Lysosomes, caspase-mediated apoptosis, and cytoplasmic activation of P21, but not cell senescence, participate in a redundant fashion in embryonic morphogenetic cell death

by Selleckchem | Jan 27 2024

Micromass cultures of embryonic limb skeletal progenitors replicate the tissue remodelling processes observed during digit morphogenesis. Here, we have employed micromass cultures in an in vitro assay to study the nature of cell degeneration events associated with skeletogenesis. In the assay, "naive" progenitors obtained from the autopod aggregate to form chondrogenic nodules and those occupying the internodular spaces exhibit intense apoptosis and progressive accumulation of larger cells, showing intense SA-β-Gal histochemical labelling that strictly overlaps with the distribution of neutral red vital staining. qPCR analysis detected intense upregulation of the p21 gene, but P21 immunolabelling showed cytoplasmic rather than the nuclear distribution expected in senescent cells. Semithin sections and transmission electron microscopy confirmed the presence of canonical apoptotic cells, degenerated cell fragments in the process of phagocytic internalization by the neighbouring cells, and large vacuolated cells containing phagosomes. The immunohistochemical distribution of active caspase 3, cathepsin D, and β-galactosidase together with the reduction in cell death by chemical inhibition of caspases (Q-VAD) and lysosomal cathepsin D (Pepstatin A) supported a redundant implication of both pathways in the dying process. Chemical inhibition of P21 (UC2288) revealed a complementary role of this factor in the dying process. In contrast, treatment with the senolytic drug Navitoclax increased cell death without changing the number of cells positive for SA-β-Gal. We propose that this model of tissue remodelling involves the cooperative activation of multiple degradation routes and, most importantly, that positivity for SA-β-Gal reflects the occurrence of phagocytosis, supporting the rejection of cell senescence as a defining component of developmental tissue remodelling.

Comments:

This passage describes a study employing micromass cultures to investigate cell degeneration events in embryonic limb skeletal progenitors during digit morphogenesis. Here's a breakdown of the key findings and interpretations:

1. **Experimental Setup**: Micromass cultures were utilized as an in vitro model to mimic tissue remodeling observed during digit development. Naive progenitor cells from the autopod aggregated, forming chondrogenic nodules. Cells between these nodules underwent intense apoptosis and showed changes in cell size, accompanied by specific histochemical staining patterns.

2. **Observations**:
    - Internodular spaces exhibited intense apoptosis.
    - Larger cells accumulated, showing specific histochemical labeling.
    - Molecular analysis revealed upregulation of the p21 gene, typically associated with cell senescence.
    - Unexpectedly, P21 immunolabeling showed a cytoplasmic distribution rather than the nuclear distribution expected in senescent cells.

3. **Microscopic Analysis**:
- Microscopic examination confirmed canonical apoptotic cells and cell fragments undergoing phagocytosis by neighboring cells.
- Large vacuolated cells containing phagosomes were observed.

4. **Biochemical Pathways**:
    - Active caspase 3, cathepsin D, and β-galactosidase were identified via immunohistochemistry.
    - Inhibition experiments (Q-VAD, Pepstatin A, UC2288) indicated involvement of multiple pathways (caspases, cathepsin D, P21) in the dying process.
    - The senolytic drug Navitoclax increased cell death but did not alter the number of cells positive for SA-β-Gal, suggesting a distinct effect on cell viability.

5. **Interpretation**:
- The study proposes a model of tissue remodeling involving multiple degradation pathways.
- SA-β-Gal positivity might reflect phagocytosis rather than indicating cell senescence, challenging its role as a defining component of developmental tissue remodeling.

The findings highlight a complex interplay of cellular events during tissue remodeling and challenge the traditional understanding of SA-β-Gal positivity as a definitive marker of cell senescence in this context. The study emphasizes the importance of multiple cellular pathways in the process and suggests a more nuanced interpretation of cellular markers in developmental biology.