Panel A shows a schematic diagram of the ana1 gene locus, highlighting the deletions in ana1a and ana1b alleles created by CRISPR/Cas9 mutagenesis. Panel B displays a western blot of protein extracts from testes of different genotypes, with bands indicating the presence of Ana1 and alpha-tubulin as a loading control. Panel C presents fluorescence microscopy images of centrioles in primary spermatocytes, stained for EGFP-Ana1, Asl, Cep135-mRuby3, and DNA, with scale bars indicating magnification. Panel D features a dot plot quantifying centriole lengths in mature primary spermatocytes for different genotypes, with each point representing the average length measured from a 16-cell cyst. The images and graph together illustrate the essential role of Ana1 in centriole duplication and its impact on centriole over-elongation in fly spermatocytes.
Ana1 is essential for centriole duplication and promotes centriole overelongation in fly spermatocytes. (A) Schematic representation of the two independent ana1 null alleles, denoted as ana1Δa and ana1Δb, created by CRISPR/Cas9 mutagenesis. Both mutants had deletion of the entire ana1 coding region. These alleles were then crossed to generate the ana1 null mutant background (referred to as ana1−/−) used throughout this study. (B) Western blot of extracts from 50 to 60 testes of indicated genotypes. Detection of Ana1 was performed using specific antibodies, with α-tubulin as a loading control for protein levels in lysates. (C) Centrioles in primary spermatocytes stained to reveal EGFP-Ana1 (green), Asl (red), Cep135-mRuby3 (red), and DNA (blue). Scale bars, 10 µm (overview image) or 2 µm (insets). (D) Graphs show the mean ± SD quantification for centriole lengths in mature primary spermatocytes, using Asl as a centriole marker for each genotype. Each point on the graph represents the average length measured from a 16-cell cyst with 10–15 centrioles scored; five independent testes were measured for each genotype with 2–3 cell cysts scored in each testis. ****P < 0.0001. Source data are available for this figure: SourceData F1.