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Apoptosis in the chick wing bud and the permanence of FGF-2 rescue
1Department of Biochemistry, Cellular and Molecular Biology, University of Tennessee, Knoxville 37996-0840, USA.
Abstract:
Two regions of programmed cell death that occur in the mesoderm of developing chick wing buds were studied in vitro. The opaque patch (OP) and posterior necrotic zone (PNZ) were examined for the presence of internucleosomal DNA degradation and for rescue by protein synthesis inhibition, two defining characteristics of apoptosis. Agarose gel electrophoresis showed that DNA from OP and PNZ tissue was cleaved into nucleosome size pieces and this cleavage was prevented by inhibition of protein synthesis with cycloheximide. Both regions showed rescue with cycloheximide as determined by the chromium release assay and examination of electron micrographs. Also, the permanence of basic fibroblast growth factor (EGF-2) rescue in the OP and NPZ was examined using the chromium release assay. While rescue in the OP was found to be permanent, rescue in the PNZ only delayed death while FGF-2 was present in the culture medium. This research shows that death in the OP and PNZ exhibits internucleosomal DNA fragmentation and is prevented by inhibition of protein synthesis with cycloheximide, biochemically characterizing this death as apoptosis. It also suggests that in vitro FGF-2 rescue is permanent in the OP but is merely a delay of cell death in the PNZ.
Insights
Programmed cell death in chick wing buds, specifically the opaque patch (OP) and posterior necrotic zone (PNZ), was confirmed as apoptosis. Protein synthesis inhibition permanently rescued OP cell death, but only temporarily delayed PNZ cell death.
Area of Science:
- Developmental Biology
- Cell Biology
- Molecular Biology
Background:
- Programmed cell death is crucial for embryonic development.
- Two distinct regions of cell death, the opaque patch (OP) and posterior necrotic zone (PNZ), are observed in developing chick wing buds.
Purpose of the Study:
- To biochemically characterize programmed cell death in the OP and PNZ of chick wing buds.
- To investigate the role of protein synthesis inhibition and FGF-2 in rescuing cell death in these regions.
Main Methods:
- In vitro culture of chick wing bud tissue.
- Agarose gel electrophoresis to detect DNA fragmentation.
- Chromium release assay to assess cell viability.
- Electron microscopy to examine cell morphology.
- Inhibition of protein synthesis using cycloheximide.
- Treatment with basic fibroblast growth factor (FGF-2).
Main Results:
- DNA from both OP and PNZ tissue exhibited internucleosomal cleavage, a hallmark of apoptosis.
- Protein synthesis inhibition with cycloheximide prevented DNA fragmentation and rescued cell death in both regions.
- Basic fibroblast growth factor (FGF-2) provided permanent rescue in the OP but only a temporary delay in cell death in the PNZ.
Conclusions:
- Cell death in the chick wing bud's OP and PNZ is biochemically characterized as apoptosis.
- Protein synthesis inhibition is a key factor in preventing apoptotic cell death.
- FGF-2 demonstrates differential rescue effects, with permanent rescue in the OP and transient rescue in the PNZ, suggesting distinct regulatory mechanisms.