Carter AD and Sible JC (2003), Loss of XChk1 function triggers apoptosis after...

XB-ART-5741

Mech Dev 2003 Mar 01;1203:315-23. doi: 10.1016/s0925-4773(02)00443-4.

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Loss of XChk1 function triggers apoptosis after the midblastula transition in Xenopus laevis embryos.

Carter AD .

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Prior to the midblastula transition (MBT), Xenopus laevis embryos do not engage cell cycle checkpoints, although overexpression of the kinase XChk1 arrests cell divisions. At the MBT, XChk1 transiently activates and promotes cell cycle lengthening. In this study, endogenous XChk1 was inhibited by the expression of dominant-negative XChk1 (DN-XChk1). Development appeared normal until the early gastrula stage, when cells lost attachments and chromatin condensed. TUNEL and caspase assays indicated these embryos died by apoptosis during gastrulation. Embryos with unreplicated DNA likewise died by apoptosis. Embryos expressing DN-XChk1 proceeded through additional rapid rounds of DNA replication but initiated zygotic transcription on schedule. Therefore, XChk1 is essential in the early Xenopus embryo for cell cycle remodeling and for survival after the MBT.

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Species referenced: Xenopus laevis
Genes referenced: chek1 gs17 parp1
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	Fig. 1. Embryos expressing DN-XChk1 develop abnormally after the MBT. Embryos were injected with luciferase or DN-XChk1 mRNA and photographed at 5 h pf (stage 8) (A, top), 9 h pf (stage 10) (A, middle, bottom) or >12 h pf when luciferase-expressing embryos were gastrulating (B). (C) Embryos were processed for immunoblotting of the FLAG epitope. (D) Embryos were processed for immunoblotting of XChk1. Positions of endogenous (wt) and exogenous (DN) XChk1 are indicated. The migration of molecular weight markers (in kDa) is indicated.
	Fig. 2. DN-XChk1 induces chromatin condensation after the MBT. Embryos were injected with luciferase or DN-XChk1 mRNA or incubated in aphidicolin at 3 h pf and fixed at 6 (A) or .12 (B) h pf, sectioned, stained with DAPI and photographed under fluorescence microscopy. arrow, mitotic figure; arrowheads, condensed chromatin; asterisks, lacy chromatin; scale bar, 25 mm.
	Fig. 3. Inhibition of endogenous XChk1 results in apoptosis at the MBT. Embryos were injected with luciferase or DN-XChk1 mRNA at the one-cell stage. (A) Albino embryos were fixed and processed for TUNEL assays when morphology of embryos expressing DN-XChk1 appeared abnormal (approximately 12 hpf). A positive TUNEL is indicated by purple staining of nuclei. In a typical experiment 0-20% of luciferase-expressing embryos and 80-100% DN-XChk1-expressing embryos scored TUNEL-positive. (B) Embryos injected with luciferase or DN-XChk1 mRNA were assayed for caspase activity by the cleavage of the exogenous substrate, PARP. Times (h pf) are indicated.
	Fig. 4. A block to DNA replication at the MBT induces apoptosis after the MBT in Xenopus embryos. (A) Embryos were incubated in 100 mg/ml aphidicolin during the MBT (6 h pf), then were photographed when controls reached stage 9 (8 h pf) and stage 12.5 (.12 h pf). In aphidicolin-treated embryos, cell cycle arrest was observed at 8 h and embryos were dying by 12 h. (B) Albino embryos were treated with aphidicolin at the MBT (5.5 h pf) and fixed and processed along with sibling controls for TUNEL assays when morphology appeared abnormal (.12 h pf). (C) Control embryos and embryos treated with aphidicolin at the MBT (6 h pf) were snap-frozen and assayed for caspase activity when controls gastrulated (approximately 12 h pf). The migration of molecular weight standards (in kDa) is indicated on the left.
	Fig. 5. Expression of DN-XChk1 results in elevated incorporation of [3H]thymidine after the MBT. Embryos were injected with luciferase or DN-XChk1 mRNA at the one-cell stage. Embryos were incubated in medium containing 3H thymidine from 70 min to 4 h pf, collected at the times indicated and assayed for the incorporation of 3H into DNA. These data, from a single experiment, are representative of the results of multiple experiments.
	Fig. 6. Expression of DN-XChk1 does not prevent the initiation of zygotic transcription of the MBT. (A) A block to zygotic transcription induces apoptosis. Embryos were injected with H2O (control) or a-amanitin at the one-cell stage and assayed for caspase activity by cleavage of PARP substrate. (B) Embryos expressing luciferase or DN-XChk1 were assayed for onset of zygotic transcription by Northern analysis of GS17 mRNA.