Fig. 4

A 14-nt deletion downstream of dfr first in-frame stop codon leads to developmental defects. a Upper panel, dfr gene structure; lower panel, nucleotide sequences of control w¹¹¹⁸ and dfr mutants. Three different dfr mutant strains were isolated, with 1, 13, and 14 nucleotide deletions downstream of the annotated stop codon, and named accordingly. The canonical stop codon is highlighted in red. The protospacer adjacent motif (PAM) sequence targeted for cleavage by the CRISPR/Cas9 system is highlighted in gray. b Percentage of larvae undergoing pupariation over time after larval hatching. In parallel, dfr14/Df was monitored (n = 5–10; N = 150–300; p**** < 0.0001; see g–h). c Upper panel: representative images of male and female adult sizes of control w¹¹¹⁸, dfr¹, dfr¹³, and dfr¹⁴. Lower panel: quantification of wet fly weight of males and females raised in non-crowded conditions (30 larvae/vial). Data represent mean + SE (n = 6). d Immunostainings of control and dfr¹⁴ BRGCs with anti-Dfr-L (upper panels) and anti-Dfr-S/L (lower panels). Dfr-L was absent in dfr¹⁴ prothoracic glands. Scale bars 100 μM. e Immunoblots of BRGC extracts (left), and whole larval extracts (right), without fat body and intestine from control and dfr¹⁴ larvae, incubated with anti-Dfr-S/L. Actin was used as a loading control. The band corresponding to Dfr-L/L* was barely detectable, while Dfr-S* increased in dfr¹⁴ mutants. f Representative anti-Dfr-S/L immunoblot prepared from BRGCs of denoted genotypes. Anti-Actin was used as loading control. +, wild type allele; Df, Df(3 L)Exel6109. g Quantification of normalized band intensities of Dfr-S* and Dfr-L*. Bar plots represents mean + SE from four independent replicates. Distinct lettering indicates significantly different intensities (p < 0.05). h Left panel: representative images of male and female adult flies of control w¹¹¹⁸ and dfr¹⁴/Df. Right panel: quantification of wet fly weight of males and females (w¹¹¹⁸, n = 10, N = 100; dfr¹⁴/Df, n = 8, N = 80; p**** < 0.0001)