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First published online 4 April 2007
doi: 10.1242/dev.02831

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grp (chk1) replication-checkpoint mutations and DNA damage trigger a Chk2-dependent block at the Drosophila midblastula transition

Saeko Takada^*, Seongae Kwak, Birgit S. Koppetsch and William E. Theurkauf

Program in Molecular Medicine, Program in Cell Dynamics, University of Massachusetts Medical School, 373 Plantation Street, Worcester, MA 01605, USA.

View larger version (77K): [in this window] [in a new window]   Fig. 1. An mnk-null mutation suppresses the cellularization- and gastrulation-defects associated with the grp mutation. (A,B) Control (w1118) and mnk-null mutant (mnk) embryos cellularize during interphase 14; a uniform monolayer of nuclei surrounded by a hexagonal network of membrane and associated actin network (insets) can be seen. (C) grp-null mutant embryos fail to cellularize and nuclei are either lost from the surface or fuse to form aggregates, and the actin cytoskeleton is disorganized (inset). (D) By contrast, mnk grp double-mutant embryos cellularize and show a near wild-type hexagonal actin network and uniform cortical nuclear monolayer (inset). However, the nuclei are significantly larger than in wild-type or mnk mutant embryos. (A-D) The actin cytoskeleton (green) was labeled with an anti--Spectrin antibody and nuclei were labeled with TOTO3 (Molecular Probes; red). Insets are enlarged images of -Spectrin staining. (E-L) Still images from transmitted light time-lapse recordings of control (w1118), mnk- and grp-null single mutant, and mnk grp double-mutant embryos. Control (E,F), mnk-null mutant (G,H) and mnk grp double-mutant (K,L) embryos cellularize and gastrulate. By contrast, grp-null mutant embryos (I,J) do not cellularize or gastrulate. Arrows indicate the cellularization front; arrowheads indicate pole cells migrating towards the anterior during gastrulation. Scale bars: 100 µm in A-L; 10 µm in insets in A-D.

View larger version (62K): [in this window] [in a new window]   Fig. 2. The mnk mutation suppresses zygotic gene transcription and Cdc2-phosphorylation defects in grp mutant embryos. (A,B) Whole-mount in situ hybridization of the segmentation gene runt (A) and the cellularization gene serendipity- (B). (A) Control (w1118) and mnk-null mutant embryos show seven-stripe runt transcript expression during interphase 14. The runt gene is never expressed at high levels in grp-null mutant embryos. By contrast, seven-stripe runt expression is clearly observed in mnk grp double-null mutant embryos. (B) serendipity- is required for cellularization and is expressed at high levels during interphase 14 in both wild-type (w1118) and mnk mutant embryos, but not in grp mutant embryos. Expression of serendipity- is also restored in mnk grp double mutants. (C) In control (w1118) and mnk mutants, inhibitory tyrosine phosphorylation of Cdc2 dramatically increases at the MBT, between 2 and 4 hours post-egg-deposition. This dramatic increase in Cdc2 phosphorylation is not observed in grp mutant embryos. In mnk grp double-mutant embryos, however, essentially wild-type Cdc2 phosphorylation is restored (at 2-3 and 3-4 hours). Western blots of embryonic lysates were probed with antibodies against phospho-Cdc2 (Tyrosine 15), and -Tubulin was used as the loading control. Embryo age in hours is indicated (0-1,1-2, 2-3 and 3-4). Scale bars: 100 µm.

View larger version (77K): [in this window] [in a new window]   Fig. 3. DNA double-strand-break accumulation in grp mutant embryos. (A-F) To assay for DNA damage in grp mutants, embryos were stained with an antibody that recognizes a phosphorylated form of histone H2AX (Drosophila homolog: Histone H2Av) (anti-H2AX), and for DNA with TOTO3. (A-C) H2AX labeling is not observed in early grp mutant embryos. (D-F) However, H2AX labeling becomes prominent in grp mutant embryos after mitosis 13. Cellularization consistently fails in these mutants (Sibon et al., 1997). (C,F) DNA, red; phospho-histone-H2Av, green. (G) Chk2 phosphorylation in grp mutant embryos. Extracts from developmentally staged wild-type (w1118) and grp mutant embryos were assayed for Chk2 expression by western blotting. Chk2 shows reduced electrophoretic mobility, characteristic of activating phosphorylation, in 3-4 hour grp mutants, but not in wild-type controls. Scale bar: 10 µm.

View larger version (92K): [in this window] [in a new window]   Fig. 4. DNA damage triggers a Chk2-dependent cellularization block. (A-H) Control (w1118) and mnk mutant embryos were injected with bleomycin, and cellularization was monitored by time-lapse transmitted light and laser scanning confocal microscopy. Rhodamine-conjugated tubulin was co-injected as a marker for cell cycle stage. Nuclei exclude fluorescent rhodamine-conjugated tubulin and thus appear as dark circles on confocal imaging (Sibon et al., 1997). Recordings were started at the beginning of interphase 14 (0 seconds, 0s). Immediately after injection, nuclei were in a uniform monolayer in both wild-type (w1118) and mnk mutant embryos (A,E). By 1200 seconds (1200s), the nuclear monolayer in w1118 controls was disorganized and some nuclei had dropped into the interior of the embryo (B), and these embryos did not cellularize (C,D). By contrast, all mnk mutant embryos injected with bleomycin consistently maintained a uniform nuclear monolayer and cellularized (E-H). (F,G) Plasma membrane invagination at the cellularization front is indicated (arrows). (D,H) Rhodamine-conjugated tubulin is excluded from interphase nuclei and was used as a cell cycle marker. (Also see Movies 1-5 in the supplementary material.) Scale bar: 50 µm.

View larger version (134K): [in this window] [in a new window]   Fig. 5. DNA damage triggers a Chk2-dependent block in the expression of a subset of zygotic genes. Enhanced fluorescence in situ hybridization for runt (A-F) and slam (G-L) during early interphase 14 is shown. Embryo stage was judged by blastoderm nuclear density (insets) and DIC images (not shown). (A,C) In w1118 and mnk mutant embryos, runt mRNA is distributed broadly over the central region of the embryo (Klingler and Gergen, 1993). (G,I) slam mRNA localizes to the invaginating membrane front, similar to the Slam protein localization reported previously (Lecuit et al., 2002), resulting in a hexagonal network structure around each nucleus. (B) In w1118 embryos treated with bleomycin, runt transcript is not detectable. (D) By contrast, similarly treated mnk mutant embryos express runt at the same levels as the embryos without DNA damage. Following bleomycin treatment, slam is expressed in both w1118 and mnk mutant embryos, although slam transcript localization is disrupted. This transcript is dispersed in w1118 embryos (H) and localizes around the large aneuploid nuclei in mnk embryos (J). runt expression is also blocked in grp mutant embryos, and expression is restored in mnk grp double mutants (E,F). By contrast, slam transcripts are detected in both grp single-mutant and mnk grp double-mutant embryos (K,L). DNA damage during the syncytial blastoderm stage and the grp mutation thus produce similar gene-specific expression defects. Scale bar: 100 µm.

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