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???displayArticle.abstract??? Limb regeneration is an excellent model for understanding organ reconstruction along PD, AP and DV axes. Re-expression of genes involved in axial pattern formation is essential for complete limb regeneration. The cellular positional information in the limbblastema has been thought to be a key factor for appropriate gene re-expression. Recently, it has been suggested that epigenetic mechanisms have an essential role in development and regeneration processes. In this review, we discuss how epigenetic mechanisms may be involved in the maintenance of positional information and the regulation of gene re-expression during limb regeneration.
Fig. 1. Limb regeneration process in urodele amphibians (see text for details). (Step 1) Wound epidermis is formed shortly after limb amputation. (Step 2) Blastema cells accumulate and they proliferate for blastema outgrowth. The blastema elongates until limb regeneration is completed. (Steps 3 and 4) Various key genes, including Fgfs, 5′ Hox and Shh, are re-expressed in the blastema. Repatterning and redifferentiation proceed.
Fig. 2. A hypothetical model of inheritable positional information for appropriate reproduction of gene expression in the limb regeneration process. (A) Morphological and structural patterns along proximal–distal (PD), anterior–posterior (AP) and dorsal–ventral (DV) axes are reconstructed in the limb regeneration process. A differentiated cell in the stump tissues (purple) maintains a characteristic positional information (×) that is composed of three positional values, a (PD), b (AP) and c (DV), in terms of the PD, AP and DV axis, respectively. This information may enable blastema cells to re-establish the lost part of a limb, in which redifferentiated cells (light blue and green) that have different positional information are included. (B) The upper part: in the dedifferentiation process, a blastema cell inherits positional information (yellow flag) from a differentiated cell in order to recognize the original position properly. Such positional information must be stable and inheritable during blastema cell proliferation and redifferentiation. Blastema cells that have the same positional information as that of their parental cells differentiate into identical tissue (purple-Re). Some genes involved in axial pattern formation newly provide a blastema cell with additional de novo positional information (blue and green flags). When de novo positional information is added, the blastema cell can differentiate into tissue distinct from the parental dedifferentiated cell (light blue-Re). The positional information is memorized also in the differentially redifferentiated cell. The middle part: blastema cells from differentiated light-blue cells inherit positional information and cannot redifferentiate into the purple cells that have the former information. If other positional information (green and red flags) is further added, the blastema cell differentiates into tissue that has a distinct property. The lower part: in the blastema cell treated with RA, de novo positional information is unwinded dose-dependently, and the dose-dependent erasing of positional information enables the cell to redifferentiate into tissue that has the former information.
Fig. 3. The concept of positional value (information) and intercalary regeneration. A sequence of positional values can be presumed in amphibian (newt) limb and insect (cockroach) leg as shown by Arabic numbers. Confrontation between normally non-adjacent positional values in the proximal–distal (PD) sequence can be produced by transplantation experiments. When the proximal and distal positional values confront with deletion of the intermediate region along the PD axis, interacalary regenerative response occurs in both newt limb (A) and cockroach leg (B). In this intercalary response, a minimum sequence of positional values will be intercalated (the shortest intercalation rule [45]) and the continuity of adjacent positional values will be restored. When the confrontation is made with duplication of the intermediate region along the PD axis, intercalary response does not occur in newt limb (C) while it occurs in cockroach leg with formation of a longer leg (D).
Fig. 4. A series of epigenetic mechanisms for gene silencing. (i) Transcriptional activation phase: DNA hypomethylation and histone acetylation in the regulatory region enable initiation complex access to nucleosomal DNA, and gene transcription is thereby activated. (ii) Transition phase: the regulatory regions are methylated by DNA methyltransferases (DNMTs) and transcriptional activity is gradually decreased. (iii) Transcriptional repression phase: MeCP2 recognizes methylated CpG sites and recruits Sin3A, histone deacetylase (HDAC) complex and histone lysine methyltransferase (HKMT) activity to the methylated sites. Histone deacetylation and histone lysine methylation occur, and MeCP2 can directly suppress activity of the initiation complex, resulting in repression of transcriptional activity. (iv) Stable gene silencing phase: heterochromatin protein 1 (HP1) or Polycomb group protein (PcG) binds chromatin, thereby maintaining the heterochromatin state and keeping transcriptional activity repressed. Modified and adapted from [116].
Fig. 5. An experiment to examine the relationship between epigenetic regulation and gene expression in the regenerating system. (A, B) To determine whether epigenetic regulation can cause the defective/sufficient Shh expression, regenerating blastema cells from Xenopus froglets were cultured in a primary culture system and treated with 5-azacytidine and TSA in vitro. The procedure used for cell culture was essentially the same as that described by Shibota et al. and Suzuki et al. [117], [118]. Samples of limb blastemas were collected from about 80 froglets at 10 days after amputation, washed, and disaggregated with collagenase/trypsin. A cell suspension was obtained by pipetting, and 1 × 105 cells were transferred with 3 ml of medium into a culture dish (35 mm) and cultured at 25 °C. The medium was changed every day and the cells were pre-cultured for 3 days before the drug treatment. For drug treatment, blastema cells were treated with 5-aza-C at a concentration of 50 μM for 9 days or first treated with 5-aza-C for 6 days and then treated with both 5-aza-C and TSA for 3 days. (C) RT-PCR analysis of the primary cultured blastema cells showed that Shh expression was strongly reactivated by treatment with both drugs, although it was undetectable in the control and in cells treated only with 5-aza-C.
