Animal Cells and Systems Vol. 16, No. 6, December 2012, 462468 Eugene Lee, Bong-Gun Ju and Won-Sun Kim* Department of Life Science, Sogang University, Sinsoo-dong 1, Mapo-gu, Seoul 121-742, Korea (Received 21 June 2012; accepted 9 September 2012) Urodeles including newt and salamander have remarkable regenerative capacity during the postembryonic life span. Some of the unique features are the formation of the well-developed wound epidermis and the active dedifferentiation process in the early phase of regeneration. These are regarded as key events for the successful regeneration since no further regenerative activity is possible without them. In this study, we investigated the role of retinoic acid (RA) in salamander limb regeneration by blocking RA synthesis using disulfiram, an inhibitor of aldehyde dehydrogenase that oxidizes retinal to RA. Disulfiram treatment resulted in delaying the limb regeneration processes via inhibition of wound epidermis formation and dedifferentiation process. When RA was administered after disulfiram treatment, the inhibitory effect of disulfiram was rescued. In addition, disulfiram treatment after the dedifferentiation stage resulted in the mild retardation of limb regeneration, suggesting that RA might also be involved in the blastema outgrowth. Furthermore, salamander MMP-9 gene expression was also inhibited by disulfiram treatment. Collectively, our findings indicate that endogenous RA may play an important role(s) in the early phase of limb regeneration by regulating the expression of molecules responsible for the modification of intracellular and extracellular environment during salamander limb regeneration. Keywords: dedifferentiation; salamander limb regeneration; retinoic acid; MMP-9 Introduction the early event of regeneration (Hay 1993; Daley et al. 2008). Regeneration is the recovery of body parts that have Retinoic acid (RA) is considered as one of the key been lost or destroyed during the postembryonic life molecules in regeneration since it has a noticeable effect span. Although mammals have a limited regenerative on the dedifferentiation state in the regenerating power, urodeles including salamander and newt possess salamander limb (Ju and Kim 1994, 1998, 2010; an outstanding regenerative ability even in the adult Shimizu-Nishikawa et al. 2001). RA is an endogenous state. The body parts to be regenerated in urodele derivative of vitamin A, which is involved in many include limbs, tail, lens, jaw and spinal cord (Carlson developmental processes including embryogenesis, 2007). Especially, limbs of urodele have been used spermatogenesis and epithelial tissue differentiation extensively as a typical model system of regeneration. (Marill et al. 2003; Clagett-Dame and Knutson 2011; Limb regeneration is composed of four steps: wound Rhinn and Dolle ´ 2012). Alcohol dehydrogenases healing, dedifferentiation, blastema formation and metabolize vitamin A (retinol) to retinal, which is, in redifferentiation (Stocum 1979). At dedifferentiation turn, converted to all-trans RA or 9-cis RA by the stage, the mature cells lose their differentiated char- retinal dehydrogenases (Duester 2000). RA modulates acteristics and re-enter cell cycle to produce blastema the gene expression through RA response element, cells. Therefore, dedifferentiation is a critical event which is mediated by RA receptors (RARs) and between the regenerating and the non-regenerating retinoid X receptors (Duong and Rochette-Egly 2011). systems (Tanaka 2003; Stocum 2004). During dediffer- In the salamander limb regeneration, exogenous entiation, extensive tissue remodeling such as modifi- supply of RA at the dose of pharmacological level cation of extracellular matrix (ECM) is undergoing in results in the extensive dedifferentiation in the stump the stump by the activities of various enzymes such as tissue and pattern duplication. This was demonstrated matrix metalloproteinases (MMPs), lysosomal enzymes by the increased expression and activity of marker and peptidases (Ju and Kim 1998, 2000, 2010; Park and enzymes of dedifferentiation such as MMP-9, cathe- Kim 1999; Yang et al. 1999; Carlson 2007; Santosh psin D and lysosomal acid phosphatase after RA et al. 2011). Since ECM is involved in the stabilization treatment both in gene expression and enzyme activity of the differentiation state of cells, the degradation of (Ju and Kim 1998, 2000, 2010; Park and Kim 1999; ECM in the mature stump tissue is likely to cause the Yang et al. 1999; Santosh et al. 2011). The pattern disorganization of the differentiated state and triggers duplication in the regenerating amphibian limbs by *Corresponding author. Email: email@example.com Present address: Eugene Lee, Department of Neuroscience, UT Southwestern Medical Center, 6000 Harry Hines Boulevard, NA4.118, Dallas, TX 75390-9111, USA. ISSN 1976-8354 print/ISSN 2151-2485 online # 2012 Korean Society for Integrative Biology http://dx.doi.org/10.1080/19768354.2012.729537 http://www.tandfonline.com DEVELOPMENTAL BIOLOGY Animal Cells and Systems 463 exogenous RA suggests that RA resets positional with disulfiram. Same volume of DMSO was injected memory of blastema cells (Kim and Stocum 1986; intraperitoneally as a control. Crawford and Stocum 1988; Ludolph et al. 1990). In the developing chick limb bud, RA differentially concentrates on the posterior region compared to the Histology anterior region (Thaller and Eichele 1987). Interest- Limbs were collected at each time course, and they ingly, local application of RA to the anterior margin were fixed in Bouin solution. Histological examination disrupts the established gradient of RA on chick wing was performed as described previously (Ju and Kim bud and induces mirror-imaged pattern duplication of 1994). The specimens were processed for paraffin wing skeletons in the anteroposterior axis (Eichele et al. embedding, serially sectioned at 10 mm and stained 1985). Furthermore, inhibition of RA synthesis sup- with Ehrlich hematoxylin and eosin. presses chick wing bud development, implying that RA also might be required for the initiation of the chick wing bud outgrowth (Stratford et al. 1996). In addition, Whole mount in situ hybridisation RA gradient is actively established in development and Limb regenerates were fixed in 4% paraformaldehyde in regeneration processes of amphibian limbs (Scadding phosphate buffered saline (PBS) at 48C for overnight. and Maden 1994; McEwan et al. 2011). The limb regenerates injected with disulfiram were In this study, we examined the role of endogenous analyzed for Hynobius MMP-9 gene expression by RA in salamander limb regeneration by inhibition of whole mount in situ hybridization. The riboprobe of RA synthesis using disulfiram, an inhibitor of aldehyde MMP-9 was transcribed by T7 RNA polymerase from dehydrogenase. Our findings indicate that RA may be Kpn I linearized MMP-9 cDNA using DIG-labeling kit involved in the wound epidermis formation and initia- (Boehringer Mannheim, Germany). The whole mount tion of dedifferentiation via up-regulation of MMP-9 in situ hybridization was performed according to the gene expression during salamander limb regeneration. Wilkinson’s protocol with some modifications (Wilkinson 1992; Ju and Kim 1998). After hybridiza- tion, color reaction was carried out using BM purple Materials and Methods alkaline phosphatase substrate (Boehringer Mannheim) Animal maintenance in a dark box with gentle rocking for several hours or Salamander (Hynobius leechii) larvae were maintained overnight. The specimens were washed 3 times with stop as described previously (Ju and Kim 1994). Briefly, the solution (1mM EDTA, 0.1% Tween 20 in PBS). larvae were individually reared in latticed cages to prevent cannibalism and fed finely chopped beef liver. At the time of amputation, the larvae were 20 to 30 mm Results in length from snout to tail. Retardation of regeneration by disulfiram treatment We investigated the role of RA in the regenerating salamander limbs using disulfiram, an inhibitor of Experimental manipulation aldehyde dehydrogenase that mediates RA synthesis. In For surgical manipulations, animals were anesthetized order to find the most effective stage of the regenera- in 0.02% benzocaine solution. The forelimbs of each tion by the disulfiram injection, Hynobius larvae were animal were amputated bilaterally through the distal intraperitoneally injected with disulfiram at 0 day (DS stylopodium (upper arm) level. Any protruding limb 0), 1 day (DS 1), 4 days (DS 4) and 8 days (DS 8) after cartilage due to the contraction of soft tissue was amputation. We first examined the days required to trimmed to make a flat cut surface immediately after reach the medium bud stage from amputation. As amputation. Disulfiram (Tetraethylthiuram disulfide, shown in Figure 1, the rate of regeneration in Sigma, USA) dissolved in dimethyl sulfoxide (DMSO) disulfiram-injected group was evidently retarded com- was injected to each salamander larvae intraperitone- pared to that in non-injected control group. The ally at the dose of 100 mg/g body weight using retardation was especially pronounced when the dis- microliter syringe (Hamilton, USA). RA (all trans, ulfiram was injected in the early stage of limb type XX, Sigma) dissolved in DMSO was injected regeneration. While control limb regenerates took for intraperitoneally at the dose of 40 mg/g body weight. To 7 days to reach the medium bud stage, disulfiram- compare the effect of the administration stage in the treated regenerates took for 20 days, 17 days, 12 days course of regeneration, animals were injected at 0 and 9 days in the groups of DS 0, DS 1, DS 4 and DS 8, (immediately), 1, 4 and 8 day(s) after amputation respectively. 464 E. Lee et al. Figure 1. Regeneration rate in the disulﬁram-treated sala- mander limbs. Amputation was performed through the level of elbow in the forelimbs of Hynobius leechii in these series of experiments. The regeneration rates of limbs in all disulﬁram- treated groups are decreased compared to that of the control. Animals were injected with disulﬁram at 0 (immediately; DS 0), 1 (DS 1), 4 (DS 4) and 8 days (DS 8) after limb amputation. The various boxes denote days corresponding to the speciﬁed regeneration stages up to the medium bud stage after amputation in the limb regenerates (Tank et al. 1976; Ju and Kim 1994). Non-injected animals are used as a control. WH, wound healing stage; DD, dedifferentiation Figure 2. Histological analysis of limb regenerates treated stage; EB, early bud stage. with disulﬁram at day 0. Salamanders were injected with DMSO (A,C,E,G) or with disulﬁram (B,D,F,H) immediately Histological analysis of disulfiram-treated limb after amputation (DS 0). Scale bar; 1mm. (A,B) Limb regenerates regenerates at 1 day after amputation (1 day after injection). Note the thinner wound epithelium (arrow) in disulﬁram- We further performed histological analysis of limb treated regenerate compared to the DMSO-treated control; regenerates throughout regeneration period. In DS 0 H, humerus; m, muscle. (C,D) Limb regenerates at 4 days group, wound epithelium of limb regenerates was after amputation (4 days after injection). In DMSO-treated thinner than that of control group at 1 day after control regenerate, dedifferentiation is extensive (bracket) amputation (1 day after injection) (Figure 2A and B). under the thickened wound epithelium (arrow). However, the While control wound epithelium thickened for a few level of dedifferentiation is low, (bracket) and the wound days after wound healed, it remained thin in disulfir- epithelium is not well-developed in disulﬁram-treated regen- erate. (E,F) Limb regenerates at 7 days after amputation (7 am-treated limb regenerates (Figure 2C and D). In days after injection). While blastema develops well in control addition, disulfiram treatment resulted in inhibition of regenerate, blastema cannot be discernible in disulﬁram- dedifferentiation process and blastema formation treated regenerate; b, blastema. (G,H) Limb regenerates at (Figure 2E and F). At 17 days after amputation (17 17 days after amputation (17 days after injection). Note the days after injection), control limb regenerates reached condensation of mesenchymal cells for digit formation to palette stage (Figure 2G). However, limb regenerate (arrows) in DMSO-treated regenerates. An abnormal cellular was still remained at the blastema formation stage in condensation is formed at the tip of the disulﬁram-treated disulfiram-injected group (Figure 2H). The histological limb. aspects of DS 1 group were similar to that of the DS 0 group (data not shown). after amputation (1 day after injection). At 12 days To examine the effect of disulfiram on the process after amputation (8 days after injection), outgrowth of of dedifferentiation, one group of animals was sub- disulfiram-treated blastema appears to be more re- jected to disulfiram injection at 4 days after amputation tarded compared to that of control regenerate (Figure (DS 4). At 4 days after amputation, the dedifferentia- 3C and D). tion is at its peak level in the normally regenerating We further tested whether disulfiram affects the limbs (Ju and Kim 1994). As shown in Figure 3A and blastema outgrowth even after it has been initiated. B, wound epithelium and blastema were not well Disulfiram was given to one group of animals at 8 days developed in disulfiram-treated regenerate at 5 days after amputation (DS 8) when the control limb Animal Cells and Systems 465 to the control (Figure 4A and B). Although the rate of regeneration in disulfiram-injected group was some- what slower than that of control group, disulfiram- treated limb regenerate reached the palette stage at 20 days after amputation (12 days after injection) (Figure 4C and D). These findings indicate that disulfiram treatment might inhibit the process of wound epithe- lium formation and dedifferentiation during salaman- der limb regeneration. RA rescues the phenotype caused by disulfiram treatment To see whether the inhibitory effect of disulfiram on limb regeneration is due to the depletion of endogenous RA, disulfiram injection was done immediately after Figure 3. Histological analysis of limb regenerates treated amputation and RA was subsequently injected at 4 with disulﬁram at day 4. Salamanders are injected with days after the disulfiram injection. Although wound DMSO (A,C) or with disulﬁram (B,D) at 4 days after epithelium formation was somewhat inhibited, dedif- amputation. Scale bar; 1mm. (A,B) Limb regenerates at 5 ferentiation was evident (Figure 5A and B). At 12 days day after amputation (1 day after injection). While exten- after amputation and disulfiram injection (8 days after sively dedifferentiated internal tissues and distal accumula- RA injection), a spike-like blastema under the well- tion of blastema cells are noted in DMSO-treated regenerate, developed wound epithelium was observed (Figure 5C). wound epithelium (arrow) is thinner and less dedifferentiated The digit formation was completed almost by 20 days tissues are observed in disulﬁram-treated regenerate. (C,D) after amputation and disulfiram injection (16 days Limb regenerates at 12 days after amputation (8 days after after RA injection) at which time point the control injection). The outgrowth of blastema appears to be retarded in disulﬁram-treated regenerate compared to control. regenerate showed similar morphology (Figure 5D). regenerate was at medium bud stage (Figure 4). The regenerate at 9 days after amputation (1 day after injection) showed a little shrunken blastema compared Figure 5. Histological analysis of limb regenerates treated with disulﬁram and RA. Disulﬁram injection was done immediately after amputation. RA was injected 4 days after disulﬁram injection. (A) Limb regenerate at 5 days after amputation and disulﬁram injection (1 day after RA injec- tion). The wound epithelium (arrow) is still hypomorphic, but Figure 4. Histological analysis of limb regenerates treated the extensive dedifferentiation process (bracket) can be seen with disulﬁram at day 8. Salamanders are injected with in the distal portion of the regenerate. (B) Limb regenerate at DMSO (A,C) or with disulﬁram (B,D) at 8 days after 8 days after amputation and disulﬁram injection (4 days after amputation. Scale bar; 1mm. (A,B) Limb regenerates at 9 RA injection). Note the active dedifferentiation in progress days after amputation (1 day after injection). The disulﬁram- (box). (C) Limb regenerate at 12 days after amputation and treated blastema (arrow) is somewhat smaller than that in the disulﬁram injection (8 days after RA injection). Note the control. (C,D) Limb regenerates at 20 days after amputation well-formed blastema (arrow). (D) Limb regenerate at 20 (12 days after injection). The digits are almost formed in the days after amputation and disulﬁram injection (16 days after control regenerate. Disulﬁram treatment resulted in slow RA injection). The digits are completely formed, and regeneration rate, but limb regenerate reached the palette regenerate showed similar morphology to the control regen- stage. erate of equivalent days. 466 E. Lee et al. These results indicate that the low level of dediffer- showed the bimodal activation during limb regenera- entiation and the retarded growth of blastema in tion (Yang et al. 1999). In the disulfiram-injected limb disulfiram-treated limb regenerates can be rescued by regenerates, the expression region of MMP-9 was not exogenous supply of RA. changed but the expression level was very low and expression region was restricted to the tip of the regenerates (Figure 6B and D). These results suggest that endogenous RA may up-regulate MMP-9 expres- The effect of disulfiram on MMP-9 gene expression sion in salamander limb regeneration. Since MMP-9 gene expression is up-regulated in the dedifferentiation stage during salamander limb regen- eration, we investigated the effects of disulfiram Discussion treatment on its expression by whole mount in situ A number of studies suggest that the RA is essential in hybridization. In control DMSO-injected group, various developmental processes including limb regen- MMP-9 gene expression started to be detected in the eration. Exogenous RA is well known to induce the wound epithelium at 2 days after amputation (1 day duplication in three cardinal axes, i.e., proximodistal, after injection) (Figure 6A). The expression level of dorsoventral and anteroposterior axes and probably to MMP-9 gene was significantly up-regulated and respecify the positional identity of the blastema cells in reached the peak level in the distal region of the the limb regeneration (Kim and Stocum 1986; mesenchymal tissue at 8 days after amputation (7 days Crawford and Stocum 1988; Ludolph et al. 1990). In after injection) (Figure 6C). As the regenerate entered addition, administration of RA both intensifies the the notch stage, the MMP-9 gene was expressed only in level of dedifferentiation and extends the duration of the edge of the hand plate (data not shown). Consistent dedifferentiation (Ju and Kim 1994, 1998, 2000, 2010; with our findings, axolotl MMP-9 gene expression Shimizu-Nishikawa et al. 2001). Although it has been reported that the inhibition of RA’s action blocks limb regeneration of urodele (Del Rinco ´ n and Scadding 2002), how the depletion of endogenous RA by disulfiram affects the process of regeneration has not been studied thoroughly at the histological and mole- cular levels. In this study, we found that disulfiram treatment results in retardation of limb regeneration processes. Especially, limb regeneration was severely inhibited when disulfiram was injected before dediffer- entiation stage, suggesting that RA is more critically involved in the wound epithelium formation and initiation of dedifferentiation process than the main- tenance of dedifferentiation state. Interestingly, it has been reported that disulfiram treatment at the initia- tion of limb bud development completely blocks the chick limb bud formation. However, the administration of disulfiram after the formation of limb bud shows little effect on the chick limb development (Stratford et al. 1996). Our findings also indicate that development of Figure 6. The gene expression pattern of MMP-9 in the wound epithelium plays important role(s) in the disulﬁram-treated regenerating salamander limbs. Salaman- process of dedifferentiation. In fact, the denervation ders were injected either with DMSO (A,C) or disulﬁram of newt limbs resulted in the formation of thin wound (B,D) 1 day after amputation. (A,B) Limb regenerate at 2 epithelium due to the lack of mitogenic effect of nerve days after amputation (1 day after injection). Low level of (Bryant et al. 1971; Satoh et al. 2008; Kumar et al. MMP-9 gene expression was detected in wound epithelium of 2010). Moreover, wound epithelium is presumed to both DMSO- or disulﬁram-treated regenerates (arrowhead), inhibit the redifferentiation of neighboring mesenchy- but the expression level is somewhat higher in the control mal cells and promotes them to re-enter the cell cycle regenerate. (C,D) Limb regenerates at 8 days after amputa- by production of signaling factors such as fibroblast tion (7 days after injection). MMP-9 gene appears to be growth factors (Stocum 1996). Therefore, the lack or expressed remarkably in the whole blastema (box). However, sub-threshold level of RA induced by disulfiram in disulﬁram-treated regenerate, the expression is low and its treatment might be involved in underdevelopment of signal is restricted to the distal region of blastema (box). Animal Cells and Systems 467 Del Rinco ´ n SV, Scadding SR. 2002. Retinoid antagonists wound epithelium, which in turn causes low activity of inhibit normal patterning during limb regeneration in the dedifferentiation in salamander limb regeneration. axolotl, Ambystoma mexicanum. J Exp Zool. 292: Although disulfiram delayed limb regeneration, we 435443. did not observe abnormal regeneration or complete Duester G. 2000. Families of retinoid dehydrogenases reg- inhibition of regeneration. This could be possible due ulating vitamin A function: production of visual pigment and retinoic acid. Eur J Biochem. 267:43154324. to the resumed supply of RA by the diminishing Duong V, Rochette-Egly C. 2011. The molecular physiology disulfiram level with time. Alternatively, it is possible of nuclear retinoic acid receptors. From health to disease. that low amount of RA was synthesized even under Biochim Biophys Acta. 1812:10231031. disulfiram treatment so that this treatment alone might Eichele G, Tickle C, Alberts BM. 1985. Studies on the not be enough to completely block RA synthesis in the mechanism of retinoid-induced pattern duplications in the early chick limb bud: temporal and spatial aspects. regenerating salamander limbs. J Cell Biol. 101:19131920. The reduced expression of MMP-9 gene by dis- Hay ED. 1993. Extracellular matrix alters epithelial differ- ulfiram treatment indicates that RA might be involved entiation. Curr Opin Cell Biol. 5:10291035. in the dedifferentiation process at the molecular level. Ju BG, Kim WS. 1994. Pattern duplication by retinoic acid In fact, RAR a (RA receptor a) binds to the promoter treatment in the regenerating limbs of Korean salaman- der larvae, Hynobius leechii, correlates well with the of MMP-9 gene when it was up-regulated by RA extent of dedifferentiation. Dev Dyn. 199:253267. treatment (Zaragoza ´ et al. 2007; Lackey and Hoag Ju BG, Kim WS. 1998. Upregulation of cathepsin D 2010). Considering that the MMPs are involved in the expression in the dedifferentiating salamander limb ECM degradation and tissue remodeling, it is possible regenerates and enhancement of its expression by retinoic that the MMP-9 regulates epidermal-mesenchymal acid. Wound Repair Regen. 6:349357. Ju BG, Kim WS. 2000. Cloning of a cDNA encoding tissue interaction by being involved in the modification cathepsin D from salamander, Hynobius leechii, and its of ECM like basement membrane. expression in the limb regenerates. DNA Seq. 11:2128. Collectively, our findings suggest that endogenous RA Ju BG, Kim WS. 2010. Lysosomal acid phosphatase mediates may play crucial role in the early events such as wound dedifferentiation in the regenerating salamander limb. epithelium formation and initiation of dedifferentiation Anim Cells Syst. 14:7381. Kim WS, Stocum DL. 1986. Retinoic acid modiﬁes positional via up-regulation of MMP-9 gene expression during memory in the anteroposterior axis of regenerating salamander limb regeneration. To better understand the axolotl limbs. Dev Biol. 114:170179. precise mechanism of endogenous RA-mediated limb Kumar A, Nevill G, Brockes JP, Forge A. 2010. A compara- regeneration, it is necessary to investigate exact expression tive study of gland cells implicated in the nerve depen- pattern of RA receptors and genome-wide analysis of dence of salamander limb regeneration. J Anat. 217: 1625. target genes regulated by RA. Lackey DE, Hoag KA. 2010. 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Animal Cells and Systems
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Published: Dec 1, 2012
Keywords: dedifferentiation; salamander limb regeneration; retinoic acid; MMP-9