RESEARCH ARTICLEAutoradiography and inmmunolabeling suggests that lizard blastema contains arginase-positive M2-like macrophages that may support tail regeneration
Graphical abstract
(A) Two likely macrophages (arrows) incorporating trititaed arginine within the blastema. (B) Arginase-positive macrophages (arrows) in the lizard tail blastema. (C) Sparse arginase-positive macrophages (red spots) within a blastema. (D) General ultrastructure of a M2-like macrophage. (E) Detail on an arginase immunogold-labeled pale vesicles in a M2-like macrophage.
Introduction
The regeneration of the tail in lizards represents the most outstanding regenerative phenomenon evolved among amniotes since most tissues present in the original tail are reformed and organized in a complex organ, the new tail, although in an anatomical simplified form (Quattrini, 1954, Bellairs and Bryant, 1985, Alibardi, 1986, Alibardi, 2010, Fisher et al., 2012, Lozito and Tuan, 2016). Tail regeneration is accomplished after the formation of a regenerating blastema, a small embryonic bud colonized by mesenchymal cells and covered by a dynamic wound epidermis (Cox, 1969, Alibardi and Sala, 1988, Alibardi, 2010, McLean and Vickaryous, 2011, Gilbert et al., 2013). The blastema becomes smaller and smaller as the regenerating tail elongates, functioning as a growth front, like the tail bud during development (Alibardi, 2017a, Alibardi, 2019a).
Recent transcriptome studies (Hutchins et al., 2014, Vitulo et al., 2017a) have shown that numerous pro-oncogenes such as wnt, shh, beta-catenin, telomerases, c-myc, etc., are up-regulated in the regenerating tail, besides tumor suppressor genes, although the precise tissue localization of these activated genes is not known. The comparison between the blastema of the tail versus that of the limb has indicated that wnts and snoRNAs are likely the key or master genes that determine tail regeneration, as these genes are not activated in the limb whereas genes sustaining inflammation and scarring are up-regulated. A following analysis has shown that a strong down-regulation of most genes of the adaptive immune system is present in the tail blastemal, but not in the blastema of the limb, destined to scar (Vitulo et al., 2017b). Previous studies on the induction of scarring after tail amputation detected an increase of gamma globulins, lymphocytes and phagocytes in the scarring tail that resemble the intense inflammation present in the amputated limb, destined to scar (Alibardi, 2013, Alibardi, 2014, Alibardi, 2018). These observations have suggested that the activation of inflammation or even of an immune response that is effective in lizards (Zimmerman et al., 2008), is largely responsible for the failure of tail regeneration. The formation of an embryonic-like blastema in connection with the normal tissues of the tail requires the re-activation of an embryonic program and immunosuppressive genes (Vitulo et al., 2017b).
The above observations sustain the hypothesis that the tail blastema is a small immune-suppressed organ, and that this condition contributes to its regeneration (Mescher et al., 2016, Mescher, 2017, Alibardi, 2018). Immune-suppression of the lizard blastema may utilize the process of immune-evasion similar to those of cancer cells in escaping from the attack of the immune system (Sica and Bonte, 2007, Gabrilovich et al., 2012). Recent studies have indicated that macrophages, later identified as M2 (healing), are necessary for regeneration in amphibians (Godwin et al., 2013, Godwin and Rosenthal, 2014), and that these cells promote wound healing in mammals (Mosser and Edwards, 2008; Sica and Mantovani, 2012, Koh and DiPietro, 2013, Simkin et al., 2017, Mescher, 2017, Krzyszczyk et al., 2018). After mesenchymal-fibroblast cells, macrophages and pigment cells are the most common cell types found in the lizard blastema (Alibardi and Sala, 1988). In the present study, the presence of M2-like macrophages (healing), identified using autoradiography, immunohistochemistry and ultrastructure, has been considered a characteristic favoring the regeneration of the tail.
Section snippets
Animals and tissue preparation
Samples from regenerating tails of 10 adult wall lizards (Podarcis muralis), obtained from fixed tissues utilized in previous studies (Alibardi, 2010, Alibardi, 2016, Alibardi, 2017b, Alibardi, 2018), were re-sectioned and employed in the present study. The adopted procedures for care and handling the animals followed the Italian guidelines (Art. 5, DL 116/92). After inducing autotomy, a natural process of tail amputation that does not harm the animals, the tail was allowed to repair for about
Histology of the regenerating blastema-cone
In a regenerating tail blastema-cone of 2–4 mm in length, the spinal cord and vertebrae of the original tail gave rise to a simple tube of ependymal cells and few axons, surrounded by a cartilaginous tube (Fig. 1A and B). Cells proliferation was observed in the ependyma at the blastema-cone stage, but also pycnotic cells (dense small nuclei) and necrotic cells (pale degeneration) were also seen in the apical (within 0.5 mm from the tip) and in more proximal ependyma (more than 1 mm from the tip) (
Blastema macrophages include the healing type (M2-like)
The autoradiographical observations have shown sparse cells incorporating tritiated arginine, a characteristic of macrophages under normal and pathological conditions (Schapira et al., 1998). Although a quantitative study was not done, the distribution of sparse arginine-labeled cells and of immunopositive cells for arginase-1 in the blastema is similar. Both M1 and M2 macrophages uptake arginine for producing NO (M1) or polyammines (M2) (Schapira et al., 1998, Koh and DiPietro, 2013), but the
Acknowledgment
The present study, including TEM and immunofluorescence analyses, was mainly supported by Comparative Histolab Padova.
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2022, Annals of AnatomyCitation Excerpt :At the same time the tail blastema-cone strongly down-regulates genes of both humoral and cellular immunity (j-chain-like, prf, mhcII, ld etc.) and up-regulates immunomodulators genes (nfact4, bcl11a, lef1 etc.). Furthermore, immunohistochemical studies have indicated that most of the macrophages present in the tail blastema belong to the type M2 (healing) while those of the limb are pro-inflammatory M1 type (Alibardi, 2013, 2020c). In contrast, the initial limb blastema, situated at few centimeters distant from that of the tail in the same individual, over-expresses genes involved in inflammation or growth inhibitors (dkk2, mecom, panx3, gas1 etc.) but keeps the activity of most immune-genes to normal levels.