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Fig. 7. Sox5 increases late myogenic regulators in Myod1-induced myogenic transcription assay. (A) Expression of more than 50 genes determined by semi-quantitative RT-PCR from RNA extracted of animal cap explants. Embryos were bilaterally injected with MyodF synthetic mRNA (100 pg) alone or co-injected with Sox5, Sox5-R, Sox5-A, Sox6, Sox6-R or Sox6-A (400 pg each). Number of PCR cycles was indicated on the left. (B) Embryos were injected unilaterally with 5 ng of moSox5 and then were submitted to in situ hybridization with Myog antisense probe at the stage 19/20. Myog labelling were compared to β-galactosidase control injected embryos. Dorsal view and anterior side on the right. β-galactosidase mRNA (blue) was co-injected to identify the injected side, indicated by an asterisk (*). Control, β-galactosidase mRNA alone. Probe is shown in the bottom right corner of each panel. (C) Embryos were injected unilaterally either with Myod1 synthetic mRNA (100 pg) alone or co-injected with Foxc1, Foxc1 plus Sox5, Foxc2, Foxc2 plus Sox5 (400 pg each), fixed at neurula stage and probed with Des and Actc1. (D) Expression of Nfib determined by semi-quantitative RT-PCR from RNA extracted of animal cap explants. Embryos were bilaterally injected with Myod1 synthetic mRNA (100 pg), Sox5, (400 pg) or both. Number of PCR cycles was indicated on the left. (E) In situ hybridization experiments showing Nfib expression in lateral view at stage 20, anterior side on the right. (F) transversal section showing Nfib expression in somites. (G) Embryos were injected unilaterally either with Nfib (400 pg), Myod1 synthetic mRNA (100 pg) alone or co-injected with Nfib, Sox5-A, Nfib plus Sox5-A, (400 pg each), fixed at neurula stage and probed with Des and Actc1. (H) Bar graph showing percentage of embryos with no, faint or strong expression (Des and Actc1) and with decreased,unchanged or increased expression (Myog). Chi-squared test analysis for Des expression: Myod1 versus Myod1+Foxc1 (P value = 0,0001), Myod1+Foxc1+Sox5 (P value = 0,0005), Myod1+Foxc2 (P value< 0,0001), Myod1+Foxc2+Sox5 (P value< 0,0001), Myod1+Foxc1 versus Myod1+Foxc1+Sox5 (P value = 0,0006), Myod1+Foxc2 versus Myod1+Foxc2+Sox5 (P value = 0,0021), Myod1 versus Myod1+Nfib (P value = 0,0001), Myod1+Sox5 (P value = 0,0001), Myod1+Sox5-A (P value< 0,0001), Myod1+Sox5-A versus Myod1+Sox5-A+Nfib (P value< 0,028), for Actc1 expression: Myod1 versus Myod1+Foxc1 ns, Myod1+Foxc1+Sox5 (P value = 0,0001), Myod1+Foxc2 ns, Myod1+Foxc2+Sox5 (P value< 0,0009), Myod1+Foxc1 versus Myod1+Foxc1+Sox5 (P value = 0,0424), Myod1+Foxc2 versus Myod1+Foxc2+Sox5 (P value = 0,049), Myod1 versus Myod1+Nfib (P value = 0,0007), Myod1+Sox5 (P value = 0,0001), Myod1+Sox5-A (P value< 0,0055), Myod1+Sox5-A versus Myod1+Sox5-A+Nfib (P value< 0,036). Chi-squared test analysis for Myog expression: Control versus moSox5 (P value< 0,0001). Numbers of injected embryos are indicated at the bottom of each bar. |
