Initiation Of Limb Regeneration Research Articles

Lmx1b activation in axolotl limb regeneration.

Axolotls can regenerate their limbs. In their limb regeneration process, developmental genes are re-expressed and reorganize the developmental axes, in which the position-specific genes are properly re-expressed. However, how such position specificity is reorganized in the regeneration processes has not been clarified. To address this issue, we focused on the reactivation process of Lmx1b, which determines the limb dorsal identity in many animals. Here, we show that Lmx1b expression is maintained in the dorsal skin before amputation and is activated after amputation. Furthermore, we demonstrate that only cells located in the dorsal side prior to limb amputation could reactivate Lmx1b after limb amputation. We also found that Lmx1b activation was achieved by nerve presence. The nerve factors, BMP2+FGF2+FGF8 (B2FF), consistently reactivate Lmx1b when applied to the dorsal skin. These results imply that the retained Lmx1b expression in the intact skin plays a role in positional memory, which instruct cells about the spatial positioning before amputation. This memory is reactivated by nerves or nerve factors that can trigger the entire limb regeneration process. Our findings highlight the role of nerves in amphibian limb regeneration, including both the initiation of limb regeneration and the reactivation of position-specific gene expression.

Midkine is a dual regulator of wound epidermis development and inflammation during the initiation of limb regeneration.

Formation of a specialized wound epidermis is required to initiate salamander limb regeneration. Yet little is known about the roles of the early wound epidermis during the initiation of regeneration and the mechanisms governing its development into the apical epithelial cap (AEC), a signaling structure necessary for outgrowth and patterning of the regenerate. Here, we elucidate the functions of the early wound epidermis, and further reveal midkine (mk) as a dual regulator of both AEC development and inflammation during the initiation of axolotl limb regeneration. Through loss- and gain-of-function experiments, we demonstrate that mk acts as both a critical survival signal to control the expansion and function of the early wound epidermis and an anti-inflammatory cytokine to resolve early injury-induced inflammation. Altogether, these findings unveil one of the first identified regulators of AEC development and provide fundamental insights into early wound epidermis function, development, and the initiation of limb regeneration.

Rethinking the Blastema

The phenomenon of tissue regeneration has been well documented across many species. Although some possess the capacity to completely restore an entire amputated limb, others are limited to just the distal digit tip. Initiation of limb regeneration has been described to start with the formation of a blastema, the composition of which has long been thought to consist of undifferentiated pluripotent cells derived through the process of dedifferentiation. Competing theories have been proposed, however, including cellular contributions through transdifferentiation and tissue-specific stem cells. Recent studies have now begun to shed light on this controversy, demonstrating tissue resident stem cells to be an evolutionarily conserved measure for limb regeneration.

Wound epidermis formation and function in urodele amphibian limb regeneration.

Upon amputation of the urodele limb, the epidermal cells surrounding the amputation plane migrate to heal the wound. The resulting wound epidermis (WE) induces the regeneration process, resulting in blastema formation, cell division, and ultimately repatterning into a new limb. Despite its central role in the initiation of limb regeneration, little is known about how the WE forms. Here we discuss various models of WE formation and the experimental data in support of each.

Initiation of limb regeneration: The critical steps for regenerative capacity

While urodele amphibians (newts and salamanders) can regenerate limbs as adults, other tetrapods (reptiles, birds and mammals) cannot and just undergo wound healing. In adult mammals such as mice and humans, the wound heals and a scar is formed after injury, while wound healing is completed without scarring in an embryonic mouse. Completion of regeneration and wound healing takes a long time in regenerative and non-regenerative limbs, respectively. However, it is the early steps that are critical for determining the extent of regenerative response after limb amputation, ranging from wound healing with scar formation, scar-free wound healing, hypomorphic limb regeneration to complete limb regeneration. In addition to the accumulation of information on gene expression during limb regeneration, functional analysis of signaling molecules has recently shown important roles of fibroblast growth factor (FGF), Wnt/beta-catenin and bone morphogenic protein (BMP)/Msx signaling. Here, the routine steps of wound healing/limb regeneration and signaling molecules specifically involved in limb regeneration are summarized. Regeneration of embryonic mouse digit tips and anuran amphibian (Xenopus) limbs shows intermediate regenerative responses between the two extremes, those of adult mammals (least regenerative) and urodele amphibians (more regenerative), providing a range of models to study the various abilities of limbs to regenerate.

Wnt/β-catenin signaling has an essential role in the initiation of limb regeneration

Anuran (frog) tadpoles and urodeles (newts and salamanders) are the only vertebrates capable of fully regenerating amputated limbs. During the early stages of regeneration these amphibians form a “blastema”, a group of mesenchymal progenitor cells that specifically directs the regrowth of the limb. We report that wnt-3a is expressed in the apical epithelium of regenerating Xenopus laevis limb buds, at the appropriate time and place to play a role during blastema formation. To test whether Wnt/β-catenin signaling is required for limb regeneration, we created transgenic X. laevis tadpoles that express Dickkopf-1 (Dkk1), a specific inhibitor of Wnt/β-catenin signaling, under the control of a heat-shock promoter. Heat-shock immediately before limb amputation or during early blastema formation blocked limb regeneration but did not affect the development of contralateral, un-amputated limb buds. When the transgenic tadpoles were heat-shocked following the formation of a blastema, however, they retained the ability to regenerate partial hindlimb structures. Furthermore, heat-shock induced Dkk1 blocked fgf-8 but not fgf-10 expression in the blastema. We conclude that Wnt/β-catenin signaling has an essential role during the early stages of limb regeneration, but is not absolutely required after blastema formation.

X-ray induced inhibition of DNA synthesis and mitosis in internal tissues during the initiation of limb regeneration in the adult newt.

Left front limbs of adult male newts were given 2000r of X-irradiation at least four weeks prior to amputation of both forelimbs. Internal stump tissues were evaluated for the ability to incorporate 3H-thymidine and accumulate colchicine-blocked mitotic figures. In otherwise uninjured limbs, irradiation stimulated low levels of DNA synthesis which did not increase significantly after amputation. Thus, as soon as DNA synthesis increased significantly in normal limbs as a result of amputation, it was demonstrably higher than in X-rayed limbs. In general, mitotic activity in both groups reflected the DNA synthetic rates. Since others have shown that denervation at the time of amputation blocks subsequent mitosis in internal stump tissues yet allows normal levels of DNA synthesis for eight days, we conclude that X-irradiation and denervation prevent cell division in potential blastema cells by different mechanisms.

Denervation effects on DNA replication and mitosis during the initiation of limb regeneration in adult newts

Histological and cellular events during the first 2 weeks after amputation of adult newt forelimbs were compared under conditions of innervation and denervation in order to ascertain the first nerve-dependent step in the regeneration process. Formation of a wound epithelium, histolysis, and dedifferentiation occurred, despite the absence of nerves, in a manner similar to that of the regenerating limbs. Autoradiography showed that cells in the denervated limb-stumps incorporate [ 3H]thymidine into DNA beginning on day 4 and that during the first week after amputation these limb stumps resembled the contralateral controls in overall labeling indices, in the distribution of the labeled nuclei, and intensity of labeling. Microspectrophotometric determinations of nuclear DNA contents in the dedifferentiated cells of denervated and innervated limbs revealed that in both cases replication of the entire chromosomal complement occurred. Cell division, which first began 6 days after amputation, and early blastema formation were observed only in the innervated limb stumps. A likely explanation of these results, consistent with the preponderance of the literature on regeneration, is that limb stumps can initiate the regeneration process in the absence of nerves but that the cells are blocked in the G 2 phase of the first cell cycle and consequently do not proliferate to form a blastema.

Cell division and ribonucleic acid synthesis during the initiation of limb regeneration in larval axolotls (Ambystoma mexicanum).

AbstractMitotic activity and RNA synthesis were examined in normal regenerating limbs and denervated non‐regenerating limbs of larval axolotls during the first six days after amputation. H3‐uridine incorporation into RNA was measured by autoradiography of deoxyribonuclease treated sections. Grain counts indicated a significant increase in RNA synthesis in both normal and denervated limbs through the six‐day time period but a greater increase was seen in normal limbs.The Mitotic Index increased significantly in normal limbs on the fifth day postamputation. In contrast, no increase in M.I. was observed in denervated limbs.

RETARDATION OF THE NEWT LIMB REGENERATION WITH SEMICARBAZIDE, AN INHIBITOR OF HISTAMINE FORMATION.

AbstractIt is widely known that the substance histamine is a noxious agent released from cells when they are injured. At the outset these experiments were intended to show whetherhistamine, persumably released by newt limb amputation, might also promote beginning steps in regeneration. In addition, according to a new view of the physiology of histamine, its formation metabolically by the decarboxylation of histidine is an attribute of the rapid growth of many tissues. These experiments were mainly designed to interfere with histamine production and thereby disrupt the rapid growth of the limb regenerate.Semicarbazide has been used successfully in mammals to inhibit histidine decarboxylase and decrease the rate of histamine formation. In these experiments, semicarbazide was administered to stumps of amputated newt limbs by microinfusion for many hours at a time in efforts to decrease histamine formation and retard growth.When semicarbazide was infused into the limb only in preblastemal stages of regeneration, it seemed to have no effect on the course of regeneration. When it was infused directly into the early mesenchymatous blastema, however, it retarded the expansion and differentiation of the blastema. From present evidence, it appears that histamine production may not be concerned in the initiation of limb regeneration but is probably concerned in the phase of rapid blastemal enlargement, whose primary histological attribute is cellular mitosis.