Smed-nf-Y gene complex is required for the proper neoblast differentiation and localization.A - WISH shows that the three Smed-nf-Y genes are expressed ubiquitously and in the cephalic ganglia, and one day after irradiation their expressions decrease. B - Double FISH of Smed-nf-YA, Smed-nf-YB-2, and Smed-nf-YC together with the neoblast marker Smed-h2b shows colocalization with the NF-Y subunits (arrowheads), demonstrating the expression of this complex in neoblasts as well as in differentiated cells (asterisk). DAPI labels the cell nuclei. See Additional file 12A to check each channel of fluorescence separately. C - Smed-nf-Y(RNAi) animals regenerate thinner blastemas with non well formed eyes and shape defects, and fail to differentiate a proper brain, with reduced cephalic ganglia as revealed with Smed-gpas. FISH with the neoblast marker Smed-h2b shows an accumulation of neoblasts in the region in front of the eyes while the early progeny marker Smed-nb.21.11e reveals a decrease of early postmitotic cells in Smed-nf-Y(RNAi) animals. D - Immunohistochemistry with the mitotic marker α-H3P shows a reduction in the number of mitosis. E - Quantification with category markers indicate a significant increase of Smed-h2b
+ cells in Smed-nf-YB-2(RNAi) and Smed-nf-YC(RNAi) animals and a significant decrease of nb.21.11e
+ cells in all of the RNAi animals, whereas Smed-agat-1
+ cells do not show significant changes (p < 0.001, t-test). Counts are referred to the whole body. ph: pharynx. All the experiments are done on bipolar regenerating trunks, at 11 days of regeneration after one round of injection.