Urodele p53 tolerates amino acid changes found in p53 variants linked to human cancer

Transgenesis promises a powerful means for assessing gene function during amphibian limb regeneration. This approach is complicated, however, by the need for embryonic appendage development to proceed unimpeded despite the genetic alterations one wishes to test later in the context of regeneration. Achieving conditional gene regulation in this amphibian has not proved to be as straightforward as in many other systems. In this report we describe a unique method for obtaining temporal control over exogenous gene expression in the axolotl. Based on technology derived from the Escherichia coli Lac operon, uninduced transgenes are kept in a repressed state by the binding of constitutively expressed Lac repressor protein (LacI) to operator sequences within the expression construct. Addition of a lactose analog, IPTG, to the swimming water of the axolotl is sufficient for the sugar to be taken up by cells, where it binds the LacI protein, thereby inducing expression of the repressed gene. We use this system to demonstrate an in vivo role for thrombospondin-4 in limb regeneration. This inducible system will allow for systematic analysis of phenotypes at defined developmental or regenerative time points. The tight regulation and robustness of gene induction combined with the simplicity of this strategy will prove invaluable for studying many aspects of axolotl biology.

Section: Methodsmentioning

confidence: 99%

Inducible genetic system for the axolotl

Whited

Lehoczky

Tabin

2012

“…In addition, it has been observed that inhibiting p53 disrupts limb regrowth in salamanders (19), although its role in this context has remained unknown. It is possible that p53 could play a role in the regulation of dedifferentiation and redifferentiation events intrinsic to vertebrate regeneration.…”

mentioning

confidence: 99%

Regulation of p53 is critical for vertebrate limb regeneration

Yun

Gates

Brockes

2013

109

101

Extensive regeneration of the vertebrate body plan is found in salamander and fish species. In these organisms, regeneration takes place through reprogramming of differentiated cells, proliferation, and subsequent redifferentiation of adult tissues. Such plasticity is rarely found in adult mammalian tissues, and this has been proposed as the basis of their inability to regenerate complex structures. Despite their importance, the mechanisms underlying the regulation of the differentiated state during regeneration remain unclear. Here, we analyzed the role of the tumor-suppressor p53 during salamander limb regeneration. The activity of p53 initially decreases and then returns to baseline. Its down-regulation is required for formation of the blastema, and its upregulation is necessary for the redifferentiation phase. Importantly, we show that a decrease in the level of p53 activity is critical for cell cycle reentry of postmitotic, differentiated cells, whereas an increase is required for muscle differentiation. In addition, we have uncovered a potential mechanism for the regulation of p53 during limb regeneration, based on its competitive inhibition by ΔNp73. Our results suggest that the regulation of p53 activity is a pivotal mechanism that controls the plasticity of the differentiated state during regeneration. myogenesis | chondrogenesis | p73 | carcinogenesis U nlike mammals, which exhibit limited regenerative abilities, the urodele amphibians-or salamanders-are capable of regenerating an extraordinary range of body structures, including ocular tissues, tail, sections of the heart, parts of the nervous system, and entire limbs (1). In salamanders, such as the newt and axolotl, limb regeneration depends on the formation of a blastema, a mound of progenitor cells of restricted potential that arises after amputation (2-4). Following a period of proliferation, blastema cells redifferentiate and restore the structures of the limb.Extensive evidence indicates that limb regeneration depends on reprogramming of cells in mature limb tissues. Upon amputation, muscle, cartilage, and connective tissue cells underneath the injury site lose their differentiated characteristics and reenter the cell cycle to give rise to the blastema (5-8). This mechanism has also been observed during zebrafish heart and fin regeneration (9, 10). In contrast, reversals of the differentiated state are rarely observed in mammalian tissues, which led to the suggestion that inability to undergo dedifferentiation could contribute to the failure of regeneration in mammals (11). Despite their significance, the mechanisms underlying regulation of the differentiated state during vertebrate regeneration remain poorly understood.Recently, the tumor suppressor p53, whose best-characterized functions are in the maintenance of genome stability (12), has been implicated in the suppression of artificial cell reprogramming to pluripotency (13-17) and the promotion of differentiation pathways in mammals (18). In addition, it has been observed that inhibiting p...

“…The p53 variations associated with environmental stresses have been described in the Mexican salamander axolotl Ambystoma mexicanum and the Israeli blind subterranean mole rat (Spalax judaei; hereafter, S.j.) (7)(8)(9).…”

mentioning

confidence: 99%

Codon 104 variation of p53 gene provides adaptive apoptotic responses to extreme environments in mammals of the Tibet plateau

Zhao

Wang

Zhang

et al. 2013

Mutational changes in p53 correlate well with tumorigenesis. Remarkably, however, relatively little is known about the role that p53 variations may play in environmental adaptation. Here we report that codon asparagine-104 (104N) and glutamic acid-104 (104E), respectively, of the p53 gene in the wild zokor (Myospalax baileyi) and root vole (Microtus oeconomus) are adaptively variable, meeting the environmental stresses of the Tibetan plateau. They differ from serine-104 (104S) seen in other rodents, including the lowland subterranean zokor Myospalax cansus, and from serine 106 (106S) in humans. Based on site-directed mutational analysis in human cell lines, the codon 104N variation in M. baileyi is responsible for the adaptive balance of the transactivation of apoptotic genes under hypoxia, cold, and acidic stresses. The 104E p53 variant in Microtus oeconomus suppresses apoptotic gene transactivation and cell apoptosis. Neither 104N nor 104E affects the cell-cycle genes. We propose that these variations in p53 codon 104 are an outcome of environmental adaptation and evolutionary selection that enhance cellular strategies for surviving the environmental stresses of hypoxia and cold (in M. baileyi and M. oeconomus) and hypercapnia (in M. baileyi) in the stressful environments of the Qinghai-Tibet plateau.T he regulatory mechanisms of p53 mutation related to tumorigenesis have been widely studied and elucidated (1, 2). Notably, however, p53 evolution and adaptation to environmental stresses have not attracted as much attention. Current studies show that p53 is a master sensor and regulator in response to various stressors, such as DNA damage and hypoxia (3-6). Activation of p53 by stresses results in cell-cycle arrest, DNA repair, senescence, or apoptosis in which a series of p53 target genes are involved to maintain genomic integrity (2). The p53 variations associated with environmental stresses have been described in the Mexican salamander axolotl Ambystoma mexicanum and the Israeli blind subterranean mole rat (Spalax judaei; hereafter, S.j.) (7-9).For animals existing on high plateaus, hypoxia and cold serve as strong environmental selective pressures generating adaptive complexes to cope with these stresses. Animals that have evolved on plateaus adopt various strategies involving multiple variations to regulate a series of genes (3, 7). The zokor (Myospalax baileyi, Thomas, 1911; hereafter M.b.) and root vole (Microtus oeconomus, Pallas, 1776; hereafter M.o.) are the dominant native mammals living on the alpine meadow of the Qinghai-Tibet Plateau of China at altitudes of 3,000-4,500 m (equivalent to 11.0-13.0% O 2 at sea level). M.b. is genetically close to Myospalax cansus (Lyon, 1907; hereafter, M.c.), which lives in subterranean burrows at a lower altitude of about 800 m in the lowland of western China. M.b. and M.c. spend their entire life cycle at 70-250 cm underground with significantly low O 2 and high CO 2 levels in their burrows (10). Since the collision of the Indian and the Eurasian plates during the...