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

Background: Our group has previously shown that disruption of TGFbeta signaling in mouse limb mesenchyme resulted in arrested tendon formation (Pryce et at, 2007). To examine the role of TGFbeta 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. 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（TGFbeta）信号通路的阻断会导致肌腱形成停滞（Pryce等，2007）。为探究转化生长因子β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，在表达肌腱转录因子Scleraxis（Scx）的细胞谱系中靶向敲除Ⅱ型转化生长因子β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，但在出生后不久，肌腱细胞（tenocytes）发生去分化：细胞丢失分化标志物，并逆转为更接近干细胞/祖细胞的状态。 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：针对出生后第7天（P7）的突变体肌腱细胞（此时多数突变细胞已发生去分化）的转录组变化，我们开展了单细胞RNA测序（scRNA-seq）分析。简要而言，我们收集了P7突变体与野生型（作为对照）幼鼠的肌腱，并通过酶解法解离得到单细胞悬液；随后利用10x Genomics平台对解离得到的细胞进行scRNA-seq分析。 实验结果：通过无监督层次聚类分析，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后发现，该结果不仅验证了我们的假说——突变体细胞丢失了分化状态，还提示这些细胞可能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信号通路对于维持肌腱细胞命运至关重要。本研究通过Illumina NextSeq（MidOutput 150）平台进行深度测序，获取了7日龄Tgfbr2;ScxCre突变体与野生型（作为对照）小鼠的肌腱mRNA表达谱。本次共提交两个样本，每个样本均为从2只幼鼠中收集并混合得到的酶解解离肌腱细胞。