Differentially expressed transcriptomes of P7 mouse tendon cells with targeted deletion of TGF-beta signaling. Differentially expressed transcriptomes of P7 mouse tendon cells with targeted deletion of TGF-beta signaling

Background: Our group has previously shown that disruption of TGFβ signaling in mouse limb mesenchyme resulted in arrested tendon formation (Pryce et at, 2007). To examine the role of TGFβ signaling in later stages of tendon development, the TGF-beta type II receptor gene (Tgfbr2) was targeted in the Scleraxis (Scx)-expressing cell lineage using the Cre-lox recombination system. We find that tendon development was not disrupted in mutant (Tgfbr2;ScxCre) embryos. However, shortly after birth tenocytes underwent dedifferentiation in which the cell lost differentiation markers and reverted to a more stem/progenitor state. Purpose: To determine gene expression changes in Tgfbr2;ScxCre mutant tendon cells. Methods: We performed scRNA-seq for transcriptome changes in P7 mutant tendon cells, a stage at which the majority of the mutant cells is dedifferentiated. Briefly, tendons from P7 mutant and wild-type (as a control) pups were harvested and enzymatically digested. The released cells were then subjected to scRNA-seq analysis using 10x Genomics platform. Results: Using unsupervised hierarchical clustering, we identified two major clusters corresponding to mutant (dedifferentiated) cells and wild-type tenocytes in the respective samples. Findings from the pairwise comparison of the gene set between the P7 wild-type tenocyte and mutant cell clusters do not only lend support to our notion that the mutant cells lost their differentiation state, but also suggest the possibility of induction of some developmental programs in these cells, a general feature in cellular dedifferentiation. Conclusions: TGF-beta signaling is critical for maintenance of the tendon cell fate. Overall design: Tendon mRNA profiles of 7-day old Tgfbr2;ScxCre mutant and wild type (as a control) mice were generated by deep sequencing using Illumina NextSeq (MidOutput 150). Two samples were submitted, in which each sample consist of enzymatically-released tendon cells that were harvested and pooled from 2 pups.

研究背景：本团队此前已证实，小鼠肢体间充质中转化生长因子β（TGFβ）信号通路的阻断会导致肌腱形成停滞（Pryce等，2007）。为探究TGFβ信号通路在肌腱发育后期的作用，本研究利用Cre-lox重组系统，在表达硬化素（Scleraxis，Scx）的细胞谱系中靶向敲除转化生长因子βⅡ型受体基因（Tgfbr2）。研究发现，突变体（Tgfbr2;ScxCre）胚胎的肌腱发育并未受到阻滞。然而，出生后不久，肌腱细胞（tenocytes）即发生去分化：细胞丧失分化标志物，逆转至更为原始的干细胞/祖细胞状态。研究目的：明确Tgfbr2;ScxCre突变体肌腱细胞中的基因表达变化。实验方法：针对出生后7天（P7）的突变体肌腱细胞，本研究开展单细胞RNA测序（scRNA-seq）以分析其转录组变化——该阶段多数突变体细胞已发生去分化。简要流程如下：收集P7突变体与野生型（对照）幼鼠的肌腱，进行酶解消化；将释放的细胞利用10x Genomics平台进行单细胞RNA测序分析。实验结果：通过无监督层次聚类，我们在对应样本中鉴定出两个主要细胞簇，分别对应突变体（去分化）细胞与野生型肌腱细胞。对P7野生型肌腱细胞簇与突变体细胞簇的基因集进行两两比较，所得结果不仅支持了“突变体细胞丧失分化状态”这一结论，还提示这些细胞可能被诱导激活了部分发育程序——这是细胞去分化的普遍特征。研究结论：转化生长因子β（TGFβ）信号通路对维持肌腱细胞命运至关重要。实验整体设计：利用Illumina NextSeq（MidOutput 150）测序平台进行深度测序，获取出生后7日龄Tgfbr2;ScxCre突变体与野生型（对照）小鼠的肌腱mRNA表达谱。本次共提交2个样本，每个样本均包含从2只幼鼠中收集并混合的酶解释放的肌腱细胞。