The zebrafish homologs of SET/I2PP2A oncoprotein: expression patterns and insights into their physiological roles during development

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.

Targeting of SET/I2PP2A oncoprotein inhibits Gli1 transcription revealing a new modulator of Hedgehog signaling

“…1 B). Similar results were obtained upon targeting both seta and setb genes in the transgenic embryos with a translation blocking morpholino (MOab) 36 (Fig. 1 S).…”

“…Previous work of our lab has shown that the zebrafish homologs of SET oncoprotein are expressed during early zebrafish development and localized in the ciliated organs of zebrafish, suggesting a potential function of SET in signaling cascades coordinated by cilia 36 . This study also demonstrated that targeting setb gene expression by injections of setb gRNA with Cas9 mRNA or seta/b morpholinos into one-cell stage zebrafish embryos resulted in several morphological abnormalities, including defects in spinal cord and eye development 36 . A systematic scoring of the eye defects in setb crispants showed that approximately 80% of the larvae with severe phenotype and 33% of the larvae with moderate phenotype exhibited cyclopia (Fig.…”

“…The generation of multiple Set knockdown clones and no clone with complete SET loss in our CRISPR experiment may indicate that mouse Set genes are essential and therefore their complete knockout was not feasible. Nevertheless, SETKD cell lines represent a cell-based experimental system complementary to zebrafish morphants and crispants that also express reduced protein levels of the zebrafish Seta/b proteins 36 .…”

Effects of the Essential Oil from Pistacia lentiscus Var. chia on the Lateral Line System and the Gene Expression Profile of Zebrafish (Danio rerio)

“…After fixation, larvae were washed twice with PBS, transferred to 100% methanol, and stored at −20 °C. Acetylated tubulin staining was performed as previously described [58].…”

CRISPR-Cas Genome Surgery in Ophthalmology

“…54,55 For example, Serifi et al 56 combined CRISPR and morpholino techniques and found that the Setb gene in zebrafish, a paralog of the human SET/I2PP2A gene, has a role in the development of the retina. 56 Another example of using SpCas9-generated knockouts and morpholinos together was shown by Taylor et al, 57 who targeted a notch-signaling pathway protein, NeuroD, to show its important role in photoceptor regeneration.…”