Rem2 in the bullfrog (Rana catesbeiana): Patterns of expression within the central nervous system and brain expression at different ontogenetic stages

Abstract

Rem2 is a member of the RGK (Rem, Rad, and Gem/Kir) subfamily of the Ras superfamily of GTP binding proteins. In mammals, Rem2 has been found to be unique in not only its structure, but also its tissue specificity, as it is the first member to be found at high levels in neuronal tissue. Because Rem2 has previously been implicated in neuronal cell proliferation, and amphibians maintain relatively high neuronal proliferative activity as adults, we sought to isolate and acquire the full-length sequence of the rem2 gene from the brain of the bullfrog (Rana catesbeiana). Furthermore, we used real time PCR (rtPCR) to characterize its tissue specificity, regional brain expression, and brain expression levels at different stages of development. Deduced amino acid sequence analysis showed that the bullfrog Rem2 protein possesses the unique 5' extension characteristic of mammalian Rem2 and the RGK subfamily to which it belongs. Tissue specificity of the bullfrog rem2 gene showed that the bullfrog is similar to both mammals and fish in that the levels of rem2 gene expression were significantly greater in the brain than all other tissues assayed. In the brain itself, differential rem2 expression patterns were observed between six major brain areas assayed and the spinal cord, with expression significantly high in the cerebrum and low in the cerebellum. Finally, examination of whole brain rem2 expression levels in bullfrogs at different stages of development revealed greater expression after metamorphic climax.

Keywords: Amphibian; Development; GTPase; Gene.

Fig. 1

Nucleotide and deduced amino acid sequences of bullfrog rem2. The nucleotide sequence is shown 5’ to 3’ and numbered to the right. The deduced amino acid sequence is in the single letter amino acid code, in boldface, and numbered to the right in italics and boldface. The start and stop codons are shaded in dark gray and underlined. The location of primers used in the Real-Time PCR gene expression assays are underlined.

Fig. 2

Amino acid sequence alignment of the bullfrog Rem2 with the Rem2 proteins of other species from different representative vertebrate classes. Included in the alignment are the Rem2 proteins of different representative vertebrate classes, as well as the representatives of the RGK family (from Human). The Rem2 proteins, listed from top to bottom below the bullfrog Rem2 protein are those of X. tropicalis (predicted; GenBank RefSeq XP_002941555), the anole (predicted; GenBank RefSeq XP_003229372.1), the zebrafish (GenBank RefSeq NP_001116518.1), and human (GenBank RefSeq NP_775798.2). The RGK members other than Rem2 then includes human RAD (GenBank RefSeq NP_004156.1), human GEM (GenBank RefSeq NP_859053.1), and human REM (GenBank RefSeq NP_054731.2). Amino acids with black background and white letters indicate 100% identity, while those shaded with gray indicate additional identity with bullfrog Rem2. Asterisks (*) indicate phosphorylation sites (Correll et al., 2008, Ghiretti et al., 2013). Sites of 14-3-3 protein interaction are indicated by the arrow ( ) (Béguin et al., 2005a). Consensus sequences for GTP-binding regions (G1–G5) and the conserved C7 sequence motif are indicated below the alignments (Finlin et al., 2000). The G3 consensus shown is that which is unique to the RGK subfamily. Bold lines below the alignment indicate N-terminal and C-terminal extensions in mammalian Rem2 (Splingard et al. 2007). The boxed amino acids contain the putative C-terminus nuclear localization sequence (NLS) based on Ghiretti et al. (2013). Amino acids are numbered to the right. Percent identity of bullfrog Rem2 with each sequence, indicated at the end of each sequence, is based on individual pairwise alignments.

Fig. 3

Tissue specificity of bullfrog rem2 using Real-Time PCR. Relative expression of the bullfrog rem2 gene was significantly different between the different tissues (One-way ANOVA; P < 0.0001). Relative expression of rem2 in the brain was significantly greater than its expression in all other tissues assayed (*: Tukey's multiple comparison; P < 0.001).

Fig. 4

Regionalization of relative rem2 expression in the bullfrog central nervous system using Real-Time PCR. The schematic of the bullfrog brain in dorsal view below the figure indicates each region assayed. The bullfrog rem2 gene showed differential expression in the central nervous system (One-way ANOVA; P < 0.0001). The relative expression of rem2 in the cerebrum was significantly greater than its expression in all other areas assayed (***: Tukey's multiple comparison; P < 0.001), as was the case for the diencephalon (**: P < 0.05), excepting being lower than the cerebrum. The midbrain had significantly greater rem2 expression levels than that of the midbrain, hindbrain, and spinal cord (*: P < 0.05). Abbreviations: OB = Olfactory bulb, CR = Cerebrum, DI = Diencephalon, MB = Midbrain, CB = Cerebellum, HB = Hindbrain, SC = Spinal cord.

Fig. 5

Relative rem2 expression in the bullfrog whole brain at different ontogenetic stages before and after metamorphic climax using Real-Time PCR. Fig. 5A The bullfrog rem2 gene was differentially expressed in the brain between the different ontogenetic stages of life (One-way ANOVA; P < 0.05). Relative expression of brain rem2 was not significantly different between any one pair of ontogenetic stages (Tukey's multiple comparison). A post test for linear trend in rem2 relative gene expression was significant (P < 0.05). Fig 5B Data from Panel A were grouped into pre-metamorphic climax stages (stages I-XV) and post-metamorphic climax stages (juvenile and adult) and analyzed for significance by t-test. Relative bullfrog brain rem2 gene expression was significantly higher post-metamorphic climax (P < 0.05; asterisk). Stages are defined in the Methods.