The Integrator complex regulates differential snRNA processing and fate of adult stem cells in the highly regenerative planarian Schmidtea mediterranea
Fig 4
ints depletion disrupts neoblast maintenance as well as tissue homeostasis and regeneration.
(A) Survival curves of homeostatic ints RNAi animals showing median survival times of 32 and 26 days after the first RNAi treatment (days of RNAi) for ints3 and ints9 RNAi, respectively. Corresponding live images are in S4A Fig. (B) Representative live images of ints RNAi fragments 7 days post amputation (7 dpa; 20 days RNAi) displaying significantly reduced blastema size, lack of eye regeneration (white arrowheads) and defective remodeling of existing tissues to form a pharynx (black arrowheads). (C) Quantification of eye regeneration in trunk and tail fragments corresponding to B (n>32; 2 independent experiments). (D) Cytometry analysis of dissociated cells derived from ints and control RNAi animals revealing a progressive loss of the X1 populations in both amputated and intact ints RNAi animals. Shown is the average proportion of X1 cells in all gated cells of 3 biological replicates. (E) FISH and immunostaining of smedwi-1 mRNA and SMEDWI-1 protein, respectively, in trunk fragments at 7 dpa (20 days of RNAi) confirming the ints RNAi-induced loss of neoblasts and their immediate progeny. The proportion of trunk fragments with the depicted phenotype are indicated (2 independent experiments; Scale bar = 100 μm). (F+G) WISH staining- and qRT-PCR time-course (2–5 dpa) showing that the expression of soxP-1, a marker of sigma neoblasts, is strongly reduced in ints RNAi fragments. The expression levels are the averages of three biological replicates normalized to those of gapdh. (two-sided t-test, * p<0.05, ** p<0.01, *** p<0.001). Error bars represent standard deviation.