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First published online 17 August 2005
doi: 10.1242/dev.01965

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Drosophila Myt1 is a Cdk1 inhibitory kinase that regulates multiple aspects of cell cycle behavior during gametogenesis

¹ Department of Biological Sciences, University of Alberta, Edmonton, AB T6G 2E9, Canada
² Program in Developmental Biology, Hospital for Sick Children, 555 University Avenue, Toronto, ON M5G 1X8, Canada

View larger version (77K): [in a new window]   Fig. 1. Genomic and molecular analysis of myt1. (A) An alignment of Myt1 sequences from Xenopus laevis, Homo sapiens and Drosophila melanogaster. Black shading indicates identical amino acids and gray shading highlights similar amino acids. The Myt1 protein sequence contains a kinase domain (purple bar), a potential trans-membrane domain (green bar) and a predicted C-terminal Cyclin B interaction motif (brown bar). (B) Physical and genetic map of myt1 and flanking genes uncovered by Df(3L)64D-F (white box). Black triangles indicate the position of the nearest lethal P-element insertions that complement Df(3L)64D-F. The black bar showing restriction enzyme sites (RI, EcoRI; SI, SalI) represents a 24 kb DNA sequence (coordinates 240,000 to 264,000) in AE003565, a genomic BAC clone. The narrow black bar represents the DNA fragment that was subcloned to generate P{myt1+}. Black arrow bars represent transcripts of myt1+ and the nearest flanking genes. Roman numerals indicate exons I to IV of the myt1 gene. (C) A single base deletion mutation in myt11 (and myt12) beginning at amino acid position 173 causes a frame-shift, producing an altered amino acid sequence in Myt1 (red letters) that ends with a premature stop codon at position 231.

View larger version (72K): [in a new window]   Fig. 2. Cell proliferation defects observed in myt1 mutant testes. (A) Phospho-histone H3 (PH3, red) antibody staining of myt1/+ controls, shows mitotic cells at the apical tip (white arrow) and a cyst of meiotic cells (blue arrow) farther along the testis. No additional cells were labeled (yellow arrow). (B) PH3 staining of mitotic cells (white arrows) at the apical tip of the testes in myt1 mutants as well as additional 16-cell cysts of PH3-positive cells farther along the testes. Isolated mitotic cells (green arrows) were also found along the testis as well as a large cluster of labeled cells at the terminal end (yellow arrow). Scale bar: 20 µm.

View larger version (81K): [in a new window]   Fig. 3. Increased numbers of myt1 mutant secondary spermatogonia. (A,D) BrdU-incorporation labels S-phase cells (green), showing that more cells are labeled in myt1 mutant testes (D) than in controls (A). (B,E) Fas3 labeling of hub cells (green, arrow) and Vasa germline-specific staining indicates the position of germline stem cells (outlined), showing that there are similar numbers of stem cells in myt1 mutants (E) and controls (B). (C,F) BamC (green) staining of secondary spermatogonia shows more spermatogonia in myt1 mutants (F) than in controls (C). Scale bars: 16 µm for A,C,D,F; 8 µm for B,E.

View larger version (108K): [in a new window]   Fig. 4. Secondary spermatogonia can undergo an extra round of mitosis in myt1 mutants. (A,B) Testes stained with anti-BamC (green) and anti-PH3 (red) antibodies. In the controls, cysts of dividing secondary spermatogonia do not contain more than eight cells (A, white arrows), whereas the myt1 mutant also contains a 16-cell cyst (B, white arrow), indicating an extra cell division. There were also many scattered PH3-positive cells not co-stained with BamC but intermingled with BamC-positive cells, in the myt1 mutants. These were probably ectopically dividing somatic cells (see Fig. 5, for explanation). (C-G) Phase-contrast and (C'-G') fluorescence (Hoechst 33258) images of cysts of: (C,C') control 16-cell primary spermatocytes, (D,D') unusual 32-cell spermatocyte cysts seen in myt1 mutants, (E,E') control 64-cell spermatid cysts, and aberrant-looking 64-cell (F,F') and 128-cell (G,G') spermatid cysts from myt1 mutants. Scale bars: in A, 20 µm for A,B; in D', 50 µm for C,D'; in G', 50 µm for E-G'.

View larger version (83K): [in a new window]   Fig. 5. Germline-associated somatic cell nuclei divide ectopically in myt1 mutant testes. (A,D) Somatic cyst cells stained with anti-Eya (green) and anti-PH3 (B, F, red) antibodies. Increased numbers of cyst cell nuclei were seen in the myt1 mutants (D, green) relative to controls (A); PH3-positive cyst cells were also detected in the mutants (D, arrows). (B,E) Cyst cells and spermatocytes stained with anti-Eya (green) and anti-Aly (red) antibodies. Extra cyst cells associated with Aly-positive spermatocytes were seen in myt1 mutants (E, arrows), when compared with controls (B). (C,F) Optical sections through seminal vesicles stained to visualize DNA (blue, arrow shows proximal end of vesicle). No sperm were present in the myt1 mutant seminal vesicle (F), in contrast to controls, which were full of mature sperm (C). Scale bar: in B, 40 µm for A,B,D,E; in C, 20 µm for C,F.

View larger version (99K): [in a new window]   Fig. 6. The myt1 mutant over-proliferation defects are due to a failure of Cdk1 inhibitory phosphorylation. (A,D) PH3 (red) staining shows that heat-shocked Cdk1AF testes (D) have extra dividing cells, compared with controls (A). (B,E) BamC (green) and PH3 (red) staining identifies dividing secondary spermatogonia. In the heat-shocked Cdk1AF testes, a 16-cell cyst undergoing ectopic mitosis (E, white arrow) is shown, not seen in the controls (B). (C,F) Eya (green) staining to label somatic cyst cells shows that heat-shocked Cdk1AF testes have more cyst cells (F) than do controls (C). Scale bar: 40 µm for A,D; 8 µm for B,E; 20 µm for C,F.

View larger version (86K): [in a new window]   Fig. 7. Mitotic over-proliferation defects seen in myt1 mutant ovaries. (A,B) Ovarioles labeled by antibodies against PH3 (red) to mark mitotic cells, Hts (green) to mark spectrosomes and stained with Hoechst 33258, marking DNA (blue). In myt1 mutants (B), germaria often contained both dividing stem cells (green arrow) and dividing cystocytes (yellow arrow), not seen in controls (compare insets: a1, b1). The myt1 mutants also had more PH3-positive somatic follicle cells surrounding the egg chambers than did controls (compare insets: a2, b2). (C,D) Centrosomin staining (Cnn, red) for centrosomes, Hts (green) staining for fusomes and DNA (blue) staining showed significantly more metaphase stem cells (green arrow) and cystocytes (yellow arrows) in myt1 mutants (D) compared with controls (C). (E,F) Stacked confocal images of a Vasa-stained germarium, showing that myt1 mutant germaria (F) contain more cysts than controls (E). The myt1 mutant germline stem cells (F, green arrows) were also slightly smaller than controls (E). Scale bar: 40 µm for A,B; 8 µm for C-F.

View larger version (98K): [in a new window]   Fig. 8. Follicle cell defects observed in myt1 mutant ovarioles at different stages of development. (A,D) Stage 2 egg chambers stained for Hts (green) and PH3 (red). Stalk cells (arrow) are PH3-positive in 90% of myt1 mutants at this stage (D), but are PH3-negative in controls (A). (B,E) Stage 7 egg chambers stained for PH3 (red). Follicle cells at each end of the egg chamber (arrows) are PH3 positive in myt1 mutants (E) but PH3 negative in controls (B). (C,F) Stage 7 egg chambers stained for Fas3 (green) and PH3 (red). Arrow indicates a PH3-positive polar cell (seen in 100% of myt1 mutants, F), not seen in controls (C). (G,J) Stage 10A egg chambers stained for Fas3 (green) and PH3 (red). Arrow in J indicates a PH3-positive border cell (seen in 20% of myt1 mutants), not present in controls (G). (H,K) Stage 10A egg chambers stained for Eya (green) and PH3 (red). Arrows indicate PH3-positive, Eya-positive stretched cells in 16% of myt1 mutants (K) not seen in controls (H). (I,L) Stage 10B egg chambers stained for DNA (cyan) and PH3 (red). Arrows indicate PH3-positive main body follicle cells in 6% of myt1 mutants (L) but not in controls (I). Scale bar: 8 µm for A,D; 40 µm for B,C,E-G,J; 20 µm for H-L.