Immunofluorescence Analysis Tissue slides (7 m) and cells were fixed with 4% of paraformaldehyde (PFA, Polysciences Polysciences, Valley Road Warrington, USA) in Phosphate Buffer Saline (PBS), permeabilized with 0.2% Triton X-100 in PBS containing 1% (w/v) of bovine serum albumin, and blocked with 1:10 donkey serum (all from Sigma-Aldrich, St. myogenic regulator and a possible target for improving skeletal muscle regeneration. The non-neural roles of Zeb2 are poorly understood. mutations in humans cause Mowat-Wilson Syndrome (OMIM #235730), involving severe intellectual disability, Hirschsprung disease, epilepsy, and other developmental defects, with some patients SB 706504 also presenting with musculoskeletal anomalies [8,12,13]. genetic inactivation in mouse embryonic stem cells (mESCs) causes defects in their pluripotency exit, making them stall as epiblast-like stem cells, and therefore compromising their neural and general differentiation . It is well known that skeletal muscle development and differentiation are regulated by myogenic basic helix-loop-helix (bHLH) proteins. Similarly, several zinc-finger proteins have been described as regulators of muscle development and specific muscle gene expression. In this context, zinc-finger repressors of transcription could compete with myogenic bHLH proteins in regulating muscle differentiation processes. In fact, it has been reported that the overexpression of LIM/double zinc-finger protein promotes myogenic differentiation in the cell line C2C12 . Zeb1 and Zeb2 have been proposed as candidate regulators and/or such competitors, based on biochemical analysis [2,16] and on phenotypes within embryonic somites in the respective knockout and compound mutant embryos and adult mice [4,17]. More recently, the presence of PW1 zinc-finger protein was reported in interstitial myogenic progenitors, and it is also required for migration ability of murine and human mesoangioblasts [18,19]. Here, we evaluated the myogenic potential of and mESCs by single-cell RNA-sequencing, and tested muscle engraftment capability of the respective myogenic progenitors. transgene (cDNA)-based expression was shown to impact positively on the myogenic differentiation potential of pluripotent stem cells and myogenic progenitors, identifying Zeb2 as a critical modulator of skeletal muscle differentiation. Strikingly, we propose, for the first time, the function of Zeb2 in skeletal muscle differentiation. Our hypothesis is that Zeb2 has a crucial role in triggering myogenic differentiation. We also evaluated the complexity of the myogenic transcriptional regulation, including the TGF/BMP system, involved in myogenic commitment and differentiation. 2. Results 2.1. The Upregulation of Zeb2 Positively ATP1B3 Affects Myogenic Markers in mESCs Subjected to Skeletal Muscle Differentiation The mESC lines used in all experiments are listed in material and methods sections [14,20]. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis confirmed the absence of Zeb2 mRNAs in mESCs, and increased mRNA in compared to controls (CTR) (Figure S1A). The expression of pluripotent markers (cMyc, Oct4, Sox2, Klf4, and SB 706504 Nanog) was confirmed in all mESC lines (Figure S1A) and lower levels of cMyc expression did not compromise the pluripotency of mESCs, as previously reported . and control (CTR) mESCs were subjected to myogenic induction (Figure 1A), using the transient transfection of MyoD expression constructs [22,23]. The qRT-PCR analysis confirmed MyoD over-expression from transfected mESC lines (Figure 1B). At day 22, an up-regulation of myogenic markers (compared to and control samples, either in the absence or in the presence of MyoD transfection (CTRMyoD, was upregulated only in compared to and control samples (Figure 1D). In addition, the MyomiR (miR-1, miR-133b, miR-208, miR-206) expression profiles were altered SB 706504 in and mESCs subjected to skeletal muscle differentiation. Interestingly, miR-1 and miR-133b, considered as markers of mesodermal transition and maturation , were up-regulated in and CTR both in the absence (Figure 1E) and in the presence of MyoD (Figure 1F). Furthermore, miR-206 which promotes myoblast differentiation , was similarly expressed in all samples in the absence of added MyoD (Figure 1E), whereas it was up-regulated in and CTR(Figure 1F). Then, miR-208 was down-regulated in and CTR in the absence of MyoD (Figure 1E). Similarly, miR-208 was highly expressed in and SB 706504 CTRMyoD (Figure 1F). Consistent with the previous findings, the expression of myomiRs is strongly enhanced in mESCs, compared to CTR cells. Open in a separate window Figure 1 Muscle gene expression and MyomiR profile in and mESCs subjected to myogenic differentiation. (A) Schematic representation of myogenic induction for mESCs. (B) The qRT-PCR analysis confirms the successful transient transfection in CTR, and mESCs. At 24, 48, and 72 h from transfection, data are represented as C= 3, *** < 0.0001. (C,D) qRT-PCR analysis for.