Fig. 5: Alteration of PG filopodia components leads to ecdysone secretion defects.
a Screening for α- and β-tubulin subunits involved in timing development by expressing RNAi under the control of phm-Gal4. b Screening for general actin-associated proteins involved in developmental timing. c Down-regulation of filopodia-associated proteins diaphanous, enabled, or α-actinin leads to a significant developmental delay or arrest. d-d” ex vivo PG confocal images of control, dia PG-downregulation, and Actn silencing showing its effect on filopodia (d, arrowheads). Quantifying filopodia length and density using PLC-GFP clonal analysis confirms that dia and Actn down-regulation significantly impair both filopodia length and density (d”). nw1118 = 9 clones, nRNAi-dia33424 = 9, nRNAi-Actn7762 = 9 (d’). Compared to a control PG, dia or Actn PG silencing does not noticeably affect overall PG shape (d”). e 20E feeding rescues developmental delay caused by silencing dia or Actn expression in the PG. Data in (c) for RNAi-Actn7762 and RNAi-dia33424 are reused in (e) as controls without ecdysone feeding. The same phm>Dcr-2 x w1118 control has been used in (a–c, e). f-f” Actn down-regulation affects ecdysone secretion, not production. For the total ecdysone measurement, the number of independent samples is the following: nphm>Dcr-2xw1118 = 3, nRNAi-Actnxw1118 = 3, nphm>Dcr-2xRNAi-Actn = 2. For the % of circulating ecdysone, the number of independent samples is: nphm>Dcr-2xw1118 = 9, nRNAi-Actnxw1118 = 9, nphm>Dcr-2xRNAi-Actn = 6. Dib expression is unaffected upon Actn silencing, as immunostaining shows (f’). Similarly, nvd and phm RNA levels remain unchanged in RNAi-Actn conditions, as assessed by qPCR (f”). Number of independent samples: nphm>Dcr-2xw1118 = 3, nRNAi-Actnxw1118 = 3, nphm>Dcr-2xRNAi-Actn = 3. White scale bars: 20 μm; yellow scale bars: 5 μm. Data are presented as follows: means of the differences ± SEM (a–c, e), median ± IQR (d’), mean ± SEM (f, f”). Except for (e), data were subjected to the Kruskal-Wallis test, followed by Dunn’s multiple comparison tests. In (e), data were subjected to the two-tailed Mann–Whitney test. (ns not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Source data are provided as a Source Data file.
