Gecko CD59 Is Implicated in Proximodistal Identity during Tail Regeneration

Citation: Wang Y, Wang R, Jiang S, Zhou W, Liu Y, et al. ( Gecko CD59 Is Implicated in Proximodistal Identity during Tail Regeneration Yongjun Wang 0 Ruili Wang 0 Shengjuan Jiang 0 Weijuan Zhou 0 Yan Liu 0 Yingjie Wang 0 Qing Gu 0 Yun Gu 0 Yingying Dong 0 Mei Liu 0 Xingxing Gu 0 Fei Ding 0 Xiaosong Gu 0 Cheng-Xin Gong, New York State Institute for Basic Research, United States of America 0 1 Key Laboratory of Neuroregeneration, Nantong University , Nantong , China , 2 College of Life Science, Anhui Science and Technology University , Fengyang , China Several adult reptiles, such as Gekko japonicus, have the ability to precisely re-create a missing tail after amputation. To ascertain the associated acquisition of positional information from blastemal cells and the underlying molecular mechanism of tail regeneration, a candidate molecule CD59 was isolated from gecko. CD59 transcripts displayed a graded expression in the adult gecko spinal cord with the highest level in the anterior segment, with a stable expression along the normal tail. After tail amputation, CD59 transcripts in the spinal cord proximal to the injury sites increased markedly at 1 day and 2 weeks; whereas in the regenerating blastema, strong CD59 positive signals were detected in the blastemal cells anterior to the blastema, with a gradual decrease along the proximodistal (PD) axis. When treated with RA following amputation, CD59 transcripts in the blastema were up-regulated. PD confrontation assays revealed that the proximal blastema engulfed the distal one after in vitro culture, and rabbit-anti human CD59 antibody was able to block this PD engulfment. Overexpression of the CD59 during tail regeneration causes distal blastemal cells to translocate to a more proximal location. Our results suggest that position identity is not restricted to amphibian limb regeneration, but has already been established in tail blastema of reptiles. The CD59, a cell surface molecule, acted as a determinant of proximal-distal cell identity. - Funding: The work was financially supported by the National Basic Research Program (973 Program) of the Ministry of Science and Technology of China (2003CB515306), the National Natural Science Foundation of China (30670283), and the Basic Research Program of Jiangsu Education Department (10KJA180041; 09KJA180005). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. The regeneration of a missing structure in adulthood is found in several classes of vertebrates, including fish, amphibians and reptiles. The urodele amphibians, characterized by extensive regenerative ability, are capable of regenerating limbs, tail, jaws, lens, and small sections of the heart [17]. Comparatively, anuran tadpoles and some reptiles have an attenuated regenerative ability with preservation of tail reconstruction after amputation or autotomy [8,9]. Re-creation of either limb or tail occurs from a proliferative zone, the blastema, in which mesenchymal stem cells dedifferentiate from internal tissues or migrate from satellite cells. The blastema retains positional identity, which is used to regenerate only correct elements. For example, a wrist blastema regenerates a hand, whereas a shoulder blastema results in an entire arm [10]. Transplantation experiments of limb blastema confirmed that proximodistal (PD) identities are already established in the earliest stages of blastema [11], and that blastemal cells are responsible for the measure of the positional information. Several assays, including blastema rotation, proximodistal blastema engulfment and grafting of distal blastema on proximal blastema, have suggested that PD identity of blastemal cells is encoded as a graded property, and expressed at the cell surface [1214]. Retinoic acid (RA) proximalizes the positional identity of blastemal cells in the proximodistal (PD) axis of regenerating urodele limbs over the small range of RA concentrations about 2.5-fold [15,16]. By screening the cDNA libraries constructed from the distal blastemas of newts treated with RA, da Silva et al. [17] identified the molecule involved in the PD positional memory, Prod1. The amino acid sequence of newt Prod1 contains the conserved motif CCXXXXCN-characteristic of the CD59/ Ly-6 family of the three-finger protein (TFP) superfamily, and eight cystine residues aligned with ten cystine residues conserved in other mammalian CD59. The protein was originally regarded as an ortholog of CD59, which interfered with the assembling MAC by preventing the binding of C9 to the C5b-8 complex [18]. The newt Prod1 is located in the cell surface with a glycosylphosphatidylinositol (GPI) anchor, and implicated in the local cell-cell interactions mediating positional identity [17]. Overexpression of Prod1 caused distal blastemal cells to proximally shift and resulted in shortening or deletion of the lower arm structures, suggesting that Prod1 is a cell surface determinant of PD cell identity [11]. Prod1 is expressed in a stable gradient along the axis in the cells of the adult limb [10]. It was hypothesized that these cells were precursors of blastemal cells and that they inherited the gradient expression of Prod1 after amputation [10]. Recent comparative analysis of the recombinant Prod1 3D solution structure to other known TFPs using phylogenetic techniques found that Prod1 was not a good match for any of the TFP families, including CD59 present in mammals [19], assuming that the role of Prod1 in encoding PD identity was restricted to the newt. However, the conclusion derived from sequence-structure bioinformatic analysis is necessarily limited by the absence of the complete sequence of an urodele genome. As an alternative, further functional verification on the positional identity of CD59 from phylogenetically adjacent species is advantageous in clarifying the association between the protein and Prod1. Although the mechanisms of limb and/or tail regeneration were distinct in their blastemal cell lineage [8,2022], it is conceivable that cells in tail blastema also retain the positional information required to form a new complex tail consisting of skin, muscle, fat, cartilage and neural tissues. The blastemal cells should obtain instructions about where to reconstruct the missing structure, and to which tissues they should differentiate, in a manner similar to that occurs in embryo development and limb regeneration. Here, we cloned the CD59 cDNA from Gekko japonicus and investigated its implication in positional identity during tail regeneration. The functional evidence of CD59 in tail-regenerative reptiles is contributable to the homologous analysis of Prod1. Isolation and analysis of gecko CD59 The cDNA clone (GenBank accession number HM208338) obtained from the brain and spinal cord (...truncated)