DEVELOPMENT AND STEM CELLS RESEARCH ARTICLE 4133

Development 139, 4133-4142 (2012) doi:10.1242/dev.079756© 2012. Published by The Company of Biologists Ltd

INTRODUCTIONRegeneration is a complex biological process by which animalsrestore the shape, structure and function of body parts lost to injuryor experiment (Brockes and Kumar, 2005; Poss, 2010). One of themost remarkable examples is heart regeneration (Ausoni andSartore, 2009; Laflamme and Murry, 2011). Several non-mammalian vertebrates, including zebrafish, possess the significantability to restore injured heart. After resectioning of the ventricularapex, zebrafish restore lost heart tissue within 1-2 months (Poss etal., 2002; Raya et al., 2003), which contrasts to the inability ofmammals to do so after the immediate postnatal period (Bergmannet al., 2009; Porrello et al., 2011). The regenerative ability is alsoobserved in response to a variety of injuries, including cryoprobe-induced injury (Chablais et al., 2011; González-Rosa et al., 2011;Schnabel et al., 2011) and genetic ablation of cardiomyocytes(CMs) by transgenic induction of toxin expression (Wang et al.,2011). It therefore provides a model system to study how an injuredheart can be repaired (Poss, 2007; Raya et al., 2004). Recentreports showed that the major source of regenerated CMs was pre-existing CMs, rather than stem/progenitor cells, that undergo partialde-differentiation, re-enter the cell cycle and proliferate to restorethe injured heart (Jopling et al., 2010; Kikuchi et al., 2010).

Simultaneously, vascularization of the regenerating area takesplace, which requires the activities of fibroblast growth factor(FGF) signaling and vascular endothelial growth factor (VEGF)signaling (Kim et al., 2010; Lepilina et al., 2006). However, it isstill unknown whether CMs are involved in other processes, inaddition to proliferation, to restore the injured heart. Morespecifically, cell migration is known to be involved in thedevelopment of a variety of organs (Friedl and Gilmour, 2009; Razand Mahabaleshwar, 2009), and organ regeneration involvesreactivation of developmental processes and genes (Iovine, 2007).

The recent advent of photoconvertible fluorescent reporterproteins provided a new approach for cell migration analysis (Starkand Kulesa, 2007). Natural and engineered green fluorescentproteins, which can be photoconverted into red fluorescentproteins, have been developed (Lukyanov et al., 2005). Suchproteins include Kaede (Ando et al., 2002), PA-GFP (Patterson andLippincott-Schwartz, 2002), KikGR (Tsutsui et al., 2005), EosEP(Nienhaus et al., 2006), PA-mRFP (Verkhusha and Sorkin, 2005)and Dendra (Gurskaya et al., 2006). Kaede is one of the foundingmembers of photoconvertible fluorescent proteins, serendipitouslyfound from a stony coral, Trachyphyllia geoffroyi (Ando et al.,2002). The unique photoconvertible characters of Kaede representseveral advantages for optic marking. First, the red state iscomparable to the green in terms of brightness and stability.Second, photoconversion is shown to result in a more than 2000-fold increase in the red/green fluorescent ratio. Third, completelyseparate wavelengths of light can be used for observation andphotoconversion. By taking advantage of these unique properties,several reports have demonstrated cell tracing in vivo, such asmonitoring cell migration during zebrafish embryogenesis (Hattaet al., 2006), cell migratory behaviors in cortical slices of mice(Mutoh et al., 2006) and T-cell migration during cutaneous immuneresponse in mice (Tomura et al., 2010; Tomura et al., 2008). The

1Department of Genetics, Cell Biology and Development, University of Minnesota,Minneapolis, MN 55455, USA. 2Stem Cell Institute, University of Minnesota,Minneapolis, MN 55455, USA. 3Health Research Institute, National Institute ofAdvanced Industrial Science and Technology, Ikeda, Osaka 563-8577, Japan.4Division of Pediatric Blood and Marrow Transplant, University of Minnesota,Minneapolis, MN 55455, USA. 5Developmental Biology Center, University ofMinnesota, Minneapolis, MN 55455, USA.

*Author for correspondence (kawak005@umn.edu)

Accepted 18 August 2012

SUMMARYAdult zebrafish possess a significant ability to regenerate injured heart tissue through proliferation of pre-existing cardiomyocytes,which contrasts with the inability of mammals to do so after the immediate postnatal period. Zebrafish therefore provide a modelsystem in which to study how an injured heart can be repaired. However, it remains unknown what important processescardiomyocytes are involved in other than partial de-differentiation and proliferation. Here we show that migration ofcardiomyocytes to the injury site is essential for heart regeneration. Ventricular amputation induced expression of cxcl12a and cxcr4b,genes encoding a chemokine ligand and its receptor. We found that cxcl12a was expressed in the epicardial tissue and that Cxcr4was expressed in cardiomyocytes. We show that pharmacological blocking of Cxcr4 function as well as genetic loss of cxcr4b functioncauses failure to regenerate the heart after ventricular resection. Cardiomyocyte proliferation was not affected but a large portionof proliferating cardiomyocytes remained localized outside the injury site. A photoconvertible fluorescent reporter-basedcardiomyocyte-tracing assay demonstrates that cardiomyocytes migrated into the injury site in control hearts but that migrationwas inhibited in the Cxcr4-blocked hearts. By contrast, the epicardial cells and vascular endothelial cells were not affected by blockingCxcr4 function. Our data show that the migration of cardiomyocytes into the injury site is regulated independently of proliferation,and that coordination of both processes is necessary for heart regeneration.

KEY WORDS: Heart regeneration, Cardiomyocytes, Zebrafish, Directed migration, CXCL12-CXCR4

Migration of cardiomyocytes is essential for heartregeneration in zebrafishJunji Itou1,2, Isao Oishi3, Hiroko Kawakami1,2, Tiffany J. Glass4, Jenna Richter1, Austin Johnson1, Troy C. Lund4, and Yasuhiko Kawakami1,2,5,*

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use of cell-type-specific promoters/enhancers has also facilitatedmonitoring specific types of cells in combination withphotoconversion technology, such as differentiation ofcardiomyocytes (de Pater et al., 2009), beta cells of the pancreas(Pisharath et al., 2007) and postmitotic neurons (Pan et al., 2012).

Here we show that migration of CMs to the injury site is essentialfor heart regeneration. We found that the chemokine ligand, cxcl12aand its receptor, cxcr4b, were induced after heart injury in zebrafish.Blocking Cxcr4 function caused mis-localization of proliferatingCMs outside of the injury site without affecting their proliferation.By localized photoconversion in hearts of the CM-specific Kaedetransgenic zebrafish, we show that CMs migrate toward the injurysite only after heart damage, and this migration requires Cxcr4function. Our data show that the migration of CMs into the injurysite is regulated independently of proliferation, and that coordinationof both processes is essential for heart regeneration.

MATERIALS AND METHODSZebrafish maintenance and surgeryZebrafish were maintained under standard conditions at around 28°C, andadult zebrafish (6 to 18 months old) were used for experiments. Ventricularamputation was performed as previously published (Raya et al., 2003).Care and experimentation were done in accordance with the InstitutionalAnimal Care and Use Committee of the University of Minnesota and theNational Institute of Advanced Industrial Science and Technology, Japan.

In situ hybridization, immunostaining and TUNEL assayIn situ hybridization on sections 14 m thick was performed as previouslydescribed (Kawakami et al., 2011). Immunostaining on sections wasperformed according to a standard procedure (Kawakami et al., 2006; Rayaet al., 2003). The primary antibodies used were anti-myosin heavy chain(MHC; Developmental Studies Hybridoma Bank, MF20, 5.14 g/ml), anti-CXCR4 (Santa Cruz Biotechnology, sc-6190, 1:100), anti-PCNA (SantaCruz Biotechnology; sc-56, 1:100), anti-MEF2 (Santa Cruz Biotechnology;sc-313, 1:50), anti-GFP (Molecular Probes, A11122, 1:500) and anti-DsRed2 (Clontech, 632496, 1:500). Secondary antibodies used were Alexafluorophore-labeled anti-mouse or rabbit or goat IgG (Invitrogen, 1:1000)or biotinylated anti-mouse IgG (Vector Laboratories, BA-9200, 1:500). AnIn Situ Cell Death Detection Kit (Roche Diagnostics) was used for TUNELassay according to the manufacturer’s instruction. Counterstaining wasdone with DAPI or hematoxylin. The number of proliferating CMs wascounted manually with the sections that represent the largest injury area atthe center of the injury site of each heart.

EdU-labeling experimentTo label proliferating cells, 20 l of 0.5 mg/ml EdU solution wasintraperitoneally injected into zebrafish at 7 days post amputation (dpa) and10 dpa. Hearts were collected at 13 dpa and subjected to standard analysisaccording to the manufacturer’s instructions (Invitrogen, Click-IT EdU cellproliferation assays).

Pharmacological blocking of Cxcr4 function and genetic loss ofcxcr4b functionFor pharmacological blocking of Cxcr4 function, each fish was maintainedin 50 ml system water with or without 40 nM FC131 after surgery (Narumiet al., 2010) (Wako Chemical, Osaka, Japan), and the water was refresheddaily. Because the carrier is H2O, nothing was added into the containers ofcontrol fish. To assist with the penetration of FC131, the pericardiac cavitywas surgically opened weekly in both control and treated fish. For geneticloss of cxcr4b function, we used the odysseus mutant fish line, whichpossesses a null mutation in the cxcr4b gene (Knaut et al., 2003). Wild-type siblings were used as controls for the odysseus mutants (hereafterodysseus mutants are referred to as cxcr4b–/–).

Generation of the cmlc2a-Kaede lineThe cmlc2a-Kaede line was established with the zebrafish cmlc2a promoter(Huang et al., 2003) and Kaede (Medical & Biological Laboratories,

Nagoya, Japan) using the Tol2 transposon system (Kawakami et al., 2004;Urasaki et al., 2006). The P0 fish were outcrossed with AB fish, and stabletransgenic lines were established after evaluating cardiac-specificexpression of Kaede.

Photoconversion of KaedeFor photoconversion of cmlc2a-Kaede transgenic fish hearts, we made asmall window by manually dissecting the pericardiac cavity afteranesthetizing fish. A compound microscope (Nikon LABPHOT) equippedwith a 100 watt mercury lamp, diachronic mirror of 380 nm and a barrierfilter of 420 nm were used to irradiate the heart for 90 seconds withapproximately 2 mm distance between the 20� objective lens and theheart. To injure the heart, we enlarged the pericardiac window andamputated the apex of the ventricle under fluorescent monitoring with aZeiss V12 stereomicroscope.

ImagingFluorescent confocal images were obtained by using a Zeiss LSM 710 laserscanning microscope system, and analyzed by ZEN2009 software. ForKaede imaging, non-fixed hearts were embedded in optimal cuttingtemperature compound and frozen. Sections at 20 m thickness were dried,washed with PBS, rinsed with water and mounted for confocal imaging.Kaede green fluorescence was excited by 514 nm light and 519–556 nmfluorescence was detected. Wavelengths for excitation and detection ofKaede red fluorescence were 562 nm and 566–674 nm, respectively.

Statistical analysisStatistical significance was analyzed by the Student’s t-test, and shown asaverage ± standard deviation. P-values are indicated within each panel.

RESULTSInduction of cxcl12a and cxcr4b expression afterheart injuryDuring expression screening of genes expressed in developing andregenerating zebrafish hearts, we found that cxcl12a, a geneencoding a chemokine ligand (also known as sdf1a), and itsreceptor cxcr4b were expressed during heart regeneration (Fig. 1).The transcripts of cxcl12a were detected between 3 dpa and 7 dpaat the surface and inside of the regenerating area (Fig. 1). Thesignals of cxcr4b became evident at 5 dpa, and persisted until 10dpa (Fig. 1). cxcr4b expression was detected at the surface layer ofthe regenerating area and around the injury site. These expressionpatterns were not detected in sham-operated hearts at 3 dpa(cxcl12a) and 7 dpa (cxcr4b), the stages at which they weredetected at high levels (n5 each, data not shown). By contrast, wedid not detect transcripts of the related ligand-receptor pair cxcl12band cxcr4a during heart regeneration by in situ hybridization(supplementary material Fig. S1). The CXCL12-CXCR4 system isknown to regulate directed cell migration during embryonicdevelopment (Friedl and Gilmour, 2009; Raz and Mahabaleshwar,2009; Schier, 2003). Thus, induction of the expression of cxcl12aand cxcr4b after heart injury suggests that directed cell migrationplays a role during heart regeneration.

CMs express cxcr4bAs a first step to investigate the possible involvement of directedcell migration during heart regeneration, we sought to identify celltypes that express cxcr4b and cxcl12a. Immunofluorescenceanalysis showed that the CM-specific nuclear signal of Mef2(supplementary material Fig. S2) is associated with membrane-localized Cxcr4 signal on the same confocal plane (Fig. 2A,B),indicating that CMs express Cxcr4b. Further analysis of the Cxcr4signal and CM-specific cmlc2a-EGFP signal also showed that 287Cxcr4-positive signals from six sections were associated with thecmlc2a-EGFP signal (Fig. 2C-E). Similarly, the Cxcr4 signal was

RESEARCH ARTICLE Development 139 (22)

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also associated with MHC and Mef2 signals (Fig. 3). Moreover,optic sectioning of Cxcr4-expressing CMs showed Cxcr4 signal inthe cytoplasm, in addition to the membrane (Fig. 3B,C), suggestingCxcl12a-dependent internalization of Cxcr4b (Orsini et al., 1999).These results demonstrate that CMs express Cxcr4. In the mouse,endothelial cells also express Cxcr4 (Gupta et al., 1998; Volin etal., 1998); however, we did not detect Cxcr4 in endothelial cells(Fig. 2F-H). Two hundred and sixty Cxcr4 signals from fivesections of fli1-EGFP transgenic fish heart were not associatedwith the endothelial-cell-specific fli1-EGFP signal.

Next, we investigated which cells express cxcl12a. Consistentwith the expression of cxcl12a at the surface of the regeneratingarea (Fig. 1), the cxcl12a signals overlapped with wt1b signals(supplementary material Fig. S3A-C�), which is expressed in theepicardial cells (Perner et al., 2007). We did not detect expressionof the cxcl12a-DsRed2 reporter (Glass et al., 2011) in MHC-positive CMs (supplementary material Fig. S3D-F) or in fli1-EGFP-positive endothelial cells (supplementary material Fig. S3G-I). These data indicate that epicardial cells express cxcl12a afterheart injury.

Cxcr4 function is required for heart regenerationTo determine the functional significance of the Cxcl12-Cxcr4system during heart regeneration, we blocked Cxcr4 function bytreating fish with an antagonist after ventricular amputation. Wefirst tested whether FC131, a recently developed selective CXCR4antagonist (Narumi et al., 2010), can block zebrafish Cxcr4

function. AMD3100, a widely used CXCR4 antagonist, was notused because our previous studies indicated that it did notefficiently block Cxcr4 function, either in the adult zebrafishhematopoietic system in vivo (Glass et al., 2011) or in themigration of primordial germ cells in zebrafish embryos (data notshown). Treating zebrafish embryos with FC131 significantlyimpaired directed migration of primordial germ cells into thegonad-forming region (supplementary material Fig. S4), a processthat has been shown to depend on cxcr4b function (Doitsidou et al.,2002; Knaut et al., 2003), demonstrating that FC131 can effectivelyblock Cxcr4 function in zebrafish. We then treated zebrafish withFC131 after amputation of the ventricle and found that this resultedin failure to regenerate the heart (Fig. 4A-F�). In controlregenerating hearts, MHC signals (CM marker) were stronglydetected in the injury site, where the heart tissue was removed, at14 dpa, and the regenerating tissue was filled with CMs at 30 dpa(Fig. 4A,A�,C,C�). At 60 dpa, the regenerated myocardiumexhibited a strong MHC signal, comparable to the non-injured area(Fig. 4E,E�). However, significantly lower levels of MHC signalswere detected in the injury site of CXCR4-antagonist-treated heartsat 14 dpa (Fig. 4B,B�, n4/5 hearts). After 30 days and 60 days theCXCR4-antagonist-treated heart lacked a sealed wall (Fig. 4D,D�,red arrows, n5/5 hearts; 4F,F�, n3/4 hearts), which was fullyformed in control hearts (Fig. 4C,C�, black arrows, n5/5 hearts;4E,E�, n5/5 hearts). These results indicate that the CXCR4-antagonist-treated heart did not regenerate. The significantreduction of MHC signals in the injury site was also observed incxcr4b–/– fish at 14 dpa (Fig. 4G,H�, n3/3 hearts). Similar to theCXCR4-antagonist-treated heart, cxcr4b–/– fish hearts failed toform the sealed wall at 30 dpa (Fig. 4J,J�, n2/2 hearts) and 60 dpa(Fig. 4L,L�, n2/2 hearts), compared with control hearts (Fig. 4I,I�,

4135RESEARCH ARTICLECardiomyocyte migration

Fig. 1. Expression of cxcl12a and cxcr4b during heartregeneration. (A-N)In situ hybridization for cxcl12a (A,C,E,G,I,K,M)and cxcr4b (B,D,F,H,J,L,N) in the regenerating heart. Time points at 0(A,B), 1 (C,D), 3 (E,F), 5 (G,H), 7 (I,J), 10 (K,L) and 14 (M,N) dpa areshown. An evident expression of cxcl12a was detected at 3 dpa to 7dpa (E,G,I). Strong expression of cxcr4b was detected at 5 dpa (H) andpersisted until 10 dpa (L). Scale bar: 50m.

Fig. 2. Cxcr4 is present on CMs. (A,B)Confocal images of Cxcr4(green) and Mef2 (magenta) double staining of control heart at 7 dpa.The Cxcr4 signal was detected at the cell surface of nuclear Mef2-positive cells in the regenerating area (arrowheads in B). B shows ahigher magnification image of the boxed area in A. The dotted lineindicates the amputation plane. (C-E)Confocal images of Cxcr4 (C) andcmlc2a-EGFP signal, detected by anti-GFP antibody (D), and a mergedimage (E). The arrowheads point to cells positive for both Cxcr4 andcmlc2a-EGFP signals. (F-H)Confocal images of Cxcr4 (F) and fli1-EGFPsignal, detected by anti-GFP antibody (G), and a merged image (H).Scale bars: 50m.

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n3/3 hearts; 4K,K�, n3/3 hearts). Fibrin clearance at 14 dpa wassimilar in control and cxcr4–/– fish (supplementary material Fig. S5,n2/2 for both control and cxcr4–/– hearts), and thus, the failure toregenerate the heart is unlikely to be caused by abnormal fibrindeposition. These data demonstrate that cxcr4b function is requiredfor heart regeneration in zebrafish. These results strongly supportour hypothesis that cxcr4b functions in CMs that contribute to heartregeneration. Although cxcr4b expression was specific to CMs inregenerating zebrafish hearts (Fig. 2), Cxcr4 is also known to beinvolved in heart development in mice (Ma et al., 1998; Tachibanaet al., 1998; Zou et al., 1998). In order to avoid complicationscaused by an unidentified role of cxcr4b in zebrafish heartdevelopment, if any, we focused our study of cxcr4b function bypharmacological antagonism using FC131.

Mis-localization of proliferating CMs by blockingCxcr4 functionGiven that the proliferation of CMs is a major factor for heartregeneration (Jopling et al., 2010; Kikuchi et al., 2010), we askedwhether blocking Cxcr4 function affected CM proliferation and/or

survival. The number of proliferating CMs, visualized byproliferating cell nuclear antigen (Pcna, an S-phase marker) andMef2 (CM marker), did not change significantly in Cxcr4-blockedhearts compared with control hearts at 7, 14, 21 and 30 dpa (Fig.5A-C, n5 hearts at each time point). This result also indicates thatthe failure to regenerate the heart by blocking Cxcr4 function is notdue to delayed CM proliferation. The number of Pcna-positivecells, which includes CMs and non-CMs, was also not changed atthese time points (supplementary material Fig. S6), indicating thatthe proliferation of other cell types was also unaffected by CXCR4antagonist treatment. TUNEL analysis did not show an increase inthe number of cell deaths at 7 dpa and 14 dpa (supplementarymaterial Fig. S7). These data indicate that the failure to regeneratethe amputated heart by blocking Cxcr4 function is not caused bydefects in proliferation or survival of CMs and other cells. A recentstudy showed that the number of genetically labeled CMs locatedat the sub-epicardial area increases in the injury site, whereas theirnumber reduces at the edge of the injury site during heartregeneration (Kikuchi et al., 2010). Thus, we compared the numberof proliferating CMs in the injury site, similar to the study byKikuchi and colleagues (Kikuchi et al., 2010). We found a changein the localization of proliferating CMs in Cxcr4-blocked hearts. Incontrol hearts we found 74.0% of proliferating CMs around thecenter of the regenerating area, and the majority of the otherproliferating CMs at the edge of the regenerating area at 14 dpa.By contrast, only 23.5% of the proliferating CMs were locatedaround the center of the injury site in Cxcr4-blocked hearts (Fig.5D). Because the total number of proliferating CMs was not altered(Fig. 5C), this observation suggests that Cxcr4 function isnecessary for localization of proliferating CMs in the regeneratingarea.

Detection of Pcna visualizes cells in the S phase of the cell cycleat the time of sample fixation. In order to further examine CM

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Fig. 3. Confocal imaging of Cxcr4-expressing cardiomyocytes.(A)Confocal images of MHC, Cxcr4 and Mef2 at 7 dpa. The yellowarrowheads point to Cxcr4-positive cells. (B)Images of a Cxcr4-expressing CM at 500 nm distance along the z-axis. Mef2 representsthe nucleus (magenta), MHC represents cytoplasm (green) and Cxcr4 isshown in yellow. The numbers in the upper area of each panel indicatepositions along the z-axis. (C)Optical section of a Cxcr4-expressing cell.High levels of Mef2 signal and MHC signal define a nucleus andcytoplasm, respectively. The open arrowheads and solid arrowheadsindicate Cxcr4 in membrane and cytoplasm, respectively. The verticalaxis represents fluorescence intensity. Scale bars: 100m in A; 10m in B.

Fig. 4. Cxcr4 function is required for heart regeneration.(A-F�) MHC (brown) and hematoxylin (purple) staining of control(A,A�,C,C�,E,E�) and CXCR4-antagonist-treated (B,B�,D,D�,F,F�) heart at14 dpa (A-B�), 30 dpa (C-D�) and 60 dpa (E-F�). (G-L�) MHC andhematoxylin staining of wild-type (G,G�,I,I�,K,K�) and cxcr4b–/–

(H,H�,J,J�,L,L�) heart at 14 dpa (G-H�), 30 dpa (I-J�) and 60 dpa (K-L�).A�-L� show close up of the boxed area in A-L, respectively. Thearrowheads in A�, B�, G� and H� point to the MHC signal in theregenerating area. The black and red arrows in C�-F� and I�-L� point tothe presence and absence of a sealed muscle wall, respectively. Dottedlines in A, B, G and H indicate the amputation planes. Scale bars:50m.

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proliferation, we labeled cells by EdU. Injection of EdU at 7 dpaand 10 dpa, followed by detection at 13 dpa, allows for labelingcells that underwent cell division during 7 dpa to 13 dpa (Fig. 5E-F�). Similar to the analysis of Pcna detection, the number ofproliferating CMs, visualized as EdU and cmlc2a-mCherry double-positive cells, were at similar levels in control and CXCR4-antagonist-treated hearts (Fig. 5G). We found that 72.4% of EdU-labeled CMs were located around the center of the regeneratingarea. By contrast, only 12.4% of EdU-labeled CMs were locatedaround the center of the injury site in Cxcr4-blocked hearts (Fig.5H), similar to the case with Pcna analysis. This EdU-labeling

experiment further confirmed a role for Cxcr4 function in thelocalization of proliferating CMs in the regenerating area.

Localized labeling of CMs by photoconversion ofthe Kaede fluorescent proteinGiven that the Cxcl12-Cxcr4 system is known to regulate directedcell migration in a variety of processes (Friedl and Gilmour, 2009;Raz and Mahabaleshwar, 2009; Schier, 2003), the abnormallocalization of proliferating CMs suggests that the migration ofproliferating CMs, rather than local activation of CMs in the injurysite, is necessary for heart regeneration. To address this hypothesis,we developed a fluorescent reporter-based CM-tracing assay. Weestablished a transgenic zebrafish line that expresses Kaede (Andoet al., 2002), driven by the CM-specific cmlc2a promoter(supplementary material Fig. S8A,B, cmlc2a-Kaede). The greenfluorescence of Kaede can be irreversibly converted into stable redfluorescence by ultraviolet irradiation for cell tracing in vivo(supplementary material Fig. S8C-H) (Ando et al., 2002; Tomuraet al., 2008). Thus, we were able to label CMs in a restricted areaof the ventricle with red fluorescence in order to trace labeled CMsduring heart regeneration (Fig. 6A-F; Fig. 7A). Maintainingcmlc2a-Kaede fish under normal breeding conditions (Fig. 6G-J,n8), as well as ventricular amputation under a fluorescentstereomicroscope (Fig. 6K-N, n8), did not induce green to redphotoconversion. For localized photoconversion of the heart, weexposed a small part of the ventricle and performed irradiation (seeMaterials and methods) (Fig. 6A-F). Photoconverted redfluorescence was observed immediately after irradiation and onlyin the irradiated area (Fig. 6O-R, n8). Seven days after irradiation,the red fluorescence was stable and remained as a cluster (Fig. 6S-V, n8), indicating that CMs labeled with red fluorescence did notmigrate under normal conditions. We also observed newlysynthesized green Kaede 7 days after irradiation such that theirradiated CMs fluoresced both red and green (compare Fig. 6Oand 6S). This indicates that the irradiation was not toxic to CMs.Also, we did not detect cell death at 1 day and 3 days afterirradiation in the irradiated area (data not shown).

Cxcr4-dependent CM migration after injuryTo analyze the migration of CMs, we amputated the apex in thenon-irradiated portions of the ventricle under a fluorescentstereomicroscope (Fig. 7A-C, n6). Three days after amputation,we did not detect CMs in the regenerating area (data not shown,n7), consistent with the lack of cxcr4b signal in the regeneratingarea at this time point (Fig. 1F). However, seven days afteramputation, CMs with both green and red fluorescence, and CMswith only green fluorescence, were detected in the regeneratingarea (Fig. 7D,E, n6). We detected 5.5% of photoconverted CMsin the regenerating area (Fig. 7P). This result demonstrates thatCMs migrated into the regenerating area by 7 dpa. The ratio ofphotoconverted CMs in the regenerating area was 10.4% and12.1% at 10 dpa (Fig. 7H,I,P, n7 hearts) and 14 dpa (Fig. 7L,M,P,n7 hearts), respectively. At 10 dpa and 14 dpa, a portion ofphotoconverted CMs exhibited strong green signals due tocontinued de novo production of Kaede. Blocking Cxcr4 functioncaused CMs to be excluded from the injury site at 7 dpa (Fig.7F,G,P, n6 hearts), 10 dpa (Fig. 7J,K,P, n6 hearts) and 14 dpa(Fig. 7N,O,P, n6 hearts). Our data revealed that the cxcl12a-cxcr4b-dependent system regulates directed migration of CMs intothe injury site, and that this process is essential for heartregeneration.

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Fig. 5. Blocking Cxcr4 function causes mis-localization ofproliferating CMs. (A-B�) Pcna (green) and Mef2 (magenta) stainingof control (A,A�) and CXCR4-antagonist-treated (B,B�) hearts at 14 dpa.The yellow arrowheads point to proliferating CMs seen as white signal.A� and B� show close up of the boxed area in A and B. For simplicity,not all proliferating CMs are labeled. (C)Quantitation of the number ofproliferating CMs in control and CXCR4-antagonist-treated hearts at 7,14, 21 and 30 dpa. The vertical axis represents the number ofproliferating CMs per section. A section that represents the largestinjury area at the center of the injury site of each heart was examined(n5). P-values at each time point are shown. (D)Quantitation of theratio of proliferating CMs in the injury site compared with the numberof entire proliferating CMs in control and CXCR4 antagonist-treatedhearts at 14 dpa. The same samples examined in C were used. (E-F�) EdU (green) and cmlc2a-mCherry (magenta) signal of control(E,E�) and CXCR4 antagonist-treated (F,F�) hearts at 13 dpa. The yellowarrowheads point to proliferating CMs seen as white signal. Forsimplicity, not all proliferating CMs are labeled. E� and F� show close upof the boxed area in E and F. (G)Quantitation of the number ofEdU/cmlc2a-mCherry double-positive cells in control and CXCR4antagonist-treated hearts at 13 dpa. Vertical axis represents the numberof proliferating CMs per section. A section that represents the largestinjury area at the center of the injury site of each heart was examined(n5). (H)Quantitation of the ratio of EdU-positive CMs in the injurysite compared with the number of entire EdU-positive CMs in controland CXCR4-antagonist-treated hearts at 13 dpa. The same samplesexamined in G were used. For D, G and H, P-values by Student’s t-testare shown. Dotted lines in A, B, E and F indicate the amputationplanes. Scale bars: 50m.DEVELO

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Epicardial cells are unlikely to be affected byblocking Cxcr4Cells other than CMs, such as epicardial cells and vascularendothelial cells, are also known to participate in heartregeneration in zebrafish (Kikuchi et al., 2011b; Kim et al.,2010; Lepilina et al., 2006). To further understand how Cxcr4regulates heart regeneration, we examined whether these cellsare also affected by blocking Cxcr4 function during heartregeneration. Expression of aldehyde dehydrogenase 1a2(aldh1a2, also known as retinaldehyde dehydrogenase 2,raldh2), a gene encoding a rate-limiting enzyme for retinoic acidsynthesis, is upregulated in the epicardial tissue after cardiacdamage (Lepilina et al., 2006), and retinoic acid signaling isrequired for heart regeneration (Kikuchi et al., 2011b). Weobserved aldh1a2 expression in a wide region of the epicardiumat 3 dpa, and at the surface of the injury site at 7 dpa, similar tothe control heart (supplementary material Fig. S9A-F). aldh1a2expression is also detected in endocardial cells after heart injury(Kikuchi et al., 2011b), which can be visualized by fli1-EGFPreporter at 3 dpa (supplementary material Fig. S9C,D). Thus,aldh1a2 expression in both epicardial and endocardial tissueappeared to be unaffected by CXCR4 antagonist treatment.Expression of wt1b marks the epicardial tissue in regeneratinghearts (González-Rosa et al., 2011; Kikuchi et al., 2011a;Schnabel et al., 2011). We detected comparable wt1b mRNAexpression in the epicardial tissue of both control and Cxcr4-blocked hearts at 3 dpa (supplementary material Fig. S9G-H�)and 7 dpa (supplementary material Fig. S9I-J�). To furtherevaluate whether epicardial responses are affected by CXCR4antagonist treatment, we measured the length of the wt1b-expressing domain (supplementary material Fig. S9G,H,I,J, bluelines). The ratio of the lengths of the wt1b-expressing domain tothe length of the surface of the regenerating area werecomparable in control and CXCR4-antagonist-treated hearts atboth time points (supplementary material Fig. S9K). Theseresults indicate that gene expression in the epicardial tissue wasnot affected by blocking Cxcr4 function during heartregeneration.

Blocking of CXCR4 function is unlikely to affectneo-vascularization in regenerating heartNeo-vascularization of the regenerating area is crucial for heartregeneration (Kim et al., 2010; Lepilina et al., 2006). AlthoughCxcr4 was not detected in endothelial cells after ventricularamputation (Fig. 2), we sought to clarify whether vascularizationwas affected by blocking Cxcr4 function. The fli1-EGFP signalwas detected similarly in control and CXCR4-antagonist-treatedhearts at 7 dpa (Fig. 8A,B, n3) and 14 dpa (Fig. 8C,D, n3).Analysis of the fli1-EGFP-positive region in the regenerating areaat 14 dpa by ImageJ software showed a similar level ofvascularization (Fig. 8E). Thus, CXCR4 antagonist-treatment isunlikely to affect neo-vascularization during heart regeneration inzebrafish.

It has been shown that FGF signaling is required for neo-vascularization of the regenerating area during heart regeneration(Lepilina et al., 2006); thus, we also examined activation of FGFsignaling. Phosphorylation of ERK, a hallmark of the activation ofFGF signaling, was detected both in control and Cxcr4-blockedhearts (Fig. 8F,G). Expression of mkp3/dusp6, a target of FGFsignaling (Kawakami et al., 2003), was also detected similarly inthe control and CXCR4-antagonist-treated heart (Fig. 8H,I). Theseresults indicate that activation of FGF signaling occurred similarly

in both control and Cxcr4-blocked hearts. Comparable activationof gene expression in the epicardial tissue (supplementary materialFig. S9) and vascularization (Fig. 8) further support the idea thatthe failure to regenerate the injured heart by blocking Cxcr4function is caused by a defect in the CMs themselves.

DISCUSSIONTwo cxcl12-cxcr4 systems in zebrafishIn this report, we identified cxcl12a-cxcr4b-dependent CM migrationas an essential mechanism for heart regeneration in zebrafish. The

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Fig. 6. CM labeling by the Kaede photoconversion shows no CMmigration in non-injured hearts. (A-F)Localized photoconversion ofthe adult cmlc2a-Kaede heart. Green (A,D), red (B,E) and mergedimages (C,F) of whole mount samples without irradiation (A-C) andimmediately after irradiation (D-F) are shown. Without irradiation,hearts show only green fluorescence (A-C). After localized irradiation,the fluorescence was converted to red in the irradiated area (E,F). (G-V)Green (G,K,O,S), red (H,L,P,T) and merged images (I,M,Q,U) ofsectioned samples are shown. J, N, R and V show higher magnificationimages of the boxed area in I, M, Q and U, respectively. (G-J)No redfluorescence was detected without irradiation and injury. (K-N)No redfluorescence was detected at 7 dpa without irradiation. The dotted lineindicates the amputation plane. Some green cells are detected in theregenerating area (N, open arrowheads). (O-R)Images immediatelyafter irradiation without injury. The green signal in the irradiated areawas lost (arrowheads, O) and red signal in the same area was detected(arrowheads, P). The merged images show boundary of green-redsignal (Q,R). The yellow arrows in R point to the photoconverted CMs.(S-V)Images 7 days after irradiation without injury. Red signal wasdetected in the irradiated area (arrowheads, T), and green signal wasdetected in the irradiated area by newly synthesized Kaede(arrowheads, S). The yellow arrows in V point to the photoconvertedCMs. Scale bars: 500m in A; 50m in G and J.

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CXCL12-CXCR4 system is a major chemokine-receptor system thatregulates directed migration of a variety of cells (Raz andMahabaleshwar, 2009). Zebrafish have two cxcl12 genes and twocxcr4 genes as a result of the teleost genome duplication during

evolution (Amores et al., 1998). This gene duplication appears tocontribute to functional segregation of the cxcl12-cxcr4 system. Forinstance, during embryonic development, the cxcl12b-cxcr4a systemfunctions for blood vessel development, endothelial cell migration(Bussmann et al., 2011; Siekmann et al., 2009), and endodermmigration during gastrulation (Mizoguchi et al., 2008; Nair andSchilling, 2008). The cxcl12a-cxcr4b system is known to functionfor primordial germ cell migration (Knaut et al., 2003; Raz, 2003),sensory ganglia assembly (Knaut et al., 2005) and lateral linemigration (Haas and Gilmour, 2006), and is also expressed inregenerating fins (Bouzaffour et al., 2009). This functionalsegregation might have contributed to the viability of the cxcr4bmutant line, because mouse mutants that lack either Cxcl12 or Cxcr4die before birth owing to a ventricular septal defect, defectiveformation of the large vessels in the gastrointestinal tract andimpaired hematopoietic development (Ma et al., 1998; Tachibana etal., 1998; Zou et al., 1998). Our data demonstrated that the cxcl12a-cxcr4b system functions to regulate CM migration, which is essentialfor heart regeneration, and that the cxcl12b-cxcr4a system might beinvolved in the regeneration of other organs.

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Fig. 7. Cxcr4 function is required for CM migration during heartregeneration. (A)Experimental strategy to assay CM migration duringheart regeneration. After localized photoconversion of the cmlc2a-Kaede heart, hearts are exposed by enlarging the pericardiac window,and the ventricular apex is amputated. At the desired time, labeledCMs are examined by imaging analysis. (B-O)Green signal representsnon-irradiated Kaede and newly synthesized Kaede after irradiation andred signal represents photoconverted Kaede. C, E, I, M, G, K and Oshow higher magnification images of boxed area in B, D, H, L, F, J andN, respectively. (B,C)Immediately after photoconversion andamputation, green signal was lost at the irradiated site (B, arrowheads),where red signal was detected (C, arrows). Ventricular amputation wasperformed in the non-irradiated area. (D,E)At 7 dpa, red signals(photoconverted CMs, white arrowheads in E) and green signals (non-photoconverted CMs, open arrowheads in E) were detected in theinjury site. (H-M)At 10 dpa (H,I) and 14 dpa (L,M), red signals andgreen signals were also detected in the injury site. (F-O)CMs were notdetected in the injury site of the CXCR4-antagonist-treated heart. Allphotoconverted CMs stayed outside the injury site (yellow arrows inG,K,O) by blocking Cxcr4 function. (P)Quantitation of the migratedCMs 7, 10 and 14 days after photoconversion and amputation. Thevertical axis represents the percentage of photoconverted CMs in theinjury site compared with the number of entire photoconverted CMs. Asection at the center of the injury site was examined from each heart.Dotted lines indicate the amputation planes. The yellow arrowheads inB, D and F point to the irradiated areas. The yellow arrows in C, E, G, I,M, K and O point to red signals outside the injury site. The openarrowheads and white arrowheads point to non-photoconverted CMsand photoconverted CMs, respectively, in the regenerating area. Theasterisks in D, J and N indicate the valves between the atrium andventricle. Scale bars: 50m.

Fig. 8. Normal neo-vascularization and activation of FGFsignaling after blocking Cxcr4 function. (A-D)fli1-EGFP (green) andcmlc2a-mCherry (magenta) signal in the control (A,C) and CXCR4-antagonist-treated (B,D) heart at 7 dpa (A,B) and 14 dpa (C,D).Arrowheads point to neo-vascularization in the regenerating area,visualized by the fli1-EGFP signal. (E)Quantitation of neo-vascularization. The fli-EGFP positive area compared with theregenerating area was measured and by ImageJ software. A section atthe center of the injury site of each heart was examined (n3). (F-I)FGFsignaling status, visualized by phospho ERK1/2 (pERK1/2)immunoreactivity (F,G) and expression of mkp3/dusp6 (H,I) at 14 dpa incontrol (F,H) and CXCR4 antagonist-treated (G,I) hearts. Thearrowheads point to the pERK1/2 (F,G) and mkp3/dusp6 (H,I) signals.The dotted lines indicate the amputation planes. Scale bars: 50m.

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Cxcr4 function is necessary in CMs during heartregeneration in zebrafishOur analysis showed that Cxcr4 functions for directed migration ofCMs toward the injury site in zebrafish. This is in contrast tomammalian myocardial infarction models (Takahashi, 2010), inwhich Cxcr4 functions in CMs (Hu et al., 2007) and bone-marrow-derived mesenchymal stromal cells (Honczarenko et al., 2006). Inmammals, Cxcr4 in CMs is shown to act for enhanced cell survivaland reduction of infarction size (Hu et al., 2007). The Cxcl12-Cxcr4system in the mesenchymal stromal cells functions forcardioprotection (Saxena et al., 2008), and a fraction of bone-marrow-derived cells can differentiate into CMs (Wojakowski et al.,2010). In amputated zebrafish hearts, CM proliferation and survivalwere not affected by blocking Cxcr4 function (Fig. 5; supplementarymaterial Fig. S7). Moreover, it is yet to be determined whethermesenchymal stromal cells in zebrafish (Lund et al., 2012) cancontribute to CMs. Nonetheless, our data highlight the requirementof Cxcr4 function in CMs during heart regeneration in zebrafish.

The Cxcl12-Cxcr4 system and neo-vascularizationOur analysis showed a lack of Cxcr4 signal in endothelial cells inregenerating hearts (Fig. 2). This contrasts to mammalianmyocardial infarction models (Takahashi, 2010), according towhich Cxcr4 is expressed in cell types involved in neo-vascularization, such as bone-marrow-derived mesenchymalstromal cells (Honczarenko et al., 2006; Yamaguchi et al., 2003)and endothelial progenitor cells (Yamaguchi et al., 2003). Althoughzebrafish stromal cells can exhibit endothelial-like properties (Lundet al., 2012), neo-vascularization seems to be unaffected inCXCR4-antagonist treated fish (Fig. 8). Studies have demonstratedthat neo-vascularization in regenerating zebrafish hearts involvesthe contribution of epicardial cells (Kim et al., 2010; Lepilina et al.,2006), which are unlikely to be affected by CXCR4 antagonisttreatment (supplementary material Fig. S9). The present study doesnot rule out the possibility that other unidentified cell types migrateto the injured heart in a Cxcr4-dependent manner and contribute toheart regeneration in zebrafish. However, data obtained in ouranalyses suggest that neo-vascularization occurs independentlyfrom Cxcr4 function during heart regeneration in zebrafish (Fig. 9).

Kaede-photoconversion system and cell tracingCell lineage analysis is an important issue to understand complexprocesses of regeneration, in which multiple cell types are involved(Tanaka and Reddien, 2011). A genetic recombination approach

using CreER transgenic lines is a powerful method, especially forlong-term lineage analysis (Jopling et al., 2010; Kikuchi et al.,2010). The advent of photoconvertible fluorescent proteins, suchas Kaede, has led to the development of an effective approach fortracing migration of specific cell types in vivo (Ando et al., 2002).Our data show that CMs labeled by Kaede photoconversionmigrate toward the injury site during heart regeneration (Fig. 7).Recent studies also showed neutrophil mobilization in zebrafishlarvae (Deng et al., 2011) and developmental timing assays duringzebrafish heart development (de Pater et al., 2009) with similarapproaches. Thus, localized photoconversion in combination withthe use of cell-type-specific promoters/enhancers would be avaluable approach for cell migration and lineage analysis in avariety of biological processes.

A role for CM migration during heartregenerationThe present study demonstrates that CM migration is an essentialmechanism for heart regeneration, in addition to CM proliferationand vasculature formation. Considering that clearing fibrin scarringwould also be important for heart regeneration, remodeling of theextracellular matrix should also be coupled to these processes. Sucha process might involve unidentified molecular systems, and is tobe studied in the future. Our data also indicate that the proliferationof CMs and neo-vascularization are regulated independently frommigration (Fig. 9). As both proliferation of CMs (Jopling et al.,2010; Kikuchi et al., 2010) and their migration (this study) arenecessary, these two events need to be coordinated for theregeneration of the injured heart. Previous studies show that genesinvolved both in cell cycle regulation and cell movement areupregulated in regenerating zebrafish hearts (Lien et al., 2006;Sleep et al., 2010), which supports the conclusions of our research.Given that the neonatal mammalian heart also possesses the abilityto regenerate after resectioning through the proliferation of pre-existing CMs (Porrello et al., 2011), the correct coordination ofmigration and proliferation may prove to be crucial for heartregeneration not only in zebrafish but also in mammalian species.

AcknowledgementsWe thank the zebrafish core facility at the University of Minnesota for generalhelp with the breeding and maintenance of zebrafish lines. We thank DrsMichael O’Connor, Yasushi Nakagawa, Angel Raya, Erez Raz and KoichiKawakami for sharing equipment or materials. We thank Drs Jonathan Slackand Naoko Koyano for critical reading, and the Developmental StudiesHybridoma Bank developed under the auspices of the NICHD and maintainedby The University of Iowa.

FundingI.O was supported by the Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C) [22570197], by the Mochida MemorialFoundation for Medical and Pharmaceutical Research and by the Kowa LifeScience Foundation. Research in Y.K.’s lab was supported by the MinnesotaMedical Foundation [4099-9216-12].

Competing interests statementThe authors declare no competing financial interests.

Supplementary materialSupplementary material available online athttp://dev.biologists.org/lookup/suppl/doi:10.1242/dev.079756/-/DC1

ReferencesAmores, A., Force, A., Yan, Y. L., Joly, L., Amemiya, C., Fritz, A., Ho, R. K.,

Langeland, J., Prince, V., Wang, Y. L. et al. (1998). Zebrafish hox clusters andvertebrate genome evolution. Science 282, 1711-1714.

Ando, R., Hama, H., Yamamoto-Hino, M., Mizuno, H. and Miyawaki, A.(2002). An optical marker based on the UV-induced green-to-red

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Fig. 9. A model of heart regeneration in zebrafish. Amputationinduces cxcl12a-cxcr4b-dependent directed migration of CMs into theinjury site. CM proliferation and neo-vascularization are regulatedindependently from CM migration. Coordinated progression of theseprocesses regenerates the injured heart. The orange circles representproliferating CMs. D

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PMENT

photoconversion of a fluorescent protein. Proc. Natl. Acad. Sci. USA 99, 12651-12656.

Ausoni, S. and Sartore, S. (2009). From fish to amphibians to mammals: insearch of novel strategies to optimize cardiac regeneration. J. Cell Biol. 184,357-364.

Bergmann, O., Bhardwaj, R. D., Bernard, S., Zdunek, S., Barnabé-Heider, F.,Walsh, S., Zupicich, J., Alkass, K., Buchholz, B. A., Druid, H. et al. (2009).Evidence for cardiomyocyte renewal in humans. Science 324, 98-102.

Bouzaffour, M., Dufourcq, P., Lecaudey, V., Haas, P. and Vriz, S. (2009). Fgfand Sdf-1 pathways interact during zebrafish fin regeneration. PLoS ONE 4,e5824.

Brockes, J. P. and Kumar, A. (2005). Appendage regeneration in adult vertebratesand implications for regenerative medicine. Science 310, 1919-1923.

Bussmann, J., Wolfe, S. A. and Siekmann, A. F. (2011). Arterial-venous networkformation during brain vascularization involves hemodynamic regulation ofchemokine signaling. Development 138, 1717-1726.

Chablais, F., Veit, J., Rainer, G. and Jazwinska, A. (2011). The zebrafish heartregenerates after cryoinjury-induced myocardial infarction. BMC Dev. Biol. 11,21.

de Pater, E., Clijsters, L., Marques, S. R., Lin, Y. F., Garavito-Aguilar, Z. V.,Yelon, D. and Bakkers, J. (2009). Distinct phases of cardiomyocytedifferentiation regulate growth of the zebrafish heart. Development 136, 1633-1641.

Deng, Q., Yoo, S. K., Cavnar, P. J., Green, J. M. and Huttenlocher, A. (2011).Dual roles for Rac2 in neutrophil motility and active retention in zebrafishhematopoietic tissue. Dev. Cell 21, 735-745.

Doitsidou, M., Reichman-Fried, M., Stebler, J., Köprunner, M., Dörries, J.,Meyer, D., Esguerra, C. V., Leung, T. and Raz, E. (2002). Guidance ofprimordial germ cell migration by the chemokine SDF-1. Cell 111, 647-659.

Friedl, P. and Gilmour, D. (2009). Collective cell migration in morphogenesis,regeneration and cancer. Nat. Rev. Mol. Cell Biol. 10, 445-457.

Glass, T. J., Lund, T. C., Patrinostro, X., Tolar, J., Bowman, T. V., Zon, L. I. andBlazar, B. R. (2011). Stromal cell-derived factor-1 and hematopoietic cellhoming in an adult zebrafish model of hematopoietic cell transplantation. Blood118, 766-774.

González-Rosa, J. M., Martín, V., Peralta, M., Torres, M. and Mercader, N.(2011). Extensive scar formation and regression during heart regeneration aftercryoinjury in zebrafish. Development 138, 1663-1674.

Gupta, S. K., Lysko, P. G., Pillarisetti, K., Ohlstein, E. and Stadel, J. M. (1998).Chemokine receptors in human endothelial cells. Functional expression ofCXCR4 and its transcriptional regulation by inflammatory cytokines. J. Biol.Chem. 273, 4282-4287.

Gurskaya, N. G., Verkhusha, V. V., Shcheglov, A. S., Staroverov, D. B.,Chepurnykh, T. V., Fradkov, A. F., Lukyanov, S. and Lukyanov, K. A. (2006).Engineering of a monomeric green-to-red photoactivatable fluorescent proteininduced by blue light. Nat. Biotechnol. 24, 461-465.

Haas, P. and Gilmour, D. (2006). Chemokine signaling mediates self-organizingtissue migration in the zebrafish lateral line. Dev. Cell 10, 673-680.

Hatta, K., Tsujii, H. and Omura, T. (2006). Cell tracking using a photoconvertiblefluorescent protein. Nat. Protoc. 1, 960-967.

Honczarenko, M., Le, Y., Swierkowski, M., Ghiran, I., Glodek, A. M. andSilberstein, L. E. (2006). Human bone marrow stromal cells express a distinctset of biologically functional chemokine receptors. Stem Cells 24, 1030-1041.

Hu, X., Dai, S., Wu, W. J., Tan, W., Zhu, X., Mu, J., Guo, Y., Bolli, R. andRokosh, G. (2007). Stromal cell derived factor-1 alpha confers protectionagainst myocardial ischemia/reperfusion injury: role of the cardiac stromal cellderived factor-1 alpha CXCR4 axis. Circulation 116, 654-663.

Huang, C. J., Tu, C. T., Hsiao, C. D., Hsieh, F. J. and Tsai, H. J. (2003). Germ-linetransmission of a myocardium-specific GFP transgene reveals critical regulatoryelements in the cardiac myosin light chain 2 promoter of zebrafish. Dev. Dyn.228, 30-40.

Iovine, M. K. (2007). Conserved mechanisms regulate outgrowth in zebrafish fins.Nat. Chem. Biol. 3, 613-618.

Jopling, C., Sleep, E., Raya, M., Martí, M., Raya, A. and Izpisúa Belmonte, J.C. (2010). Zebrafish heart regeneration occurs by cardiomyocytededifferentiation and proliferation. Nature 464, 606-609.

Kawakami, K., Takeda, H., Kawakami, N., Kobayashi, M., Matsuda, N. andMishina, M. (2004). A transposon-mediated gene trap approach identifiesdevelopmentally regulated genes in zebrafish. Dev. Cell 7, 133-144.

Kawakami, Y., Rodríguez-León, J., Koth, C. M., Büscher, D., Itoh, T., Raya, A.,Ng, J. K., Esteban, C. R., Takahashi, S., Henrique, D. et al. (2003). MKP3mediates the cellular response to FGF8 signalling in the vertebrate limb. Nat. CellBiol. 5, 513-519.

Kawakami, Y., Rodriguez Esteban, C., Raya, M., Kawakami, H., Martí, M.,Dubova, I. and Izpisúa Belmonte, J. C. (2006). Wnt/beta-catenin signalingregulates vertebrate limb regeneration. Genes Dev. 20, 3232-3237.

Kawakami, Y., Marti, M., Kawakami, H., Itou, J., Quach, T., Johnson, A.,Sahara, S., O’Leary, D. D., Nakagawa, Y., Lewandoski, M. et al. (2011).Islet1-mediated activation of the -catenin pathway is necessary for hindlimbinitiation in mice. Development 138, 4465-4473.

Kikuchi, K., Holdway, J. E., Werdich, A. A., Anderson, R. M., Fang, Y.,Egnaczyk, G. F., Evans, T., Macrae, C. A., Stainier, D. Y. and Poss, K. D.(2010). Primary contribution to zebrafish heart regeneration by gata4(+)cardiomyocytes. Nature 464, 601-605.

Kikuchi, K., Gupta, V., Wang, J., Holdway, J. E., Wills, A. A., Fang, Y. andPoss, K. D. (2011a). tcf21+ epicardial cells adopt non-myocardial fates duringzebrafish heart development and regeneration. Development 138, 2895-2902.

Kikuchi, K., Holdway, J. E., Major, R. J., Blum, N., Dahn, R. D., Begemann, G.and Poss, K. D. (2011b). Retinoic acid production by endocardium andepicardium is an injury response essential for zebrafish heart regeneration. Dev.Cell 20, 397-404.

Kim, J., Wu, Q., Zhang, Y., Wiens, K. M., Huang, Y., Rubin, N., Shimada, H.,Handin, R. I., Chao, M. Y., Tuan, T. L. et al. (2010). PDGF signaling is requiredfor epicardial function and blood vessel formation in regenerating zebrafishhearts. Proc. Natl. Acad. Sci. USA 107, 17206-17210.

Knaut, H., Werz, C., Geisler, R., Tübingen 2000 Screen Consortium andNüsslein-Volhard, C. (2003). A zebrafish homologue of the chemokinereceptor Cxcr4 is a germ-cell guidance receptor. Nature 421, 279-282.

Knaut, H., Blader, P., Strähle, U. and Schier, A. F. (2005). Assembly of trigeminalsensory ganglia by chemokine signaling. Neuron 47, 653-666.

Laflamme, M. A. and Murry, C. E. (2011). Heart regeneration. Nature 473, 326-335.

Lepilina, A., Coon, A. N., Kikuchi, K., Holdway, J. E., Roberts, R. W., Burns, C.G. and Poss, K. D. (2006). A dynamic epicardial injury response supportsprogenitor cell activity during zebrafish heart regeneration. Cell 127, 607-619.

Lien, C. L., Schebesta, M., Makino, S., Weber, G. J. and Keating, M. T.(2006). Gene expression analysis of zebrafish heart regeneration. PLoS Biol. 4,e260.

Lukyanov, K. A., Chudakov, D. M., Lukyanov, S. and Verkhusha, V. V. (2005).Innovation: Photoactivatable fluorescent proteins. Nat. Rev. Mol. Cell Biol. 6,885-890.

Lund, T. C., Glass, T. J., Somani, A., Nair, S., Tolar, J., Nyquist, M., Patrinostro,X. and Blazar, B. R. (2012). Zebrafish stromal cells have endothelial propertiesand support hematopoietic cells. Exp. Hematol. 40, 61-70 e1.

Ma, Q., Jones, D., Borghesani, P. R., Segal, R. A., Nagasawa, T., Kishimoto,T., Bronson, R. T. and Springer, T. A. (1998). Impaired B-lymphopoiesis,myelopoiesis, and derailed cerebellar neuron migration in CXCR4- and SDF-1-deficient mice. Proc. Natl. Acad. Sci. USA 95, 9448-9453.

Mizoguchi, T., Verkade, H., Heath, J. K., Kuroiwa, A. and Kikuchi, Y. (2008).Sdf1/Cxcr4 signaling controls the dorsal migration of endodermal cells duringzebrafish gastrulation. Development 135, 2521-2529.

Mutoh, T., Miyata, T., Kashiwagi, S., Miyawaki, A. and Ogawa, M. (2006).Dynamic behavior of individual cells in developing organotypic brain slicesrevealed by the photoconvertable protein Kaede. Exp. Neurol. 200, 430-437.

Nair, S. and Schilling, T. F. (2008). Chemokine signaling controls endodermalmigration during zebrafish gastrulation. Science 322, 89-92.

Narumi, T., Hayashi, R., Tomita, K., Kobayashi, K., Tanahara, N., Ohno, H.,Naito, T., Kodama, E., Matsuoka, M., Oishi, S. et al. (2010). Synthesis andbiological evaluation of selective CXCR4 antagonists containing alkene dipeptideisosteres. Org. Biomol. Chem. 8, 616-621.

Nienhaus, G. U., Nienhaus, K., Hölzle, A., Ivanchenko, S., Renzi, F., Oswald,F., Wolff, M., Schmitt, F., Röcker, C., Vallone, B. et al. (2006).Photoconvertible fluorescent protein EosFP: biophysical properties and cellbiology applications. Photochem. Photobiol. 82, 351-358.

Orsini, M. J., Parent, J. L., Mundell, S. J., Marchese, A. and Benovic, J. L.(1999). Trafficking of the HIV coreceptor CXCR4. Role of arrestins andidentification of residues in the c-terminal tail that mediate receptorinternalization. J. Biol. Chem. 274, 31076-31086.

Pan, Y. A., Caron, S. J. and Schier, A. F. (2012). BAPTI and BAPTISM Birthdatingof Neurons in Zebrafish. Cold Spring Harb. Protoc. 2012, 87-92.

Patterson, G. H. and Lippincott-Schwartz, J. (2002). A photoactivatable GFP forselective photolabeling of proteins and cells. Science 297, 1873-1877.

Perner, B., Englert, C. and Bollig, F. (2007). The Wilms tumor genes wt1a andwt1b control different steps during formation of the zebrafish pronephros. Dev.Biol. 309, 87-96.

Pisharath, H., Rhee, J. M., Swanson, M. A., Leach, S. D. and Parsons, M. J.(2007). Targeted ablation of beta cells in the embryonic zebrafish pancreas usingE. coli nitroreductase. Mech. Dev. 124, 218-229.

Porrello, E. R., Mahmoud, A. I., Simpson, E., Hill, J. A., Richardson, J. A.,Olson, E. N. and Sadek, H. A. (2011). Transient regenerative potential of theneonatal mouse heart. Science 331, 1078-1080.

Poss, K. D. (2007). Getting to the heart of regeneration in zebrafish. Semin. CellDev. Biol. 18, 36-45.

Poss, K. D. (2010). Advances in understanding tissue regenerative capacity andmechanisms in animals. Nat. Rev. Genet. 11, 710-722.

Poss, K. D., Wilson, L. G. and Keating, M. T. (2002). Heart regeneration inzebrafish. Science 298, 2188-2190.

Raya, A., Koth, C. M., Büscher, D., Kawakami, Y., Itoh, T., Raya, R. M.,Sternik, G., Tsai, H. J., Rodríguez-Esteban, C. and Izpisúa-Belmonte, J. C.

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DEVELO

PMENT

4142

(2003). Activation of Notch signaling pathway precedes heart regeneration inzebrafish. Proc. Natl. Acad. Sci. USA 100 Suppl. 1, 11889-11895.

Raya, A., Consiglio, A., Kawakami, Y., Rodriguez-Esteban, C. and Izpisúa-Belmonte, J. C. (2004). The zebrafish as a model of heart regeneration. CloningStem Cells 6, 345-351.

Raz, E. (2003). Primordial germ-cell development: the zebrafish perspective. Nat.Rev. Genet. 4, 690-700.

Raz, E. and Mahabaleshwar, H. (2009). Chemokine signaling in embryonic cellmigration: a fisheye view. Development 136, 1223-1229.

Saxena, A., Fish, J. E., White, M. D., Yu, S., Smyth, J. W., Shaw, R. M.,DiMaio, J. M. and Srivastava, D. (2008). Stromal cell-derived factor-1alpha iscardioprotective after myocardial infarction. Circulation 117, 2224-2231.

Schier, A. F. (2003). Chemokine signaling: rules of attraction. Curr. Biol. 13, R192-R194.

Schnabel, K., Wu, C. C., Kurth, T. and Weidinger, G. (2011). Regeneration ofcryoinjury induced necrotic heart lesions in zebrafish is associated with epicardialactivation and cardiomyocyte proliferation. PLoS ONE 6, e18503.

Siekmann, A. F., Standley, C., Fogarty, K. E., Wolfe, S. A. and Lawson, N. D.(2009). Chemokine signaling guides regional patterning of the first embryonicartery. Genes Dev. 23, 2272-2277.

Sleep, E., Boué, S., Jopling, C., Raya, M., Raya, A. and Izpisua Belmonte, J. C.(2010). Transcriptomics approach to investigate zebrafish heart regeneration. J.Cardiovasc. Med. 11, 369-380.

Stark, D. A. and Kulesa, P. M. (2007). An in vivo comparison of photoactivatablefluorescent proteins in an avian embryo model. Dev. Dyn. 236, 1583-1594.

Tachibana, K., Hirota, S., Iizasa, H., Yoshida, H., Kawabata, K., Kataoka, Y.,Kitamura, Y., Matsushima, K., Yoshida, N., Nishikawa, S. et al. (1998). Thechemokine receptor CXCR4 is essential for vascularization of the gastrointestinaltract. Nature 393, 591-594.

Takahashi, M. (2010). Role of the SDF-1/CXCR4 system in myocardial infarction.Circ. J. 74, 418-423.

Tanaka, E. M. and Reddien, P. W. (2011). The cellular basis for animalregeneration. Dev. Cell 21, 172-185.

Tomura, M., Yoshida, N., Tanaka, J., Karasawa, S., Miwa, Y., Miyawaki, A.and Kanagawa, O. (2008). Monitoring cellular movement in vivo with

photoconvertible fluorescence protein “Kaede” transgenic mice. Proc. Natl.Acad. Sci. USA 105, 10871-10876.

Tomura, M., Honda, T., Tanizaki, H., Otsuka, A., Egawa, G., Tokura, Y.,Waldmann, H., Hori, S., Cyster, J. G., Watanabe, T. et al. (2010). Activatedregulatory T cells are the major T cell type emigrating from the skin during acutaneous immune response in mice. J. Clin. Invest. 120, 883-893.

Tsutsui, H., Karasawa, S., Shimizu, H., Nukina, N. and Miyawaki, A. (2005).Semi-rational engineering of a coral fluorescent protein into an efficienthighlighter. EMBO Rep. 6, 233-238.

Urasaki, A., Morvan, G. and Kawakami, K. (2006). Functional dissection of theTol2 transposable element identified the minimal cis-sequence and a highlyrepetitive sequence in the subterminal region essential for transposition.Genetics 174, 639-649.

Verkhusha, V. V. and Sorkin, A. (2005). Conversion of the monomeric redfluorescent protein into a photoactivatable probe. Chem. Biol. 12, 279-285.

Volin, M. V., Joseph, L., Shockley, M. S. and Davies, P. F. (1998). Chemokinereceptor CXCR4 expression in endothelium. Biochem. Biophys. Res. Commun.242, 46-53.

Wang, J., Panáková, D., Kikuchi, K., Holdway, J. E., Gemberling, M., Burris,J. S., Singh, S. P., Dickson, A. L., Lin, Y. F., Sabeh, M. K. et al. (2011). Theregenerative capacity of zebrafish reverses cardiac failure caused by geneticcardiomyocyte depletion. Development 138, 3421-3430.

Wojakowski, W., Tendera, M., Kucia, M., Zuba-Surma, E., Milewski, K.,Wallace-Bradley, D., Kazmierski, M., Buszman, P., Hrycek, E., Cybulski, W.et al. (2010). Cardiomyocyte differentiation of bone marrow-derived Oct-4+CXCR4+SSEA-1+ very small embryonic-like stem cells. Int. J. Oncol. 37, 237-247.

Yamaguchi, J., Kusano, K. F., Masuo, O., Kawamoto, A., Silver, M.,Murasawa, S., Bosch-Marce, M., Masuda, H., Losordo, D. W., Isner, J. M.et al. (2003). Stromal cell-derived factor-1 effects on ex vivo expandedendothelial progenitor cell recruitment for ischemic neovascularization.Circulation 107, 1322-1328.

Zou, Y. R., Kottmann, A. H., Kuroda, M., Taniuchi, I. and Littman, D. R.(1998). Function of the chemokine receptor CXCR4 in haematopoiesis and incerebellar development. Nature 393, 595-599.

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