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Phenotypic characterization of Drosophila ida mutants: defining the role of APC5 in cell cycle progression

¹ Department of Molecular Pharmacology and Biological Chemistry, Robert H. Lurie Comprehensive Cancer Center, Northwestern University, 303 East Chicago Avenue, Chicago, IL 60611-3093, USA
² Department of Molecular Biology and Genetics, Biotechnology Building, Cornell University, Ithaca, NY 14853-2703, USA

View larger version (64K): [in a new window]   Fig. 1. ida mutants die during metamorphosis and are `imaginal discs arrested'. Wild-type (A) and ida107 (B) animals are pictured at 48 hours after puparium formation. The arrow marks the position of a stalled gas bubble (B). ß-gal activity is detected in dissected tissues from esg-lacZ; ida107/+ (C) and esg-lacZ; ida107/ida107 (D) wandering third-instar larvae. Arrows point to proliferation zones in the optic lobes that are reduced in size in ida mutants (D). Arrowheads mark the position of excorporate imaginal discs in (C), and rudimentary imaginal discs in (D). Hoechst staining of the anterior gut in wild-type (E) and ida107 (F) larvae is shown. An arrow marks the presence (E) and absence (F) of the imaginal ring. ß-gal staining in the anterior gut of an esg-lacZ; ida107/+ (G) and an esg-lacZ; ida107/ida107 (H) animal is presented. Note the severe reduction of ß-gal positive cells (arrows, H) in the imaginal ring in ida107 animals. ß-gal staining in the anterior dorsal (ad) and posterior dorsal (pd) histoblast nests from esg-lacZ; ida107/+ (I) and esg-lacZ; ida107/ida107 (J) animals are indicated. Adjacent segments are presented for each genotype. No differences are detected in primordial cell number.

View larger version (30K): [in a new window]   Fig. 2. A single 2.3 kb mRNA is disrupted in ida mutants. (A) A schematic of the 63F genetic locus is presented. The position of three subclones from the 63F walk and dSc2, ida, and mge gene structures are shown. Exons are demarcated by open rectangles, and small arrows mark the direction of transcription. Genomic DNA missing in ida107 and Df(3L)449 deficiency stocks is indicated by gaps marked with parenthesis. A solid line represents genomic DNA present in the P[w+ 63F/k1] transgene (see text for details). The proximal boundary of the P[w+ 63F/k1] transgene is off the scale of the figure. (B) Three identical northern blots with samples isolated from homozygous animals of the genotypes indicated were prepared. The probes used are designated above each blot. A strand-specific riboprobe generated against the full-length p63F.15 subclone using T3 polymerase hybridizes to ida and dSc2 transcripts. A riboprobe generated using T7 polymerase recognizes mge. Note that mge and dSc2 transcript levels are unaffected in ida mutants. A blot hybridized for rp49 serves as a loading and blotting control.

View larger version (67K): [in a new window]   Fig. 3. ida encodes an APC5 homolog. (A) IDA coding information is in uppercase letters. 5'-untranslated, 3'-untranslated, and the DNA for the single intron are in lowercase letters. Amino acid residues are in three-letter code. Numbers to the right represent nucleotide position (upper) relative to the end of dSc2, and amino acid position (lower) relative to the putative IDA start. The presumed polyA signal is bold and underlined. A blue box outlines the putative TPR domain. A pink box demarcates putative phosphorylation sites that are lost in the nonsense mutation of the idab4 allele (asterisk, see text for details). The GenBank accession number for ida is AF312026. (B) An alignment of putative TPR domains in APC5 homologs from Drosophila, H. sapiens, S. cerevisiae and C. elegans is presented. Red residues are identical in all species, orange residues indicate that at least two species share identity, and yellow indicates similar residues.

View larger version (113K): [in a new window]   Fig. 4. ida mutants display abnormal chromosome cytology. Mitotic figures of wild-type prometaphase (A), wild-type anaphase (B), ida107 prometaphase (C), and ida107 anaphase (D) are presented. Prometaphase figures show that ida cells contain an increased number of chromatin structures (compare A with C). Aberrant anaphase figures with lagging chromosomes are observed in ida cells (arrow, D). Wild-type (E) and ida107 (F) cells show normal staining patterns for tubulin (white) and centrosomin (green). Hoechst staining (blue) shows the position of chromosomes along the spindle.

View larger version (129K): [in a new window]   Fig. 5. ida cells are aneuploid. Mitotic figures from idak1-449 show examples of aneuploidy, with one cell containing 21 unfragmented chromosomes (A), and another containing 39 (B). Aneuploidy is also observed in null ida alleles. All individual Hoechst-labeled chromatin figures in an ida107 cell (C) have a corresponding Bub1 antibody signal (D). Note that some of the Bub1 staining is not in the plane of focus.

View larger version (54K): [in a new window]   Fig. 6. Bub1 is detected at ida kinetochores throughout mitosis. Hoechst staining (A-C) shows chromosome morphology and Bub1 staining (D-F) marks kinetochore positions. In all panels, asterisks mark prometaphase, arrows mark metaphase, and brackets mark anaphase. Two fields of ida107 cells are shown (B,C,E,F). Bub1 staining is present on prometaphase figures from both wild-type (D) and ida107 cells (E,F). During anaphase, Bub1 staining decreases in wild-type (bracket, D). The staining intensity is the same at both stages in ida107 cells (brackets, E,F). Note that some ida mutant cells are outside the plane of focus.

View larger version (50K): [in a new window]   Fig. 7. Sister-chromatid separation occurs in cells depleted of IDA. Sister-chromatid separation is observed in brain squashes from an idak1-449 mutant in which chromosomes are not hypercondensed (arrows, A). Hoechst staining of untreated Kc cells (B) is compared with cells treated with dsRNA specific for ida (C). Control cells retain normal chromosome morphology (B), while RNAi treated cells contain chromosomes with hypercondensed morphology and separated sister chromatids (arrowheads, C). A rarely occurring four-lobed structure, or unseparated chromatid pair is designated by an arrow (C). RNAi phenocopies the chromosome hypercondensation observed in ida null mutants.

View larger version (53K): [in a new window]   Fig. 8. Cyclin B levels remain high in ida cells with anaphase chromosome positioning. Hoechst staining (A-D) and cyclin B staining (E-H) in wild-type (A,E,C,G), and ida107 (B,F,D,H) cells is presented. In wild-type, cyclin B levels are high at prometaphase (E), but are reduced at anaphase (G). ida107 mutant cells contain high cyclin B levels during prometaphase (F) in a pattern similar to wild-type. High cyclin B levels are retained during anaphase in most ida107 cells (H).