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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Curr Biol. Author manuscript; available in PMC May 22, 2012.
Published in final edited form as:
PMCID: PMC3236601
NIHMSID: NIHMS341807

Sexually dimorphic fin regeneration in zebrafish controlled by androgen/GSK3 signaling

Summary

Teleost fish and urodele amphibians regenerate entire fins and limbs after amputation, while such potential is absent in avians and limited in mammals to digit tips [1, 2]. Additionally, regenerative success can change during life stages. Anuran tadpoles gradually lose the capacity to regenerate limbs [3, 4], and digit regeneration occurs more effectively in fetal mice and human children than adults [58]. Little is known about mechanisms that control regenerative capacity. Here, we identified an unexpected difference between male and female zebrafish in the regenerative potential of a major appendage. Males displayed regenerative defects in amputated pectoral fins, caused by impaired blastemal proliferation. This regenerative failure emerged after sexual maturity, was mimicked in androgen-treated females, and was suppressed in males by androgen receptor antagonism. Androgen signaling maintained expression of dkk1b and igfbp2a, encoding secreted inhibitors of Wnt and Igf signaling, respectively. Furthermore, the regulatory target of Wnts and Igfs, GSK3β, was inefficiently inactivated in male fin regenerates compared with females. Pharmacological inhibition of GSK3 in males increased blastemal proliferation and restored regenerative pattern. Our findings identify a natural sex bias in appendage regenerative capacity, and indicate an underlying regulatory circuit in which androgen locally restricts key morphogenetic programs after amputation.

Results and Discussion

Pectoral Fin Regeneration in Zebrafish Is Sexually Dimorphic

To identify possible modifiers of appendage regenerative capacity, we assessed fin regeneration in 6 month-old adult zebrafish of several commonly used strains. Zebrafish have 2 sets of paired fins, the pectoral and pelvic fins, and 3 unpaired fins, the anal, caudal, and dorsal fins (Figure 1A). Fish of all strains displayed reliable regeneration of amputated anal, caudal, dorsal, and pelvic fins at 5 dpa (Figure S1A). By contrast, approximately half of the fish from the EK and AB strains, and lower frequencies of WIK (29%) and Tubingen (14%) fish, displayed severe anteroposterior (AP) patterning defects in regenerated pectoral fin tissue. Unexpectedly, only males showed impaired pectoral fin regeneration (Figure S1B–D). Regenerative defects in males of the EK strain, on which we focused our subsequent experiments, were mainly present in anterior and anteromedial rays. While ranging somewhat in severity, male regenerates were on average 79% shorter at 5 days post-amputation (dpa) than the corresponding female regenerates (Figure 1B, C). By 10 dpa, when all female zebrafish had nearly completed regeneration, less than 15% of males had regenerated a fanned pattern with multiple bone segments in each fin ray (Figure 1B). These results indicated that regeneration of pectoral fins is sexually dimorphic in adult zebrafish.

Figure 1
Sexually Dimorphic Regeneration of Zebrafish Pectoral Fins

To test whether regenerative capacity is normal prior to acquisition of male sexual characteristics, we amputated pectoral fins of juvenile animals that displayed only subtle sexual features. We found that all 8-week old females and 93% of 8-week old males regenerated fins of normal length and pattern (Figure 1D; P = 0.25, Fisher-Irwin exact test), indicating that regenerative capacity is present in pectoral fins of young male zebrafish, but then diminishes during maturation.

As in other vertebrates, androgen levels rise as male teleosts grow and sexually mature [9]. We postulated that androgen production was responsible for the stage-specific regenerative defects observed in male pectoral fins. To test this idea, we treated adult female zebrafish by bath incubation with the androgen norethindrone acetate (NA; 1 μg/mL) for 4 days after pectoral fin amputation [10]. Anterior pectoral fin regenerates of NA-treated animals were 62% shorter than those of vehicle-treated fish, while posterior regenerates were 43% shorter (Figure 2A, B). NA inhibited regeneration of female pectoral fins more robustly than caudal fins, which showed just a 12% reduction in regenerative length (Figure S2A). NA treatment further decreased male pectoral fin regeneration by 56% (Figure S2B). These experiments indicated negative effects of androgen on pectoral fin regeneration.

Figure 2
Androgen Signaling Inhibits Blastemal Proliferation

To experimentally decrease androgen levels, we removed the majority of testes tissue from male fish and amputated both pectoral fins 2 days later. In these experiments, 38% of animals showed morphologically normal regeneration in both pectoral fins at 5 dpa (Figure 1E; P < 0.05, Fisher-Irwin exact test). This result suggested that any structural features of male pectoral fins acquired during maturation do not preclude regeneration, and implicated circulating androgens in control of regeneration. To specifically inhibit androgen receptors during fin regeneration, we treated male animals with fenitrothion (FEN), a competitive androgen receptor inhibitor [11, 12]. Bath treatment with FEN (1 μg/mL) increased the length of regenerating male anterior rays by 52% at 4 dpa compared to vehicle-treated animals. Bath treatment with other androgen receptor inhibitors, vinclozolin (250 ng/mL) and flutamide (250 ng/mL) [13], increased male regenerative length by 44% (P < 0.05) and 38% (P = 0.063), respectively (Figure S2C). Androgen receptor antagonism did not significantly affect the lengths of male posterior regenerates or female regenerates (Figure 2B; Figure S2D). These findings indicated that androgen presence inhibits regeneration in male animals, and that regenerative potential can be recovered by impeding androgen signaling.

Androgen Signaling Regulates Blastemal Proliferation

Appendage regeneration is initiated by formation of a specialized epidermis after amputation. This regeneration epidermis stimulates the creation and maintenance of a blastema, a mass of proliferative tissue that is comprised at least in part by cell type-restricted progenitor cells [14, 15]. To determine the cellular basis of sexually dimorphic regeneration, we assessed blastemal cell proliferation in regenerating anterior pectoral fin rays of male and female animals. Anterior blastemas of regenerating male pectoral fins had 66% fewer cells positive for phosphorylated Histone 3-positive (H3P), a marker of mitosis, than the corresponding female regions at 4 dpa (Figure 2C, D). NA treatment reduced the number of H3P+ cells by 53% and 40% in anterior and posterior blastemas, respectively, of female fin regenerates (Figure 2C, E). Conversely, androgen receptor blockade increased the number of H3P+ cells in the anterior blastemas of regenerating male pectoral fins by 112%, with no significant effects on posterior blastemal proliferation (Figure 2C, F). These results indicate that androgen signaling inhibits cell proliferation in the appendage blastema.

Androgen Signaling Regulates Wnt and Igf Signaling Inhibitors During Pectoral Fin Regeneration

To define molecular differences between male and female regenerative responses, we performed gene expression microarrays with adult EK female or male pectoral fins. Examination of uninjured fins as well as 4 dpa regenerates permitted identification of genes that are differentially regulated by sex and/or regeneration. We identified 700 genes with significant, sex-specific expression differences in the absence of injury. A total of 4653 genes displayed differential expression between uninjured and 4 dpa samples, including 400 of the 700 sexually dimorphic genes (Figure 3A; data available on NCBI GEO website, entry GSE31871). This subgroup of sexually dimorphic, regeneration-responsive genes represented diverse cellular and molecular functions. In particular, genes that participate in DNA replication were induced at greater levels upon injury and regeneration in female fins compared with male fins, consistent with the sex-specific differences in blastemal proliferation that we observed (data not shown). Thus, uninjured and regenerating male and female fins exhibit distinct gene expression profiles.

Figure 3
Sexually Dimorphic Gene Expression and GSK3β Regulation in Regenerating Fins

Previous studies of zebrafish caudal fin regeneration identified many locally secreted factors that influence blastemal proliferation, including Fgfs, Wnts, retinoic acid, Bmps, Activinβ-A, Shh, and Igf2 [1623]. We examined the microarray dataset to detect sex-specific differences in regulation of these upstream factors and identified male-specific expression of dkk1b and igfbp2a, encoding secreted inhibitors of Wnt and Igf signaling, respectively. In previous studies, ectopic expression of dkk1 decreased blastemal proliferation and blocked fin or limb regeneration [17, 19], as did pharmacological inhibition of Igf signaling [16]. Quantitative PCR using uninjured pectoral fin anterior tissue revealed that male dkk1b and igfbp2a expression levels were 48- and 4.6-fold higher than those of females (Figure S3A). Expression of these inhibitors decreased in males after amputation, but remained 8.2-fold and 7.2-fold, respectively, higher than those of regenerating female fins (Figure 3B). These inhibitors were present at low or undetectable levels in the posterior rays of male pectoral fins, and were similarly diminished in male caudal fins (Figure S3B). To assess whether androgen signaling influences dkk1b and igfbp2a during regeneration, we treated females with NA and males with FEN for 4 days after fin amputation. Anterior fin regenerates from NA-treated females had dkk1b and igfbp2a levels that were 2.5- and 21-fold, respectively, higher than those from vehicle-treated animals (Figure 3C). The particularly high expression of these inhibitory factors after NA treatment might explain its effects on regeneration across the AP axis of female pectoral fins (see Figure 2B, E). NA treatment did not significantly induce dkk1b and igfbp2a expression in female caudal fins (Figure S3C). FEN treatment of males reduced dkk1b and igfbp2a expression in anterior pectoral fin regenerates by 47% and 57%, respectively (Figure 3C). Thus, secreted inhibitors of key pathways required for blastemal proliferation are positively regulated in male pectoral fins by androgen signaling.

GSK3 Activity Is a Regulatory Target of Androgen Signaling During Regeneration

A common mode of Wnt and Igf signaling activity is inhibition of GSK3β, a multifunctional kinase that, among other regulatory roles, targets β-catenin, cyclin D, and other protein substrates for degradation [2428]. A recent study indicated that Wnts inhibit GSK3β through its sequestration in endosomes [29], while Igfs have been shown to inactivate GSK3β through phosphorylation of serine 9 [30, 31]. At 4 dpa, we found that amounts of inactive P-GSK3β were present at 2.7-fold higher levels in female anterior pectoral fin regenerates than in males (Figure 3D, E). Treatment of males with FEN was able to increase by 1.7-fold the amount of P-GSK3β in the regenerate (Figure 3D, E). These experiments indicated that GSK3β activity is a regulatory target of androgen signaling during fin regeneration, likely via control of dkk1b and igfbp2a expression.

To determine the significance of GKS3β activity on sexually dimorphic regenerative capacity, we treated male zebrafish with the GSK3 inhibitor, (2′Z, 3′E)-6-Bromoindirubin-3′-oxime (BIO), a manipulation expected to be epistatic to influences of Dkk1b or Igfbp2a [32]. Animals were treated with 100 nM BIO by bath incubation following amputation and assessed for blastemal proliferation at 4 dpa. This treatment increased blastemal mitoses by 56% in male regenerates (Figure 4A, B). BIO treatment had no significant effect on female blastemal proliferation, suggesting that its proliferative effect in males is specific to normal functions of GSK3β signaling during regeneration (Figure 4B). We also examined regenerates from male animals that had undergone 4 days of BIO treatment after amputation, plus an additional 3 days in the absence of BIO. GSK3 inhibitor treatment markedly improved regeneration, frequently restoring normal or near-normal fin pattern and increasing its regenerate length by 47% compared to vehicle-treated animals (Figure 4C, D). This extent of regenerative recovery was similar to the effects of the same treatment regimen with FEN instead of BIO (Figure 4C, D). Thus, transient pharmacological inhibition of GSK3 signaling in zebrafish was sufficient to de-repress the regenerative responses of male pectoral fins.

Figure 4
GSK3 Inhibition Rescues Fin Regeneration

Together, our findings support a model in which the sex- and age-specific systemic factor, androgen, influences the regenerative potential of appendage tissue through modulation of its GSK3β activity. Locally, amputation and wound healing trigger synthesis of Igf2, Wnts, and possibly other ligands that contribute to inactivating the GSK3β pool and enabling blastemal proliferation. Androgen counters these effects in male pectoral fins through the maintenance of ligand antagonists, repressing GSK3β inactivation mechanisms and blunting regenerative capacity. Notably, androgen signaling has been implicated in multiple contexts of tissue homeostasis and regeneration. These include positive effects on neuron survival and bone density [33, 34], and negative effects on wound healing and hair follicle maintenance [35, 36]; androgens impact antler regeneration in red deer in both positive and negative fashions [37, 38]. Thus, it will be important to determine the range of functions performed by androgen/GSK3 interactions in fins and other tissues, and what may be the physiological consequences of sexually dimorphic regeneration. Interestingly, we have observed that atrophied pectoral fins are much more common in aging male zebrafish than females (Figure S4). Homeostatic maintenance of zebrafish fin structures has been shown to rely on factors important for amputation-induced regeneration [39]; therefore, this sex-biased phenotype might be caused by reduced regenerative capacity.

We found that androgen-regulated gene expression and diminished regenerative capacity were mainly localized to anterior pectoral fin structures of male zebrafish. While androgen receptor expression was slightly higher in anterior pectoral fin regions than posterior regions, it was expressed at similar levels in all fin types (Figure S3D). Thus, we suspect that differential expression and/or activity of androgen receptor cofactors or downstream regulatory targets underlie tissue-specific effects within fins and fin regions. Such differential expression or activity may be related to positional memory, a poorly understood mechanism by which cellular positional identities are maintained, restoring appendage size and pattern after amputation.

In addition to appendages, stage- or age-dependent losses in regenerative potential have been described for mammalian tissues like the heart, blood, and pancreas [4042]. Murine skeletal muscle regeneration, which is also less effective in old animals versus young, can be modulated by an unidentified circulating factor(s) whose presence changes with age [43]. An implicated target of this factor is Wnt signaling, which displays an age-dependent increase in myogenic cells that is associated with conversion to a fibrogenic lineage and inhibition of regeneration [44]. In the current study, we found that pharmacological blockade of activities of either circulating androgen or a target within appendage tissue, GSK3, considerably increased the regenerative capacity of amputated zebrafish fins. Approaches to retain or increase the regenerative capacity of injured human tissues remain challenging, but these studies suggest that elucidating and modulating interactions between systemic factors and local regenerative programs will aid this important goal.

Highlights

  • Pectoral fin regeneration is frequently defective in sexually mature male zebrafish
  • Androgen signaling restricts cell proliferation in pectoral fin blastemas
  • Androgen regulates Wnt and Igf signaling components during fin regeneration
  • Pharmacological inhibition of GSK3 enhances fin regeneration in male zebrafish

Supplementary Material

01

Acknowledgments

We thank J. Burris, A. Eastes, and P. Williams for zebrafish care, V. Yin for discussions, and R. Baugh, B. Hogan, and Poss lab members for comments on the manuscript. K.D.P. is an Early Career Scientist of the Howard Hughes Medical Institute. This work was supported by grants from NIH (GM074057), American Federation for Aging Research, and Pew Charitable Trusts to K.D.P.

Footnotes

Supplemental Information

Supplemental Information includes four figures, one table, and Experimental Procedures. Microarray files are available on the NCBI GEO website as series entry GSE31871 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE31871).

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