Ivanova AS et al. (2013), Agr genes, missing in amniotes, are involved in...

XB-ART-46704

Sci Rep 2013 Jan 01;3:1279. doi: 10.1038/srep01279.

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Agr genes, missing in amniotes, are involved in the body appendages regeneration in frog tadpoles.

Ivanova AS , Tereshina MB , Ermakova GV , Belousov VV , Zaraisky AG .

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Previous studies have shown that Agr genes, which encode thioredoxin domain-containing secreted proteins, play a critical role in limb regeneration in salamanders. To determine the evolutionary conservation of Agr function, it is important to examine whether Agrs play a similar role in species with a different type of regeneration. Here, we refined the phylogeny of Agrs, revealing three subfamilies: Ag1, Agr2 and Agr3. Importantly, we established that Ag1 was lost in higher vertebrates, which correlates with their decreased regeneration ability. In Xenopus laevis tadpoles (anamniotes), which have all three Agr subfamilies and a high regenerating capacity, Agrs were activated in the stumps of tails and hindlimb buds that were amputated at stage 52. However, Agrs were not up-regulated when the hindlimb buds were amputated at stage 57, the stage at which their regeneration capacity is lost. Our findings indicate the general importance of Agrs for body appendages regeneration in amphibians.

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Species referenced: Xenopus laevis
Genes referenced: ag1 agr2 agr3 ahr ankmy2 bzw2 gnas nbl1 odc1 snx13 tspan13 txndc12

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	Figure 4. Analysis of Agrs expression by whole-mount in situ hybridisation.(A) and (B), (F) and (G), (K) and (L). Intact hindlimb buds and hindlimb buds amputated at stage 52 and hybridised at 1 dpa with Xag2, Xagr2a and Xagr3a probes, respectively; distal to the top, ventral side to the right. (C), (H) and (M). Sagittal sections of amputated hindlimb buds shown on (B), (G) and L; ep – epidermis, bl – blastema. (D) and (E), (I) and (J), (N) and (O). Left side view and frontal sections (the level of section is indicated by dotted lines on (D), (I) and (N)) of tails amputated at stage 52 and hybridised at 1 dpa with Xag2 and Xagr2a probes; distal to the bottom, ventral side to the left. (P) and (Q). Stage 25 tailbud embryo hybridized with Xag3a antisense probe. Frontal and right side view respectively. Dorsal to the top. Anterior on B to the right. (R) and (S). Intact and amputated hindlimb buds operated at stage 52 and hybridized at 1 dpa with Xag2 sense (control) probe; distal to the top, ventral side to the right. (T). Sagittal sections of amputated hindlimb buds operated at stage 52 and hybridized at 1 dpa with Xag2 sense (control) probe. (U) and (V). Left side view and frontal sections (the level of section is indicated by dotted line on (U) of amputaited tail operated at stage 52 and hybridized at 1 dpa with Xag2 sense (control) probe; distal to the bottom, ventral side to the left. Bars – 250 microns.
	Figure 5. Analysis of Xag2 expression by transgenic tadpoles.(A) and (A′), (B) and (B′). Amputated and intact hindlimb buds of the same transgenic embryo, 12 h post-amputation at stage 52; distal to the right, dorsal to the top. (C) and (C′). Amputated tail, 12 h post-amputation at stage 52; distal to the bottom, ventral to the right. (D) and (D′), (E) and (E′). Amputated and intact hindlimb buds of the same transgenic embryo, 1 dpa at stage 57; distal to the right, dorsal to the top. (F) and (F′). Amputated tail, 1 dpa at stage 57; distal to the right, ventral to the bottom. Arrows indicate cells expressing EGFP. Bars – 500 microns.
	Figure 1. Clustal alignment of Agr genes.Putative cleavage sites for signalling peptides predicted by Signal-P program (http://www.cbs.dtu.dk/services/SignalP/) are indicated by a red dotted line. Highly conservative aa positions are marked by black, and less conservative aa positions are marked by grey shading. Different classes of vertebrates, whose proteins were aligned, are highlighted by different colours (see decoding of colours at bottom). Abbreviations: Amb – Ambistoma maculatum (salamander) (GeneBank accession numbers: Ag1 - KC253227, Agr2 - KC253228; Agr3 - KC253230; TLP19 - KC253229); Ano – Anolis carolinesis (lizard) (GeneBank accession numbers: Agr2 - JX854521; Agr3 - JX854522; TLP19 - JX854523); Dan –Danio rerio (fish) (GeneBank accession numbers: Ag1 - JX566722, Agr2 - AY796218; TLP19 – DQ28661); Gal – Gallus gallus (chick) (GeneBank accession numbers: Agr2 - XM418698, Agr3 - NM001199613, TLP19 – XM001235372); Gas –Casterosteus aculeatus (fish) (GeneBank accession numbers: Ag1 - JX854524, Agr2 - JX854525; TLP19 – JX854526); Hom – Homo sapiens (GeneBank accession numbers: Agr2 - BT007048, Agr3 - AY069977, TLP19 – BC008913; Not – Notophthalmus viridescens (newt) (GeneBank accession numbers: Agr2 - EF667357); Pel – Pelodiscus sinensis (turtle) (GeneBank accession numbers: Agr2 - JX854518, Agr3 - JX854519, TLP19 – JX854520); and Xen – Xenopus tropicalis (frog) (GeneBank accession numbers: Ag1 - NM213699, Agr2 - NM001016627; Agr3 - NM001006908; TLP19 - CT030507).
	Figure 2. Phylogeny of Agr genes.(A). Phylogenetic unrooted tree with branch length for Ag1, Agr2, Agr3 and TLP19 proteins shown in Fig. 1. (B). Evolution of different subfamilies of Agrs in vertebrates.
	Figure 3. Analysis of Agrs expression by qRT-PCR.(A). Scheme of experiments. (B). QRT-PCR analysis of Xag2, Xagr2a and Xagr3a expression in tissue samples of 1st -3rd types as indicated in (A). Days after amputation are in brackets. All graphs represent means of triplicate experiments. Bars indicate standard deviations. The geometric mean of expression of ornithine decarboxylase (ODC)16 and elongation factor 1alpa (EF-1alpha)16 was used for normalisation of experimental values. The value of normalised PCR signal in sample 2(0) in each series was taken as an arbitrary unit.

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