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.

Agr family includes three groups of genes, Ag1, Agr2 and Agr3, which encode the thioredoxin domain-containing secreted proteins and have been shown recently to participate in regeneration of the amputated body appendages in amphibians. By contrast, higher vertebrates have only Agr2 and Agr3, but lack Ag1, and have low ability to regenerate the body appendages. Thus, one may hypothesize that loss of Ag1 in evolution could be an important event that led to a decline of the regenerative capacity in higher vertebrates. To test this, we have studied now the expression and role of Ag1 in the regeneration of fins of a representative of another large group of lower vertebrates, the fish Danio rerio. As a result, we have demonstrated that amputation of the Danio fins, like amputation of the body appendages in amphibians, elicits an increase of Ag1 expression in cells of the stump. Furthermore, down-regulation of DAg1 by injections of Vivo-morpholino antisense oligonucleotides resulted in a retardation of the fin regeneration. These data are in a good agreement with the assumption that the loss of Ag1 in higher vertebrates ancestors could lead to the reduction of the regenerative capacity in their modern descendants.

In contrast to amniotes (reptiles, birds and mammals), anamniotes (fishes and amphibians) can effectively regenerate body appendages such as fins, limbs and tails. Why such a useful capability was progressively lost in amniotes remains unknown. As we have hypothesized recently, one of the reasons for this could be loss of some genes regulating the regeneration in evolution of amniotes. Here, we demonstrate the validity of this hypothesis by showing that genes of small GTPases Ras-dva1 and Ras-dva2, that had been lost in a stepwise manner during evolution of amniotes and disappeared completely in placental mammals, are important for regeneration in anamniotes. Both Ras-dva genes are quickly activated in regenerative wound epithelium and blastema forming in the amputated adult Danio rerio fins and Xenopus laevis tadpoles’ tails and hindlimb buds. Down-regulation of any of two Ras-dva genes in fish and frog resulted in a retardation of regeneration accompanied by down-regulation of the regeneration marker genes. On the other hand, Ras-dva over-expression in tadpoles’ tails restores regeneration capacity during the refractory period when regeneration is blocked due to natural reasons. Thus our data on Ras-dva genes, which were eliminated in amniotes but play role in anamniotes regeneration regulation, satisfy our hypothesis.

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We previously found that the small GTPase Ras-dva1 is essential for the telencephalic development in Xenopus laevis because Ras-dva1 controls the Fgf8-mediated induction of FoxG1 expression, a key telencephalic regulator. In this report, we show, however, that Ras-dva1 and FoxG1 are expressed in different groups of cells; whereas Ras-dva1 is expressed in the outer layer of the anterior neural fold, FoxG1 and Fgf8 are activated in the inner layer from which the telencephalon is derived. We resolve this paradox by demonstrating that Ras-dva1 is involved in the transduction of Fgf8 signal received by cells in the outer layer, which in turn send a feedback signal that stimulates FoxG1 expression in the inner layer. We show that this feedback signal is transmitted by secreted Agr proteins, the expression of which is activated in the outer layer by mediation of Ras-dva1 and the homeodomain transcription factor Otx2. In turn, Agrs are essential for maintaining Fgf8 and FoxG1 expression in cells at the anterior neural plate border. Our finding reveals a novel feedback loop mechanism based on the exchange of Fgf8 and Agr signaling between neural and non-neural compartments at the anterior margin of the neural plate and demonstrates a key role of Ras-dva1 in this mechanism.