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Updated: Jul 20, 2026
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.
Abstract:
Mammalian CYPHER (Oracle, KIA0613), a member of the PDZ-LIM family of proteins (Enigma/LMP-1, ENH, ZASP/Cypher, RIL, ALP, and CLP-36), has been associated with cardiac and muscular myopathies. Targeted deletion of Cypher in mice is neonatal lethal possibly caused by myopathies. To further investigate the role of cypher in development, we have cloned the zebrafish orthologue. We present here the gene, domain structure, and expression pattern of zebrafish cypher during development. Cypher was not present as a maternal mRNA and was absent during early development. Cypher mRNA was first detected at the 3-somite stage in adaxial somites, and as somites matured, cypher expression gradually enveloped the whole somite. Later, cypher expression was also found in the heart, in head and jaw musculature, and in the brain. We further identified 13 alternative spliced forms of cypher from zebrafish heart and skeletal muscle tissue, among them a very short form containing the PDZ domain but lacking the ZM (ZASP-like) motif and the LIM domains. Targeted gene knock-down experiments using cypher antisense morpholinos led to severe defects, including truncation of the embryo, deformation of somites, dilatation of the pericardium, and thinning of the ventricular wall. The phenotype could be rescued by a cypher form, which contains the PDZ domain and the ZM motif, but lacks all three LIM domains. These findings indicate that a PDZ domain protein is important for normal somite formation and in normal heart development. Treatment of zebrafish embryos with cyclopamine, which disrupts hedgehog signaling, abolished cypher expression in 9 somite and 15-somite stage embryos. Taken together, our data suggest that cypher may play a role downstream of sonic hedgehog, in a late stage of somite development, when slow muscle fibers differentiate and migrate from the adaxial cells.
Insights
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.
