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Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish
Published on: April 22, 2017
Protein O-mannosylation is necessary for normal embryonic development in zebrafish
Eriko Avsar-Ban1, Hisayoshi Ishikawa, Hiroshi Manya
1Department of Life Science, Mie University Graduate School of Bioresources, 1577 Kurimamachiya, Tsu, Mie 514-8507, Japan.
Glycobiology
|May 15, 2010
Summary
Two zebrafish protein O-mannosyltransferase (POMT) genes, zPOMT1 and zPOMT2, are essential for normal embryonic development. Both genes are required for full enzymatic activity, suggesting conserved O-mannosylation machinery across species.
Area of Science:
- Biochemistry
- Developmental Biology
- Genetics
Background:
- Protein O-mannosylation is a critical post-translational modification.
- The roles of specific O-mannosyltransferase (POMT) genes in vertebrate development are not fully understood.
- Zebrafish offer a powerful model for studying gene function during embryogenesis.
Purpose of the Study:
- To clone and characterize two zebrafish POMT genes, zPOMT1 and zPOMT2.
- To investigate the expression patterns and functional roles of zPOMT1 and zPOMT2 during zebrafish development.
- To determine the requirement of zPOMT1 and zPOMT2 for protein O-mannosyltransferase activity.
Main Methods:
- Cloning of zPOMT1 and zPOMT2 cDNAs.
- Quantitative gene expression analysis during zebrafish embryogenesis and in adult tissues.
- mRNA injection of EGFP-fused constructs to study mRNA distribution.
- Antisense morpholino oligonucleotide knockdown to assess gene function.
- Immunohistochemistry using anti-glycosylated alpha-dystroglycan antibody (IIH6).
- Enzymatic activity assays in human embryonic kidney 293T cells.
Main Results:
- zPOMT1 and zPOMT2 are expressed throughout zebrafish embryonic development and in adult tissues.
- Knockdown of zPOMT1 or zPOMT2 resulted in severe developmental phenotypes, including body bending and abnormal eye pigmentation.
- zPOMT2 knockdown exhibited more severe phenotypes than zPOMT1 knockdown, with loss of IIH6 reactivity in both.
- Full protein O-mannosyltransferase activity required co-expression of both zPOMT1 and zPOMT2, or heterologous combinations with human POMT genes.
Conclusions:
- zPOMT1 and zPOMT2 are essential for zebrafish embryonic development.
- Both zebrafish POMT genes are necessary for full protein O-mannosyltransferase enzymatic activity.
- The protein O-mannosylation machinery is conserved between zebrafish and humans, highlighting zebrafish as a valuable model for studying this process.
