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Developmental expression of functional cyclooxygenases in zebrafish
Tilo Grosser1, Shamila Yusuff, Ellina Cheskis
1Center for Experimental Therapeutics, University of Pennsylvania School of Medicine, 153 Johnson Pavilion, 3620 Hamilton Walk, Philadelphia, PA 19104, USA.
Abstract:
Study of the cyclooxygenases (COXs) has been limited by the role of COX-2 in murine reproduction and renal organogenesis. We sought to characterize COX expression and function in zebrafish (z). Full-length cDNAs of zCOX-1 and zCOX-2 were cloned and assigned to conserved regions of chromosomes 5 and 2, respectively. The deduced proteins are 67% homologous with their human orthologs. Prostaglandin (PG) E(2) is the predominant zCOX product detected by mass spectrometry. Pharmacological inhibitors demonstrate selectivity when directed against heterologously expressed zCOX isoforms. Zebrafish thrombocyte aggregation ex vivo and hemostasis in vivo are sensitive to inhibition of zCOX-1, but not zCOX-2. Both zCOXs were widely expressed during development, and knockdown of zCOX-1 causes growth arrest during early embryogenesis. zCOX-1 is widely evident in the embryonic vasculature, whereas zCOX-2 exhibits a more restricted pattern of expression. Both zCOX isoforms are genetically and functionally homologous to their mammalian orthologs. The zebrafish affords a tractable model system for the study of COX biology and development.
Insights
Zebrafish cyclooxygenase (COX) enzymes, zCOX-1 and zCOX-2, are homologous to human forms and crucial for development. Zebrafish offer a valuable model for studying COX biology and prostaglandin production.
Area of Science:
- Molecular Biology
- Developmental Biology
- Pharmacology
Background:
- Cyclooxygenase (COX) research is limited by COX-2's role in mammalian reproduction and organogenesis.
- Zebrafish (Danio rerio) present a potential model system to overcome these limitations.
- Understanding COX function is vital for developmental and physiological processes.
Purpose of the Study:
- To characterize cyclooxygenase (COX) expression and function in zebrafish.
- To compare zebrafish COX isoforms (zCOX-1 and zCOX-2) with their mammalian orthologs.
- To establish zebrafish as a model for studying COX biology.
Main Methods:
- Cloning of full-length zebrafish COX-1 and COX-2 cDNAs.
- Chromosomal assignment of zCOX genes.
- Mass spectrometry for prostaglandin product identification.
- Pharmacological inhibition studies using selective inhibitors.
- Gene knockdown experiments (morpholino) to assess functional roles.
- Whole-mount in situ hybridization for expression pattern analysis.
Main Results:
- Zebrafish COX-1 and COX-2 proteins show 67% homology to human orthologs.
- Prostaglandin E2 is the primary product of zebrafish COX activity.
- zCOX-1 inhibition affects zebrafish thrombocyte aggregation and hemostasis, while zCOX-2 inhibition does not.
- zCOX-1 and zCOX-2 are widely expressed during zebrafish development.
- zCOX-1 knockdown leads to growth arrest in early embryogenesis.
- zCOX-1 expression is prominent in embryonic vasculature; zCOX-2 has a more restricted pattern.
Conclusions:
- Zebrafish COX-1 and COX-2 isoforms are genetically and functionally homologous to mammalian orthologs.
- Zebrafish provide a tractable and valuable model system for investigating COX biology and its role in development.
- Differential roles of zCOX-1 and zCOX-2 in zebrafish physiology and development are highlighted.