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. 2024 Jun 8;12(6):1274. doi: 10.3390/biomedicines12061274

Figure 4.

Figure 4

Impact of β-catenin(RNAi) on Sp5 expression in Hv-Basel and HySp5-3169:GFP transgenic animals. (A) Schematic view of the procedure: After one, two or three electroporations (EP1, EP2 and EP3) with scramble or β-catenin siRNAs, animals were either dissected in apical and body column regions for RNA extraction (grey triangles), or fixed for whole-mount in situ hybridization (ISH, blue triangles) or imaged live (red triangles) at indicated time-points (d pEP: day(s) post-EP). (B) Q-PCR analysis of Sp5, β-catenin and GFP transcript levels in apical (100–80%, left) and body column (80–0%, right) regions of epidermal (left) and gastrodermal (right) HySp5-3169:GFP transgenic animals taken one or two days after EP1 (days 1 and 2), after EP2 (days 3 and 4) or one day after EP3 (day 5). In each panel, the colored line corresponds to the Fold Change (FC) values between β-catenin RNAi animals (continuous grey lines) and control animals exposed to scramble siRNAs (dotted grey lines). Values are each expressed as FC relative to non-electroporated animals at time 0, just before EP1. See Figure S10. (C) Sp5 expression pattern and phenotypic changes in scramble and β-catenin(RNAi) Hv_Basel animals at indicated time-points. Red arrows point to Sp5-expressing patches along the body column, yellow arrows to bud-like structures that express Sp5 and become multiple two days pEP3 without differentiating apical structures. See Figure S11. (D) GFP (green) and mCherry (red) fluorescence in β-catenin(RNAi) HySp5-3169:GFP transgenic live animals. GFP fluorescence in apical regions is either normal (white arrows) or missing (red arrows). Note the transient bud-like structures that develop after β-catenin(RNAi) (red arrowheads) and express GFP in gastrodermal_HySp5-3169:GFP animals (green triangles). See Figure S12. (E) Immunodetection of GFP and mCherry in β-catenin(RNAi) HySp5-3169:GFP animals 6 days post-EP2 (6d pEP2). Apical regions of scramble and β-catenin(RNAi) animals are magnified on the right. White and red arrows as in (D). See Figure S13. Scale bar: 250 µm.