Fig. 3. ofs mutants exhibit altered timing of Cyclin A, Boule and Twine accumulation. (A-D) Whole-mount immunofluorescence. (A,B) ofs clones (GFP+, green) in a heterozygous background. (A) ofs clones precociously exhibit nuclear Cyclin A (CycA, arrows, red), whereas heterozygous spermatocytes are only beginning to accumulate Cyclin A, and nuclear entry occurs only in cysts entering the G₂-M transition (arrowhead). (Inset i) In early spermatocytes, the degree of chromatin condensation in ofs clones (outlined, upper right) is similar to heterozygous cells (left). (Inset ii) By contrast, older mutant cysts retain less condensed chromatin (outlined, upper right), whereas heterozygotes begin to condense chromatin as they approach meiosis (left-most and lower cells). (B) Midsection of the testis (tip is up) showing persistence of nuclear Cyclin A in clones (white arrows) past the meiotic region and its eventual degradation (yellow arrow). The position of the most advanced Cyclin A-containing heterozygous spermatocytes (located in a different focal plane) is outlined (merged panel). (C,D) In contrast to heterozygotes (C, arrow), in ofs/Df(3R)mbc-R1 mutants, Cyclin A (green) does not overlap with Boule (red) expression (D, line). Notice that ofs/Df has a slightly different (perhaps stronger) Cyclin A phenotype in which all spermatocyte staining is nuclear. (E,F) twine-lacZ reporter, X-Gal activity staining. (E) In heterozygotes (boxed area magnified on right) expression first appears faintly in nuclei of mature primary spermatocytes (arrow), becomes stronger during the meiotic divisions and is strongest in spermatids (arrowhead). Asterisk labels nonspecific, endogenous enzymatic activity. (F) In ofs mutants, expression is first detected relatively more distal in the testis tube (arrow) and resembles the mature primary spermatocyte signal.