Figure 2. Expression of genes previously identified in studies of gene activity during the early phase of Xenopus limb regeneration, shown by RT-PCR at different times PA during the transition from regeneration-complete to –incomplete in stages 53, 55, and 57.

Tissue from 20 limb stumps was used for each time point and expression of the loading control ornithine decarboxylase (ODC) is shown in the top row. (a) Genes with primarily inflammation-related function: Expression of interleukin-1β (IL-1β) appeared maximal by 6 hPA but then was rapidly diminished at all stages. Expression of factors involved in the resolution of inflammation, including annexin-A1 (ANXA1), fibrinogen-like protein 2 (FGL2), suppressors of cytokine signaling (SOCS) 1 and 3, galectin-1, and the key marker for regulatory T cells, FoxP3, were also all up-regulated by 6hPA and then diminished in limbs of all 3 stages, but more slowly in regeneration-incomplete limbs suggesting that inflammation-related activity persists after amputation in the more developed limbs. Low expression of many of these pro-resolution factors, including FoxP3, was seen already at the time of amputation in regeneration-deficient limbs, indicating the presence of immune cells not found in limbs at early regeneration-complete stages. (b) Genes with primarily cell reprogramming and organ patterning function: Expression of SALL4 and TBX3, both involved in cell reprogramming during early blastema formation, are up-regulated by amputation at all 3 stages, but much more slowly and at apparently lower levels in regeneration-incomplete limbs. Expression of genes required for blastema patterning, including Shh, Msx1, SALL1, and HOXA13, occurred at various times PA in stage 53 limbs, but was increasingly reduced and delayed in regeneration-deficient limbs, results consistent with the failure of the latter limbs to regenerate with normal patterns. Cycle number for ODC PCR was 25; FoxP3 required 40 cycles and all other genes 30.