Activity of Immune Response/Inflammation-Related Genes During Limb Regeneration
Recent evidence has begun to shed light on the potential importance of both the innate and adaptive immune responses on cellular dedifferentiation and how these responses may affect the onset of tissue regeneration as well as regenerative capacity itself (Harty et al., 2003; Mescher and Neff, 2005, 2006). The influence of the immune system on regeneration was considered over 20 years ago (Sicard, 1985), but a detailed examination of the expression of immune system regulatory and regulated genes had yet to be undertaken. Our screen has shown that many genes previously identified as being important for immune responses are indeed differentially expressed during regeneration-complete and -incomplete stages of limb development (see Master Table in Supplementary Materials). We find that expression of several select immune genes is elevated from day 1 to day 5 postamputation during the regeneration-incomplete stage (Fig. 4, and Supplementary Master Table). The adaptive immune system undergoes considerable development in the perimetamorphic period in Xenopus (Rollins-Smith, 1998), and it is possible that the observed expression differences are reflective only of this maturation process and not directly related to regenerative capacity. To address this possibility, we chose to examine expression of these genes in the intact limb. Although expression may be seen in the intact limb, this finding may not necessarily indicate that the expression domain is in the midzeugopodial region where amputations were performed. This latter possibility can be addressed with 0dPA tissue. However, as we have hypothesized (Harty et al., 2003; Mescher and Neff, 2005, 2006), the increased complexity of the adult Xenopus immune system may be directly correlated with regenerative capacity. We have focused on several immune-related genes in our validation studies, either because previously published data implicated these genes in regeneration or because of their striking patterns of expression during the process of regeneration.
Several genes known to be involved in the immune response and/or inflammation that have been reported previously as playing a role in regeneration were found to be differentially expressed in our assay. These genes include FKBP4 (Avramut and Achim, 2003), FN1 (Christensen and Tassava, 2000; Tazaki et al., 2005), SOCS3 (Campbell et al., 2001), Vimentin (VIM; Ribotta et al., 2004), C3 (Del Rio-Tsonis et al., 1998), LTF (Tazaki et al., 2005), and ANXA2 (Tazaki et al., 2005). In the regeneration-complete (st53) tissues, there is a widespread down-regulation of most immune response markers, including SOCS3, VIM, FN1, EGR1, ETS1, MMP13, ANXA2, RAF1, and PTAFR, from 1dPA to 5dPA. However, some genes, including THBS1, INHBA, TIMP2, and CXCL12, are up-regulated in the st53 blastemas (see Supplementary Figure S3, including high-resolution images). We selected several immune/inflammation related genes for further analysis by RT-PCR and qPCR, and we discuss the potential implications of their expression patterns below.
The complement system, with its classic, lectin-mediated and alternative activation pathways, plays a central role in the inflammatory response and tissue/organ injury response. Traditionally, the function of complement has been linked to the innate immune system's role in the recognition and elimination of pathogens through direct killing and stimulation of phagocytosis. Complement factor 3 (C3) is a central player in each of the complement activation pathways. C3 is synthesized locally by cells of regenerating urodele limbs (Del Rio-Tsonis et al., 1998) and was shown to be upregulated during Xenopus tail regeneration (Tazaki et al., 2005). Importantly, we have extended this observation by showing that C3 is expressed at higher levels in regeneration-complete blastemas compared to regeneration-incomplete pseudoblastemas. Moreover, the following stimulatory as well as inhibitory components of the complement system were found to be up-regulated in our microarray screen: C4 beta chain, C8 beta chain, C1s, C1q, C9, complement component factor H, and complement factor D (see Supplementary Materials). Other components of the complement system have also been associated with tissue and organ regeneration: C5 (Del Rio-Tsonis et al., 1998), complement factor B (Tazaki et al., 2005), and CD59 (da Silva et al., 2002). The expression of so many components of the classic, lectin-mediated and alternative activators and inhibitors of the complement pathway in several different regeneration systems suggests that the complement system in these cases is doing more than defense against pathogens and may be playing an active role in establishing a permissive or tolerant immune environment influencing epimorphic regeneration (Mescher and Neff, 2005, 2006).
The signaling of cytokines inside cells is regulated, in part, by suppressors of cytokine signaling (SOCS). SOCS3 is a member of this family that is a potent regulator of signaling by proinflammatory cytokines such as IFN-γ, TNF-α, and IL-6 (Alexander, 2002). Expression of SOCS3 is rapidly and transiently induced after limb amputation and is significantly higher 1dPA at st53 than at st57. Suppression of cytokine signaling by SOCS3 early in the postamputation regeneration-complete limb may be a critical event allowing for immune tolerance of dedifferentiating cells and regeneration to proceed. Consistent with this possibility, other models have shown that SOCS3 may play a role in both wound healing and regeneration. Xenopus SOCS3 is rapidly and transiently induced in response to tadpole epithelial wounds and mitogen-activated protein kinase inhibitors did not block the induction (Kuliyev et al., 2005). Additionally, SOCS3 is rapidly up-regulated after partial hepatectomy where it appears to be regulated by IL-6 (Campbell et al., 2001). This outcome may be a necessary response, allowing for liver regeneration to proceed.
Consistent with Katogi et al. (2004) who found SOCS3 (identified as olrf30i08) expressed at higher levels at day 3 postamputation vs. day 10 postamputation in regenerating Medaka fins, we find that the induced expression of SOCS3 declines 14-fold from 1dPA to 5dPA at the regeneration-complete stage. However, at the regeneration-incomplete stage, the decline in expression is only 1.3-fold (Katogi et al., 2004). Continued expression of SOCS3 at the regeneration-incomplete stage compared to the regeneration-complete stage may indicate an attempt to negatively regulate the chronic cytokine-induced inflammation of st57 5dPA pseudoblastemas relative to st53 blastemas (Schreiber et al., 2002).
Like SOCS3, the other modulators of immune responses that we have examined, galectin-Ia (LGALS1), MyD88, gp96, and FGL2, each show similar patterns of expression in our model. These four genes are expressed at higher levels in st57 intact limbs relative to st53 limbs, consistent with the maturation of the immune system between these two stages. In addition, all four genes are expressed at higher levels in the regeneration-incomplete pseudoblastema relative to the -complete blastema.
LGALS1 is a member of the galectin family of β-galactoside–binding endogenous lectins (Barondes et al., 1994). LGALS1 appears to play a role in a broad array of cell functions, including cell adhesion, migration, apoptosis, differentiation, nerve and muscle regeneration, and immunoregulation (Almkvist and Karlsson, 2004; Watt et al., 2004; Jiang et al., 2005; Liu, 2005). LGALS1 has also been associated with an anti-inflammatory function (Chung et al., 2000; La et al., 2003). LGALS1 could be part of the general anti-inflammatory mechanism in st57 pseudoblastemas to counter the effects of proinflammatory components such as MyD88, gp96, and FGL2. Interpretation of the positive correlation of LGALS1 expression and incomplete limb regeneration will have to await localization of both the message and protein in normal stage 57 limbs and pseudoblastemas.
MyD88 is an adapter protein involved in signal transduction initiated by IL-1 and Toll-like receptors (TLRs), resulting in the production of many proinflammatory cytokines in a MyD88-dependent manner. TLR10, which signals by means of MyD88 (Brown et al., 2006; Obhrai and Goldstein, 2006) was shown to be differentially expressed in st57 1dPA pseudoblastemas in our array (see Supplementary Materials).The early expression of the MyD88/TLR pathway is essential for normal liver regeneration (Seki et al., 2005) and repair of acute lung injury (Jiang et al., 2005).
The endoplasmic reticulum chaperone gp96 (also designated as Grp94—glucose regulated protein-94 and TRA1) is a member of the HSP90 family that is instrumental in initiation of both innate as well as adaptive immunity (Yang and Li, 2005). Gp96 has been localized to the cell surface in Xenopus lymphocytes (Robert et al., 2004) and shown to have proinflammatory properties that are dependent on MyD88 (Liu et al., 2003).
The prothrombinase fibroleukin (fibrinogen-like protein 2, FGL2) is related to the fibrinogen beta and gamma chains, which can directly cleave prothrombin to thrombin (Chan et al., 2002). FGL2 appears to exist in two forms: a membrane-linked FGL2 with prothrombinase activity, and a soluble FGL2 that has potent immunosuppressive activity (Chan et al., 2003). FGL2 is expressed in several subsets of T-lymphocytes, including gut mucosal lymphocytes (Ruegg and Pytela, 1995) as well as activated hepatic endothelial cells and macrophages (Marsden et al., 2003). FGL2 is induced in type 1 lymphocytes by IFN signaling pathways, including irf1 (Hancock et al., 2004), which we found up-regulated in our array screen (see Supplementary Materials). Conflicting data from FGL2-deficient mice experiments show that FGL2 may or may not contribute to immunologically mediated thrombosis (Marsden et al., 2003; Hancock et al., 2004). Thrombin, proposed to be activated by tissue factor (TF) appears to couple injury and regeneration (lens and limb) in urodele amphibians (Imokawa et al., 2004). Because FGL2 bypasses the TF/factor VII extrinsic thrombin activation pathway (Marsden et al., 2003), we speculate that FGL2 may lead to thrombin activation in anuran amphibians by a TF-independent pathway. Given that expression of FGL2 and gp96 in amputated hindlimbs is similar and that gp96 is also induced by IFN, it is tempting to speculate that these expression patterns are indicative of IFN stimulation of other proinflammatory signals.
Patterning Pathways and Regeneration
Several patterning and growth-related genes that are known to play crucial roles in the specification of the axes of the limb are differentially expressed during limb regeneration (e.g., SHH, MSX2, TBX3, FGF8). As might be expected, our data demonstrate that each of these genes is expressed in regeneration-complete limbs and blastemas. By contrast, these patterning genes show little or no expression in regeneration-incomplete limbs or pseudoblastemas. Amputation of a st57 hindlimb is known to result in a pattern-deficient spike of cartilage; thus, the observed pattern of expression of this family of genes is not surprising.
Sonic hedgehog (Shh) is a critical limb morphogen that plays a role in anterior–posterior patterning of the limb, particularly the autopod (Zakany et al., 2004). Our data confirm the observation that Xenopus Shh is only expressed in blastemas at the regeneration-complete stage (Endo et al., 1997). It is not known if failure of Shh expression in regeneration-incomplete pseudoblastemas is responsible for complete regeneration failure. However, the observation that st57 amputated limbs give rise to a single spike and that Shh−/− mice produce a single digit (Chiang et al., 2001) suggests that the principal role of this gene is in the patterning process rather than in creating conditions that allow for regeneration.
The homeobox-containing genes Msx1 and Msx2 are expressed in multiple tissue–tissue interactions during development, including limbs (Lallemand et al., 2005), digit tips, mouse digit tip regeneration (Han et al., 2003), and during axolotl wound healing and limb regeneration (Carlson et al., 1998). Unlike Xmsx1, which is expressed in both regeneration-complete blastemas and regeneration-incomplete pseudoblastemas and pseudoblastemas (Endo et al., 2000; our unpublished observations), Xenopus Msx2 is shown to be preferentially expressed in regeneration-complete 1dPA and 5dPA blastemas.
Analysis of Tbx3 was carried out because it is required for normal development of the posterior structures of the limb (Davenport et al., 2003) and appears to be involved in the positioning of the limb along the craniocaudal axis (Rallis et al., 2005). The higher level of expression of Tbx3 in st53 limbs and st53 1dPA and 5dPA blastemas, relative to st57 tissues, is consistent with its role in patterning.
FGF8 is expressed throughout the apical ectodermal ridge during normal limb development, where it is involved in the maintenance of limb bud outgrowth and patterning (Moon and Capecchi, 2000). FGF8 and FGF4 are also expressed in the intermediate mesoderm, where they are involved in regulating limb bud initiation and outgrowth (Boulet et al., 2004). FGF8 is expressed in axolotl limb blastemas (Christensen et al., 2002) and in Xenopus blastemas at the regeneration-complete stage as well in pseudoblastemas at the regeneration-incomplete stage (Endo et al., 2000). Consistent with the published data, FGF8 was expressed in both regeneration-complete and -incomplete 1dPA and 5dPA blastemas. However, it is expressed at a higher level in regeneration-complete blastemas relative to regeneration-incomplete pseudoblastemas.
Nrp-1 (Musashi-1) encodes an RNA-binding protein originally thought to be a neurogenesis marker (Richter et al., 1990) and plays a major role in controlling stem cell maintenance in the brain and other tissues (Okano et al., 2005). Consistent with its function as a stem cell marker, it is expressed in regeneration-complete developing limbs at a much higher level than in the more differentiated regeneration-incomplete limbs. Nrp-1 is also expressed at much higher levels in regeneration-complete 1dPA and 5dPA blastemas than regeneration-incomplete pseudoblastemas. It is tempting to speculate that nrp-1–positive cells represent multipotential stem cells within the developing limb and blastemas.
Neuronal nitric oxide synthase (nNOS or NOS1) is highly expressed in some central and peripheral neuronal cells and is also widely expressed elsewhere, including epithelial cells and smooth and skeletal muscle cells (Forstermann et al., 1998). In contrast to some of the other limb growth/patterning genes such as FGF8, TBX3, nrp-1, SHH, and MSX2, nNOS is expressed at much lower levels in st53 one dPA blastemas, but like the other genes, its expression is up-regulated in st53 five dPA blastemas. Because nNOS knockout mice show no gross limb abnormalities and most of the defects are related to nervous system development (Nelson et al., 1995), it is unclear what the function of nNOS is during st53 limb development or in regeneration-complete st53 5dPA blastemas. However, the observation that nNOS is an essential factor for peripheral nerve regeneration (Keilhoff et al., 2003) and that it is up-regulated during mouse skin repair (Boissel et al., 2004) indicates that nNOS may not be involved in patterning of the regenerate, but instead in the regeneration response itself in the st53 limb. Of interest, during mouse skin wound healing, nNOS expression is down-regulated at 1 day after skin injury, followed by dramatic up-regulation at 3 days (Boissel et al., 2004).