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First published online May 5, 2004
doi: 10.1242/10.1242/dev.01108

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dMyc is required for larval growth and endoreplication in Drosophila

Sarah B. Pierce^*, Cynthia Yost^*, Jessica S. Britton, Lenora W. M. Loo, Erin M. Flynn, Bruce A. Edgar and Robert N. Eisenman

Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, PO Box 19024, Seattle, WA 98109-1024, USA

View larger version (27K): [in a new window]   Fig. 1. Mutants with reduced dMyc have a larval growth defect. (A) dm gene. The dm4 deletion, the positions of the P-elements used in the excision screen (in red), and the P-element and gypsy element that define the hypomorphic alleles dmP1/P0 and dm1, respectively, are indicated. Black boxes, coding region; arrow, the transcription start site. (B) Comparison of age-matched dm4/Y hemizygous mutants with control dm1-14-2/Y (see text) hemizygous males. (C) RT-PCR analysis of RNA from larvae of the indicated genotypes, harvested at 24 hours AED, using primers to detect regions across the exon 2/3 boundary and within exon 3 of dMyc. CaMKII is a loading control. (D) Western blot of extracts from larvae of the indicated genotypes, harvested 24 hours AED.

View larger version (59K): [in a new window]   Fig. 2. dMyc and dMnt are expressed in endoreplicating tissues. (A) Salivary glands from wandering third instar larvae were stained with antibodies to dMyc or dMnt (left panels), and DAPI (right panels). dMyc is expressed at higher levels in distal cells while dMnt is expressed at higher levels in proximal cells. (B) RT-PCR to detect dMyc transcript in gut or fat body from whole wandering third instar w; iso2,3 larvae. Rp49 is a loading control.

View larger version (64K): [in a new window]   Fig. 3. dm4 mutants have reduced nuclear size. (A) DAPI stained fat body (arrows) and salivary glands (arrowheads) from dm4/Y hemizygous mutants or control dm1-14-2/Y hemizygous males, harvested at the indicated times AED. Lower magnification (72 hr-low mag) shows the difference in salivary gland size by 72 hours AED. (B-I) Control dm1-24-2/Y (B,C,F,G) and dm4/Y (D,E,H,I) larvae were fed BrdU from 30 to 48 hours AED. Midgut (B-E) and fat body (F-I) were stained with anti-BrdU (B,D,F,H) and DNA was visualized with propidium iodide (C,E,G,I). (J) The percentage of BrdU-positive cells in control (white bars) or dm4/Y (black bars) fat body and midgut tissue was determined for the experiment above. Fifteen to 21 tissue samples were analyzed for each bar of the graph, each consisting of an average of 50 cells.

View larger version (46K): [in a new window]   Fig. 4. dMyc promotes and dMnt blocks endoreplication. Flp/Gal4 was used to clonally express dMyc (A-C,G-K) or dMnt (D-F) with GFP (green). DNA is stained with DAPI (blue). (A-F) Heat shock-induced clones in fat body from third instar larvae; DNA and GFP images are merged in C and F. (G) Quantification of nuclear DAPI fluorescence. DAPI stained fat body nuclei from larvae of the indicated ages were analyzed by deconvolution microscopy and heat shock-independent GFP marked dMyc-expressing cells were compared with neighboring non-GFP control cells. (H-K) Fat body from a second instar larva containing a heat shock-independent dMyc expressing FLP/Gal4 clone [marked with GFP (I) and outlined in white] was stained with DAPI (H) and anti-fibrillarin (J). DAPI and anti-fibrillarin images are merged in K.

View larger version (71K): [in a new window]   Fig. 5. DNA replication and Cyclin E protein levels oscillate in dMyc-expressing cells. (A-D) Control larvae (A,C) and larvae expressing dMyc driven by Adh-Gal4 (B,D) were fed BrdU for 5 hours and dissected at late second instar. Fat body was stained with anti-BrdU (A,B) and DNA was visualized with DAPI (C,D). Arrowheads indicate nuclei that did not label with BrdU. (E,F) Fat body from third instar control larvae (E,G) or larvae expressing dMyc driven by Adh-Gal4 (F,H) was stained with anti-Cyclin E (E,F) and DAPI (G,H). Cyclin E is expressed at high (arrows) and low (arrowheads) levels in control and dMyc-expressing cells.

View larger version (104K): [in a new window]   Fig. 6. dMyc induced growth and endoreplication is blocked by Cyclin E and cdk overexpression but only partially by loss of PI3K activity. (A-P) DAPI stained salivary glands with attached fat body (FB) from wandering third instar larvae with or without (control) the indicated transgenes driven by ptc-Gal4. (I-P) Nuclei double stained with DAPI (blue) and anti-fibrillarin (green) are shown at higher magnification. Numbers in the lower right corner (A-H) indicate the ratio of median DAPI fluorescent intensity of posterior salivary gland nuclei to a single fat body nucleus.