Transient and lineage-restricted requirement of Ebf3 for sternum ossification

Osteoblasts arise from bone-surrounding connective tissue containing tenocytes and fibroblasts. Lineages of these cell populations and mechanisms of their differentiation are not well understood. Screening enhancer-trap lines of zebrafish allowed us to identify Ebf3 as a transcription factor marking tenocytes and connective tissue cells in skeletal muscle of embryos. Knockout of Ebf3 in mice had no effect on chondrogenesis but led to sternum ossification defects as a result of defective generation of Runx2+ pre-osteoblasts. Conditional and temporal Ebf3 knockout mice revealed requirements of Ebf3 in the lateral plate mesenchyme cells (LPMs), especially in tendon/muscle connective tissue cells, and a stage-specific Ebf3 requirement at embryonic day 9.5-10.5. Upregulated expression of connective tissue markers, such as Egr1/2 and Osr1, increased number of Islet1+ mesenchyme cells, and downregulation of gene expression of the Runx2 regulator Shox2 in Ebf3-deleted thoracic LPMs suggest crucial roles of Ebf3 in the onset of lateral plate mesoderm differentiation towards osteoblasts forming sternum tissues. Thoracic LPM mRNA profiles of E10.5 Hetero (Prx1-Cre Ebf3 flox/+) and Homo KO (Prx1-Cre Ebf3 flox/flox) mice were generated by RNA sequencing, in triplicate, using Illumina NextSeq500.成骨细胞起源于包绕骨骼的结缔组织，该组织内含肌腱细胞（tenocytes）与成纤维细胞。目前对于这些细胞类群的谱系及其分化机制尚不明晰。通过筛选斑马鱼增强子陷阱（enhancer-trap）品系，我们鉴定出Ebf3作为一种转录因子，可标记胚胎骨骼肌中的肌腱细胞与结缔组织细胞。在小鼠中敲除Ebf3不会对软骨发生（chondrogenesis）产生影响，但会因Runx2阳性前成骨细胞生成障碍，导致胸骨骨化缺陷。条件性与时间依赖性Ebf3敲除小鼠模型显示，Ebf3在侧板间充质细胞（lateral plate mesenchyme cells, LPMs）中发挥必需作用，尤其在肌腱/肌肉结缔组织细胞中，且其必需性具有发育阶段特异性，即胚胎发育第9.5-10.5天。在Ebf3缺失的胸侧板间充质细胞中，结缔组织标志物（如Egr1/2与Osr1）的表达上调、Islet1阳性间充质细胞数量增加，且Runx2调控因子Shox2的基因表达下调，这表明Ebf3在侧板中胚层分化为形成胸骨组织的成骨细胞的起始过程中发挥关键作用。本研究采用Illumina NextSeq500测序平台，对胚胎发育第10.5天的杂合子（Prx1-Cre Ebf3 flox/+）与纯合敲除（Prx1-Cre Ebf3 flox/flox）小鼠的胸侧板间充质细胞进行了三次重复的RNA测序，以获取其mRNA表达谱。