Inhibition of limb regeneration in the axolotl after treatment of the skin with actinomycin D

Abstract

In this experiment actinomycin D was used to explore the action of the wound epidermis on underlying tissues during limb regeneration. In axolotl forelimbs the skin was removed from the elbow to the shoulder. Skin from the right limbs was soaked for three hours in actinomycin D (5.0 or 10.0 ug/ml 0.6% NaC1). For controls, skin from left limbs was soaked in 0.6% NaCl for the same period of time. Each piece of skin was orthotopically replanted, and both limbs were amputated through the treated skin, proximal to the elbow. After an initial healing period, the control limbs regenerated normally. Except for a slightly paler color, limbs bearing actinomycin-treated skin were indistinguishable from the controls, both grossly and histologically, during the first week following amputation. While the control limbs formed early blastemas, no grossly visible evidence of regeneration was apparent in the experimental limbs, but histologically some dedifferentiation was occurring. Normally three to four digits were seen in the control regenerates before blastemas appeared on the experimental limbs. By 3 5 4 0 days blastemas had appeared on most experimental limbs. These developed very rapidly, and within a short time many of them had attained levels of development close to the controls. Actinomycin D temporarily suppresses formation of the apical epidermal cap and the subsequent aggregation of dedifferentiated cells into a blastema. When the effect wears off, an apical cap forms and the dedifferentiated cells quickly organize into a blastema and begin to differentiate.The outgrowth of a developing vertebrate extremity, whether it be in an embryo (ZwiUing, '61) or a regenerating limb (Thornton, '65), appears to be the morphological expression of a continuous interaction between the epidermal covering and the underlying mesodermal components. Of particular importance is a thickening of the epidermis over the tip of the growing limb. In the regenerating urodele limb, Thornton ('54) has called this structure the apical epidermal cap, and it seems to play a vital role in the distal migration of dedifferentiated cells and their eventual accumulation into a blastema (Steen and Thornton, '63; Thornton and Steen, '62). The factors which bring about the apical epidermal thickening and the manner in which the epidermis influences the underlying tissues have continued to elude characterization. The present experiment is an outgrowth of some previous work on the effect of actinomycin D upon limb regeneration in newts (Carlson, '66, '67a; Wolsky and VanDoi, '65). In animals given systemic doses of actinomycin D, certain disturbances were noted in the pattern of epithelialization of the wound surface of an ANAT. REC., 163: 389-402. amputated limb. These disturbances consisted primarily of a tremendous overgrowth and disarray of the wound epithelium. Since in systemically treated animals the wound epidermis was underlain by considerable cellular debris, it was not possible to exclude the indirect effects of general toxicity, namely ...

Macrophages are required to coordinate mouse digit tip regeneration

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Proteomic analysis of fibroblastema formation in regenerating hind limbs of Xenopus laevis froglets and comparison to axolotl

“…Methylation of this amino acid leads to transcriptional silencing; maintenance or up regulation of KDM3B expression would counter this. The fact that KDM3B is strongly down regulated instead suggests that the genes it regulates are transcriptionally silenced as part of the shift in transcriptional activity leading to repression of genes associated with tissue differentiation and the activation of progenitor cell and more embryonic ECM genes [73-76]. …”

Mechanisms of urodele limb regeneration

“…Inhibition of these transcriptional changes by actinomycin D does not affect histolysis, but does prevent or retard dedifferentiation, leading to regenerative failure or delay (Carlson, 1969). Dedifferentiated cells express a more limb bud‐like ECM in which type II collagen synthesis is suppressed, type I collagen synthesis remains the same, and fibronectin, tenascin, and hyaluronate accumulate (Ashahina, Obara, & Yoshizato, 1999; Gulati, Zakewski, & Reddi, 1983; Mescher & Munaim, 1986; Onda, Poulin, Tassava, & Chiu, 1991).…”