Fig. 3

Apontic limits the Zfh-1+ population. a Total number of Zfh-1+ cells in adult testes from flies of the specified genotypes, represented by a box and whisker plot: diamond gives the mean, horizontal line indicates the median (see Methods). For a single testis, cell quantification was capped at 100, so graphs likely represent an underestimate. b-c Stacks of optical sections, projected into 2D, of testes stained with the following antibodies: Zfh-1 expression (magenta) marks CySCs and immediate daughters, Eyes absent (Eya, white) labels mature cyst cells, and Fas3 indicates the hub (white, blue outline). Insets display Zfh-1 or Eya/Fas3 staining alone. b In control testes (apt ¹⁶⁷/+), wild-type numbers of Zfh-1+ cells are found proximal to the hub. Arrows indicate Zfh-1+/Eya+ distal somatic cells. c In testes from an apt ¹⁶⁷ /apt ^KG05830 mutant male, the Zfh-1+/Eya- population is increased around and distal to the hub, unlike in wild type (arrowheads). d Total percent of Zfh-1+ cells that did not co-express the differentiation marker Eya in testes from the experiment in (a). e-f Total numbers of Zfh-1+ cells in adult testes from flies of the specified genotypes. Males from all genotypes tested in (f) (except c587; aptRNAi; tsGal80 –RNAi-OFF) were incubated for 4 days at 30 °C prior to dissection. g-h Stacks of optical sections, projected into 2D, of testes from males incubated at 30 °C for 4 days stained with antibodies directed against Zfh-1 (magenta), Eya (white), and NCad (white). DAPI (blue) labels nuclei. Insets show Zfh-1 or Eya/NCad channels. g Control males display wild-type numbers and arrangements of the Zfh-1+ population. h A testis from a c587; aptRNAi; tsGal80 male kept at the tsGal80 restrictive temperature, reducing apt in CySCs and early cyst cells, shows an expanded Zfh-1+ population (RNAi-ON). i Total percent of Zfh-1+ cells that did not co-express the differentiation marker Eya in testes of the indicated genotypes from experiment in (f). For (a, e, f), two-tailed t-tests measured significance of the difference compared to apt ¹⁶⁷/+ (a) or c587; mGFP (e, f) unless otherwise indicated by a bar. For (d, i) two-tailed Fisher’s exact tests measured significance of the difference compared to apt ¹⁶⁷/+ (d) or mGFP control (i), unless otherwise indicated by bar. For all statistical tests: **p<0.01, ***p<0.005, ****p<0.0001; n.s., not significant. “n” indicates number of testes scored. For all images scale bars = 20 μm