1. Cell Biology
  2. Developmental Biology
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Tgfβ signaling is critical for maintenance of the tendon cell fate

  1. Guak-Kim Tan
  2. Brian A Pryce
  3. Anna Stabio
  4. John V Brigande
  5. ChaoJie Wang
  6. Zheng Xia
  7. Sara F Tufa
  8. Douglas R Keene
  9. Ronen Schweitzer  Is a corresponding author
  1. Shriners Hospital for Children, United States
  2. Oregon Health & Science University, United States
Research Article
Cite this article as: eLife 2020;9:e52695 doi: 10.7554/eLife.52695
8 figures, 5 tables, 1 data set and 4 additional files

Figures

Figure 1 with 4 supplements
Tendon phenotypes manifested in Tgfbr2;ScxCre mutants.

(A–D) Whole-mount imaging in fluorescent ScxGFP signal or brightfield. (A) Dorsally viewed embryo forelimb shows the formation of a complete network of tendons in both mutant and heterozygous control by E14.5. (B) Tendons of mutant pups appeared intact at birth, but by P3 lateral tendons disintegrated and were eventually eliminated (yellow arrowheads), whereas the majority of other tendons persisted with a substantial loss of the ScxGFP signal (white arrowheads). (C) Mutant pups appeared normal at birth but showed physical abnormalities including abducted paw and splayed limb (black arrowheads) by P3. (D–E) Substantial loss of ScxGFP signal was also detected in all tendons and related tissues. (D) Tail tendons and annulus fibrosus of the intervertebral disc (white arrowheads) in P7 pups. (E) Collateral ligaments of the metacarpophalangeal joint imaged in transverse section through the joints of heterozygous control and mutant pups at P7 (white arrowhead). Scale bar, 100 μm. Mutant: CKO, Heterozygous: Het.

Figure 1—figure supplement 1
Verification of the Tgfbr2 knockdown efficiency in mutant cells.

(A) Transverse sections through the extensor digitorium communis tendons from an E16.5 ScxCre;RosaT embryo. While some of the tenocytes express the RosaT Cre reporter others do not (arrowheads), reflecting that ScxCre activity is not uniform in embryonic stages. (B–E) Immunostaining for TGFβ type II receptor (TGFBRII) demonstrates a nearly complete loss of receptor expression in (C) Tgfbr2;ScxCre and (E) Tgfbr2;RosaCreERT2 mutant cells, as compared with the robust expression in (B,D) wild-type tendons. (F) Tenocytes in Tgfbr2;RosaCreERT2 mutant retained expression of the tendon markers ScxGFP and tenomodulin (Tnmd), suggesting that a mere loss of TGFβ signaling was not sufficient to cause tenocyte dedifferentiation. All mice also carried the tendon reporter ScxGFP. Dotted lines in (B–E) demarcate tendons. Figures not to scale.

Figure 1—figure supplement 2
Gradual loss of tendon marker ScxGFP in Tgfbr2;ScxCre mutants at post-natal stages.

Cryosections of the forelimbs of Tgfbr2;ScxCre mutant pups showed positive ScxGFP signal in both wild-type and mutant tendons at P0. A gradual loss of the ScxGFP signal in mutant tendons started around P2-P3, that is before the manifestation of the physical abnormalities in the mutant pups. All mutant tendon cells lost ScxGFP at P7. Most analyses were therefore performed in tendons from this fully-phenotypic stage. Dotted lines demarcate mutant tendons in P3 and P7 pups. Mutant: CKO; Wild-type: WT. Figures not to scale.

Figure 1—figure supplement 3
Fragmentation and elimination of lateral tendons in Tgfbr2;ScxCre mutant neonates.

(A) Rapid disruption of lateral extensor tendons in neonatal stages of mutant pups revealed by examination of forelimb tendons using the tendon reporter ScxGFP. The extensor carpi radialis longus tendon (yellow arrowheads) is present in a mutant pup at P0 but lost in a P1 mutant. (B) TEM images of the extensor carpi radialis longus tendon at wrist level. The mutant tendon shows signs of fragmentation already at P0, and by P3 the tendon appears disintegrated accompanied by complete loss of the epitenon and structural definition of the tendon circumference. The red dotted line in (B) demarcates the mutant tendon. Mutant: CKO, Heterozygous: Het.

Figure 1—figure supplement 4
Disruption of the flexor carpi radialis tendon in Tgfbr2;ScxCre mutant embryos.

Examination of flexor tendons in E16.5 (A) mutant and (B) heterozygous littermates using the tendon reporter ScxGFP. Boxed regions in (A) and (B) are shown enlarged in (A’) and (B’). While most tendons appeared normal in mutant embryos, starting at E16.5 the flexor carpi radialis tendon (red arrowheads) was consistently torn in mutant embryo. Mutant: CKO, Heterozygous: Het. Figures not to scale.

Figure 2 with 1 supplement
Tendon development in Tgfbr2;ScxCre mutant embryos was not perturbed through embryogenesis.

(A) ScxGFP signal and (B) tenomodulin (Tnmd) immunofluorescence on transverse sections at wrist level of E16.5 mutant embryos demonstrate that the pattern and expression of prototypic tenocyte markers was not disrupted in mutant tendons. (C) Tnmd immunofluorescence in E16.5 wild-type tenocytes. (A’), (B’) and (C’) are higher magnifications of extensor digitorium communis tendons as boxed in (A), (B) and (C). (D) In situ hybridization for Col1a1 on transverse sections of the forelimb from E15.5 mutant and wild-type littermates reveals that expression of the major matrix genes was not altered in mutant embryos (black arrowhead). (E,F) TEM images of tendons from forelimbs of E18.5 mutant and wild-type embryos reveals that organization and accumulation of the tendon extracellular matrix was not disrupted in the mutant. (E’,F’) Higher magnification views of (E) and (F) for direct visualization of the collagen fibers. Scale bars, 200 μm (A–C) and 20 μm (A’–C’). Mutant: CKO, Wild-type: WT.

Figure 2—figure supplement 1
Evaluating cell death, proliferation and transdifferentiation in Tgfbr2;ScxCre mutant tendons.

(A) TUNEL assay did not detect significant cell death in mutant tendons throughout the developmental stages from E14.5 to P7. Shown here is a transverse section of P7 mutant forelimb, in which white line demarcates the extensor digitorium communis tendons. Inset in (A) shows a transverse section of E14.5 forelimb paw that serves as a positive control for TUNEL staining. As expected, cell death is detected only at the distal edge of the autopod, but not in tendons (ScxGFP-positive) at this stage. (B) EdU labeling of proliferating cells in transverse sections of the forelimb from P3 pups. The rate of proliferation was also not altered in mutant tendons compared with the wild-type littermates, an observation that also found in E14.5 to P10 samples. The pups were injected i.p. with 100 μg of EdU in PBS and tissues were harvested 2 hr post-injection. (C) Histological staining for the prototypic markers of chondrocytes (toluidine blue), osteocytes (alizarin red) and adipocytes (oil-red-o) showed that the loss of tendon markers in mutant tenocytes was also not due to transdifferentiation. The positive control tissues for the respective staining are cartilage, adipose tissue and bone from the same section. Dotted lines demarcate tendons. Mutant: CKO, Wild-type: WT. Figures not to scale.

Tendon degeneration observed in Tgfbr2;ScxCre mutants only at later postnatal stages.

TEM images of tendons from forelimbs of mutant and wild-type littermates at P3, P7 and P13. (A,B) Despite detectable functional defects starting around P3 in mutant pups, collagen matrix organization in mutant neonates was indistinguishable from that of their wild-type littermates. (C–E) By P7, the mutant tendon began to show signs of matrix degradation compared to the wild-type tendon. Collagen fibrils remained intact in some areas (D) and showed signs of deterioration in other areas (E). (F,G) Apparent collagen degradation and disrupted epitenon structures (white arrowhead) could be detected in tendons of P13 mutant pups. Black arrowhead indicates epitenon in wild-type pups. Boxed region in (G) is shown enlarged in (G’). Insets show transverse section TEM images of entire tendons at low-magnification (not to scale). Mutant: CKO, Wild-type: WT.

Deletion of Tgfbr2 in Scx-expressing cells (Tgfbr2;ScxCre) results in loss of tenocyte differentiation markers.

(A–D) Transverse sections of extensor digitorium communis tendons of wild-type and mutant pups at wrist level. (A) In P7 wild-type pups, all tenocytes were positive for tendon reporter ScxGFP signal. (B) Conversely, most cells in P7 mutant tendons lost the ScxGFP signal (white arrowhead), whereas the cells positive for ScxGFP signal are newly recruited cells (yellow arrowhead) (Tan et al. in preparation). (C) In situ hybridization shows that the mutant cells also lost gene expression of tendon markers Col1a1 and Tnmd (images not to scale). (D) Lineage tracing using ScxCre shows that all ScxGFP-negative cells in (B) were positive for Ai14 Rosa26-tdTomato (RosaT) Cre reporter (white arrowhead), proving that the ScxGFP-negative cells in mutant tendons were derived from tenocytes. Dashed lines demarcate the mutant tendons. Scale bar, 20 μm. Mutant: CKO, wild-type: WT.

Figure 5 with 1 supplement
Tgfbr2;ScxCre mutant tenocytes acquired stem/progenitor features.

(A) The colony-forming efficiency of P7 wild-type and heterozygous tenocytes as well as mutant tendon cells were evaluated by seeding one cell per well of the FACS-sorted cells in 96-well plates, and colonies formed were visualized with crystal violet staining. Mutant tenocytes exhibited significantly higher clonogenic capacity compared to wild-type and heterozygous controls. The results shown are mean ± SD (n = 5–6, **p<0.01). (B) Immunofluorescence staining for stem/progenitor markers in transverse sections of mutant tendons shows that mutant tendon cells acquired in postnatal stages expression of stem cell antigen-1 (Sca-1) and CD44. (C) In wild-type littermate controls, expression of both markers was detected in epitenon (white arrowhead), but not in tenocytes. Dashed line demarcates the mutant tendon. Scale bars, 10 μm. Mutant: CKO, Wild-type: WT, Heterozygous: Het.

Figure 5—figure supplement 1
Expression of Sca-1 and CD44 during embryonic tendon development.

(A–D) Immunofluorescence staining for Sca-1 and CD44 on wrist-level transverse sections from E14.5 ScxGFP-carrying wild-type embryos. Robust expression of (B) Sca-1 and (D) CD44 was detected in cells that surround the tendons at E14.5 (boxed areas), likely the precursors of the epitenon/paratenon. (B’,D’) Higher magnification views of the boxed areas in (B) and (D). The epitenon/paratenon layer is indicated by white arrowheads. Note that both markers were not expressed by the tenocytes at E14.5, the onset of tenocyte differentiation or at any other stages during embryonic tendon development (not shown). Scale bars, 100 μm (A–D) and 25 μm (B’,D’).

Molecular profile of the dedifferentiated mutant tenocytes.

(A) tSNE plots (K-means clustering) of enzymatically released cells from P7 wild-type and Tgfbr2;ScxCre mutant tendons reveals two major clusters corresponding to tenocytes and dedifferentiated mutant cells in the respective samples. Other cell type assignments are provided in the plots. See Supplementary file 1 for the list of genes highly expressed in these two clusters relative to other clusters. (B) Upregulated expression of Cd34 gene in P7 mutant tenocytes as revealed by scRNASeq analysis (see also Table 2) was determined using immunostaining. Transverse section of forelimb tendons shows that CD34 was indeed expressed by mutant tenocytes, while in wild-type controls CD34 was detected only in epitenon cells (white arrowhead). Dashed line demarcates the mutant tendon. (C,D) Gene ontology (GO) enrichment analysis in terms of biological processes associated with the (C) upregulated and (D) downregulated genes in P7 mutant compared with wild-type tenocytes. Selected GO terms are included in this figure, and genes annotated to the GO terms are available in Supplementary file 3. Scale bar, 10 μm. Mutant: CKO, Wild-type: WT.

Figure 7 with 1 supplement
Tenocyte dedifferentiation is dependent on cell autonomous loss of TGFβ signaling.

(A) AAV1-FLEX-Tgfbr2-V5 virus contains the reverse-complement sequence of Tgfbr2 with a C-terminal V5 epitope tag. Cre activity will lead to a permanent inversion of the cassette that will then express the V5-tagged TGFβ type II receptor. (B) Targeted expression of TGFβ type II receptor in E16.5 mutant tendon cells using the AAV1-FLEX-Tgfbr2-V5 prevented the loss of tendon markers in the infected tenocytes. The forelimb of E16.5 mutant embryos was infected with AAV1-FLEX-Tgfbr2-V5 virus and harvested at P6. Transverse forelimb sections were stained with antibodies for V5 (red) to detect AAV-infected cells and tenomodulin (Tnmd; yellow), a prototypic tendon marker expressed by (C) all tenocytes in the wild-type tendon at this stage. Dashed line demarcates the mutant tendon. (D) Quantification shows that about 95–98% of the AAV-infected (V5-positive) mutant tendon cells retained or re-expressed tendon differentiation markers after viral injection at different developmental stages (n = 3 pups for each stage). Note that the embryonic infection was performed with Cre-activated AAV1-FLEX-Tgfbr2-V5 virus and the P1 infection was performed with the constitutive AAV1-Tgfbr2-FLAG virus. Scale bar, 10 μm. Mutant: CKO, Wild-type: WT.

Figure 7—figure supplement 1
Induction of tendon markers by TGFβ signaling is context dependent.

AAV1-Tgfbr2-FLAG viral infection resulted in constitutive expression Tgfbr2-FLAG expression in cells both within and outside of tendons. The virus was injected locally into P1 mutant limbs and the limbs were harvested at P7. Sections from infected limbs were stained with antibodies to FLAG (yellow) to detect infected cells, and TGFβ type II receptor (TGFBRII) to confirm the re-expression of the receptor. ScxGFP signal and tenomodulin (Tnmd) antibody staining were used to identify induction of tendon markers. (A) Infected mutant tendon cells expressed the tendon markers ScxGFP and Tnmd. (B) In cells located outside of tendons (demarcated lines), the viral infection as detected by positive FLAG and TGFBRII immunofluorescence did not result in induction of the tendon markers ScxGFP and Tnmd. Figures not to scale.

Proposed roles of TGFβ signaling in the maintenance of tendon cell fate.

Targeted disruption of the TGFβ type II receptor (Tgfbr2) by ScxCre resulted in tenocyte dedifferentiation in early postnatal stages. Tenocyte dedifferentiation was reversed by reactivation of TGFβ signaling in individual mutant cells, demonstrating a cell autonomous role for TGFβ signaling for maintenance of the cell fate. Conversely, a mere loss of the receptor in individual tendon cell was not sufficient to cause tenocyte dedifferentiation, suggesting that external factors may also play a critical role in this process. We therefore propose that maintenance of the tendon cell fate is dependent on a combination of a cell autonomous function of TGFβ signaling and an additional, likely non-cell autonomous factor, for example the microenvironment of the tendon in the Tgfbr2;ScxCre mutant (cell-matrix interaction, mechanical loading, cell-cell contacts etc).

Tables

Table 1
Top 25 downregulated genes in P7 Tgfbr2;ScxCre mutant cells compared with P7 wild-type tenocytes (≥2 fold change, adjusted p<0.05).

See also Supplementary file 2 for a complete list of the downregulated genes.

Gene symbolGene nameFold change
Wif1Wnt inhibitory factor 1157.4
Col11a2#Collagen, type XI, alpha 292.0
Scx#Scleraxis66.2
Col2a1δCollagen, type II, alpha 158.9
Car9Carbonic anhydrase 958.1
Sema3bSema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3B43.9
Cgref1Cell growth regulator with EF hand domain 133.2
Fmod#Fibromodulin27.9
Cilp2Cartilage intermediate layer protein 224.7
Matn4Matrilin 419.3
P4ha1δProcollagen-proline, 2-oxoglutarate 4-dioxygenase (proline 4-hydroxylase), alpha one polypeptide13.5
Pcolce2δProcollagen C-endopeptidase enhancer 211.8
Tpm1Tropomyosin 1, alpha10.0
Wisp1WNT1 inducible signaling pathway protein 19.7
Tnmd#Tenomodulin8.5
Loxl2δLysyl oxidase-like 28.3
1500015O10RikRIKEN cDNA 1500015O10 gene7.1
Col11a1#Collagen, type XI, alpha 17.1
PdgfrlδPlatelet-derived growth factor receptor-like7.0
Mfap4Microfibrillar-associated protein 46.5
Col1a1#Collagen, type I, alpha 16.4
PtgisProstaglandin I2 (prostacyclin) synthase6.4
Col1a2#Collagen, type I, alpha 26.2
Itgbl1Integrin, beta-like 15.7
Tpm2Tropomyosin 2, beta5.4
  1. Note:

    1) #=Tendon differentiation or specific marker; δ = genes related to tendons.

  2. 2) Note that the expression level detected for Scx also included that of ScxGFP, and therefore do not reflect the expression level of endogenous Scx.

Table 2
Top 25 upregulated genes in P7 Tgfbr2;ScxCre mutant cells compared with P7 wild-type tenocytes (≥2 fold change, adjusted p<0.05).

See also Supplementary file 2 for a complete list of the downregulated genes.

Gene symbolGene nameFold change
Dlk1Delta-like one homolog (Drosophila)137.9
Serpine2Serine (or cysteine) peptidase inhibitor, clade E, member 2118.2
DptDermatopontin95.7
Ly6aLymphocyte antigen six complex, locus A54.3
H19H1951.1
Cd34CD34 antigen47.8
LumLumican36.6
LgmnLegumain31.8
Cxcl12Chemokine (C-X-C motif) ligand 1226.1
Mfap5Microfibrillar associated protein 522.5
Ly6c1Lymphocyte antigen six complex, locus C121.7
Igf2Insulin-like growth factor 221.4
Serping1Serine (or cysteine) peptidase inhibitor, clade G, member 119.2
Mgst1Microsomal glutathione S-transferase 118.3
AspnAsporin15.9
Mt1Metallothionein 115.4
Mgst3Microsomal glutathione S-transferase 313.1
Col3a1δCollagen, type III, alpha 113.0
PostnPeriostin, osteoblast specific factor13.0
Itm2aIntegral membrane protein 2A12.7
PtnPleiotrophin10.3
Rps18-ps3Ribosomal protein S18, pseudogene 39.7
GsnGelsolin8.3
Ifitm3Interferon induced transmembrane protein 38.2
Col5a1δCollagen, type V, alpha 18.1
  1. Note: δ = genes related to tendons.

Table 3
PANTHER protein class differentially expressed in P7 Tgfbr2;ScxCre mutant cells compared with P7 wild-type tenocytes.

A complete list of differentially expressed genes (≥2 fold change, adjusted p<0.05) used for the analysis is available in Supplementary file 2.

(A) Downregulated protein class
Protein classGene list
ReceptorPdgfrl, Col6a3, Kdelr3, Col6a1, Kdelr2, Itgbl1, Ssc5d, Col6a2, Ssr4, Col12a1, Matn4
Signaling moleculeSdc1, Wisp1, Sparc, Mfap4, Sema3b, Angptl2, Tgfbi
Membrane traffic proteinSec13, Kdelr3, Copz2, Kdelr2, Rabac1, Lman1
Extracellular matrix proteinSdc1, Crtap, Clec11a, P3h3, Sparc, P3h4
(B) Upregulated protein class
Protein classGene list
Nuclei acid bindingNdn, Eif3f, Rpl39, Rpl36a, Rpl3, Rpl9-ps6, Rpl22l1, Rps27, Rps4x, Cirbp, Rps19, Eif3e, Rps18, Rps5, Junb
Enzyme modulatorFstl1, Dbi, Sfrp2, Ctsb, Serpine2, Serping1, Igfbp3, Igfbp4
Cytoskeletal proteinGsn, Map1lc3b, Tuba1b, Arpc1b, Emp1, Tubb5
Signaling moleculeS100a16, Ptn, Dlk1, Efemp2, Postn, Sfrp2
Transcription factorEif3h, Naca, Fos, Id3, Junb
Table 4
PANTHER pathway analysis of upregulated genes in P7 Tgfbr2;ScxCre mutant cells compared with P7 wild-type tenocytes.
PANTHER pathwayPANTHER accessionGene list
Integrin signaling pathwayP00034Arpc2, Col4a1, Rac1, Col5a2, Rap1b, Cdc42, Arpc5, Col5a1, Rap1a, Rhoc, Fn1, Arpc1b, Col3a1
Inflammation mediated by chemokine and cytokine signaling pathwayP00031Arpc2, Rac1, Cdc42, Nfkbia, Arpc5, Rhoc, Arpc1b, Arpc4, Jun, Junb
Wnt signaling pathwayP00057Fstl1, Sfrp2, Ppp3ca, Csnk1a1
Insulin/IGF pathwayP00032, P00033Igf1, Igf2, Fos
  1. Note:

    1A complete list of differentially expressed genes (DEGs) used for the analysis is available in Supplementary file 2.

  2. 2Different values of the filter parameter (mean UMI count and fold change) were applied for enriching DEGs in P7 mutant cells. Only pathways that stood out as relevant for this study are listed.

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional
information
Genetic reagent
(M. musculus)
Tgfbr2f/f(Chytil et al., 2002)NANA
Genetic reagent
(M. musculus)
ScxCre(Blitz et al., 2013)NANA
Genetic reagent
(M. musculus)
RosaCreERT(Hameyer et al., 2007)NANA
Genetic reagent
(M. musculus)
ScxGFP(Pryce et al., 2007)NANA
Genetic reagent
(M. musculus)
Ai14 Rosa26-tdTomato (RosaT)(Madisen et al., 2010)NANA
Recombinant DNA reagentpAAV1-FLEX-Tgfbr2-V5GenScriptThis paperNA
Recombinant DNA reagentpAAV1-Tgfbr2-FLAGGenScriptThis paperNA
AntibodyRat anti-CD34 (Clone RAM34)BD BiosciencesCat# 553731
RRID:AB_395015
IF(1:200), Antigen retrieval
AntibodyRat anti-CD44 (Clone IM7)BD BiosciencesCat# 550538
RRID:AB_393732
IF(1:40), Pre-treated with cold acetone for 10 min at −20°C
AntibodyRabbit anti-FLAG (DYKDDDDK)Thermo Fisher ScientificCat# 740001
RRID:AB_2610628
IF(1:200), Antigen retrieval
AntibodyRat anti-FLAG (DYKDDDDK)Novus BiologicalsCat# NBP1-06712SS
RRID:AB_1625982
IF(1:100), Antigen retrieval
AntibodyGoat anti-Sca-1/Ly6R and D SystemsCat# AF1226
RRID:AB_354679
IF(1:80)
AntibodyRat anti-Sca-1/Ly6R and D SystemsCat# MAB1226
RRID:AB_2243980
IF(1:50)
AntibodyGoat anti-tenomodulin
(Clone C-20)
Santa Cruz BiotechnologyCat# sc-49324
RRID:AB_2205971
IF(1:50), Antigen retrieval
AntibodyRabbit anti-TGFβ type II receptorBioworld IncCat# BS1360
RRID:AB_1663474
IF(1:250)
AntibodyRabbit anti-V5AbcamCat# ab206566
RRID:AB_2819156
IF(1:500), Antigen retrieval
AntibodyRat anti-V5AbcamCat# ab206570
RRID:AB_2819157
IF(1:500), Antigen retrieval
AntibodyCy5 donkey anti-goat secondaryJackson ImmunoResearchCat# 705-175-147
RRID:AB_2340415
IF(1:500)
AntibodyAlexaFluor647 donkey anti-rabbit secondaryJackson ImmunoResearchCat# 711-607-003
RRID:AB_2340626
IF(1:400)
AntibodyCy3 donkey anti-rabbit secondaryJackson ImmunoResearchCat# 711-166-152
RRID:AB_2313568
IF(1:800)
AntibodyAlexaFluor647 donkey anti-rat secondaryJackson ImmunoResearchCat# 712-606-153
RRID:AB_2340696
IF(1:800)
AntibodyCy3 donkey anti-rat secondaryJackson ImmunoResearchCat# 712-166-150
RRID:AB_2340668
IG(1:800)
Commercial assay or kitIn situ cell death detection kitRocheCat# 12156792910Follow the manufacturer’s instruction
Commercial assay or kitClick-iT EdU kitLife TechnologiesCat# C10340Follow the manufacturer’s instruction
OtherDAPI stainThermo Fisher ScientificD1306
RRID:AB_2629482
1 μg/ml
  1. Note:

    * Antigen retrieval: Incubated with warm citrate buffer (10 mM sodium citrate with 0.05% Tween 20, pH 6) at 550W, 50°C for 5 min using a PELCO BioWave.

Data availability

All data generated or analyzed during this study are included in the manuscript and Supplementary Files. Single cell RNA-Seq data has been deposited onto GEO under accession code GSE139558.

The following data sets were generated
  1. 1
    NCBI Gene Expression Omnibus
    1. G Tan
    2. C Wang
    3. Z Xia
    4. R Schweitzer
    (2020)
    ID GSE139558. Differentially expressed transcriptomes of P7 mouse tendon cells with targeted deletion of TGF-beta signaling.

Additional files

Supplementary file 1

Signature genes in tenocytes and dedifferentiated mutant cells in comparison with other clusters.

See also Figure 6A for the tSNE plots of the sample. (A) Top 25 genes highly expressed in the tenocyte cluster relative to other clusters in the P7 wild-type tendon sample (≥1.5 fold change, adjusted p<0.05). (B) Top 25 genes highly expressed in the dedifferentiated mutant cell cluster relative to other clusters in the P7 Tgfbr2;ScxCre mutant tendon sample (≥1.5 fold change, adjusted p<0.05).

https://cdn.elifesciences.org/articles/52695/elife-52695-supp1-v2.docx
Supplementary file 2

Differentially expressed genes in P7 Tgfbr2;ScxCre mutant tendon cells compared with P7 wild-type tenocytes (≥2 fold change, adjusted p<0.05).

Note that the expression level detected for Scx also included that of ScxGFP, and therefore do not reflect the expression level of endogenous Scx.

https://cdn.elifesciences.org/articles/52695/elife-52695-supp2-v2.xlsx
Supplementary file 3

Gene Ontology (GO) term enrichment of differentially expressed genes in P7 Tgfbr2;ScxCre mutant cells compared with P7 wild-type tenocytes.

A complete list of differentially expressed genes (2 fold change, p<0.05) used for the analysis is available in Supplementary file 2.

https://cdn.elifesciences.org/articles/52695/elife-52695-supp3-v2.docx
Transparent reporting form
https://cdn.elifesciences.org/articles/52695/elife-52695-transrepform-v2.docx

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