Myogenic Differentiation of Mouse Embryonic Stem Cells That Lack a Functional Pax7 Gene

The transcription factor Pax7 plays a key role during embryonic myogenesis and sustains the proper function of satellite cells, which serve as adult skeletal muscle stem cells. Overexpression of Pax7 has been shown to promote the myogenic differentiation of pluripotent stem cells. However, the effects of the absence of functional Pax7 in differentiating embryonic stem cells (ESCs) have not yet been directly tested. Herein, we studied mouse stem cells that lacked a functional Pax7 gene and characterized the differentiation of these stem cells under conditions that promoted the derivation of myoblasts in vitro. We analyzed the expression of myogenic factors, such as myogenic regulatory factors and muscle-specific microRNAs, in wild-type and mutant cells. Finally, we compared the transcriptome of both types of cells and did not find substantial differences in the expression of genes related to the regulation of myogenesis. As a result, we showed that the absence of functional Pax7 does not prevent the in vitro myogenic differentiation of ESCs

Biological sources and sinks of nitrous oxide and strategies to mitigate emissions

Nitrous oxide (N 2 O) is a powerful atmospheric greenhouse gas and cause of ozone layer depletion. Global emissions continue to rise. More than two-thirds of these emissions arise from bacterial and fungal denitrification and nitrification processes in soils, largely as a result of the application of nitrogenous fertilizers. This article summarizes the outcomes of an interdisciplinary meeting, ‘Nitrous oxide (N 2 O) the forgotten greenhouse gas’, held at the Kavli Royal Society International Centre, from 23 to 24 May 2011. It provides an introduction and background to the nature of the problem, and summarizes the conclusions reached regarding the biological sources and sinks of N 2 O in oceans, soils and wastewaters, and discusses the genetic regulation and molecular details of the enzymes responsible. Techniques for providing global and local N 2 O budgets are discussed. The findings of the meeting are drawn together in a review of strategies for mitigating N 2 O emissions, under three headings, namely: (i) managing soil chemistry and microbiology, (ii) engineering crop plants to fix nitrogen, and (iii) sustainable agricultural intensification. </jats:p

Adhesion proteins--an impact on skeletal myoblast differentiation.

Formation of mammalian skeletal muscle myofibers, that takes place during embryogenesis, muscle growth or regeneration, requires precise regulation of myoblast adhesion and fusion. There are few evidences showing that adhesion proteins play important role in both processes. To follow the function of these molecules in myoblast differentiation we analysed integrin alpha3, integrin beta1, ADAM12, CD9, CD81, M-cadherin, and VCAM-1 during muscle regeneration. We showed that increase in the expression of these proteins accompanies myoblast fusion and myotube formation in vivo. We also showed that during myoblast fusion in vitro integrin alpha3 associates with integrin beta1 and ADAM12, and also CD9 and CD81, but not with M-cadherin or VCAM-1. Moreover, we documented that experimental modification in the expression of integrin alpha3 lead to the modification of myoblast fusion in vitro. Underexpression of integrin alpha3 decreased myoblasts' ability to fuse. This phenomenon was not related to the modifications in the expression of other adhesion proteins, i.e. integrin beta1, CD9, CD81, ADAM12, M-cadherin, or VCAM-1. Apparently, aberrant expression only of one partner of multiprotein adhesion complexes necessary for myoblast fusion, in this case integrin alpha3, prevents its proper function. Summarizing, we demonstrated the importance of analysed adhesion proteins in myoblast fusion both in vivo and in vitro

Expression of mRNAs encoding integrin alpha3 and other adhesion proteins at 24 and 48 hours after transfection of MPCs-derived myoblasts with siRNA-alpha3. sqRT-PCR analysis.

<p>NT – control, not transfected myoblasts, siRNA-alpha3 – myoblasts transfected with siRNA downregulating the expression of integrin alpha3. Optical densities of representative bands are shown as a percentage of GAPDH band density taken as a 100%.</p

Changes in expression and localization of adhesion proteins during <i>Soleus</i> muscle regeneration.

<p>A - level of mRNAs encoding adhesion proteins analyzed by sqRT-PCR. Optical densities of representative bands are shown as a percentage of GAPDH band density taken as a 100%. B – localization of integrin alpha3 (red) in intact and in regenerating muscle at day 3, 5, 7, 14. Nuclei – blue. White arrows show degenerating myofibers. Scale bars 50 µm.C –colocalization of Pax7, MyoD, myogenin (MG) (red) with integrin alpha3 (green) in intact muscle and at day 1, 3 and 5 of regeneration (intact, d1, d3, d5 respectively). White arrows - myoblasts, pink – muscle fiber nuclei, yellow - MyoD, myogenin and integrin alpha3 negative cells. D – control staing with secondary antibodies only. Nuclei – blue. Scale bars 50 µm. E - localization of integrin beta1, ADAM12, CD9, or M-cadherin (green) at day 3 of regeneration. Nuclei – blue. Scale bars 50 µm. F – immunoblotting analysis of M-cadherin (M-cad), integrin beta1, ADAM12 (AD12), CD9 and integrin alpha3 level during muscle regeneration (days 1, 3, 7, 14).</p

Downregulation of alpha3 integrin expression reduces fusion of MPCs-derived myoblasts.

<p>A - Pappenheim's staining of fusing myoblasts. B – total number of nuclei calculated at day 12 of myoblast culture. C –index of fusion analyzed at day 12 of culture in control and experimental myoblasts shown as a percentage of myotube nuclei per number of all nuclei. D - proportion of 2–4, 5–7, 8–10 and >11 nuclear myotubes per number of all myotubes, respectively. NT – control, not transfected myoblasts, TR – control myoblasts cultured in medium supplemented with transfection reagent, siRNA-cont - control myoblasts transfected with scramble siRNA, siRNA-alpha3 – myoblasts transfected with siRNA downregulating the expression of integrin alpha3. Error bars indicate SEM, results were analyzed by Kruskal-Wallis test. Kruskal-Wallis One Way Analysis of Variance showed differences between experimental groups which were considered statistically significant when p<0.05 (marked with asterisks). * p≤0.05.</p

Downregulation of integrin alpha3 reduces the C2C12 ability to fuse with control myoblasts.

<p>A – control - myoblasts transfected with scramble siRNA-AlexaRed (red) cultured with myoblasts stained with QTracker (green), experiment+siRNA-alpha3– myoblasts cotransfected with scramble siRNA-AlexaRed (red) and siRNA-alpha3 cultured with control myoblasts stained with QTracker (green). Hybrid myotubes are yellow. Scale bars 20 µm. B –number of hybrid myotubes formed by control myoblasts transfected with scramble siRNA-AlexaRed and control myoblasts stained with QTracker (control) compared with the number of hybrid myotubes formed by myoblasts co-transfected with scramble siRNA-AlexaRed and siRNA-alpha3 and control myoblasts stained with QTracker (siRNA-alpha3) (day 11 of culture). Error bars indicate SEM, results were analyzed by Student's test and differences were considered statistically significant when p<0.05 (marked with asterisks). ** p≤0.01.</p