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Updated: Mar 13, 2026
High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function
Published on: October 19, 2013
The zebrafish homologs of SET/I2PP2A oncoprotein: expression patterns and insights into their physiological roles
Iliana Serifi1,2, Eleni Tzima1,2, Katerina Soupsana1,2
1Laboratory of Biological Chemistry, Medical School, University of Ioannina, Ioannina, Greece.
Abstract:
The oncoprotein SET/I2PP2A (protein phosphatase 2A inhibitor 2) participates in various cellular mechanisms such as transcription, cell cycle regulation and cell migration. SET is also an inhibitor of the serine/threonine phosphatase PP2A, which is involved in the regulation of cell homeostasis. In zebrafish, there are two paralogous set genes that encode Seta (269 amino acids) and Setb (275 amino acids) proteins which share 94% identity. We show here that seta and setb are similarly expressed in the eye, the otic vesicle, the brain and the lateral line system, as indicated by in situ hybridization labeling. Whole-mount immunofluorescence analysis revealed the expression of Seta/b proteins in the eye retina, the olfactory pit and the lateral line neuromasts. Loss-of-function studies using antisense morpholino oligonucleotides targeting both seta and setb genes (MOab) resulted in increased apoptosis, reduced cell proliferation and morphological defects. The morphant phenotypes were partially rescued when MOab was co-injected with human SET mRNA. Knockdown of setb with a transcription-blocking morpholino oligonucleotide (MOb) resulted in phenotypic defects comparable with those induced by setb gRNA (guide RNA)/Cas9 [CRISPR (clustered regularly interspaced short palindromic repeats)-associated 9] injections. In vivo labeling of hair cells showed a significantly decreased number of neuromasts in MOab-, MOb- and gRNA/Cas9-injected embryos. Microarray analysis of MOab morphant transcriptome revealed differential expression in gene networks controlling transcription in the sensory organs, including the eye retina, the ear and the lateral line. Collectively, our results suggest that seta and setb are required during embryogenesis and play roles in the zebrafish sensory system development.
Insights
Zebrafish seta and setb genes are crucial for sensory system development, regulating cell homeostasis and embryogenesis. Their loss-of-function leads to developmental defects and impacts sensory organ formation.
Area of Science:
- Developmental Biology
- Molecular Biology
- Genetics
Background:
- The oncoprotein SET/I2PP2A inhibits protein phosphatase 2A (PP2A), a key regulator of cell homeostasis.
- Zebrafish possess two paralogous set genes, seta and setb, encoding highly similar proteins.
Purpose of the Study:
- To investigate the expression patterns and functional roles of seta and setb during zebrafish embryogenesis.
- To elucidate the involvement of seta and setb in the development of sensory organs.
Main Methods:
- In situ hybridization and immunofluorescence for expression analysis.
- Antisense morpholino oligonucleotides and CRISPR/Cas9 for loss-of-function studies.
- Microarray analysis to assess transcriptomic changes.
Main Results:
- Seta and setb are co-expressed in sensory organs like the eye, ear, and lateral line system.
- Loss of seta and setb function caused increased apoptosis, reduced proliferation, and morphological defects.
- Knockdown resulted in fewer neuromasts and altered gene expression in sensory organ development pathways.
Conclusions:
- Zebrafish seta and setb genes are essential for proper embryogenesis and sensory system development.
- SET/I2PP2A function is critical for maintaining cell homeostasis and development in sensory organs.
