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Modulation of tissue repair by regeneration enhancer elements

Nature volume 532, pages 201–206 (2016)Cite this article

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Abstract

How tissue regeneration programs are triggered by injury has received limited research attention. Here we investigate the existence of enhancer regulatory elements that are activated in regenerating tissue. Transcriptomic analyses reveal that leptin b (lepb) is highly induced in regenerating hearts and fins of zebrafish. Epigenetic profiling identified a short DNA sequence element upstream and distal to lepb that acquires open chromatin marks during regeneration and enables injury-dependent expression from minimal promoters. This element could activate expression in injured neonatal mouse tissues and was divisible into tissue-specific modules sufficient for expression in regenerating zebrafish fins or hearts. Simple enhancer-effector transgenes employing lepb-linked sequences upstream of pro- or anti-regenerative factors controlled the efficacy of regeneration in zebrafish. Our findings provide evidence for ‘tissue regeneration enhancer elements’ (TREEs) that trigger gene expression in injury sites and can be engineered to modulate the regenerative potential of vertebrate organs.

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Figure 1: Activation of lepb regulatory sequences during tissue regeneration.
Figure 2: A DNA element upstream of lepb directs regeneration-dependent gene expression.
Figure 3: LEN activity in neonatal mice.
Figure 4: LEN is separable into tissue-specific elements.
Figure 5: LEN controls fin regeneration when paired with Fgf effectors.
Figure 6: Enhancer-directed nrg1 expression boosts cardiomyocyte proliferation.

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Acknowledgements

We thank J. Burris, N. Lee, and T. Thoren for zebrafish care; A. Knecht, and J. Savage for technical advice or assistance; and M. Bagnat, C. Chen, F. Conlon, D. Fox, and M. Mokalled for comments on the manuscript. J.H. was supported by an AHA postdoctoral fellowship (12POST11920060), R.K. by an NIH Clinical Investigator Award (K08 HL116485), V.A.T. by an NSF Graduate Research Fellowship (1106401), and J.A.G. by an NIH postdoctoral fellowship (F32 HL120494). This work was supported by NIH grants to B.L.B. (R01 HL089707 and R01 HL064658) and K.D.P. (R01 GM074057 and R01 HL081674), who acknowledges support from HHMI.

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Authors and Affiliations

  1. Department of Cell Biology, Duke University Medical Center, Durham, 27710, North Carolina, USA

    Junsu Kang, Amy L. Dickson, Valerie A. Tornini, Gregory Nachtrab, Matthew Gemberling, Joseph A. Goldman & Kenneth D. Poss

  2. Cardiovascular Research Institute, University of California, San Francisco, San Francisco, 94143, California, USA

    Jianxin Hu & Brian L. Black

  3. Department of Medicine, Duke University Medical Center, Durham, 27710, North Carolina, USA

    Ravi Karra

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Contributions

J.K. and K.D.P. designed the experimental strategy, analysed data, and prepared the manuscript. J.K. generated transgenic zebrafish and performed regeneration experiments and analysis. J.H. and B.L.B. generated and analysed transgenic mice. R.K. generated and analysed sequencing datasets. A.L.D. performed surgeries and histology. V.A.T. generated and analysed mutant zebrafish. G.N., M.G. and J.A.G. contributed unpublished results and methodology. All authors commented on the manuscript.

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Correspondence to Kenneth D. Poss.

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Extended data figures and tables

Extended Data Figure 1 lepb transcripts sharply increase during fin and heart regeneration.

a, Venn diagram displaying numbers of genes with significantly increased transcript levels during fin and heart regeneration. b, RT–PCR of samples from 2 days post-fertilization (dpf) and 4 dpf embryos, and uninjured and regenerating adult tissues. lepb was not detected during embryogenesis and in uninjured tissues, but induced during regeneration. β-act2 is used as loading control. Uninj, Uninjured. c, Left: relative expression of lepb in uninjured, 1, 2, and 4 dpa fin regenerates. lepb transcript levels are increased at 1 and 2 dpa. Right: relative expression of lepb in uninjured or 3 dpa cardiac ventricles, assessed by qPCR. d, e, Endogenous lepb expression assessed by in situ hybridization in sections of fins (d) and cardiac ventricle and atrium (e). Arrowhead, amputation plane. Arrows, endocardial lepb expression. Left: uninjured tissues, Right: regenerating tissues. dpa: days post-amputation. f, g, F0 animals, injected with the transgenic lepb:eGFP BAC reporter construct at the one-cell stage, induced eGFP after larval fin fold amputation (f) and during adult fin regeneration (g). Note that lepb:eGFP is mosaically expressed. Arrowheads, amputation planes. h, i, Expression pattern of lepb:eGFP stable transgenic animals. lepb:eGFP was not detected in fin and heart during embryogenesis (2 dpf (h); 4 dpf (i)). Below ‘i’ are enlargements of the boxed areas, which show heart (left) and fin fold (right). Dotted line, outline of fin fold. The yolk is autofluorescent. j, k, Section images of lepb:eGFP caudal fin regenerates at 2 dpa (j) and 4 dpa (k). The majority of lepb:eGFP-positive cells are mesenchymal cells, overlapping partially with cells that incorporate EdU (collected 60 min after injection; red). l, m, Lack of detectable expression of lepb:eGFP in hearts of uninjured (l) or sham-operated (m) lepb:eGFP animals. n = 8 and 5 for uninjured and sham-operated hearts, respectively. Arrowheads, amputation planes. Scale bars represent 10 μm (d, f, h–k); 50 μm (e, l, m); 500 μm (g).

Extended Data Figure 2 Leptin signalling during fin and heart regeneration.

a–e, Expression pattern of lepr:lepr–mCherry BAC reporter line. a, Schematic of the lepr:lepr–mCherry BAC transgenic construct. mCherry is fused at the C terminus of Lepr. b, mCherry fluorescence in the lepr:lepr–mCherry BAC reporter strain is induced during fin regeneration. n = 5; all animals displayed a similar expression pattern. c, Section images of 4 dpa lepr:lepr–mCherry caudal fin regenerates. The majority of Lepr–mCherry+ cells are epidermal cells, overlapping partially with p63+ basal and suprabasal cells (left). In addition, some putative vascular cells in the intra-ray region have Lepr–mCherry signals (right). d, e, Confocal images of sections through uninjured (d) and regenerating (e) lepr:lepr–mCherry hearts. Lepr–mCherry fluorescence co-localizes with MHC+ cardiomyocytes in uninjured and 3 dpa hearts (arrows). Note that these expression patterns are similar to leptin receptor expression in mice (see Supplementary Information). n = 7 and 6 for uninjured and 3 dpa hearts, respectively. f–j, Analysis of fin and heart regeneration in lepbpd94 mutants. f, A schematic representation of Lepb, showing the effects of the pd94 mutation. Lepb is composed of 5 alpha-helix domains. lepbpd94 has a 19 bp insertion and a 3 bp deletion at the third α-helix (helix C). g, Sequencing of wild-type and lepbpd94 alleles revealed an indel (red highlight). h, A comparison of the amino acid sequences of leptin genes of human, mice, and zebrafish. The predicted amino acid sequence of the lepbpd94 gene product is shown at the bottom, with the predicted truncation sites indicated in red. The predicted lepbpd94 protein product lacks the majority of C-terminal amino acids. Asterisk indicates identical amino acid residue between three species. i, Quantification of regenerated fin lengths from lepbpd94 and wild type siblings at 4 dpa. n = 12 each of lepbpd94 and wild-type. j, Quantification of cardiomyocyte proliferation at 7 dpa. n = 7 (lepbpd94) and 8 (wild-type). Data are represented as mean ± s.e.m. NS, not significant.

Extended Data Figure 3 Identification of LEN and tests of regulatory sequences near lepb.

a, Schematic depicting the genomic region surrounding lepb (corresponding to the lepb BAC used in this study) with the profiles of RNA-sequencing and H3K27ac marks from uninjured and regenerating heart tissues. b, Enlargement of the boxed area in a. lepb is the only upregulated gene in this genomic region during regeneration. H3K27ac-enriched peaks in regenerating samples are present in a ~1 kb region (red bar) that is ~7 kb upstream of the start codon. c, Schematic representation of transgenes to examine regulatory sequence activity. Fin and endocardial expression during regeneration and the number of stable lines are indicated. Asterisk indicates that one LENP2:eGFP line showed occasional, weak endocardial eGFP expression in uninjured hearts, whereas eGFP signal in this line was broad and strong during regeneration. EC, endocardial cells. d, Images of representative 0 dpa fins from lines indicated in c. eGFP fluorescence is not detectable in fins at 0 dpa or in uninjured fins, but is induced in regenerating ray blastemas in P7:eGFP and LENP2:eGFP lines. P6:eGFP regenerates displayed weak eGFP expression below the amputation plane during regeneration, with very weak or undetectable expression in regenerating portions (see Fig. 2c). e, LENP2:eGFP expression pattern during fin regeneration. eGFP is detectable as early as 12 hpa, but is undetectable at 30 dpa. n = 5; all animals displayed a similar expression pattern. Arrowheads, amputation planes. f, Section images of representative uninjured and regenerating hearts from P2:eGFP, P6:eGFP, P7:eGFP, and LENP2:eGFP animals. eGFP fluorescence is rarely detectable in uninjured P2:eGFP, P6:eGFP, P7:eGFP, or LENP2:eGFP hearts, except in one line of LENP2:eGFP (mentioned above). Upon injury, P2 drove weak, occasional expression in cardiomyocytes and epicardium but not in endocardium, whereas P7 and LEN drove endocardial eGFP expression in ventricle and atrium. i, ii, enlargements of boxes areas in regenerating ventricle and atrium, respectively. Scale bars: 500 μm (d, e); 50 μm (f).

Extended Data Figure 4 Additional putative regeneration enhancer elements.

a, Cartoon depicting the distal upstream regions of il11a, cd248b, vcana, and plek. RNA-sequencing profiles indicate that these genes are upregulated during heart regeneration. The red bar indicates putative enhancer regions that are enriched with H3K27ac marks in regenerating tissue. Two of these putative enhancers, near il11a and vcana, showed primary sequence conservation in other non-mammalian vertebrates but not in mammals. b, Scheme depicting assays in injected F0 transgenic animals. At 4 dpf, eGFP expression in the uninjured fin fold was examined, and then the fin fold was amputated. eGFP expression near the amputation plane was examined at 5 dpf. c, Table indicating injected constructs and the number of animals with eGFP+ cells near the amputation plane. d, Images of representative 4 dpf (uninjured) and 5 dpf (regenerating) fin folds from animals in c. e, Vista plot of genomic regions from mir129 to lepb based on LAGAN alignment with reference sequence zebrafish. Sequence comparison indicates that this region is not highly conserved between zebrafish and mammals. Arrowheads, amputation planes.

Extended Data Figure 5 Transient transgenic assays examining lepb-linked regeneration enhancer fragments in combination with different promoters (fin regeneration).

a, Scheme depicting assays in injected F0 transgenic animals. Transgene-positive larvae were selected by detection of eGFP in response to fin fold amputation (lepb promoter), in cardiomyocytes (cmlc2 promoter), or in lenses (α-cry promoter). Caudal fins of F0 transgenic positive zebrafish were amputated at 60–90 days post-fertilization (dpf), and eGFP expression was examined at 2 dpa. b, Schematic representation of the transgenic constructs to examine fin regeneration enhancer activity. Expression during fin regeneration and the number of assessed F0 animals are indicated. Many embryos transgenic for LEN(1–850), LEN(450–1000), LEN(450–850), and LEN(660–850) coupled with the lepb or cmlc2 promoter showed activity during fin regeneration. One of eleven LENα-cry:eGFP animals displayed fin eGFP expression, but LEN(1–850)α-cry:eGFP and LEN(450–1000)α-cry:eGFP did not drive eGFP expression during fin regeneration, indicating that there may be repressive motifs in the α-cry promoter fragment that affect fin regeneration enhancer activity (See also Extended Data Fig. 9). ND, not determined.

Extended Data Figure 6 X-gal staining in stable transgenic mouse lines.

a, Additional whole mount images of X-gal stained hearts from neonatal LEN-hsp68::lacZ (line 13, presented in Fig. 3) and control animals injured at postnatal day 1 and assessed at postnatal day 4. X-gal staining is undetectable in sham-operated hearts of LEN-hsp68::lacZ mice (n = 6; representative image shown) and injured hearts of control mice, but strong in partially resected hearts of LEN-hsp68::lacZ mice (arrows). Dashed red lines indicate injury area, positioned facing the front. Arrows, injury-dependent β-galactosidase expression. dpi, days post-injury. b, Whole-mount images of X-gal stained hearts and paws from LEN-hsp68::lacZ line 6, which exhibited vascular endothelial expression in uninjured hearts and paws. Scale bars represent 1 mm.

Extended Data Figure 7 Transgenic assays examining lepb-linked regeneration enhancer fragments in combination with lepb P2 (fin regeneration).

a, Schematic representation of the transgenic constructs to examine LEN fragments that direct expression during fin regeneration. Expression during fin regeneration and the number of stable lines is indicated. b, Images of representative 0 dpa and 2 dpa fins from a. eGFP fluorescence is rarely detectable in uninjured fins. LEN(1–850), LEN(450–1000), LEN(450–850), and LEN(660–850) coupled with P2 directed eGFP expression during fin regeneration. *LEN(830–1350)P2:eGFP lines exhibited very weak eGFP expression in fin regenerates, detectable with long exposure times and at high magnification (data not shown), suggesting the possibility of minor fin regeneration enhancer elements in 850–1000. At least 5 fish from each transgenic line were examined, and all animals displayed a similar expression pattern. Arrowheads, amputation planes.

Extended Data Figure 8 Images of heart sections from uninjured and regenerating transgenic lines that employ lepb-linked enhancer fragments.

a–h, eGFP fluorescence is rarely detectable in uninjured hearts in all transgenic lines. One exception is LEN(1000–1350)P2:eGFP, which showed occasional, weak endocardial eGFP expression in uninjured hearts. LEN(1–850)P2:eGFP (a), LEN(450–1000)P2:eGFP (b), LEN(450–850)P2:eGFP (d), and LEN(660–850)P2:eGFP (g) transgenic lines, which include distal LEN elements, directed eGFP expression from promoters in a subset of epicardial cells and/or cardiomyocytes, but not endocardial cells. LEN(450–660)P2:eGFP lines (e) showed regeneration-dependent enhancer activity in cardiomyocytes near the injury site, but not in endocardial cells. Our data indicated that the activities of LEN(1–850)P2:eGFP (a), LEN(450–1000)P2:eGFP (b), and LEN(450–850)P2:eGFP (d) lines were not as strong as LEN(450–660)P2:eGFP (e), suggesting that there might be repressive elements for cardiomyocyte expression outside of sequences 450–660. LEN(830–1350) (c) and LEN(1000–1350) (h), which did not activate expression from promoters during fin regeneration, could direct endocardial expression in both ventricle and atrium during regeneration, similar to the reference reporters lepb:eGFP and LENP2:eGFP. Arrows in c, h, endocardial eGFP. i, ii, Enlargements of the boxed areas in regenerating ventricle and atrium, respectively. At least 5 fish from each transgenic line were examined, and all animals displayed a similar expression pattern. Scale bars represent 50 μm.

Extended Data Figure 9 Transgenic assays to examine lepb-linked enhancer fragment activity in combination with cmlc2 and α-cry promoters.

a, Schematic representation of the transgenic constructs to examine enhancer fragment activity in combination with the cmlc2 promoter. Expression during fin regeneration and the number of stable lines is indicated. b, Images of representative 0 dpa and 2 dpa fins from a. eGFP fluorescence was very weak or undetectable in 0 dpa or uninjured fins. (1–850), (450–1000), (450–850), and (660–850) LEN fragments coupled with the cmlc2 promoter activated blastemal eGFP fluorescence (arrows) during fin regeneration. One LEN(1–850)cmlc2:eGFP line did not show fin regeneration enhancer activity. Arrowheads, amputation planes. At least five fish from each transgenic line were examined, and all animals displayed a similar expression pattern except for the following: For two strains of LEN(450–850)cmlc2:eGFP, 4 of 5 animals induced eGFP fluorescence at 2 dpa; For LEN(660–850)cmlc2:eGFP, 4 of 7 animals induced eGFP fluorescence at 2 dpa. c, Left: schematic diagram of the LEN(1000–1350)cmlc2:eGFP transgenic construct. Right: images of sections from uninjured and regenerating LEN(1000–1350)cmlc2:eGFP hearts. eGFP is expressed mosaically in cardiomyocytes via the cmlc2 promoter. Uninjured hearts had no detectable endocardial eGFP fluorescence, whereas 3 dpa hearts displayed induced endocardial eGFP fluorescence (arrows). Arrowheads indicate cardiomyocyte eGFP fluorescence driven by cmlc2 promoter activity. d–h, Schematic representation of the transgenic constructs to examine enhancer fragment activity in combination with the α-cry promoter. Expression during fin regeneration and injury-activated endocardial expression, and the number of stable lines are indicated. At least 5 fish from each transgenic line were examined, and all animals displayed a similar expression pattern. EC, endocardial cells. One LENα-cry:eGFP line showed regeneration-dependent expression (arrows) in fins (e); yet, unlike when coupled with lepb and cmlc2 promoters, the LEN(450–1000) fragment did not direct expression during fin regeneration (d and data not shown). This suggests a possible repressive motif within α-cry sequences. Asterisk indicates that one LENα-cry:eGFP line showed weak endocardial eGFP expression in uninjured hearts, but the eGFP signal (arrows) was stronger and broader during regeneration (g). Two LEN(830–1350)α-cry:eGFP lines had no detectable eGFP fluorescence in regenerating fins (f) or uninjured hearts (h), but displayed induced endocardial eGFP fluorescence (arrows) during heart regeneration (h). i, ii, Enlargements of the boxed areas in regenerating ventricle and atrium, respectively. i, LEN sequences annotated with putative binding sites in fin (663–854) and cardiac (1034–1350) regeneration enhancer modules. j, A predicated AP-1 binding site is necessary for fin regeneration enhancer activity. Top, schematic representation of the LEN(450–850-AP-1mut)P2 transgenic construct, in which the predicted AP-1 binding site (TGACTCA) is mutated to AAAAAA. Two LEN(450–850-AP-1mut)P2 lines had no detectable eGFP fluorescence in regenerating fins. Scale bars represent 50 μm.

Extended Data Figure 10 Pairing LEN with potent developmental influences can control regenerative capacity.

a, Images of representative F0 transgenic zebrafish injected with P2:dnfgfr1 (left) or LENP2:dnfgfr1 (right) constructs, shown at 3 dpa. The dn-fgfr1 cassette is fused in frame to eGFP. Whereas zero of 27 P2:dnfgfr1 F0 animals displayed defective regeneration, 7 of 67 LENP2:dnfgfr1 F0 zebrafish had impaired fin regeneration in some fin rays, corresponding to eGFP fluorescence (arrow). b, Additional examples of LENP2:dnfgfr1 fins at 30 dpa, from experiments with a stable line. Inset in b, high magnification view of the boxed area, showing eGFP fluorescence. c, Quantification of regenerated area from dob; LENP2:fgf20a F0 transgenic zebrafish (n = 45, 44 at 5, 10 dpa, respectively), dob mutants (n = 19, 19 at 5, 10 dpa, respectively), and dob; P2:fgf20a F0 transgenic zebrafish (n = 40, 40 at 5, 10 dpa, respectively) at 5 dpa and 10 dpa. Dotted line indicates 500,000 μm2. d, Images of representative dob; LENP2:fgf20a F0 transgenic zebrafish, dob mutants, and dob; P2:fgf20a F0 transgenic zebrafish at 5 dpa. e, Confocal images of tissue sections of 3 dpa fin regenerates. Mosaic regenerates indicate expression of the linked ef1α:nls–mCherry marker construct (red), and EdU incorporation (collected 60 min after injection; green). DAPI, blue. F0 mosaic dob; LENP2:fgf20a regenerates show evidence of distal growth and blastemal EdU incorporation. Arrow, blastema. Dotted lines, amputation planes. i, ii, Enlargements of the boxed areas. f, In situ hybridization in sections of 3 dpa fin regenerates from dob; P2:fgf20a (left) and F0 mosaic dob; LENP2:fgf20a (right) animals, indicating LEN-induced fgf20a expression in mesenchymal cells and regenerative growth (arrows). fgf20a is rarely detected in dob; P2:fgf20a regenerates. Arrowheads, amputation planes.

Supplementary information

Supplementary Table 1

RNA-sequencing data of differentially expressed genes during fin regeneration. (XLSX 1494 kb)

Supplementary Table 2

RNA-sequencing data of differentially expressed genes during heart regeneration. (XLSX 1362 kb)

Supplementary Data

Lists of reagents, additional experimental data, and references. (PDF 518 kb)

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Kang, J., Hu, J., Karra, R. et al. Modulation of tissue repair by regeneration enhancer elements. Nature 532, 201–206 (2016). https://doi.org/10.1038/nature17644

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