YAP and TAZ couple osteoblast precursor mobilization to angiogenesis and mechanoregulation in fetal bone development. YAP and TAZ couple osteoblast precursor mobilization to angiogenesis and mechanoregulation in fetal bone development

Fetal bone development occurs by endochondral conversion of avascular cartilage to vascularized bone at the growth plate. This requires coordinated mobilization of osteoblast precursors with blood vessels. In adult bone, vessel-adjacent osteoblast precursors are maintained by mechanical stimuli; however, the mechanisms by which these cells mobilize and respond to mechanical cues during embryonic development are unknown. Here, we unify osteoblast precursor co-mobilization and mechanotransduction in a single signaling pathway, via the mechanosensitive transcriptional regulators YAP and TAZ. We show that YAP and TAZ spatially couple osteoblast precursor mobilization to angiogenesis, regulate vascular loop morphogenesis to control growth plate remodeling, and mediate mechanoregulation of load-induced osteogenesis in embryonic bone. Mechanistically, YAP and TAZ uniquely regulate a subset of osteoblast-lineage cells, identified by single-cell RNA sequencing as vessel-associated osteoblast precursors. Among these cells, YAP and TAZ regulate expression of the angiogenic chemokine, Cxcl12. Functionally, in 3D human cell co-culture, CXCL12 treatment rescued angiogenesis impaired by stromal cell YAP/TAZ depletion. Together, these data establish new functions of the vessel-associated osteoblast precursors in endochondral bone development. Overall design: Fetal forelimbs were generated from E17.5 mice from 3 genotypes - 2 control and 1 experimental. 1) YAPfl/fl; TAZfl/fl - hereafter WT(fl/fl) (n = 4 embryos), 2).YAPWT/WT; TAZWT/WT; Osx-cre - hereafter WT(Osx-cre) (n=4 embryos), 3) YAPfl/fl; TAZfl/fl; Osx-Cre - hereafter YAP/TAZ Osx cKO (n=3 embryos)

胎儿骨发育通过生长板（growth plate）处无血管软骨向血管化骨的软骨内转化（endochondral conversion）实现。该过程需要成骨细胞前体（osteoblast precursors）与血管的协同动员。在成体骨骼中，血管邻近的成骨细胞前体由机械刺激维持；然而，这些细胞在胚胎发育过程中的动员及对机械信号的响应机制仍未明确。
本研究通过机械敏感性转录调控因子YAP与TAZ，将成骨细胞前体协同动员与机械转导（mechanotransduction）整合至单一信号通路中。我们证实，YAP与TAZ可在空间上将成骨细胞前体动员与血管生成（angiogenesis）耦联，调控血管环形态发生以控制生长板重塑，并介导胚胎骨骼中负荷诱导成骨的机械调控过程。
从机制层面而言，YAP与TAZ可特异性调控成骨细胞谱系细胞的一个亚群，该亚群经单细胞RNA测序（single-cell RNA sequencing）鉴定为血管相关成骨细胞前体。在这群细胞中，YAP与TAZ可调控促血管生成趋化因子Cxcl12的表达。
功能实验中，在三维人细胞共培养体系内，CXCL12处理可挽救因基质细胞（stromal cell）YAP/TAZ耗竭所受损的血管生成过程。综上，本研究数据明确了血管相关成骨细胞前体在软骨内骨发育中的全新功能。
实验整体设计：样本取自3种基因型的E17.5（胚胎第17.5天）小鼠胎鼠前肢，其中2组为对照组，1组为实验组：1）YAPfl/fl; TAZfl/fl，后续记为WT(fl/fl)（n=4枚胎鼠）；2）YAPWT/WT; TAZWT/WT; Osx-cre，后续记为WT(Osx-cre)（n=4枚胎鼠）；3）YAPfl/fl; TAZfl/fl; Osx-Cre，后续记为YAP/TAZ Osx条件性敲除（cKO）组（n=3枚胎鼠）