Cathepsin B differential expression and enzyme processing and activity during Fundulus heteroclitus embryogenesis

Area of Science:

• Developmental Biology
• Molecular Biology
• Fish Embryology

Background:

• Lysosomal proteases like cathepsin B (Ctsb) and cathepsin L (Ctsla) are crucial for yolk metabolism in fish oocytes.
• The specific function of Ctsb during marine teleost embryogenesis remains largely uncharacterized.

Purpose of the Study:

• To investigate the spatio-temporal expression, enzymatic activities, processing, and cellular localization of Ctsb and Ctsla in developing killifish (Fundulus heteroclitus) embryos.
• To elucidate the role of FhCtsb during early embryogenesis and its potential involvement in gastrulation.

Main Methods:

• Quantitative analysis of fhctsb and fhctsla transcript levels.
• Assay of cathepsin B- and L-like enzymatic activities.
• Detection of active FhCtsb forms (single and double chain).
• Electrophoretic analysis of yolk protein processing.
• Immunohistochemical localization of FhCtsb protein.

Main Results:

• Both fhctsb and fhctsla transcript levels and cathepsin B/L activities increased from the 2-4 cell stage to 7 days post-fertilization.
• An increase in active FhCtsb single chain preceded a rise in cathepsin B activity during the morula to gastrula transition, suggesting post-transcriptional regulation.
• Cathepsin B activity showed an 8-fold increase during neurulation, with cathepsin L activity moderately increasing and reaching a 6-fold enhancement by 7 dpf.
• Increased protease activities correlated with yolk protein modifications and strong FhCtsb expression in the yolk syncytial layer.

Conclusions:

• FhCtsb is likely involved in the initiation of gastrulation in F. heteroclitus embryos.
• FhCtsb and FhCtsla may have complementary roles in the breakdown of yolk reserves during embryonic development.
• Post-transcriptional mechanisms significantly regulate FhCtsb activity during killifish embryogenesis.