Developmental defects observed in hypomorphic anaphase-promoting complex mutants are linked to cell cycle abnormalities - PubMed (original) (raw)

Developmental defects observed in hypomorphic anaphase-promoting complex mutants are linked to cell cycle abnormalities

Diane C Shakes et al. Development. 2003 Apr.

Abstract

In C. elegans, mutants in the anaphase-promoting complex or cyclosome (APC/C) exhibit defects in germline proliferation, the formation of the vulva and male tail, and the metaphase to anaphase transition of meiosis I. Oocytes lacking APC/C activity can be fertilized but arrest in metaphase of meiosis I and are blocked from further development. To examine the cell cycle and developmental consequences of reducing but not fully depleting APC/C activity, we analyzed defects in embryos and larvae of mat-1/cdc-27 mutants grown at semi-permissive temperatures. Hypomorphic embryos developed to the multicellular stage but were slow to complete meiosis I and displayed aberrant meiotic chromosome separation. More severely affected embryos skipped meiosis II altogether and exhibited striking defects in meiotic exit. These latter embryos failed to produce normal eggshells or establish normal asymmetries prior to the first mitotic division. In developing larvae, extended M-phase delays in late-dividing cell lineages were associated with defects in the morphogenesis of the male tail. This study reveals the importance of dosage-specific mutants in analyzing molecular functions of a ubiquitously functioning protein within different cell types and tissues, and striking correlations between specific abnormalities in cell cycle progression and particular developmental defects.

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Figures

Table 1

mat-1 temperature-allelic series

Fig. 1

Phenotypic analysis of mat-1 mutant and cdc-27 RNAi embryos. Micrographs show tubulin (A-D) and DAPI (A′-D′) staining of individual metaphase I embryos of wild type (N2) (A,A′), mat-1(ax144) at 25°C (B,B′), mat-1(ax212) at 25.5°C (C,C′) and cdc-27 RNAi (late) (D,D′). White arrows indicates oocyte chromosomes. Only wild-type embryos progress to anaphase I and exit meiosis. (E-H) Micrographs of H2B::GFP embryos within the uteri of wild type (E), cdc-27 RNAi (F,G) and mat-1(ax144) at 25°C (H). Non-viable multicellular embryos produced after 20-24 hours in cdc-27 RNAi feeding experiments [(cdc-27 RNAi (early); white arrowhead] and meiotic one-cell arrested embryos produced after 24 hours in RNAi feeding experiments [cdc-27 RNAi (late)]. (I-L) DIC micrographs of embryos in the uteri of hermaphrodites incubated at 20°C. Wild type, mat-1(ax212) [permissive; 98.3% hatch], mat-1(ax161) [ semi-permissive; 16.3% hatch] and mat-1(ax520) [ restrictive; 0.7% hatch]. Meiotic one-cell embryos (asterisks) accumulate at all temperature regimes for all of the mat-1 alleles (Table 1) but at permissive (J) and semi-permissive temperatures (K), the mutant embryos are able to exit meiosis and divide mitotically. At semi-permissive temperatures, the vast majority of the multicellular embryos (K) produced by mat-1 hermaphrodites die prior to morphogenesis. The average embryo is ∼50 μm in length.

Fig. 2

Sequence alignment of C. elegans mat-1/cdc-27 and its homologs from humans, Drosophila, A. nidulans and S. cerevisae. Identical residues are highlighted in black, conservative changes in gray, and the mutant lesions are indicated in red. Tetratricopeptide repeat (TPR) domains are underlined and numbered. Hs, H. sapiens; Dm, Drosophila melanogaster; An, A. nidulans; Sc, S. cerevisiae and Ce, C. elegans. Numbers on left represent amino acid positions.

Fig. 3

Reductions in APC/C activity result in meiotic defects. DAPI (A-K) and tubulin (A′-K′) localization of meiotic chromosomes and spindles in wild type (A-D) and mutant mat-1 (E-K) embryos. The dotted white lines represent the approximate position of the plasma membrane. After fertilization, oocytes progress through MI metaphase (A,A′), anaphase A (B,B′), anaphase B (C,C′) and telophase (D,D′; only chromosomes within the polar body can be seen in this focal plane). During oocyte meiosis, the metaphase to anaphase transition promotes a 90° rotation of the anastral spindle axis (A,B,A′,B′; see cartoon below. The dotted black line represents the long axis of the spindle). Consequently, one pole of the spindle abuts the plasma membrane (B′; see cartoon below). The cartoon series shows the organization of the meiotic chromosomes and spindles during wild-type meiosis. (E-K′) mat-1 mutants incubated at restrictive and semi-permissive temperatures have meiotic defects. (E,E′) A metaphase plate and spindle from a metaphase arrested mat-1(ax212) embryo. (F′) Spindle rotation without chromosome separation (F; gray arrowhead) from a mat-1(ax72) embryo at 16°C. (G,H) Examples of MI anaphase bridges (white arrows) in mat-1(ax227) at 20°C (G) and mat-1(ax72) at 16°C (H). (I, Ι′) In the small percentage of the embryos that hatch at semi-permissive temperatures, normal meiosis I and II figures are seen [i.e., metaphase II (I,I′; the first polar body is outside of the field)]. (J,J′) Despite spindle rotation (J′), chromosome separation during meiosis II is not always normal (gray arrowhead). (K,K′) An example of an abnormal elongated meiotic spindle and an abnormally small array of meiosis II chromosomes [_mat-1(ax161)_]. Scale bar: (in D′) 2 μm.

Fig. 4

Developmental consequences of the reduction in APC/C activity in the early embryo. DAPI and tubulin localization during the pronuclear stage and two- to four-cell stage in wild-type embryos (A-E) and mat-1 2PB class (F-J) and 1PB class embryos (K-O). (A-C) Wild-type pronuclear stage embryo in which the sperm pronucleus (A; right white arrowhead) is anchored in the peripheral cortex by the sperm asters (B, white arrow). The female pronucleus (A; left white arowhead) is positioned more centrally. The centrosomes of the sperm aster lie on opposite sides of the sperm pronucleus (B, other centrosome is below the focal plane) and each centrosome has multiple microtubules emanating from the centrosome to the cortex (C). (D) Dividing wild-type two-cell embryos have two polar bodies (black arrowheads; one polar body is outside the focal plane), a larger blastomere (left) and a smaller blastomere (right). The second mitotic division is asynchronous and the individual spindles set up perpendicular to each other (E). (F-J) In the less severely affected 2PB class embryos (F,I; black arrowheads indicate position of the two polar bodies), the relative position of the female pronucleus is normal (F; left white arrowhead). In addition, the sperm asters and microtubules extend normally from each centrosome to the cortex (G,H). (I,J) Although the relative blastomere size and cleavage orientation of the two blastomeres are similar to wild type, the mutant blastomeres tend to divide more synchronously. (K-O) Under semi-permissive temperature conditions when MI predominates (K,N; black arrowhead indicates single polar body), embryos exit meiosis but zygotic development is severely compromised. The pronuclear stage of this 1PB class is characterized by abnormalities in the relative positions of the pronuclei (K) and the maturation of the sperm asters (L,M). The first division in this 1PB class is symmetric (N). In the second division, the blastomeres divide synchronously with both spindles perpendicular to the long axis of the embryo (N,O). C, H and M are enlargements (4×) of the sperm asters in B, G and L. The carets in E, J and O indicate the orientation of the mitotic spindle of the right blastomere of two- to four-cell embryos. The average embryo is ∼50 μm in length.

Fig. 5

sep-1 RNAi mimics the 1PB APC/C phenotype. (A,B) H2B::GFP micrographs of individual post-meiotic one-cell stage zygotes. The wild-type zygote in A has completed both meiotic divisions (carets point to the two polar bodies, one is out of the focal plane) and is at the pronuclear migration stage of zygotic development. The female pronucleus (left) travels from the far left end of the zygote to meet up with the male pronucleus (right). Pronuclear meeting normally occurs within the right end of the embryo (arrow). The sep-1 RNAi zygote in B has completed only a single meiotic division prior to developing pronuclei (arrowheads point to the single polar body). Like the APC/C 1PB class, SEP-1 depleted zygotes remain in a meiosis I stage and fail to progress to meiosis II. The sep-1 RNAi zygote transitions directly into an abnormal pronuclear stage embryo (cortical contractions and flows are absent). As in the 1PB class, the female pronucleus and the male pronucleus meet centrally within the cell (arrow). The chromosomes in both pronuclei appear more condensed than in wild-type zygotes.

Fig. 6

Meiotic exit is compromised in embryos with reduced APC/C activity. Localization of tubulin, DAPI and phospho-histone H3 (p-H3) in wild type (A-L) and mat-1 meiotic hypomorphs (M-R). Wild-type telophase II is characterized by the presence of a single polar body (white arrow in A,C,E,I) and a MII spindle (A; white arrowhead) that lies between two p-H3 staining (E) haploid chromosome sets (C). In early post-meiotic embryos, the compact MII spindle remnant lies at the surface of the plasma membrane (G; white arrowhead). Both the first (I, white arrow) and second (I, gray arrowhead) polar bodies can be seen, but only the second stains with p-H3 (K). At this stage, both the female (I, black arrowhead) and male pronucleus (J) have formed. The female, but not the male, pronucleus stains weakly with p-H3 (K,L). mat-1 hypomorphs (1PB class) display variable meiotic exit defects, with post-meiotic mutant embryos retaining many meiotic characteristics. Typical defects include large, disorganized meiotic spindle remnants (M; white arrowhead) co-existing with the developing sperm aster (N; caret). When the pronuclei form, only a single polar body (O; white arrow) is present and both the female pronucleus (O; black arrowhead) and polar body stain brightly with p-H3 (Q). In some 1PB class embryos, the male pronucleus (P; black arrowhead) was found to abnormally stain for p-H3 (R; black arrowhead).

Fig. 7

Germline defects in mat-1 hermaphrodites. Animals were shifted from 15°C to 25°C as L1 larvae and prepared for whole-mount DAPI staining as young adults. Images show one arm of a bilobed gonad (A,C), one arm plus the entire uterus (B), or the entire gonad (D,E). Oocytes can be identified by their diakinetic chromosomes (white arrows). In wild-type hermaphrodites (A), the gonad extends and reflexes so that the distal tips lie dorsally (gray arrowhead; distal tip is out of the plane of focus) over the vulva. Wild-type sperm with their highly condensed, haploid nuclei can be seen in the upper regions of the spermatheca (white arrowhead). Left of the spermatheca, a meiotic one-cell embryo (asterisk) and progressively older embryos lie within the uterus (white line). (B) A non-sterile, ax212 hermaphrodite in which the distal tips overlap (gray arrowhead). The mutant sperm lack DNA (spermatheca; white arrowhead), and the uterus (white line) contains a mixture of viable and dead embryos. (C) In ax227 hermaphrodites, the gonad is only slightly shorter than wild type but excess metaphase nuclei can be seen in the distal region of the gonad (C′; black arrow). The uterus contains only meiotic one-cell embryos (C; asterisks). In ax144 and ax520, the gonad is significantly reduced (D) and amorphous (E) germlines lack both oocytes and sperm.

Fig. 8

Somatic defects in mat-1 mutants. DIC images of tails from wild-type (A) or mat-1 (B-D) young adult males that had been shifted from 15°C to 25°C as L1 larvae. ax161 tails (C) are essentially normal, ax212 tails (B) exhibit moderate defects including missing or fused rays, and the severely reduced ax144 tails (D) typically lack rays altogether. Posterior regions of wild-type (E,F) and ax144 (G,H) males that were shifted to 25°C as L1 larvae and processed for immunofluorescence as L4 larvae for DAPI (E,G) and p-H3 (F,H). In comparison to the wild-type controls (E,F), ax144 animals exhibit reduced cell proliferation as assessed by DAPI-staining (G) and increased numbers of M-phase nuclei as assessed by anti-p-H3 immunofluorescence (H). (I) Wild-type vulva (white arrowhead). (J) Everted vulva (white arrowhead). (K) Quantitation of excess p-H3-staining somatic nuclei within the posterior of mat-1 L4 males (30-36 hours after the L1 upshift).

Fig. 9

Summary of meiotic and developmental defects observed in mat-1 hypomorphs. Under semi-permissive temperatures (rows 1-3), three different classes of meiotic and developmental phenotypes are observed for mat-1 mutants: normal meiosis (top row), the 2PB class (row 2), and the 1PB class (row 3). In the 2PB class, both MI and MII defects occur, leading to asymmetric but more synchronous divisions in the two- to four-cell stage. In the 1PB class, MI defects occur, MII is skipped, and meiotic exit defects are apparent. These defects lead to a symmetric first cleavage and a synchronous second mitosis in which the orientations of divisions are abnormal. Eggshell formation is also defective. In the fully restrictive conditions (row 4), all embryos arrest at metaphase of MI and make weak, incompletely hardened eggshells.

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Developmental defects observed in hypomorphic anaphase-promoting complex mutants are linked to cell cycle abnormalities - PubMed (original) (raw)