Regeneration in starved planarians depends on TRiC/CCT subunits modulating the unfolded protein response (2021)
Figure 4

Figure 4. cct3A RNAi down-regulates the UPR components xbp1 and atf6 whose down-regulation leads to a similar phenotype as cct3A.

(A) Volcano plot displaying DEGs in starved cct3A(RNAi) planarians compared to controls at 72hR (q-value < 0.05). Y-axis indicates the negative log10 of the false discovery rate (FDR) (q- value). X-axis indicates the beta values (b), a biased estimator of the fold change. For better visualization, 13 dots with -log10 (q-value) > 20 are not displayed. This includes cct3A with q = 0. Notice that when down-regulating cct3A in starved planarians, xbp1, atf6, bip-1 and a repertoire of ER chaperones are down-regulated, whereas dnajb9, a repressor of the UPR, and other ccts are up-regulated.

(B) Relative expression of xbp1 and atf6 related to gfp control at 72 hours and 13 days of regeneration during either starving or feeding conditions in cct3A(RNAi) animals. The graph shows that cct3A RNAi down-regulates xbp1 and atf6 specifically during starvation at 72hR. n = 3 replicates (5 planarians each) per time point.

(C) The graph shows that both cct3A RNAi down-regulates xbp1, atf6 and bip-1 specifically at 72hR during starvation in X1 stem cells. 50 planarians per replicate (3 replicates) were used to obtain the X1 population (stem cells in S and G2/M).

(D) Live images show that xbp1/atf6(RNAi) planarians in starved conditions form a minimal blastema when compared to controls while at the feeding condition most of them can regenerate as controls. The remaining planarians in the starved condition are dead by the time point of regeneration shown. In the feeding condition, the remaining planarians at 8-10dR are either dead (13/145) or have a tiny blastema and died or regenerated later (40/145) while at 21-27dR are dead.

(E) Mitotic numbers during different time points of regeneration in starved conditions for xbp1/atf6 RNAi and controls. n ≥ 7 planarians per time point.

(F) Mitotic numbers in feeding conditions for xbp1/atf6 RNAi and controls. n ≥ 9 planarians per time point.

(G) Maximum projections of representative regenerating trunks after FISH for smedwi-1 in starved conditions. The dashed line delimits the body of the planarian.

(H) Relative expression of smedwi-1 related to controls at 13 and 20 days of regeneration of xpb1/atf6 RNAi in starved conditions. n = 3 replicates (5 planarians each) per time point.

(I) TUNEL maximum projections of representative blastemas from regenerating trunks in starved conditions. The dashed line delimits the body of the planarian. The graph shows the number of TUNEL+ cells per mm2 at the same time points in anterior (A) and posterior (P) blastemas. n ≥ 6 planarians per time point.

(J) Quantification of the percentage of stem cells in different mitotic phases and the percentage of defective mitotic figures in 72hR anterior blastemas of xbp1/atf6 RNAi and controls in starved conditions after double immunostaining with anti-tyrosine-tubulin and anti-H3P (****P < 0.0001, ***P < 0.001, *P < 0.05, n.s. indicates not significant using two-sided Chi-square test); n ≥ 5 planarians. Representative images are shown. Arrows indicate abnormal organization or number of spindle poles. j8 displays asymmetrical distribution of chromosome content.

Data information: In (b, c e, f, h and i), error bars are s.d. from the mean and asterisks indicate P < 0.0001 (four asterisks), P < 0.001 (three asterisks), P < 0.01 (two asterisks), P < 0.05 (one asterisk) and n.s. indicates not significant using two-tailed Student's test with equal variance; at the bottom are the number of planarians with the phenotype shown. dR, days of regeneration; hR, hours of regeneration. Scale bars, 500 µm (D and G), 100 µm (I) and 10 µm (J).

Source data are available online for this figure.