Drosophila growth and development in the absence of dMyc and dMnt

Abstract

Myc oncoproteins are essential regulators of the growth and proliferation of mammalian cells. In Drosophila the single ortholog of Myc (dMyc), encoded by the dm gene, influences organismal size and the growth of both mitotic and endoreplicating cells. A null mutation in dm results in attenuated endoreplication and growth arrest early in larval development. Drosophila also contains a single ortholog of the mammalian Mad/Mnt transcriptional repressor proteins (dMnt), which is thought to antagonize dMyc function. Here we show that animals lacking both dMyc and dMnt display increased viability and grow significantly larger and develop further than dMyc single mutants. We observe increased endoreplication and growth of larval tissues in these double mutants and disproportionate growth of the imaginal discs. Gene expression analysis indicates that loss of dMyc leads to decreased expression of genes required for ribosome biogenesis and protein synthesis. The additional loss of dMnt partially rescues expression of a small number of dMyc and dMnt genes that are primarily involved in rRNA synthesis and processing. Our results indicate that dMnt repression is normally overridden by dMyc activation during larval development. Therefore the severity of the dm null phenotype is likely due to unopposed repression by dMnt on a subset of genes critical for cell and organismal growth. Surprisingly, considerable growth and development can occur in the absence of both dMyc and dMnt.

Figure 1

Loss of dMnt partially rescues the dMyc null mutant growth defect. Larvae grown on standard fly food (A) or yeast paste (B–D) are shown at the following ages: (A) 5 days AED, (B) maximum larval size, 5 days AED for dm⁴ and control and 12 days AED for dm⁴dmnt¹, (C) 1 day after puparium formation, 6 days AED for control and 13 days AED for dm⁴dmnt¹. (D) The development of 60 larvae of the indicated genotypes was assessed by scoring the number of larvae at each stage on the indicated days.

Figure 2

Endoreplication is partially rescued in dm⁴dmnt¹ mutant larvae. (A) DAPI stained salivary glands (arrows) and DAPI and phalloidin stained fat body from control (5 days AED), dm⁴dmnt¹ (12 days AED), and dm⁴ (5 days AED) larvae are shown at the indicated magnification. (B) Larvae were fed BrdU from 30–48 hr AED and the percentage of BrdU positive cells in the indicated tissues was determined. Ten to 25 tissue samples, containing an average of ~80 cells, were analyzed for each bar of the graph.

Figure 3

Imaginal discs from dm⁴dmnt¹ mutant larvae are correctly patterned. Wing and eye discs isolated from control (5 days AED) and dm⁴dmnt¹ (12 days AED) larvae were stained with anti-Wg or anti-ELAV antibodies, or DAPI, as indicated.

Figure 4

Imaginal tissues in dm⁴dmnt¹ mutant larvae grow disproportionately relative to endoreplicating tissues. Tissues were isolated from control (A, B, E, F) and dm⁴dmnt¹ mutant larvae (C, D, G, H) within one hour of the molt to third instar (A, C; 3 and 5 days AED for control and dm⁴dmnt¹ respectively) or at maximum larval size (B, D, E–H; 5 and 12 days AED for control and dm⁴dmnt¹, respectively). (A–D) DAPI stained salivary glands (SG) and wing discs (insets, WD). (E–H) BrdU incorporation to indicate S phase cells in eye-antennal discs at lower (E, G) and higher (F, H). BrdU labeled eye discs are shown at 200x magnification.

Figure 5

dm⁴ and dm⁴dmnt¹ mutant cells proliferate slowly. (A) Clones of dmnt¹, dm⁴dmnt¹, and dm⁴ homozygous mutant cells (GFP negative, outlined in yellow) with homozygous wild type twin spot clones (distinguished by brighter GFP than surrounding heterozygous cells, outlined in red). (B) Area of homozygous mutant clones relative to their homozygous wild type twin spot clones. The number of clones analyzed per genotype is indicated (n). Area was calculated for clone pairs in which a mutant clone was detectable.

Figure 6

The expression of rRNA processing genes is rescued by loss of dMnt in dMyc mutant larvae. Expression levels of the indicated genes in dm⁴ (blue), dm⁴dmnt¹ (red), and dmnt¹ (yellow) mutant larvae, relative to control larvae, were determined by real-time PCR. Genes for which expression in dm⁴ and dm⁴dmnt¹ larvae is significantly different are indicated (*, p<0.05; **, p<0.005; t-test). Genes that contain an E box within the 5’ untranslated region or 500 bp upstream of the transcriptional start site are indicated by #. The functional categories to which the genes belong are shown at the top.