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Fig. 1. Sox5 enhances muscle gene expression and acts as a transrepressor in Myod1-induced myogenic transcription assay. (A) In situ hybridization experiments of Sox5 and Myod1 at stage 18 show that Sox5 is expressed at dorsomedial and ventrolateral region of the somites like Myod1. Lateral views. The anterior side of the embryos is on the left. (B) Whole-mount in situ hybridization of embryos unilaterally injected with 100 pg of synthetic mRNAs coding for Myod1 alone or co-injected with Sox5 (400 pg), fixed at stages 17/18 and probed with Des, Actc1, MyhE3, Ckm and endogenous Myod1. (C) Embryos were injected unilaterally either with Myod1 synthetic mRNA alone (100 pg), or co-injected with Sox5, Sox5-R or Sox5-A (400 pg each), fixed at neurula stage and submitted to in situ hybridization for Des and Actc1. (D) Same experiments as in (C) on animal cap explants. (E) Embryos were injected unilaterally either with Myod1-R synthetic mRNA (400 pg), with Myod1 synthetic mRNA (100 pg) alone or co-injected with Sox5-R or Myod1-R (400 pg each), fixed at neurula stage and probed with Des. The arrowhead indicates ectopic expression. Probe is shown in the bottom right corner of each panel. (F) Embryos were injected unilaterally either with Myod1 synthetic mRNA (100 pg), Sox5, Sox5-R or Sox5-A (400 pg each), fixed at neurula stage and submitted to in situ hybridization for Des and Actc1. (G) Expression of Des, Actc1, Ckm, and MyhE3 determined by semi-quantitative RT-PCR from RNA extracted of animal cap explants. Embryos were bilaterally injected with Myod1 synthetic mRNA (100 pg) or with Sox5. Number of PCR cycles was indicated on the left. (H) Bar graph showing percentage of embryos with no, faint or strong ectopic expression. Chi-squared test analysis for ectopic Des expression: Myod1 versus Sox5 (P value< 0,0001), Sox5-R (P value< 0,0001), Sox5-A (P value< 0,0001), for ectopic Actc1 expression: : Myod1 versus Sox5 (P value< 0,0001), Sox5-R (P value< 0,0001), Sox5-A (P value< 0,0001), for ectopic MyhE3 expression: Myod1 versus Sox5 (P value< 0,0001), for ectopic Ckm expression: Myod1 versus Sox5 (P value< 0,0001), for ectopic Myod1 expression: Myod1 versus Sox5 (P value< 0,0002), for Des expression in animal caps: Myod1 versus Sox5 (P value= 0,0228), Sox5-R (P value< 0,0001), Sox5-A (P value< 0,0001), for ectopic Actc1 expression in animal cap: Myod1 versus Sox5 (P value< 0,0001), Sox5-R (P value< 0,0001), Sox5-A (P value= 0,0140), for ectopic Des expression of Myod1-R construct: Myod1 versus Sox5EF (P value= 0,0110), Myod1-R (P value= 0,0011). Numbers of injected embryos are indicated at the bottom of each bar. |
