Developmental and cell cycle progression defects in Drosophila hybrid males

Abstract

Matings between D. melanogaster females and males of sibling species in the D. melanogaster complex yield hybrid males that die prior to pupal differentiation. We have reexamined a previous report suggesting that the developmental defects in these lethal hybrid males reflect a failure in cell proliferation that may be the consequence of problems in mitotic chromosome condensation. We also observed a failure in cell proliferation, but find in contrast that the frequencies of mitotic figures and of nuclei staining for the mitotic marker phosphohistone H3 in the brains of hybrid male larvae are extremely low. We also found that very few of these brain cells in male hybrids are in S phase, as determined by BrdU incorporation. These data suggest that cells in hybrid males are arrested in either the G(1) or G(2) phases of the cell cycle. The cells in hybrid male brains appear to be particularly sensitive to environmental stress; our results indicate that certain in vitro incubation conditions induce widespread cellular necrosis in these brains, causing an abnormal nuclear morphology noted by previous investigators. We also document that hybrid larvae develop very slowly, particularly during the second larval instar. Finally, we found that the frequency of mitotic figures in hybrid male larvae mutant for Hybrid male rescue (Hmr) is increased relative to lethal hybrid males, although not to wild-type levels, and that chromosome morphology in Hmr(-) hybrid males is also not completely normal.

Figure 1.—

Developmental progress of hybrid males. (A) Second instar hybrid males are smaller than pure species of the same age, but they can continue to grow and become much larger than wild-type second instars. The smaller and larger hybrid male second instar larvae (bottom) are 6 and 12 days post-egg deposition, respectively. (B) Third instar male larvae are smaller than those of either pure species, but considerably larger than second instar hybrids. The hybrid third instar is 12 days post-egg deposition. Bar, 1 mm for A and B. (C) Brains from hybrid second instar larvae are slightly smaller than those of the corresponding pure-species stage (2.4 vs. 2.6 mm in length on average; n > 20). (D) By the third instar, the discrepancy in brain size becomes more obvious (3.2 vs. 5.3 mm in length on average; n > 20). Hybrid male third instar larvae also lack the imaginal discs seen in the pure species (arrows). Bar, 0.5 mm for C and D.

Figure 2.—

Chromosome morphology in hybrid brains. In A–D, DNA is shown in blue and PH3 in red. (A) D. simulans mitotic figure with condensed and clearly defined chromosomes. (B) Many (>50%) mitotic figures in Df(1)Hmr⁻ hybrid male larval brains show a diffuse thread-like chromosome morphology. (C) In FM7/X_sim hybrid females, mitotic chromosomes are generally undercondensed, despite their positive PH3 signal. (D) Df(1)Hmr⁻/X_sim hybrid female with normal-appearing mitotic chromosomes. Bar, 5 μm.

Figure 3.—

Hybrid male larval brains lack cells in S phase. (A and B) Wild-type D. melanogaster brains incorporate substantial BrdU in the developing optic lobe (long arrows; antibody against BrdU is in green). Some scattered neuroblasts are also observed to undergo DNA replication (short arrows). As a counterstain, differentiated neurons are marked by anti-Elav (red). (C and D) Brains from third instar hybrid larvae have no (C) or few (short arrows in D) BrdU-incorporating cells. Because hybrid male brains have poorly developed optic lobes, the outline of the brain tissue in D is shown with white dots. Incubation of dissected brains in the presence of BrdU was done in Grace's media to avoid potential artifacts (see Figure 5; Table 5). Bar, 0.5 mm.

Figure 4.—

Cells with aberrant nuclear morphology seen in the cultured brains of hybrid males are not in mitosis. (A, B, E, and F) Control D. melanogaster third instar larval brains. (C, D, G, and H) Hybrid male third instar larval brains. The brains in A, C, E, and G were squashed immediately after dissection, while those in B, D, F, and H were examined after a 1-hr incubation in colchine. (A–D) Orcein-stained brain squashes. The mitotic index in pure-species brains increases after colchicine treatment (A and B). Approximately 25% of the cells in hybrid male larval brains develop an abnormally dense chromatin morphology after the same treatment (C and D). Bar, 5 μm for A–D. (E–G) Brain squashes stained for DNA (blue) and PH3 (red) to visualize mitotic chromosomes. The mitotic index as measured by PH3 staining increases in pure species with colchicine incubation (E and F), but few cells in hybrids stain with PH3 in the presence or absence of colchicine (G and H). Bar, 15 μm for E–H.

Figure 5.—

Cell death is increased in 0.7% NaCl cultured third instar hybrid male brains. (A–D) Trypan blue staining of dead cells. The surface of the central region of a single brain hemisphere is shown in A–H. (A) D. melanogaster brain showing no cell death after incubation in 0.7% NaCl for 1 hr. (B) Hybrid male brains incubated under the same conditions show massive cell death. (C) As a positive control, D. melanogaster brains cultured in 10 mm cycloheximide for 1 hr and allowed to recover for 30 min to induce cell death similarly show intensive staining with Trypan blue. (D) Hybrid male brains incubated in Grace's media for 1 hr instead of 0.7% NaCl. The number of dead cells is greatly diminished. Single-species brains incubated in Grace's media had virtually no Trypan blue staining (data not shown). Bar, 50 μm for A–D. (E–H) Annexin V staining to visualize apoptosis. Neither D. melanogaster (E) nor hybrid brains incubated in 0.7% NaCl (F) or Grace's media (G) display many apoptotic cells. (H) A positive control in which apoptosis was induced in D. melanogaster brains by cycloheximide. Bar, 1 mm for E–H.

Figure 6.—

Naturally forming melanotic tumors from third instar hybrid males. These tumors form primarily in gut tissue (A–C), although they also develop in salivary glands (not shown). Bar, 100 μm for A and B; 250 μm for C. (D) Lower magnification of several gut tumors in a single larvae.