Characterization of mt-mmpA(RNAi) phenotypes in regenerating S. mediterranea.

(A) Bright–field images of head and tail fragments, after two rounds of dsRNA injections and amputation, at 5 days of regeneration. Control: β-gal(RNAi) fragments. Following Smed-mt-mmpA(RNAi) the fragments show a significantly reduced blastema. Relative blastema size between Smed-mt-mmpA(RNAi) and β-gal(RNAi) fragments was calculated as percentage of the whole fragment, considered 100%. Consistent with the delayed regeneration phenotype detected at the morphological level, immunostaining with anti-synapsin antibody of Smed-mt-mmpA(RNAi) tail fragments (green signal) reveals a regenerating brain smaller than that of controls. The dashed yellow lines indicate the border between the regenerating area and the stump. Anterior is up. Scale bars: 1 mm. (B) Expression level of different markers (Smedwi-1, Smed-prog1, Smed-AGAT1, Smed-syt) after Smed-mt-mmpA(RNAi), analyzed by Real Time RT-PCR in regenerating fragments. No significant variation in the expression level of different markers was observed. In the Real Time RT-PCR experiments the expression level is indicated in relative units, assuming as unitary the value of the controls. Each value is the mean ± s.d. of three independent samples, carried out in duplicate. c: β-gal(RNAi) controls; mt-mmpA(RNAi): Smed-mt-mmpA(RNAi).