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. Author manuscript; available in PMC: 2015 Apr 25.
Published in final edited form as: Gene. 2014 Feb 24;540(1):37–45. doi: 10.1016/j.gene.2014.02.030

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

Megan M DeRocher 1, Faris H Armaly 1, Cara J Lepore 1, David M Hollis 1,†
PMCID: PMC4005034  NIHMSID: NIHMS570342  PMID: 24576576

1. Introduction

The RGK (Rem, Rad, and Gem/Kir) subfamily of small molecule GTPases belong to the Ras superfamily of GTP binding proteins. The RGK proteins are recognized by a number of characteristics that distinguish them among the Ras superfamily, which includes N-terminal and C-terminal extensions beyond the Ras core as well as a unique G3 motif (Finlin et al., 2000), regulation at the transcriptional level (Maguire et al., 1994; Cohen et al., 1994; Vanhove et al., 1997; Leone et al., 2001; Suyama et al., 2003; Ohsugi et al., 2004; Warton et al., 2004; Finlin et al., 2005; Paradis et al., 2007), a lack of lipid modification for membrane anchorage (Del Villar et al., 1996), and binding of calmodulin and 14-3-3 protein (Béguin et al., 2005a, 2005b, 2006). Functionally, the RGK proteins are associated with modulation of Ca2+ currents (Seu and Pitt, 2006), as well as changes in cytoskeletal arrangement (Leone et al., 2001; Béguin et al., 2005a, Fu et al., 2005; Krey et al., 2013).

Among the RGK family, Rem2 is further distinguished by its high expression levels in neural tissue (Finlin et al. 2000). Within neural tissue Rem2 plays a role in very diverse functions including excitatory and inhibitory synapse development (Paradis et al., 2007; Ghiretti and Paradis, 2010), survival of embryonic stem cells (Edel et al., 2010a, 2010b), and regulation of dendrite morphology (Ghiretti and Paradis, 2010, Ghiretti et al., 2014). Like other RGK members, Rem2 has been implicated in Ca2+ current modulation (Chen et al., 2005, Finlin et al., 2005, Seu and Pitt, 2006, Leyris et al., 2009, Yang et al., 2012).

In mammals, Rem2 has been well-characterized with the original clone of mammalian Rem2 described in rat (Finlin et al., 2000). There is a high level of conservation among the Ras core of Rem2 with other members of the RGK subfamily. However, the extended N-terminus of Rem2 appears distinct and may be responsible for functions unique from other RGK subfamily members (Seu and Pitt, 2006). Across vertebrate classes, Rem2 appears conserved as it has been isolated in two species of fish (Edel et al., 2010b; Hollis et al., 2012). Rem2 has also been isolated in amphibians Xenopus laevis, X. tropicalis (Klein et al., 2002). However, unlike in fish, its characterization including tissue specificity in amphibians has yet to be elucidated. A comparative examination of Rem2 gene expression has shown that in both mammals and fish, the pattern of high Rem2 expression in the brain relative to other tissues is conserved, yet the general distribution of Rem2 in the central nervous system between these vertebrate classes is very distinct. In mammals (mice), expression of Rem2 appears limited to the forebrain (Becker et al., 2008), however, in the brains of adult fish it has been found in every major region of the brain as well as the spinal cord in (Hollis et al., 2012). In both mammals and fish, Rem2 has been shown to influence the central nervous system regarding neural tissue development, synapse formation, cell proliferation and apoptosis, and stem cell survival (Paradis et al., 2007; Edel et al., 2010a, 2010b; Ghiretti and Paradis, 2010). Unlike mammals, adult fish possess an enormous capacity for neurogenesis and brain repair (Zupanc, 1999) and have zones of cell proliferation throughout the brain and spinal cord (Zupanc et al., 2005). Mammals on the other hand, have zones of proliferation limited to within restricted regions of the telencephalon (reviewed in Ferretti, 2011). Thus, when looking at the comparative brain cell proliferation and plasticity, an important question is what molecular components promote widespread adult neurogenesis in some species rather than others? Whether the distribution of Rem2 expression has any reflection on these abilities in the vertebrate brain is unknown.

Like fish, adult anuran amphibians are a vertebrate class with known zones of cell proliferation in the telencephalon, diencephalon and mesencephalon and, to a much lesser extent, in the rhombencephalon (Raucci et al., 2006; Almli and Wilczynski, 2007; Simmons et al., 2008). However, unlike fish (and urodele amphibians), the adult anuran amphibian central nervous system appears to lack neurogenesis in the spinal cord and also lacks the capacity for spontaneous regeneration overall (Yoshino and Tochinai, 2004; Endo et al., 2007). To further examine rem2 expression for phylogenetic analysis, this study characterized Rem2 and examined its gene expression in the adult bullfrog (Rana catesbeiana). Furthermore, because there is an ontogenetic decline in the ability of the central nervous system to regenerate in anuran amphibians, (Sims, 1962; Forehand & Farel, 1982; Beattie et al., 1990; Ten Donkelaar, 2000), we also examined rem2 expression in the brain of the bullfrog at different stages of development. The present study characterizes Rem2 in an anuran amphibian species, the bullfrog. Furthermore, we examine the tissue specificity for the amphibian rem2 gene as well as its expression profile across major regions of the central nervous system and finally its relative brain expression levels at different stages of development.

2. Materials and methods

2.1 Animals

Bullfrogs at different stages of life were obtained in the spring (mid-March) from The Sullivan Company (Nashville, TN), and additional adults were purchased in the late spring (early June) from both The Sullivan Company and Ward’s Science (Rochester, NY). The life stages were based on observations in Ranids (R. pipiens) as described in Rugh (1951), while the boundary for metamorphic climax was based on Duellman and Trueb (1986) and Shi (2000). In brief, metamorphic climax was defined as the period when a dramatic transformation of tadpole organs into their adult form occurs, including resorption and/or degeneration of tadpole-specific structures (such as the tail and larval mouthparts), as well as completed development of adult-specific tissues such as the limbs an jaw. The life stages obtained were divided into tadpole stages before metamorphic climax, which included those with no limbs, or hind limb buds with a foot paddle, but no distinction of digits (between Stages I-VII) and those tadpoles with small hind limbs and distinguishable digits on the foot paddle (Stages X-XV). The latter stages have yet to develop fore legs. The stages after metamorphic climax included small, fully metamorphosed juveniles (mass less than 40g) and large adults (mass between 300 and 575g). Immediately upon arrival, bullfrogs were anesthetized in 10% ethyl 4-aminobenzoate (benzocaine) and then sacrificed by rapid decapitation.

2.2 Tissue collection and RNA isolation

After sacrifice, tissues were collected, weighed, and immediately homogenized in QIAzol® Reagent (QIAGEN) for total RNA isolation using the QIAzol® RNeasy Lipid Tissue Mini Kit (QIAGEN) along with the RNase-Free DNase Set (QIAGEN) to remove genomic DNA. For the isolation of the full-length bullfrog rem2 gene, whole brain tissue was collected. For tissue specificity of the bullfrog rem2 gene, tissue was collected from six bullfrogs and included the brain, liver, stomach, intestine, skeletal muscle, spleen, lung, ventricle, conus arteriosus, pancreas, and kidney. For the regional distribution of gene expression of the rem2 genes in the bullfrog central nervous system, brains were removed from six bullfrogs and the olfactory bulb, cerebrum, diencephalon, midbrain, cerebellum, and hindbrain were isolated from each as was a portion of the spinal cord.

2.3 Molecular cloning and sequencing of full-length rem2 genes

Bullfrog RNA was reverse transcribed from one microgram of total RNA using Improm II Reverse Transcriptase (Promega). The cycling conditions were 25°C (5 min), 42°C (60 min), and 70°C (15 min). Samples were stored at −20°C. To initially isolate a fragment of the bullfrog rem2 transcript using end point PCR, primers were designed based on the predicted rem2 sequence of another anuran amphibian, Xenopus tropicalis (GenBank RefSeq XM_002941509.1). Primers used in this and all subsequent assays are listed in Table 1. The cycling conditions for end point PCR were 94°C (5 min) followed by 35 cycles of 94°C (30 sec), 60°C (30 sec), 72°C (1 min). Sequence analysis (described below) of the initial isolated fragment was then used to design bullfrog rem2 gene-specific primers which were paired with primers that were, again, specific to the same predicted rem2 sequence of X. tropicalis for use in standard end point PCR to acquire a larger bullfrog rem2 fragment. After isolating this larger fragment of the bullfrog rem2 gene (verified through sequence analysis), 3’ and 5’ RACE assays were performed using the RNA ligase-mediated Rapid Amplification of cDNA Ends (RLM-RACE) Amplification kit (BD Biosciences) according to the manufacturer's instructions. Four primers were designed (primary and nested) and the full-length bullfrog rem2 gene was isolated. The RLM-RACE cycling conditions were 94 °C (3 min) followed by 35 cycles of 94 °C (30 s), 59 °C (30 s) for 3’Race and 60°C (30 s) for 5’Race, 72 °C (1 min), followed by 72 °C (7 min). Finally, verification of the full length open reading frame (ORF) was performed using primers specific for the extreme 5’ and 3’ end of the ORF.

Table 1.

Primers and the assays in which they were used.

Assay (Usage)
    Primer Sets 		Primer sequence
End point PCR (Isolate bullfrog rem2 fragment)
    Degenerate primer (forward) 5′-AGTATGCCCTTGCCTTACAAGCACCAGCT-3′
    Degenerate primer (reverse) 5′-TCACCTTTATTGCCAACAAGGATGATGGG-3′
End point PCR (Extend isolated fragment)
    Gene specific primer (forward)* 5′-ACTGGAGTAGGAAAGACAACACTTGCT-3′
    Gene specific primer (reverse) * 5′-TGACAAGACTTCGATCTTTGCTTGAAGA-3′
Nested RLM-RACE PCR (Obtain full-length sequence)
    rem2 3’ RACE (forward-outer) 5′-TGTCCATTCAGGGTCATCAGACTCT-3′
    rem2 3’ RACE (forward-inner) 5′-AGTAGGAAAGACAACACTTGCTGGGA-3′
    rem2 5’ RACE (reverse-inner) 5′-TCCCAGCAAGTGTTGTCTTTCCTACT-3′
    rem2 5’ RACE (reverse-outer) 5′-AGAGTCTGATGACCCTGAATGGACA-3′
End point PCR (Obtain full-length ORF)
    rem2 (forward) 5′-ATCTGTCACAGAGAAGACCCAGGCAGAAGA-3′
    rem2 (reverse) 5′-TGGCATGATTTCGACCTCTGCTTGAAGA-3′
Real-Time PCR (Relative gene expression levels)
  rem2
    Gene specific primer (forward) 5′-TCTGCTCGTCCACACCGC-3′
    Gene specific primer (reverse) 5′-GAGACATCTGCTGCCCTTCACC-3′
  rpl8 (Reference gene)
    Gene specific primer (forward) 5′-GCTGTTGACTTCGCAGAAAGGCAT-3′
    Gene specific primer (reverse) 5′-ATGGATACCCTCAGCCGCAATGA-3′
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*

Paired with a primer specific to the predicted X. tropicalis rem2 nucleotide sequence (GenBank RefSeq XM_002941509.1)

For cloning, PCR products were separated on agarose gels, visualized under UV light and the appropriate sized band was cut and cloned into TOPO pCR 2.1 (Invitrogen). Plasmid preparations were made from randomly selected positive colonies which were then prepped for sequencing using the Purelink Quick Plasmid Mini Prep Kit (Invitrogen). Sequencing of the isolated DNA was performed on an automated sequencer (ABI 3730) at the Arizona State University DNA Laboratory (Tempe, AZ, USA). Each product was cloned and sequenced a minimum of 20 times. Four to five additional sequences were also obtained by directly sequencing DNA from PCR reactions when a single product of expected size was acquired. The DNA was purified directly from the PCR reaction using the ExoSAP-It kit (USB/Affymetrix) following the manufacturers’ instructions. If multiple transcripts were present in gel electrophoresis analysis, the appropriate sized band was cut and gel purified using the QIAquick Gel Extraction kit (QIAgen) following the manufacturers’ instructions. Direct sequencing of the isolated DNA was also performed on an automated sequencer (ABI 3730) at the Arizona State University DNA Laboratory.

Sequence files were analyzed locally using the Basic Local Alignment Search Tool (BLAST: http://blast.ncbi.nlm.nih.gov/Blast.cgi), specifically, blastx against the NCBI nonredundant (nr) protein database and blastn against the NCBI nucleotide (nt) database. Nucleotide and deduced amino acid alignments were performed using an online ClustalW2 alignment program (http://www.ebi.ac.uk/Tools/msa/clustalw2/).

2.4 Gene expression analysis

For tissue specificity, tissue samples, including whole brains, were taken from six individual adult bullfrogs. Likewise, for the central nervous system distribution assay, the brains of six individual adult bullfrogs were each portioned into the major regions previously mentioned. For the ontogenetic assay, whole brains were acquired from three individual tadpoles or frogs from each developmental stage. Gene-specific primers were used for real time PCR (rtPCR) (see Table 1). rtPCR was carried out following the manufacturer’s instructions for Power SYBR Green RNA-to-Ct 1-Step kit rtPCR method (Applied Biosystems). A total of 60ng of total RNA was used per 20µl reaction (3ng/µl per reaction). All reactions were performed in triplicate. Reverse transcription was carried out using ArrayScript™ UP Reverse Transcriptase (Applied Biosystems) and the generated cDNA was used as a template for PCR amplification. The cycling conditions were, for the reverse transcription, 48°C (30 min), which was then followed by PCR consisting of 95°C (10 min), then 40 cycles of 95°C (15 sec), and 60°C (1 min). Reverse transcription and PCR amplification were performed on an Eppendorf Mastercycler® ep realplex2 thermal cycler. Gene expression was normalized using a 154bp fragment of the ribosomal protein L8 (rpl8) gene using gene-specific primers following the methods of Hasebe et al. (2008) and Bilesimo et al. (2011). Like rem2, expression of rpl8 was performed using real-time PCR following the manufacturer’s instructions for the Power SYBR Green RNA-to-Ct 1-Step kit rtPCR method (Applied Biosystems). The cycling conditions for rpl8 were as those for rem2. Standard curves for both rem2 and rpl8 were generated from total RNA of bullfrog brain tissue. Individual 20µl reactions ranged from 10ng to 5000ng in total RNA (2ng/µl to 250ng/µl) performed in triplicate, which were used to compute expression values for both genes.

Statistical significance for differences in expression levels across tissues, central nervous system regions, and stages of life was evaluated by One-Way ANOVA (P < 0.05) followed with either Tukey’s multiple comparison test (P < 0.05) or post-test for linear trend (P < 0.05), as well as t-test (P < 0.05). All statistics were performed using GraphPad Prism software.

3. Results

3.1 Molecular cloning and sequencing of the full-length bullfrog rem2 gene

The full-length bullfrog rem2 gene was isolated by cDNA cloning of PCR products from 5’-RACE and 3’-RACE and had an ORF of 876bp (Fig. 1). BLAST searches (blastx) in the GenBank database resulted in a deduced amino acid sequence having highest identity (80%) with that of the predicted X. tropicalis Rem2 protein (GenBank RefSeq XP_002941555.1) followed by the Rem2 proteins of different vertebrates, of which shared between 55% and 63% identity. Thus, the nucleotide sequence coded for an amphibian Rem2 protein and therefore, was named R. catesbeiana rem2 (GenBank RefSeq KJ026766). The full-length cDNA sequence of bullfrog rem2 was 1299bp, including a 162bp 5’ untranslated region (UTR), an 876bp ORF, and a 261bp 3’ UTR (excluding the poly (A)+ tail).

Fig. 1.

Fig. 1

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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.

3.2 Deduced amino acid sequence alignment

The bullfrog Rem2 protein consisted of 292 deduced amino acids (see Fig. 1). When aligned in pairwise comparisons with the mammalian RGK subfamily members (using those of Homo sapiens), the bullfrog Rem2 protein aligned most closely with that of REM2 (61.3% identity. GenBank RefSeq NP_775798.2), as compared to RAD (47.6%. GenBank RefSeq number NP_004156.1), GEM (45.2%. GenBank RefSeq NP_859053.1), or REM (43.8%. GenBank RefSeq NP_054731.2). The bullfrog Rem2 protein shares conservation with the Ras superfamily G1, G3, G4, and G5 GTP binding sites, but like the RGK subfamily, lacks the threonine residue of the G2 GTP binding domain that is conserved throughout the rest of the Ras superfamily (Thr35 in H-RAS; Colicelli, 2004) (Fig. 2). In contrast, RGK subfamily members are distinct from the Ras superfamily in that they possess altered residues (Glu84 in GEM, Pro89 in REM and Pro100 in RAD, and Ser123 in REM2) of the residue equivalent to the highly conserved Gly12 in H-RAS within the G1 domain (Colicelli, 2004; Correll et al. 2008). Consistent with this, rather than a glycine residue, the bullfrog Rem2 protein possesses a threonine (Thr81) in this position when aligned with mammalian (Human) Rem2, as does the Rem2 proteins of other non-mammalian vertebrates as seen in that of the zebrafish Rem2 protein (Asn83) and the predicted anole Rem2 protein (Ser66) (see Fig. 2). Furthermore, like the RGK members, within the G3 box, where the Ras superfamily possess a conserved glycine (Gly60 in H-RAS) the bullfrog Rem2 protein possesses a glutamic acid residue (Glu130). Thus, while bullfrog Rem2 protein shares the conserved DXWE G3 motif with the rest of the RGK subfamily members, it is divergent from the consensus DTAGQ motif found in the rest of the Ras superfamily. However, the bullfrog does possess the Ras superfamily consensus glutamine (Q: Gln131) as seen with mammalian Rem2 (Gln173) and Rad (Gln148). The Rem2 proteins of other vertebrates also follow this pattern (zebrafish; Gln143, predicted anole; Gln116; see Fig. 2).

Fig. 2.

Fig. 2

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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.

Within the N-terminal extension of the RGK members, the initial four amino acids of the bullfrog, Met1Thr2Leu3Asn4, are conserved with those of the predicted Rem2 protein of Xenopus as well as human RAD, GEM, and REM. However, unlike the other members of the RGK family, the corresponding amino acids of human REM2 are preceded by an additional 37 amino acids and only the threonine and leucine (Thr39Leu40) are conserved. Furthermore, the predicted anole and zebrafish Rem2 proteins lack homology with regard to the initial four amino acids on the N-terminal end with the exception of the initial methionine. In contrast, in the alignment between the bullfrog Rem2 protein and the Rem2 proteins of the other vertebrates, there is high homology in the N-terminal extension between the bullfrog Rem2 residues Arg27 and Pro51. Pairwise alignments of this region of the bullfrog Rem2 protein with those of the other vertebrate Rem2 proteins (alignments not shown) indicated higher identity than the pairwise comparisons of the entire proteins (predicted Xenopus; 92%, predicted anole; 83%, zebrafish; 72%, human, 96%). On the other hand, identity between this same region of the bullfrog Rem2 protein and the other RGK family members either did not deviate much from the pairwise comparisons of the entire protein (RAD; 46%), or was much lower (GEM; 26%, REM; 23%). On the C-terminal end, the bullfrog Rem2 protein conformed to the conserved CHXLXVL C7 motif with the rest of the RGK subfamily members.

3.3 Tissue specificity of the bullfrog rem2 gene

The pattern of expression of the bullfrog rem2 gene showed the brain as having significantly greater levels of expression than any other tissue (Fig. 3). Relative to the brain, all tissues were extremely low in rem2 expression with no other tissue significantly greater or lower than any other tissue (save for the brain). After the brain, the kidney and the pancreas showed the highest levels of rem2 expression, again, though not significant. The lowest level of expression observed was in the skeletal muscle tissue.

Fig. 3.

Fig. 3

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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).

3.4 Central nervous system distribution of the bullfrog rem2 gene

In general, there was differential expression of the rem2 gene between the different regions of the bullfrog central nervous system (P < 0.0001). The level of expression for the rem2 gene in the central nervous system was highest in the more anterior regions with the cerebrum showing significantly higher levels of expression than any other region (P < 0.001; Fig. 4). Apart from the cerebrum, the diencephalon also had significantly greater levels of rem2 expression than the other regions (P < 0.001, excepting the midbrain at P < 0.05). In contrast, the cerebellum, hindbrain, and spinal cord had very low levels of rem2 expression. The midbrain and olfactory bulb had similar levels of rem2 expression, however, only the midbrain had significantly greater levels when compared to the very low levels in the cerebellum, hindbrain, and spinal cord (P < 0.05).

Fig. 4.

Fig. 4

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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.

3.5 Brain expression of rem2 at different ontogenetic stages

Because anurans have distinct stages of development, we examined the levels of rem2 gene expression in the whole brains of these animals at different stages of development (as was able to be provided) to examine whether brain rem2 expression levels change as the frogs metamorphose (Fig. 5). A significant change in brain rem2 gene expression was observed throughout the different stages assayed (P < 0.05; Fig. 5A). Furthermore, there was a significant linear trend identified as well (P < 0.05; Fig. 5A). In this case, the tadpole stages, which had similar levels of rem2 expression, had lower levels of rem2 expression relative to those of the fully metamorphosed juveniles and adults. When the different stages were re-grouped into two separate stages based on stage relative to metamorphic climax, in other words, pre-metamorphic climax (I-VII and X-XV grouped) versus post-metamorphic climax (juveniles and adults grouped), there was a significant increase in rem2 expression levels at post-metamorphic climax (t-test, P < 0.05; Fig. 5B).

Fig. 5.

Fig. 5

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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.

4. Discussion

4.1 Molecular cloning and sequencing of the full-length bullfrog rem2 gene

In this study, we isolated, cloned, and characterized a rem2 gene from the adult bullfrog brain. The Rem2 protein was first described in mammals (Finlin et al., 2000), and to date, has been identified in a large number of other mammalian species. Furthermore, the rem2 gene has also been isolated and characterized in teleost fish (Edel et al., 2010b, Hollis et al., 2012). Recent work isolated the rem2 gene from the brain of another anuran amphibian, X. laevis (Ghiretti et al. 2014). Using RACE PCR, we acquired both the 5’ and 3’ ends of the bullfrog rem2 gene and to our knowledge, are the first to report the full-length sequence of the rem2 gene from the bullfrog central nervous system.

4.2 The deduced amino acid sequences of bullfrog Rem2

At the deduced amino acid level, bullfrog Rem2 was highly conserved, sharing conservation with the Ras superfamily G1, G3, G4, and G5 GTP binding sites, and like the RGK subfamily, it lacks the threonine residue of the G2 GTP binding domain that is conserved throughout the rest of the Ras superfamily (Thr35 in H-RAS; Colicelli, 2004). Previous work in non-mammals, specifically fish, has shown a deviation from a very highly conserved alanine found within the G5 GTP binding domain (Edel et al., 2010b, Hollis et al., 2012). This was not the case with the bullfrog (nor is this identified in the predicted proteins of Xenopus and the anole). Thus, deviation of this nucleotide in Rem2 proteins deduced from isolated genes so far remains within fish.

The Rem2 protein possesses N-terminal and C-terminal extensions that flank the Ras-homologous G domain (Finlin et al., 2000). These extensions are characteristic of the RGK subfamily. It has been speculated that the effects of mammalian Rem2 upon Ca2+ channel kinetics is due to its extended N-terminus and C-terminus (Seu and Pitt, 2006), while the C-terminal end possesses a functional Ca2+/CaM binding site (Finlin et al., 2005). Thus, these extensions may provide further functional distinctions among RGK proteins yet to be elucidated. The bullfrog Rem2 protein possessed both extensions. With regard to the N-terminal extension, that of mammals is unique compared to that of other RGK subfamily members in that it is extended with signal transduction binding sites, which likely allows Rem2 to be a transcription regulator as is speculated in its ability to hold human embryonic stem cells in a state of pleuropotency by blocking p53 and cyclinD1 transcription (Edel et al., 2010a). Within the N-terminus of the bullfrog Rem2 protein there is a specific region of high homology with only the Rem2 proteins of the other vertebrates (from Arg27 to Pro51), but is lacking with the other RGK members. Within this region resides a phosphorylation site (Correll et al., 2008; Ghiretti et al., 2013), as well as a site of 14-3-3 protein interaction (Béguin et al., 2005a). Whether other functional regions reside in this area remains to be elucidated.

Like the deduced amino acid sequences of Rem2 of other non-mammalian vertebrates, the N-terminal extension of the bullfrog Rem2 deduced amino acid sequence lacks a large portion of the mammalian Rem2 N-terminal extension, as based on Seu and Pitt (2006). In the case of our alignment, the mammalian Rem2 deduced amino acid sequence precedes the bullfrog amino acid sequence on the 5’ end by 37 residues. This makes up the largest regional amino acid number difference between the two, as mammalian Rem2 has a total of 48 additional residues compared to the bullfrog. This discrepancy occurs just upstream of the aforementioned, highly conserved regions of the Rem2 proteins.

On the C-terminal end, the bullfrog Rem2 protein conformed to the conserved CHXLXVL C7 motif with the rest of the RGK subfamily members, while the C-terminal phosphorylation sites (Correll et al., 2008; Ghiretti et al., 2013), as well as the site of 14-3-3 protein interaction (Béguin et al. 2005a), were conserved as well. The bullfrog Rem2 protein offers further phylogenetic insight into the RGK subfamily, particularly with regard to areas of conservation in the distinct N-terminal and C-terminal extensions of Rem2. On the other hand, areas of discrepancy may require further comparative studies, particularly involving urodeles and birds, so as to offer a more comprehensive phylogenetic perspective into putative functional domains.

4.3 Tissue specificity of the bullfrog rem2 gene

The bullfrog brain had the highest levels of rem2 gene expression which is consistent with other vertebrates (Finlin et al., 2000; Becker et al., 2008; Edel et al., 2010b; Hollis et al., 2012). In fact, though rem2 gene expression was found in all eleven tissues assayed, only the brain showed significant levels of the rem2 gene as it was exceptionally low in every other tissue. This is a bit of a contrast with other adult vertebrates. For example, in the rat, while Rem2 expression is highest in the brain, the kidney also has very high basal levels as well (Finlin et al., 2000). In the bullfrog, kidney levels were very low, however, relative to other tissues, it did have a higher level than most (though not significant). Interestingly, in the mouse, Rem2 expression levels in the kidney are also low (Becker et al., 2008). Thus, the differences in tissue expression level patterns may be more species-specific than simply typical of any one vertebrate class. In another anamniotic vertebrate, the rainbow trout, though the highest levels of rem2 were in the brain, the spleen also had high levels relative to other tissues (Hollis et al., 2012). This contrasts with the bullfrog where levels of rem2 gene expression were exceptionally low. Thus, while rem2 gene expression consistently appears at high levels in neural tissue relative to other tissues, there appears to be greater variation with regard to tissues outside the central nervous system.

4.4 The expression of the bullfrog rem2 gene in the central nervous system

The relative expression levels of bullfrog rem2 in the different brain regions varied with the higher expression levels occurring in the more anterior regions. More specifically, there were relatively higher levels of rem2 in the olfactory bulb, cerebrum, diencephalon and midbrain, whereas the cerebellum, hindbrain, and spinal cord were very low. This is the first such description of the general distribution of rem2 in an amphibian brain. An overall similar pattern is seen in the distribution of rem2 in the salmonid brain (Hollis et al., 2012), which also has the higher levels of rem2 expression occurring in the anterior regions, with the highest levels occurring in the cerebrum. Also similarly, the lower levels of rem2 expression in the posterior regions are typified with the cerebellum having the very lowest levels. The one exception however, is that in the bullfrog brain, the posterior regions had barely detectable levels of rem2, whereas the rainbow trout has higher levels of rem2 relative to the anterior regions, particularly in the hindbrain and spinal cord. Because both fish and amphibians maintain strong proliferative capacity in their central nervous systems as adults (reviewed in Grandel and Brand, 2013), coupled with the fact that Rem2 can promote cell proliferation (Bierings et al., 2008), maintain stem cell survival (Edel et al., 2010a), and promote neuronal survival (Edel et al., 2010b), it is possible that the distribution of the rem2 gene in the adult central nervous systems of anamniotes may reflect a role for Rem2 in regional cell proliferative and or neurogenic capacities. For example, in the hylid brain, the greatest proliferative activity resides within the telencephalon followed by the diencephalon, whereas a tremendous drop off occurs from the midbrain to the hindbrain (Almli and Wilczynski, 2007). A similar pattern has been found in the adult bullfrog where relatively high levels of cell proliferation occur in the telencephalon, diencephalon (hypothalamus), and mesencephalon (Simmons et al., 2008). Furthermore, neurogenesis, which is lacking in the posterior brain of anuran amphibians, remains in fish (reviewed in Ferretti, 2013), which follows the discrepancy pattern seen in the relative rem2 level differences between the bullfrog and past work in the rainbow trout. However, in contrast, cell proliferation also occurs in the adult anuran (R. exculenta) cerebellum (Raucci et al., 2006), which would appear to contradict the idea that rem2 patterns of expression might reflect proliferative zones, as previously discussed in fish (Hollis et al., 2012). While the involvement of Rem2 in adult anuran amphibian brain cell proliferation capability and neurogenesis is unknown, given the number of different functions that have been attributed to Rem2 in nervous tissue, the differential expression of the rem2 gene between the different brain regions likely indicates a significant functional role for this small molecule GTPase in the amphibian central nervous system as supported by recent evidence in X. laevis (Ghiretti et al., 2014).

4.5 The expression of bullfrog brain rem2 at different ontogenetic stages

We examined the relative rem2 expression levels in the brain of the bullfrog across different stages of its development and found increased expression of brain rem2 as the animals went from tadpole to adult. Most striking is the considerable increase in brain rem2 expression from pre- to post-metamorphic climax (see Fig. 5B). It is possible that the shift in rem2 expression observed occurs during metamorphic climax where anurans transition from an aquatic to an amphibious existence. As mentioned, evidence suggests an influence of Rem2 on cell proliferation (Bierings et al., 2008). Shifts in cell proliferation have been observed in the brain of the bullfrog as it moves from the tadpole stages to the postmetamorphic froglet (juvenile) stage. In certain brain regions, such as the torus semicircularis, increases in mitotic index increase significantly from early tadpole stages to metamorphic climax and froglet stages (Simmons et al., 2006). Additionally, the superior olivary nucleus and dorsolateral nucleus experience cell proliferation increases at metamorphic climax as well (Chapman et al., 2006). In this study, the adults we observed maintained high levels of rem2 expression as seen in the juveniles, however, it has been noted that while there is an overall reduction in proliferative potential of adults, there is, in contrast, a regional increase in the anterior preoptic area (Raucci et al., 2006).

Beyond proliferation, metamorphic climax is characterized by central anatomical changes marked with connection plasticity thought to reflect the development, maturation, and increase in activity from the periphery as described in the anuran auditory path (Horowitz et al., 2007). Recent work on Rem2 has implicated its role in neuronal plasticity, dendritic morphology, and synapse development (Flynn et al., 2012; Ghiretti and Paradis, 2010; Ghiretti et al., 2013; Moore et al., 2013, Ghiretti et al., 2014). In Xenopus, for example, high levels of central plasticity in the visual system (binocular inputs) are observed early post-metamorphosis (Udin and Keating, 1981; Titmus et al., 1999). Furthermore, neural plasticity in both early post-metamorphic and adult frog visual systems (pretectal) have been shown to be influenced by NMDA receptors (Cline and Constantine-Paton 1990; Jardon and Bonaventure, 1992, 1997), while in postmetamorphic frogs, there is evidence that within developing retinotectal projections, maintenance of arbor branches requires stable synapses associated with local inhibition within the remodeling terminal (Cline and Constantine-Paton, 1990). Rem2 is known to promote the development of dendritic spines (sites of NMDA clusters) and excitatory synapses and also functions to inhibit dendritic branching (Ghiretti and Paradis, 2010). In fact, it was recently found that transcriptional regulation of Rem2 inhibits dendritic arborization in the visual system of X. laevis (Ghiretti et al., 2014). Thus, the changes seen in rem2 brain expression in bullfrog development may also reflect shifts in its role in regional changes in central plasticity. Furthermore, because Rem2 is known to be regulated at the transcriptional level (Finlin et al., 2005, Bierings et al. 2008), the question arises as to how such changes in rem2 brain gene expression in development are governed regarding the physiological state of the animal.

4.6 Conclusion

The Rem2 protein is unique among the small molecule GTPases in its tissue specificity and its functional role in different neural processes such as neural development and synapse formation. In this study, we isolated and characterized Rem2 in an adult amphibian, and give the first report of rem2 gene expression in the amphibian brain at different stages of its development. The tissue specificity of the rem2 gene in the bullfrog indicates a conserved pattern across vertebrates from the different classes examined thus far, while the distribution of the rem2 gene in the central nervous system indicates a putatively conserved pattern in anamniotic vertebrates. Finally, the shift in rem2 expression in the bullfrog brain across different stages of development may be indicative of a shift in its role as a putative mechanism of amphibian brain plasticity and/or proliferative capacity, which vary in the lifetime of a species. Due to the relatively high cell proliferative capacity of adult anamniotic vertebrates as compared to mammals, the characterization of Rem2 in the bullfrog brain is important as it will allow further investigation into whether this protein is a mechanism intrinsic to the proliferative discrepancies across the central nervous systems of vertebrates.

Highlights.

We acquired the full-length sequence of the rem2 gene from the bullfrog brain

Bullfrog rem2 gene expression was much greater in the brain than other tissues

The bullfrog rem2 gene is differentially expressed between the major brain regions

An increase in rem2 brain expression occurs after metamorphic climax

Tissue and brain region rem2 expression patterns appear conserved in adult anamniotes

Acknowledgments

This project was supported by grants from the National Center for Research Resources (5 P20 RR016461) and the National Institute of General Medical Sciences (8 P20 GM103499) from the National Institutes of Health. Further support came from Furman University’s Research and Professional Growth (RPG) and Furman Advantage awards. Special thanks to John F. Wheeler, Jason S. Rawlings, Wade B. Worthen, Katrina M. Morgan, and the Arizona State University DNA Lab.

Abbreviations list

bp

base pairs

cDNA

DNA complimentary to RNA

DNase

deoxyribonuclease

NMDA

N-methyl-D-aspartate

ORF

open reading frame

RNase

ribonuclease

UTR

untranslated region(s)

‘(prime)

denotes a truncated gene at the indicated side

Footnotes

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The origin and development of an individual organism from embryo to adult.

References

  1. Almli LM, Wilczynski W. Regional distribution and migration of proliferating cell populations in the adult brain of Hyla cinerea (Anura, Amphibia) Brain Res. 2007;1159:112–118. doi: 10.1016/j.brainres.2007.05.020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Beattie MS, Bresnahan JC, Lopate G. Metamorphosis alters the response to spinal cord transection in Xenopus laevis frogs. J. Neurobiol. 1990;21:1108–1122. doi: 10.1002/neu.480210714. [DOI] [PubMed] [Google Scholar]
  3. Becker JAJ, Befort K, Blad C, Filliol D, Ghate A, Dembele D, Thibault C, Koch M, Muller J, Lardenois A, Poch O, Kieffer BL. Transcriptome analysis identifies genes with enriched expression in the mouse central Extended Amygdala. Neuroscience. 2008;156(4):950–965. doi: 10.1016/j.neuroscience.2008.07.070. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Béguin P, Mahalakshmi RN, Nagashima K, Cher DH, Kuwamura N, Yamada Y, Seino Y, Hunziker W. Roles of 14-3-3 and calmodulin binding in subcellular localization and function of the small G-protein Rem2. Biochem. J. 2005a;390:67–75. doi: 10.1042/BJ20050414. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Béguin P, Mahalakshmi RN, Nagashima K, Cher DH, Takahashi A, Yamada Y, Seino Y, Hunziker W. 14-3-3 and calmodulin control subcellular distribution of Kir/Gem and its regulation of cell shape and calcium channel activity. J. Cell Sci. 2005b;118:1923–1934. doi: 10.1242/jcs.02321. [DOI] [PubMed] [Google Scholar]
  6. Béguin P, Mahalakshmi RN, Nagashima K, Cher DH, Ikeda H, Yamada Y, Seino Y, Hunziker W. Nuclear sequestration of beta-subunits by Rad and Rem is controlled by 14-3-3 and calmodulin and reveals a novel mechanism for Ca2+ channel regulation. J. Mol. Biol. 2006;355(1):34–46. doi: 10.1016/j.jmb.2005.10.013. [DOI] [PubMed] [Google Scholar]
  7. Bierings R, Beato M, Edel MJ. An endothelial cell genetic screen identifies the GTPase Rem2 as a suppressor of p19ARF expression that promotes endothelial cell proliferation and angiogenesis. J. Biol. Chem. 2008;283(7):4408–4416. doi: 10.1074/jbc.M707438200. [DOI] [PubMed] [Google Scholar]
  8. Bilesimo P, Jolivet P, Alfama G, Buisine N, Le Mevel S, Havis E, Demeneix BA, Sachs LM. Specific histone lysine 4 methylation patterns define TR-binding capacity and differentiate direct T3 responses. Mol. Endocrinol. 2011;25(2):225–237. doi: 10.1210/me.2010-0269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chapman JA, Weinstein JL, Simmons AM. Cell proliferation in the Rana catesbeiana auditory medulla over metamorphic development. J. Neurobiol. 2006;66(2):115–133. doi: 10.1002/neu.20209. [DOI] [PubMed] [Google Scholar]
  10. Chen H, Puhl HL, III, Niu S-L, Mitchell DC, Ikeda SR. Expression of Rem2, an RGK family small GTPase, reduces N-type calcium current without affecting channel surface density. J. Neurosci. 2005;25(42):9762–9772. doi: 10.1523/JNEUROSCI.3111-05.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cline HT, Constantine-Paton M. NMDA receptor agonist and antagonists alter retinal ganglion cell arbor structure in the developing frog retinotectal projection. J. Neurosci. 1990;10(4):1197–1216. doi: 10.1523/JNEUROSCI.10-04-01197.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cohen L, Mohr R, Chen YY, Huang M, Kato R, Dorin D, Tamanoi F, Goga A, Afar D, Rosenberg N. Transcriptional activation of a ras-like gene (kir) by oncogenic tyrosine kinases. Proc. Natl. Acad. Sci. USA. 1994;91(26):12448–12452. doi: 10.1073/pnas.91.26.12448. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Colicelli J. Human RAS superfamily proteins and related GTPases. Sci. STKE. 2004;2004(250):RE13. doi: 10.1126/stke.2502004re13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Correll RN, Pang C, Niedowicz DM, Finlin BS, Andres DA. The RGK family of GTP-binding proteins: regulators of voltage-dependent calcium channels and cytoskeleton remodeling. Cell Signal. 2008;20(2):292–300. doi: 10.1016/j.cellsig.2007.10.028. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Del Villar K, Dorin D, Sattler I, Urano J, Poullet P, Robinson N, Mitsuzawa H, Tamanoi F. C-terminal motifs found in Ras-superfamily G-proteins: CAAX and C-seven motifs. Biochem. Soc. Trans. 1996;24(3):709–713. doi: 10.1042/bst0240709. [DOI] [PubMed] [Google Scholar]
  16. Duellman WE, Trueb L. Biology of Amphibians. New York: The McGraw-Hill Book Company, Inc.; 1986. [Google Scholar]
  17. Edel MJ, Menchon C, Menendez S, Consiglio A, Raya A, Izpisua Belmonte JC. Rem2 GTPase maintains survival of human embryonic stem cells as well as enhancing reprogramming by regulating p53 and cyclin D1. Genes Dev. 2010a;24(6):561–573. doi: 10.1101/gad.1876710. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Edel MJ, Boue S, Menchon C, Sánchez-Danés A, Izpisua Belmonte JC. Rem2 GTPase controls proliferation and apoptosis of neurons during embryo development. Cell Cycle. 2010b;9:3414–3422. doi: 10.4161/cc.9.17.12719. [DOI] [PubMed] [Google Scholar]
  19. Endo T, Yoshino J, Kado K, Tochinai S. Brain regeneration in anuran amphibians. Dev. Growth Differ. 2007;49(2):121–129. doi: 10.1111/j.1440-169X.2007.00914.x. [DOI] [PubMed] [Google Scholar]
  20. Ferretti P. Is there a relationship between adult neurogenesis and neuron generation following injury across evolution? Eur. J. Neurosci. 2011;34(6):951–962. doi: 10.1111/j.1460-9568.2011.07833.x. [DOI] [PubMed] [Google Scholar]
  21. Finlin BS, Shao H, Kadono-Okuda K, Guo N, Andres DA. Rem2, a new member of the Rem/Rad/Gem/Kir family of Ras-related GTPases. Biochem. J. 2000;347:223–231. [PMC free article] [PubMed] [Google Scholar]
  22. Finlin BS, Mosley AL, Crump SM, Correll RN, Ozcan S, Satin J, Andres DA. Regulation of L-type Ca2+ channel activity and insulin secretion by the Rem2 GTPase. J. Biol. Chem. 2005;280(51):41864–41871. doi: 10.1074/jbc.M414261200. [DOI] [PubMed] [Google Scholar]
  23. Flynn R, Labrie-Dion E, Bernier N, Colicos MA, De Koninck P, Zamponi GW. Activity-dependent subcellular cotrafficking of the small GTPase Rem2 and Ca2+/CaM-dependent protein kinase IIα. PLoS One. 2012;7(7):e41185. doi: 10.1371/journal.pone.0041185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Forehand CJ, Farel PB. Anatomical and behavioral recovery from the effects of spinal cord transection: dependence on metamorphosis in anuran larvae. J. Neurosci. 1982;2:654–662. doi: 10.1523/JNEUROSCI.02-05-00654.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Fu M, Zhang J, Tseng YH, Cui T, Zhu X, Xiao Y, Mou Y, De Leon H, Chang MM, Hamamori Y, Kahn CR, Chen YE. Rad GTPase attenuates vascular lesion formation by inhibition of vascular smooth muscle cell migration. Circulation. 2005;111(8):1071–1077. doi: 10.1161/01.CIR.0000156439.55349.AD. [DOI] [PubMed] [Google Scholar]
  26. Ghiretti AE, Paradis S. The GTPase Rem2 regulates synapse development and dendritic morphology. Dev. Neurobiol. 2010;71(5):374–389. doi: 10.1002/dneu.20868. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Ghiretti AE, Kenny K, Marr MT, 2nd, Paradis S. CaMKII-dependent phosphorylation of the GTPase Rem2 is required to restrict dendritic complexity. J. Neurosci. 2013;33(15):6504–6515. doi: 10.1523/JNEUROSCI.3861-12.2013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Ghiretti AE, Moore AR, Brenner RG, Chen LF, West AE, Lau NC, Van Hooser SD, Paradis S. Rem2 is an activity-dependent negative regulator of dendritic complexity in vivo. J. Neurosci. 2014;34(2):392–407. doi: 10.1523/JNEUROSCI.1328-13.2014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Grandel H, Brand M. Comparative aspects of adult neural stem cell activity in vertebrates. Dev Genes Evol. 2013;223(1–2):131–147. doi: 10.1007/s00427-012-0425-5. [DOI] [PubMed] [Google Scholar]
  30. Hasebe T, Kajita M, Shi YB, Ishizuya-Oka A. Thyroid hormone-up-regulated hedgehog interacting protein is involved in larval-to-adult intestinal remodeling by regulating sonic hedgehog signaling pathway in Xenopus laevis. Dev. Dyn. 2008;237(10):3006–3015. doi: 10.1002/dvdy.21698. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Hollis DM, Sawa Y, Wagoner A, Rawlings JS, Goetz FW. Isolation and molecular characterization of Rem2 isoforms in the rainbow trout (Oncorhynchus mykiss): Tissue and central nervous system expression. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 2012;161(2):93–101. doi: 10.1016/j.cbpb.2011.09.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Horowitz SS, Chapman JA, Simmons AM. Plasticity of auditory medullary-midbrain connectivity across metamorphic development in the bullfrog, Rana catesbeiana. Brain Behav. Evol. 2007;69(1):1–19. doi: 10.1159/000095027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Jardon B, Bonaventure N. N-methyl-D-aspartate antagonists suppress the development of frog symmetric monocular optokynetic nystagmus observed after unilateral visual deprivation. Brain Res. Dev. Brain Res. 1992;67(1):67–73. doi: 10.1016/0165-3806(92)90026-s. [DOI] [PubMed] [Google Scholar]
  34. Jardon B, Bonaventure N. Involvement of NMDA in a plasticity phenomenon observed in the adult frog monocular optokinetic nystagmus. Vision Res. 1997;37(11):1511–1124. doi: 10.1016/s0042-6989(96)00291-x. [DOI] [PubMed] [Google Scholar]
  35. Klein SL, Strausberg RL, Wagner L, Pontius J, Clifton SW, Richardson P. Genetic and genomic tools for Xenopus research: The NIH Xenopus initiative. Dev. Dyn. 2002;225(4):384–391. doi: 10.1002/dvdy.10174. [DOI] [PubMed] [Google Scholar]
  36. Krey JF, Paşca SP, Shcheglovitov A, Yazawa M, Schwemberger R, Rasmusson R, Dolmetsch RE. Timothy syndrome is associated with activity-dependent dendritic retraction in rodent and human neurons. Nat. Neurosci. 2013;16(2):201–209. doi: 10.1038/nn.3307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Leone A, Mitsiades N, Ward Y, Spinelli B, Poulaki V, Tsokos M, Kelly K. The Gem GTP-binding protein promotes morphological differentiation in neuroblastoma. Oncogene. 2001;20(25):3217–3225. doi: 10.1038/sj.onc.1204420. [DOI] [PubMed] [Google Scholar]
  38. Leyris JP, Gondeau C, Charnet A, Delattre C, Rousset M, Cens T, Charnet P. RGK GTPase-dependent CaV2.1 Ca2+ channel inhibition is independent of CaVbeta-subunit-induced current potentiation. FASEB J. 2009;23(8):2627–2638. doi: 10.1096/fj.08-122135. [DOI] [PubMed] [Google Scholar]
  39. Maguire J, Santoro T, Jensen P, Siebenlist U, Yewdell J, Kelly K. Gem: An induced, immediate early protein belonging to the Ras family. Science. 1994;265(5169):241–244. doi: 10.1126/science.7912851. [DOI] [PubMed] [Google Scholar]
  40. Moore AR, Ghiretti AE, Paradis S. A loss-of-function analysis reveals that endogenous Rem2 promotes functional glutamatergic synapse formation and restricts dendritic complexity. PLoS One. 2013;8(8):e74751. doi: 10.1371/journal.pone.0074751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Ohsugi M, Cras-Méneur C, Zhou Y, Warren W, Bernal-Mizrachi E, Permutt MA. Glucose and insulin treatment of insulinoma cells results in transcriptional regulation of a common set of genes. Diabetes. 2004;53(6):1496–1508. doi: 10.2337/diabetes.53.6.1496. [DOI] [PubMed] [Google Scholar]
  42. Paradis S, Harrar DB, Lin Y, Koon AC, Hauser JL, Griffith EC, Zhu L, Brass LF, Chen C, Greenberg ME. An RNAi-based approach identifies molecules required for glutamatergic and GABAergic synapse development. Neuron. 2007;53(2):217–232. doi: 10.1016/j.neuron.2006.12.012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Raucci F, Di Fiore MM, Pinelli C, D'Aniello B, Luongo L, Polese G, Rastogi RK. Proliferative activity in the frog brain: a PCNA-immunohistochemistry analysis. J. Chem. Neuroanat. 2006;32(2–4):127–142. doi: 10.1016/j.jchemneu.2006.08.001. [DOI] [PubMed] [Google Scholar]
  44. Rugh R. The Frog: Its Reproduction and Development. New York: The McGraw-Hill Book Company, Inc.; 1951. Chronological summary of organ anlagen appearance of Rana pipiens; pp. 251–260. [Google Scholar]
  45. Seu L, Pitt GS. Dose-dependent and isoform-specific modulation of Ca2+ channels by RGK GTPases. J. Gen. Physiol. 2006;128(5):605–613. doi: 10.1085/jgp.200609631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Shi Y-B. Amphibian Metamorphosis: From Morphology to Molecular Biology. New York: Wiley-Liss; 2000. [Google Scholar]
  47. Simmons AM, Chapman JA, Brown RA. Developmental changes in cell proliferation in the auditory midbrain of the bullfrog, Rana catesbeiana . J. Neurobiol. 2006;66(11):1212–1224. doi: 10.1002/neu.20301. [DOI] [PubMed] [Google Scholar]
  48. Simmons AM, Horowitz SS, Brown RA. Cell proliferation in the forebrain and midbrain of the adult bullfrog, Rana catesbeiana. Brain Behav. Evol. 2008;71(1):41–53. doi: 10.1159/000108610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Sims RT. Transection of the spinal cord in developing Xenopus laevis. J. Embryol. Exp. Morphol. 1962;10:115–126. [PubMed] [Google Scholar]
  50. Splingard A, Ménétrey J, Perderiset M, Cicolari J, Regazzoni K, Hamoudi F, Cabanié L, El Marjou A. Biochemical and structural characterization of the gem GTPase. J. Biol. Chem. 2007;282(3):1905–1915. doi: 10.1074/jbc.M604363200. [DOI] [PubMed] [Google Scholar]
  51. Suyama E, Kawasaki H, Nakajima M, Taira K. Identification of genes involved in cell invasion by using a library of randomized hybrid ribozymes. Proc. Natl. Acad. Sci. USA. 2003;100(10):5616–5621. doi: 10.1073/pnas.1035850100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Ten Donkelaar HJ. Development and regenerative capacity of descending supraspinal pathways in tetrapods: a comparative approach. Adv. Anat. Embryol. Cell Biol. 2000;154:1–145. doi: 10.1007/978-3-642-57125-1. [DOI] [PubMed] [Google Scholar]
  53. Titmus MJ, Tsai HJ, Lima R, Udin SB. Effects of choline and other nicotinic agonists on the tectum of juvenile and adult Xenopus frogs: a patch-clamp study. Neuroscience. 1999;91(2):753–769. doi: 10.1016/s0306-4522(98)00625-3. [DOI] [PubMed] [Google Scholar]
  54. Udin SB, Keating MJ. Plasticity in a central nervous pathway in Xenopus: anatomical changes in the isthmotectal projection after larval eye rotation. J. Comp. Neurol. 1981;203(4):575–594. doi: 10.1002/cne.902030403. [DOI] [PubMed] [Google Scholar]
  55. Vanhove B, Hofer-Warbinek R, Kapetanopoulos A, Hofer E, Bach FH, de Martin R. Gem, a GTP-binding protein from mitogen-stimulated T cells, is induced in endothelial cells upon activation by inflammatory cytokines. Endothelium. 1997;5(1):51–61. doi: 10.3109/10623329709044158. [DOI] [PubMed] [Google Scholar]
  56. Warton K, Foster NC, Gold WA, Stanley KK. A novel gene family induced by acute inflammation in endothelial cells. Gene. 2004;342(1):85–95. doi: 10.1016/j.gene.2004.07.027. [DOI] [PubMed] [Google Scholar]
  57. Yang T, Puckerin A, Colecraft HM. Distinct RGK GTPases differentially use α1- and auxiliary β-binding-dependent mechanisms to inhibit CaV1.2/CaV2.2 channels. PLoS One. 2012;7(5):e37079. doi: 10.1371/journal.pone.0037079. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Yoshino J, Tochinai S. Successful reconstitution of the non-regenerating adult telencephalon by cell transplantation in Xenopus laevis. Dev. Growth Differ. 2004;46(6):523–534. doi: 10.1111/j.1440-169x.2004.00767.x. [DOI] [PubMed] [Google Scholar]
  59. Zupanc GK. Neurogenesis, cell death and regeneration in the adult gymnotiform brain. J. Exp. Biol. 1999;202(Pt 10):1435–1446. doi: 10.1242/jeb.202.10.1435. [DOI] [PubMed] [Google Scholar]
  60. Zupanc GK, Hinsch K, Gage FH. Proliferation, migration, neuronal differentiation, and long-term survival of new cells in the adult zebrafish brain. J. Comp. Neurol. 2005;488(3):290–319. doi: 10.1002/cne.20571. [DOI] [PubMed] [Google Scholar]

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