The expression analysis of Sfrs10 and Celf4 during mouse retinal development

Abstract

Processing of mRNAs including, alternative splicing (AS), mRNA transport and translation regulation are crucial to eukaryotic gene expression. For example, >90% of the gene in the human genome are known to undergo alternative splicing thereby expanding the proteome production capacity of a limited number of genes. Similarly, mRNA export and translation regulation plays a vital role in regulating protein production. Thus, it is important to understand how these RNA binding proteins including alternative splicing factors (ASFs) and mRNA transport and translation factors regulate these processes. Here we report the expression of an ASF, Serine-arginine rich splicing factor 10 (Sfrs10) and a mRNA translation regulation factor, CUGBP, elav like family member 4 (Celf4) in the developing mouse retina. Sfrs10 was expressed throughout postnatal (P) retinal development and was observed progressively in newly differentiating neurons. Immunofluorescence (IF) showed Sfrs10 in retinal ganglion cells (RGCs) at P0, followed by amacrine and bipolar cells, and at P8 it was enriched in red/green cone photoreceptor cells. By P22, Sfrs10 was observed in rod photoreceptors in a peri-nuclear pattern. Like Sfrs10, Celf4 was also observed in the developing retina, but with two distinct retinal isoforms. In situ hybridization (ISH) showed progressive expression of Celf4 in differentiating neurons, which was confirmed by IF that showed a dynamic shift in Celf4 localization. Early in development Celf4 expression was restricted to the nuclei of newly differentiating RGCs and later (E16 onwards) it was observed in the initial segments of RGC axons. Later, during postnatal development, Celf4 was observed in amacrine and bipolar cells, but here it was predominantly cytoplasmic and enriched in the two synaptic layers. Specifically, at P14, Celf4 was observed in the synaptic boutons of rod bipolar cells marked by Pkc-α. Thus, Celf4 might be regulating AS early in development besides its known role of regulating mRNA localization/translation. In all, our data suggests an important role for AS and mRNA localization/translation in retinal neuron differentiation.

Dysregulation of CELF4 Splicing Factor in Pancreatic Neuroendocrine Tumors Enhances Aggressiveness and Alters Mtor Pathway and Everolimus Response

“…These ndings on CELF4 are completely original, as only a marginal nding of unexplored signi cance has identi ed a methylation-related expression of this gene in PanNETs (24). Actually, while the role of CELF4 in alternative splicing and neurological pathophysiology is well established (20,(22)(23)(24)(25), its precise implication in cancer is still poorly understood. In colorectal cancer, bioinformatic analysis of open databases suggested a prognostic role involving an intronic variant (26), reviewed in (21), while, in endometrial cancer, CELF4 expression seems to be downregulated due to hypermethylation and may also provide prognostic information (28).…”

“…CELF4 (CUGBP ELAV-like family member 4) is one of the 6 members of the CELF family of RNA binding proteins associated with regulation of pre-RNA alternative splicing (20). Earlier studies on CELF4 expression were con icting, suggesting either a broad tissue expression or more restricted to nervous tissue (21), while reports on gene mutations and variants in humans and experimental studies on rodents associated this gene to neurological, neurodevelopmental, and behavioral defects (22)(23)(24)(25). To date, only a limited number of studies have linked CELF4 to cancer (colorectal and endometrial cancer, and glioma) (21,(26)(27)(28), but no reports have studied CELF4 in NETs.…”

Analysis of Transgene Expression

“…However, if the gene of interest is expressed in only a small subset of cells within a tissue or embryo, but its mRNA is relatively abundant within that small group of cells, then the in situ analysis would give an unequivocally positive signal on a few of the cells in the section, whereas other detection methods (with the exception of RT-PCR) might give a negative result. The combined use of in situ hybridization for transcript localization along with protein immunolocalization is a powerful method for gene expression analysis (Karunakaran et al, 2013).…”