Zic4 is required for tentacle development and tentacle maintenance

 

Zic4 is required for tentacle development and tentacle maintenance

To explore Zic4 function, we knocked down Zic4 by RNAi. We measured the efficiency of Zic4 RNAi at different time points during the procedure and detected a significant down-regulation of Zic4 starting 1 day after the first electroporation (EP1) and lasting until at least 11 days after the procedure has been initiated (Fig. 3A). Thereafter, we consistently performed experiments within this time window. Three days after EP3, intact Zic4(RNAi) animals exhibit tentacles with half the length of those in control animals, while the overall tentacle number is not affected (100%; n = 100) (Fig. 3B and fig. S5A). In contrast, apical-regenerating Zic4(RNAi) animals regenerate not only shorter but also 25% fewer tentacles (100%; n = 99) (Fig. 3C and fig. S5B). Overactivation of Wnt signaling through ALP treatment leads normally to ectopic tentacle formation (35). We found that when ALP treatment is combined with Zic4 down-regulation through RNAi, the development of ectopic tentacles along the body column is strongly impaired, pointing out to a strong genetic interaction between Wnt/β-catenin signaling and Zic4 (Fig. 3D and fig. S5C). We also noticed that the expression of two components of the head organizer, Wnt3 and HyBra1 (34), is not affected in intact or regenerating Zic4(RNAi) animals, suggesting that Zic4 is required neither for the maintenance nor for the formation of a functional head organizer (Fig. 3E and fig. S6). When we transplanted apical tissue from control and Zic4(RNAi) animals into actin:GFP transgenic animals, we observed a similar induction of a secondary body axis, a hallmark of a functional head organizer (Fig. 3F). We concluded that Zic4 operates downstream of the head organizer and is required for tentacle maintenance and tentacle formation.

Fig. 3. HyZic4 is required for tentacle maintenance and tentacle development but not for head organizer activity.

(A) Zic4 knockdown efficiency detected by qPCR. Vertical black arrows indicate time points of RNA extraction. Each data point represents one biological replicate. (B to E) Impact of Zic4 knockdown in intact (B, E), apical-regenerating (C, E), and ALP-treated (D) Hv_Basel animals electroporated three times (3x) with siRNAs. Zic4(RNAi) animals exhibit shorter (B to D) and fewer tentacles (C and D). Each data point represents one animal (see more animals in fig. S5). White arrows: ectopic tentacles. **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. (E) Wnt3 and Bra1 expression in intact (left) and head-regenerating (right) Zic4(RNAi) animals fixed on day 7 (intact) and day 9 (apical regenerating) (see fig. S6). (F) Homeostatic head organizer activity in Zic4(RNAi) animals . Hypostomal tissue and a tentacle of intact nontransgenic Hv_AEP2 animals electroporated three times (3x) with scramble or Zic4 siRNAs were transplanted laterally onto the body column of a GFP+ hoTG host (white arrows). All hosts have grown ectopic axes (red arrows) 4 days post-grafting (dpg). Scale bars, 200 μm (B to E) and 250 μm (F).