Baek S and Seeling JM (2007), Identification of a novel conserved mixed-isofo...

XB-ART-36971

BMC Dev Biol 2007 May 31;7:139. doi: 10.1186/1471-213X-7-139.

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Identification of a novel conserved mixed-isoform B56 regulatory subunit and spatiotemporal regulation of protein phosphatase 2A during Xenopus laevis development.

Baek S , Seeling JM .

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Wnt signaling is a key regulator of development and tumorigenesis. Protein phosphatase 2A (PP2A), which consists of a catalytic C, a structural A, and a regulatory B subunit, plays diverse roles in Wnt signaling through its B56 subunits. B56 is a multigene family encoding for proteins with a conserved core domain and divergent amino- and carboxy-termini. Ectopic B56alpha and B56gamma reduce beta-catenin abundance and B56alpha reduces Wnt-dependent transcription, suggesting that B56alpha and B56gamma inhibit Wnt signaling. In contrast, B56epsilon is required for Wnt signaling. Knowledge of where and when B56 subunits are expressed during Xenopus development will aid in our understanding of their roles in Wnt signaling. We have undertaken expression analyses of B56alpha and B56gamma in Xenopus laevis. We cloned Xenopus B56alpha; it is 88% identical to human B56alpha. Xenopus B56gamma is 94% identical with human B56gamma, however, a novel evolutionarily conserved mixed-isoform transcript was identified that contains a B56delta-like amino-terminal domain and a B56gamma core domain. The B56delta-like variable domain exon is located upstream of the B56gamma variable domain exon at the human B56gamma locus, suggesting that the mixed-isoform transcript is due to alternative splicing. B56gamma transcripts with different 3' ends were identified that lack or possess a 35 base pair sequence, resulting in either a transcript similar to human B56gamma1, or an uncharacterized evolutionarily conserved sequence. Real time RT-PCR analyses revealed that B56alpha is expressed at moderate levels before the midblastula transition (MBT), at reduced levels during gastrulation and neurulation, and at high levels during organogenesis, while B56gamma is expressed at low levels until organogenesis. B56alpha is enriched in the ventral hemisphere pre-MBT, while B56gamma is ventrally enriched post-MBT. Aalpha, Abeta, Calpha and Cbeta are expressed in early Xenopus development, suggesting the presence of a functional heterotrimer. Our data suggest that B56 functional diversity is achieved in part through the synthesis of a novel mixed-isoform B56delta/gamma transcript. Our data also suggest that B56alpha functions pre-MBT, inhibiting Wnt signaling on the ventral side of the embryo, and again during organogenesis, while B56gamma functions primarily post-MBT.

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Species referenced: Xenopus laevis
Genes referenced: gdf1 nodal6 odc1 pnma2 ptpa

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	Figure 1. Alignment of Xenopus laevis and human B56α and B56γ proteins. Dark blue regions signify identity, light blue regions signify similarity, and dashes signify gaps. The amino-terminal variable domain is boxed in green, while the carboxyl-terminal variable domain is boxed in red. A. Sequences of Xenopus laevis (Xl) and human (Hs) B56α proteins are aligned. B. Sequences of Xenopus laevis (Xl) and human (Hs) B56γ proteins are aligned.
	Figure 2. Alignment of B56γ amino- and carboxy-terminal variable domains. Sequence alignments are annotated as described in Figure 1. A. Alignment of Xenopus laevis B56δ/γ and human B56δ amino-terminal variable domains. B. Alignment of Xenopus laevis B56γ/γ and human B56γ amino-terminal variable domains. C. Alignment of Xenopus laevis (Xl), Xenopus tropicalis (Xt, GI:39645667), Pan troglodyte (Pt, GI:114654932), Macaca mulatta (Mm2, GI:109084945), human (Hs, GI:47077243), Mus musculus (Mm1, GI:37359748), Rattus norvegicus (Rn, GI:109478743), and Danio rerio (Dn, GI:113674011) B56δ/γ sequences from the B56δ-like amino-terminal variable region through the first ten amino acids of the B56γ core domain. The division between the variable domain and the core domain is marked by an arrowhead. D. Alignment of an alternative long form of the B56γ carboxy-terminal variable domain with the species described in C; Xenopus tropicalis (GI:45361341), Mus musculus (GI:71153488), Rattus norvegicus (GI:109478743), Macaca mulatta (GI:109084945), human (GI:31083259), Danio rerio (GI:113674011), Pan troglodyte (GI:114607473).
	Figure 3. The B56δ-like amino-terminal variable domain is distinct from B56δ and encoded at the B56γ locus. A. Human B56δ and human B56δ-like amino-terminal variable domains are 51% identical. Sequence alignments are annotated as described in Figure 1. B. The exon encoding the B56δ-like amino-terminal variable domain (Exon 1a, light blue and dark blue bars represent noncoding and coding sequences, respectively) is upstream of the exon encoding the B56γ amino-terminal variable domain (Exon 1b, light green and dark green bars represent noncoding and coding sequences, respectively) in the human B56γ gene (chromosome 14p32.2). The introns are represented by lines and are shown in scale with one another, but not in scale with the exons.
	Figure 4. B56α and B56γ are differentially expressed during early Xenopus development. RNA was purified from Xenopus laevis embryos at the indicated stages, and real time RT-PCR was carried out. A standard curve was done from stage 32 embryos, and signals were normalized to that stage. A. B56α is expressed at approximately 45% of the stage 32 value prior to MBT, at approximately 10% of the stage 32 value during gastrula and neurula stages, and its expression gradually increases to its highest expression level during organogenesis. B. The expression level of B56ε is relatively unchanged from the egg to stage 32. C. The expression level of B56γ is highest during organogenesis and is approximately 25% of that value during earlier stages (unfertilized egg to stage 19). D. The mixed-isoform B56δ/γ transcript is expressed at moderate levels until organogenesis (dark blue bars), while B56γ/γ is not significantly expressed until neurulation (light blue bars); both B56δ/γ and B56γ/γ display increased expression during organogenesis. E. A dose response curve of real time RT-PCR amplification data shows that B56α, B56γ, and B56ε primer pairs are isoform specific. Input plasmid DNA concentrations are plotted against their concentration calculated from the amplification of the correct primer pair/template combination.
	Figure 5. PP2A Aα, Aβ, Cα, and Cβ are differentially expressed during early Xenopus development. RNA was purified from Xenopus laevis embryos at the indicated stages, and real time RT-PCR was carried out. A standard curve was done from stage 32 embryos, and signals were normalized to that stage. A. Aα is expressed at low levels until organogenesis. Aβ (B) and Cα (C) are expressed at moderate levels through gastrulation, and at progressively increasing levels during neurulation and organogenesis. D. Cβ is expressed at similar levels from the egg to stage 32, with the exception of its reduced expression during neurulation. E. A dose response curve of real time RT-PCR amplification data shows that Aα, Aβ, Cα, and Cβ primer pairs are isoform specific. Input plasmid DNA concentrations are plotted against their concentration calculated from the amplification of the correct primer pair/template combination.
	Figure 6. PP2A subunits are enriched in the animal hemisphere of Xenopus embryos. Xenopus laevis embryos at stages 7 and 10 were hemisected into animal and vegetal halves. RNA was purified from the hemisected halves and real time RT-PCR was carried out. The data is presented as the percentage of the total signal that is present in the specified hemisphere ± SD. B56α (A), B56γ (B), B56ε (C), Aα (F), Aβ (G), Cα (H), and Cβ (I) are enriched in the animal hemisphere of stage 7 and 10 Xenopus embryos. Dissection control ODC (D) is enriched animally, while Vg1 (E) is enriched vegetally. A = animal, V = vegetal.
	Figure 7. The dorsoventral distribution of PP2A subunits in Xenopus embryos. Xenopus laevis embryos at stages 3, 7, and 10 were hemisected into dorsal and ventral halves. RNA was purified from the hemisected halves and real time RT-PCR was carried out. The data were normalized to ODC expression levels, and are presented as the percentage of the total signal present in the specified hemisphere. PP2A A and C subunits Aα (E), Aβ (F), Cα (G), and Cβ (H) are relatively equally distributed dorsoventrally or slightly enriched dorsally. However, B56α (A) at stage 3 and B56γ (B) at stage 10 are more abundant ventrally. Dissection controls Vg1 (stage 3) and Xnr6 (stages 7 and 10) are enriched dorsally (D). D = dorsal, V = ventral.

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