May 24, 2024

Pursuing transfer, membranes had been obstructed in 5% dairy in Tris-buffered saline (TBS)-T (TBS plus 0

Pursuing transfer, membranes had been obstructed in 5% dairy in Tris-buffered saline (TBS)-T (TBS plus 0.02% Tween20) for 1?hr in room temperatures. to repression from the slow-twitch phenotype in zebrafish embryos. These data show useful co-operation between Sox6 and Nfix in regulating MyHC-I expression during prenatal muscle development. gene) and of the embryonic MyHC isoform (MyHC-emb, encoded by in skeletal muscle (Calabria et?al., 2009, Messina et?al., 2010). We have also reported that the zebrafish (by binding to the 5-upstream region in two different binding sites. The first is located ?200?bp from the Rabbit Polyclonal to MIA transcription start site (TSS) in the proximal promoter and is sufficient for Sox6-dependent repression in fetal myotubes (Hagiwara et?al., 2007, An et?al., 2011), and the second is located ?2,900?bp from the TSS in a distal muscle enhancer that is required for full promoter activity (Giger et?al., 2000, Blow et?al., 2010). As a consequence, and the transcription factor expression via transcriptional activation of Mef2C. As a consequence, Sox6 deficiency in embryonic muscle leads to a strong downregulation of in a complex in which Nfix is necessary for the proper binding of Sox6 to the promoter in fetal myotubes. Finally, we show that Nfixa and Sox6 together regulate sMyHC in zebrafish embryos, revealing an evolutionarily conserved mechanism that is required for the acquisition of normal muscle phenotype. Results Sox6 Transcriptionally Promotes MyHC-I Expression during Embryonic Myogenesis Sox6 has been intensively studied as an inhibitor of slow muscle phenotype during the fetal period. However, we observed that Sox6 is also expressed during embryonic myogenesis, which is mainly characterized by the expression of typical slow genes such as the slow MyHC isoform MyHC-I. embryos and fetuses were collected at E12.5 or E16.5, and GFP-positive myoblasts were isolated via fluorescence-activated cell sorting (FACS) as previously described (Messina et?al., 2010). Using quantitative real-time PCR and western blot, we found that levels do not significantly change, whereas allele (hence referred to as null in comparison to wild-type (WT) muscle (Figures 1D and 1E). Importantly, no differences in total MyHC content were assessed in embryonic muscle groups of gain-of-function embryo (Figures 1F and 1I), in which the Nfix2 splice variant is ectopically expressed in SCH 563705 muscle cells from E11.5, leading to a fetal-like muscle phenotype (Kelly et?al., 1997, Messina et?al., 2010). Importantly, Nfix expression in embryonic skeletal muscle is not altered in the absence of Sox6 at both the protein and mRNA levels (Figures S2ACS2D). Taken together, these results show that Sox6 is expressed at equal levels in skeletal muscle during embryonic and fetal myogenesis and that deletion of during the embryonic period unexpectedly leads to downregulation of embryonic (E12.5) and fetal (E16.5) myoblasts showing relative expression of and transcripts in the two populations. (B) Western blot on lysates from freshly isolated embryonic and fetal myoblasts. -Tubulin was used to normalize the amount of protein loaded. (C) Quantitative densitometry of the protein expression levels of Sox6 and Nfix at SCH 563705 E12.5 and E16.5. (DCI) Immunofluorescence on E12.5 muscle sections from WT (D and G), null (E and H), and Tg:Mlc1f(F and I) mice stained with anti-MyHC-I (DCF) or anti-MyHCs (MF20) antibodies (G-I). Dashed lines highlight the forelimb anlagen contour. Scale bars, 100?m. (J) Western blot on E12.5 muscle samples from WT, null, and Tg:Mlc1fmice. Vinculin was used to normalize the amount of loaded proteins. (K) Quantitative real-time PCR on E12.5 muscle tissue from WT and Promoter In order to define a possible mechanism by which SCH 563705 Sox6 regulates the transcription of MyHC-I during embryonic myogenesis, we performed chromatin immunoprecipitation (ChIP) for Sox6 in differentiated embryonic myoblasts. We found that Sox6 does not significantly bind either to the proximal or to the distal regulative regions upstream of (Figure?2A), thus suggesting that Sox6 is not able to directly regulate transcription in embryonic muscle. To confirm this hypothesis, we performed luciferase assays on WT and full 5-upstream region (MyHC-I 3500), the 408-bp proximal promoter sequence (MyHC-I 408), or the mutated forms of the?distal and proximal canonical Sox6 binding sites, MyHC-I 3500?m and MyHC-I 408?m (Figure?S3A) (Hagiwara et?al., 2007, An et?al., 2011). As expected, in the absence of Sox6, we found a significant reduction of firefly luciferase activity in SCH 563705 all the conditions with the only notable exception of the 408 WT construct (Figure?2B), suggesting that Sox6 is promoting MyHC-I expression in embryonic myocytes without direct binding to its canonical binding sites. In order to identify a possible indirect mechanism by which Sox6 enhances MyHC-I expression in embryonic muscle, we focused on the transcription factor Mef2C, a known positive regulator of the slow phenotype (Wu et?al., 2000, Potthoff.