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First published online 14 January 2004
doi: 10.1242/dev.00932

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Drosophila dMyc is required for ovary cell growth and endoreplication

Jean Z. Maines^1,*, Leslie M. Stevens^1,2,*, Xianglan Tong² and David Stein^1,3,

¹ Section of Molecular, Cell and Developmental Biology, and Institute for Cellular and Molecular Biology, The University of Texas at Austin, 1 University Station C-0930, Austin, TX 78712-0253, USA
² Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA
³ Department of Molecular Genetics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA

View larger version (29K): [in a new window]   Fig. 1. Structure of the dMyc protein and gene. (A) Segments of the dMyc protein that are similar to stretches of the mouse Myc protein are shown in blue. The leucine zipper (LZ) and basic helix-loop-helix (bHLH) motifs are indicated. The arrow marks the position at which the protein is truncated by the dm2 mutation. (B) dm exons are indicated by boxes, with amino acid coding regions in yellow. The positions of transposon insertions responsible for the dm1 and dmPG45 alleles are indicated, as is the site of the dm2 point mutation (asterisk). (C) The dm2/Y pupa (left) is significantly smaller than the dm2/+ pupa (right).

View larger version (66K): [in a new window]   Fig. 2. Expression of dMyc in the ovary, and morphology of dm2 germline clones. (A) In wild-type ovaries, dMyc is present in the nuclei of germline and somatic cells at most stages of oogenesis. (B,C) Nuclear dMyc is absent from follicle cells homozygous for dm1 (white outlines), identified by their blue color in the hGFP/DAPI overlay. (D,E) Nuclear dMyc is also absent from follicle cells homozygous for dmPG45 (white outlines; identified by their blue color in the hGFP/DAPI overlay). (F,H) Egg chambers with a dm2 mutant germline (arrowhead), identified by the absence of hGFP, appear smaller than normal, on the basis of their position in the ovariole. (G,I) Oocyte specification is not apparently affected in dm2 mutant germ cells. The oocyte nucleus labels faintly with DAPI (arrowhead), and is positioned at the posterior of this stage 4 egg chamber.

View larger version (72K): [in a new window]   Fig. 3. Homozygous dm2/dm2 nurse cells present in mosaic egg chambers exhibit cell-autonomous growth defects. (A,B) The nuclei of dm2/dm2 mutant germ cells, which stain with DAPI but do not exhibit hGFP-associated fluorescence (arrowheads), are significantly smaller than the nuclei of their wild-type sister nurse cells. (C,D) dm2 mutant nurse cell nuclei, visualized with DAPI (arrowheads), often exhibit a blob-like morphology indicating the presence of polytene chromosomes, in the same egg chambers as wild-type nuclei exhibiting the more dispersed polyploid DNA morphology, characteristic of relatively more mature nurse cells.

View larger version (94K): [in a new window]   Fig. 4. dm2 mutant nurse cell nuclei undergo DNA endoreplication. (A-C) dm2 mutant nuclei (arrowhead) incorporate BrdU. (D-F) dm2 mutant germ cells (arrowhead) express Cyclin E periodically. (G-I) dm2 mutant germ cells (arrowhead) also express Dacapo periodically.

View larger version (66K): [in a new window]   Fig. 5. dMyc is a dose-dependent regulator of follicle cell and nuclear size. (A) An overlay of DAPI/hGFP images shows a large clone of dm2 follicle cells with nuclei of unusually small size. (B-D) DAPI (B), hGFP (C) and merged (D) images of a field of follicle cells with a clone of dm2/dm2 mutants (yellow outline) and twin-spot derived +/+ cells (white outline). The nuclei of +/+ cells (carrying two copies of the hGFP gene and therefore distinguishable from dm2/+ cells by their increased fluorescent intensity) are larger than the nuclei of their dm2/+ sister cells, which are larger than the nuclei of the dm2/dm2 mutant cells. (E) Measurement of the area of nuclei indicates that homozygous wild-type nuclei are twice the size of dm2/+ nuclei, and tenfold larger than the nuclei of dm2/dm2 mutant cells. See Materials and methods for details on size determination. (F) A DAPI-stained follicle at stage 5, prior to the time at which follicle cell endoreplication initiates. (G) At higher magnification, the lack of hGFP nuclear staining (white outline) indicates the position of a clone of dm2/dm2 mutant follicle cells. (H) Anti-Fas III staining, which weakly stains the periphery of all follicle cells, demonstrates that the homozygous mutant cells (asterisks) are considerably smaller than the neighboring follicle cells. Thus, dMyc regulates follicle cell growth prior to the onset of endoreplication.

View larger version (91K): [in a new window]   Fig. 6. Follicle cell DNA endoreplication, but not gene amplification, is perturbed by mutations in dm. (A) An overlay of DAPI/hGFP images shows a clone of homozygous dm2/dm2 mutant cells outlined in white. (B) An overlay of hGFP/anti-BrdU images of the same field of follicle cells shows that a much smaller proportion of dm2 mutant follicle cells than wild-type cells incorporate BrdU. (C) An overlay of DAPI/hGFP images shows a large clone of dm2/dm2 mutant cells, present in a stage 11 egg chamber, outlined in white. (D) In the overlay of DAPI/BrdU images of the same mutant clone, gene amplification can be detected by the presence of puncta of BrdU incorporation in both the heterozygous and dm2 mutant cells.

View larger version (50K): [in a new window]   Fig. 7. Growth of germline and somatic tissues in the ovary is tightly coordinated. (A,B) An egg chamber with a dm2 mutant germline (arrowhead) exhibits abnormal perdurance of Fas III expression in the overlying wild-type follicle cells. (C,D) By contrast, an egg chamber with a dm2 mutant germline (arrowhead) expresses Broad Complex (BR-C) in the follicle cell layer on the basis of its age or position in the ovariole, rather than on its apparent maturity based on size. Note that the adjacent anterior egg chamber (arrow) is larger, but younger, and does not express BR-C. (E,F) An egg chamber in which all follicle cells are mutant for dm2 (arrow) is abnormally small, with small nurse cells.