Pioneering function of Yap and Taz in promoting osteogenesis of neural crest cells by preventing chondrogenesis. Pioneering function of Yap and Taz in promoting osteogenesis of neural crest cells by preventing chondrogenesis

Neural crest cells (NCCs) are multipotent stem cells with a remarkable ability to differentiate into multiple cell lineages, including osteoblasts and chondrocytes. NCCs contribute to the majority of craniofacial skeleton, yet the molecular mechanisms regulating NCCs diversification into osteoblasts or chondrocytes remain poorly understood. We found that Yap and Taz function redundantly as key determinants of the osteogenesis versus chondrogenesis fate decision and differentiation in NCCs in vitro, ex vivo and in vivo, and Yap/Taz-deficiency in NCCs resulted in a switch from osteogenesis to chondrogenesis. Comprehensive analysis of unbiased datasets including CUT&RUN-seq and RNA-seq indicated that Yap/Taz directly regulate key genes that govern osteogenesis and chondrogenesis. During NCC-derived osteogenesis, Yap/Taz promote expression of osteogenic genes such as Runx2 and Sp7 but repress expression of chondrogenic genes such as Sox9 and Col2a1. Further, we found Yap/Taz directly interact with β-catenin in NCCs to coordinately promote osteoblast differentiation and repress chondrogenesis. Together our data indicate that Yap/Taz promote osteogenesis in NCCs by preventing chondrogenesis, partly through interactions with the Wnt-β-catenin pathway. Overall design: 1. We performed CUT&RUN in O9-1 neural crest cells using Yap, IGG and H3K27me3 antibodies, followed by sequencing. 2. O9-1 cells were cultured under conditioned basal medium. Then the cells were seperated into 2 group: one group used for Yap/Taz dKD and the other group used as control. For the siRNA-mediated KD of Yap and Taz, O9-1 cells were plated at 50% confluency in the conditioned basal media and transfected with siRNA SMART pools of Yap (Dharmacon, L-046247-01-0005), Taz (Dharmacon, L-041057-01), and nontargeting siRNA (Dharmacon, D-001810-04-05) for 48 h according to the guidelines of the RNAiMAX transfection procedure (Life Technologies, 13778075) and cultured to 80% confluency. Then the cells were harvested and used for CUT&Tag experiment.

神经嵴细胞（Neural crest cells, NCCs）是一类多能干细胞，具备分化为包括成骨细胞、软骨细胞在内的多种细胞谱系的卓越潜能。神经嵴细胞构成了颅面骨骼的绝大部分，但调控其向成骨细胞或软骨细胞分化的分子机制仍不甚明晰。本研究发现，Yap与Taz存在功能冗余，作为调控神经嵴细胞体外、离体及体内成骨-软骨细胞命运决定与分化的关键调控因子；神经嵴细胞中Yap/Taz缺失会导致细胞命运从成骨向软骨转化。对包括CUT&RUN测序（CUT&RUN-seq）与RNA测序（RNA-seq）在内的无偏数据集进行综合分析后发现，Yap/Taz可直接调控成骨与软骨细胞分化的关键基因。在神经嵴细胞源性成骨过程中，Yap/Taz可促进成骨相关基因（如Runx2与Sp7）的表达，同时抑制软骨相关基因（如Sox9与Col2a1）的表达。进一步研究表明，Yap/Taz可在神经嵴细胞中直接与β-连环蛋白（β-catenin）结合，协同促进成骨细胞分化并抑制软骨细胞生成。综上，本研究数据证实，Yap/Taz可通过抑制软骨细胞生成（部分依赖于与Wnt-β-连环蛋白通路的相互作用），促进神经嵴细胞的成骨分化。

实验设计：
1. 采用Yap抗体、IGG抗体及H3K27me3抗体对O9-1神经嵴细胞开展CUT&RUN实验，随后进行测序。
2. 将O9-1细胞置于条件化基础培养基中培养，随后分为两组：一组用于Yap/Taz双敲低（dKD）实验，另一组作为空白对照。针对Yap与Taz的siRNA介导敲低实验：将O9-1细胞以50%汇合度接种于条件化基础培养基内，参照RNAiMAX转染试剂（"Life Technologies, 13778075"）的操作指南，分别转染Yap的siRNA SMARTpool（"Dharmacon, L-046247-01-0005"）、Taz的siRNA SMARTpool（"Dharmacon, L-041057-01"）及非靶向对照siRNA（"Dharmacon, D-001810-04-05"），转染48小时后继续培养至细胞汇合度达80%，随后收集细胞用于CUT&Tag实验。