First published online 14 January 2004
doi: 10.1242/dev.00932
Development 131, 775-786 (2004)
Published by The Company of Biologists 2004
Drosophila dMyc is required for ovary cell growth and endoreplication
Jean Z. Maines1,*,
Leslie M. Stevens1,2,*,
Xianglan Tong2 and
David Stein1,3,
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
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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).
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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.
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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.
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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.
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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.
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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.
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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.
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© The Company of Biologists Ltd 2004
