Changes in the Inflammatory Response to Injury and Its Resolution during the Loss of Regenerative Capacity in Developing Xenopus Limbs

Abstract

Tissue and organ regeneration, unlike development, involves an injury that in postembryonic animals triggers inflammation followed by resolution. How inflammation affects epimorphic regeneration is largely uninvestigated. Here we examine inflammation and its resolution in Xenopus laevis hindlimb regeneration, which declines during larval development. During the first 5 days postamputation, both regeneration-competent stage 53 and regeneration-deficient stage 57 hindlimbs showed very rapid accumulation of leukocytes and cells expressing interleukin-1β and matrix metalloproteinase 9. Expression of genes for factors mediating inflammatory resolution appeared more persistent at stages 55 and 57 than at stage 53, suggesting changes in this process during development. FoxP3, a marker for regulatory T cells, was upregulated by amputation in limbs at all three stages but only persisted at stage 57, when it was also detected before amputation. Expression of genes for cellular reprogramming, such as SALL4, was upregulated in limbs at all 3 stages, but markers of limb patterning, such as Shh, were expressed later and less actively after amputation in regeneration-deficient limbs. Topical application of specific proinflammatory agents to freshly amputated limbs increased interleukin-1β expression locally. With aqueous solutions of the proinflammatory metal beryllium sulfate, this effect persisted through 7 days postamputation and was accompanied by inhibition of regeneration. In BeSO4-treated limbs expression of markers for both inflammation and resolution, including FoxP3, was prolonged, while genes for cellular reprogramming were relatively unaffected and those for limb patterning failed to be expressed normally. These data imply that in Xenopus hindlimbs postamputation inflammation and its resolution change during development, with little effect on cellular dedifferentiation or reprogramming, but potentially interfering with the expression of genes required for blastema patterning. The results suggest that developmental changes in the larval anuran immune system may be involved in the ontogenetic loss of epimorphic regeneration in this system.

Beryllium nitrate inhibits fibroblast migration to disrupt epimorphic regeneration

“…Because we did not observe complete inhibition of limb regeneration, our results probably reflect differences in size and stage (larvae versus juvenile) of animals across studies where larval limbs contain far fewer connective tissue fibroblasts. Previous studies showing that beryllium completely inhibits regeneration in other Ambystoma species, newts and Xenopus larvae (Thornton, 1949(Thornton, , 1950(Thornton, , 1951Scheuing and Singer, 1957;Tsonis et al, 1991;Mescher et al, 2013), have also documented that the BeN effect is size dependent (for a given concentration), with reduced effects observed on larger organisms (Thornton, 1949). Axolotls used in the present experiments were larger (7-9 cm) compared with larval salamanders and tadpoles used in all previous experiments, suggesting that a similar concentration of BeN may be insufficient to achieve complete inhibition.…”

“…Beryllium is the only alkali or alkaline earth metal that specifically inhibits regeneration without disrupting normal physiological function (Needham, 1941). A recent study using Xenopus suggests that beryllium acts as a potentiator of inflammation to inhibit larval limb regeneration in Anurans, while another study suggests that beryllium inhibits limb regeneration by antagonizing the PIP3 inositol pathway and subsequent proliferation (Tsonis et al, 1991;Mescher et al, 2013). Here, we investigate how beryllium inhibits blastema-based regeneration using axolotl (Ambystoma mexicanum) limb amputation as a model system.…”

Mechanisms of urodele limb regeneration

“…The innate immune system has long been postulated as a major factor that determines whether appendages can regenerate or not (Harty, Neff, King, & Mescher, 2003; Mescher & Neff, 2006; Mescher, Neff, & King, 2013; for reviews). Urodeles, which can regenerate limbs as larvae and adults, have a much less developed immune system than anurans (frogs and toads), which regenerate limbs only as early tadpoles, and mammals, which have no limb regenerative power except for digit tips.…”

Deciphering the complexity of macrophage biology and function with the zebrafish model

“…After an injury, although the inflammatory response is required for limiting the area of tissue damage and debris clearance, it might be deleterious if it persists during the whole process of tissue repair. Indeed, in larval Xenopus which is known to regenerate only during its early developmental stages, hindlimb amputation triggers a transient inflammatory response followed by an increased expression of blastemal markers that signs the regeneration process [42] . In contrast, when the limb amputation is performed after metamorphosis it results in a persistent inflammatory response marked by the absence of blastemal marker expression.…”