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High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function
Published on: October 19, 2013
Zebrafish cypher is important for somite formation and heart development.
David L M van der Meer1, Ines J Marques, Jelani T D Leito
1Institute of Biology, Department of Integrative Zoology University of Leiden, Kaiserstraat 3, 2311 GN, The Netherlands.
Developmental Biology
|September 20, 2006
Summary
Zebrafish cypher, a PDZ-LIM protein, is crucial for embryonic development, somite formation, and heart development. Its expression is regulated by sonic hedgehog signaling, impacting muscle fiber differentiation.
Area of Science:
- Developmental Biology
- Molecular Genetics
- Cardiovascular Research
Background:
- Mammalian CYPHER (Oracle, KIA0613) is linked to cardiac and muscular myopathies, with its absence causing neonatal lethality in mice.
- Investigating the zebrafish orthologue of cypher is essential to understand its developmental roles.
Purpose of the Study:
- To clone and characterize the zebrafish cypher orthologue.
- To elucidate the gene structure, domain organization, and developmental expression pattern of zebrafish cypher.
- To investigate the functional significance of cypher in embryonic development and heart formation.
Main Methods:
- Cloning of zebrafish cypher orthologue.
- Analysis of gene structure, domain composition, and alternative splicing.
- Whole-mount in situ hybridization to determine expression patterns.
- Antisense morpholino-mediated gene knockdown to assess functional roles.
- Rescue experiments using specific cypher variants.
- Pharmacological inhibition of hedgehog signaling pathway.
Main Results:
- Zebrafish cypher mRNA is not maternally provided and first appears at the 3-somite stage in adaxial somites, later expanding to the entire somite, heart, head/jaw musculature, and brain.
- Thirteen alternative spliced forms of cypher were identified, including a short form with only a PDZ domain.
- Gene knockdown resulted in severe developmental defects: embryonic truncation, somite deformation, pericardial dilatation, and ventricular wall thinning.
- A rescue construct containing PDZ and ZM motifs, but lacking LIM domains, restored normal development.
- Disruption of hedgehog signaling abolished cypher expression, indicating its role downstream of this pathway.
Conclusions:
- Zebrafish cypher is vital for proper somite formation and heart development.
- A PDZ domain-containing protein is essential for these processes.
- Cypher functions downstream of sonic hedgehog signaling in late somite development, influencing muscle fiber differentiation and migration.
