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Developmental activation of the capability to undergo checkpoint-induced apoptosis in the early zebrafish embryo
R Ikegami1, P Hunter, T D Yager
1Division of Developmental Biology and Research Institute, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, M5G 1X8, Canada.
Abstract:
In this study, we demonstrate the developmental activation, in the zebrafish embryo, of a surveillance mechanism which triggers apoptosis to remove damaged cells. We determine the time course of activation of this mechanism by exposing embryos to camptothecin, an agent which specifically inhibits topoisomerase I within the DNA replication complex and which, as a consequence of this inhibition, also produces strand breaks in the genomic DNA. In response to an early (pre-gastrula) treatment with camptothecin, apoptosis is induced at a time corresponding approximately to mid-gastrula stage in controls. This apoptotic response to a block of DNA replication can also be induced by early (pre-MBT) treatment with the DNA synthesis inhibitors hydroxyurea and aphidicolin. After camptothecin treatment, a high proportion of cells in two of the embryo's three mitotic domains (the enveloping and deep cell layers), but not in the remaining domain (the yolk syncytial layer), undergoes apoptosis in a cell-autonomous fashion. The first step in this response is an arrest of the proliferation of all deep- and enveloping-layer cells. These cells continue to increase in nuclear volume and to synthesize DNA. Eventually they become apoptotic, by a stereotypic pathway which involves cell membrane blebbing, "margination" and fragmentation of nuclei, and cleavage of the genomic DNA to produce a nucleosomal ladder. Fragmentation of nuclei can be blocked by the caspase-1,4,5 inhibitor Ac-YVAD-CHO, but not by the caspase-2,3,7[, 1] inhibitor Ac-DEVD-CHO. This suggests a functional requirement for caspase-4 or caspase-5 in the apoptotic response to camptothecin. Recently, Xenopus has been shown to display a developmental activation of the capability for stress- or damaged-induced apoptosis at early gastrula stage. En masse, our experiments suggest that the apoptotic responses in zebrafish and Xenopus are fundamentally similar. Thus, as for mammals, embryos of the lower vertebrates exhibit the activation of surveillance mechanisms, early in development, to produce the selective apoptosis of damaged cells.
Insights
Zebrafish embryos activate a cell-death surveillance system to eliminate damaged cells during development. This mechanism, triggered by DNA replication inhibitors, involves specific caspases and is similar to responses in Xenopus.
Area of Science:
- Developmental Biology
- Cell Biology
- Genetics
Background:
- Developing organisms possess mechanisms to remove damaged cells.
- Apoptosis, or programmed cell death, is crucial for normal development and tissue homeostasis.
- Understanding the early activation of these surveillance pathways is key to developmental biology.
Purpose of the Study:
- To investigate the developmental activation of a cell-death surveillance mechanism in zebrafish embryos.
- To determine the timeline and molecular players involved in apoptosis induction following DNA damage.
- To compare the zebrafish apoptotic response to that observed in other vertebrates like Xenopus.
Main Methods:
- Zebrafish embryos were treated with DNA-damaging agents (camptothecin, hydroxyurea, aphidicolin) at various developmental stages.
- Apoptosis was monitored through morphological changes and DNA fragmentation.
- Inhibitors of specific caspases (e.g., Ac-YVAD-CHO, Ac-DEVD-CHO) were used to identify key proteases involved.
- Cell-autonomous apoptosis was assessed across different embryonic cell layers.
Main Results:
- Exposure to DNA replication inhibitors (camptothecin, hydroxyurea, aphidicolin) before gastrulation induced apoptosis at the mid-gastrula stage.
- Apoptosis occurred in a cell-autonomous manner in the enveloping and deep cell layers, but not the yolk syncytial layer.
- DNA replication was arrested, but DNA synthesis and nuclear volume increased before cells underwent apoptosis.
- Nuclear fragmentation was inhibited by a caspase-1,4,5 inhibitor, suggesting a role for caspase-4 or -5.
- The apoptotic pathway shares similarities with that observed in Xenopus embryos.
Conclusions:
- Zebrafish embryos possess a developmentally regulated surveillance mechanism that triggers apoptosis to eliminate cells with DNA damage.
- This response is initiated by blocks in DNA replication and involves specific caspases, likely caspase-4 or -5.
- The findings highlight fundamental similarities in stress-induced apoptosis pathways across vertebrate development, from fish to mammals.