Reprimo tissue-specific expression pattern is conserved between zebrafish and human

Abstract

Reprimo (RPRM), a member of the RPRM gene family, is a tumor-suppressor gene involved in the regulation of the p53-mediated cell cycle arrest at G2/M. RPRM has been associated with malignant tumor progression and proposed as a potential biomarker for early cancer detection. However, the expression and role of RPRM, as well as its family, are poorly understood and their physiology is as yet unstudied. In this scenario, a model system like the zebrafish could serve to dissect the role of the RPRM family members in vivo. Phylogenetic analysis reveals that RPRM and RPRML have been differentially retained by most species throughout vertebrate evolution, yet RPRM3 has been retained only in a small group of distantly related species, including zebrafish. Herein, we characterized the spatiotemporal expression of RPRM (present in zebrafish as an infraclass duplication rprma/rprmb), RPRML and RPRM3 in the zebrafish. By whole-mount in situ hybridization (WISH) and fluorescent in situ hybridization (FISH), we demonstrate that rprm (rprma/rprmb) and rprml show a similar spatiotemporal expression profile during zebrafish development. At early developmental stages rprmb is expressed in somites. After one day post-fertilization, rprm (rprma/rprmb) and rprml are expressed in the notochord, brain, blood vessels and digestive tube. On the other hand, rprm3 shows the most unique expression profile, being expressed only in the central nervous system (CNS). We assessed the expression patterns of RPRM gene transcripts in adult zebrafish and human RPRM protein product in tissue samples by RT-qPCR and immunohistochemistry (IHC) staining, respectively. Strikingly, tissue-specific expression patterns of the RPRM transcripts and protein are conserved between zebrafish and humans. We propose the zebrafish as a powerful tool to elucidate the both physiological and pathological roles of the RPRM gene family.

Expression of RPRM/rprm in the Olfactory System of Embryonic Zebrafish (Danio rerio)

“…Our study, together with previous evidence (Figures 1 , 2 and Figueroa et al, 2017 ), show that the rprma transcript is prominently expressed in the PNS at the OP/OS. To investigate whether RPRM proteins are expressed in different subsets of cells within the peripheral and central nervous system (PNS/CNS), we performed IF coupled with confocal microscopy using a rabbit polyclonal anti-human RPRM antibody, which was generated against an immunogenic 13 amino acidic sequence that is partly conserved between zebrafish and human (Figure 3 ).…”

“…At 72 hpf, rprmb transcript is now located at the TeO and Tg (Figures 2K,L ). (Figueroa et al, 2017 ). The spatiotemporal expression patterns for rprma, rprmb and rprml indicate that these genes are expressed differentially during zebrafish neural development (Figures 1 , 2 ).…”

“…Our previous studies in human revealed that RPRM protein was expressed in the brain (Figueroa et al, 2017 ). Moreover, mice embryos showed strong RPRM transcriptional expression in the OE, using ISH and/or the mouse reporter transgenic line TG(BAC-180MB-RPRM-EGFP) .…”

“…Although RPRM genes have a function during the cell cycle and the neurons in general does not experience cell division, their expression in the adult nervous tissues could be seen as a protective measure within this organ (Zohrabian et al, 2007 ). More recently, we determined the RPRM expression profiles during embryonic development using zebrafish as a model system (Figueroa et al, 2017 ). In agreement with our in silico analysis, RPRM genes are expressed in the developing brain but also in digestive tube and blood vessels (Figueroa et al, 2017 ).…”

“…Recently, we showed that RPRM / rprm genes are expressed in the zebrafish brain and that the expression domains are conserved between fish and mammals (Figueroa et al, 2017 ). Since teleost fish underwent an extra round of whole-genome duplication, zebrafish have retained duplicated copies of RPRM genes: rprma and rprmb (Wichmann et al, 2016 ).…”