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G2/M checkpoint gene expression in developing germ cells
Suzanne Hasthorpe1, Kellie Tainton, Melissa Peart
1Germ Cell Research, Murdoch Childrens Research Institute, Parkville, Victoria, Australia. shirley.dcruz@mcri.edu.au
Abstract:
Cell cycle progression is prevented by signal transduction pathways known as checkpoints which are activated in response to replication interference and DNA damage. We cloned a G2/M cell cycle phase-related checkpoint gene from a neonatal mouse testis cDNA library which was identified as mouse claspin, a proposed adaptor protein for Chk1. As part of a study on germ cell differentiation we examined the expression of the checkpoint gene, Chk1, and claspin at 12.5 and 14.5 days post coitum (dpc) and in the post-natal phase. Chk1 mRNA expression increased from 12.5 to 14.5 dpc in female gonads and was strong in males at both time points. Claspin however, was not detected until 14.5 dpc. This suggests there may be some dissociation of claspin expression from Chk1 in fetal germ cell development. Chk1 and claspin expression was also studied in testis over the first 3 days following birth, when apoptosis regulates germ stem cell number. We modulated checkpoint-related gene expression in testis using the anti-metabolite, 5-fluorouracil, resulting in increased apoptosis and upregulation of Chk1 (P<0.0001) and Cdc2 (P<0.02) mRNA. Although we do not fully understand the role checkpoint gene expression has during mammalian germ cell development this report is the first to show the expression of checkpoint-related genes in early mammalian germ cells.
Insights
This study investigates cell cycle checkpoint genes, Chk1 and claspin, during mouse germ cell development. Findings reveal distinct expression patterns, suggesting complex regulation of these critical genes in early development.
Area of Science:
- Cell Biology
- Developmental Biology
- Genetics
Background:
- Cell cycle checkpoints are crucial for preventing progression with DNA damage or replication interference.
- Checkpoint proteins like Chk1 and its proposed adaptor claspin play vital roles in cell cycle regulation.
- Understanding these pathways is essential for comprehending germ cell differentiation and development.
Purpose of the Study:
- To investigate the expression patterns of cell cycle checkpoint genes, specifically Chk1 and claspin, during mouse germ cell differentiation.
- To examine the relationship between Chk1 and claspin expression in fetal and neonatal mouse gonads.
- To explore the impact of DNA damage on checkpoint gene expression in the developing testis.
Main Methods:
- Cloning of mouse claspin from a neonatal mouse testis cDNA library.
- Quantitative analysis of Chk1 and claspin mRNA expression in gonads at 12.5 and 14.5 days post coitum (dpc) and in the postnatal phase.
- Treatment of neonatal mouse testis with 5-fluorouracil to modulate checkpoint gene expression and assessment of apoptosis.
Main Results:
- Chk1 mRNA expression was detected early in female gonads and strongly in males from 12.5 dpc.
- Claspin expression was not detected until 14.5 dpc, indicating a potential dissociation from Chk1 during fetal development.
- 5-fluorouracil treatment led to increased apoptosis and upregulation of Chk1 and Cdc2 mRNA in the neonatal testis.
Conclusions:
- Chk1 and claspin exhibit distinct temporal expression profiles during mouse germ cell development.
- These findings suggest a complex regulation of cell cycle checkpoints in early mammalian germ cells.
- This study provides the first evidence of checkpoint gene expression in early mammalian germ cells, opening avenues for further research.
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