To identify which specific cellular processes are regulated by dAkt in the ovary, we analyzed the loss-of-function phenotype using the embryonic lethal allele dAkt1q. This allele encodes an inactive form of the dAkt kinase carrying the amino acid replacement F327I in a core residue of the kinase catalytic domain (Staveley et al., 1998). dAkt1q mutant is homozygous lethal. To examine the mutational effects in the follicle cells, an adult tissue, genetic mosaic analysis was used. Homozygous dAkt1q clones, genetically marked by the absence of GFP, were obtained through somatic recombination using the FLP/FRT system (Golic, 1991; Xu and Rubin, 1993) and were induced by driving FLP under the control of a heat shock promoter (Xu and Harrison, 1994). To induce mutant clones during oogenesis, heat shocks were applied to adult females that were dissected 48–72 hours later. It should be noted that the follicular mutant clones are most likely generated early because follicular mitotic cycle ends at stage 6. To analyze follicle cell morphology, we performed immunostaining of mosaic ovaries for alpha-spectrin, which localizes to the apicolateral follicle cell contacts (Lee et al., 1997) and participates in the organization and formation of the membrane cytoskeleton (Hudson and Cooley, 2002). Nuclei are marked by propidium iodide staining. Figure 2 shows the confocal analysis of stage 8 (Fig. 2A–H) and stage 9 (Fig. 2I–P) egg chambers each containing two mutant clones (Fig. 2B,J, boxed areas). The follicle cells homozygous for the dAkt1q mutation, marked by the absence of GFP, are greatly reduced in size compared with both the parental heterozygous cells and the sister wild-type clones (Fig. 2C,F,K,N in which asterisks mark sister wild-type cells that can be distinguished from heterozygous cells by higher levels of GFP). Follicle cell nuclei in mosaic clones are also reduced in size compared with wild-type and heterozygous cells, as assessed by propidium iodide staining (Fig. 2D,G,L,O) and 4′,6-diamidine-2-phenylidole-dihydrochloride (DAPI) staining (not shown). In addition, the propidium iodide and DAPI staining show no chromatin condensation in dAkt1q clones, indicating that loss of dAkt function do not cause apoptosis. Thus, loss of dAkt function causes a cell-autonomous effect on follicle cell growth, because only mutant follicle cells show reduced cell size (Fig. 2E,H,M,P).
Figure 2. Analysis of dAkt loss of function in follicle cells. A–P: Stage 8 (A–H) and stage 9 (I–P) dAkt1q mosaic egg chambers labeled for alpha-spectrin (blue) and propidium iodide (red). Absence of green fluorescent protein (GFP) expression (green) marks the cells that are homozygous for dAkt1q, and the asterisks indicate sister wild-type cells, which show stronger GFP staining than the neighboring heterozygous counterparts. A,B,I,J: A and I are cross-sections and B and J are surface sections. Mutant follicle cell clones are marked by the boxes in B and J. C–E,F–H: Higher magnification views of the left and right boxed area in B, respectively. K–M,N–P: Higher magnification views of the left and right boxed area in J, respectively. C,F,K,N: Merged images of the GFP and alpha-spectrin signals. D,G,L,O: Propidium iodide staining of nuclei. E,H,M,P: Merged GFP, alpha-spectrin, and propidium iodide signals. Q–T: Cross-section of a stage 9 egg chamber stained for alpha-spectrin (blue), propidium iodide (red), and GFP (green). R–T: Enlarged view of the boxed area in Q; mutant cells (lack of GFP in S) are narrower, but the height remains the same as the wild-type neighbors. U–X: Egg chambers labeled for anti-PH3 (red). U: Wild-type egg chambers. V–X: The same dAkt1q mosaic egg chambers are shown in anti-PH3 alone (V) or merged images of anti-PH3 and GFP (W,X), and in optical cross- section (V,W) or surface section (X). U,W,X: In wild-type egg chambers (U) and dAkt1q follicular clones (W,X), no PH3 staining can be seen after stage 6. Anterior is up in panels Q–T and is toward the left in all other panels.
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To ensure that the observed reduced surface area of the dAkt mutant cells is not due to cell elongation and constriction, cross-sections of the mutant clones were analyzed (Fig. 2Q–T). The basal–apical length of the mutant cells remains constant, suggesting that the smaller cell size results from reduced cell diameter.
Reduction of the follicle cell size could arise from an extension of the proliferative program of these cells beyond stage 6 of oogenesis without accompanying cell growth. However, by counting the number of cells in mutant and wild-type twin spots of stage 10 egg chambers it appears that follicle cell proliferation is not affected by the loss of dAkt kinase. We calculated the ratio of the cell number within the dAkt1q mutant clones per cell number within the sister wild-type clones (number of clones examined = 18) and obtained a mean value of 0.94 (± 0.17 standard deviation). Furthermore, the mosaic ovaries were stained with antibodies against phosphohistone H3 (PH3) that are specific for the phosphorylated form of histone H3 present only in mitotic nuclei (anti-PH3; Hendzel et al., 1997). In mosaic dAkt1q egg chambers (Fig. 2V–X) as in wild-type ovaries (Fig. 2U) no PH3-positive cells were detected beyond stage 6, indicating that loss of dAkt function in follicular clones does not induce detectable extension of the proliferative program of the follicle cell population. In addition, considering that the number of cells in dAkt1q mutant and sister clones is the same, it is possible to rule out the possibility that inappropriate cytokinesis without DNA replication causes reduced cell size in dAkt1q mutant clones.
Alternatively, the reduced cell size could be due to perturbation of the three endoreplication cell cycles (Lilly and Spradling, 1996) through which the follicle cells become polyploid and increase their size by the end of stage 10B (Calvi et al., 1998). We note that the intensity of propidium iodide staining per unit area of the nuclei is not altered in the mutant clones (Fig. 2D,G,L,O). Because the overall size of the mutant nucleus is smaller, the total DNA content within the mutant cell is proportionally reduced. This in turn indicates that the number of endoreplication cycles is reduced in mutant cells.
The results described above define for the first time a role for the dAkt kinase in egg chambers development. This kinase is required for proper growth of follicle cells, and loss of its function alters follicle cell development without altering proliferation and death of these cells.