Yap/Taz transcriptional activity in endothelial cells promotes intramembranous ossification via the BMP pathway

Osteogenesis is categorized into two groups based on developmental histology, intramembranous and endochondral ossification. The role of blood vessels during endochondral ossification is well known, while their role in intramembranous ossification, especially the intertissue pathway, is poorly understood. Here, we demonstrate endothelial Yap/Taz is a novel regulator of intramembranous ossification in zebrafish. Appropriate blood flow is required for Yap/Taz transcriptional activation in endothelial cells and intramembranous ossification. Additionally, Yap/Taz transcriptional activity in endothelial cells specifically promotes intramembranous ossification. BMP expression by Yap/Taz transactivation in endothelial cells is also identified as a bridging factor between blood vessels and intramembranous ossification. Furthermore, the expression of Runx2 in pre-osteoblast cells is a downstream target of Yap/Taz transcriptional activity in endothelial cells. Our results provide novel insight into the relationship between blood flow and ossification by demonstrating intertissue regulation.


Inhibition of Yap/Taz transcriptional activity in endothelial cells downregulates intramembranous ossification. Because of the location of vessels and intramembranous bones, we hypothesized that
Yap/Taz transcriptional activity in endothelial cells regulates intramembranous ossification. To confirm our model, we generated two different dominant negative mutants, hTead2Δ N (amino acids 159-450) and hYapN (amino acids . The hTead2Δ N contains only C-terminal, Tead binding domain for Yap/Taz, causing endogenous Yap/Taz to fail to bind to endogenous Tead for transactivation. The hYapN consists of only the Tead binding domain and not the transactivation domain with endogenous Yap/Taz, failing to bind to authentic Tead for transactivation. To confirm that the truncated hTead2Δ N or hYapN inhibit Yap/Taz transcriptional activity, we measured the transcriptional activity of Yap/Taz based on the Gal4/UAS system ( Fig. 1a and Supplemental Fig. S1) 23 . 293T cells were transfected with pFR-Luc and pcDNA3.1-Gal4-hTead2Δ N for measuring the transcriptional activities of zYap or zTaz cells. Compared with control cells introduced pFR-Luc and pcDNA3.1-Gal4-hTead2Δ N, overexpression of zYap or zTaz dramatically enhanced the luciferase activity (lane 3 and 5 in each panel), representing that Gal4-hTead2Δ N/UAS can represents the transcriptional activity of zebrafish Yap/ Taz with luciferase activity. Based on these results, we also tested whether overexpression of GFP-hTead2Δ N and GFP-hYapN can inhibit Yap/Taz transcriptional activity (lane 4 and 6 in each panel). As expected, enhancements by zYap/zTaz were completely canceled by co-expressing hTead2Δ N or hYapN, representing that excess expression of hTead2Δ N or hYapN work as dominant negative mutants for zYap/zTaz transcriptional activity.
Next, we reduced Yap/Taz transcriptional activity in endothelial cells by specifically overexpressing hTead2Δ N (Fig. 1b). In this experiments, we prepared two transgenic fishlines, Tg(fli1: gal4-vp16) and Tg(UAS: GFP-htead2Δ N). By crossing these two lines, we obtained the fishline overexpressing GFP-hTead2Δ N in endothelial cells specifically. Intramembranous ossification was retarded in the opercle and cleithrum in embryos expressing hTead2Δ N compared to control Tg(fli1: gal4-vp16) (Fig. 1c). Meanwhile, the diameter of otoliths was little affected by overexpression of hTead2Δ N (Fig. 1c, right panel). Notably, angiogenesis in the head, especially in the opercular artery (ORA), was also little affected by expression of hTead2Δ N ( Supplementary Fig. S2a). Collectively, these results suggest that Yap/Taz transcriptional activity in endothelial cells promotes early intramembranous ossification.
We further investigated the role of Yap/Taz transcriptional activity during intramembranous ossification by overexpressing hYapN. Embryos of Tg(fli1: gal4-vp16): (UAS: GFP-hYapN) expressing hYapN in endothelial cells also showed similar results as Fig. 1b,c (Fig. 1d,e and Supplementary Fig. S2b). These results support our hypothesis that the inhibition of Yap/Taz transcriptional activity in endothelial cells downregulates early intramembranous ossification.

Overexpression of Yap/Taz in endothelial cells promotes intramembranous ossification.
To confirm that endothelial Yap/Taz is responsible for intramembranous ossification, we tested whether forced expression of Yap/Taz in endothelial cells affects early intramembranous ossification, because dominant-negatives could also affect pathways other than Yap/Taz transactivation. In these experiments, we focused on the size of the opercle, because it is one of the earliest intramembranous ossifications in zebrafish (Fig. 2). Embryos overexpressing Yap and Taz in endothelial cells showed larger opercle volumes in very early ossification compared to controls. Notably, cartilaginous tissues in head were little affected by expression of hTead2Δ N or Yap in endothelial cells ( Supplementary Fig. S6a). Thus, we concluded that Yap/Taz transcriptional activity in endothelial cells promotes intramembranous ossification.   Blood flow is required for Yap/Taz transcriptional activation in ORA and intramembranous ossification. Next we endeavored to verify whether Yap/Taz transcriptional activity does indeed occur in endothelial cells during opercle formation. For this purpose, we generated a fish line for monitoring Yap/Taz transcriptional activity specifically in endothelial cells. The fish line expresses Gal4-hTead2Δ N under the control of the fli1 promoter. We crossed this line with Tg(UAS: GFP): (fli1: myr-mCherry) to visualize the activity of Yap/ Taz only in endothelial cells. Since endogenous Yap/Taz can bind to hTead2Δ N, Yap/Taz is recruited on GFP coding region via Gal4/UAS binding. These Tg(fli1: gal4-htead2Δ N): (UAS: GFP): (fli1: myr-mCherry) exhibited GFP signals in the opercular artery (ORA) during opercle development between 48 to 96 hpf (Fig. 3a), indicating that Yap/Taz transcriptional activity increased in the ORA during opercle generation.
Since Yap/Taz transcriptional activity is regulated by mechanical stress factors, we hypothesized that shear stress derived from circulation regulates Yap/Taz transcriptional activity in the ORA (Fig. 3b). We treated embryos with 2, 3-Butanedione monoxime (BDM), which is an inhibitor of myosin ATPase and suppresses cardiac contraction to decrease circulation volume and blood pressure. Embryos treated with 3 mM and 6 mM BDM from 54 hpf to 60 hpf and to 72 hpf showed less GFP signal in the ORA, indicating that Yap/Taz transcriptional activity in the ORA is upregulated by blood flow. Furthermore, in similar conditions, opercle formation was abrogated by BDM treatment (Fig. 3c), while cartilaginous tissues in head were little affected ( Supplementary  Fig. S6b). Thus, we concluded that blood flow is required for Yap/Taz transcriptional activation in the ORA and intramembranous ossification.
Yap/Taz transcriptional activation in endothelial cells promotes runx2 expression. Since our results demonstrated that endothelial Yap/Taz regulates early intramembranous ossification, we hypothesized that Yap/Taz affects differentiation during osteogenesis. To confirm this model, we tested the expression levels of runx2 and osterix as markers for pre-osteoblasts and osteoblasts, respectively (Fig. 4a). To manipulate Yap/Taz transcriptional activity in endothelial cells, we prepared embryos expressing hTead2Δ N or Yap in endothelial cells at 60 hpf and performed whole mount in situ hybridization (WISH). Compared to the controls, hTead2Δ N-expressing fish showed less runx2 signals. Furthermore, Yap-overexpressing fish showed stronger runx2 signals, indicating that Yap/Taz transcriptional activation in endothelial cells promotes runx2 expression. In the case of osterix, the signal in Yap-overexpressing fish was comparable to the control, though hTead2Δ N-expressing fish showed less osterix signal. These data support the hypothesis that runx2 is essential for osterix expression but not sufficient. In summary, Yap/Taz transcriptional activation in endothelial cells promotes runx2 expression specifically during intramembranous ossification. We also performed experiments with BDM, which suppresses Yap/Taz transcriptional activity in ORA, similar to those shown in Fig. 3. Treatment with BDM from 54 to 60 hpf reduced the expression levels of runx2 and osterix in a dose dependent manner (Fig. 4b), supporting our model that Yap/Taz transcriptional activation in endothelial cells is required for runx2 expression in pre-osteoblast cells.
BMP pathway upregulates runx2 expression during opercle formation. We next investigated the bridging molecules/pathway between blood vessels and intramembranous ossification. Given the above results, we hypothesized that a secreted downstream factor of Yap/Taz might promote osteogenesis. The bone morphogenetic protein (BMP) is a family of secreted proteins that are well known osteogenic factors and promote runx2 expression 25 . Hence, we tested whether the BMP pathway regulates runx2 expression in the opercle by using DMH1, which is an inhibitor of the BMP receptor (Fig. 5a). Compared to the controls, DMH1 treatment significantly reduced the expression level of runx2, showing that the BMP pathway is essential for runx2 expression. To confirm the inhibitory effect of DMH1 with a complimentary approach, we generated Tg (runx2enhancer: gal4-vp16): (UAS: kaede) to monitor the enhancer activity of runx2. Kaede is a fluorescence protein, which changes color from green to red by photoconversion under ultra violet (UV) radiation. Using this property of Kaede, we were able to improve the temporal resolution in our monitored fish. Embryos were treated with DMH1 from 54 hpf and irradiated with UV at 60 hpf, and then Kaede-green intensity was measured at 72 hpf in the opercle area (Fig. 5b). Compared to the controls, DMH1 treatment significantly reduced Kaede-green intensity. These results further support our hypothesis that the BMP pathway is essential for runx2 expression in opercle development.

Yap/Taz transcriptional activity is required for bmp expression in endothelial cells. We next
tested whether the BMP family is indeed expressed in the ORA via Yap/Taz transcriptional activity. Since Bmp2a, 2b, and 4 have already been reported as secreted proteins from blood vessels, we focused on these homologs. In these experiments, we used glutaraldehyde for fixation because vascular structure in embryos fixed with paraformaldehyde collapsed during WISH experiments (Fig. 6a,b). At 72 hpf, signals from bmp2a, 2b, and 4 were detected specifically in the ORA using control embryos, while these signals were diminished in embryos expressing hTead2Δ N in endothelial cells. Notably, there were no remarkable signals in the opercle, suggesting that bmp expression is not derived from the opercle itself. Thus, our results demonstrate that Yap/Taz transcriptional activation in the ORA promotes bmp expression from endothelial cells during intramembranous ossification.

Bmp4 expression from endothelial cells promotes opercle formation. Finally, we investigated
which Bmp from endothelial cells is responsible for opercle formation. We prepared embryos overexpressing Bmp2a, 2b, and 4 in endothelial cells, and measured the volume of opercles at 72 hpf (Fig. 7). Compared to controls, Bmp4 expression in endothelial cells clearly enlarged the opercle, however the overexpression of Bmp2a and 2b did not. Thus, we concluded that Bmp4 is a bridging molecule between blood vessels and intramembranous ossification.
To determine that endothelial Yap/Taz-Bmp4 pathway is a sufficient factor in intramembranous ossification, we tested whether the effect of endothelial GFP-htead2Δ N could be cancelled by Bmp4 overexpression (data not

Discussion
In this paper, we demonstrated the intertissue relationship between blood flow and osteogenesis in zebrafish. Circulation activates Yap/Taz transcriptional activity in endothelial cells and transactivated Yap/Taz promotes Bmp4 expression in endothelial cells, and Runx2 expression in precursor cells of the opercle for its ossification.
The relationship between blood vessels and intramembranous ossification is poorly understood 2 . Due to the location of vessels and intramembranous bones, the linkage between these two has been implicated that vessels regulates intramembranous bone generations 26 . A previous genetic study also showed the importance of blood vessels for intramembranous ossification 6 . Thus, vascular structure has been regarded as a regulator for intramembranous ossification. However, though most studies have shown defects in intramembranous ossification related to undeveloped vascular structure, the molecular linkages between blood vessels and intramembranous ossification remained unidentified. Our results demonstrate, for the first time, that endothelial Yap/Taz is a key factor in intramembranous ossification but has little effect on angiogenesis, at least in the opercular artery.
In our study, manipulation of Yap/Taz transactivation in endothelial cells affected the very early phase of intramembranous ossification but the effect was negated in later stages. We hypothesize that Yap/Taz transactivation has two roles, promoting early intramembranous ossification and inhibiting bone maturation in later phase. Indeed, GFP expression of Tg(fli1: gal4-htead2Δ N): (UAS: GFP) in the ORA was transient. These results imply that Yap/Taz transactivation may be tightly regulated during fish development, and should be downregulated at certain stages. The expression of Runx2 as a target of Yap/Taz transactivation may explain the bipolar function of Yap/Taz transactivation in endothelial cells during intramembranous ossification. Runx2 has been shown to be a master regulator of osteogenesis including intramembranous ossification 27,28 , while forced expression of Runx2 in osteoblast cells results in defective osteogenesis presenting as osteopenia 29 . Our results thus suggest another The role of Yap/Taz transactivation in osteoblast cells has remained undefined. Although several studies showed the requirement of Yap/Taz in osteogenesis 17,18,30 , most in in vivo studies in mice, by depleting Taz showed subtle effects on osteogenesis [19][20][21][22] . Here, we succeeded in detecting the transactivation of Yap/Taz in osteoblast cells during intramembranous ossification. Meanwhile, the size of the opercle was similar to the controls in early intramembranous osteogenesis by upregulation and downregulation of Yap/Taz transactivation in osteoblast cells. It is possible that Yap/Taz transactivation is required in a later phase of homeostasis of bone formation. Further studies are required to understand the role of Yap/Taz transactivation in osteoblasts.
We hypothesized that Yap/Taz transactivation in endothelial cells may be also involved in pathological ossification, such as arteriosclerosis. Since lesions of arteriosclerosis are frequently found near the branching point in the aorta where turbulent flow occures [31][32][33] , several studies have implied that turbulent flow is a factor in arteriosclerosis. Additionally, recent studies have shown that Yap/Taz transactivation was dramatically enhanced by mechanical stress including flow 15,34,35 . Notably, our results show that endothelial BMP4 and osteoblast Runx2 are bridging factors between blood vessels and osteogenesis. Similar to our model, BMP in endothelial cells 36,37 and Runx2 in vascular smooth muscle 38,39 are also key factors for arteriosclerosis in mice. In summary, our results provide clues towards understanding the relationship between turbulent flow and ectopic ossification.

Materials and Methods
Zebrafish husbandry. Zebrafish (Danio rerio) strains were maintained under standard conditions. We used a fish medium containing 0.03% sea salt and 0.006% methylene blue as an antiseptic agent. Embryo stages were determined from hpf at 28 °C 40 . Microinjections and chemical treatment of embryos were undertaken as described below. All experiments using zebrafish were carried out with approval from the institutional ethics committee of the University of Tokyo, Japan, strictly following the guidelines set for the usage of animals by this committee.
Transgenic zebrafish lines. Tol2 transposase mRNA was synthesized in vitro with SP6 RNA polymerase from a NotI linearized pCS-TP vector. To generate transgenic zebrafish lines, the corresponding Tol2-based DNAs (100 ng/μ l) were microinjected along with Tol2 transposase mRNA (25 ng/μ l) into one-cell stage embryos of the wild type strain AB. We generated Tg Image acquisition, processing, and quantification. Zebrafish embryos were dechorionated and mounted in 1% low-melting agarose on a 35 mm glass bottomed dish (Asahi Techno Glass) with 0.016% tricaine (Sigma-Aldrich) in fish medium as described previously 48 . The dish was submerged in fish medium containing 0.001% tricaine. Bone staining with alizarin red s (Wako) was performed as per the manufacturer's protocol.
Confocal images were obtained with an FV1000 confocal upright microscope system (Olympus) equipped with a 20× water-immersion lens (XLUMPlanFL, NA 1.0). 473 nm and 559 nm laser lines were employed for green fluorescence protein (GFP and Kaede-green), and red fluorescence molecules (mCherry and alizarin red s), respectively. Image files were processed and analyzed using FLUOVIEW Viewer software (Olympus), MetaMorph (Molecular Devices), and Volocity (perkinelmer).
Chemical treatment of zebrafish embryos. Zebrafish embryos were treated with BDM and DMH1 (Sigma-Aldrich) in fish medium. Whole-mount in situ hybridization. Whole-mount in situ hybridization of zebrafish embryos was performed as described previously 49 . Pigmentation of embryos was inhibited by 0.04 mM 1-phenyl-2-thiourea (PTU) (Sigma-Aldrich) from 8 hpf onwards.
Microinjection of plasmid to zebrafish embryos. Plasmid DNA (100 ng/μ l) mixed with injection buffer (120 mM KCl, 20 mM Hepes, 0.25% phenol red) was microinjected into embryos in their one-cell stage.
Statistical analysis. Data are expressed as the average ± SD. The statistical significance for paired samples was determined using Student's t-test.