Specific NuRD components are required for fin regeneration in zebrafish
© Pfefferli et al.; licensee BioMed Central Ltd. 2014
Received: 6 November 2013
Accepted: 23 April 2014
Published: 29 April 2014
Epimorphic regeneration of a missing appendage in fish and urodele amphibians involves the creation of a blastema, a heterogeneous pool of progenitor cells underneath the wound epidermis. Current evidence indicates that the blastema arises by dedifferentiation of stump tissues in the vicinity of the amputation. In response to tissue loss, silenced developmental programs are reactivated to form a near-perfect copy of the missing body part. However, the importance of chromatin regulation during epimorphic regeneration remains poorly understood.
We found that specific components of the Nucleosome Remodeling and Deacetylase complex (NuRD) are required for fin regeneration in zebrafish. Transcripts of the chromatin remodeler chd4a/Mi-2, the histone deacetylase hdac1/HDAC1/2, the retinoblastoma-binding protein rbb4/RBBP4/7, and the metastasis-associated antigen mta2/MTA were specifically co-induced in the blastema during adult and embryonic fin regeneration, and these transcripts displayed a similar spatial and temporal expression patterns. In addition, chemical inhibition of Hdac1 and morpholino-mediated knockdown of chd4a, mta2, and rbb4 impaired regenerative outgrowth, resulting in reduction in blastema cell proliferation and in differentiation defects.
Altogether, our data suggest that specialized NuRD components are induced in the blastema during fin regeneration and are involved in blastema cell proliferation and redifferentiation of osteoblast precursor cells. These results provide in vivo evidence for the involvement of key epigenetic factors in the cellular reprogramming processes occurring during epimorphic regeneration in zebrafish.
KeywordsNuRD Blastema Fin Regeneration Zebrafish
In contrast to mammals, some vertebrates such as urodeles and teleost fish benefit from exceptional regeneration mechanisms. Zebrafish are able to regenerate different organs after injury, including heart, fins, retina, liver, and spinal cord, and have become a powerful model organism for regenerative studies [1–5]. The caudal fin displays rapid and robust regeneration, and therefore provides a well-established system to study appendage regeneration in vertebrates [4, 6, 7].
The caudal fin of zebrafish is constituted of 16 to 18 bony fin rays (lepidotrichia), covered by an epidermis, and interconnected by soft inter-ray mesenchymal tissue . Each individual bony ray consists of two concave hemirays that enclose a mesenchymal compartment composed of blood vessels, nerves, pigment cells, fibroblasts, and osteoblasts.
Upon amputation, the caudal fin is fully restored after approximately 3 weeks. This type of regeneration, called epimorphic regeneration, involves the formation of a blastema, a population of proliferating progenitor cells that arise from dedifferentiation of mesenchymal cells in the stump [4, 8]. Regeneration of the caudal fin proceeds through three main steps: 1) wound healing, 2) blastema formation, and 3) regenerative outgrowth, including differentiation and patterning. Upon fin amputation, epidermal cells rapidly migrate to protect the wound and form a wound epidermis. Mesenchymal tissues in the stump then become disorganized, and cells start to proliferate and migrate distally, forming a blastema after approximately 24 to 48 hours post-amputation (hpa). During regenerative outgrowth, the blastema progenitor cells are maintained at the distal margin, while their daughter cells progressively redifferentiate in the proximal part of the fin regenerate. During this later phase, the fin regenerate can be subdivided into several compartments with distinct cellular and molecular properties [9–11].
The exact origin of blastema cells still remains unresolved. Recently, genetic cell-fate tracing studies have shown that the blastema is composed of a heterogeneous population of cells with restricted lineage fate and different tissue origin [12–14]. Thus, regeneration is achieved without cellular transdifferentiation. However, genetic ablation studies of osteoblasts prior to amputation have revealed that new bones are able to regenerate from non-osteoblast cells, suggesting that other cell types are plastic, and can transdifferentiate into osteoblasts to promote bone regeneration .
Animals with robust regenerative capacities are characterized by their flexibility to change gene expression in response to amputation. This cellular plasticity allows temporal suppression of differentiation genes and reactivation of developmental signaling pathways, which are required for the reconstitution of lost tissues [11, 16–28].
Regulation of the chromatin structure is an important epigenetic mechanism, which has a direct influence on many biological processes. The Nucleosome Remodeling and Deacetylase (NuRD) complex is a multi-subunit complex widely expressed and evolutionarily conserved in animals and plants . This complex is able to couple two important enzymatic functions: an ATP-dependent nucleosome remodeling activity catalyzed by the chromodomain helicase DNA binding proteins CHD3/4, also called Mi-2α/β, and a deacetylase activity executed by the histone deacetylases HDAC1/2 [30–33]. Additionally, the NuRD complex is also constituted of other non-catalytic subunits, including the methyl-CpG-binding domain proteins MBD2/3, the retinoblastoma-binding proteins RBBP7/4, and the metastasis-associated proteins MTA1/2/3 . The composition of the NuRD complex can also be changed by the incorporation of unique subunits, raising the possibility of functional specialization for these distinct complexes .
The NuRD complex has been shown to play important developmental roles in cell fate determination . In Caenorhabditis elegans, the Mi-2 homolog LET-418 is required for proper differentiation of the vulva  and for repression of germline-specific genes in somatic cells . In Drosophila melanogaster, dMi-2 is essential for embryogenesis and germ cell development . Yoshida et al.  have demonstrated that in mammals, Mi-2β functions in self-renewal and lineage choice of hematopoietic stem cells . In addition, embryonic stem cells deficient in mbd3 can initiate differentiation, but are not able to commit to specific lineages .
In this study, we investigated the potential role of the Mi-2/NuRD complex during fin regeneration in zebrafish. The zebrafish genome encodes several orthologs for every member of the vertebrate NuRD complex. However, we found that only one of each is expressed during fin regeneration. The orthologs of the NuRD components chd4a/Mi-2, hdac1/HDAC1/2, rbb4/RBBP4/7, and mta2/MTA are all induced in the distal blastema during regeneration of the adult and embryonic caudal fin, and display similar expression patterns. Additionally, inhibition of these genes impairs regenerative outgrowth. Our data suggest that putative NuRD components are induced in the blastema during fin regeneration, and are involved in the maintenance of blastema cell proliferation and in redifferentiation during the regenerative outgrowth phase.
One of the three Mi-2 orthologs, chd4a, is specifically expressed in the blastema during fin regeneration
Mi-2, which is the core ATPase of the NuRD complex, is essential for regeneration and neoblast differentiation in the planarian Schmidtea mediterranea. We therefore investigated whether Mi-2 could also be involved in zebrafish fin regeneration. A BLAST search of the zebrafish genome database (National Center for Biotechnology Information) identified three genes, chd4a (Gene ID: 558344), chd4b (Gene ID: 560622), and chd3 (Gene ID: 568230), which encode polypeptides with high similarity to human Mi-2 proteins, also called CHD4 (or Mi-2β) and CHD3 (or Mi-2α). Sequence alignment revealed high similarity between the three zebrafish Mi-2 homologs, with the main functional domains being conserved (see Additional file 1: Figure S1). Chd4a and Chd4b share 82% identity, while Chd3 shares 66% identity with Chd4a and Chd4b. Moreover, Chd4a contains an additional domain, the AP endonuclease family 2 domain (AP2Ec) (see Additional file 1: Figure S1), which is not present in other Mi-2 orthologs. This evolutionarily conserved domain is associated with DNA damage repair and maintenance of genome stability .
Early zebrafish larvae are also able to regenerate their caudal fin folds after amputation with a similar mechanism to that of regenerating adult caudal fins [43, 44]. Interestingly, chd4a, but neither chd4b nor chd3, was expressed in the mesenchymal cells of regenerating larval fin folds at 1 dpa (Figure 1E-G). Expression of chd4a mRNA is specific for regenerating fins, as it was not detected in uncut fin folds at the same developmental stage (3 days post-fertilization) (data not shown). Altogether, these results show that one of the three Mi-2 orthologs, chd4a, is transcriptionally induced in the blastema of regenerating adult and embryonic fins.
Specific NuRD component orthologs are expressed in the blastema of regenerating fins
qRT-PCR data were confirmed by ISH on cryosections of adult caudal fins at 3 dpa. A single RNA antisense probe was designed for the two RBBP4/7 orthologs rbb4 and rbb4l because of their high RNA (75%) and amino acid (94%) sequence similarity. Positive signals for hdac1, rbb4, and mta2 transcripts were detected in the blastema of adult regenerating fins, with an expression pattern similar to that of chd4a (Figure 2B-D). No signals were detected for the orthologs whose expression was not upregulated by qRT-PCR (data not shown). Furthermore, hdac1, rbb4, and mta2 transcripts were also expressed in mesenchymal cells of regenerating larval fin folds at 1 dpa (Figure 2E-G). Thus, the overlapping expression pattern of some NuRD orthologs in fin regenerates raises the possibility that the expression of a specialized NuRD complex composed of Chd4a, Rbb4/Rbb4l, Hdac1, and Mta2 is specifically induced in the blastema during fin regeneration.
Morpholino-mediated knockdown of chd4a, mta2, and the two RBBP4 orthologs rbb4 and rbb4limpairs fin regeneration
Specific HDAC1 inhibition affects regenerative outgrowth
Interestingly, treatment of regenerating fins with 5 μM MGCD0103 for 10 days resulted in a substantial reduction in regenerative growth (Figure 4B-E). However, the early stages of the regeneration process seemed not to be affected because wound healing was properly completed and a seemingly normal blastema was formed (Figure 4B,C), suggesting that Hdac1 activity is not essential for the earliest phases of regeneration. Regenerative outgrowth was impaired, starting from 3 dpa, and the regeneration process was progressively blocked and finally stopped (Figure 4D,E). Indeed, MGCD0103 treatment for 10 days resulted in the formation of abnormal curled fin-like structures, suggesting differentiation defects. To test whether Hdac1 inhibition also affects fin regeneration after blastema formation, fish were treated with MGCD0103 for 4 days starting at 3 dpa. As expected, we found that regenerative growth was blocked, similar to fins that were continuously treated from the time of amputation (see Additional file 1: Figure S8). This result confirmed that Hdac1 inhibition affects regeneration from the onset of regenerative outgrowth.
To test whether MGCD0103 treatment is reversible, fish were exposed to MGCD0103 for 10 days from the time of amputation, and then transferred to normal water for 10 additional days. In general, fins failed to restart the initially blocked regenerative process properly, indicating that the effects of Hdac1 inhibition on caudal fin regeneration are irreversible (see Additional file 1: Figure S9). However, occasionally, a few rays resumed regrowth (see Additional file 1: Figure S9), suggesting that some residual blastema cells retained their original regenerative potential despite the prolonged inhibition of regeneration.
Taken together, these data indicate that MGCD0103-mediated inhibition of Hdac1 does not affect wound healing and initial blastema formation, but impairs progression of fin regeneration during the regenerative outgrowth phase.
The NuRD components hdac1, chd4a, mta2, and rbb4are required for blastema cell proliferation during the regenerative outgrowth phase
MGCD0103 treatment resulted in a noticeable increase in wound epidermis (Figure 5B,D). However, no increase in cell proliferation was detected in the epidermis of MGCD0103-treated fins (Figure 5I,J). MGCD0103 treatment did not alter expression of the wound epidermis markers wnt5b and lef1, indicating that hdac1 is not required for the correct specification of the wound epidermis (see Additional file 1: Figure S11). The enlargement of the epidermis in MGCD0103 regenerates could be the result of an abnormal migration of epithelial cells from the stump. As this phenotype was not observed in MO-injected fin regenerates, it is possible that Hdac1 plays an additional role independent of the Mi-2/NuRD complex during fin regeneration.
Depletion of the NuRD components hdac1, chd4a, mta2, and rbb4results in abnormal patterning of actinotrichia during regeneration
To evaluate the molecular specification of the blastema in fin regenerates deficient in NuRD components, we analyzed the expression of msxb by ISH. msxb is a molecular marker of the distal blastema and is required for blastema cell proliferation during fin regeneration . We found that msxb transcripts were correctly expressed in MGCD0103-treated and in chd4a MO-injected fin regenerates (see Additional file 1: Figure S11), indicating that the distal blastema is correctly specified.
Finally, we analyzed the expression of Actinodin 1, a marker for actinotrichia-forming cells . Actinotrichia are non-mineralized structural components that mechanically support the larval fin fold and the blastema of the fin regenerate [54, 55]. The expression pattern of Actinodin 1 was completely disorganized at 4 dpa in fin regenerates treated with MGCD0103, compared with control fins (Figure 6C,D), indicating an abnormal patterning of actinotrichial fibers. A similarly disorganized expression pattern of Actinodin 1 was also observed in fins deficient in chd4a, mta2, or the two rbb4 orthologs (Figure 6E-H). Altogether, these data suggest that depletion of the NuRD components results in cellular defects after the onset of regenerative outgrowth. Thus, these epigenetic factors are not essential for mesenchymal reorganization or initial blastema formation, but they are required for growth and correct patterning of the blastema during regenerative outgrowth.
Hdac1 inhibition impairs osteoblast differentiation
Next we used transgenic fish lines expressing fluorescent proteins to examine the expression of the bone differentiation markers runx2, osterix, and osteocalcin, which are sequentially activated during osteoblast differentiation [10, 12]. In control fish, expression of the pre-osteoblast marker runx2 and the intermediate osteoblast marker osterix is relatively low in unamputated fins, and it becomes strongly activated in the blastema during fin regeneration  (Figure 7D,F). In MGCD0103-treated fins, runx2:GFP and osterix:mCherry were both reactivated normally in the blastema at 3 dpa (Figure 7D-G), indicating that osteoblast dedifferentiation was not affected by Hdac1 inhibition. However, expression of runx2 and osterix persisted in the proximal zone at 7 dpa, whereas it was progressively downregulated in the proximal differentiating zone in control fins (Figure 7D-G). This indicates a delay in the redifferentiation process in MGCD0103-treated fins.
The late bone differentiation marker osteocalcin, which labels mature osteoblasts, is downregulated in the stump of amputated fins and then robustly re-expressed in the proximal differentiated regenerate . Interestingly, osteocalcin:GFP expression was not reactivated in fin regenerates treated with MGCD0103 at 7 dpa (Figure 7H-I). Furthermore, osteocalcin:GFP expression was also strongly reduced in the blastema of regenerating fins treated with MGCD0103, starting at 3 dpa, demonstrating that inhibiting Hdac1 after the blastema has been formed also blocks osteocalcin reactivation (see Additional file 1: Figure S14A). In uninjured fins, MGCD0103 treatment did not alter the expression of osteocalcin:GFP in mature bones (see Additional file 1: Figure S14B), indicating that Hdac1 inhibition specifically blocks the reactivation of osteocalcin:GFP expression in the differentiating blastema during fin regeneration. Taken together, our results indicate that Hdac1 inhibition prevents redifferentiation of osteoblast precursor cells. However, Hdac1 is not required for osteoblast dedifferentiation following fin amputation.
Hdac1 inhibition results in the upregulation of regeneration marker and two pluripotency-associated genes
Here we show evidence for the role of putative NuRD components during fin regeneration in zebrafish. We propose a model in which a specialized Mi-2/NuRD complex could be involved in blastema cell proliferation and redifferentiation during regenerative outgrowth. The zebrafish genome encodes orthologs for every subunit of the vertebrate NuRD complex. However, we found that transcripts of the putative NuRD components chd4a/Mi-2, hdac1/HDAC1/2, rbb4/RBB4/7, and mta2/MTA were specifically co-induced in the blastema during adult and embryonic fin regeneration, and displayed similar spatial and temporal expression patterns. Although there are several homologs for each NuRD component encoded by the genome of zebrafish (with the exception of hdac1), only one of each seems to be present in the putative NuRD complex involved in fin regeneration. Thus, the combinatorial assembly of the different paralogs of each NuRD subunit may define its specific function.
We also found that disruption of these putative 'regenerating' NuRD components impaired fin regeneration. Chemical inhibition of Hdac1 by MGCD0103 and morpholino-mediated knockdown of chd4a, mta2, and the two rbb4 orthologs resulted in the reduction in blastema cell proliferation during regenerative outgrowth. However, these putative NuRD components seem not to be required for the earliest stages of fin regeneration. This is demonstrated by the facts that inhibition of Hdac1 starting from the time of amputation had no influence on wound healing and blastema formation. In addition, Tenascin C, an early mesenchymal marker, and msxb, a marker of the distal blastema, were normally expressed in chd4a-deficient and hdac1-deficient fin regenerates.
The wound epidermis was noticeably enlarged in hdac1-deficient fin regenerates. It is likely that the increase in the epidermis size resulted from the migration of epithelial cells from the stump, as no increase in cell proliferation was detected in the wound epidermis of MGCD0103-treated fins. Although Hdac1 inhibition reduced cell proliferation in the blastema, epithelial cells might continue to migrate and accumulate, forming an enlarged wound epidermis. This phenotype was not observed in fins deficient in the other NuRD components chd4a, mta2, and rbb4. As HDAC1 is also known to be a catalytic subunit of other multiprotein complexes in mammals, such as CoREST and Sin3 complexes , we cannot exclude that Hdac1 plays additional roles independent of the NuRD complex during fin regeneration. Further experiments are needed to identify direct interacting partners of these proteins in regenerating fins.
We found that on addition to the proliferation defects of blastema cells during regenerative outgrowth, Hdac1 inhibition and knockdown of chd4a, mta2, and the two rbb4 orthologs resulted in an abnormal expression pattern of Actinodin 1, a component of structural fibers called actinotrichia. During development, actinotrichia support the fragile fin fold of the larvae. During regeneration, actinotrichia are formed between the epidermis and the blastema prior to lepidotrichia regrowth, and are probably required for shaping the regenerate [53, 55]. Consistently, osteoblast proliferation and differentiation were also impaired in hdac1-deficient fin regenerates. Analysis of the bone differentiation markers runx2, osterix, and osteocalcin, which are sequentially expressed during fin regeneration , indicated that Hdac1 inhibition did not interfere with osteoblast dedifferentiation. However, expression of the late bone differentiation marker osteocalcin, expressed only in mature bones, was not reactivated in the redifferentiating proximal fin regenerates after Hdac1 inhibition, suggesting that Hdac1 is essential for redifferentiation of osteoblast precursor cells. Indeed, expression of runx2 and osterix persisted in the proximal blastema of MGCD0103-treated fins, indicating that blastema cells were blocked in an intermediate state.
The effects of morpholino-mediated knockdown of the other NuRD components were not persistent, and regeneration resumed 48 hours post-injection. Morpholino injection has some limitations and is not an appropriate technique to analyze differentiation defects of bone-forming cells. Therefore, we were not able to analyze the consequences of morpholino-mediated knockdown of chd4a, mta2, and rbb4 on osteoblast regeneration.
Somewhat reminiscent to our findings in zebrafish, the planarian ortholog Smed-CHD4 is also essential for regeneration and neoblast differentiation in Schmidtea mediterranea. Smed-CHD4 expression is induced in neoblasts after wounding, and CHD4(RNAi) worms fail to regenerate following amputation or even to maintain normal tissue turnover. In CHD4-depleted animals, the number of neoblast progeny cells is reduced because neoblasts are unable to produce progeny cells committed to differentiation . It is, however, not clear whether Smed-CHD4 also acts as a member of a NuRD complex.
Recently, an elegant model has been proposed in which the NuRD complex binds to the promoters of numerous pluripotency genes in embryonic stem cells (ESCs), probably to fine-tune the transcription levels of the genes and to maintain the differentiation responsiveness of the ESCs . In the absence of a functional NuRD complex, expression of these genes is increased above a threshold, thereby blocking the response of ESCs to developmental cues and preventing them from exiting from the self-renewal state .
We hypothesize that the Mi-2/NuRD complex might have a similar function during fin regeneration in zebrafish. This is suggested by our findings that the NuRD components were all expressed in the proliferative zone of the blastema during regenerative outgrowth and that their depletion resulted in a reduction in blastema proliferation and an increase in cellular differentiation defects. In addition, Hdac1 inhibition leads to the upregulation of the two pluripotency-associated genes, myca and klf4, and genes encoding regeneration markers associated with dedifferentiation. The histone deacetylase Hdac1 might be required to downregulate the expression of these genes, thereby promoting the responsiveness of blastema cells to regenerative signals in order to ensure correct reconstitution of lost tissues. In the absence of Hdac1, expression of these genes continues to be high, resulting in the blocking of blastema cells in an undifferentiated or partially differentiated state. Further experiments are needed to determine whether Hdac1 represses the expression of these genes in a NuRD-dependent context.
Epigenetic mechanisms are critical for the regulation of gene expression and lineage specification during development . A previous study has shown that H3K27me3 demethylase is required for caudal fin regeneration in zebrafish . Stewart et al. established that many developmental regulatory genes involved in fin regeneration are poised in a bivalent H3K4me3/H3K27me3 chromatin domain, and that the demethylation of H3K27me3 enables activation of expression of these genes in response to injury. It is possible that the zebrafish maintains key developmental regulatory genes in a dormant state to allow rapid switching of their expression profile through epigenetic mechanisms in response to amputation.
Our study provides further in vivo evidence for the involvement of key epigenetic factors in epimorphic fin regeneration in zebrafish. We propose a model in which a specialized Mi-2/NuRD complex is induced in the blastema of regenerating fins to coordinate proliferation and differentiation and thus reform the missing tissues. Even though different animals may be endowed with different regenerative capacities, crucial regeneration markers are conserved in all vertebrate species. Thus, fin regeneration constitutes an excellent in vivo system to study the epigenetic mechanisms regulating regeneration, and to elucidate how this process is maintained in some vertebrates.
The experimental animal research was approved by the cantonal veterinary office of Fribourg (Switzerland).
Zebrafish and fin amputation
The following zebrafish strains were used in this study: AB wild-type strain, and the osterix:mCherry (OlSp7:mCherryzf131) , runx2:GFP (Has.RUNX2-Mmu.Fos:EGFPzf259), and osteocalcin:GFP (Ola.Osteocalcin.1:EGFPhu4008) fish lines . 6-24 month-old adult fish were anesthetized in 0.1% tricaine, and the caudal fins were amputated with a razor blade. Animals were allowed to regenerate at 28.5°C. Larval fin folds were amputated as previously described . Larvae were allowed to regenerate at 28.5°C and were collected at 1 dpa for further analysis. For proliferation assays, fish were incubated for 6 hours before fin collection in fish water containing 50 μg/ml BrdU (Sigma-Aldrich, Buchs, Switzerland).
MGCD0103 (Selleckchem, Houston, USA) was dissolved in DMSO at 10 mM stock concentration and then added to the fish water at a final concentration of 5 μM. 0.05% DMSO was added to the water of control fish.
where D is the area of the dorsal side and V is the area of the ventral side of the fin regenerate. Statistical significance was determined with the Student’s t-test, and significance was set at P < 0.01.
Whole-mount in situ hybridization and in situ hybridization on fin cryosections was performed as previously described [22, 69]. Normarski imaging was performed with a Zeiss Axioplan microscope. The following primers were used to generate ISH probes:
chd4a (NM_001044858.1): F GTTCCCAAAGCAGAAGATGC, R TTCGTTAAGAATGGCGAACC (735 bp),
chd4b (XM_680607.5): F GGTGAAAGGCTCCAGACAG, R GCGGCTCTCTCTTCATTCTG (513 bp),
chd3 (XM_691549.5): F CTGACAAGACGGAGAAGAGC, R CCTGAAAGCAGCCAGAAGTC (730 bp),
hdac1 (NM_173236.1): F CATTAACTGGGCAGGAGGTC, R GGCTATCCGCTTATCGTGAG (847 bp),
mta2 (NM_214695.1): F CAACCAGATCACAGCACCTG, R CCACAAACACCACAGGATTG (791 bp),
rbb4 (NM_212595.1): F ATTTGGTGGTTTTGGCTCAG, R CCCATGGTTCATTTGGATTC (854 bp),
wnt5b (NM_130937.1): F CAAGTGTCATGGCGTCTCAG, R CAACAGCAAGGTGGAGTGTG (850 bp)
lef1 (NM_131426.1): F ATGCACGCTGAAGGAGAG, R GAACCCAAGATGTCGAGGAG (801 bp)
msxb (NM_131260): F GAGAATGGGACATGGTCAGG, R GCGGTTCCTCAGGATAATAAC (721 bp)
Fins were fixed in 4% paraformaldehyde in PBS, embedded in OCT, and cryosectioned. Antibody staining was performed as previously described . The following primary antibodies were used: rat anti-BrdU (1:200), rabbit anti-active-caspase 3 (1:10000) (both Abcam), rabbit anti-Tenascin C (1:500; US Biological), mouse anti-Zns5 (1:100; Zebrafish International Resource Center), rabbit anti-And1 (1:5000; Eurogentec). The following secondary antibodies were used at a concentration of 1:500: goat anti-rat Alexa Fluor 488 (Molecular Probes), and goat anti-rabbit Cy3-conjugated and anti-mouse Cy5-conjugated antibodies (Jackson ImmunoResearch).
For proliferation assay, BrdU-positive cells distal to the amputation plane were counted in the mesenchyme and epidermis, and the number of BrdU-positive cells was normalized to the total number of DAPI-stained nuclei. Fluorescent pictures were taken with a confocal microscope (TCS SP5; Leica) and Image J 1.43q software was used for the measurements.
Quantitative real-time PCR
Fin regenerates were collected and total RNA was extracted using Qiazol (Qiagen, Basel, Switzerland). cDNA was synthesized using the QuantiTect Reverse Transcription Kit (Qiagen, Basel, Switzerland). Quantitative real-time PCR was performed in triplicate using the SensiMix SYBR No-ROX Kit (Bioline, Luckenwalde, Germany). Relative expression levels were normalized to β-actin levels. At least two independent experiments were performed for each target, and data were pooled to generate mean normalized RNA levels. The following primers were used for qPCR experiments:
bactin1 (NM_131031.1): F ACATCAGGGAGTGATGGTTG, R TCACAATACCGTGCTCAATG,
chd4a (NM_001044858.1): F GAGAAAGTGCCAAAGACAGC, R AATTCGGTGAATCCTCCATC,
chd4b (XM_680607.5): F CATGGGAGACGATATCGAAG, R CGTTTGCTAGTCCTGCTTTC,
chd3 (XM_691549.5): F ACATCCCTGAGTTTGCTCTG, R CGTTCTCCTCTCTCCTCCTC,
hdac1 (NM_173236.1): F TGACAAACGCATCTCCATTC, R TCTTCACTCGTTTTGGCTTC
mta1 (XM_001333237.3): F CGTACACACCTGTCAACACC, R TGCGCCTCGAGATATCTAAC
mta2 (NM_214695.1): F AAAGATTTGGCCATTCAAGC, R AAATGACCTCCAGCATTGTC
mta3 (NM_199912.2): F CTGCACCTAACGAATCACG, R GTCTTCATGGAGGATTTTGG
rbb4 (NM_212595.1): F TATCCATGGAGGCCATACAG, R TAGATGTTCTCCGCCATCTG
rbb4l (NM_212610.1): F AAGTATGGCAGATGGCTGAG, R TGTGAAGAGTAAGAAGGGGTTG
mbd3a (NM_212769.1): F AGACATGCTGGCACACATC, R GTTCAGCCTCTCATCTGATTG
mbd3b (NM_212580.1): F AGCACAGGTATTTAGATGTGTCTG, R GCTAATCTGGGAGATGAAAATG
mbd2 (NM_212768.1): F CTGCAAAGCGTTCAGTGTTAC, R GCCTGTGGGATCTCTCTAAAC
klf4 (NM_131723.1): F GACGCACACAGGTGAGAAG, R GTCCGGTGTGTTTCCTGTAG
myca (NM_131412.1): F GGCAGCGATTCAGAAGATG, R CTTTTCTGTCGCTTTTCCAC
cebpb (NM_131884.2): F GACGCGAGAGGAACAATCTC, R GCTTCTGTAACCGGTCGTTC
ctsd (NM_131710.1): F CATCGGCAGTGGACTATCTC, R CCATGTACTCTCCCTGCATC
ctsba (NM_213336.2): F TTTGGGAAGACGTCCTACAG, R AGCAGGAAATCCTCATAGACC
junba (NM_213556.3): F AGTACCACCACCATCACCAC, R GTCTGTGGCTCCTCTTTCAG
sdha (NM_200910.1): F TGTGTGGAACACTGATCTGG, R TCCACACGATCCTTGAAGTC
For MOSP efficacy, segments of the correctly spliced chd4a mRNA around the exon 8 were amplified with the following primers:
prCP46: F TCCTTATCGTGACAGGCCTAC
prCP47: R GGAGTAGGGCCCTTTCAATC
prCP82: R AAGCAGACCATGTGATAGGC
Fin regenerates were disrupted using glass beads in a mixture of 240 mM Tris HCl pH 6.8, 8% SDS, 40% glycerol, 0.01% bromophenol blue, and 1.4 M β-mercaptoethanol. Then 20 μg of total proteins were loaded per lane and separated by SDS-PAGE (12%). Even loading was verified by staining with Ponceau S and with β-actin antibodies (1:2000; Sigma). Proteins were transferred onto nitrocellulose membranes, and blots were incubated in 5% milk with rabbit anti-Histone H3 (1:2000), rabbit anti-acetyl-histone H3 (1:1000), anti-acetyl-histone H4 (1:1000) (all Millipore) and β-actin (1:2000; Sigma). Secondary HRP anti-rabbit and anti-mouse antibodies (Sigma) were used at 1:10,000.
Splice blocking vivo-morpholino
Translational blocking vivo-morpholino
quantitative real-time polymerase chain reaction
We are grateful to Franscesco Hofmann, Novartis Pharma AG for providing the NPV-AEW541 compounds, to Claire Jacob for providing the MGCD0103 compound, and to Gilbert Weidinger for providing the transgenic fish. We thank V. Zimmerman for excellent fish care; S. Käser-Pébernard for critical reading of the manuscript; and Y. Molleyres and L. Bulliard for excellent technical help. This work was funded by the Swiss National Funds grants 31003A_125577 of F. M. and C. W. and 310030_138062 of A. J.
- Becker T, Wullimann MF, Becker CG, Bernhardt RR, Schachner M: Axonal regrowth after spinal cord transection in adult zebrafish. J Comp Neurol. 1997, 377: 577-595.PubMedView ArticleGoogle Scholar
- Bernhardt RR, Tongiorgi E, Anzini P, Schachner M: Increased expression of specific recognition molecules by retinal ganglion cells and by optic pathway glia accompanies the successful regeneration of retinal axons in adult zebrafish. J Comp Neurol. 1996, 376: 253-264.PubMedView ArticleGoogle Scholar
- Poss KD, Wilson LG, Keating MT: Heart regeneration in zebrafish. Science. 2002, 298: 2188-2190.PubMedView ArticleGoogle Scholar
- Poss KD, Keating MT, Nechiporuk A: Tales of regeneration in zebrafish. Dev Dyn. 2003, 226: 202-210.PubMedView ArticleGoogle Scholar
- Sadler KC, Krahn KN, Gaur NA, Ukomadu C: Liver growth in the embryo and during liver regeneration in zebrafish requires the cell cycle regulator, uhrf1. Proc Natl Acad Sci U S A. 2007, 104: 1570-1575.PubMed CentralPubMedView ArticleGoogle Scholar
- Akimenko M-A, Marí-Beffa M, Becerra J, Géraudie J: Old questions, new tools, and some answers to the mystery of fin regeneration. Dev Dyn. 2003, 226: 190-201.PubMedView ArticleGoogle Scholar
- Iovine MK: Conserved mechanisms regulate outgrowth in zebrafish fins. Nat Chem Biol. 2007, 3: 613-618.PubMedView ArticleGoogle Scholar
- Gurley KA, Alvarado AS: Stem cells in animal models of regeneration. StemBook. 2008, ed. The Stem Cell Research Community, StemBook, doi/10.3824/stembook.1.32.1Google Scholar
- Nechiporuk A, Keating MT: A proliferation gradient between proximal and msxb-expressing distal blastema directs zebrafish fin regeneration. Development. 2002, 129: 2607-2617.PubMedGoogle Scholar
- Brown AM, Fisher S, Iovine MK: Osteoblast maturation occurs in overlapping proximal-distal compartments during fin regeneration in zebrafish. Dev Dyn. 2009, 238: 2922-2928.PubMed CentralPubMedView ArticleGoogle Scholar
- Lee Y, Hami D, De Val S, Kagermeier-Schenk B, Wills AA, Black BL, Weidinger G, Poss KD: Maintenance of blastemal proliferation by functionally diverse epidermis in regenerating zebrafish fins. Dev Biol. 2009, 331: 270-280.PubMed CentralPubMedView ArticleGoogle Scholar
- Knopf F, Hammond C, Chekuru A, Kurth T, Hans S, Weber CW, Mahatma G, Fisher S, Brand M, Schulte-Merker S, Weidinger G: Bone regenerates via dedifferentiation of osteoblasts in the zebrafish fin. Dev Cell. 2011, 20: 713-724.PubMedView ArticleGoogle Scholar
- Kragl M, Knapp D, Nacu E, Khattak S, Maden M, Epperlein HH, Tanaka EM: Cells keep a memory of their tissue origin during axolotl limb regeneration. Nature. 2009, 460: 60-65.PubMedView ArticleGoogle Scholar
- Tu S, Johnson SL: Fate restriction in the growing and regenerating zebrafish fin. Dev Cell. 2011, 20: 725-732.PubMed CentralPubMedView ArticleGoogle Scholar
- Singh SP, Holdway JE, Poss KD: Regeneration of amputated zebrafish fin rays from de novo osteoblasts. Dev Cell. 2012, 22: 879-886.PubMed CentralPubMedView ArticleGoogle Scholar
- Chablais F, Jazwinska A: IGF signaling between blastema and wound epidermis is required for fin regeneration. Development. 2010, 137: 871-879.PubMedView ArticleGoogle Scholar
- Grotek B, Wehner D, Weidinger G: Notch signaling coordinates cellular proliferation with differentiation during zebrafish fin regeneration. Development. 2013, 140: 1412-1423.PubMedView ArticleGoogle Scholar
- Jaźwińska A, Badakov R, Keating MT: Activin-betaA signaling is required for zebrafish fin regeneration. Curr Biol. 2007, 17: 1390-1395.PubMedView ArticleGoogle Scholar
- Lee Y, Grill S, Sanchez A, Murphy-Ryan M, Poss KD: Fgf signaling instructs position-dependent growth rate during zebrafish fin regeneration. Development. 2005, 132: 5173-5183.PubMedView ArticleGoogle Scholar
- Münch J, González-Rajal A, de la Pompa JL: Notch regulates blastema proliferation and prevents differentiation during adult zebrafish fin regeneration. Development. 2013, 140: 1402-1411.PubMedView ArticleGoogle Scholar
- Poss KD, Shen J, Nechiporuk A, McMahon G, Thisse B, Thisse C, Keating MT: Roles for Fgf signaling during zebrafish fin regeneration. Dev Biol. 2000, 222: 347-358.PubMedView ArticleGoogle Scholar
- Poss KD, Shen J, Keating MT: Induction of lef1 during zebrafish fin regeneration. Dev Dyn. 2000, 219: 282-286.PubMedView ArticleGoogle Scholar
- Smith A, Avaron F, Guay D, Padhi BK, Akimenko MA: Inhibition of BMP signaling during zebrafish fin regeneration disrupts fin growth and scleroblasts differentiation and function. Dev Biol. 2006, 299: 438-454.PubMedView ArticleGoogle Scholar
- Stoick-Cooper CL, Weidinger G, Riehle KJ, Hubbert C, Major MB, Fausto N, Moon RT: Distinct Wnt signaling pathways have opposing roles in appendage regeneration. Development. 2007, 134: 479-489.PubMedView ArticleGoogle Scholar
- Tawk M, Tuil D, Torrente Y, Vriz S, Paulin D: High-efficiency gene transfer into adult fish: a new tool to study fin regeneration. Genesis. 2002, 32: 27-31.PubMedView ArticleGoogle Scholar
- Thummel R, Bai S, Sarras MP, Song P, McDermott J, Brewer J, Perry M, Zhang X, Hyde DR, Godwin AR: Inhibition of zebrafish fin regeneration using in vivo electroporation of morpholinos against fgfr1 and msxb. Dev Dyn. 2006, 235: 336-346.PubMedView ArticleGoogle Scholar
- White JA, Boffa MB, Jones B, Petkovich M: A zebrafish retinoic acid receptor expressed in the regenerating caudal fin. Development. 1994, 120: 1861-1872.PubMedGoogle Scholar
- Whitehead GG, Makino S, Lien C-L, Keating MT: fgf20 is essential for initiating zebrafish fin regeneration. Science. 2005, 310: 1957-1960.PubMedView ArticleGoogle Scholar
- Denslow SA, Wade PA: The human Mi-2/NuRD complex and gene regulation. Oncogene. 2007, 26: 5433-5438.PubMedView ArticleGoogle Scholar
- Tong JK, Hassig CA, Schnitzler GR, Kingston RE, Schreiber SL: Chromatin deacetylation by an ATP-dependent nucleosome remodelling complex. Nature. 1998, 395: 917-921.PubMedView ArticleGoogle Scholar
- Wade PA, Gegonne A, Jones PL, Ballestar E, Aubry F, Wolffe AP: Mi-2 complex couples DNA methylation to chromatin remodelling and histone deacetylation. Nat Genet. 1999, 23: 62-66.PubMedGoogle Scholar
- Xue Y, Wong J, Moreno GT, Young MK, Côté J, Wang W: NURD, a novel complex with both ATP-dependent chromatin-remodeling and histone deacetylase activities. Mol Cell. 1998, 2: 851-861.PubMedView ArticleGoogle Scholar
- Zhang Y, LeRoy G, Seelig HP, Lane WS, Reinberg D: The dermatomyositis-specific autoantigen Mi2 is a component of a complex containing histone deacetylase and nucleosome remodeling activities. Cell. 1998, 95: 279-289.PubMedView ArticleGoogle Scholar
- Allen HF, Wade PA, Kutateladze TG: The NuRD architecture. Cell Mol Life Sci. 2013, 70: 3513-3524.PubMed CentralPubMedView ArticleGoogle Scholar
- Bowen NJ, Fujita N, Kajita M, Wade PA: Mi-2/NuRD: multiple complexes for many purposes. Biochim Biophys Acta. 2004, 1677: 52-57.PubMedView ArticleGoogle Scholar
- Von Zelewsky T, Palladino F, Brunschwig K, Tobler H, Hajnal A, Müller F: The C. elegans Mi-2 chromatin-remodelling proteins function in vulval cell fate determination. Development. 2000, 127: 5277-5284.PubMedGoogle Scholar
- Unhavaithaya Y, Shin TH, Miliaras N, Lee J, Oyama T, Mello CC: MEP-1 and a homolog of the NURD complex component Mi-2 act together to maintain germline-soma distinctions in C. elegans. Cell. 2002, 111: 991-1002.PubMedView ArticleGoogle Scholar
- Kehle J, Beuchle D, Treuheit S, Christen B, Kennison JA, Bienz M, Müller J: dMi-2, a hunchback-interacting protein that functions in polycomb repression. Science. 1998, 282: 1897-1900.PubMedView ArticleGoogle Scholar
- Yoshida T, Hazan I, Zhang J, Ng SY, Naito T, Snippert HJ, Heller EJ, Qi X, Lawton LN, Williams CJ, Georgopoulos K: The role of the chromatin remodeler Mi-2beta in hematopoietic stem cell self-renewal and multilineage differentiation. Genes Dev. 2008, 22: 1174-1189.PubMed CentralPubMedView ArticleGoogle Scholar
- Kaji K, Caballero IM, MacLeod R, Nichols J, Wilson VA, Hendrich B: The NuRD component Mbd3 is required for pluripotency of embryonic stem cells. Nat Cell Biol. 2006, 8: 285-292.PubMedView ArticleGoogle Scholar
- Scimone ML, Meisel J, Reddien PW: The Mi-2-like Smed-CHD4 gene is required for stem cell differentiation in the planarian Schmidtea mediterranea. Development. 2010, 137: 1231-1241.PubMed CentralPubMedView ArticleGoogle Scholar
- Zakaria C, Kassahun H, Yang X, Labbé J-C, Nilsen H, Ramotar D: Caenorhabditis elegans APN-1 plays a vital role in maintaining genome stability. DNA Repair (Amst). 2010, 9: 169-176.View ArticleGoogle Scholar
- Kawakami A, Fukazawa T, Takeda H: Early fin primordia of zebrafish larvae regenerate by a similar growth control mechanism with adult regeneration. Dev Dyn. 2004, 231: 693-699.PubMedView ArticleGoogle Scholar
- Yoshinari N, Ishida T, Kudo A, Kawakami A: Gene expression and functional analysis of zebrafish larval fin fold regeneration. Dev Biol. 2009, 325: 71-81.PubMedView ArticleGoogle Scholar
- Stadler JA, Shkumatava A, Norton WHJ, Rau MJ, Geisler R, Fischer S, Neumann CJ: Histone deacetylase 1 is required for cell cycle exit and differentiation in the zebrafish retina. Dev Dyn. 2005, 233: 883-889.PubMedView ArticleGoogle Scholar
- Nambiar RM, Ignatius MS, Henion PD: Zebrafish colgate/hdac1 functions in the non-canonical Wnt pathway during axial extension and in Wnt-independent branchiomotor neuron migration. Mech Dev. 2007, 124: 682-698.PubMed CentralPubMedView ArticleGoogle Scholar
- Ignatius MS, Unal Eroglu A, Malireddy S, Gallagher G, Nambiar RM, Henion PD: Distinct functional and temporal requirements for zebrafish Hdac1 during neural crest-derived craniofacial and peripheral neuron development. PLoS One. 2013, 8: e63218-PubMed CentralPubMedView ArticleGoogle Scholar
- Yamaguchi M, Tonou-Fujimori N, Komori A, Maeda R, Nojima Y, Li H, Okamoto H, Masai I: Histone deacetylase 1 regulates retinal neurogenesis in zebrafish by suppressing Wnt and Notch signaling pathways. Development. 2005, 132: 3027-3043.PubMedView ArticleGoogle Scholar
- Harrison MRM, Georgiou AS, Spaink HP, Cunliffe VT: The epigenetic regulator Histone Deacetylase 1 promotes transcription of a core neurogenic programme in zebrafish embryos. BMC Genomics. 2011, 12: 24-PubMed CentralPubMedView ArticleGoogle Scholar
- Cunliffe VT: Histone deacetylase 1 is required to repress Notch target gene expression during zebrafish neurogenesis and to maintain the production of motoneurones in response to hedgehog signalling. Development. 2004, 131: 2983-2995.PubMedView ArticleGoogle Scholar
- Fournel M, Bonfils C, Hou Y, Yan PT, Trachy-Bourget M-C, Kalita A, Liu J, Lu A-H, Zhou NZ, Robert M-F, Gillespie J, Wang JJ, Ste-Croix H, Rahil J, Lefebvre S, Moradei O, Delorme D, Macleod AR, Besterman JM, Li Z: MGCD0103, a novel isotype-selective histone deacetylase inhibitor, has broad spectrum antitumor activity in vitro and in vivo. Mol Cancer Ther. 2008, 7: 759-768.PubMedView ArticleGoogle Scholar
- Khan N, Jeffers M, Kumar S, Hackett C, Boldog F, Khramtsov N, Qian X, Mills E, Berghs SC, Carey N, Finn PW, Collins LS, Tumber A, Ritchie JW, Jensen PB, Lichenstein HS, Sehested M: Determination of the class and isoform selectivity of small-molecule histone deacetylase inhibitors. Biochem J. 2008, 409: 581-589.PubMedView ArticleGoogle Scholar
- Zhang J, Wagh P, Guay D, Sanchez-Pulido L, Padhi BK, Korzh V, Andrade-Navarro MA, Akimenko M-A: Loss of fish actinotrichia proteins and the fin-to-limb transition. Nature. 2010, 466: 234-237.PubMedView ArticleGoogle Scholar
- Santamaría JA, Becerra J: Tail fin regeneration in teleosts: cell-extracellular matrix interaction in blastemal differentiation. J Anat. 1991, 176: 9-21.PubMed CentralPubMedGoogle Scholar
- Durán I, Marí-Beffa M, Santamaría JA, Becerra J, Santos-Ruiz L: Actinotrichia collagens and their role in fin formation. Dev Biol. 2011, 354: 160-172.PubMedView ArticleGoogle Scholar
- Sousa S, Afonso N, Bensimon-Brito A, Fonseca M, Simões M, Leon J, Roehl H, Cancela ML, Jacinto A: Differentiated skeletal cells contribute to blastema formation during zebrafish fin regeneration. Development. 2011, 138: 3897-3905.PubMedView ArticleGoogle Scholar
- Johnson SL, Weston JA: Temperature-sensitive mutations that cause stage-specific defects in Zebrafish fin regeneration. Genetics. 1995, 141: 1583-1595.PubMed CentralPubMedGoogle Scholar
- Kidder BL, Palmer S: HDAC1 regulates pluripotency and lineage specific transcriptional networks in embryonic and trophoblast stem cells. Nucleic Acids Res. 2012, 40: 2925-2939.PubMed CentralPubMedView ArticleGoogle Scholar
- Reynolds N, Latos P, Hynes-Allen A, Loos R, Leaford D, O’Shaughnessy A, Mosaku O, Signolet J, Brennecke P, Kalkan T, Costello I, Humphreys P, Mansfield W, Nakagawa K, Strouboulis J, Behrens A, Bertone P, Hendrich B: NuRD suppresses pluripotency gene expression to promote transcriptional heterogeneity and lineage commitment. Cell Stem Cell. 2012, 10: 583-594.PubMed CentralPubMedView ArticleGoogle Scholar
- Ishida T, Nakajima T, Kudo A, Kawakami A: Phosphorylation of Junb family proteins by the Jun N-terminal kinase supports tissue regeneration in zebrafish. Dev Biol. 2010, 340: 468-479.PubMedView ArticleGoogle Scholar
- Ju BG, Kim WS: Upregulation of cathepsin D expression in the dedifferentiating salamander limb regenerates and enhancement of its expression by retinoic acid. Wound Repair Regen. 1998, 6: 349-357.PubMedView ArticleGoogle Scholar
- Mathew LK, Sengupta S, Franzosa JA, Perry J, La Du J, Andreasen EA, Tanguay RL: Comparative expression profiling reveals an essential role for raldh2 in epimorphic regeneration. J Biol Chem. 2009, 284: 33642-33653.PubMed CentralPubMedView ArticleGoogle Scholar
- Greenbaum LE, Li W, Cressman DE, Peng Y, Ciliberto G, Poli V, Taub R: CCAAT enhancer- binding protein beta is required for normal hepatocyte proliferation in mice after partial hepatectomy. J Clin Invest. 1998, 102: 996-1007.PubMed CentralPubMedView ArticleGoogle Scholar
- Yang X-J, Seto E: The Rpd3/Hda1 family of lysine deacetylases: from bacteria and yeast to mice and men. Nat Rev Mol Cell Biol. 2008, 9: 206-218.PubMed CentralPubMedView ArticleGoogle Scholar
- Reynolds N, O’Shaughnessy A, Hendrich B: Transcriptional repressors: multifaceted regulators of gene expression. Development. 2013, 140: 505-512.PubMedView ArticleGoogle Scholar
- Barrero MJ, Boué S, Izpisúa Belmonte JC: Epigenetic mechanisms that regulate cell identity. Cell Stem Cell. 2010, 7: 565-570.PubMedView ArticleGoogle Scholar
- Stewart S, Tsun Z-Y, Izpisua Belmonte JC: A histone demethylase is necessary for regeneration in zebrafish. Proc Natl Acad Sci U S A. 2009, 106: 19889-19894.PubMed CentralPubMedView ArticleGoogle Scholar
- Spoorendonk KM, Peterson-Maduro J, Renn J, Trowe T, Kranenbarg S, Winkler C, Schulte-Merker S: Retinoic acid and Cyp26b1 are critical regulators of osteogenesis in the axial skeleton. Development. 2008, 135: 3765-3774.PubMedView ArticleGoogle Scholar
- Smith A, Zhang J, Guay D, Quint E, Johnson A, Akimenko MA: Gene expression analysis on sections of zebrafish regenerating fins reveals limitations in the whole-mount in situ hybridization method. Dev Dyn. 2008, 237: 417-425.PubMedView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.