A Cd9+Cd271+ stem/progenitor population and the SHP2 pathway contribute to neonatal-to-adult switching that regulates tendon maturation
Cd9 + Cd271 + 干细胞/祖细胞群体和 SHP2 通路对调控肌腱成熟的新生儿-成年转换的贡献
Graphical abstract
Keywords
Research topic(s)
Introduction
Developmental biology is the template for tissue regeneration. Most adult mammalian tissues cannot achieve complete regeneration after injury (Iismaa et al., 2018), and repaired tissue often displays immature characteristics and physiological functions (Eming et al., 2014). Hence, more promising regenerative therapies require a deeper understanding of tissue maturation. As a dense connective tissue that transmits the mechanical force exerted by the muscles to the bones (Kjær, 2004; Nourissat et al., 2015), a tendon mainly consists of a complex network of highly specialized extracellular matrices maintained by tenocytes (Subramanian et al., 2018). Tendon injuries are common, accounting for almost 50% of musculoskeletal disorders (James et al., 2008). Because of the low regeneration capacity of mature tenocytes, an injured tendon seldom recovers completely. In the repaired tendon, only small, fetal-like collagen fibrils can be generated, which have inferior biomechanical properties and a high re-rupture rate (Andarawis-Puri et al., 2015). Therefore, restoring a functional tendon requires a more comprehensive understanding of the cellular and molecular mechanisms of the tendon maturation.
发育生物学是组织再生的模板。大多数成年哺乳动物组织在受伤后无法实现完全再生（ Iismaa et al., 2018 ），修复的组织常常表现出不成熟的特征和生理功能（ Eming et al., 2014 ）。因此，更有前景的再生疗法需要对组织成熟过程有更深入的理解。肌腱作为一种将肌肉产生的机械力传递给骨骼的致密结缔组织（ Kjær, 2004 ； Nourissat et al., 2015 ），主要由腱细胞维持的高度专业化细胞外基质复杂网络组成（ Subramanian et al., 2018 ）。肌腱损伤很常见，约占肌肉骨骼疾病的 50%（ James et al., 2008 ）。由于成熟腱细胞再生能力低，受伤肌腱很少能完全恢复。在修复的肌腱中，只能生成小型的、类胎儿样胶原纤维，这些纤维具有较差的生物力学特性和较高的再断裂率（ Andarawis-Puri et al., 2015 ）。因此，恢复功能性肌腱需要对肌腱成熟的细胞和分子机制有更全面的理解。
32.
Iismaa, S.E. ∙ Kaidonis, X. ∙ Nicks, A.M. ...
Comparative regenerative mechanisms across different mammalian tissues
Npj Regen. Med. 2018; 3:619.
Eming, S.A. ∙ Martin, P. ∙ Tomic-Canic, M.
Wound repair and regeneration: mechanisms, signaling, and translation
Sci. Transl. Med. 2014; 6, 265sr638.
Kjær, M.
Role of extracellular matrix in adaptation of tendon and skeletal muscle to mechanical loading
Physiol. Rev. 2004; 84:649-69846.
Nourissat, G. ∙ Berenbaum, F. ∙ Duprez, D.
Tendon injury: from biology to tendon repair
Nat. Rev. Rheumatol. 2015; 11:223-23355.
Subramanian, A. ∙ Kanzaki, L.F. ∙ Galloway, J.L. ...
Mechanical force regulates tendon extracellular matrix organization and tenocyte morphogenesis through TGFbeta signaling
ELife. 2018; 7, e3806934.
James, R. ∙ Kesturu, G. ∙ Balian, G. ...
Tendon: biology, biomechanics, repair, growth factors, and evolving treatment options
J. Hand Surg. 2008; 33:102-1123.
Andarawis-Puri, N. ∙ Flatow, E.L. ∙ Soslowsky, L.J.
Tendon basic science: development, repair, regeneration, and healing
J. Orthop. Res. 2015; 33:780-784发育生物学是组织再生的模板。大多数成年哺乳动物组织在受伤后无法实现完全再生（ Iismaa et al., 2018
32.
Iismaa, S.E. ∙ Kaidonis, X. ∙ Nicks, A.M. ...
Comparative regenerative mechanisms across different mammalian tissues
Npj Regen. Med. 2018; 3:619.
Eming, S.A. ∙ Martin, P. ∙ Tomic-Canic, M.
Wound repair and regeneration: mechanisms, signaling, and translation
Sci. Transl. Med. 2014; 6, 265sr638.
Kjær, M.
Role of extracellular matrix in adaptation of tendon and skeletal muscle to mechanical loading
Physiol. Rev. 2004; 84:649-69846.
Nourissat, G. ∙ Berenbaum, F. ∙ Duprez, D.
Tendon injury: from biology to tendon repair
Nat. Rev. Rheumatol. 2015; 11:223-23355.
Subramanian, A. ∙ Kanzaki, L.F. ∙ Galloway, J.L. ...
Mechanical force regulates tendon extracellular matrix organization and tenocyte morphogenesis through TGFbeta signaling
ELife. 2018; 7, e3806934.
James, R. ∙ Kesturu, G. ∙ Balian, G. ...
Tendon: biology, biomechanics, repair, growth factors, and evolving treatment options
J. Hand Surg. 2008; 33:102-1123.
Andarawis-Puri, N. ∙ Flatow, E.L. ∙ Soslowsky, L.J.
Tendon basic science: development, repair, regeneration, and healing
J. Orthop. Res. 2015; 33:780-784Compared with our in-depth understanding of tendon development during the embryonic stage, maturation of postnatal tendons is less understood. The key stage and mechanisms for tendon maturation are less discussed, and most studies focus on morphology or mechanical changes. Previous studies have demonstrated that tendon development is dependent on regulation of collagen fibrillogenesis, including assembly, deposition, and organization of the collagen fibers (Zhang et al., 2005). The fibril-forming collagens (collagen I, collagen III, and collagen V), fibril-associated collagens (collagen XII and collagen XIV), and small leucine-rich repeat proteoglycans (SLRPs; FMOD, LUM, DCN, and BGN) are key regulators of these processes (Birk and Zycband, 1994; Izu et al., 2021; Zhang et al., 2005). Key transcription factors (such as Scx [Brent et al., 2003; Huang et al., 2019; Wang et al., 2011], Mkx [Liu et al., 2010], and Egr1 [Guerquin et al., 2013; Liu et al., 2010]) and signaling pathways (including transforming growth factor β [TGFβ; Edom-Vovard and Duprez, 2004; Havis et al., 2016; Murtaugh et al., 2001], fibroblast growth factor [FGF; Brent and Tabin, 2004], and connective tissue growth factor [CTGF; Li et al., 2019]) required for tendon formation and repair have also been investigated, and most of these molecules are engaged in the onset of tendon differentiation. The cellular and molecular mechanisms of maturation of tendon are less understood.
与我们对胚胎期肌腱发育的深入理解相比，出生后肌腱的成熟过程研究较少。肌腱成熟的关键阶段和机制讨论不多，大多数研究集中在形态或力学变化上。先前研究表明，肌腱发育依赖于胶原纤维形成的调控，包括胶原纤维的组装、沉积和组织（ Zhang et al., 2005 ）。成纤维胶原（I 型胶原、III 型胶原和 V 型胶原）、纤维相关胶原（XII 型胶原和 XIV 型胶原）以及小亮氨酸富集重复蛋白多糖（SLRPs；FMOD、LUM、DCN 和 BGN）是这些过程的关键调节因子（ Birk and Zycband, 1994 ； Izu et al., 2021 ； Zhang et al., 2005 ）。对肌腱形成和修复所需的关键转录因子（如 Scx [ Brent et al., 2003 ； Huang et al., 2019 ； Wang et al., 2011 ]、Mkx [ Liu et al., 2010 ]和 Egr1 [ Guerquin et al., 2013 ； Liu et al., 2010 ]）和信号通路（包括转化生长因子β [TGFβ； Edom-Vovard and Duprez, 2004 ； Havis et al., 2016 ； Murtaugh et al., 2001 ]、成纤维细胞生长因子[FGF； Brent and Tabin, 2004 ]和结缔组织生长因子[CTGF； Li et al., 2019 ]）也已被研究，这些分子大多参与肌腱分化的起始过程。肌腱成熟的细胞和分子机制研究较少。
65.
Zhang, G. ∙ Young, B.B. ∙ Ezura, Y. ...
Development of tendon structure and function: regulation of collagen fibrillogenesis
J. Musculoskelet. Neuronal Interact. 2005; 5:5-217.
Birk, D.E. ∙ Zycband, E.
Assembly of the tendon extracellular matrix during development
J. Anat. 1994; 184:457-46333.
Izu, Y. ∙ Adams, S.M. ∙ Connizzo, B.K. ...
Collagen XII mediated cellular and extracellular mechanisms regulate establishment of tendon structure and function
Matrix Biol. 2021; 95:52-6765.
Zhang, G. ∙ Young, B.B. ∙ Ezura, Y. ...
Development of tendon structure and function: regulation of collagen fibrillogenesis
J. Musculoskelet. Neuronal Interact. 2005; 5:5-2110.
Brent, A.E. ∙ Schweitzer, R. ∙ Tabin, C.J.
A somitic compartment of tendon progenitors
Cell. 2003; 113:235-24829.
Huang, A.H. ∙ Watson, S.S. ∙ Wang, L. ...
Requirement for Scleraxis in the recruitment of mesenchymal progenitors during embryonic tendon elongation
Development Dev. 2019;, 18278258.
Wang, L. ∙ Bresee, C.S. ∙ Jiang, H. ...
Scleraxis is required for differentiation of the stapedius and tensor tympani tendons of the middle ear
J. Assoc. Res. Otolaryngol. 2011; 12:407-42141.
Liu, W. ∙ Watson, S.S. ∙ Lan, Y. ...
The atypical homeodomain transcription factor mohawk controls tendon morphogenesis
Mol. Cell. Biol. 2010; 30:4797-480725.
Guerquin, M.-J. ∙ Charvet, B. ∙ Nourissat, G. ...
Transcription factor EGR1 directs tendon differentiation and promotes tendon repair
J. Clin. Invest. 2013; 123:3564-357641.
Liu, W. ∙ Watson, S.S. ∙ Lan, Y. ...
The atypical homeodomain transcription factor mohawk controls tendon morphogenesis
Mol. Cell. Biol. 2010; 30:4797-480717.
Edom-Vovard, F. ∙ Duprez, D.
Signals regulating tendon formation during chick embryonic development
Dev. Dyn. 2004; 229:449-45727.
Havis, E. ∙ Bonnin, M.-A. ∙ de Lima, J.E. ...
TGFβ and FGF promote tendon progenitor fate and act downstream of muscle contraction to regulate tendon differentiation during chick limb development
Development. 2016;, 13624244.
Murtaugh, L.C. ∙ Zeng, L. ∙ Chyung, J.H. ...
The chick transcriptional repressor Nkx3.2 acts downstream of Shh to promote BMP-dependent axial chondrogenesis
Dev. Cell. 2001; 1:411-4229.
Brent, A.E. ∙ Tabin, C.J.
FGF acts directly on the somitic tendon progenitors through the Ets transcription factors Pea3 and Erm to regulate scleraxis expression
Development. 2004; 131:3885-389640.
Li, X. ∙ Pongkitwitoon, S. ∙ Lu, H. ...
CTGF induces tenogenic differentiation and proliferation of adipose-derived stromal cells
J. Orthop. Res. 2019; 37:574-582与我们对胚胎期肌腱发育的深入理解相比，出生后肌腱的成熟过程研究较少。肌腱成熟的关键阶段和机制讨论不多，大多数研究集中在形态或力学变化上。先前研究表明，肌腱发育依赖于胶原纤维形成的调控，包括胶原纤维的组装、沉积和组织（ Zhang et al., 2005
65.
Zhang, G. ∙ Young, B.B. ∙ Ezura, Y. ...
Development of tendon structure and function: regulation of collagen fibrillogenesis
J. Musculoskelet. Neuronal Interact. 2005; 5:5-217.
Birk, D.E. ∙ Zycband, E.
Assembly of the tendon extracellular matrix during development
J. Anat. 1994; 184:457-46333.
Izu, Y. ∙ Adams, S.M. ∙ Connizzo, B.K. ...
Collagen XII mediated cellular and extracellular mechanisms regulate establishment of tendon structure and function
Matrix Biol. 2021; 95:52-6765.
Zhang, G. ∙ Young, B.B. ∙ Ezura, Y. ...
Development of tendon structure and function: regulation of collagen fibrillogenesis
J. Musculoskelet. Neuronal Interact. 2005; 5:5-2110.
Brent, A.E. ∙ Schweitzer, R. ∙ Tabin, C.J.
A somitic compartment of tendon progenitors
Cell. 2003; 113:235-24829.
Huang, A.H. ∙ Watson, S.S. ∙ Wang, L. ...
Requirement for Scleraxis in the recruitment of mesenchymal progenitors during embryonic tendon elongation
Development Dev. 2019;, 18278258.
Wang, L. ∙ Bresee, C.S. ∙ Jiang, H. ...
Scleraxis is required for differentiation of the stapedius and tensor tympani tendons of the middle ear
J. Assoc. Res. Otolaryngol. 2011; 12:407-42141.
Liu, W. ∙ Watson, S.S. ∙ Lan, Y. ...
The atypical homeodomain transcription factor mohawk controls tendon morphogenesis
Mol. Cell. Biol. 2010; 30:4797-480725.
Guerquin, M.-J. ∙ Charvet, B. ∙ Nourissat, G. ...
Transcription factor EGR1 directs tendon differentiation and promotes tendon repair
J. Clin. Invest. 2013; 123:3564-357641.
Liu, W. ∙ Watson, S.S. ∙ Lan, Y. ...
The atypical homeodomain transcription factor mohawk controls tendon morphogenesis
Mol. Cell. Biol. 2010; 30:4797-480717.
Edom-Vovard, F. ∙ Duprez, D.
Signals regulating tendon formation during chick embryonic development
Dev. Dyn. 2004; 229:449-45727.
Havis, E. ∙ Bonnin, M.-A. ∙ de Lima, J.E. ...
TGFβ and FGF promote tendon progenitor fate and act downstream of muscle contraction to regulate tendon differentiation during chick limb development
Development. 2016;, 13624244.
Murtaugh, L.C. ∙ Zeng, L. ∙ Chyung, J.H. ...
The chick transcriptional repressor Nkx3.2 acts downstream of Shh to promote BMP-dependent axial chondrogenesis
Dev. Cell. 2001; 1:411-4229.
Brent, A.E. ∙ Tabin, C.J.
FGF acts directly on the somitic tendon progenitors through the Ets transcription factors Pea3 and Erm to regulate scleraxis expression
Development. 2004; 131:3885-389640.
Li, X. ∙ Pongkitwitoon, S. ∙ Lu, H. ...
CTGF induces tenogenic differentiation and proliferation of adipose-derived stromal cells
J. Orthop. Res. 2019; 37:574-582Another prerequisite for deciphering the regulation of tendon maturation is to resolve its dynamic cellular composition. Researchers have identified several tendon stem/progenitor cells (TSPCs) with different cell markers, including embryonic Scx+ TSPCs (Bi et al., 2007), perivascular CD146+ TSPCs (Harvey et al., 2019) that have enriched the seed cell repertoire for tissue engineering. Emerging single-cell sequencing technology has also provided prospects for characterizing new cell types (Kumar et al., 2017; Papalexi and Satija, 2018). With this technique, regeneration-related Nes+ (Yin et al., 2016) and Tppp3+ (Harvey et al., 2019) tendon stem cells were identified. However, the critical cell populations and their dynamic changes, focusing on tendon maturation, are less understood.
解析肌腱成熟调控的另一个前提是解决其动态细胞组成问题。研究人员已经鉴定了几种具有不同细胞标记的肌腱干/祖细胞（TSPCs），包括胚胎期 Scx + TSPCs（ Bi et al., 2007 ）、血管周围 CD146 + TSPCs（ Harvey et al., 2019 ），这些发现丰富了组织工程的种子细胞库。新兴的单细胞测序技术也为鉴定新细胞类型提供了前景（ Kumar et al., 2017 ； Papalexi and Satija, 2018 ）。利用这一技术，研究者鉴定了与再生相关的 Nes + （ Yin et al., 2016 ）和 Tppp3 + （ Harvey et al., 2019 ）肌腱干细胞。然而，关注肌腱成熟的关键细胞群体及其动态变化的研究仍然较少。
6.
Bi, Y. ∙ Ehirchiou, D. ∙ Kilts, T.M. ...
Identification of tendon stem/progenitor cells and the role of the extracellular matrix in their niche
Nat. Med. 2007; 13:1219-122726.
Harvey, T. ∙ Flamenco, S. ∙ Fan, C.-M.
A Tppp3+Pdgfra+ tendon stem cell population contributes to regeneration and reveals a shared role for PDGF signalling in regeneration and fibrosis
Nat. Cell Biol. 2019; 21:1490-150339.
Kumar, P. ∙ Tan, Y. ∙ Cahan, P.
Understanding development and stem cells using single cell-based analyses of gene expression
Development. 2017; 144:17-3247.
Papalexi, E. ∙ Satija, R.
Single-cell RNA sequencing to explore immune cell heterogeneity
Nat. Rev. Immunol. 2018; 18:35-4563.
Yin, Z. ∙ Hu, J. ∙ Yang, L. ...
Single-cell analysis reveals a nestin+ tendon stem/progenitor cell population with strong tenogenic potentiality
Sci. Adv. 2016; 2, e160087426.
Harvey, T. ∙ Flamenco, S. ∙ Fan, C.-M.
A Tppp3+Pdgfra+ tendon stem cell population contributes to regeneration and reveals a shared role for PDGF signalling in regeneration and fibrosis
Nat. Cell Biol. 2019; 21:1490-1503解析肌腱成熟调控的另一个前提是解决其动态细胞组成问题。研究人员已经鉴定了几种具有不同细胞标记的肌腱干/祖细胞（TSPCs），包括胚胎期 Scx + TSPCs（ Bi et al., 2007
6.
Bi, Y. ∙ Ehirchiou, D. ∙ Kilts, T.M. ...
Identification of tendon stem/progenitor cells and the role of the extracellular matrix in their niche
Nat. Med. 2007; 13:1219-122726.
Harvey, T. ∙ Flamenco, S. ∙ Fan, C.-M.
A Tppp3+Pdgfra+ tendon stem cell population contributes to regeneration and reveals a shared role for PDGF signalling in regeneration and fibrosis
Nat. Cell Biol. 2019; 21:1490-150339.
Kumar, P. ∙ Tan, Y. ∙ Cahan, P.
Understanding development and stem cells using single cell-based analyses of gene expression
Development. 2017; 144:17-3247.
Papalexi, E. ∙ Satija, R.
Single-cell RNA sequencing to explore immune cell heterogeneity
Nat. Rev. Immunol. 2018; 18:35-4563.
Yin, Z. ∙ Hu, J. ∙ Yang, L. ...
Single-cell analysis reveals a nestin+ tendon stem/progenitor cell population with strong tenogenic potentiality
Sci. Adv. 2016; 2, e160087426.
Harvey, T. ∙ Flamenco, S. ∙ Fan, C.-M.
A Tppp3+Pdgfra+ tendon stem cell population contributes to regeneration and reveals a shared role for PDGF signalling in regeneration and fibrosis
Nat. Cell Biol. 2019; 21:1490-1503Here we applied a systematic pipeline that combines the in vivo maturation model, single-cell transcriptomics, and functional validation to resolve the cellular and molecular dynamics during postnatal tendon maturation. We first found that days 7–14 are the crucial transition stage for tendon maturation. We then identified a nerve growth factor (NGF)-secreting Cd9+Cd271+ stem/progenitor cell population and the receptor tyrosine kinase (RTK) family signaling pathways dominating the maturation trajectory by single-cell analysis. Manipulation of SHP2, the key constituent of RTK signaling, affects tendon maturation. The results and pipeline provided here for studying tendon maturation could facilitate future studies of tendon regeneration and enable other developmental processes to identify new therapeutic targets for tissue regeneration.
在此，我们应用了结合体内成熟模型、单细胞转录组学和功能验证的系统方法来解析出生后肌腱成熟过程中的细胞和分子动态。我们首先发现第 7-14 天是肌腱成熟的关键转变阶段。随后，通过单细胞分析，我们鉴定了一种分泌神经生长因子（NGF）的 Cd9 + Cd271 + 干/祖细胞群体，以及主导成熟轨迹的受体酪氨酸激酶（RTK）家族信号通路。调控 RTK 信号关键成分 SHP2 会影响肌腱成熟。本研究提供的结果和方法可促进未来肌腱再生研究，并使其他发育过程能够识别组织再生的新治疗靶点。
在此，我们应用了结合体内成熟模型、单细胞转录组学和功能验证的系统方法来解析出生后肌腱成熟过程中的细胞和分子动态。我们首先发现第 7-14 天是肌腱成熟的关键转变阶段。随后，通过单细胞分析，我们鉴定了一种分泌神经生长因子（NGF）的 Cd9 + Cd271 + 干/祖细胞群体，以及主导成熟轨迹的受体酪氨酸激酶（RTK）家族信号通路。调控 RTK 信号关键成分 SHP2 会影响肌腱成熟。本研究提供的结果和方法可促进未来肌腱再生研究，并使其他发育过程能够识别组织再生的新治疗靶点。
Results
Post-natal day 7 (P7) to P14 is the critical stage for tendon maturation
出生后第 7 天（P7）至第 14 天（P14）是肌腱成熟的关键阶段
Postnatal tendon maturation is regulated in a time-sequential manner, including transformation from proliferative growth to homeostasis status (Grinstein et al., 2019). Although many studies have focused on tendon development (Ansorge et al., 2011; Zhang et al., 2005), the critical transitional stage during the maturation process has remained less understood. To systematically evaluate tendon maturation, we compared the morphological, molecular, and mechanical properties of the Achilles tendon at P1, P4, P7, P10, P14, and P28.
出生后肌腱成熟以时序方式调控，包括从增殖生长到稳态状态的转变（ Grinstein et al., 2019 ）。虽然许多研究关注肌腱发育（ Ansorge et al., 2011 ； Zhang et al., 2005 ），但成熟过程中的关键转变阶段仍未得到充分理解。为系统评估肌腱成熟，我们比较了出生后第 1 天（P1）、第 4 天（P4）、第 7 天（P7）、第 10 天（P10）、第 14 天（P14）和第 28 天（P28）跟腱的形态学、分子和力学特性。
23.
Grinstein, M. ∙ Dingwall, H.L. ∙ O’Connor, L.D. ...
A distinct transition from cell growth to physiological homeostasis in the tendon
ELife. 2019; 8, e486894.
Ansorge, H.L. ∙ Adams, S. ∙ Birk, D.E. ...
Mechanical, compositional, and structural properties of the post-natal mouse Achilles tendon
Ann. Biomed. Eng. 2011; 39:1904-191365.
Zhang, G. ∙ Young, B.B. ∙ Ezura, Y. ...
Development of tendon structure and function: regulation of collagen fibrillogenesis
J. Musculoskelet. Neuronal Interact. 2005; 5:5-21出生后肌腱成熟以时序方式调控，包括从增殖生长到稳态状态的转变（ Grinstein et al., 2019
23.
Grinstein, M. ∙ Dingwall, H.L. ∙ O’Connor, L.D. ...
A distinct transition from cell growth to physiological homeostasis in the tendon
ELife. 2019; 8, e486894.
Ansorge, H.L. ∙ Adams, S. ∙ Birk, D.E. ...
Mechanical, compositional, and structural properties of the post-natal mouse Achilles tendon
Ann. Biomed. Eng. 2011; 39:1904-191365.
Zhang, G. ∙ Young, B.B. ∙ Ezura, Y. ...
Development of tendon structure and function: regulation of collagen fibrillogenesis
J. Musculoskelet. Neuronal Interact. 2005; 5:5-21We first examined H&E (hematoxylin and eosin) staining results from the mid-substance of the Achilles tendon of mice (Figure 1A). At P1, round nuclear cells infiltrated the neonatal tendon. From P1 to P4, cells exhibited a progressively elongated nuclear morphology. The number of cells gradually decreased from P1 to P14. By P28, there were only a few cells per area remaining. The extracellular matrix also displayed compelling alterations during postnatal tendon growth; typical crimp patterns began to be visible only at P7 and became evident at P14; the fibers gradually became more parallel and denser with concurrent development. Histology scores of the tendon at all time points are documented in Figure 1E. To explore the maturation process of the collagen fibers, we evaluated the histological sections using polarized light microscopy (Figure 1B). At P1 and P4, the tendon could hardly be visualized. At P7, the tissue started to produce a light golden yellow color, suggesting that the tendon structure had been determined but remained immature. At P14, golden yellow mature collagen fibers with parallel patterns began to be seen, with more prominent patterns at P28. Quantitative analysis revealed a gradually increased crimp wavelength from P7 to P28 (Figure 1F), corresponding to the progressive maturation process. To examine the collagen fibril changes, we analyzed transmission electron microscopy (TEM) images of the Achilles tendons (Figure 1C). Statistics results demonstrated that fibril diameter increased during postnatal tendon development, with the average fibril diameter increasing from 34.41 nm at P1 to 49.54 nm at P7 and eventually reaching 110.92 nm at P28 (Figure 1D; Table S1). Notably, the variation of the fiber diameter also increased during development (Figure 1D; Table S1). The neonatal tendon only comprised homogeneous collagen fibers. During development, a bimodal distribution of diameters was initiated at P10 and became evident at P28, as described previously (Parry et al., 1978). We evaluated the interfibrillar spacing changes along with the tendon maturation process. The center-to-center distance and the surface-to-surface distance showed an increasing trend (Figures S1A–S1D).
我们首先检查了小鼠跟腱中部的 H&E（苏木精和伊红）染色结果（图 1A）。在 P1 阶段，圆形核细胞浸润新生儿腱。从 P1 到 P4，细胞逐渐表现出细长的核形态。细胞数量从 P1 到 P14 逐渐减少。到 P28 时，每个区域仅剩少量细胞。在出生后腱的生长过程中，细胞外基质也呈现出明显变化；典型的波纹图案仅在 P7 开始可见，并在 P14 变得明显；随着发育的进行，纤维逐渐变得更加平行且密集。腱在所有时间点的组织学评分记录在图 1E 中。为了探索胶原纤维的成熟过程，我们使用偏振光显微镜评估了组织学切片（图 1B）。在 P1 和 P4 时，腱几乎不可见。在 P7 时，组织开始呈现浅金黄色，表明腱结构已经确定但仍不成熟。在 P14 时，开始出现具有平行图案的金黄色成熟胶原纤维，到 P28 时图案更加明显。定量分析显示从 P7 到 P28 波纹波长逐渐增加（图 1F），与逐步成熟过程相对应。为了检查胶原原纤维的变化，我们分析了跟腱的透射电镜（TEM）图像（图 1C）。统计结果表明，原纤维直径在出生后腱发育过程中增加，平均原纤维直径从 P1 的 34.41 nm 增加到 P7 的 49.54 nm，最终在 P28 达到 110.92 nm（图 1D；表 S1）。值得注意的是，纤维直径的变异性也在发育过程中增加（图 1D；表 S1）。新生腱仅由均质胶原纤维组成。在发育过程中，双峰直径分布在 P10 开始出现，并在 P28 变得明显，如先前所述（ Parry et al., 1978 ）。我们评估了腱成熟过程中纤维间间距的变化。纤维中心到中心的距离和表面到表面的距离均呈增加趋势（图 S1A-S1D）。
48.
Parry, D.A. ∙ Barnes, G.R. ∙ Craig, A.S.
A comparison of the size distribution of collagen fibrils in connective tissues as a function of age and a possible relation between fibril size distribution and mechanical properties
Proc. R. Soc. Lond. B Biol. Sci. 1978; 203:305-321我们首先检查了小鼠跟腱中部的 H&E（苏木精和伊红）染色结果（图 1A）。在 P1 阶段，圆形核细胞浸润新生儿腱。从 P1 到 P4，细胞逐渐表现出细长的核形态。细胞数量从 P1 到 P14 逐渐减少。到 P28 时，每个区域仅剩少量细胞。在出生后腱的生长过程中，细胞外基质也呈现出明显变化；典型的波纹图案仅在 P7 开始可见，并在 P14 变得明显；随着发育的进行，纤维逐渐变得更加平行且密集。腱在所有时间点的组织学评分记录在图 1E 中。为了探索胶原纤维的成熟过程，我们使用偏振光显微镜评估了组织学切片（图 1B）。在 P1 和 P4 时，腱几乎不可见。在 P7 时，组织开始呈现浅金黄色，表明腱结构已经确定但仍不成熟。在 P14 时，开始出现具有平行图案的金黄色成熟胶原纤维，到 P28 时图案更加明显。定量分析显示从 P7 到 P28 波纹波长逐渐增加（图 1F），与逐步成熟过程相对应。为了检查胶原原纤维的变化，我们分析了跟腱的透射电镜（TEM）图像（图 1C）。统计结果表明，原纤维直径在出生后腱发育过程中增加，平均原纤维直径从 P1 的 34.41 nm 增加到 P7 的 49.54 nm，最终在 P28 达到 110.92 nm（图 1D；表 S1）。值得注意的是，纤维直径的变异性也在发育过程中增加（图 1D；表 S1）。新生腱仅由均质胶原纤维组成。在发育过程中，双峰直径分布在 P10 开始出现，并在 P28 变得明显，如先前所述（ Parry et al., 1978
48.
Parry, D.A. ∙ Barnes, G.R. ∙ Craig, A.S.
A comparison of the size distribution of collagen fibrils in connective tissues as a function of age and a possible relation between fibril size distribution and mechanical properties
Proc. R. Soc. Lond. B Biol. Sci. 1978; 203:305-321
Figure 1 P7–P14 is the critical stage for postnatal tendon maturation
图 1 P7-P14 是出生后腱成熟的关键阶段
图 1 P7-P14 是出生后腱成熟的关键阶段
We also examined changes in gene expression during this process (Figure 1H), focusing on genes that are crucial for tenocyte differentiation (Scx, Mkx, Egr1, and Nes) and matrix production (Col1a1 and Tnmd). The expression levels of Scx, Mkx, Tnmd, and Col1a1 gradually decreased after 4 days. Nes, a progenitor marker (Yin et al., 2016), demonstrated the highest expression at P1. In comparison, the expression of Egr1 peaked at P28. Previous studies of the mechanical properties of the tendon, including load versus displacement curves and the stiffness modulus test, demonstrated that most parameters showed a significant increase with age (Ansorge et al., 2011).
我们还检查了这一过程中基因表达的变化（图 1H），重点关注对腱细胞分化（Scx、Mkx、Egr1 和 Nes）和基质产生（Col1a1 和 Tnmd）至关重要的基因。Scx、Mkx、Tnmd 和 Col1a1 的表达水平在 4 天后逐渐下降。作为祖细胞标记的 Nes（ Yin et al., 2016 ）在 P1 时表达最高。相比之下，Egr1 的表达在 P28 达到峰值。先前关于腱的机械特性研究，包括载荷与位移曲线和刚度模量测试，表明大多数参数随年龄显著增加（ Ansorge et al., 2011 ）。
63.
Yin, Z. ∙ Hu, J. ∙ Yang, L. ...
Single-cell analysis reveals a nestin+ tendon stem/progenitor cell population with strong tenogenic potentiality
Sci. Adv. 2016; 2, e16008744.
Ansorge, H.L. ∙ Adams, S. ∙ Birk, D.E. ...
Mechanical, compositional, and structural properties of the post-natal mouse Achilles tendon
Ann. Biomed. Eng. 2011; 39:1904-1913我们还检查了这一过程中基因表达的变化（图 1H），重点关注对腱细胞分化（Scx、Mkx、Egr1 和 Nes）和基质产生（Col1a1 和 Tnmd）至关重要的基因。Scx、Mkx、Tnmd 和 Col1a1 的表达水平在 4 天后逐渐下降。作为祖细胞标记的 Nes（ Yin et al., 2016
63.
Yin, Z. ∙ Hu, J. ∙ Yang, L. ...
Single-cell analysis reveals a nestin+ tendon stem/progenitor cell population with strong tenogenic potentiality
Sci. Adv. 2016; 2, e16008744.
Ansorge, H.L. ∙ Adams, S. ∙ Birk, D.E. ...
Mechanical, compositional, and structural properties of the post-natal mouse Achilles tendon
Ann. Biomed. Eng. 2011; 39:1904-1913Combining all morphological, molecular, and mechanical results, we performed a principal-component analysis (PCA) at different time points (Figure 1G). The first two components appropriately separated the tendon tissues at different time points (P4 and P7 versus P14 and P28), with P10 located in the middle. The PCA plot also displayed a transitional trajectory across the P7–P14 timeline. Our findings suggested that P7–P14 is the crucial transition state for the postnatal tendon maturation process.
结合所有形态学、分子和机械结果，我们在不同时间点进行了主成分分析（PCA）（图 1G）。前两个主成分恰当地区分了不同时间点（P4 和 P7 与 P14 和 P28）的腱组织，P10 位于中间位置。PCA 图还显示了横跨 P7-P14 时间线的转变轨迹。我们的发现表明，P7-P14 是出生后腱成熟过程的关键转变阶段。
结合所有形态学、分子和机械结果，我们在不同时间点进行了主成分分析（PCA）（图 1G）。前两个主成分恰当地区分了不同时间点（P4 和 P7 与 P14 和 P28）的腱组织，P10 位于中间位置。PCA 图还显示了横跨 P7-P14 时间线的转变轨迹。我们的发现表明，P7-P14 是出生后腱成熟过程的关键转变阶段。
Single-cell RNA sequencing (scRNA-seq) analysis identified cellular heterogeneity of the early postnatal tendon
单细胞 RNA 测序（scRNA-seq）分析鉴定了早期出生后腱的细胞异质性
To precisely dissect the tendon maturation process in vivo, we performed scRNA-seq of mouse tail tendon cells at P7 and P14. Tendon cells were prepared using the Fluidigm C1 system, which has a great balance of resolution and throughput. The transcriptome profile was obtained from 800 individual cells. After stringent quality control filtering, 734 cells were projected onto the t-SNE (t-distributed stochastic neighbor embedding) plot and grouped into six distinct clusters (Figures 2A and 2D). Based on the annotated biological functions of the DEGs (differentially expressed genes) (Figures 2E and 2F) and the expression of the tendon-related genes (Figure 2G), we defined six clusters: tendon fibroblast 1 (cluster 0), tendon fibroblast 2 (cluster 1), tendon fibroblast 3 (cluster 2), stem cells (cluster 3), proliferation cells (cluster 4) and neuronal cells (cluster 5).
为了精确解析体内腱成熟过程，我们对 P7 和 P14 小鼠尾腱细胞进行了单细胞 RNA 测序（scRNA-seq）。腱细胞使用 Fluidigm C1 系统制备，该系统在分辨率和通量之间取得了良好平衡。我们获得了 800 个单细胞的转录组谱。经过严格的质量控制筛选后，734 个细胞被投射到 t-SNE（t-分布随机邻域嵌入）图上并分为六个不同的簇（图 2A 和 2D）。基于差异表达基因（DEGs）的注释生物功能（图 2E 和 2F）以及腱相关基因的表达（图 2G），我们定义了六个簇：腱成纤维细胞 1（簇 0）、腱成纤维细胞 2（簇 1）、腱成纤维细胞 3（簇 2）、干细胞（簇 3）、增殖细胞（簇 4）和神经细胞（簇 5）。
为了精确解析体内腱成熟过程，我们对 P7 和 P14 小鼠尾腱细胞进行了单细胞 RNA 测序（scRNA-seq）。腱细胞使用 Fluidigm C1 系统制备，该系统在分辨率和通量之间取得了良好平衡。我们获得了 800 个单细胞的转录组谱。经过严格的质量控制筛选后，734 个细胞被投射到 t-SNE（t-分布随机邻域嵌入）图上并分为六个不同的簇（图 2A 和 2D）。基于差异表达基因（DEGs）的注释生物功能（图 2E 和 2F）以及腱相关基因的表达（图 2G），我们定义了六个簇：腱成纤维细胞 1（簇 0）、腱成纤维细胞 2（簇 1）、腱成纤维细胞 3（簇 2）、干细胞（簇 3）、增殖细胞（簇 4）和神经细胞（簇 5）。
Figure 2 scRNA-seq analysis identified cellular heterogeneity of the mouse postnatal tendon
图 2 单细胞 RNA 测序分析鉴定了小鼠出生后腱的细胞异质性
图 2 单细胞 RNA 测序分析鉴定了小鼠出生后腱的细胞异质性
The three tendon fibroblast clusters highly expressed collagen type I (Col1a1 and Col1a2; Figure 2G), the fundamental building blocks of the fibrillar extracellular matrix (ECM) (Huang et al., 2015; Kannus, 2000). Because the largest cluster, cluster 0 (n = 255), mainly consisted of P7 tendon cells (Figures 2B and S2A) and highly expressed the embryonic gene Pbx4 (Figure 2F), we assigned this group as an immature cell cluster. Gene Ontology (GO) enrichment analysis also demonstrated that cells from this cluster are involved in ribonucleoprotein complex biosynthesis and regulation of the mRNA metabolic process, suggesting active biosynthesis features of early postnatal tendon cell growth (Figure 2E). Tendon fibroblast 2 (cluster 1, n = 187) and tendon fibroblast 3 (cluster 2, n = 158) mainly consisted of cells from P14, and both showed high expression of classic ECM genes (Jelinsky et al., 2010; Rees et al., 2002) such as Tnmd, Fmod, and Thbs4. We assigned these two groups as mature cell clusters. Cells in cluster 1 also highly expressed Fbn1 and Mfap5 (Figure 2F), which are associated with collagen fibril organization. In contrast, cells in cluster 2 demonstrated enrichment mainly in response to mechanical stimulation, corresponding to the highest expression of Sparc and Serpinf1 (Figure 2F). The remaining three clusters were mostly from P7 tendons. We annotated cluster 3 (n = 66) as stem cells with high expression of stem-related genes: Pou3f1 (Oct6), Thy1 (Cd90), Nes, and Ly6a (Sca-1) (Figures 2F and 2G). The DEGs of this cluster were also significantly enriched in the “stem cell population maintenance” GO term (Figure 2E). Cluster 4 (n = 37) was assigned as proliferation cells with specific expression of the proliferation marker Mki67 (Figure S2C) and the highest score for the G2M cell cycle stage (Figure 2C). The neuronal cells (cluster 5 (n = 31) highly expressed the neuronal markers Sox2 (Graham et al., 2003) and Nrgn (Nielsen et al., 2009) (Figure S2C), and GO analysis displayed enrichment of the “regulation of neurogenesis” biological process (Figure 2E). Aldh8a1, which regulates the metabolism of the amino acid tryptophan (Davis et al., 2018), was also explicitly identified in this neuronal cluster (Figure S2C).
三个腱成纤维细胞簇高表达 I 型胶原(Col1a1 和 Col1a2；图 2G)，这是纤维状细胞外基质(ECM)的基本构建单元( Huang et al., 2015 ； Kannus, 2000 )。由于最大的簇，即簇 0(n = 255)，主要由 P7 腱细胞组成(图 2B 和 S2A)，并高表达胚胎基因 Pbx4(图 2F)，我们将该组定义为未成熟细胞簇。基因本体论(GO)富集分析也表明，该簇的细胞参与核糖核蛋白复合体生物合成和 mRNA 代谢过程的调控，这表明早期出生后腱细胞生长具有活跃的生物合成特征(图 2E)。腱成纤维细胞 2(簇 1，n = 187)和腱成纤维细胞 3(簇 2，n = 158)主要由 P14 的细胞组成，两者都表现出经典 ECM 基因的高表达( Jelinsky et al., 2010 ； Rees et al., 2002 )，如 Tnmd、Fmod 和 Thbs4。我们将这两组定义为成熟细胞簇。簇 1 中的细胞还高表达 Fbn1 和 Mfap5(图 2F)，这些与胶原纤维组织有关。相比之下，簇 2 的细胞主要表现为对机械刺激的反应富集，对应于 Sparc 和 Serpinf1 的最高表达(图 2F)。其余三个簇主要来自 P7 腱。我们将簇 3(n = 66)注释为干细胞，其高表达干细胞相关基因：Pou3f1(Oct6)、Thy1(Cd90)、Nes 和 Ly6a(Sca-1)(图 2F 和 2G)。该簇的差异表达基因在"干细胞群体维持"GO 术语中也显著富集(图 2E)。簇 4(n = 37)被定义为增殖细胞，特异性表达增殖标志物 Mki67(图 S2C)，并在 G2M 细胞周期阶段得分最高(图 2C)。神经元细胞(簇 5，n = 31)高表达神经元标志物 Sox2( Graham et al., 2003 )和 Nrgn( Nielsen et al., 2009 )(图 S2C)，GO 分析显示"神经发生调控"生物过程的富集(图 2E)。调节色氨酸代谢的 Aldh8a1( Davis et al., 2018 )也在这一神经元簇中被明确鉴定(图 S2C)。
28.
Huang, A.H. ∙ Lu, H.H. ∙ Schweitzer, R.
Molecular regulation of tendon cell fate during development
J. Orthop. Res. 2015; 33:800-81237.
Kannus, P.
Structure of the tendon connective tissue
Scand. J. Med. Sci. Sports. 2000; 10:312-32035.
Jelinsky, S.A. ∙ Archambault, J. ∙ Li, L. ...
Tendon-selective genes identified from rat and human musculoskeletal tissues
J. Orthop. Res. 2010; 28:289-29751.
Rees, S.G. ∙ Davies, J.R. ∙ Tudor, D. ...
Immunolocalisation and expression of proteoglycan 4 (cartilage superficial zone proteoglycan) in tendon
Matrix Biol. 2002; 21:593-60221.
Graham, V. ∙ Khudyakov, J. ∙ Ellis, P. ...
SOX2 functions to maintain neural progenitor identity
Neuron. 2003; 39:749-76545.
Nielsen, A.A. ∙ Kjartansdóttir, K.R. ∙ Rasmussen, M.H. ...
Activation of the brain-specific neurogranin gene in murine T-cell lymphomas by proviral insertional mutagenesis
Gene. 2009; 442:55-6215.
Davis, I. ∙ Yang, Y. ∙ Wherritt, D. ...
Reassignment of the human aldehyde dehydrogenase ALDH8A1 (ALDH12) to the kynurenine pathway in tryptophan catabolism
J. Biol. Chem. 2018; 293:9594-9603三个腱成纤维细胞簇高表达 I 型胶原(Col1a1 和 Col1a2；图 2G)，这是纤维状细胞外基质(ECM)的基本构建单元( Huang et al., 2015
28.
Huang, A.H. ∙ Lu, H.H. ∙ Schweitzer, R.
Molecular regulation of tendon cell fate during development
J. Orthop. Res. 2015; 33:800-81237.
Kannus, P.
Structure of the tendon connective tissue
Scand. J. Med. Sci. Sports. 2000; 10:312-32035.
Jelinsky, S.A. ∙ Archambault, J. ∙ Li, L. ...
Tendon-selective genes identified from rat and human musculoskeletal tissues
J. Orthop. Res. 2010; 28:289-29751.
Rees, S.G. ∙ Davies, J.R. ∙ Tudor, D. ...
Immunolocalisation and expression of proteoglycan 4 (cartilage superficial zone proteoglycan) in tendon
Matrix Biol. 2002; 21:593-60221.
Graham, V. ∙ Khudyakov, J. ∙ Ellis, P. ...
SOX2 functions to maintain neural progenitor identity
Neuron. 2003; 39:749-76545.
Nielsen, A.A. ∙ Kjartansdóttir, K.R. ∙ Rasmussen, M.H. ...
Activation of the brain-specific neurogranin gene in murine T-cell lymphomas by proviral insertional mutagenesis
Gene. 2009; 442:55-6215.
Davis, I. ∙ Yang, Y. ∙ Wherritt, D. ...
Reassignment of the human aldehyde dehydrogenase ALDH8A1 (ALDH12) to the kynurenine pathway in tryptophan catabolism
J. Biol. Chem. 2018; 293:9594-9603When we investigated the cell proliferation rates, a decrease in the total percentage of G2M phase proliferation cells, from 38.5% at P7 to 14.4% at P14, was observed (Figures 2C and S2B), which was consistent with previous findings using bromodeoxyuridine (BrdU) labeling assays (Grinstein et al., 2019). We performed binary regulon (transcription factors [TFs] and their predicted target genes) activity matrix-based unsupervised clustering analysis (Aibar et al., 2017; Figure S2D) and generated highly consistent clusters compared with the above results based on the gene expression matrix (Figure S2E), demonstrating the robustness and consistency of our analysis.
当我们研究细胞增殖率时，观察到 G2M 期增殖细胞的总百分比从 P7 的 38.5%下降到 P14 的 14.4%(图 2C 和 S2B)，这与先前使用溴脱氧尿苷(BrdU)标记实验的发现一致( Grinstein et al., 2019 )。我们进行了基于二元调控子(转录因子[TFs]及其预测的靶基因)活性矩阵的无监督聚类分析( Aibar et al., 2017 ；图 S2D)，并与上述基于基因表达矩阵的结果相比，生成了高度一致的聚类(图 S2E)，证明了我们分析的稳健性和一致性。
23.
Grinstein, M. ∙ Dingwall, H.L. ∙ O’Connor, L.D. ...
A distinct transition from cell growth to physiological homeostasis in the tendon
ELife. 2019; 8, e486892.
Aibar, S. ∙ González-Blas, C.B. ∙ Moerman, T. ...
SCENIC: single-cell regulatory network inference and clustering
Nat. Methods. 2017; 14:1083-1086当我们研究细胞增殖率时，观察到 G2M 期增殖细胞的总百分比从 P7 的 38.5%下降到 P14 的 14.4%(图 2C 和 S2B)，这与先前使用溴脱氧尿苷(BrdU)标记实验的发现一致( Grinstein et al., 2019
23.
Grinstein, M. ∙ Dingwall, H.L. ∙ O’Connor, L.D. ...
A distinct transition from cell growth to physiological homeostasis in the tendon
ELife. 2019; 8, e486892.
Aibar, S. ∙ González-Blas, C.B. ∙ Moerman, T. ...
SCENIC: single-cell regulatory network inference and clustering
Nat. Methods. 2017; 14:1083-1086The Cd9+Cd271+ TSPC population and RTK signaling pathways were found in the P7 and P14 tendon niches
在 P7 和 P14 腱微环境中发现了 Cd9 + Cd271 + 腱干/祖细胞(TSPC)群体和 RTK 信号通路
To further characterize the differences between immature and mature tendons, we performed a refined clustering analysis. Together with the stem clusters, the immature tendon cell cluster (cluster 0) could be further subcategorized into 6 clusters (Figure 3A). Cells in subclusters 0 and 1 demonstrated high expression of typical tenocyte genes such as Col1a1, Fmod, Lum, and Tnmd (Figures S3C and S3D). Subcluster 0 also presented high expression of the mesenchymal progenitor marker Cd9 (Figures 3B and S3A) and, thus, was annotated as the Cd9+ cluster. Expression of chondrocyte-related genes, such as Sox9, Acan, and Col2a1, was also enriched in this cluster (Figure S3C). The GO-enriched results revealed that Cd9+ cells were mainly engaged in response to hypoxia, ECM organization, and chondrocyte hypertrophy (Figure S3B). The Cd9+ cluster was the only shared subgroup in P7 and P14 tendons (Figure 3C). The gene set enrichment analysis (GSEA)-enriched result revealed that Cd9+ cells were mainly enriched in the cell cycle arrest, stem cell differentiation, embryonic morphogenesis, and development maturation pathways (Figure 3D), implying their potential roles in tendon maturation. The Txnip+ cluster (subcluster1) demonstrated the highest expression of the redox regulation gene Txnip (Figure 3B). It also exhibited high expression levels of Scx, Mkx, and Tnc (Figure S3C), representative markers of early tenocytes. Subcluster2 and subcluster3 highly expressed mesenchymal stem markers such as Thy1 (Cd90), Cd44, and Ly6a (Sca-1) (Figure S3C). Subcluster 2 also highly expressed the tendon stem marker Nes (Yin et al., 2016; Figures 3B, S3C, and S3D) and, therefore, was assigned as Nes+ TSPCs. Subcluster 3 demonstrated high expression of endothelial markers such as Cd34 and Pdgfra and, thus, was annotated as the endothelial cluster (Figures 3B and S3C). We identified an epithelial cluster (subcluster 4) with exclusively high expression of Krt2, Krt10, and Krt1 and an immune cell cluster (subcluster 5) in immature tendon cells with high expression of the macrophage marker Cd68 and chemokine Ccl9 (Figures 3B and S3C).
为了进一步表征未成熟和成熟腱之间的差异，我们进行了精细的聚类分析。连同干细胞簇，未成熟腱细胞簇(簇 0)可进一步细分为 6 个亚簇(图 3A)。亚簇 0 和 1 中的细胞表现出腱细胞典型基因如 Col1a1、Fmod、Lum 和 Tnmd 的高表达(图 S3C 和 S3D)。亚簇 0 还呈现间充质祖细胞标志物 Cd9 的高表达(图 3B 和 S3A)，因此被注释为 Cd9 + 簇。该簇中还富集了软骨细胞相关基因的表达，如 Sox9、Acan 和 Col2a1(图 S3C)。GO 富集结果显示，Cd9 + 细胞主要参与低氧反应、ECM 组织和软骨细胞肥大(图 S3B)。Cd9 + 簇是 P7 和 P14 腱中唯一共享的亚群(图 3C)。基因集富集分析(GSEA)富集结果显示，Cd9 + 细胞主要富集于细胞周期阻滞、干细胞分化、胚胎形态发生和发育成熟通路(图 3D)，暗示了它们在腱成熟中的潜在作用。Txnip + 簇(亚簇 1)展示了氧化还原调节基因 Txnip 的最高表达(图 3B)。它还表现出 Scx、Mkx 和 Tnc 的高表达水平(图 S3C)，这些是早期腱细胞的代表性标志物。亚簇 2 和亚簇 3 高表达间充质干细胞标志物，如 Thy1(Cd90)、Cd44 和 Ly6a(Sca-1)(图 S3C)。亚簇 2 还高表达腱干细胞标志物 Nes( Yin et al., 2016 ；图 3B、S3C 和 S3D)，因此被指定为 Nes + 腱干/祖细胞(TSPCs)。亚簇 3 表现出内皮标志物如 Cd34 和 Pdgfra 的高表达，因此被注释为内皮簇(图 3B 和 S3C)。我们在未成熟腱细胞中鉴定了一个上皮簇(亚簇 4)，其专一性高表达 Krt2、Krt10 和 Krt1，以及一个免疫细胞簇(亚簇 5)，其高表达巨噬细胞标志物 Cd68 和趋化因子 Ccl9(图 3B 和 S3C)。
63.
Yin, Z. ∙ Hu, J. ∙ Yang, L. ...
Single-cell analysis reveals a nestin+ tendon stem/progenitor cell population with strong tenogenic potentiality
Sci. Adv. 2016; 2, e1600874为了进一步表征未成熟和成熟腱之间的差异，我们进行了精细的聚类分析。连同干细胞簇，未成熟腱细胞簇(簇 0)可进一步细分为 6 个亚簇(图 3A)。亚簇 0 和 1 中的细胞表现出腱细胞典型基因如 Col1a1、Fmod、Lum 和 Tnmd 的高表达(图 S3C 和 S3D)。亚簇 0 还呈现间充质祖细胞标志物 Cd9 的高表达(图 3B 和 S3A)，因此被注释为 Cd9 + 簇。该簇中还富集了软骨细胞相关基因的表达，如 Sox9、Acan 和 Col2a1(图 S3C)。GO 富集结果显示，Cd9 + 细胞主要参与低氧反应、ECM 组织和软骨细胞肥大(图 S3B)。Cd9 + 簇是 P7 和 P14 腱中唯一共享的亚群(图 3C)。基因集富集分析(GSEA)富集结果显示，Cd9 + 细胞主要富集于细胞周期阻滞、干细胞分化、胚胎形态发生和发育成熟通路(图 3D)，暗示了它们在腱成熟中的潜在作用。Txnip + 簇(亚簇 1)展示了氧化还原调节基因 Txnip 的最高表达(图 3B)。它还表现出 Scx、Mkx 和 Tnc 的高表达水平(图 S3C)，这些是早期腱细胞的代表性标志物。亚簇 2 和亚簇 3 高表达间充质干细胞标志物，如 Thy1(Cd90)、Cd44 和 Ly6a(Sca-1)(图 S3C)。亚簇 2 还高表达腱干细胞标志物 Nes( Yin et al., 2016
63.
Yin, Z. ∙ Hu, J. ∙ Yang, L. ...
Single-cell analysis reveals a nestin+ tendon stem/progenitor cell population with strong tenogenic potentiality
Sci. Adv. 2016; 2, e1600874
Figure 3 The Cd9+Cd271+ cell subpopulation and RTK signaling pathways were found in the P7 and P14 tendon niches
图 3 在 P7 和 P14 腱微环境中发现了 Cd9 + Cd271 + 细胞亚群和 RTK 信号通路
图 3 在 P7 和 P14 腱微环境中发现了 Cd9 + Cd271 + 细胞亚群和 RTK 信号通路
Because most immature tendon cell clusters were from the P7 tendon, we attempted to decipher the cellular cross-talk in the natural P7 tendon milieu. Combined with the previously identified proliferation and neuronal clusters in the P7 tendon, we performed potential cell-cell interaction inference based on the recently developed receptor/ligand database (Efremova et al., 2020). The cell-cell interaction landscape of the P7 tendon revealed that the Nes+ TSPC, Txnip+, and Cd9+ clusters exhibited the strongest cellular communications (Figures 3C and S3E). Among these, the Nes+ TSPC and Txnip+ clusters showed the most cell-cell interactions (Figure S3E). In contrast, the immune and epithelial cell clusters displayed the weakest interactions with other clusters, suggesting their environment support roles (Morrissey and Sherwood, 2015). When we further investigated the specific ligand and receptor interactions, the COL1A1-a11b1 interaction stood out. Prominent growth factor signaling pathways were revealed (Figures 3E and S3F). The FGF18 pathway exhibited the most abundant signaling interactions between the Nes+/Txnip+ cluster and other clusters (Figure 3E). The Txnip+ cluster also interacted strongly with other cell subpopulations through TGFB2-TGFBR1, which confirmed the crucial roles of the TGF-β signaling pathway in early development of tendons (Zehender et al., 2018). When we thoroughly examined the expression of tenogenesis-related genes, Tgfb2 showed the highest expression in Txnip+ cells (Figure 3F). Notably, RTK family growth factors, including Fgf5 and Pdgfa, showed relatively high expression in the Cd9+ subpopulation and the proliferation cluster, implying their critical roles in tendon growth (Figures 3F and S3F). Specifically, the Cd9+ cluster exclusively expressed Ngf and also exhibited high expression of its receptor, Ngfr, so we renamed this cell cluster the Cd9+Cd271+ cluster. Immunofluorescence staining revealed that NGFR (CD271)-positive cells were mainly located in the peritenon of the tendon (Figure 3G).
由于大多数未成熟腱细胞簇来自 P7 腱，我们尝试解析 P7 腱自然环境中的细胞间对话。结合先前在 P7 腱中鉴定的增殖和神经元簇，我们基于最近开发的受体/配体数据库( Efremova et al., 2020 )进行了潜在细胞-细胞相互作用推断。P7 腱的细胞-细胞相互作用全景图显示，Nes + TSPC、Txnip + 和 Cd9 + 簇表现出最强的细胞间通讯(图 3C 和 S3E)。其中，Nes + TSPC 和 Txnip + 簇展示了最多的细胞-细胞相互作用(图 S3E)。相比之下，免疫和上皮细胞簇与其他簇的相互作用最弱，表明它们在环境支持中的作用( Morrissey and Sherwood, 2015 )。当我们进一步研究特定的配体和受体相互作用时，COL1A1-a11b1 相互作用尤为突出。研究揭示了显著的生长因子信号通路(图 3E 和 S3F)。FGF18 通路在 Nes + /Txnip + 簇与其他簇之间展现了最丰富的信号相互作用(图 3E)。Txnip + 簇还通过 TGFB2-TGFBR1 与其他细胞亚群强烈互动，证实了 TGF-β信号通路在腱早期发育中的关键作用( Zehender et al., 2018 )。当我们全面检查腱发生相关基因的表达时，Tgfb2 在 Txnip + 细胞中表现出最高表达(图 3F)。值得注意的是，RTK 家族生长因子，包括 Fgf5 和 Pdgfa，在 Cd9 + 亚群和增殖簇中表现出相对高的表达，暗示它们在腱生长中的关键作用(图 3F 和 S3F)。具体而言，Cd9 + 簇独特地表达 Ngf，并且还表现出其受体 Ngfr 的高表达，因此我们将这个细胞簇重命名为 Cd9 + Cd271 + 簇。免疫荧光染色显示，NGFR(CD271)阳性细胞主要位于腱的周腱组织中(图 3G)。
18.
Efremova, M. ∙ Vento-Tormo, M. ∙ Teichmann, S.A. ...
CellPhoneDB: inferring cell–cell communication from combined expression of multi-subunit ligand–receptor complexes
Nat. Protoc. 2020; 15:1484-150643.
Morrissey, M.A. ∙ Sherwood, D.R.
An active role for basement membrane assembly and modification in tissue sculpting
J. Cell Sci. 2015;, jcs.16802164.
Zehender, A. ∙ Huang, J. ∙ Györfi, A.-H. ...
The tyrosine phosphatase SHP2 controls TGFβ-induced STAT3 signaling to regulate fibroblast activation and fibrosis
Nat. Commun. 2018; 9:3259由于大多数未成熟腱细胞簇来自 P7 腱，我们尝试解析 P7 腱自然环境中的细胞间对话。结合先前在 P7 腱中鉴定的增殖和神经元簇，我们基于最近开发的受体/配体数据库( Efremova et al., 2020
18.
Efremova, M. ∙ Vento-Tormo, M. ∙ Teichmann, S.A. ...
CellPhoneDB: inferring cell–cell communication from combined expression of multi-subunit ligand–receptor complexes
Nat. Protoc. 2020; 15:1484-150643.
Morrissey, M.A. ∙ Sherwood, D.R.
An active role for basement membrane assembly and modification in tissue sculpting
J. Cell Sci. 2015;, jcs.16802164.
Zehender, A. ∙ Huang, J. ∙ Györfi, A.-H. ...
The tyrosine phosphatase SHP2 controls TGFβ-induced STAT3 signaling to regulate fibroblast activation and fibrosis
Nat. Commun. 2018; 9:3259We also re-clustered the mature tendon clusters fibroblast 2 and fibroblast 3, which are mainly composed of P14 tendon cells. Three groups with different levels of the classic tendon molecular collagen were identified (Figure S4A). Cells in subcluster 1 showed the highest expression of Col1a1 and, thus, were annotated as the Col1a1+ cluster (Figure S4C). Tnmd and Lox were also highly expressed in this cluster, corresponding to the collagen fibril and ECM organization functions. Subcluster 2 cells demonstrated the highest expression of another critical molecular collagen, Col3a1, and expression of immune-related genes such as Cxcl2, Cd68, and Il1rn (Figure S4C and S4D). Therefore, we described this cluster as the Col3a1+ cluster. Unexpectedly, a subcluster (Sca-1+ cluster) with the highest expression of stem cell markers of Ly6a (Sca-1) and Cd44 enriched in the ribosome biogenesis biological function was also identified in the P14 tendon (Figures S4B–S4D). When we examined the cell-cell communications between all cell subclusters of the P14 tendon, Col3a1+ tenocytes displayed the strongest communications with other groups (Figures 3C and S4E). The interactions of the growth factor signaling pathways played major part, especially for the FGF18-FGFR1 signaling pathway (Figure S4F). Pdgfa and Igf1 maintained relatively highly expression in all of these cell subclusters, corresponding to enrichment of the RTK signaling pathway of the GO annotation of the P14 tendon subclusters (Figures S4B). It should be noted that Ngf-positive cells expanded to all of the clusters in the P14 tendons (Figure S4G) compared with only the Cd9+ tenocyte cluster of the P7 tendon (Figure 3F). Compared with the P7 tendon, the P14 tendon clusters demonstrated higher ECM and integrin interactions, concomitant with their increased adaptability to transmit the mechanic stimulus (Sun et al., 2016) (Figure S4F). Including the Col1A1-a11b1 interaction, the Col1a1+, Col3a1+, and Cd9+ tenocyte clusters also displayed strong COL1A1-a1b1/a10b1 complex interactions (Figures S4F and S4H). COL5A1 and a1b1 complex interactions were mainly enriched in the Col3a1+ cluster. In contrast, MMP2 and aVb3 complex interactions were mainly detected in the Sca-1+ cluster, suggesting their function in promoting adhesion and migration. Through cellular communication analysis, activated RTK signaling pathways and increased collagen and ECM interactions were identified, suggesting their potential roles during tendon maturation.
我们还对主要由 P14 腱细胞组成的成熟腱簇成纤维细胞 2 和成纤维细胞 3 进行了重新聚类。我们识别出三个具有不同水平经典腱分子胶原蛋白的群体(图 S4A)。亚簇 1 中的细胞显示出 Col1a1 最高表达，因此被注释为 Col1a1 + 簇(图 S4C)。Tnmd 和 Lox 在该簇中也高度表达，对应于胶原纤维和细胞外基质组织功能。亚簇 2 细胞展示了另一种关键分子胶原蛋白 Col3a1 的最高表达，以及免疫相关基因如 Cxcl2、Cd68 和 Il1rn 的表达(图 S4C 和 S4D)。因此，我们将该簇描述为 Col3a1 + 簇。出乎意料的是，在 P14 腱中还鉴定出一个亚簇(Sca-1 + 簇)，其表达最高的干细胞标记物为 Ly6a(Sca-1)和 Cd44，富集在核糖体生物合成生物学功能中(图 S4B-S4D)。当我们检查 P14 腱所有细胞亚簇之间的细胞-细胞通讯时，Col3a1 + 腱细胞与其他组别展示了最强的通讯(图 3C 和 S4E)。生长因子信号通路的相互作用发挥了主要作用，尤其是 FGF18-FGFR1 信号通路(图 S4F)。Pdgfa 和 Igf1 在所有这些细胞亚簇中保持相对高表达，对应于 P14 腱亚簇 GO 注释中 RTK 信号通路的富集(图 S4B)。需要注意的是，与 P7 腱中仅在 Cd9 + 腱细胞簇中存在的情况(图 3F)相比，Ngf 阳性细胞在 P14 腱中扩展到了所有簇(图 S4G)。与 P7 腱相比，P14 腱簇展示出更高的细胞外基质和整合素相互作用，伴随其传递机械刺激的适应性增强( Sun et al., 2016 )(图 S4F)。包括 Col1A1-a11b1 相互作用在内，Col1a1 + 、Col3a1 + 和 Cd9 + 腱细胞簇也显示出强烈的 COL1A1-a1b1/a10b1 复合物相互作用(图 S4F 和 S4H)。COL5A1 和 a1b1 复合物相互作用主要富集在 Col3a1 + 簇中。相比之下，MMP2 和 aVb3 复合物相互作用主要在 Sca-1 + 簇中检测到，表明它们在促进粘附和迁移方面的功能。通过细胞通讯分析，我们识别出激活的 RTK 信号通路以及增加的胶原蛋白和细胞外基质相互作用，表明它们在腱成熟过程中的潜在作用。
56.
Sun, Z. ∙ Guo, S.S. ∙ Fässler, R.
Integrin-mediated mechanotransduction
J. Cell Biol. 2016; 215:445-456我们还对主要由 P14 腱细胞组成的成熟腱簇成纤维细胞 2 和成纤维细胞 3 进行了重新聚类。我们识别出三个具有不同水平经典腱分子胶原蛋白的群体(图 S4A)。亚簇 1 中的细胞显示出 Col1a1 最高表达，因此被注释为 Col1a1 + 簇(图 S4C)。Tnmd 和 Lox 在该簇中也高度表达，对应于胶原纤维和细胞外基质组织功能。亚簇 2 细胞展示了另一种关键分子胶原蛋白 Col3a1 的最高表达，以及免疫相关基因如 Cxcl2、Cd68 和 Il1rn 的表达(图 S4C 和 S4D)。因此，我们将该簇描述为 Col3a1 + 簇。出乎意料的是，在 P14 腱中还鉴定出一个亚簇(Sca-1 + 簇)，其表达最高的干细胞标记物为 Ly6a(Sca-1)和 Cd44，富集在核糖体生物合成生物学功能中(图 S4B-S4D)。当我们检查 P14 腱所有细胞亚簇之间的细胞-细胞通讯时，Col3a1 + 腱细胞与其他组别展示了最强的通讯(图 3C 和 S4E)。生长因子信号通路的相互作用发挥了主要作用，尤其是 FGF18-FGFR1 信号通路(图 S4F)。Pdgfa 和 Igf1 在所有这些细胞亚簇中保持相对高表达，对应于 P14 腱亚簇 GO 注释中 RTK 信号通路的富集(图 S4B)。需要注意的是，与 P7 腱中仅在 Cd9 + 腱细胞簇中存在的情况(图 3F)相比，Ngf 阳性细胞在 P14 腱中扩展到了所有簇(图 S4G)。与 P7 腱相比，P14 腱簇展示出更高的细胞外基质和整合素相互作用，伴随其传递机械刺激的适应性增强( Sun et al., 2016
56.
Sun, Z. ∙ Guo, S.S. ∙ Fässler, R.
Integrin-mediated mechanotransduction
J. Cell Biol. 2016; 215:445-456The intermediate Cd9+Cd271+ TSPC population drives the immature to mature tendon transition through the RTK signaling pathways
中间型 Cd9 + Cd271 + TSPC 群体通过 RTK 信号通路驱动腱从未成熟到成熟的转变
To systematically dissect the tendon maturation process, we next reconstructed the tendon maturation process based on single-cell transcriptome profiling data. The pseudo-temporal trajectory well matched the clustering results and the developmental course (Figures 4A–4C). The proliferation and stem cell populations of P7 tendons were designated at the beginning, whereas the mature tendon fibroblast 2 and tendon fibroblast 3 clusters of P14 were ascribed at the end of the trajectory.
为了系统地解析腱成熟过程，我们接下来基于单细胞转录组分析数据重构了腱成熟过程。伪时序轨迹与聚类结果和发育过程很好地匹配(图 4A-4C)。P7 腱的增殖和干细胞群体被指定在轨迹起点，而 P14 的成熟腱成纤维细胞 2 和腱成纤维细胞 3 簇则被归属于轨迹终点。
为了系统地解析腱成熟过程，我们接下来基于单细胞转录组分析数据重构了腱成熟过程。伪时序轨迹与聚类结果和发育过程很好地匹配(图 4A-4C)。P7 腱的增殖和干细胞群体被指定在轨迹起点，而 P14 的成熟腱成纤维细胞 2 和腱成纤维细胞 3 簇则被归属于轨迹终点。
Figure 4 Temporal transcriptomics dynamics along the tendon maturation trajectory
图 4 沿腱成熟轨迹的时序转录组动态变化
图 4 沿腱成熟轨迹的时序转录组动态变化
To gain deep insight into the sequential dynamics of gene expression along the tendon maturation process, we clustered the DEGs and obtained five groups (Figure 4D). Genes in cluster 1 declined from the beginning and were mainly assigned to regulation of cell division (Mki67, Top2a, and Prc1) and developmental growth involved in morphogenesis (Gal) (Figures 4E and 4F). Genes in cluster 2 were mainly engaged in regulating cell differentiation (Thy1 and Nes) and apoptotic processes (Bax and Cav1), demonstrating a gradually downregulated pattern along the pseudo-time axis (Figures 4E and 4F). Cluster 3 and 4 genes were initially upregulated and then downregulated, related to epithelial-to-mesenchymal transition (Notch1, Foxa1, and Fuz) and activation of mitogen-activated protein kinase (MAPK) activity (Cybb, Mapk311, Ret, and Dusp5), respectively. Finally, genes in cluster 5 peaked at the end of the maturation process, with strong involvement in ECM organization (Ctsk, Prg4, and Thbs4), response to hypoxia (Ang and Loxl2), and cellular response to mechanical stimulation (Dcn and Serpine2). Immunofluorescence staining results confirmed that the expression of PRG4 and THBS4 was increased from day 7 to day 14 (Figure 4G). We also examined the continuous time-serial expression pattern of known tenogenic genes (Figure S5A). Nes was downregulated along the pseudo-time path. Simultaneously, the expression of Col1a1 and Col3a1 exhibited an upregulation pattern (Figure S5A).
为深入了解肌腱成熟过程中基因表达的时序动态，我们对差异表达基因进行聚类分析，得到了五个群组（图 4D）。第 1 类基因从一开始就呈下降趋势，主要与细胞分裂调控（Mki67、Top2a 和 Prc1）以及形态发生相关的发育生长（Gal）有关（图 4E 和 4F）。第 2 类基因主要参与细胞分化（Thy1 和 Nes）和细胞凋亡过程（Bax 和 Cav1）的调控，在拟时间轴上呈现逐渐下调的模式（图 4E 和 4F）。第 3 类和第 4 类基因起初上调后下调，分别与上皮-间充质转化（Notch1、Foxa1 和 Fuz）和丝裂原活化蛋白激酶（MAPK）活性的激活（Cybb、Mapk311、Ret 和 Dusp5）相关。最后，第 5 类基因在成熟过程末期达到峰值，主要参与细胞外基质组织（Ctsk、Prg4 和 Thbs4）、低氧响应（Ang 和 Loxl2）以及细胞对机械刺激的响应（Dcn 和 Serpine2）。免疫荧光染色结果证实 PRG4 和 THBS4 的表达从第 7 天到第 14 天呈上升趋势（图 4G）。我们还检测了已知肌腱发生相关基因的连续时序表达模式（图 S5A）。Nes 在拟时间路径上呈下调趋势，同时 Col1a1 和 Col3a1 的表达呈现上调模式（图 S5A）。
为深入了解肌腱成熟过程中基因表达的时序动态，我们对差异表达基因进行聚类分析，得到了五个群组（图 4D）。第 1 类基因从一开始就呈下降趋势，主要与细胞分裂调控（Mki67、Top2a 和 Prc1）以及形态发生相关的发育生长（Gal）有关（图 4E 和 4F）。第 2 类基因主要参与细胞分化（Thy1 和 Nes）和细胞凋亡过程（Bax 和 Cav1）的调控，在拟时间轴上呈现逐渐下调的模式（图 4E 和 4F）。第 3 类和第 4 类基因起初上调后下调，分别与上皮-间充质转化（Notch1、Foxa1 和 Fuz）和丝裂原活化蛋白激酶（MAPK）活性的激活（Cybb、Mapk311、Ret 和 Dusp5）相关。最后，第 5 类基因在成熟过程末期达到峰值，主要参与细胞外基质组织（Ctsk、Prg4 和 Thbs4）、低氧响应（Ang 和 Loxl2）以及细胞对机械刺激的响应（Dcn 和 Serpine2）。免疫荧光染色结果证实 PRG4 和 THBS4 的表达从第 7 天到第 14 天呈上升趋势（图 4G）。我们还检测了已知肌腱发生相关基因的连续时序表达模式（图 S5A）。Nes 在拟时间路径上呈下调趋势，同时 Col1a1 和 Col3a1 的表达呈现上调模式（图 S5A）。
To further investigate the regulatory mechanisms along with tendon maturation, we divided the cells into three different states: early, intermediate, and late (Figure 5A). Notably, when projecting all subgroup tendon cells onto the pseudo-time trajectory, a considerably increased proportion of the Cd9+Cd271+ cluster was recognized at the intermediate transition state and then decreased at the maturation late state (Figure 5A). Subsequently, we analyzed the critical signaling pathways involved in the tendon maturation event. The TGF-β and RTK signaling pathways were mainly enriched in the intermediate and late stages. Specifically, the FGF signaling pathway, platelet-derived growth factor (PDGF) signaling pathway, and NGF pathway were enriched in the intermediate state compared with the early state (Figure 5B). In contrast, only the insulin growth factor (IGF) signaling pathway was enhanced in the late stage compared with the intermediate stage. Accordingly, we systematically investigated the expression patterns of genes related to these signaling pathways (Figure 5C). Specifically, Tgfb2 was downregulated from the early to the late stage. Pdgfra was upregulated from the intermediate stage to the late stage (Figure 5C), whereas its ligand Pdgfa showed decreased expression (Figure S5B). Ngf, Igf1, and Fgfr1 demonstrated consistent upregulation in the intermediate and late states, suggesting their involvement in promoting tendon maturation (Figure 5C).
为进一步研究肌腱成熟过程中的调控机制，我们将细胞分为早期、中期和晚期三个状态（图 5A）。值得注意的是，当将所有亚群肌腱细胞投射到拟时间轨迹上时，我们发现 Cd9 + Cd271 + 簇在中期过渡状态显著增加，而在成熟晚期则减少（图 5A）。随后，我们分析了参与肌腱成熟事件的关键信号通路。TGF-β和 RTK 信号通路主要在中期和晚期富集。具体而言，与早期相比，成纤维细胞生长因子（FGF）信号通路、血小板源性生长因子（PDGF）信号通路和 NGF 通路在中期状态富集（图 5B）。相比之下，与中期相比，只有胰岛素样生长因子（IGF）信号通路在晚期得到增强。据此，我们系统研究了这些信号通路相关基因的表达模式（图 5C）。具体来说，Tgfb2 从早期到晚期呈下调趋势。Pdgfra 从中期到晚期呈上调趋势（图 5C），而其配体 Pdgfa 则表现出表达下降（图 S5B）。Ngf、Igf1 和 Fgfr1 在中期和晚期状态持续上调，表明它们参与促进肌腱成熟（图 5C）。
为进一步研究肌腱成熟过程中的调控机制，我们将细胞分为早期、中期和晚期三个状态（图 5A）。值得注意的是，当将所有亚群肌腱细胞投射到拟时间轨迹上时，我们发现 Cd9 + Cd271 + 簇在中期过渡状态显著增加，而在成熟晚期则减少（图 5A）。随后，我们分析了参与肌腱成熟事件的关键信号通路。TGF-β和 RTK 信号通路主要在中期和晚期富集。具体而言，与早期相比，成纤维细胞生长因子（FGF）信号通路、血小板源性生长因子（PDGF）信号通路和 NGF 通路在中期状态富集（图 5B）。相比之下，与中期相比，只有胰岛素样生长因子（IGF）信号通路在晚期得到增强。据此，我们系统研究了这些信号通路相关基因的表达模式（图 5C）。具体来说，Tgfb2 从早期到晚期呈下调趋势。Pdgfra 从中期到晚期呈上调趋势（图 5C），而其配体 Pdgfa 则表现出表达下降（图 S5B）。Ngf、Igf1 和 Fgfr1 在中期和晚期状态持续上调，表明它们参与促进肌腱成熟（图 5C）。
Figure 5 The intermediate Cd9+Cd271+ cell cluster bridged the immature-to-mature tendon transition through the RTK signaling pathways
图 5 中期 Cd9 + Cd271 + 细胞簇通过 RTK 信号通路架起了肌腱从未成熟到成熟状态的过渡桥梁
图 5 中期 Cd9 + Cd271 + 细胞簇通过 RTK 信号通路架起了肌腱从未成熟到成熟状态的过渡桥梁
In addition to gene expression, we examined the dynamic gene-regulatory networks along with tendon maturation. In total, 338 regulons (TFs and their predicted target genes) were predicted based on the transcriptome profile. Highly active developmental-stage-specific regulons were also identified (Figures S5C and S5D). To further investigate regulation patterns along the tendon maturation process, we constructed the TF correlation network along the early, intermediate, and late stages (Figure S5E). The intermediate subnetwork in which Sox10 activated many RTK family growth factors highlighted the sequential transcriptional switch (Figure S5E), suggesting an essential role of Sox10 in bridging early to late maturation states.
除了基因表达外，我们还研究了肌腱成熟过程中的动态基因调控网络。基于转录组谱，我们预测出了 338 个调控子（转录因子及其预测的靶基因）。我们还识别出了高度活跃的发育阶段特异性调控子（图 S5C 和 S5D）。为进一步研究肌腱成熟过程中的调控模式，我们构建了早期、中期和晚期的转录因子相关网络（图 S5E）。中期亚网络中，Sox10 激活了多个 RTK 家族生长因子，突显了序贯性转录开关（图 S5E），表明 Sox10 在连接早期和晚期成熟状态中发挥着重要作用。
除了基因表达外，我们还研究了肌腱成熟过程中的动态基因调控网络。基于转录组谱，我们预测出了 338 个调控子（转录因子及其预测的靶基因）。我们还识别出了高度活跃的发育阶段特异性调控子（图 S5C 和 S5D）。为进一步研究肌腱成熟过程中的调控模式，我们构建了早期、中期和晚期的转录因子相关网络（图 S5E）。中期亚网络中，Sox10 激活了多个 RTK 家族生长因子，突显了序贯性转录开关（图 S5E），表明 Sox10 在连接早期和晚期成熟状态中发挥着重要作用。
Our findings delineated the sequential transcriptome dynamics that contribute to tendon maturation and identified that the novel intermediate Cd9+Cd271+ cell subpopulation drove the transition from an immature state to a maturation state. Notably, the enrichment of the RTK signaling pathways in the intermediate state also showed their potential for governing early tenocyte maturation.
我们的研究描绘了促进肌腱成熟的序贯性转录组动态，并发现新型的中期 Cd9 + Cd271 + 细胞亚群驱动了从未成熟状态到成熟状态的转变。值得注意的是，中期状态中 RTK 信号通路的富集也显示了它们在调控早期肌腱细胞成熟方面的潜力。
我们的研究描绘了促进肌腱成熟的序贯性转录组动态，并发现新型的中期 Cd9 + Cd271 + 细胞亚群驱动了从未成熟状态到成熟状态的转变。值得注意的是，中期状态中 RTK 信号通路的富集也显示了它们在调控早期肌腱细胞成熟方面的潜力。
NGF can effectively promote tendon maturation
NGF 能有效促进肌腱成熟
Based on the single-cell data, several RTK family growth factor signaling pathways were activated during tendon maturation. We then selected FGF, PDGF-AA, IGF1, and NGF to validate their roles in the tendon maturation process. To closely retain the physiological, biochemical, and biomechanical cues of the tendon, we adopted the tissue explant model (Figure 5D).
基于单细胞数据，我们发现多个 RTK 家族生长因子信号通路在肌腱成熟过程中被激活。我们随后选择了 FGF、PDGF-AA、IGF1 和 NGF 来验证它们在肌腱成熟过程中的作用。为了最大程度地保持肌腱的生理、生化和生物力学特征，我们采用了组织外植体模型（图 5D）。
基于单细胞数据，我们发现多个 RTK 家族生长因子信号通路在肌腱成熟过程中被激活。我们随后选择了 FGF、PDGF-AA、IGF1 和 NGF 来验证它们在肌腱成熟过程中的作用。为了最大程度地保持肌腱的生理、生化和生物力学特征，我们采用了组织外植体模型（图 5D）。
We first examined the expression of the tendon-related genes in these growth-factor-treated groups. The qRT-PCR results showed increased expression of Tnmd in all growth-factor-treated groups (Figure S6A). However, only the NGF-treated group displayed significantly increased expression of the tendon master TF Scx (Figure 5E), which was further validated by the immunofluorescence staining results (Figure 5F). NGF treatment also upregulated the expression of other tendon-related genes, such as Col1a1, Col3a1, Bgn, and Dcn (Figures 5E and S6A). The lubricin Prg4, which is mainly expressed in mature tenocytes, was also significantly upregulated. After treatment with PDGF-AA, Col3a1 was upregulated with 2-fold changes, although less than the NGF group (Figure 5E). To further verify the role of NGF in collagen fibrillogenesis, we conducted TEM experiments (Figure 5G). The quantitative results demonstrated that the average diameter of collagen fibrils in the NGF group was significantly larger than that of the control group (Figures 5G and S6B). The distribution range of the collagen fibril diameter was also increased (Figure 5H). These findings comprehensively demonstrated that NGF signaling could effectively promote tendon maturation.
我们首先检测了这些生长因子处理组中肌腱相关基因的表达。qRT-PCR 结果显示所有生长因子处理组中 Tnmd 的表达均有增加（图 S6A）。然而，只有 NGF 处理组显著增加了肌腱主要转录因子 Scx 的表达（图 5E），这一结果通过免疫荧光染色得到进一步验证（图 5F）。NGF 处理还上调了其他肌腱相关基因的表达，如 Col1a1、Col3a1、Bgn 和 Dcn（图 5E 和 S6A）。主要在成熟肌腱细胞中表达的润滑蛋白 Prg4 也显著上调。经 PDGF-AA 处理后，Col3a1 表达上调了 2 倍，但低于 NGF 组（图 5E）。为进一步验证 NGF 在胶原纤维形成中的作用，我们进行了 TEM 实验（图 5G）。定量结果表明，NGF 组胶原纤维的平均直径显著大于对照组（图 5G 和 S6B）。胶原纤维直径的分布范围也有所增加（图 5H）。这些发现全面证明了 NGF 信号通路能有效促进肌腱成熟。
我们首先检测了这些生长因子处理组中肌腱相关基因的表达。qRT-PCR 结果显示所有生长因子处理组中 Tnmd 的表达均有增加（图 S6A）。然而，只有 NGF 处理组显著增加了肌腱主要转录因子 Scx 的表达（图 5E），这一结果通过免疫荧光染色得到进一步验证（图 5F）。NGF 处理还上调了其他肌腱相关基因的表达，如 Col1a1、Col3a1、Bgn 和 Dcn（图 5E 和 S6A）。主要在成熟肌腱细胞中表达的润滑蛋白 Prg4 也显著上调。经 PDGF-AA 处理后，Col3a1 表达上调了 2 倍，但低于 NGF 组（图 5E）。为进一步验证 NGF 在胶原纤维形成中的作用，我们进行了 TEM 实验（图 5G）。定量结果表明，NGF 组胶原纤维的平均直径显著大于对照组（图 5G 和 S6B）。胶原纤维直径的分布范围也有所增加（图 5H）。这些发现全面证明了 NGF 信号通路能有效促进肌腱成熟。
SHP2 is involved in transduction of signals triggered by NGF during tendon maturation
SHP2 参与神经生长因子（NGF）在肌腱成熟过程中的信号转导
To further elucidate the mechanism that regulates the RTK signaling pathway during tendon maturation, we investigated the key regulator of the RTK signaling pathway: Shp2. As the main regulator of phosphorylation status of RTKs, SHP2 is involved in activating growth factor signaling cascades and regulating cellular invasion, migration, and proliferation (Idrees et al., 2019). The STRING network revealed the correlation between SHP2 (PTPN11)- and RTK family-related growth factors (Figure 6A). To further define the cellular events of Shp2 involved during early postnatal tendon maturation, we examined its expression distribution across cells. The transcriptome results demonstrated that Shp2 was only detected in the Txnip+ and Cd9+ cell groups of the P7 tendon (Figure S6D), suggesting its role in tenogenesis. In the P14 tendon, all subgroups expressed Shp2, whereas the Sca1+ tenocyte demonstrated the highest expression (Figure S6E). The pseudo-time analysis also revealed an upregulated expression pattern of Shp2 along the maturation trajectory (Figure 6B). The immunofluorescence (IF) results confirmed the expression pattern of Shp2 during tendon maturation (Figure 6D). GSEA revealed that the expression of Shp2 in P7 and P14 was positively correlated with the tendon gene sets (Figure 6C) and several tendon development-related pathways, including the TGF-β signaling pathway, focal adhesion, and the mTOR signaling pathway (Figure S6C).
为进一步阐明调控肌腱成熟过程中 RTK 信号通路的机制，我们研究了 RTK 信号通路的关键调节因子 SHP2。作为 RTK 磷酸化状态的主要调节因子，SHP2 参与激活生长因子信号级联反应并调控细胞侵袭、迁移和增殖（ Idrees et al., 2019 ）。STRING 网络分析揭示了 SHP2（PTPN11）与 RTK 家族相关生长因子之间的相关性（图 6A）。为进一步明确 Shp2 在早期出生后肌腱成熟过程中参与的细胞事件，我们检测了其在各细胞中的表达分布。转录组结果显示，在 P7 肌腱中 Shp2 仅在 Txnip + 和 Cd9 + 细胞群中被检测到（图 S6D），提示其在肌腱发生中的作用。在 P14 肌腱中，所有亚群均表达 Shp2，其中 Sca1 + 肌腱细胞显示最高表达（图 S6E）。拟时序分析也揭示了 Shp2 沿成熟轨迹呈上调表达模式（图 6B）。免疫荧光（IF）结果证实了 Shp2 在肌腱成熟过程中的表达模式（图 6D）。GSEA 分析显示，P7 和 P14 中 Shp2 的表达与肌腱基因集（图 6C）以及多个肌腱发育相关通路（包括 TGF-β信号通路、局部粘附和 mTOR 信号通路）呈正相关（图 S6C）。
31.
Idrees, M. ∙ Xu, L. ∙ Song, S.-H. ...
PTPN11 (SHP2) is indispensable for growth factors and cytokine signal transduction during bovine oocyte maturation and blastocyst development
Cells. 2019; 8:1272为进一步阐明调控肌腱成熟过程中 RTK 信号通路的机制，我们研究了 RTK 信号通路的关键调节因子 SHP2。作为 RTK 磷酸化状态的主要调节因子，SHP2 参与激活生长因子信号级联反应并调控细胞侵袭、迁移和增殖（ Idrees et al., 2019
31.
Idrees, M. ∙ Xu, L. ∙ Song, S.-H. ...
PTPN11 (SHP2) is indispensable for growth factors and cytokine signal transduction during bovine oocyte maturation and blastocyst development
Cells. 2019; 8:1272
Figure 6 SHP2 is involved in the relay of signals triggered by NGF during tendon maturation
图 6 SHP2 参与神经生长因子（NGF）在肌腱成熟过程中的信号传递
图 6 SHP2 参与神经生长因子（NGF）在肌腱成熟过程中的信号传递
GeneMANIA (Warde-Farley et al., 2010) and gene-regulatory network analyses predicted a notable correlation between the NGF signaling pathway and Shp2 (Figure S6F and S6G). When we pre-treated TSPCs with PHPS1, which can inhibit the phosphatase activity of SHP2, and then treated them with NGF, the expression of Bgn, Fmod, Sox9, and Thbs4 was downregulated compared with the NGF addition group (Figure 6I). The IF results also demonstrated downregulation of SCX expression of the PHPS1-treated group (Figure 6H). The diameter of the collagen fibrils of the tendon explant was also reduced in the NGF and PHPS1 groups compared with the NGF group (Figures 6E–6G). Therefore, SHP2 is involved in regulation of tendon maturation via NGF.
GeneMANIA（ Warde-Farley et al., 2010 ）和基因调控网络分析预测 NGF 信号通路与 Shp2 之间存在显著相关性（图 S6F 和 S6G）。当我们用能够抑制 SHP2 磷酸酶活性的 PHPS1 预处理 TSPCs，随后用 NGF 处理时，与单纯 NGF 添加组相比，Bgn、Fmod、Sox9 和 Thbs4 的表达均下调（图 6I）。免疫荧光结果也显示 PHPS1 处理组的 SCX 表达下调（图 6H）。与 NGF 组相比，NGF 和 PHPS1 组中肌腱外植体的胶原原纤维直径也减小（图 6E-6G）。因此，SHP2 通过 NGF 参与调控肌腱成熟。
59.
Warde-Farley, D. ∙ Donaldson, S.L. ∙ Comes, O. ...
The GeneMANIA prediction server: biological network integration for gene prioritization and predicting gene function
Nucleic Acids Res. 2010; 38:W214-W220GeneMANIA（ Warde-Farley et al., 2010
59.
Warde-Farley, D. ∙ Donaldson, S.L. ∙ Comes, O. ...
The GeneMANIA prediction server: biological network integration for gene prioritization and predicting gene function
Nucleic Acids Res. 2010; 38:W214-W220SHP2 is indispensable for promoting tendon maturation
SHP2 对促进肌腱成熟不可或缺
We further examined the function of Shp2 on TSPCs. When TSPCs were pre-treated with PHPS1, downregulation of tendon-related genes was observed (Figure 6L). In addition, the diameter of collagen fibrils of TSPC sheets was markedly reduced in the PHPS1 group (Figures 6J, 6K, and S6I). We also evaluated the multi-lineage differentiation capacity of TSPCs. We found that chondrogenesis, adipogenesis, and osteogenesis of PHPS1-treated TSPCs were enhanced (Figure S6H). Tenogenesis was diminished. Shp2 is essential for tendon stem cell phenotype maintenance and tenogenesis.
我们进一步研究了 Shp2 对 TSPCs 的功能。当 TSPCs 经 PHPS1 预处理后，观察到肌腱相关基因表达下调（图 6L）。此外，PHPS1 组中 TSPC 片层的胶原原纤维直径明显减小（图 6J、6K 和 S6I）。我们还评估了 TSPCs 的多向分化能力。我们发现 PHPS1 处理的 TSPCs 的软骨形成、脂肪形成和成骨分化能力增强（图 S6H）。而肌腱发生能力减弱。Shp2 对维持肌腱干细胞表型和肌腱发生至关重要。
我们进一步研究了 Shp2 对 TSPCs 的功能。当 TSPCs 经 PHPS1 预处理后，观察到肌腱相关基因表达下调（图 6L）。此外，PHPS1 组中 TSPC 片层的胶原原纤维直径明显减小（图 6J、6K 和 S6I）。我们还评估了 TSPCs 的多向分化能力。我们发现 PHPS1 处理的 TSPCs 的软骨形成、脂肪形成和成骨分化能力增强（图 S6H）。而肌腱发生能力减弱。Shp2 对维持肌腱干细胞表型和肌腱发生至关重要。
To rigorously investigate the roles of Shp2 in tendon development, we constructed tendon-specific Shp2 knockout mice (Figure 7A). General tendon hypoplasia was observed in conditional knockout mice. Specifically, compared with the control group, the Achilles tendons of the ScxCre,Shp2fl/fl mice were considerably thinner (Figure 7B) and displayed a more disordered and loose collagen fiber organization (Figure 7D). Impaired collagen maturation with a light yellow color was also identified, based on the polarized image (Figure S7A). Similar results were observed in the tail tendon and patellar tendon (Figures 7B and 7D). The wire hanging test results of Shp2 CKO (conditional knockout) mice also demonstrated their weaker movement ability (Figure 7E). To further investigate the role of Shp2 in collagen fibrillogenesis, we analyzed TEM images of the Achilles tendons (Figure 7C). The statistical results indicated that conditional Shp2 knockout mice displayed a smaller average collagen fibril diameter (Figures 7C, 7F, and S7B). The distribution range of the collagen fibril diameter in ScxCre,Shp2fl/fl mice was also narrowed compared with the control group (Figure 7F). When examining the TEM results after cuprolinic blue staining, we found that glycosaminoglycan (GAG) was significantly reduced (Figures 7C and 7G) and that the interfibrillar distance was also decreased in Shp2 CKO mice (Figures S7C–S7F). Thus, conditional knockout of Shp2 could impair collagen fibrillogenesis and fibril spacing during the postnatal tendon maturation process.
为严格研究 Shp2 在肌腱发育中的作用，我们构建了肌腱特异性 Shp2 敲除小鼠（图 7A）。在条件性敲除小鼠中观察到普遍的肌腱发育不全。具体而言，与对照组相比，Scx Cre 、Shp2 fl/fl 小鼠的跟腱明显变薄（图 7B），并呈现更加紊乱和疏松的胶原纤维组织（图 7D）。基于偏振图像，还发现胶原成熟受损，呈浅黄色（图 S7A）。尾腱和髌腱也观察到类似结果（图 7B 和 7D）。Shp2 条件性敲除（CKO）小鼠的悬丝测试结果也显示其运动能力较弱（图 7E）。为进一步研究 Shp2 在胶原原纤维形成中的作用，我们分析了跟腱的 TEM 图像（图 7C）。统计结果表明，条件性 Shp2 敲除小鼠显示较小的平均胶原原纤维直径（图 7C、7F 和 S7B）。与对照组相比，Scx Cre 、Shp2 fl/fl 小鼠的胶原原纤维直径分布范围也变窄（图 7F）。在检查铜蓝染色后的 TEM 结果时，我们发现糖胺聚糖（GAG）显著减少（图 7C 和 7G），且 Shp2 CKO 小鼠的纤维间距也减小（图 S7C-S7F）。因此，条件性敲除 Shp2 可能损害出生后肌腱成熟过程中的胶原原纤维形成和纤维间距。
为严格研究 Shp2 在肌腱发育中的作用，我们构建了肌腱特异性 Shp2 敲除小鼠（图 7A）。在条件性敲除小鼠中观察到普遍的肌腱发育不全。具体而言，与对照组相比，Scx Cre 、Shp2 fl/fl 小鼠的跟腱明显变薄（图 7B），并呈现更加紊乱和疏松的胶原纤维组织（图 7D）。基于偏振图像，还发现胶原成熟受损，呈浅黄色（图 S7A）。尾腱和髌腱也观察到类似结果（图 7B 和 7D）。Shp2 条件性敲除（CKO）小鼠的悬丝测试结果也显示其运动能力较弱（图 7E）。为进一步研究 Shp2 在胶原原纤维形成中的作用，我们分析了跟腱的 TEM 图像（图 7C）。统计结果表明，条件性 Shp2 敲除小鼠显示较小的平均胶原原纤维直径（图 7C、7F 和 S7B）。与对照组相比，Scx Cre 、Shp2 fl/fl 小鼠的胶原原纤维直径分布范围也变窄（图 7F）。在检查铜蓝染色后的 TEM 结果时，我们发现糖胺聚糖（GAG）显著减少（图 7C 和 7G），且 Shp2 CKO 小鼠的纤维间距也减小（图 S7C-S7F）。因此，条件性敲除 Shp2 可能损害出生后肌腱成熟过程中的胶原原纤维形成和纤维间距。
Figure 7 Depletion of Shp2 in the tendon led to defective maturation of the postnatal tendon
图 7 肌腱中 Shp2 的缺失导致出生后肌腱成熟缺陷
图 7 肌腱中 Shp2 的缺失导致出生后肌腱成熟缺陷
We also performed bulk RNA-seq on the Achilles tendons of Shp2 CKO and control mice at 6 weeks of age to decipher the molecular changes upon Shp2 disruption. Based on the KEGG analysis of the DEGs (Figure 7H), we observed downregulation of the PI3K-AKT signaling pathway, focal adhesion, ECM-receptor interaction, and response to NGF signaling in the CKO mouse group (Figure 7I). We also found that the expression levels of the NGF-signaling related genes Ngf and Ngfr were downregulated (Figure S7G). This suggests that Shp2 does not only participate in transmission of signals from growth factor receptors but also plays a specific role in control of their expression (Lu et al., 1998). GO analysis revealed that the upregulated genes were mainly engaged in mitotic cell cycle regulation, mRNA processing, and positive regulation of chromatin organization, corresponding with the biological enrichment function of the early state of the pseudo-time of tendon maturation (Figure 4E; Table S2). However, the biological functions involved in the late state, such as ECM organization and collagen metabolic processes, were only enriched in the control group (Table S2), illustrating failed transition of the mature tendon in ScxCre,Shp2fl/fl mice. Specifically, Shp2 deletion significantly downregulated the expression of genes that regulate ECM structure and collagen assembly, including Col1a1, Tnmd, and Fmod, as well as the critical tenogenic TF Scx, all confirmed by qRT-PCR (Figure 7J). These results indicated the indispensable roles of Shp2 in promoting postnatal tendon maturation.
我们还对 6 周龄 Shp2 CKO 和对照组小鼠的跟腱进行了整体 RNA-seq 测序，以解析 Shp2 缺失后的分子变化。基于对差异表达基因(DEGs)的 KEGG 分析(图 7H)，我们观察到 CKO 小鼠组中 PI3K-AKT 信号通路、局部粘附、ECM-受体相互作用以及对 NGF 信号的响应均下调(图 7I)。我们还发现 NGF 信号相关基因 Ngf 和 Ngfr 的表达水平降低(图 S7G)。这表明 Shp2 不仅参与生长因子受体信号的传递，还在控制其表达方面发挥特定作用( Lu et al., 1998 )。GO 分析揭示上调的基因主要参与有丝分裂周期调节、mRNA 处理和染色质组织的正向调控，这与肌腱成熟伪时间早期阶段的生物富集功能相对应(图 4E；表 S2)。然而，与晚期阶段相关的生物功能，如 ECM 组织和胶原代谢过程，仅在对照组中富集(表 S2)，说明 Scx Cre ，Shp2 fl/fl 小鼠中成熟肌腱的转变失败。具体而言，Shp2 缺失显著下调了调控 ECM 结构和胶原组装的基因表达，包括 Col1a1、Tnmd 和 Fmod，以及关键的肌腱生成转录因子 Scx，这些都通过 qRT-PCR 得到了确认(图 7J)。这些结果表明 Shp2 在促进出生后肌腱成熟中具有不可或缺的作用。
42.
Lu, X. ∙ Qu, C.-K. ∙ Shi, Z.-Q. ...
Downregulation of platelet-derived growth factor receptor-β in Shp-2 mutant fibroblast cell lines
Oncogene. 1998; 17:441-448我们还对 6 周龄 Shp2 CKO 和对照组小鼠的跟腱进行了整体 RNA-seq 测序，以解析 Shp2 缺失后的分子变化。基于对差异表达基因(DEGs)的 KEGG 分析(图 7H)，我们观察到 CKO 小鼠组中 PI3K-AKT 信号通路、局部粘附、ECM-受体相互作用以及对 NGF 信号的响应均下调(图 7I)。我们还发现 NGF 信号相关基因 Ngf 和 Ngfr 的表达水平降低(图 S7G)。这表明 Shp2 不仅参与生长因子受体信号的传递，还在控制其表达方面发挥特定作用( Lu et al., 1998
42.
Lu, X. ∙ Qu, C.-K. ∙ Shi, Z.-Q. ...
Downregulation of platelet-derived growth factor receptor-β in Shp-2 mutant fibroblast cell lines
Oncogene. 1998; 17:441-448Discussion
Promoting the transition from the repaired fetal-like status into a functionally mature tendon is the key to achieving tendon regeneration. In this study, we applied single-cell transcriptome analysis to decipher the naturally programmed maturation of tendons at an unprecedentedly high resolution. Being a quantitative high-throughput measurement approach, the single-cell transcriptome analysis method is revolutionizing the field of developmental and regenerative biology (Xia and Yanai, 2019). Novel cell types and transient cell states are being identified. Combined with the computational lineage-inferring method, scRNA-seq can also identify continuous cellular dynamics along with the developmental and regeneration axes and help dissect the underlying regulatory mechanisms (Gerber et al., 2018; Plass et al., 2018). Here, using the scRNA-seq technique, we delineated the heterogeneity tenocyte map and reconstructed the molecular cascades underlying the natural tendon maturation process. The NGF-secreting Cd9+Cd271+ TSPC population and the dominating RTK signaling pathways in the transitional maturation state were revealed.
促进修复的胎儿样状态向功能性成熟肌腱转变是实现肌腱再生的关键。在本研究中，我们应用单细胞转录组分析以前所未有的高分辨率解析肌腱自然程序化成熟过程。作为一种定量高通量测量方法，单细胞转录组分析正在革新发育和再生生物学领域( Xia and Yanai, 2019 )。新的细胞类型和瞬时细胞状态正被鉴定出来。结合计算谱系推断方法，scRNA-seq 还能识别沿发育和再生轴的连续细胞动态，并帮助剖析潜在的调控机制( Gerber et al., 2018 ； Plass et al., 2018 )。在此，我们利用 scRNA-seq 技术描绘了异质性肌腱细胞图谱，并重建了自然肌腱成熟过程的分子级联。研究揭示了分泌 NGF 的 Cd9 + Cd271 + 肌腱干/祖细胞(TSPC)群体以及在过渡性成熟状态中占主导地位的 RTK 信号通路。
61.
Xia, B. ∙ Yanai, I.
A periodic table of cell types
Development. 2019; 146:dev16985420.
Gerber, T. ∙ Murawala, P. ∙ Knapp, D. ...
Single-cell analysis uncovers convergence of cell identities during axolotl limb regeneration
Science. 2018; 362, eaaq068149.
Plass, M. ∙ Solana, J. ∙ Wolf, F.A. ...
Cell type atlas and lineage tree of a whole complex animal by single-cell transcriptomics
Science. 2018; 360, eaaq1723促进修复的胎儿样状态向功能性成熟肌腱转变是实现肌腱再生的关键。在本研究中，我们应用单细胞转录组分析以前所未有的高分辨率解析肌腱自然程序化成熟过程。作为一种定量高通量测量方法，单细胞转录组分析正在革新发育和再生生物学领域( Xia and Yanai, 2019
61.
Xia, B. ∙ Yanai, I.
A periodic table of cell types
Development. 2019; 146:dev16985420.
Gerber, T. ∙ Murawala, P. ∙ Knapp, D. ...
Single-cell analysis uncovers convergence of cell identities during axolotl limb regeneration
Science. 2018; 362, eaaq068149.
Plass, M. ∙ Solana, J. ∙ Wolf, F.A. ...
Cell type atlas and lineage tree of a whole complex animal by single-cell transcriptomics
Science. 2018; 360, eaaq1723Tenocytes and tendon-derived stem cells are two major cell types in tendon tissues. The heterogeneous nature of these cells has been distinguished previously mainly by their anatomical locations with limited characteristic markers. Further investigation is urgently needed to decipher the phenotypical differences and the specific markers to discriminate these cell populations (Dyment and Galloway, 2015). In this project, different stem, proliferation, and maturation tendon cells were characterized, enriching our knowledge of the tendon cell repertoire. Notably, a convergence tendency of cell identities was identified throughout the maturation process, corresponding with the initial developmental plasticity (Subramanian and Schilling, 2015) to a final maturation homogeneity of tendons. When we compared the cellular composition, only Cd9+Cd271+ cells remained in the P14 tendon, implying their key transition role in bridging TSPCs to mature tendon cells. Quantitative assessment of ligand-receptor interactions during this process revealed decreased cellular communication accompanied by reduced cell density and cell types. Cell-cell junctions also allow direct chemical communication between adjacent cytoplasm (Richardson et al., 2007) and participate in modulating tendon development and injury (Theodossiou et al., 2020). A previous study has demonstrated a relatively stable number of postnatal cell-cell junctions using microscopy (Kalson et al., 2015). However, in our transcriptomics data, an increase in Cdh13 and a decrease in Cdh7 reflected the dynamic cellular gap junction communications.
肌腱细胞和肌腱源性干细胞是肌腱组织中的两种主要细胞类型。以往对这些细胞异质性的区分主要基于它们的解剖位置，且特征性标记有限。迫切需要进一步研究来解析这些细胞群体的表型差异并确定特异性标记( Dyment and Galloway, 2015 )。在本项目中，我们对不同的干细胞、增殖和成熟肌腱细胞进行了表征，丰富了我们对肌腱细胞谱系的认识。值得注意的是，我们在整个成熟过程中发现了细胞身份的趋同趋势，这与初始发育可塑性( Subramanian and Schilling, 2015 )到最终肌腱成熟同质性相对应。在比较细胞组成时，我们发现仅 Cd9 + Cd271 + 细胞在 P14 肌腱中保留，暗示它们在连接肌腱干/祖细胞(TSPCs)到成熟肌腱细胞的转变中具有关键作用。对这一过程中配体-受体相互作用的定量评估显示，伴随细胞密度和细胞类型的减少，细胞间通讯也减弱。细胞-细胞连接还允许相邻细胞质之间的直接化学通讯( Richardson et al., 2007 )，并参与调节肌腱发育和损伤( Theodossiou et al., 2020 )。先前的研究已使用显微镜技术证明出生后细胞-细胞连接数量相对稳定( Kalson et al., 2015 )。然而，在我们的转录组学数据中，Cdh13 的增加和 Cdh7 的减少反映了动态的细胞间隙连接通讯。
16.
Dyment, N.A. ∙ Galloway, J.L.
Regenerative biology of tendon: mechanisms for renewal and repair
Curr. Mol. Biol. Rep. 2015; 1:124-13154.
Subramanian, A. ∙ Schilling, T.F.
Tendon development and musculoskeletal assembly: emerging roles for the extracellular matrix
Development. 2015; 142:4191-420452.
Richardson, S.H. ∙ Starborg, T. ∙ Lu, Y. ...
Tendon development requires regulation of cell condensation and cell shape via cadherin-11-mediated cell-cell junctions
Mol. Cell. Biol. 2007; 27:6218-622857.
Theodossiou, S.K. ∙ Murray, J.B. ∙ Schiele, N.R.
Cell-cell junctions in developing and adult tendons
Tissue Barriers. 2020; 8:169549136.
Kalson, N.S. ∙ Lu, Y. ∙ Taylor, S.H. ...
A structure-based extracellular matrix expansion mechanism of fibrous tissue growth
ELife. 2015; 4, e05958肌腱细胞和肌腱源性干细胞是肌腱组织中的两种主要细胞类型。以往对这些细胞异质性的区分主要基于它们的解剖位置，且特征性标记有限。迫切需要进一步研究来解析这些细胞群体的表型差异并确定特异性标记( Dyment and Galloway, 2015
16.
Dyment, N.A. ∙ Galloway, J.L.
Regenerative biology of tendon: mechanisms for renewal and repair
Curr. Mol. Biol. Rep. 2015; 1:124-13154.
Subramanian, A. ∙ Schilling, T.F.
Tendon development and musculoskeletal assembly: emerging roles for the extracellular matrix
Development. 2015; 142:4191-420452.
Richardson, S.H. ∙ Starborg, T. ∙ Lu, Y. ...
Tendon development requires regulation of cell condensation and cell shape via cadherin-11-mediated cell-cell junctions
Mol. Cell. Biol. 2007; 27:6218-622857.
Theodossiou, S.K. ∙ Murray, J.B. ∙ Schiele, N.R.
Cell-cell junctions in developing and adult tendons
Tissue Barriers. 2020; 8:169549136.
Kalson, N.S. ∙ Lu, Y. ∙ Taylor, S.H. ...
A structure-based extracellular matrix expansion mechanism of fibrous tissue growth
ELife. 2015; 4, e05958Here, we identified NGF as a promising maturation promotion factor for tendon growth. NGF is one of the neurotrophins, which is essential for the growth and remodeling phases in different types of tissues besides nerve cells. Previous studies have shown that ovine tendon (Bagge et al., 2009) and human Achilles tendon (Russo et al., 2015) cells are sources of NGF. A group of tendon-related genes, Col1a1, Prg4, and Bgn, were enhanced in our NGF-treated tendon explant samples. We also found that Shp2 is involved in transduction of signals triggered by NGF during tendon maturation. The cross-talk between NGF and other signaling pathways is essential for tendon stem cell phenotype maintenance and tenogenesis, such as the TGF-β and the mTOR signaling pathways, requires further in-depth investigation.
在此，我们确定了 NGF 作为肌腱生长的一种有前景的成熟促进因子。NGF 是神经营养因子之一，除神经细胞外，它对不同类型组织的生长和重塑阶段都至关重要。先前研究表明，绵羊肌腱( Bagge et al., 2009 )和人跟腱( Russo et al., 2015 )细胞是 NGF 的来源。在我们的 NGF 处理的肌腱外植体样本中，一组肌腱相关基因（Col1a1、Prg4 和 Bgn）的表达增强。我们还发现 Shp2 参与了肌腱成熟过程中由 NGF 触发的信号转导。NGF 与其他信号通路之间的交叉对话对肌腱干细胞表型维持和肌腱发生至关重要，例如与 TGF-β和 mTOR 信号通路之间的相互作用，这需要进一步深入研究。
5.
Bagge, J. ∙ Lorentzon, R. ∙ Alfredson, H. ...
Unexpected presence of the neurotrophins NGF and BDNF and the neurotrophin receptor p75 in the tendon cells of the human Achilles tendon
Histol. Histopathol. 2009; 24:839-84853.
Russo, V. ∙ Mauro, A. ∙ Martelli, A. ...
Cellular and molecular maturation in fetal and adult ovine calcaneal tendons
J. Anat. 2015; 226:126-142在此，我们确定了 NGF 作为肌腱生长的一种有前景的成熟促进因子。NGF 是神经营养因子之一，除神经细胞外，它对不同类型组织的生长和重塑阶段都至关重要。先前研究表明，绵羊肌腱( Bagge et al., 2009
5.
Bagge, J. ∙ Lorentzon, R. ∙ Alfredson, H. ...
Unexpected presence of the neurotrophins NGF and BDNF and the neurotrophin receptor p75 in the tendon cells of the human Achilles tendon
Histol. Histopathol. 2009; 24:839-84853.
Russo, V. ∙ Mauro, A. ∙ Martelli, A. ...
Cellular and molecular maturation in fetal and adult ovine calcaneal tendons
J. Anat. 2015; 226:126-142We also revealed a role of Shp2 in regulating tendon maturation. Being a non-receptor tyrosine phosphatase protein, Shp2 is an indispensable and positive governor of signals mediated by various receptors and involved in phosphatidylinositol 3-kinase (PI3K), MAPK/P38, Jak/Stat, NFAT, and nuclear factor κB (NF-κB) signaling in a receptor- and cell-type-specific manner (Grossmann et al., 2010). In connective tissue, SHP2 has been identified as a molecular checkpoint for skin fibrosis (Zehender et al., 2018) and as a critical regulator of cartilage homeostasis and bone skeletal cell lineage differentiation (Bowen et al., 2014; Yang et al., 2013; Zuo et al., 2018). Loss of Shp2 in muscle could lead to a deficit in postnatal muscle growth by impairing proliferation of the satellite cells (Griger et al., 2017). In our tendon-specific Shp2 knockout mouse model, generally impaired tendon phenotypes were characterized. Tendon development is dependent on regulation of collagen fibrillogenesis and assembly, deposition, and organization of collagen fibers. In relation to the significant decrease in fibril-forming collagen molecular collagen I, ScxCre,Shp2fl/fl mice exhibited a dramatic decrease in tendon thickness compared with wild-type mice, concomitant with less parallel fibers. TEM images of ScxCre,Shp2fl/fl mice also demonstrated a decreased number of large-diameter fibrils. The interfibrillar spacing was also significantly diminished in the conditional knockout mouse group, consistent with reduced expression of SLRPs such as Dcn, Fmod, and Lum, which are key regulators of GAG side chains. There is a similar phenotype in Shp2 CKO and the fibromodulin/lumican double knockout model (Zhang et al., 2005). The mean diameter of the collagen fibrils was significantly decreased compared with the control group, with a disproportionate increase in small diameter, immature collagen fibrils, and a lack of progression to mature, large-diameter fibrils. Expression of Fmod was significantly decreased in Shp2 CKO mice, whereas expression of Lum remained unaltered. Thus, loss of fibromodulin mainly drove the immature fibril phenotype in the fibromodulin/lumican double knockout model. Multiple dysregulated signaling pathways, including the PI3K-AKT signaling pathway, focal adhesion, and ECM-receptor interaction, were also involved in fibrillogenesis failure. Contrary to muscle stem cells, loss of Shp2 in TSPCs did not affect cellular proliferation. In contrast, teno-lineage differentiation was impaired in TSPCs when SHP2 activity was inhibited.
我们还揭示了 Shp2 在调控肌腱成熟过程中的作用。作为一种非受体酪氨酸磷酸酶蛋白，Shp2 是多种受体介导信号的必需正向调节因子，以受体和细胞类型特异的方式参与磷脂酰肌醇 3-激酶(PI3K)、MAPK/P38、Jak/Stat、NFAT 和核因子κB(NF-κB)信号通路( Grossmann et al., 2010 )。在结缔组织中，SHP2 被确认为皮肤纤维化的分子检查点( Zehender et al., 2018 )，以及软骨稳态和骨骼细胞谱系分化的关键调节因子( Bowen et al., 2014 ; Yang et al., 2013 ; Zuo et al., 2018 )。肌肉中 Shp2 的缺失会通过损害卫星细胞增殖导致出生后肌肉生长缺陷( Griger et al., 2017 )。在我们的肌腱特异性 Shp2 敲除小鼠模型中，观察到普遍受损的肌腱表型。肌腱发育依赖于胶原原纤维形成、组装、沉积和胶原纤维组织的调控。与成纤维胶原分子胶原 I 的显著减少相关，与野生型小鼠相比，Scx Cre ,Shp2 fl/fl 小鼠表现出肌腱厚度的显著降低，同时平行纤维减少。Scx Cre ,Shp2 fl/fl 小鼠的 TEM 图像也显示大直径原纤维数量减少。条件性敲除小鼠组中的原纤维间距也显著减小，这与 SLRP（如 Dcn、Fmod 和 Lum）表达降低一致，这些是 GAG 侧链的关键调节因子。Shp2 CKO 和纤维连接蛋白/亮氨酸富集蛋白双敲除模型中存在相似的表型( Zhang et al., 2005 )。与对照组相比，胶原原纤维的平均直径显著减小，小直径未成熟胶原原纤维比例不成比例地增加，且缺乏向成熟的大直径原纤维发展的进程。Shp2 CKO 小鼠中 Fmod 的表达显著降低，而 Lum 的表达保持不变。因此，纤维连接蛋白的缺失主要导致了纤维连接蛋白/亮氨酸富集蛋白双敲除模型中的未成熟原纤维表型。多个失调的信号通路，包括 PI3K-AKT 信号通路、局部粘附和 ECM-受体相互作用，也参与了原纤维形成失败。与肌肉干细胞不同，TSPCs 中 Shp2 的缺失并不影响细胞增殖。相反，当 SHP2 活性被抑制时，TSPCs 的肌腱谱系分化受到损害。
24.
Grossmann, K.S. ∙ Rosário, M. ∙ Birchmeier, C. ...
The tyrosine phosphatase Shp2 in development and cancer
Adv. Cancer Res. 2010; 106:53-8964.
Zehender, A. ∙ Huang, J. ∙ Györfi, A.-H. ...
The tyrosine phosphatase SHP2 controls TGFβ-induced STAT3 signaling to regulate fibroblast activation and fibrosis
Nat. Commun. 2018; 9:32598.
Bowen, M.E. ∙ Ayturk, U.M. ∙ Kurek, K.C. ...
SHP2 regulates chondrocyte terminal differentiation, growth plate Architecture and skeletal cell fates
PLoS Genet. 2014; 10, e100436462.
Yang, W. ∙ Wang, J. ∙ Moore, D.C. ...
Ptpn11 deletion in a novel progenitor causes metachondromatosis by inducing hedgehog signalling
Nature. 2013; 499:491-49567.
Zuo, C. ∙ Wang, L. ∙ Kamalesh, R.M. ...
SHP2 regulates skeletal cell fate by modifying SOX9 expression and transcriptional activity
Bone Res. 2018; 6:1222.
Griger, J. ∙ Schneider, R. ∙ Lahmann, I. ...
Loss of Ptpn11 (Shp2) drives satellite cells into quiescence
ELife. 2017; 6, e2155265.
Zhang, G. ∙ Young, B.B. ∙ Ezura, Y. ...
Development of tendon structure and function: regulation of collagen fibrillogenesis
J. Musculoskelet. Neuronal Interact. 2005; 5:5-21我们还揭示了 Shp2 在调控肌腱成熟过程中的作用。作为一种非受体酪氨酸磷酸酶蛋白，Shp2 是多种受体介导信号的必需正向调节因子，以受体和细胞类型特异的方式参与磷脂酰肌醇 3-激酶(PI3K)、MAPK/P38、Jak/Stat、NFAT 和核因子κB(NF-κB)信号通路( Grossmann et al., 2010
24.
Grossmann, K.S. ∙ Rosário, M. ∙ Birchmeier, C. ...
The tyrosine phosphatase Shp2 in development and cancer
Adv. Cancer Res. 2010; 106:53-8964.
Zehender, A. ∙ Huang, J. ∙ Györfi, A.-H. ...
The tyrosine phosphatase SHP2 controls TGFβ-induced STAT3 signaling to regulate fibroblast activation and fibrosis
Nat. Commun. 2018; 9:32598.
Bowen, M.E. ∙ Ayturk, U.M. ∙ Kurek, K.C. ...
SHP2 regulates chondrocyte terminal differentiation, growth plate Architecture and skeletal cell fates
PLoS Genet. 2014; 10, e100436462.
Yang, W. ∙ Wang, J. ∙ Moore, D.C. ...
Ptpn11 deletion in a novel progenitor causes metachondromatosis by inducing hedgehog signalling
Nature. 2013; 499:491-49567.
Zuo, C. ∙ Wang, L. ∙ Kamalesh, R.M. ...
SHP2 regulates skeletal cell fate by modifying SOX9 expression and transcriptional activity
Bone Res. 2018; 6:1222.
Griger, J. ∙ Schneider, R. ∙ Lahmann, I. ...
Loss of Ptpn11 (Shp2) drives satellite cells into quiescence
ELife. 2017; 6, e2155265.
Zhang, G. ∙ Young, B.B. ∙ Ezura, Y. ...
Development of tendon structure and function: regulation of collagen fibrillogenesis
J. Musculoskelet. Neuronal Interact. 2005; 5:5-21Our findings delineate the sequential transcriptome dynamics that contribute to tendon maturation and identify the novel intermediate Cd9+Cd271+ stem/progenitor cell population that drives the immature state to the maturation state. Notably, enrichment of the RTK signaling pathways in the intermediate state also indicates their potential to govern early tenocyte maturation.
我们的研究描绘了促进肌腱成熟的序列性转录组动态，并识别了推动从未成熟状态向成熟状态转变的新型中间态 Cd9 + Cd271 + 干细胞/祖细胞群。值得注意的是，中间态中 RTK 信号通路的富集也表明了它们调控早期肌腱细胞成熟的潜能。
我们的研究描绘了促进肌腱成熟的序列性转录组动态，并识别了推动从未成熟状态向成熟状态转变的新型中间态 Cd9 + Cd271 + 干细胞/祖细胞群。值得注意的是，中间态中 RTK 信号通路的富集也表明了它们调控早期肌腱细胞成熟的潜能。
Limitations of the study 研究局限性
Although our data revealed that Shp2 is involved in regulation of signals triggered by NGF during tendon maturation, we cannot rule out pro-maturation effects of other RTK family growth factors because Shp2 is downstream of virtually all RTKs. The defective tendon maturation of Shp2 CKO mice could be the effect of all RTK growth factors. The mechanisms and the critical molecule that regulate NGF/SHP2 signaling in promoting tendon maturation need further investigation and validation.
尽管我们的数据表明 Shp2 参与调控肌腱成熟过程中 NGF 触发的信号，但我们无法排除其他 RTK 家族生长因子的促成熟作用，因为 Shp2 是几乎所有 RTK 的下游因子。Shp2 CKO 小鼠的肌腱成熟缺陷可能是所有 RTK 生长因子的综合效应。调控 NGF/SHP2 信号促进肌腱成熟的机制和关键分子需要进一步研究和验证。
尽管我们的数据表明 Shp2 参与调控肌腱成熟过程中 NGF 触发的信号，但我们无法排除其他 RTK 家族生长因子的促成熟作用，因为 Shp2 是几乎所有 RTK 的下游因子。Shp2 CKO 小鼠的肌腱成熟缺陷可能是所有 RTK 生长因子的综合效应。调控 NGF/SHP2 信号促进肌腱成熟的机制和关键分子需要进一步研究和验证。
STAR★Methods
Key resources table
| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
| Antibodies | ||
| Anti-PRG4 | Abcam | Cat# ab28484; RRID:AB_776089 |
| Anti-THBS4 | Abcam | Cat# ab176116 |
| Anti-SHP2 | Santa Cruz | Cat# sc-280; RRID:AB_632401 |
| Anti-CD271 | Abcam | Cat# ab52987; RRID:AB_881682 |
| Coat-anti-rab Alexa Fluor 488 兔抗体 Alexa Fluor 488 标记物 | Thermo Fisher | Cat# A-11034; RRID:AB_2576217 |
| Chemicals, peptides, and recombinant proteins 化学品、多肽和重组蛋白 | ||
| Collagenase | Gibco | 17100017 |
| low-glucose Dulbecco's modified Eagle's medium 低糖杜尔伯科改良鹰培养基 | Gibco | C11885500BT |
| Trizol reagent | TAKARA | 9109 |
| SuperScript II reverse transcriptase SuperScript II 反转录酶 | Invitrogen | 18064022 |
| IGF1 | Genescript | Z03177-100 |
| bFGF | Peprotech | AF-100-18B |
| PDGF-AA | Genescript | Z03276-10 |
| NGF | Peprotech | 450-01 |
| PHPS1 | Sigma | P0039 |
| TRIzol | Invitrogen | 15596026 |
| TB Green Premix Ex Taq | TakaRa | RR420A |
| Critical commercial assays 关键商业试剂盒 | ||
| NEBNext mRNA second strand synthesis kit NEBNext mRNA 双链合成试剂盒 | NEB | E6111S |
| Nextera XT kit DNA Sample Preparation Kit Nextera XT 试剂盒 DNA 样品制备套件 | Illumina | FC-131-1024 |
| Deposited data | ||
| Raw data | This paper | GSA: CRA003892 |
| Experimental models: Organisms/strains 实验模型：生物体/菌株 | ||
| Mouse: Scx-GFP | Dr. R. Schweitzer (Oregon Health & Science University, Oregon, USA) R. Schweitzer 博士（美国俄勒冈健康与科学大学，美国俄勒冈州） | N/A |
| Mouse: ScxCre | Dr. R. Schweitzer (Oregon Health & Science University, Oregon, USA) R. Schweitzer 博士（美国俄勒冈健康与科学大学，美国俄勒冈州） | N/A |
| Mouse: Shp2fl/fl | Dr. Yi Wang (Zhejiang University) 王毅博士（浙江大学） | N/A |
| Mouse: ScxCre,Shp2fl/fl 小鼠：Scx Cre ，Shp2 fl/fl | Dr. Xiao Chen (Zhejiang University) 陈晓博士（浙江大学） | N/A |
| Software and algorithms 软件与算法 | ||
| Seurat (v Seurat_2.3.4) | (Butler et al., 2018 11. Butler, A. ∙ Hoffman, P. ∙ Smibert, P. ... Integrating single-cell transcriptomic data across different conditions, technologies, and species Nat. Biotechnol. 2018; 36:411-420 | https://satijalab.org/seurat/ |
| CellphoneDB | (Efremova et al., 2020 18. Efremova, M. ∙ Vento-Tormo, M. ∙ Teichmann, S.A. ... CellPhoneDB: inferring cell–cell communication from combined expression of multi-subunit ligand–receptor complexes Nat. Protoc. 2020; 15:1484-1506 | https://github.com/Teichlab/cellphonedb |
| Monocle (v2.10.1) | (Qiu et al., 2017 50. Qiu, X. ∙ Hill, A. ∙ Packer, J. ... Single-cell mRNA quantification and differential analysis with Census Nat. Methods. 2017; 14:309-315 | http://cole-trapnell-lab.github.io/monocle-release/ |
| Metascape | (Zhou et al., 2019 66. Zhou, Y. ∙ Zhou, B. ∙ Pache, L. ... Metascape provides a biologist-oriented resource for the analysis of systems-level datasets Nat. Commun. 2019; 10:1523 | https://metascape.org |
| SCENIC | (Aibar et al., 2017 2. Aibar, S. ∙ González-Blas, C.B. ∙ Moerman, T. ... SCENIC: single-cell regulatory network inference and clustering Nat. Methods. 2017; 14:1083-1086 | https://github.com/aertslab/SCENIC |
| bigSCale2 | (Iacono et al., 2019 30. Iacono, G. ∙ Massoni-Badosa, R. ∙ Heyn, H. Single-cell transcriptomics unveils gene regulatory network plasticity Genome Biol. 2019; 20:110 | https://github.com/iaconogi/bigSCale2 |
| R v3.4.1 | The R Foundation for Statistical Computing R 语言统计计算基金会 | https://www.r-project.org/ |
Resource availability 资源可获取
Lead contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Xiao Chen (chenxiao-610@zju.edu.cn).
有关资源和试剂的进一步信息和请求应直接联系第一作者 Xiao Chen (chenxiao-610@zju.edu.cn)，并将由其 fulfillment 提供。
有关资源和试剂的进一步信息和请求应直接联系第一作者 Xiao Chen (chenxiao-610@zju.edu.cn)，并将由其 fulfillment 提供。
Materials availability 材料可获取
This study did not generate new unique reagents.
本研究未产生新的独特试剂。
本研究未产生新的独特试剂。
Experimental model and subject details
实验模型与研究对象详情
Animals
Experiments were performed in 1-, 4-, 7-, 10-, 14-, and 28-day-old female C57BL/6 mice, 7-day-old Scx-GFP female mice, or 5- to 6-week-old ScxCre,Shp2fl/fl female mice. The Scx-GFP and ScxCremice were kindly provided by Dr. R. Schweitzer (Oregon Health & Science University, Oregon, USA). The Shp2fl/fl mice were kindly provided by Dr. Yi Wang (Zhejiang University). All animal studies were approved by the Institutional Animal Care and Use Committee of Zhejiang University and followed by the regulations.
实验分别在 1、4、7、10、14 和 28 天龄的雌性 C57BL/6 小鼠、7 天龄的 Scx-GFP 雌性小鼠，以及 5-6 周龄的 Scx Cre 、Shp2 fl/fl 雌性小鼠中进行。Scx-GFP 和 Scx Cre 小鼠由美国俄勒冈健康与科学大学的 R. Schweitzer 博士惠赠。Shp2 fl/fl 小鼠由浙江大学王毅博士惠赠。所有动物实验均经浙江大学实验动物管理和使用委员会批准并遵循相关规定。
实验分别在 1、4、7、10、14 和 28 天龄的雌性 C57BL/6 小鼠、7 天龄的 Scx-GFP 雌性小鼠，以及 5-6 周龄的 Scx Cre 、Shp2 fl/fl 雌性小鼠中进行。Scx-GFP 和 Scx Cre 小鼠由美国俄勒冈健康与科学大学的 R. Schweitzer 博士惠赠。Shp2 fl/fl 小鼠由浙江大学王毅博士惠赠。所有动物实验均经浙江大学实验动物管理和使用委员会批准并遵循相关规定。
Method details
Single cell capture, cDNA library preparation and sequencing
单细胞捕获、cDNA 文库构建与测序
Single-cell experiments were performed with the C57 mouse tail tendons on day 7 and day 14. Samples from three mice were pooled at each time point. Firstly, the tissues were minced into small pieces and digested in 0.2% collagenase I (Gibco) diluted in low-glucose Dulbecco’s modified Eagle’s medium (Gibco) at 37°C for 2 h. Then the suspension was filtered through a 70 μm strainer to remove the incompletely digested clumps and adjusted to a concentration of 4 × 105 cells/mL. Single-cell capture, RNA extraction and cDNA preparation were performed following the methods described in the Fluidigm protocol (PN 100-9886, C1 High-Throughput IFC to Generate Single-Cell cDNA Libraries for mRNA Sequencing). cDNA products were quantified using the Qubit and subsequently diluted to a final concentration of 0.2 ng/mL using the C1 Harvest Reagent. The diluted cDNA products were then converted into mRNA-seq libraries using the Nextera XT DNA Sample Preparation Kit (Illumina, FC-131-1096) following the manufacturer's instructions. The qualified libraries were then sequenced on the Illumina Hiseq X Ten platform.
在第 7 天和第 14 天分别对 C57 小鼠尾腱进行单细胞实验。每个时间点合并三只小鼠的样本。首先，将组织切成小块，在 37°C 下用 0.2%胶原酶 I（Gibco）稀释的低糖 DMEM 培养基（Gibco）中消化 2 小时。随后将悬液通过 70 μm 筛网过滤以去除未完全消化的组织块，并调整细胞浓度至 4 × 10 5 个/mL。按照 Fluidigm 协议（PN 100-9886，C1 高通量 IFC 用于生成单细胞 cDNA 文库进行 mRNA 测序）进行单细胞捕获、RNA 提取和 cDNA 制备。使用 Qubit 对 cDNA 产物进行定量，并用 C1 收获试剂将其稀释至终浓度 0.2 ng/mL。按照制造商说明，使用 Nextera XT DNA 样品制备试剂盒（Illumina，FC-131-1096）将稀释的 cDNA 产物转化为 mRNA-seq 文库。合格的文库随后在 Illumina Hiseq X Ten 平台上进行测序。
在第 7 天和第 14 天分别对 C57 小鼠尾腱进行单细胞实验。每个时间点合并三只小鼠的样本。首先，将组织切成小块，在 37°C 下用 0.2%胶原酶 I（Gibco）稀释的低糖 DMEM 培养基（Gibco）中消化 2 小时。随后将悬液通过 70 μm 筛网过滤以去除未完全消化的组织块，并调整细胞浓度至 4 × 10 5 个/mL。按照 Fluidigm 协议（PN 100-9886，C1 高通量 IFC 用于生成单细胞 cDNA 文库进行 mRNA 测序）进行单细胞捕获、RNA 提取和 cDNA 制备。使用 Qubit 对 cDNA 产物进行定量，并用 C1 收获试剂将其稀释至终浓度 0.2 ng/mL。按照制造商说明，使用 Nextera XT DNA 样品制备试剂盒（Illumina，FC-131-1096）将稀释的 cDNA 产物转化为 mRNA-seq 文库。合格的文库随后在 Illumina Hiseq X Ten 平台上进行测序。
scRNA-seq data analysis 单细胞 RNA 测序数据分析
Raw sequencing reads were processed with Perl scripts to ensure the quality of data used in further analysis. We first removed the adaptor-polluted reads (reads containing more than 5 adapter-polluted bases) and the low-quality reads (reads with the number of Quality value less than 19 accounting for more than 15% of total bases). Reads with number of N bases accounting for more than 5% were also discarded. The obtained clean data were mapped to the mm10 genome release with bowtie2 using default parameters. Reads count for each gene in each sample was counted by HTSeq. After obtaining the digital gene expression (DE) data matrix, we used Seurat (Butler et al., 2018) (V2.3.4) for dimension reduction, clustering and differential gene expression analysis. For quality control, we excluded cells in which expression of less than 1,200 or more than 7,000 genes were detected. Cells with less than 25,000 or more than 1,000,000 total counts were also filtered out. Genes that were ascertained in more than 2 cells were kept. The Dimensional reduction was performed and cell clusters were identified based on the most significant principal components. Genes significantly enriched in each cell cluster were identified using the default algorithm in Seurat. Functional annotation of the resulting marker gene lists relative to Gene Ontology terms was performed using Metascape (Zhou et al., 2019). We also used SCENIC(Aibar et al., 2017) to construct the regulons to cluster the cells based on the same expression data matrix. Ligand-Receptor was analyzed by Cellphonedb (Efremova et al., 2020); trajectory analysis was performed using Monocle (v2.10.1) (Qiu et al., 2017). Gene regulatory networks based on the single cell transcriptomic data were generated using the R package bigSCale2 (Iacono et al., 2019) as previously reported (Adams et al., 2020).
使用 Perl 脚本处理原始测序读段以确保后续分析所用数据的质量。首先去除接头污染读段（含有超过 5 个接头污染碱基的读段）和低质量读段（质量值低于 19 的碱基占总碱基数超过 15%的读段）。含 N 碱基数超过 5%的读段也被剔除。将获得的清洁数据使用默认参数通过 bowtie2 比对到 mm10 基因组。使用 HTSeq 计算每个样本中每个基因的读段数。获得数字基因表达（DE）数据矩阵后，使用 Seurat（ Butler et al., 2018 ）（V2.3.4）进行降维、聚类和差异基因表达分析。质量控制时，我们排除了检测到少于 1,200 个或多于 7,000 个基因表达的细胞。总计数少于 25,000 或多于 1,000,000 的细胞也被过滤掉。保留在 2 个以上细胞中确认的基因。基于最显著的主成分进行降维并识别细胞簇。使用 Seurat 默认算法识别每个细胞簇中显著富集的基因。使用 Metascape（ Zhou et al., 2019 ）对所得标记基因列表进行基于 Gene Ontology 术语的功能注释。我们还使用 SCENIC（ Aibar et al., 2017 ）基于相同的表达数据矩阵构建调控子以聚类细胞。使用 Cellphonedb（ Efremova et al., 2020 ）分析配体-受体；使用 Monocle（v2.10.1）（ Qiu et al., 2017 ）进行轨迹分析。如先前报道（ Adams et al., 2020 ）所述，使用 R 包 bigSCale2（ Iacono et al., 2019 ）基于单细胞转录组数据生成基因调控网络。
11.
Butler, A. ∙ Hoffman, P. ∙ Smibert, P. ...
Integrating single-cell transcriptomic data across different conditions, technologies, and species
Nat. Biotechnol. 2018; 36:411-42066.
Zhou, Y. ∙ Zhou, B. ∙ Pache, L. ...
Metascape provides a biologist-oriented resource for the analysis of systems-level datasets
Nat. Commun. 2019; 10:15232.
Aibar, S. ∙ González-Blas, C.B. ∙ Moerman, T. ...
SCENIC: single-cell regulatory network inference and clustering
Nat. Methods. 2017; 14:1083-108618.
Efremova, M. ∙ Vento-Tormo, M. ∙ Teichmann, S.A. ...
CellPhoneDB: inferring cell–cell communication from combined expression of multi-subunit ligand–receptor complexes
Nat. Protoc. 2020; 15:1484-150650.
Qiu, X. ∙ Hill, A. ∙ Packer, J. ...
Single-cell mRNA quantification and differential analysis with Census
Nat. Methods. 2017; 14:309-31530.
Iacono, G. ∙ Massoni-Badosa, R. ∙ Heyn, H.
Single-cell transcriptomics unveils gene regulatory network plasticity
Genome Biol. 2019; 20:1101.
Adams, T.S. ∙ Schupp, J.C. ∙ Poli, S. ...
Single-cell RNA-seq reveals ectopic and aberrant lung-resident cell populations in idiopathic pulmonary fibrosis
Sci. Adv. 2020; 6, eaba1983使用 Perl 脚本处理原始测序读段以确保后续分析所用数据的质量。首先去除接头污染读段（含有超过 5 个接头污染碱基的读段）和低质量读段（质量值低于 19 的碱基占总碱基数超过 15%的读段）。含 N 碱基数超过 5%的读段也被剔除。将获得的清洁数据使用默认参数通过 bowtie2 比对到 mm10 基因组。使用 HTSeq 计算每个样本中每个基因的读段数。获得数字基因表达（DE）数据矩阵后，使用 Seurat（ Butler et al., 2018
11.
Butler, A. ∙ Hoffman, P. ∙ Smibert, P. ...
Integrating single-cell transcriptomic data across different conditions, technologies, and species
Nat. Biotechnol. 2018; 36:411-42066.
Zhou, Y. ∙ Zhou, B. ∙ Pache, L. ...
Metascape provides a biologist-oriented resource for the analysis of systems-level datasets
Nat. Commun. 2019; 10:15232.
Aibar, S. ∙ González-Blas, C.B. ∙ Moerman, T. ...
SCENIC: single-cell regulatory network inference and clustering
Nat. Methods. 2017; 14:1083-108618.
Efremova, M. ∙ Vento-Tormo, M. ∙ Teichmann, S.A. ...
CellPhoneDB: inferring cell–cell communication from combined expression of multi-subunit ligand–receptor complexes
Nat. Protoc. 2020; 15:1484-150650.
Qiu, X. ∙ Hill, A. ∙ Packer, J. ...
Single-cell mRNA quantification and differential analysis with Census
Nat. Methods. 2017; 14:309-3151.
Adams, T.S. ∙ Schupp, J.C. ∙ Poli, S. ...
Single-cell RNA-seq reveals ectopic and aberrant lung-resident cell populations in idiopathic pulmonary fibrosis
Sci. Adv. 2020; 6, eaba198330.
Iacono, G. ∙ Massoni-Badosa, R. ∙ Heyn, H.
Single-cell transcriptomics unveils gene regulatory network plasticity
Genome Biol. 2019; 20:110RNA-seq experiments and data analysis
RNA 测序实验与数据分析
RNA-seq was modified based on a previous method (Chen et al., 2009). In brief, total RNA was extracted from tissue samples using Trizol reagent (TAKARA), reverse transcription was conducted by SuperScript II reverse transcriptase (Invitrogen), double-stranded cDNA was generated using the NEBNext mRNA second strand synthesis kit (NEB) and cleaned by AMPure XP beads (Beckman Coulter, a63881), sequencing library was constructed using the Nextera XT kit (Illumina) and sequenced on the Illumina X-Ten platform. Sequencing reads were mapped to the reference genome mm10 using bowtie2 with default parameters, and per gene, counts were calculated using HTSeq. All the statistical analyses were conducted using R statistical programming languages. Digital expression data were converted to counts per million by dividing with the total number of reads and multiplying by 106. We used DESeq2 to identify DEGs in each tissue from the control and Shp2 CKO mice. In our analyses, a gene was considered to be expressed in a sample if its count value was equal to or greater than 1 in the sample. Genes with count values of zero across all samples were removed. DEGs were defined as fold change ≥2 and p value ≤ 0.05. Gene Ontology and KEGG analysis was performed using Metascape.
RNA 测序方法基于先前方法（ Chen et al., 2009 ）改进。简而言之，使用 Trizol 试剂（TAKARA）从组织样本中提取总 RNA，使用 SuperScript II 反转录酶（Invitrogen）进行反转录，使用 NEBNext mRNA 第二链合成试剂盒（NEB）生成双链 cDNA 并用 AMPure XP 磁珠（Beckman Coulter，a63881）纯化，使用 Nextera XT 试剂盒（Illumina）构建测序文库并在 Illumina X-Ten 平台上测序。使用 bowtie2 默认参数将测序读段比对到参考基因组 mm10，并使用 HTSeq 计算每个基因的计数。所有统计分析均使用 R 统计编程语言进行。通过除以总读段数并乘以 106 将数字表达数据转换为每百万计数。我们使用 DESeq2 识别对照组和 Shp2 CKO 小鼠各组织中的差异表达基因（DEGs）。在我们的分析中，如果样本中某基因的计数值大于或等于 1，则认为该基因在该样本中表达。删除所有样本中计数值为零的基因。将差异表达基因定义为倍数变化≥2 且 p 值≤0.05。使用 Metascape 进行 Gene Ontology 和 KEGG 分析。
14.
Chen, X. ∙ Song, X.-H. ∙ Yin, Z. ...
Stepwise differentiation of human embryonic stem cells promotes tendon regeneration by secreting fetal tendon matrix and differentiation factors
Stem Cells. 2009; 27:1276-1287RNA 测序方法基于先前方法（ Chen et al., 2009
14.
Chen, X. ∙ Song, X.-H. ∙ Yin, Z. ...
Stepwise differentiation of human embryonic stem cells promotes tendon regeneration by secreting fetal tendon matrix and differentiation factors
Stem Cells. 2009; 27:1276-1287PCA analysis
The prcomp function in R was used to perform the PCA analysis. The input variables included: 4 histological scores (fiber structure, fiber arrangement, nuclear roundness and number of cells), 2 crimp scores (crimp wavelength and crimp angle), 9 mechanical properties (Ansorge et al., 2011) (percent relax, transitional relax, transitional load, transitional stress, transitional strain, toe stiffness, toe modulus, linear stiffness, linear modulus and angular deviation), 6 mRNA quantification results (Scx, Mkx, Tnmd, Nes, Egr1 and Col1a1) and the mean collagen fibril diameter.
使用 R 中的 prcomp 函数进行 PCA 分析。输入变量包括：4 个组织学评分（纤维结构、纤维排列、细胞核圆度和细胞数量）、2 个卷曲评分（卷曲波长和卷曲角度）、9 个力学特性（ Ansorge et al., 2011 ）（松弛百分比、过渡松弛、过渡载荷、过渡应力、过渡应变、趾部刚度、趾部模量、线性刚度、线性模量和角度偏差）、6 个 mRNA 定量结果（Scx、Mkx、Tnmd、Nes、Egr1 和 Col1a1）以及平均胶原纤维直径。
4.
Ansorge, H.L. ∙ Adams, S. ∙ Birk, D.E. ...
Mechanical, compositional, and structural properties of the post-natal mouse Achilles tendon
Ann. Biomed. Eng. 2011; 39:1904-1913使用 R 中的 prcomp 函数进行 PCA 分析。输入变量包括：4 个组织学评分（纤维结构、纤维排列、细胞核圆度和细胞数量）、2 个卷曲评分（卷曲波长和卷曲角度）、9 个力学特性（ Ansorge et al., 2011
4.
Ansorge, H.L. ∙ Adams, S. ∙ Birk, D.E. ...
Mechanical, compositional, and structural properties of the post-natal mouse Achilles tendon
Ann. Biomed. Eng. 2011; 39:1904-1913Tissue explant culturing 组织外植体培养
Both the limb and tail were dissected from the 7-day C57 mouse, and the skin and surrounding soft tissues were carefully removed. The Achilles tendon and tail tendon were cultured together on the culture dish with different growth factors and chemical inhibitors, including IGF1 (Genescript, Z03177-100, 100 ng/mL), bFGF (Peprotech, AF-100-18B 100 ng/mL), PDGF-AA (Genescript, Z03276-10, 100 ng/mL), NGF (Peprotech, 450-01, 200 ng/mL), PHPS1 (Sigma, P0039, 20 μg/ml). Culture media was changed every two days. On day 7, the samples were collected for subsequent analyses.
从 7 日龄 C57 小鼠身上解剖取出肢体和尾部，仔细剥离皮肤和周围软组织。将跟腱和尾腱一起培养在培养皿中，添加不同的生长因子和化学抑制剂，包括 IGF1（Genescript，Z03177-100，100 ng/mL）、bFGF（Peprotech，AF-100-18B，100 ng/mL）、PDGF-AA（Genescript，Z03276-10，100 ng/mL）、NGF（Peprotech，450-01，200 ng/mL）和 PHPS1（Sigma，P0039，20 μg/ml）。培养基每两天更换一次。在第 7 天，收集样本进行后续分析。
从 7 日龄 C57 小鼠身上解剖取出肢体和尾部，仔细剥离皮肤和周围软组织。将跟腱和尾腱一起培养在培养皿中，添加不同的生长因子和化学抑制剂，包括 IGF1（Genescript，Z03177-100，100 ng/mL）、bFGF（Peprotech，AF-100-18B，100 ng/mL）、PDGF-AA（Genescript，Z03276-10，100 ng/mL）、NGF（Peprotech，450-01，200 ng/mL）和 PHPS1（Sigma，P0039，20 μg/ml）。培养基每两天更换一次。在第 7 天，收集样本进行后续分析。
Histology and immunofluorescence imaging
组织学和免疫荧光成像
Tissue specimens were harvested at desired ages and fixed in 4% (v/v) PFA for 24 h at room temperature. After dehydration with gradient alcohol, the samples were embedded in paraffin and sectioned at 6 μm, and performed hematoxylin & eosin (H&E) staining and immunostaining. The measurement method of fiber structure is based on our previous study (Chen et al., 2010). Adapted from a previous study (Chen et al., 2007), the fiber structure was scored by a blinded scorer using a semi-quantitative scoring system based on H&E staining results. In more details, 0 to 3 points were used for evaluating the fiber structure. 0 point represented the bundled continuous dense fiber structure with a typical large wave shape. 1 point represented the bundled fibrous structure with small wave shape. 2 points corresponded to the scatted, unbundled fiber structure and 3 points scored maximally abnormal structure with no fiber to be seen. Five samples per group were evaluated, and each sample was randomly selected from three sections for evaluation. For the IF experiment, the following antibodies were used: PRG4 (Abcam, ab28484), THBS4 (Abcam, ab176116), SHP2(Santa Cruz, sc-280), CD271 (Abcam, ab52987) and secondary antibodies conjugated with Alexa Fluor 488 fluorescent dyes (Thermo Fisher Scientific). The stained specimens were photographed digitally under a confocal microscope (Olympus BX61).
在预定年龄收获组织标本并在室温下用 4%（v/v）PFA 固定 24 小时。经梯度酒精脱水后，样本被包埋在石蜡中并切片至 6 μm 厚，然后进行苏木精&伊红（H&E）染色和免疫染色。纤维结构的测量方法基于我们之前的研究（ Chen et al., 2010 ）。参考先前的研究（ Chen et al., 2007 ），纤维结构由一位不知情的评分者使用基于 H&E 染色结果的半定量评分系统进行评分。具体而言，使用 0 至 3 分评估纤维结构。0 分代表具有典型大波浪形状的成束连续致密纤维结构。1 分代表具有小波浪形状的成束纤维结构。2 分对应于散乱、非成束的纤维结构，3 分评为最大异常结构，看不到纤维。每组评估五个样本，每个样本随机从三个切片中选择进行评估。对于 IF 实验，使用了以下抗体：PRG4（Abcam，ab28484）、THBS4（Abcam，ab176116）、SHP2（Santa Cruz，sc-280）、CD271（Abcam，ab52987）和与 Alexa Fluor 488 荧光染料（Thermo Fisher Scientific）结合的二抗。染色后的标本在共聚焦显微镜（Olympus BX61）下进行数字拍照。
12.
Chen, J.L. ∙ Yin, Z. ∙ Shen, W.L. ...
Efficacy of hESC-MSCs in knitted silk-collagen scaffold for tendon tissue engineering and their roles
Biomaterials. 2010; 31:9438-945113.
Chen, J.M. ∙ Willers, C. ∙ Xu, J. ...
Autologous tenocyte therapy using porcine-derived bioscaffolds for massive rotator cuff defect in rabbits
Tissue Eng. 2007; 13:1479-1491在预定年龄收获组织标本并在室温下用 4%（v/v）PFA 固定 24 小时。经梯度酒精脱水后，样本被包埋在石蜡中并切片至 6 μm 厚，然后进行苏木精&伊红（H&E）染色和免疫染色。纤维结构的测量方法基于我们之前的研究（ Chen et al., 2010
12.
Chen, J.L. ∙ Yin, Z. ∙ Shen, W.L. ...
Efficacy of hESC-MSCs in knitted silk-collagen scaffold for tendon tissue engineering and their roles
Biomaterials. 2010; 31:9438-945113.
Chen, J.M. ∙ Willers, C. ∙ Xu, J. ...
Autologous tenocyte therapy using porcine-derived bioscaffolds for massive rotator cuff defect in rabbits
Tissue Eng. 2007; 13:1479-1491Cell isolation, multipotent differentiation and cell sheets culture
细胞分离、多能分化和细胞片培养
TSPCs were isolated and cultured as previously described (Bi et al., 2007). The osteogenic, chondrogenic and adipogenic differentiation potential of TSPCs from the control and the PHPS1 treated groups was assessed according to previous methods (Yin et al., 2016). The multilayered cell sheet was harvested after 2 weeks in culture as in the previous study before subsequent use in the TEM experiments.
TSPCs 的分离和培养按照先前描述的方法进行（ Bi et al., 2007 ）。对照组和 PHPS1 处理组的 TSPCs 的成骨、软骨和脂肪分化潜能根据先前的方法进行评估（ Yin et al., 2016 ）。多层细胞片在培养 2 周后收获，如之前研究中所述，随后用于 TEM 实验。
6.
Bi, Y. ∙ Ehirchiou, D. ∙ Kilts, T.M. ...
Identification of tendon stem/progenitor cells and the role of the extracellular matrix in their niche
Nat. Med. 2007; 13:1219-122763.
Yin, Z. ∙ Hu, J. ∙ Yang, L. ...
Single-cell analysis reveals a nestin+ tendon stem/progenitor cell population with strong tenogenic potentiality
Sci. Adv. 2016; 2, e1600874TSPCs 的分离和培养按照先前描述的方法进行（ Bi et al., 2007
6.
Bi, Y. ∙ Ehirchiou, D. ∙ Kilts, T.M. ...
Identification of tendon stem/progenitor cells and the role of the extracellular matrix in their niche
Nat. Med. 2007; 13:1219-122763.
Yin, Z. ∙ Hu, J. ∙ Yang, L. ...
Single-cell analysis reveals a nestin+ tendon stem/progenitor cell population with strong tenogenic potentiality
Sci. Adv. 2016; 2, e1600874QRT-PCR
RNA was isolated by sample lysis in TRIzol (Invitrogen). PCR was performed using a TB Green Premix Ex Taq (TakaRa) on a Light Cycler apparatus (ABI 7900HT). The relative expression level of each target gene was calculated using the 2-ΔΔCt method. Table S3 details the primers used.
RNA 通过在 TRIzol（Invitrogen）中裂解样本进行分离。PCR 使用 TB Green Premix Ex Taq（TakaRa）在 Light Cycler 仪器（ABI 7900HT）上进行。每个目标基因的相对表达水平使用 2-ΔΔCt 方法计算。表 S3 详细列出了所用引物。
RNA 通过在 TRIzol（Invitrogen）中裂解样本进行分离。PCR 使用 TB Green Premix Ex Taq（TakaRa）在 Light Cycler 仪器（ABI 7900HT）上进行。每个目标基因的相对表达水平使用 2-ΔΔCt 方法计算。表 S3 详细列出了所用引物。
Transmission electron microscopy and cuprolinic blue staining
透射电子显微镜和茜素蓝染色
Tissue specimens were fixed by standard procedures for TEM to assess collagen fibril diameters and alignment. Briefly, samples were pre-fixed with 2% (w/v) glutaraldehyde for 2 h at 4°C and then washed twice with PBS at 4°C followed by post-fixation with 1% (w/v) osmic acid for 2 h at 4°C. After two washes in PBS, the samples were dehydrated with increasing ethanol gradient solutions and dried to a critical point. The samples were subsequently mounted and sputter-coated with gold for viewing under TEM (Quanta 10 FEI). To image the GAG chains, we performed cuprolinic blue staining according to the protocol (Watanabe et al., 2016) and examined the longitude section under a TEM. About 300 or 500 collagen fibrils of each sample were measured and assessed.
组织标本通过标准程序固定，用于透射电镜(TEM)评估胶原纤维直径和排列。简言之，样本先用 2%(w/v)戊二醛在 4°C 下预固定 2 小时，然后在 4°C 下用 PBS 清洗两次，随后用 1%(w/v)锇酸在 4°C 下后固定 2 小时。PBS 清洗两次后，样本用递增梯度乙醇溶液脱水并干燥至临界点。随后将样本安装并喷金，在透射电镜(Quanta 10 FEI)下观察。为了成像糖胺聚糖(GAG)链，我们按照协议( Watanabe et al., 2016 )进行铜蓝染色，并在透射电镜下检查纵切面。每个样本测量和评估了约 300 或 500 根胶原纤维。
60.
Watanabe, T. ∙ Kametani, K. ∙ Koyama, Y. ...
Ring-mesh model of proteoglycan glycosaminoglycan chains in tendon based on three-dimensional reconstruction by focused ion beam scanning electron microscopy
J. Biol. Chem. 2016; 291:23704-23708组织标本通过标准程序固定，用于透射电镜(TEM)评估胶原纤维直径和排列。简言之，样本先用 2%(w/v)戊二醛在 4°C 下预固定 2 小时，然后在 4°C 下用 PBS 清洗两次，随后用 1%(w/v)锇酸在 4°C 下后固定 2 小时。PBS 清洗两次后，样本用递增梯度乙醇溶液脱水并干燥至临界点。随后将样本安装并喷金，在透射电镜(Quanta 10 FEI)下观察。为了成像糖胺聚糖(GAG)链，我们按照协议( Watanabe et al., 2016
60.
Watanabe, T. ∙ Kametani, K. ∙ Koyama, Y. ...
Ring-mesh model of proteoglycan glycosaminoglycan chains in tendon based on three-dimensional reconstruction by focused ion beam scanning electron microscopy
J. Biol. Chem. 2016; 291:23704-23708Quantification and statistical analysis
定量和统计分析
Statistical analysis 统计分析
One-way ANOVA and Student’s t-test were performed to assess whether there were statistically significant differences in the results between groups. Values of p < 0.05 were considered to be significantly different. The significance level is presented as ∗p < 0.05 or ∗∗p < 0.01.
采用单因素方差分析和 Student's t 检验评估各组结果间是否存在统计学显著差异。p < 0.05 被视为有显著差异。显著性水平表示为 ∗ p < 0.05 或 ∗∗ p < 0.01。
采用单因素方差分析和 Student's t 检验评估各组结果间是否存在统计学显著差异。p < 0.05 被视为有显著差异。显著性水平表示为 ∗ p < 0.05 或 ∗∗ p < 0.01。
Acknowledgments
We thank Dr. Ronen Schweitzer (Oregon Health & Science University) for providing ScxCre and Scx-GFP mice. We thank Dr. Yi Wang (Zhejiang University) for providing Shp2fl/fl mice. We acknowledge the Core Facilities of Zhejiang University School of Medicine, Analysis Center of Agrobiology, and Environment Sciences of Zhejiang University for technical assistance. We thank Ms. Liu from the core facility platform of Zhejiang University School of Medicine for technical support. We thank Beibei Wang in the Center of Cryo-Electron Microscopy (CCEM), Zhejiang University for technical assistance with transmission electron microscopy. This work was supported by the National Key R&D Program of China (grant 2021YFA1100500), the National Natural Sciences Foundation of China (grants T2121004, 81972099, 81772418, 81871764, and 82072463), and the Zhejiang Provincial Natural Science Foundation of China (LZ22H060002 and LR20H060001).
我们感谢俄勒冈健康与科学大学的 Ronen Schweitzer 博士提供 Scx Cre 和 Scx-GFP 小鼠。感谢浙江大学的王毅博士提供 Shp2 fl/fl 小鼠。我们感谢浙江大学医学院核心设施、浙江大学农业生物学和环境科学分析中心的技术支持。感谢浙江大学医学院核心设施平台的刘女士提供的技术支持。感谢浙江大学冷冻电镜中心(CCEM)的王贝贝在透射电镜方面提供的技术协助。本研究获得中国国家重点研发计划(2021YFA1100500)、中国国家自然科学基金(T2121004、81972099、81772418、81871764 和 82072463)以及浙江省自然科学基金(LZ22H060002 和 LR20H060001)的资助。
我们感谢俄勒冈健康与科学大学的 Ronen Schweitzer 博士提供 Scx Cre 和 Scx-GFP 小鼠。感谢浙江大学的王毅博士提供 Shp2 fl/fl 小鼠。我们感谢浙江大学医学院核心设施、浙江大学农业生物学和环境科学分析中心的技术支持。感谢浙江大学医学院核心设施平台的刘女士提供的技术支持。感谢浙江大学冷冻电镜中心(CCEM)的王贝贝在透射电镜方面提供的技术协助。本研究获得中国国家重点研发计划(2021YFA1100500)、中国国家自然科学基金(T2121004、81972099、81772418、81871764 和 82072463)以及浙江省自然科学基金(LZ22H060002 和 LR20H060001)的资助。
Author contributions 作者贡献
Conceptualization, X.C., Z.Y., and W.S.; supervision, X.C., Z.Y., and W.S.; investigation, C.F., Y.C., Y.Z., T.Q., J.L., and S.H.; formal analysis, C.F., Y.C., and R.Y.; methodology, T.L., Y.X., and T.W.; writing – original draft, C.F.; writing – review & editing, Y.C., W.S., Z.Y., X.C., S.G., and H.O.; funding acquisition, X.C. and Z.Y. All authors have read and approved the manuscript.
概念化，X.C.、Z.Y.和 W.S.；监督，X.C.、Z.Y.和 W.S.；研究调查，C.F.、Y.C.、Y.Z.、T.Q.、J.L.和 S.H.；正式分析，C.F.、Y.C.和 R.Y.；方法学，T.L.、Y.X.和 T.W.；写作-初稿，C.F.；写作-审阅与编辑，Y.C.、W.S.、Z.Y.、X.C.、S.G.和 H.O.；资金获取，X.C.和 Z.Y.。所有作者均已阅读并批准本稿件。
概念化，X.C.、Z.Y.和 W.S.；监督，X.C.、Z.Y.和 W.S.；研究调查，C.F.、Y.C.、Y.Z.、T.Q.、J.L.和 S.H.；正式分析，C.F.、Y.C.和 R.Y.；方法学，T.L.、Y.X.和 T.W.；写作-初稿，C.F.；写作-审阅与编辑，Y.C.、W.S.、Z.Y.、X.C.、S.G.和 H.O.；资金获取，X.C.和 Z.Y.。所有作者均已阅读并批准本稿件。
Declaration of interests 利益声明
The authors declare no competing interests.
作者声明不存在利益冲突。
作者声明不存在利益冲突。
Supplemental information (2)
补充信息（2）
Document S1. Figures S1–S7 and Tables S1–S3
文档 S1. 图 S1-S7 和表 S1-S3
文档 S1. 图 S1-S7 和表 S1-S3
Document S2. Article plus supplemental information
文档 S2. 文章及补充信息
文档 S2. 文章及补充信息
Data and code availability
数据和代码可用性
•
All raw sequencing data have been deposited in the Genome Sequence Archive (GSA: CRA003892).
所有原始测序数据已存储于基因组序列档案库（GSA：CRA003892）。
所有原始测序数据已存储于基因组序列档案库（GSA：CRA003892）。
•
This paper does not report original code.
本文未报告原创代码。
本文未报告原创代码。
•
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request
如需获取重新分析本文所报告数据所需的任何其他信息，可向通讯作者提出申请。
如需获取重新分析本文所报告数据所需的任何其他信息，可向通讯作者提出申请。
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肌腱损伤：从生物学到肌腱修复
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单细胞 RNA 测序探索免疫细胞异质性
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通过单细胞转录组学构建完整复杂动物的细胞类型图谱和谱系树
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使用 Census 进行单细胞 mRNA 定量和差异分析
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肌腱发育需要通过钙黏蛋白-11 介导的细胞-细胞连接来调控细胞聚集和细胞形态
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Scleraxis is required for differentiation of the stapedius and tensor tympani tendons of the middle ear
硬肌腱蛋白对中耳镫骨肌和鼓膜张肌腱的分化是必需的
J. Assoc. Res. Otolaryngol. 2011; 12:407-421硬肌腱蛋白对中耳镫骨肌和鼓膜张肌腱的分化是必需的
耳鼻咽喉研究协会杂志 2011; 12:407-421
Warde-Farley, D. ∙ Donaldson, S.L. ∙ Comes, O. ...
The GeneMANIA prediction server: biological network integration for gene prioritization and predicting gene function
GeneMANIA 预测服务器：用于基因优先级排序和预测基因功能的生物网络整合
Nucleic Acids Res. 2010; 38:W214-W220GeneMANIA 预测服务器：用于基因优先级排序和预测基因功能的生物网络整合
核酸研究 2010; 38:W214-W220
Watanabe, T. ∙ Kametani, K. ∙ Koyama, Y. ...
Ring-mesh model of proteoglycan glycosaminoglycan chains in tendon based on three-dimensional reconstruction by focused ion beam scanning electron microscopy
基于聚焦离子束扫描电子显微镜三维重建的肌腱蛋白多糖糖胺聚糖链环网模型
J. Biol. Chem. 2016; 291:23704-23708基于聚焦离子束扫描电子显微镜三维重建的肌腱蛋白多糖糖胺聚糖链环网模型
生物化学杂志 2016; 291:23704-23708
Xia, B. ∙ Yanai, I.
A periodic table of cell types
细胞类型周期表
Development. 2019; 146:dev169854细胞类型周期表
发育 2019; 146:dev169854
Yang, W. ∙ Wang, J. ∙ Moore, D.C. ...
Ptpn11 deletion in a novel progenitor causes metachondromatosis by inducing hedgehog signalling
在新型祖细胞中删除 Ptpn11 通过诱导刺猬信号通路导致异常软骨瘤病
Nature. 2013; 499:491-495在新型祖细胞中删除 Ptpn11 通过诱导刺猬信号通路导致异常软骨瘤病
自然 2013; 499:491-495
Yin, Z. ∙ Hu, J. ∙ Yang, L. ...
Single-cell analysis reveals a nestin+ tendon stem/progenitor cell population with strong tenogenic potentiality
单细胞分析揭示具有强大成腱潜能的 nestin + 肌腱干/祖细胞群
Sci. Adv. 2016; 2, e1600874单细胞分析揭示具有强大成腱潜能的 nestin + 肌腱干/祖细胞群
Zehender, A. ∙ Huang, J. ∙ Györfi, A.-H. ...
The tyrosine phosphatase SHP2 controls TGFβ-induced STAT3 signaling to regulate fibroblast activation and fibrosis
酪氨酸磷酸酶 SHP2 通过调控 TGFβ诱导的 STAT3 信号通路来控制成纤维细胞活化和纤维化
Nat. Commun. 2018; 9:3259酪氨酸磷酸酶 SHP2 通过调控 TGFβ诱导的 STAT3 信号通路来控制成纤维细胞活化和纤维化
Zhang, G. ∙ Young, B.B. ∙ Ezura, Y. ...
Development of tendon structure and function: regulation of collagen fibrillogenesis
肌腱结构与功能的发育：胶原纤维形成的调控
J. Musculoskelet. Neuronal Interact. 2005; 5:5-21肌腱结构与功能的发育：胶原纤维形成的调控
J. Musculoskelet. Neuronal Interact. 2005; 5:5-21
Zhou, Y. ∙ Zhou, B. ∙ Pache, L. ...
Metascape provides a biologist-oriented resource for the analysis of systems-level datasets
Metascape 为生物学家提供了一个用于分析系统级数据集的资源
Nat. Commun. 2019; 10:1523Metascape 为生物学家提供了一个用于分析系统级数据集的资源
Zuo, C. ∙ Wang, L. ∙ Kamalesh, R.M. ...
SHP2 regulates skeletal cell fate by modifying SOX9 expression and transcriptional activity
SHP2 通过调控 SOX9 的表达和转录活性来调节骨骼细胞命运
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