Notch Signaling Slows Down the Progression of Embryonic Myogenic Differentiation in Landrace

were randomly selected for analysis. The fusion index was calculated as the percentage of nuclei in fused myotubes out of the total nuclei. Data are presented as mean±sem or mean from at least three independent experiments. Statistical differences between groups were tested using an unpaired two-tailed Student’s t-test. Values of P<0.05 was considered as signicance.

Molecular mechanisms concerning determination and differentiation of myoblasts during porcine embryonic development remain to be further explored. Studies in mouse have reported that Notch and Wnt signaling pathways are involved in this process. Notch signaling pathway is implicated as an important regulator of proliferation and differentiation of myogenic progenitor cells [9,10]. In addition, it is also very important in maintaining satellite cells quiescence [11,12]. Notch signaling activation requires physical interaction between a ligand (delta1/4 or jagged 1/2) and one of the four Notch receptors (notch [1][2][3][4]. This interaction leads to the release of the Notch intracellular domain (NICD) which then translocates into nucleus where it binds to the Rbpj transcription factor and induce downstream effectors, such as the Hes/Hey family [13,14]. Wnt signaling has been demonstrated to be crucial for the maintenance of fetal muscle progenitors in mouse [15,16], and also has been proved to play a great role in the expansion of satellite cells [16,17]. In addition, AKT/mTOR signaling also regulates the differentiation of myoblasts [18].
Increasing evidence proved that pigs with less meat production showed more intense embryonic myogenesis. For example, the myo ber density and diameter were signi cantly higher in Meishan pigs (fatty, a Chinese indigenous breed) compared to Large White pigs (a lean breed) at 35dpc [19]. In our previous study, we found that Wuzhishan pigs (fatty) showed earlier myoblast differentiation than that of Landrace pigs (LR, lean) in embryonic stages [20]. Primary muscle bers appear earlier in Lantang (LT, fatty) than that in LR, at 35dpc and 42dpc respectively [21]. These observations suggest that the progression of embryonic myogenic differentiation is faster in obese pig breeds, which result in precocious myo ber formation. To verify this possibility, in the present study, we compared the dorsal myogenesis progression of LT and LR pigs at 35dpc, as well as the differentiation capacity of their myogenic progenitors in vitro. Further, for the rst time, the molecular mechanism concerning their differences was linked to Notch signaling.

Results
Myogenesis progression is more intense in LT than that in LR at 35dpc To study early embryonic myogenesis, the expression of embryonic myosin heavy chain (eMyHC, a marker for fully differentiated myocytes) was analyzed in longissimus dorsi muscle (LDM) of LT and LR pigs at 35dpc. There were more eMyHC + cells in LT ( Fig. 1a and b), which is consistent with previous reports, indicating precocious terminal differentiation of myoblasts in LT [20]. To de ne the progression of myogenic differentiation, dual immunostaining of Pax7 and MyoD was conducted. As a result, the percents of committed myoblasts (Pax7 + /MyoD + ) and differentiated myoblasts (Pax7 -/MyoD + ) were both higher in LT ( Fig. 1c and d). The percent of undifferentiated progenitors (Pax7 + /MyoD -) was higher in LR.
Accordingly, higher level of Pax7 protein and lower level of MyoD protein were found in LR (Fig. 1e).
Altogether, these results demonstrate that myogenesis process is more intense in LT than that in LR at 35dpc, which is well explained by the stronger myogenic differentiation tendency of muscle progenitors in LT.

Embryonic muscle progenitors from LT express MyoD earlier and have stronger differentiation capacity in vitro
To further investigate myogenic potentials of embryonic muscle progenitors in these two pig breeds, we isolated them from LDM by collagenase digestion combined with differential adherent puri cation. Identi cation of isolated cells by immunofluorescence staining showed that the Pax7-positive cells and Desmin-positive cells accounted for more than 80% of total cells respectively, both for LT and LR (Fig. 2a, b and c). In vivo study proved that muscle progenitors in LT showed a stronger myogenic differentiation tendency, we supposed that MyoD expression in LR progenitors may be slowed down, which subsequently blocked myogenic differentiation. To test this hypothesis, newly isolated LT and LR progenitors were cultured in growth medium, and time course expression of MyoD was tested by immuno uorescence staining (Fig. 2d). With the extension of culture time, MyoD + cell numbers gradually increased both in LT and LR progenitors. Compared with LR, more MyoD + cells appeared earlier in LT progenitors (Fig. 2e). Then, immuno uorescence staining for eMyHC was performed to compare the differentiation ability of two kinds of progenitors at day 6 after differentiation. Signi cantly, eMyHC + myotubes generated from LT progenitors were more and larger than that from LR progenitors (Fig. 2f). The statistical results indicated that LT progenitor showed a higher fusion index than LR progenitors (Fig.   2g). Congruent with these ndings, protein levels of MyoD and MyoG were higher in LT progenitors (Fig.  2h). Collectively, these results indicate that embryonic muscle progenitors from LT showed earlier expression of MyoD, and have stronger differentiation capacity in vitro.
Notch signaling is more active in LR myogenic progenitors To explore the molecular mechanism of different differentiation property between LT and LR progenitors, we detected Wnt, AKT/mTOR and Notch signaling pathways. The expression of total protein level and active protein level of β-catenin, an important mediator of canonical Wnt signaling pathway, were comparable between LT and LR progenitors (Fig. 3a). To examine the activity of the AKT/mTOR pathway, the expression levels of total and phosphorylated protein of AKT and S6K1 were measured. Results revealed that the levels of AKT, pAKT, S6K1 and pS6K1 showed no difference between two breeds ( Fig. 3b and c). Interestingly, qPCR results revealed that Notch genes, including ligand (Jagged1), Notch receptors (Notch1 and Notch3), Rbpj transcription factor (Rbpj) and downstream effectors (Hey1, HeyL and Hes1) had higher mRNA levels in LR progenitors (Fig. 3d). In line with these results, Western blotting demonstrated the protein levels of Jagged1, Hey1 and Hes1 were higher in LR (Fig. 3e). Immuno uorescence staining proved that there were more Jagged1 protein expressed in LR dorsal cells, whether it is a Pax7 + cell or a Pax7cell (Fig. 3f). Collectively, Notch signaling was more active in LR myogenic cells.
Boosted Notch signaling prevents C2C12 myoblast differentiation To further clarify the regulatory function of Notch signaling on myogenic differentiation, C2C12 cells were treated with the peptide of Jagged1, a Notch ligand known to activate Notch signaling in skeletal muscle cells. After 2 days of Jagged1 treatment in GM, the expression of Pax7 was up-regulated while MyoD was down-regulated ( Fig. 4a). In addition, the percentages of Pax7 + /MyoD + and Pax7 -/MyoD + cells were decreased, and the percentage of Pax7 + /MyoDcells was increased ( Fig. 4b and c), which implied that Notch signaling prevents myogenic differentiation. Then, C2C12 cells were induced to differentiate in DM supplemented with Jagged1 peptide, followed by differentiation assays. As a result, the percentage of MyoG + cells was signi cantly reduced after 1 day treatment ( Fig. 4d and e). When induced to differentiate for 3 days, as expected, cells treated with jagged1 formed less myotubes (Fig. 4f), which was proved by a decreased fusion index (Fig. 4g). In line with this, the expression of MyoG and MHC were down-regulated, indicating there is a differentiation defect in Jagged1 treated myoblasts (Fig. 4h).

Inhibition of Notch signaling promotes the differentiation of embryonic myogenic progenitors in vitro
Given that Notch signaling was over-activated in LR progenitors and it has inhibitory effect on myoblast differentiation, we speculated that this signaling contributes to the difference of differentiation ability between two kinds of muscle progenitors. LR progenitors were cultured in growth medium containing 20 μΜ γ-secretase inhibitor DAPT an inhibitor of Notch signaling to inhibit Notch activity. As expected, the expressions of Notch effectors Hey1, HeyL and Hes1 were decreased after DAPT treatment ( Fig. 5a and  b). In addition, inhibition of Notch signaling led to down-regulated Pax7 expression and up-regulated MyoD expression ( Fig. 5c and d). Furthermore, time course expression of MyoD was tested by immuno uorescence staining. As a result, there were more MyoD + cells in the DAPT group at each indicated time ( Fig. 5e and f). Immuno uorescence staining and Western blotting for eMyHC at day 6 after differentiation revealed a lower cell fusion index together with a decreased eMyHC protein level in DAPT-treated groups (Fig. 5g, h and i). Taken together, these ndings indicate that Notch inhibition prevents Pax7 but promotes MyoD expression as well as myogenic differentiation.

Pax7 inhibits terminal differentiation of myoblasts
The above assays have proved that Notch signaling promotes the expression of Pax7. Then, in order to verify that the regulatory effect of Notch signaling on myogenic differentiation is mediated through Pax7, we explored the speci c in uences of Pax7 on myoblasts differentiation. C2C12 cells were transfected with Pax7 siRNAs to knock down Pax7 expression. As a result, MyoD level was not changed, but Myf5 level was down-regulated ( Fig. 6a and b). Although Pax7 knockdown resulted in loss of Myf5 expression in C2C12 cells cultured in a low-density, it promoted MyoG expression in cells differentiated for 1day ( Fig.   6c and d). Accordingly, Pax7 knockdown cells formed more MyHC + myotubes with increased fusion index after 3 days differentiation ( Fig. 6e and f), and showed up-regulated protein levels of MyoG and MyHC (Fig. 6g). To further con rm the function of Pax7, Pax7 expression vector was transfected into C2C12 cells. As a result, the mRNA and protein levels of Myf5 were partially increased ( Fig. 6h and i).
Immuno uorescence assay showed that over expression of Pax7 in C2C12 cells reduced the percentage of cells expressing MyoG at differentiation 1 day (Fig. 6j and k), and MyHC expression was inhibited, which resulted in a decrease of fusion index at differentiation 3 day (Fig. 6l, m and n). These observations proved that Pax7 inhibits terminal differentiation of myoblasts.

Notch signaling slows down myogenic differentiation of muscle progenitors in embryo limbs
In order to verify the correlation between earlier progression of myogenic differentiation and repressed Notch signaling in LT with less muscle, an ex vivo limb culture system was employed [22,23]. Embryo forelimbs were separated at 35dpc and cultured for 30 hours, with or without 20 µM DAPT. qPCR analysis showed that Notch genes were successfully suppressed after DAPT treatment (Fig. 7a and c). In addition, inhibition of Notch signaling led to reduced numbers of Pax7 + /MyoDcells, whereas the MyoD + cell population was increased (Fig. 7d and e), con rming the robustness of our ex vivo model. Accordingly, we found decreased expression level of Pax7 and increased level of MyoD in DAPT-treated samples (Fig. 7b  and c). Altogether, these results demonstrate that in embryonic muscle progenitor cells Notch signaling antagonizes myogenic differentiation by promoting Pax7 expression and preventing MyoD expression.

Experimental animals and tissues
Six LT sows and six LR sows were arti cially inseminated with semen from the same breed boars, respectively. All sows were slaughtered at 35 dpc after insemination, and embryos were collected as previously described. For each embryo, the longissimus dorsi muscle (LDM) tissues were isolated, then, digested to isolate embryonic muscle progenitors or xed to prepare para n sections of tissue, or frozen in liquid nitrogen for further use.

Embryonic muscle progenitors isolation and culture condition
Embryonic muscle progenitors were isolated from the LDM of embryos of two breeds at 35 dpc. The isolated LDM was cleaned free of connective tissues, minced and digested with 0.2% Collagenase type I (Sigma, Shanghai, China) solution at 37°C water bath for 2h to get su cient cells. Mixed cells was preplated 2 h in 5.0 ml growth media on a culture dish to remove broblasts and then transferred to a new culture dish for attachment. Cells were cultured in Dulbecco's modi ed Eagle medium (DMEM) supplemented with 20% (v/v) fetal bovine serum (FBS), 1% penicillin-streptomycin antibiotics and 0.5% chicken essential extract (growth medium, GM). For experiments, muscle progenitors were sub-cultured onto 12-well plates at densities of 1.0 × 10 4 cells per well. Cells were switched into DMEM with 2% horse serum (differentiation medium, DM) after reaching 100% con uence to induce differentiation.
C2C12 cells were purchased from American Tissue Culture Collection (CATCC), cultured in DMEM with 10% FBS, and 1% penicillin-streptomycin (growth medium, GM) at subcon uent density. To induce differentiation, cells were switched into DM after reaching 100% con uence. All cells were cultured at 37°C in a humidi ed atmosphere of 5% CO 2 .

siRNAs, plasmids and transfection
For RNA interference, negative control siRNAs (siNC) and three stealth mouse Pax7 siRNAs were purchased from Invitrogen (Thermo Fisher Scienti c, USA) (Table. S1). siPax7-2 was the most e cient (Fig. S1). So, it was used in all of the following analysis. For Pax7 expression vector, mouse Pax7 CDS sequence was inserted into pcDNA3.1 vector (Invitrogen, Shanghai, China). C2C12 cells were transfected with siRNAs or plasmids using Lipofectamine 3000 (Invitrogen) according to the manufacturer's instruction. All transfections were performed in triplicate for each experiment.

Explant
Forelimbs from LR 35 dpc embryos were cultured in 12-well plates in BGJb medium (Life Technologies), without FBS, with 200 µg/ml ascorbic acid (Life Technologies) and 1% penicillin-streptomycin antibiotics. For Notch inhibition, forelimbs originating from the same embryo were immediately treated with 20 µM DAPT or DMSO carrier for 30 h. Then, treated and control forelimbs were cleaned and collected for further analysis.

Western blot
Protein extracts were obtained from incubating cultured cells, LDM or forelimbs homogenates in lysis buffer (150 mmol/L NaCl, 50 mmol/L Tris, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, pH 8.0) supplemented with protease inhibitor phenylmethanesulfonyl uoride (PMSF, Thermo Scienti c) on ice until protein was released completely. Total extracts were separated by SDS-PAGE on 10% (w/v) polyacrylamide gels, transferred onto 0.45 μm PVDF membrane (Roche). After being blocked in 4% bovine serum albumin (BSA) for 1-2 h, the membranes were incubated with speci c primary antibodies at 4 °C overnight, then incubated with secondary antibodies for 1 h at room temperature. Blots were visualized using an enhanced chemiluminescence (ECL) detection kit (FDbio, Hangzhou, China). β-Tubulin and GAPDH were used as internal controls. Antibodies are listed in Table S2. RNA extraction and real-time quantitative PCR Total RNA was extracted from cultured cells, LDM or forelimbs according to the instructions of TRIzol® Reagent (Invitrogen, Shanghai, China), then cDNA was synthesized from 1 μg total RNA using a reversetranscription Kit (Promega, Beijing, China). The real-time quantitative PCR (qPCR) was performed using a SYBR Green qPCR Kit (Genestar, Beijing, China), detected on the LightCycler 480 II system (Roche, Basel, Switzerland). The primers used for qPCR were given in Table S3. Gapdh was used as internal control and all reactions were run in triplicate.

Immuno uorescence
Cultured cells were xed in 4% paraformaldehyde for 10 minutes, permeabilized in 0.5% Triton X-100 for 15-20 minutes. After being blocked with 4% BSA in Tris-buffered saline with Tween (TBST) for 1 hour, the cells were incubated with primary antibodies overnight at 4°C, followed by incubation with secondary antibodies for 1 hour at room temperature. The nuclei were counterstained with 4′,6-diamidino-2phenylindole (DAPI; 1:1000 in PBS). Antibodies are listed in Table S2. Immunostaining images were obtained via uorescent reverse microscopy (Nikon, Tokyo, Japan).
Immunohistochemistry LDM or forelimbs were xed in 4% paraformaldehyde for 19 h at 4°C, then dehydrated using gradient alcohol and embedded with para n. Para n embedded samples were cut into 5-μm sections. The para n sections were placed in an oven at 64°C for 30 minutes and immediately moved to xylene for dewaxing. Rehydrated in gradient alcohol, antigen repaired using citrate antigen retrieval solution. Finally, immuno uorescence was performed using IHC Kit (Abcam, Cambridge, England) according to the manufacturer's instruction. Immunostaining images were obtained via uorescent reverse microscopy (Nikon).

Statistical test
Immunostainings were performed on at least three embryos of each group. Images of immunostainings were randomly selected for analysis. The fusion index was calculated as the percentage of nuclei in fused myotubes out of the total nuclei. Data are presented as mean±sem or mean from at least three independent experiments. Statistical differences between groups were tested using an unpaired twotailed Student's t-test. Values of P<0.05 was considered as signi cance.

Discussion
Different pig breeds vary in muscle mass because of differences in muscle development. In recent years, several studies revealed that pigs with less muscle showed earlier formation of primary myo bers, which arise from earlier expression of myogenic genes [20]. In the present report, we found more eMyHC-positive cells in LT LDM at 35 dpc, indicating precocious terminal differentiation of myoblasts in LT. MyoD, which can initiate the process of multiple non-muscle cell lineages into muscle cell lineages, is a crucial master switch in regulating muscle-speci c gene transcription [27,28]. Its higher expression in LT re ected that dermomyotome-derived muscle progenitors differentiated more rapidly, leading to a larger percentage of differentiating myoblasts and differentiated myocytes in LT. Consistently, in vitro experiments, the MyoDpositive cells appeared earlier in LT progenitors, resulting in more eMyHC-positive myotubers with a higher fusion index. Collectively, these analyses suggested that LT muscle progenitors showed a stronger myogenic differentiation tendency in early embryonic stage which is relevant to earlier and higher MyoD expression.
Signals mediated by the Notch pathway is involved in the regulation of myogenic differentiation in vertebrate embryos and cultured cell lines [26,29,30]. Activation of Notch signaling in C2C12 myoblasts can repress MRFs as well as other muscle-speci c genes expression, and block myotube formation [31,32,33]. In mouse embryos, the Notch ligand Delta1 (Dll1) controls both maintenance of myogenic progenitors and early differentiation of myoblasts [34]. In Xenopus embryos, MyoD regulates expression of Dll1, suggesting a feedback loop between Notch signaling and myogenic basic helix-loop-helix proteins during vertebrate myogenesis and a potential role of Notch in myogenic determination [35].
However, overexpression of Dll1 in chick embryos did not affect early steps of myogenesis, but it blocked the differentiation of postmitotic myogenic cells [29]; and Notch in zebra sh embryos controls the segmental arrangement of myogenic cells, but it does not affect their commitment or differentiation [36,37,38,39]. Taken together, Notch signaling plays varying roles during embryonic myogenesis in different vertebrate species. In this study, the higher expression levels of Notch genes in LT LDM suggesting it may function in porcine skeletal muscle development and contributes to the differences in embryonic myogenesis between pig breeds. Then, using in vitro Notch manipulation and an ex vivo limb culture system, we found that Notch signaling facilitates maintenance of myogenic progenitor cells and antagonizes myogenic differentiation by promoting Pax7 expression but preventing MyoD expression.
The precise in uence of Pax7 on myogenic progression remains a controversial debate. Pax7 -/mice have no evident defects in muscle formation [40]. However, it shows a progressive loss of satellite cells in multiple muscle groups [41]. In adult muscle, quiescent satellite cells show a high expression of Pax7, whereas Myf5 and MyoD is almost nondetectable [42]. Once activated, Pax7 protein persists at lower levels in proliferating satellite cells and decreases rapidly in cells that commit to terminal differentiation [43]. In cultures, Pax7 is not detectable in differentiated myotubes, but persists in undifferentiated and mitotically inactive myogenic cells that reduce MyoD expression [44]. Moreover, ectopic expression of Pax7 represses the MyoD-dependent conversion of mesenchymal cells to myoblasts [45]. However, plated satellite cell-derived myoblasts exhibit an increase in the number of cells containing MyoD in the presence of constitutive Pax7 [46]. Primary myoblasts where Pax7 was deleted exhibited a reduction in the levels of Myf5 and MyoD expression, and no change in myogenin expression [47]. Here, when myoblasts were cultured at a low-density in GM, the expression of Myf5 but bot MyoD was positively in uenced by Pax7. When cultured at a high-density in DM, Pax7 knockdown cells up-regulated MyoG expression and cells differentiated precociously. On the contrary, overexpression of Pax7 hindered myogenic differentiation by repressing MyoG expression. These results demonstrate that Pax7 inhibits precocious terminal differentiation, suggesting that pressed Pax7 expression resulted from weaker Notch signals contributes to intense myogenic differentiation progression in LT progenitors.
Embryonic and fetal muscle development depends on a su cient population of myogenic progenitors that are characterized by expression of the paired-box transcription factors Pax3 and Pax7 [48,49]. By dual immunostaining of Pax7 and MyoD, we con rmed that the myogenic differentiation progression in LT embryos was more rapidly than that in LR embryos, which resulted in more differentiated myocytes (eMyHC+) at 35 dpc but a serious depletion of progenitor cells (Pax7 + /MyoD -). Previous study has reported that premature myogenic differentiation and depletion of progenitor cells cause severe muscle hypotrophy [9]. We speculated that the severe loss of muscle progenitors caused by intense myogenesis at early embryonic stage should be one of the reasons for the less meat production of LT pigs, for there are not su cient progenitors differentiating into muscle bers in the later stage of embryo, resulting in a small total number of muscle bers.

Conclusion
In summary, we show here that myogenic differentiation is more rapidly in LT than that in LR at 35dpc. In addition, embryonic muscle progenitors from LT have stronger differentiation capacity in vitro. Mechanically, as shown in Fig. 8, the stronger Notch signaling in LR myogenic progenitors facilitates the maintenance of myogenic progenitor cells and antagonizes myogenic differentiation by promoting Pax7 expression but preventing MyoD expression. The results presented here may provide new insight into studying the mechanisms of difference in meat production between different pig breeds.