Smed-smg-1(RNAi) leads to lethal outgrowths during planarian homeostasis.

A, B. Panels and table show Smed-smg-1(RNAi) phenotype progression compared to controls. Black arrows show epidermal hyperplasia and green arrows show outgrowths. B. Progression of the RNAi phenotypic characteristics at various time points (days) after the last injection. C. Mitotic numbers at 9 days after the last injection (n≥6 per condition). D. Volume of nuclei in 10 µm paraffin sections. Equivalent regions for at least 6 different planarians per condition were measured. E. Single confocal section of H3P⁺ cells (arrows) observed in an outgrowth (arrow head) for a Smed-smg-1(RNAi) animal at 9 days after the last injection (n = 7/8 displaying outgrowths had mitotic cells visible; see Video S3 for a whole worm view). F. The cartoons show how the sagittal paraffin sections were performed. Panels display the region of the orange square in the cartoons. Panels are maximum confocal projections of slices performed on 10 µm paraffin sections showing a monostratified dorsal and ventral epidermis in control animals and a multistratified dorsal epidermis in the case of Smed-smg-1 (RNAi) animals (yellow arrowheads). Smed-smg-1(RNAi) animals show Smedwi-1⁺ cells in outgrowths (white arrows; n = 2/4 planarians), h2b⁺ cells infiltrating to the epidermis (green arrow) and disrupting the integrity of the basement membrane (dotted white line) and at the multilayered epidermis (white arrows; n = 3/4 planarians). G. Dorsal-most confocal sections of the planarian head showing the subepidermal nerve plexus stained by synapsin where several disruptions can be observed in Smed-smg-1(RNAi) animals (arrows). Asterisks indicate the position of the eyes. H. The panels and graph show that Smed-smg-1(RNAi) animals have bigger ventral epidermal cells (hypertrophy) than controls. Panels display the ventral epidermis (deconvolved confocal maximum projections of slices performed on 10 µm paraffin sections). Double arrows show the distance from the basement membrane until the outer membrane of the epidermal cells (stained with anti-BCAT-2). See Material and methods for details about quantification method. Error bars for all the graphs are s.d from the mean and asterisks indicate P<0.01 using two-tailed Student's test with equal sample variance. Black scale bars indicate 300 µm and white scale bars indicate 50 µm.