised of blood vessel associated cells and the surrounding extracellular 58 matrix (ECM) (1,2). Compromised vascular integrity can result in devastating human diseases, such 59 as aneurysms, vascular malformations, and hemorrhagic strokes (3-6). However, how blood vessels 60 are stabilized by different perivascular components is still poorly understood. 61The prevailing model is that vascular stability is maintained at three different levels: endothelial 62 cells, mural cells and the surrounding ECM (2). First, blood vessels are lined by endothelial cells. 63Adherens and tight junctions between endothelial cells provide the primary barrier to passage o...

Despite their importance in tissue maintenance and repair, fibroblast diversity and plasticity are poorly understood. Using single-cell RNA sequencing, we uncover six distinct sclerotome-derived fibroblast populations in the zebrafish trunk, including progenitor-like perivascular/interstitial fibroblasts, and specialized fibroblasts such as tenocytes. To determine fibroblast plasticity in vivo, we develop a tendon regeneration model. Lineage tracing reveals that laser-ablated tenocytes can be quickly regenerated by pre-existing fibroblasts. Combining live imaging and clonal analysis, we demonstrate that perivascular/interstitial fibroblasts actively migrate to the injury site, where they proliferate and give rise to new tenocytes. Strikingly, other specialized fibroblast subtypes derived from the same sclerotome lineage, including uninjured tenocytes, show no regenerative response. Interestingly, active Hedgehog (Hh) signaling is required for the proliferation and differentiation of activated fibroblasts. 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.