Beryllium nitrate inhibits fibroblast migration to disrupt epimorphic regeneration

Abstract

Epimorphic regeneration proceeds with or without formation of a blastema, as observed for the limb and skin, respectively. Inhibition of epimorphic regeneration provides a means to interrogate the cellular and molecular mechanisms that regulate it. In this study, we show that exposing amputated limbs to beryllium nitrate disrupts blastema formation and causes severe patterning defects in limb regeneration. In contrast, exposing full-thickness skin wounds to beryllium only causes a delay in skin regeneration. By transplanting full-thickness skin from ubiquitous GFP-expressing axolotls to wild-type hosts, we demonstrate that beryllium inhibits fibroblast migration during limb and skin regeneration in vivo. Moreover, we show that beryllium also inhibits cell migration in vitro using axolotl and human fibroblasts. Interestingly, beryllium did not act as an immunostimulatory agent as it does in Anurans and mammals, nor did it affect keratinocyte migration, proliferation or re-epithelialization, suggesting that the effect of beryllium is cell type-specific. While we did not detect an increase in cell death during regeneration in response to beryllium, it did disrupt cell proliferation in mesenchymal cells. Taken together, our data show that normal blastema organogenesis cannot occur without timely infiltration of local fibroblasts and highlights the importance of positional information to instruct pattern formation during regeneration. In contrast, non-blastemal-based skin regeneration can occur despite early inhibition of fibroblast migration and cell proliferation.

Macrophages and fibroblasts during inflammation and tissue repair in models of organ regeneration

“…() suggests that sustained production of PDGF to enhance fibroblast migration is a required activity of the local macrophages critical for blastema formation and distal outgrowth instead of scarring. This possibility is supported by the observations that fibroblast migration in amputated limbs of axolotls is severely reduced both by treatment with either a PDGFR inhibitor (Currie et al., ) or Be (Cook & Seifert, ), since exposure of macrophages to Be induces a persistent inflammatory phenotype, upregulating production of TNF‐α and reactive oxygen species, and is proapoptotic for these cells (Sawyer et al., ). Both of these new regeneration studies with axolotl limbs support the view that PDGF production by macrophages is one of their functions required for the fibroblastic movements which orchestrate growth and development of other lineage‐specific blastema cells, a function at least partially inhibited by persistent inflammation in the presence of Be, interfering with successful regeneration.…”

“…As discussed previously (Mescher et al, 2013(Mescher et al, , 2017, urodeles' greater tolerance of Be, like their longer tolerance of allografts compared to adult frogs or mammals, probably reflects their relatively weaker cellular immunity (Barlow, DiMarzo, & Cohen, 1981;Cohen, 1971;Tournefier et al, 1998), which includes their low MHC II diversity and weak T helper cells (Sammut et al, 1999). Cook and Seifert (2016) further examined the inhibitory effects of 100 mM BeN on Ambystoma limb regeneration, immersing larger (7−8 cm) animals for 2 min immediately after amputation. Unlike our results with the much smaller larvae (Mescher et al, 2013;Tsonis et al, 1991), limbs in the larger axolotls did regenerate after Be exposure but with severely perturbed skeletal patterns.…”

“…Cook and Seifert () further examined the inhibitory effects of 100 mM BeN on Ambystoma limb regeneration, immersing larger (7−8 cm) animals for 2 min immediately after amputation. Unlike our results with the much smaller larvae (Mescher et al, ; Tsonis et al., ), limbs in the larger axolotls did regenerate after Be exposure but with severely perturbed skeletal patterns.…”

“…Unlike our results with the much smaller larvae (Mescher et al, ; Tsonis et al., ), limbs in the larger axolotls did regenerate after Be exposure but with severely perturbed skeletal patterns. Leukocyte infiltration of the regenerating limbs was not stimulated by Be which, however, was found to inhibit migration and proliferation of both dermal fibroblasts and blastema cells, with no increase in apoptosis and without affecting either migration or growth of epidermal cells (Cook & Seifert, ). The results were interpreted to suggest that in urodeles Be disrupts regenerative patterning of limbs by directly interfering with mesenchymal cell or fibroblast migration and growth, a conclusion consistent with the effects of Be infused into regenerating limbs of adult newts (Scheuing & Singer, ).…”

Mechanisms of urodele limb regeneration

“…Following amputation and histolysis, blastema cells having the value of their PD level of origin migrate centripetally from different positions on the limb circumference (Gardiner, Muneoka, & Bryant, 1986) and use short range interactions to intercalate a complete cross‐section of APDV identities which adopt the next distal PD value. Beryllium treatment of amputated limbs induces pattern abnormalities by interfering with the migration and interaction of blastema cells derived from different circumferential positions on the skin (Cook & Seifert, 2016). Successive rounds of migration and intercalation after amputation restore the normal APDV and PD nearest neighbor map.…”