Suppression of the immune response potentiates tadpole tail regeneration during the refractory period

Many animal species possess the ability to regenerate their lost appendages, although this ability varies depending on the animal species,organs and appendages, and the developmental stage of the animals(Slack, 2003). Although some molecules are reported to stimulate regenerative abilities in various animals(Whitehead et al., 2005; Kawakami et al., 2006; Adams et al., 2007), the reason for the variable regenerative ability remains a mystery.

Xenopus laevis tadpoles have the ability to regenerate whole tail tissues after tail amputation. Recently, however, Xenopus tadpoles were reported to lose their regenerative ability transiently at particular developmental stages (stages 45-47), called the `refractory period'(Beck et al., 2003). During the refractory period, tadpoles fail to generate a regeneration bud (blastema) and thus fail to regenerate their lost tails(Beck et al., 2003). The refractory period appears between the pre- and post-refractory `regeneration periods'. We intended to utilize this unique characteristic to analyze the regeneration-specific processes as well as to analyze the molecular basis of the developmental stage-specific regenerative abilities.

In the present study, we compared gene expression profiles in the wound tail stumps between the refractory and regeneration periods using the differential display method, and found that tail amputation evoked different immune responses between these two periods. Furthermore, suppression of the immune response restored regenerative ability during the refractory period,indicating that immune responses play crucial roles in determining the regenerative ability of Xenopus tadpole tails.

Xenopus laevis tadpoles were kept at 20°C in 0.2% salt water. Stage 39-41, 46-47 and 52-53 tadpoles were used for the pre-refractory regeneration period, refractory period and post-refractory regeneration period, respectively.

The differential display method was performed as described previously(Ishino et al., 2003), using total RNA extracted from wound stumps dissected at 0, 2 and 15 hours post amputation (hpa) during the refractory and post-refractory regeneration periods.

To obtain partial cDNA, degenerate primers were designed for the conserved forkhead domain of X. laevis FOXP1, 2 and 4, and polymerase chain reaction (PCR) was performed with cDNA from thymi of stage 52 tadpoles. The rapid amplification of cDNA ends method (FirstChoice RLM-RACE; Ambion) was performed to obtain the full-length cDNA (GenBank AB359948).

For expression analysis using the wound stumps, we collected four sets of wound stumps for pre-refractory regeneration period (n=15-16),refractory period (n=20-24) and post-refractory regeneration period(n=5-6) at 0, 5, 10, 15, 24 and 48 hpa. For developmental expression analysis, we collected four sets of intact tadpoles (n=4-5) at 4- to 9-days post fertilization (dpf). qRT-PCR was performed with gene-specific primers. Accession numbers of X. laevis chemokine genes were obtained from the Cytokine Family Database(http://cytokine.medic.kumamoto-u.ac.jp/). In situ hybridization on wax sections was performed using standard procedures.

Tadpoles with tails amputated during the refractory period were exposed to 50 ng/ml Celastrol, 1 μM IKK inhibitor VII, 3 μM FK506, 10 μM cyclosporin A (chemicals were from Calbiochem), or 0.1% dimethyl sulfoxide(vehicle control), in 0.33× De Boer solution. The solutions were refreshed every other day and tail regeneration was checked 7 days post amputation (dpa). The regenerative abilities were classified into four groups:excellent, whole tail structure was regenerated including fin, muscle,notochord and spinal cord; good, whole tail was regenerated, but length was shorter; partial, tail was regenerated, but lacked some tissues or had a curved axis; none, no regeneration was observed.

We injected 4.6 nl of 1 mM (PU.1-MO1 and 5mis PU.1-MO1) or 0.75 mM(PU.1-MO2 and 5mis PU.1-MO2) translation-blocking antisense MO (GeneTools)into both blastomeres of 2-cell-stage embryos, essentially as described previously (Iijima et al.,2008; Eisen and Smith,2008). Injected embryos were maintained at 20°C in 0.1×Steinberg's solution. The tails were amputated at stage 46 and tail regeneration was checked 7 dpa. The sequences of MOs used were: PU.1-MO1,5′-TGTTGTGATATAAACACTCCTCGTC-3′; and PU.1-MO2,5′-ATAGGGGTATGGAGTATTCATCACA-3′, which were designed corresponding to -24 to +1 and -49 to -25 of the PU.1 translation initiation site,respectively; and 5mis PU.1-MO1, 5′-AGTTGAGATAAAAACAGTCCTGGTC-3′;5mis PU.1-MO2, 5′-ATACGGGTTTGGACTATTGATCAGA-3′, with five mismatches in the PU.1-MO1 and PU.1-MO2, respectively. qRT-PCR was performed with total RNA extracted from 3-4 sets of 8-17 MO-injected or noninjected tadpoles at stage 46.

To analyze the regenerative processes, we focused on wound epidermis formation, which has important roles in appendage regeneration(Thornton, 1957; Brockes, 1997). In our experimental conditions, wound epidermis coverage completed approximately 8 to 12 hpa (see Fig. S1 in the supplementary material), and proliferating cells appeared approximately 18 to 24 hpa (see Fig. S2 in the supplementary material). Therefore, we intended to compare gene expression profiles in the wound stumps at two time points: one at which the wound epidermis just covered the stump (2 hpa) and the other at which wound epidermis coverage was complete, but proliferating cells had not yet appeared (15 hpa).

We used the differential display method to detect 36 candidate bands, the intensities of which at 15 hpa differed only during the post-refractory regeneration period, but not during the refractory period, when compared with those at 2 hpa. qRT-PCR confirmed the differential expression of 6 of the 36 candidate genes. cDNA cloning identified three of them as Xenopushomologs of cathelicidin-like, CXCR2 and RIN3, respectively. Two other genes contained no significant open reading frames and had frequent one-base substitutions, implying their function as non-coding RNAs, and the last gene had an open reading frame, which showed no significant sequence similarity with any other known proteins (data not shown). We focused on the three coding genes for further analysis. Expression of all of these three genes in the wound stump drastically increased until 10 to 15 hpa and then gradually decreased during the post-refractory regeneration period, whereas there were no such obvious changes during the refractory period(Fig. 1A-C). Their expression levels were below the quantification threshold during the pre-refractory regeneration period (data not shown). Next, to identify cell types that express these genes, we performed in situ hybridization using sections of wound stumps at 10 hpa during the post-refractory regeneration period'. cathelicidin-like was expressed in leukocyte-like cells, which invade the wound stump (Fig. 1D,E),and CXCR2 was expressed in both the wound epidermis and in leukocyte-like cells (Fig. 1F,G). In mammals, cathelicidins are antimicrobial peptides expressed in wound epidermis or in neutrophils(Bals and Wilson, 2003). CXCR2 is a receptor for CXC chemokines, which elicit chemotaxis of neutrophils and monocytes that express the receptors(Chuntharapai et al., 1994). Therefore, it is plausible that the induction of cathelicidin-likeand CXCR2 expression during the post-refractory regeneration period was due to invasion of the leukocyte-like cells expressing these genes in the wound stump. RIN3 is a RAB5-binding protein that has an important role in endocytosis (Kajiho et al.,2003), and is expressed in monocytes or natural killer cells(Su et al., 2002). RIN3 expression in the wound stump was, however, not detected by in situ hybridization, possibly due to its low expression level (data not shown).

As all of these three genes were related to immune responses, we next compared expression profiles of the other immune-related genes between the refractory and pre-/post-refractory regeneration periods. We analyzed 17 chemokine (CXCLs and CCLs), four chemokine receptor, interleukin-1β(Zou et al., 2000) and the major histocompatibility complex (MHC) genes(Liu et al., 2002; Bos and Waldman, 2006), the sequences of which were available from databases. Expression of seven chemokine genes and MHC class II differed between the refractory and post-refractory regeneration periods. Among them, CXCLb, CXCLe and CCLf showed immediate early upregulation (within 5 hours) and subsequent downregulation only during the post-refractory regeneration period,whereas there was no obvious change during the refractory period(Fig. 2A-C). By contrast, CXCLh and MHC class II were slowly upregulated only during the refractory period, whereas there was no obvious change or the genes were downregulated during the post-refractory regeneration period(Fig. 2D,H). CCLb,CCL5L1 and CCL5L2 were upregulated at the later stages (15-24 hpa) during the refractory period and their expression was sustained until 48 hpa, but were immediately (until 5-10 hpa) downregulated during the post-refractory regeneration period (Fig. 2E-G). During the pre-refractory regeneration period, the expression of only two genes, CXCLe and CCLf, was detected,but no obvious change in expression was observed(Fig. 2B,C). Expression of the other genes did not differ significantly between the refractory and post-refractory regeneration periods or were too low to be detected (data not shown). These findings suggest that the immune responses in the wound stumps differ between the refractory and pre-/post-refractory regeneration periods:prolonged or delayed responses occur during the refractory period, whereas no obvious induction or transient induction occurs during the pre-/post-refractory regeneration periods, respectively.

As the immune responses in the refractory period seemed chronic, we hypothesized that these chronic immune responses were responsible for the impaired regenerative ability during this period. To test this, we first examined the effect of four immunosuppressants on regenerative ability during the refractory period. We first used Celastrol, a cell-permeable inhibitor-κB kinase (IKK) inhibitor(Pinna et al., 2004; Lee et al., 2006) because IKK stimulates nuclear factor-κB signaling, which has a pivotal role in immune and inflammatory responses. In untreated groups, almost all of the tadpoles failed to regenerate their lost tails(Fig. 3A,C). By contrast, the Celastrol-treated group significantly recovered their regenerative ability(Fig. 3B,D). A complete tail tissue structure was regenerated in a typical Celastrol-treated tadpole tail(Fig. 3E). We repeated this experiment seven times and obtained six reproducible results(Fig. 3F). Treatment with IKK inhibitor VII (Waelchli et al.,2006) also significantly restored the regenerative ability(Fig. 3F). In addition,treatment with two other immunosuppressants, FK506 and cyclosporin A, which bind to FK-binding protein and cyclophilin, respectively, and inhibit the nuclear factor of activated T cell pathway-dependent cytokine expression(Bierer et al., 1993), also significantly restored the regenerative ability(Fig. 3F). In two experiments(experiment 4 for Celastrol-treated and experiment 10 for cyclosporin A-treated tadpoles; Fig. 3F),the inhibitors failed to restore the regenerative ability, possibly due to physiological conditions or a polymorphism of IKK, cyclophilin, or other target molecules, affecting the efficiency of the inhibitors. FK506 treatment repressed induction of the immune-related genes during the refractory period in amputated tails (see Fig. S3 in the supplementary material), suggesting that the inhibitor actually affected immune responses in Xenopustadpole tails.

We next investigated whether the depletion of immune cells during the refractory period restores the regenerative ability by injecting two kinds of antisense MOs (PU.1-MO1 and MO2) designed for transcription factor PU.1. PU.1-null mice show abnormal neutrophil development, delayed T cell development and have no detectable monocytes/macrophages and B cells(McKercher et al., 1996). Xenopus embryos injected with MOs were maintained until the refractory period and their tails were amputated. PU.1-MO1- or PU.1-MO2-injected groups showed significantly higher regenerative abilities than control 5mis PU.1-MO1- or 5mis PU.1-MO2-injected, or noninjected groups(Fig. 3G-K). Furthermore, both PU.1-MO1 and PU.1-MO2 injections significantly reduced the expression of CD45 (Fig. 3L,M), a pan-leukocyte marker (Turpen et al.,1997), at stage 46, indicating that they effectively depleted leukocytes. These results strongly suggest that immune cells that require PU.1 for their development impair regenerative ability during the refractory period. The recovery of regenerative ability induced by the injection of PU.1-MO was lower than that induced by immunosuppressant treatment, in part because MO injection itself had some negative effects on regenerative ability (Fig. 3J,K; see Fig. S4 in the supplementary material).

Finally, to investigate the possible relationship between regenerative ability and immune system development in X. laevis tadpoles, we examined the development of the T cell population around the onset of the refractory period. We quantified by qRT-PCR the amounts of transcripts for T cell markers: T cell antigen receptor (TCR) α and γ subunits(Haire et al., 2002) and the CD3γ/δ subunit (Dzialo and Cooper, 1988), using total RNAs extracted from the whole intact tadpoles at 4 to 9 dpf. In our experimental condition, the refractory period started around 6 dpf(Fig. 4A). The timings of TCRα, TCRγ and CD3γ /δ induction (Fig. 4B,C,D, respectively) and the decrease in regenerative ability largely coincided (around 6 dpf; Fig. 4A), suggesting that the development of the immune cell population is related to the decrease in regenerative ability during the refractory period.

As tadpoles regain their regenerative ability at the post-refractory regeneration period, we also hypothesized that `regulatory T cells' that suppress the function of various immune cells(Piccirillo, 2008) are related to this period. To test this, we analyzed expression of the X. laevishomolog of FOXP3, a key regulatory gene of regulatory T cells(Hori et al., 2003). qRT-PCR revealed that transient FOXP3 induction in the wound stump was more prominent in the post-refractory regeneration period than in the refractory period (Fig. 2I), implying that regulatory T cells play a role in regaining the regenerative ability during the post-refractory regeneration period. Taken together, it is plausible that there are only a few immune cells that impair the regenerative ability during the pre-refractory regeneration period, whereas such immune cells develop to impair the ability during the refractory period, and then an immune regulatory system is established, which again permits regeneration, during the post-refractory regeneration period (see Fig. S5 in the supplementary material).

Although there has been significant discussion on the possible link between immune responses and the regenerative abilities in some animal species(Arai et al., 1998; Mescher and Neff, 2005; Kanao and Miyachi, 2006), our research provides the first direct evidence for this link in X. laevis tadpole tails, and provides a clue for understanding not only developmental stage-specific regenerative ability, but also the molecular mechanisms of early epimorphic regenerative processes.