Canonical and noncanonical TGF-β signaling regulate fibrous tissue differentiation in the axial skeleton

Previously, we showed that embryonic deletion of TGF-β type 2 receptor in mouse sclerotome resulted in defects in fibrous connective tissues in the spine. Here we investigated how TGF-β regulates expression of fibrous markers: Scleraxis, Fibromodulin and Adamtsl2. We showed that TGF-β stimulated expression of Scleraxis mRNA by 2 h and Fibromodulin and Adamtsl2 mRNAs by 8 h of treatment. Regulation of Scleraxis by TGF-β did not require new protein synthesis; however, protein synthesis was required for expression of Fibromodulin and Adamtsl2 indicating the necessity of an intermediate. We subsequently showed Scleraxis was a potential intermediate for TGF-β-regulated expression of Fibromodulin and Adamtsl2. The canonical effector Smad3 was not necessary for TGF-β-mediated regulation of Scleraxis. Smad3 was necessary for regulation of Fibromodulin and Adamtsl2, but not sufficient to super-induce expression with TGF-β treatment. Next, the role of several noncanonical TGF-β pathways were tested. We found that ERK1/2 was activated by TGF-β and required to regulate expression of Scleraxis, Fibromodulin, and Adamtsl2. Based on these results, we propose a model in which TGF-β regulates Scleraxis via ERK1/2 and then Scleraxis and Smad3 cooperate to regulate Fibromodulin and Adamtsl2. These results define a novel signaling mechanism for TGFβ-mediated fibrous differentiation in sclerotome.

www.nature.com/scientificreports/ skeletal malformation, including short stature and limb abnormalities. Mice with loss of ERK1/2 in Col2a-positive cells (Col2a Cre-ERK1 −/− ; ERK2 f/f ) died immediately after birth, likely due to deformity of the rib cage. These mice also demonstrated kyphosis in the spine, which is also seen in mice with Tgfbr2 mutation 17 suggesting ERK1/2 may have overlapping roles with Tgfbr2 in skeletal development.
Here, we address the mechanism of how TGF-β signaling affects fibrous tissue differentiation in sclerotome through canonical and non-canonical pathways. First, we showed that TGF-β regulates Scleraxis (Scx) mRNA, a transcription factor that regulates fibrous tissue differentiation, by 2 h after treatment. In contrast, Fibromodulin (Fmod) and Adamtsl2 mRNAs, coding for ECM markers of fibrous tissue differentiation, were expressed later at 8 h after treatment with TGF-β. Second, we showed that TGF-β-mediated expression of Scx mRNA did not require new protein synthesis. In addition, neither Smad2 nor Smad3 were required for TGF-β-mediated regulation of Scx. Next, we showed that new protein synthesis and Smad3 were required for TGF-β-mediated stimulation of Fmod and Adamtsl2 mRNAs. Nevertheless, exogenous Smad3 in conjunction with TGF-β treatment was not sufficient to super-regulate these markers suggesting another effector was involved. We then demonstrated that a non-canonical TGF-β signaling pathway involving ERK is required for expression of Scx, Fmod and Adamtsl2 mRNAs. Furthermore, we showed that Scx is required for full regulation of Fmod and Adamtsl2 in response to TGF-β. In summary, we have defined a new TGF-β signaling pathway in sclerotome that promotes differentiation of fibrous tissues in the axial skeleton.

Results
TGF-β regulates fibrous tissue markers in the sclerotome. We previously identified, through microarray screens, several markers for fibrous tissues in the axial skeleton including Scx, Fmod, and Adamtsl2. These markers are enriched in the putative AF and regulated by TGF-β in sclerotome 18 . Here we further analyzed the timing of TGF-β-mediated induction of these gene's mRNA after 2 h and 8 h of TGFβ1 treatment using quantitative real time RT-PCR (qPCR) analyzed using REST software. TGFβ1 induced Scx but not Fmod or Adamtsl2 after 2 h of treatment. However, when cells were treated with TGFβ1 for 8 h, expression of Fmod and Adamtsl2 was increased (Fig. 1A, Table S1). We then wanted to determine if TGF-β-mediated regulation of Scx, Fmod, and Adamtsl2 required new protein synthesis. Sclerotome cells were treated with cycloheximide (CHX) for 30 min to block protein synthesis, and then treated with vehicle control or TGFβ1 for 2 h or 8 h. A previous study using limb mesenchyme showed that Scx was induced after treatment with CHX, suggesting the existence of a labile protein repressor 19 . In addition, Scx mRNA was induced by TGF-β in the presence of CHX, suggesting direct regulation of Scx expression by TGF-β 19 . Similarly, in this experiment, Scx mRNA was increased in sclerotome in the presence of CHX and was induced after treatment with TGFβ1 in the presence of CHX (Fig. 1B, Table S2). The level of Scx mRNA induction after treatment with TGFβ1 in the presence of CHX was, however, attenuated relative to induction with TGFβ1 treatment alone (Fig. 1B, Table S2). Together the results indicate that TGF-β, at least in part, regulates Scx mRNA levels directly without new protein synthesis. In contrast, when cells were incubated with TGFβ1 for 8 h in the presence of CHX, Fmod and Adamtsl2 were not regulated after TGFβ1 treatment (Fig. 1C, Table S3). The results indicate that new protein synthesis is required for TGF-β-meditated expression of Fmod and Adamtsl2 and suggest that an intermediate factor is involved in their regulation.

Role of Smad-mediated signaling in regulation of fibrous tissue differentiation. Smad2 and
Smad3 mediate TGF-β signaling by binding to regulatory regions of target DNA acting as transcription factors 10 . We tested the hypothesis that Smads are involved in TGF-β-mediated regulation of fibrous tissue differentiation by measuring expression of Scx, Fmod, and Adamtsl2 via qPCR while manipulating Smad2 and Smad3 activity. First, we determined the time course for activation of Smad2 and Smad3 after treatment with TGFβ1. Phosphorylated Smad2 and Smad3 (pSmad2 and pSmad3) were measured by western blot to show activation of signaling ( Fig. 2A). Quantification shows that pSmad2 and pSmad3 were increased when sclerotome was treated with TGFβ1 after 2 and 8 h while total levels of Smad2/3 were not altered (Fig. 2B,C). We then used the Smad3 inhibitor, SIS3, and qPCR to determine if Smad3 was required for TGF-β-mediated regulation of the markers. SIS3 is Figure 1. TGF-β regulates expression of fibrous tissue markers. (A) Primary sclerotome was treated with vehicle control or TGFβ1 for 2 h or 8 h. qPCR was used to determine the relative levels of Scx, Fmod and Adamtsl2 mRNA. (B, C) Sclerotome was treated with cycloheximide (CHX) for 30 min then treated either with or without TGFβ1 for 2 h (B) or 8 h (C). Relative levels of fibrous tissue markers were determined by qPCR. All mRNA levels were normalized to Hprt and then compared to vehicle control using REST software. (*) indicates significance, p < 0.05, n = 3. Detailed results from qPCR REST analysis are shown in Table S1 www.nature.com/scientificreports/ known to specifically inhibit Smad3 and affects Smad3 target genes 20 . Primary sclerotome was pretreated with vehicle or two concentrations of SIS3 (5 and 10 µM) for 24 h at which time the cells were treated with TGFβ1 for 8 h. Western blot confirmed that SIS3 inhibited activation of pSmad3 after treatment with 5 and 10 µM TGFβ1 (Fig. 2D,E). Prg4 mRNA expression was also used as a positive control for SIS3 efficacy since we and others previously showed that Prg4 mRNA is regulated by Smad3 21 . Consistent with previous studies, inhibition of Smad3 with SIS3 blocked full induction of Prg4 by TGFβ1 in a dose dependent manner (Fig. 2F, Table S4). Induction of Fmod and Adamtsl2 by TGFβ1 were also attenuated following treatment of SIS3 (Fig. 2F, Table S4). These results suggest that Smad3 is required for TGF-β-mediated expression of Fmod and Adamtsl2. In contrast, SIS3 did not block TGF-β-mediated induction of Scx suggesting that regulation of Scx occurs through a non-canonical pathway or alternatively through pSmad2. Since it has been shown that Smad2 and Smad3 can regulate different downstream targets 22 , we tested a potential role for Smad2 in regulating Scx. Sclerotome was transduced with a dominant-negative form of Smad2 (Ad-DNSmad2). This point mutation in Smad2 was previously shown to dominantly attenuate signaling by both Smad2 and Smad3 due to its inability to be released from Tgfbr1 22 . Ad-DNSmad2 activity was confirmed as reduced pSmad2 and pSmad3 levels after treatment with TGF-β when compared to Ad-GFP control cells (Fig. 2G,H,I). As a positive control and as expected, infection with Ad-DNSmad2 Immunoblot was used to determine the levels of pSmad3, pSmad2 and total Smad2/3. α-Tubulin was used as a general loading control. (B) Quantification of pSmad3 and (C) pSmad2 levels relative to total Smad2/3 are shown. Immunoblots were quantified using ImageJ. (*) indicates significance, p < 0.05, n = 3 for each (D) Sclerotome was pretreated with vehicle control or SIS3 (5 µM and 10 µM) for 24 h and then treated with or without TGFβ1 for 8 h. pSmad3 and Smad2/3 protein levels were analyzed via immunoblot. α-Tubulin was used as a general loading control. (E) Smad3 activity was quantified from the immunoblots using ImageJ as the relative levels of pSmad3 normalized to total Smad2/3. (*) indicates significance, p < 0.05, n = 3 (F) Relative levels of Scx, Fmod, Adamtls2 and Prg4 mRNA were determined by qPCR after indicated treatment with SIS3. All mRNA levels were normalized to the housekeeping gene Hprt and then analyzed for significance using REST (*). Results are shown relative to TGFβ1 treated cells. n = 3 (G) Cells were infected with AdDNSmad2 or AdGFP control virus 48 h before cells were treated with or without TGFβ1 for 8 h. Immunoblot was used to visualize relative levels of pSmad3, pSmad2 and total Smad2/3. α-Tubulin is shown as a general loading control. (H) pSmad3 and (I) pSmad2 activity were quantified using ImageJ as the level of each phosphoSmad normalized to total Smad2/3. (*) indicates significance, p < 0.05, n = 3 (J) RNA was extracted from cells that were infected with AdDNSmad2 or AdGFP and treated or untreated with TGFβ1. Relative levels of Scx and Adamtsl2 mRNA were determined by qPCR. Expression was normalized to the housekeeping gene Hprt. Results are shown relative to the untreated control (*) indicates significance, p < 0.05, n = 5 (K) Cells were infected with AdSmad3 or AdGFP for 48 h and Smad3 expression was verified via immunoblot. (L) Immunoblots were quantified using ImageJ. ( www.nature.com/scientificreports/ attenuated induction of Adamtsl2 mRNA in response to TGF-β ( Fig. 2J, Table S5). In contrast, Scx was still regulated by TGF-β to a statistically similar level as in the cells infected with the control (ad-GFP) virus (Fig. 2J, Table S5). The results suggested that TGFβ-mediated regulation of Scx did not require Smad2 or Smad3. Next, to determine whether exogenous Smad3 was sufficient to super-induce Scx, Fmod, or Adamtsl2 in the presence of TGFβ1, adenovirus encoding Smad3 (Ad-Smad3) was used to over-express Smad3 in sclerotome cultures. Immunoblot was used to confirm Smad3 overexpression in Ad-Smad3 infected cells (Fig. 2K,L). Cells were infected with Ad-Smad3 or Ad-GFP, as a control, for 48 h and then treated with or without TGFβ1 for 8 h. As expected, Prg4 mRNA levels were increased after treatment with TGFβ1 alone and super-induced in Ad-Smad3 infected cultures after Smad3 activation with TGFβ1 ( Fig. 2M). In contrast, while TGFβ1 regulated expression of Scx, Fmod, and Adamtsl2, Smad3 did not super-induce expression after activation with TGFβ1 ( Fig. 2M, Table S6). Since Smad3 was required for full TGF-β-mediated induction of Adamtsl2 and Fmod but not sufficient to super-induce expression after activation with TGFβ1, we propose an additional pathway is also required. This led us to investigate non-canonical signaling pathways.

Non-canonical pathways are regulated by TGF-β in the sclerotome. TGF-β transduces signals
through not only Smad2/3 but also other kinases such as ERK1/2, p38, and AKT 12 . These Smad-independent pathways are often referred to as non-canonical TGF-β signaling pathways. To determine if TGF-β regulated fibrous tissue differentiation through non-canonical pathways, we determined the time course of activation of ERK1/2, p38, and AKT after treatment of sclerotome with TGFβ1 (Fig. 3A). Activated forms of ERK1/2 (pERK1/2) were observed after 2, 8, and 24 h of TGFβ1 treatment (Fig. 3B). Total levels of ERK1/2 were not affected by TGFβ1. Activation of p38 as measured by the level of phosphorylated p38 (pp38) was observed at 2 and 8 h of TGFβ1 treatment (Fig. 3C). Total p38 levels were not affected by TGFβ1 treatment. Increase in the phosphorylated form of AKT (p-AKT) was observed after TGFβ1 treatment for 2 and 8 h (Fig. 3D). Total AKT was not affected. These data indicate that TGF-β can activate non-canonical signaling pathways in sclerotome.
p38 and AKT are not required for TGF-β-mediated regulation of fibrous tissue differentiation. We then tested whether activation of p38 is required for TGFβ-mediated fibrous tissue differentiation using a p38 antagonist, doramapimod (BIRB796), that inhibits all isoforms of p38 23 . TAK1/p38 signaling is activated by TGF-β in many cell types and mice with loss of the Tak1 gene have a similar skeletal phenotype to www.nature.com/scientificreports/ mice deleted for Tgfbr2 24 . Isolated sclerotome was treated with BIRB796 or vehicle control, DMSO, for 24 h and then treated with TGFβ1 or vehicle control for an additional 8 h. The efficacy of the antagonist was measured by immunoblot of pMAPK-APK2, a direct downstream target of p38 25 . Activated MAPK-APK2 has multiple bands since p38 phosphorylates multiple residues to regulate activity 26,27 (Fig. 4A). Treatment with TGFβ1 resulted in increased pMAPK-APK2 levels. Treatment with various concentrations of BIRB796 blocked the increase in pMAPK-APK2 in response to TGFβ1 (Fig. 4A,B). Using the same concentrations of BIRB796, we next measured Scx, Fmod, and Adamtsl2 expression in response to TGFβ1 using qPCR (Fig. 4C, Table S7). TGFβ1 was still able to up-regulate Scx, Fmod, and Adamtsl2 in the presence of the p38 antagonist suggesting that p38 signaling is not required for TGFβ1 to regulate fibrous tissue. We then tested whether activation of AKT signaling was required for TGF-β-mediated fibrous tissue differentiation by using an AKT inhibitor, MK2206 28 . The PI3K/AKT pathway is known to have an important role in cartilage development and is also activated by TGF-β in other cell types 29 . The concentration of MK2206 necessary to block activity was determined by immunoblots of the active form of AKT. As expected, MK2206 attenuated TGF-β-induced phosphorylated AKT in the sclerotome (Fig. 4D,E). Sclerotome was then treated with MK2206 and TGFβ1 so that the effects of AKT on fibrous tissue differentiation could be determined as expression of fibrous tissue markers by qPCR. mRNA levels of fibrous tissue markers were the same after treatment with TGF-β in the presence or absence of the AKT inhibitor, MK2206 (Fig. 4F, Table S8). These results suggest that AKT was not involved in TGF-β-mediated induction of fibrous tissue differentiation.
Role of ERK1/2 in fibrous tissue differentiation and TGFβ-mediated inhibition of chondrogenesis. Next, we investigated whether ERK1/2 signaling mediates the effect of TGFβ1 on fibrous tissue differentiation. We used a MEK inhibitor, PD184352, to inhibit ERK1/2 activity 30 . MEK is directly upstream of www.nature.com/scientificreports/ ERK1/2 and inhibition of MEK is a common method to inhibit ERK1/2 activity. Mice with deletion of ERK1 and ERK2 in their axial skeletons demonstrate kyphotic deformity in the thoracic spine 17 . To determine the necessary drug concentration, sclerotome was treated with PD184352 in presence or absence of TGFβ1. Treatment with TGFβ1 resulted in an increase in phosphorylated ERK1/2, as previously shown (Fig. 5A,B). Efficacy of 1, 5 and 10 µg/ml doses of PD184352 was confirmed via immunoblot as a dose dependent reduction in the levels of TGFβ1-induced phosphorylated ERK1/2 (pERK1/2) (Fig. 5A,B). Next, expression of markers for fibrous tissue differentiation were measured via qPCR after TGFβ1 treatment. Induction of Scx, Fmod, and Adamtls2 by TGF-β were all attenuated in a dose dependent manner after treatment with PD184352 (Fig. 5C, Table S9). The results indicated that ERK1/2, but not p38 or AKT, is required for TGFβ1-mediated fibrous tissue differentiation. Next, we tested the specificity of the response and determined whether TGFβ1 utilizes ERK1/2 signaling to down-regulate the expression of early markers for chondrogenesis, the alternate cell fate of sclerotome. Previously, our laboratory showed that TGFβ1 inhibits chondrogenesis and down-regulates EBF1 and cMAF, vertebrae enriched genes that are involved in chondrogenesis, in the sclerotome within 8 h of treatment 31 . EBF1 and cMAF mRNA levels were measured by qPCR after treatment with TGFβ1 and PD184352. Inhibition of ERK1/2 did not affect TGFβ1-mediated down-regulation of these genes, indicating that ERK1/2 signaling is specific for TGFβ-mediated regulation of fibrous differentiation (Fig. 5D, Table S10).

Scx is required for TGFβ-mediated regulation of Fmod and
Adamtsl2. Finally, we tested the hypothesis that Scx is involved in subsequent TGFβ-mediated expression of the ECM fibrous markers Fmod and Adamtsl2. Scx is a basic helix loop helix (bHLH) transcription factor 8 that is regulated earlier than the other fibrous tissue markers ( Fig. 2A) and is up-regulated at least in part by TGF-β even in the absence of new protein synthesis suggesting direct regulation by TGF-β Fig. 2B). To determine if Scx expression was required to regulate Fmod and Adamtsl2, we inhibited Scx synthesis by transducing C 3 H 10 T 1/2 cells, a murine mesenchymal stem cell line, with 30 pM of Scx specific siRNA or a 30 pM of Scrambled (Scram) siRNA as a control. We and others have previously shown that fibrous genes are regulated by TGFβ1 in C 3 H 10 T 1/2 cells 32,33 . Western blot analysis showed a significant decrease in Scx protein expression in Scx siRNA transduced cells when compared to Scram control (Fig. 6A,B). qPCR determined that mRNA levels of Scx were also significantly decreased with Scx siRNA treatment (Fig. 6C, Table S11). We then wanted to test whether a decrease in Scx expression affected expression of Fmod and Adamtsl2. As expected, qPCR analysis determined that in Scram control siRNA transduced cells treatment with TGFβ1 resulted in an increase in marker expression. Basal Fmod expression was significantly downregulated in Scx siRNA transduced cells when compared to Scram siRNA suggesting Scx is important for regulation of Fmod in general. Furthermore, Fmod was not up-regulated by TGFβ1 in Scx siRNA transduced www.nature.com/scientificreports/ cells, signifying that Scx expression is also required for TGF-β mediated regulation of Fmod (Fig. 6D, Table S12). Inhibtion of Scx with siRNA did not affect basal levels of Adamtsl2 mRNA, but, induction of Adamtl2 by TGFβ1 was blocked, indicating that Scx is also required for TGF-β mediated regulation of Adamtsl2 (Fig. 6D, Table S12).

Discussion
We previously identified several TGF-β regulated genes for which expression is enriched in the putative IVD region of the embryonic sclerotome, including Adamtsl2, Fmod, and Scx 18,31,34 . Localization to a patterned subset of sclerotome was previously confirmed by in-situ hybridization 18,35 . Mutations in Adamtls2 result in a condition called Geleophysic dysplasia, which is characterized in part by short stature and joint contractures. It was recently shown that the joint contractures and short limb phenotype were mediated by Adamtsl2 expression in Scx-positive cells 35 . Phenotype in the spine was not discussed. Likewise, Fmod is required for development of tendons and other fibrous tissues 36 . Almost nothing is known about upstream regulators of Adamtsl2 or Fmod expression. We previously showed that both are strongly regulated by TGF-β, which is also involved in development of tendon and other fibrous tissues 8,18,31,34 . Here we show that Fmod and Adamtsl2 mRNAs are regulated by non-canonical and canonical signaling pathways, ERK1/2 and Smad3. Furthermore, experiments using a protein synthesis inhibitor, CHX, showed new protein synthesis was required for TGF-β-mediated up-regulation of Fmod and Adamtsl2 mRNAs. These results suggest that an intermediary protein must be made so that TGF-β can regulate the expression of these ECM proteins. Scx, a basic helix loop helix (bHLH) transcription factor 8 , was up-regulated, at least in part, by TGF-β even in the absence of new protein synthesis suggesting direct regulation by TGF-β. Induction of Scx by TGF-β in CHX treated cells was, however, attenuated suggesting that there may be two mechanisms for TGF-β-mediated regulation of Scx. One that does not require de novo protein synthesis, perhaps regulating the immediate response to TGF-β, and another that requires new protein, perhaps regulating maintenance of Scx expression in fibrous cells. In addition, Scx required ERK1/2 but not Smad3 for TGF-βmediated regulation. We then showed that Scx was required to regulate Fmod and Adamtsl2. We propose that TGF-β regulates Fmod and Adamtsl2 through induction of one or more transcription factors, including Scx. Deletion of Scx results in alterations in the development of long tendons of the spine 37 . More recently, it was shown that loss of Scx disrupts maturation of AF, and the loss of Sox9 in Scx expressing cells disrupts formation of the AF and chondro-tendinous/ligamentous junctions 38,39 . Scx expression is up-regulated by TGF-β in mesenchymal cells including primary sclerotome and C 3 H 10 T 1/2 cells and down-regulated in tissues in which Tgfbr2 has been conditionally deleted 8,31,34 . bHLH transcription factors like Scx generally bind to DNA as dimers www.nature.com/scientificreports/ to elements known as E-boxes 40 . It has also been shown that Scx can bind to Smad3 to regulate down-stream gene expression 41,42 .
A little is known about how Scx expression is regulated. FGF was the first factor shown to regulate Scx during development 43 . It was shown that FGF secreted from the myotome acted on the adjacent sclerotome to induce Scx expression and fibrous differentiation. FGF is known to signal though ERK1/2 and it was shown that ERK1/2 is required for Scx expression in chick axial and appendicular skeleton [43][44][45] . TGF-β is known to activate ERK1/2 through a non-canonical pathway 46,47 . In this study we showed that ERK1/2 is required for TGF-β-mediated expression of Scx; however canonical signaling through Smad was not necessary to regulate Scx mRNA in sclerotome. Likewise, regulation of Scx in cardiac fibroblasts is Smad3-independent 48 and Scx expression is not depleted in tendons of embryonic Smad3 knock out mice 41 . However, in contrast to these observations, one study showed that TGF-β-mediated expression of Scx in limb was independent of ERK1/2 and dependent on Smad3 45 . This discrepancy may be due to different cell type, microenvironment, or developmental stage.
Since Smad3 was necessary but not sufficient to super-induce expression of Fmod and Adamtsl2, we hypothesized that TGF-β likely uses a combination of signaling pathways in addition to Smad3 (Fig. 7). Noncanonical TGF-β signaling pathways, which are Smad-independent, use various kinases like p38, AKT and ERK1/2 12 . TGF-β differentially activates these non-canonical pathways based on cell type and other circumstances. Previous studies showed that p38 mediates TGF-β signaling in articular chondrocytes 49 . Also, mice lacking TAK1, which lies upstream of p38, have defects in joint formation, similar to that observed in the absence of Tgfbr2 24 . However, the results here indicated that TGF-β does not utilize p38 signals to regulate fibrous tissue differentiation. Next, we tested the role of ERK1/2, since ERK1/2 is also known to mediate TGF-β signaling 12 . Results indicated that ERK1/2 plays an important role in mediating fibrous tissue differentiation by TGF-β Inhibition of ERK1/2 did not alter the response of cartilage genes to TGF-β treatment, indicating that the role of ERK1/2 is specific to fibrous tissue differentiation.
This study demonstrates that canonical and noncanonical TGF-β signaling pathways regulate fibrous tissue differentiation in sclerotome. We propose that TGF-β acting through ERK1/2 regulates expression of Scx. Subsequently, Scx and Smad3 cooperate to regulates expression of Fmod and Adamtsl2 (Fig. 7). This study begins to define a novel signaling pathway for TGF-β in sclerotome that regulates fibrous tissue differentiation.

Materials and methods
All experiments with animals were approved by the University of Alabama Institutional Animal Care and Use Committee (IACUC) under protocol number 09713. All experiments with animals and all methods were carried out in accordance with relevant guidelines and regulations.
Sclerotome isolation and cell culture. Sclerotome was isolated from wild type mouse embryos at E11.5 days as previously described 18 . Embryos were generated by mating C57BL/6 male mice to ICR female mice. The tissue was digested with Collagenase D, dissociated into individual cells, and plated. Cells were plated Figure 7. A schematic of the proposed mechanism of TGF-β-mediated regulation of fibrous tissue differentiation from sclerotome. We hypothesize that TGFβ regulates fibrous tissue differentiation by first regulating non-canonical signaling through ERK1/2 (1), which is required for Scx mRNA expression (2). We propose that subsequently, Scx and Smad3 cooperate to regulate the ECM markers, Fmod and Adamtsl2 (3).  52 . REST analysis provides statistical testing for nonparametric data using a pair-wise fixed reallocation randomization test to determine significance (p > 0.05). "n" denotes the number of biological replicates. HPRT was used as a reference/normalization control. Primer sequences for qPCR are shown in Table S13.
Western blot. Cultured sclerotome was lysed with RadioImmunoPrecipitationAssay (RIPA) buffer containing phosphatase and protease inhibitors (Roche). The concentration of total protein was measured using a DC Protein Assay kit (Bio-Rad Laboratories). 25-30 μg of protein lysate per sample was loaded in 4-20% polyacrylamide gels (Bio-Rad Laboratories) and separated protein was transferred from gels to polyvinylidene fluoride membranes (PVDF) using a Trans-Blot Turbo Transfer system (Bio-Rad Laboratories). The membrane was blocked with 5% Bovine Serum Albumin (Sigma-Aldrich) and incubated with various primary antibodies. Membranes were then washed with Tris-buffered saline containing 0.1% Tween 20 (TBST) and incubated with various secondary antibodies. The chemiluminescent detection of the horseradish peroxidase on the secondary antibodies was detected by the Supersignal West Dura kit (Thermo Scientific). Images and of western blots were acquired on a ChemiDoc MP system (Bio-Rad Laboratories) and quantification of blots was performed on ImageJ. All statistical analyses of immunoblots was performed using GraphPad Prism either using an unpaired t-test or a one-way ANOVA with a Tukey's post hoc analysis. All immunoblots are n = 3 (biological replicates) and asterisks denote p < 0.05. siRNA. Murine C 3 H 10 T 1/2 cells (ATCC) were plated in 6-well tissue culture treated plates (Corning Costar) at a density of 3 × 10 5 cells/well in cell culture medium (88% DMEM, 10% heat-inactivated FBS, 1% 0.5 mM L-glutamine, 1% Penicillin/Streptomycin,) for 24 h. After 24 h, culture medium was replaced with MEM Alpha 1X (Gibco) and cells were transfected using Lipofecatime RNAiMax reagent (ThermoFisher Scientific) with either 30 pM of Scleraxis siRNA Catalog #sc-153257 (Santa Cruz) or 30 pM of Scrambled Control siRNA (FITC Conjugate)-A Catalog #sc-36869 (Santa Cruz) for 24 h. Cells were then lysed with RIPA Buffer for western blot analysis or TRIzol reagent for qPCR. Western blot data n = 3 and qPCR data n = 6. "n" denotes number of biological replicates. www.nature.com/scientificreports/ Received: 10 February 2020; Accepted: 10 November 2020