Nesd is required for viability and spermatocyte cytokinesis. (A) Analysis of the lethality of nesd¹ mutants. Homozygous nesd¹ females were crossed to either Df(2L)ED1305/CyO (Df/CyO) or Df(2L)ED1305/CyO;nesd::GFP (Df/CyO;nesd::GFP) males, and the resulting progeny is indicated in the left and right pie charts, respectively. The male and female progenies are indicated in light/dark blue and light/dark pink, respectively. At least 400 animals per each cross were counted. (B) Western blot analysis of Nesd protein levels in testes dissected from wild-type, nesd¹/Df(2L)ED1305 (nesd¹/Df), and Df(2L)ED1305/+ (Df/+) adult males. The arrow indicates the band correspondent to Nesd protein, whereas the asterisk marks a nonspecific band used as a loading control. (C) Phase-contrast micrographs of onion-stage spermatids dissected from Df(2L)ED1305/+ (control), nesd¹/Df(2L)ED1305, and nesd¹/Df(2L)ED1305;nesd::GFP (nesd¹/Df;nesd::GFP) males. Multiple white nuclei associated with a large dark Nebenkern were observed in nesd¹/Df(2L)ED1305 hemizygous mutants (arrowheads). Bars, 50 µm. (D) Quantification of the number of multinucleate spermatids observed in testes dissected from flies of the genotypes described in C and also from nesd homo- (nesd¹) and hemizygous (nesd¹/Df(2L)ED1305) mutants carrying either a GFP::Pav-KLP transgene (GFP::Pav) or the pav^B200 allele. More than 700 onion-stage spermatids per genotype were counted in three separate experiments; the error bars indicate standard deviations.