[temporary] major contribution of cardiac regeneration through gata41 heart muscle zebra Fish

Recent studies have shown that mammals, including humans, main-some of the ability to renew myocardial cells throughout the postpartum life1,2. However, there is little or no significant myocardial damage after regenera-ash such as acute myocardial infarction3. By contrast, zebra fish efficiently regenerates lost myocardium, providing a model for understanding how natural heart regenera-may be clogged or enhanced4,5. The cell origin of the new regenerative myocardium is still unclear in the absence of a tracking technique suitable for adult zebrafish lineage. Using the new genetic fate mapping method, here we determine the population of cardiac muscle cells that are activated and prominent after resection of the ventricles of the heart to become active and protruding. Through the use of genetically modified strains reported, we found that cardiac myocytes in the entire week of trauma embry-onic heart gene GATA4 of the outer membrane ventricular layer trigger expression, the anterior expression is located in the peripheral proliferative myocardium cells and injury site. CRE-recombinant enzyme-based pedigree-tracing cells express GATA4 significantly before the regeneration, or cell-expressed gene contraction CMLC2 injured before the subsequent regeneration of each dozen into most of the myocardium. Through the optical voltage mapping of the myocardium on the entire ventricular surface, we found that the electrical conductivity was re-established between the existing and regenerated cardiomyocytes between 2 and 4 weeks after the injury. After injury and prolonged fibroblast growth factor receptor inhibition arrests the heart regeneration, so that the scar formation, the experimental release of the signal block caused GATA4 expression and injury vent-ricular wall morphology improvement, no scar tissue loss. Our results show that the regeneration after electrical-coupled myocardial resection is mainly by activating and amplifying the cardiomyo-cyte population. These findings affect the regeneration of the heart of the injured person.
after removing the apex of their heart ventricles, the zebra fish replace the excision myocardium4,5. Although these events involve the proliferation of cardiovascular muscle cells, it is unclear whether proliferating cardiomyo-nuclei are derived from existing muscle cells or from non-cardiac cell sources such as stem cells. The contribution of determining embryonic or regenerative organ cells is also usually involved in the genetic mode of irreversible labeling of different cell types and tracking their progeny6-11.
while looking for the regeneration of molecular marker information, we identified GATA4 's upstream regulation of the conservative sequence-driven unique expression of a transcription factor gene in the development of embryonic heart expression and the normal heart pattern part of ING and vascularization12-17 essential. Through the use of TG (GATA4:EGFP) AE1 reporter Line18, we found that the fluorescence of green fluorescent protein (EGFP) in the non-injured adult ventricle basically does not exist. However, after resection of the heart of the ventricles, GATA4 drives the EGFP during the transfer period from 3-7 days after amputation to induce a high percentage of the outer dense layer of the cell's entire ventricular myocardium (DPA) (Figure 1a,d, f,g). GATA4:EGFP expressed in many cells and lesions around the site by 2 weeks of injury (figure 1H), and at all points in time is limited to the cell-positive muscle cell markers (Figure 1b, c) and negative markers epicardial, a vas-culogenic cortical covering in compact muscles (Fig. 1e). A study of-labelling (5-bromo-bromide) showed that many gata4:egfp1 cells were at the wound at 7 DPA, and that the DNA synthesis (Figure 1j,k) had recently occurred on the lateral edge of the wound at the site of DPA. In 30 DPA, most areas of the reclaimed chamber wall are still labeled GATA4:EGFP fluorescence (Figure 1I). At these stages, GATA4:EGFP expresses the main marker of the compact cardiomyo-nucleus cells, which usually occupy a larger portion of regeneration than Nechoran cardiomyocytes4. Fluorescent vent-ricular muscle regeneration, at different stages of histology, quantitatively represents the expression of GATA4-driven EGFP, which is progressively introduced from the edge of the wound to the lesion site (supplementary figure 2a,b).

Fig. 1 | Myocardial markers GATA4:EGFP are activated, proliferate, at the site of injury. One, GATA4:EGFP induces cells all around in the compact muscle 7 DPA (right). Amplifiers. Amputation. B,C,GATA4:EGFP (in C-arrows) co-tagged with cardiac markers MEF2 (b) and myosin heavy Chain (MHC) (c). Two-dapi,49,6-phenyl-indole. D,GATA4:EGFP (arrows) are co-stained with B-catenin, which indicates a EGFP limitation in the myocardial wall. E,GATA4:EGFP does not co-locate a 3x vehicle, D.p.a.dapi EGFPB 3x vehicle, the D.P.A. with epicardial RALDH2 protein (arrow). F-Ⅰ,GATA4:EGFP expression (arrow) is not injured and regenerates the ventricles. The dashed line represents the cut approximation plane. J,k,brdu Markers and 7 (j) and 14 immunofluorescence (K) of the DPA GATA4:EGFP ventricle. Arrows indicate a common label. Single-focus slices are shown in b-e and illustrations in J and K. The 7-or 8-mm Z-stack confocal protrusions are shown in J and K. Scale 50 mm. 　

Figure 2| GATA41 myocardial cells contribute significantly to heart regeneration. One, Gata4:ercreer; B-ACT2:RSG Animals in 9 (center; N5) Injections once a day the medium (left) or the serotonin in 5-7 DPA, collected before or 14 (right; N5 6) DPA EGFP fluorescence (arrow) at the injured boundary 9 DPA observation and damage site D.p.a. The dotted line indicates
Cut the approximate plane. B,CMLC2: Rose Cooper Cui; B-ACT2:RSG animal Vehicles (left) or 4-ht are injected once a day for 3 days before being injured. The 4-ht Ventricular (center) and the most recovered myocardium (right; N510). Illustrations, the red fluorescent protein channel is used for the calculation of marker efficiency. Displays a single confocal slice. Scale 50 mm.

To clarify the dynamics of these events, we have produced new genetically modified strains to promote induction, CRE-recombinant enzymes-based pedigree tracking from gata41 cells. We created a line pd39 with tamoxifen--induced CRE recombinant estrogen receptor fusion by GATA4 modulating sequence, TG (gata4:ercreer) driving protein, and an indicator line resection, The EGFP fluorescence of the permissible cardiac myocytes is visible loxp the flanking stop sequence, TG (BACTIN2:LOXP red fluorescent protein-STOP-LOXP EGFP) S928 (B-ACT2:RSG; supplement Figure 3). We inject once a day, 4-hydroxy (4-HT) or vehicles into the gata4:ercreer; B-act2:5-7 DPA, an RSG animal that detects GATA4-driven EGFP fluorescence at the site of the lesion before the time. Injection of GATA4: Ercreer; B-ACT2:RSG animal 4-ht, but not a car, playing into what presumably represents the muscle cells, a subset of the fluorescence emitted in the GATA4: the EGFP line, exposing a small portion of the EGFP1 myocardium bordering the wound 9 dpa Additionally, the EGFP1 myocardial continuous area can be located at the lesion site 14 DPA detection (FIG. 2a), represents a significant expansion of 20D.PA in the labeled Myocardium quanti-fiably (Supplementary figure 2a,c). These results indicate a mechanism in which epicardial cells in the entire ventricular damage response are induced by induction of GATA4 expression, and the proportion of cells adjacent to the injured site prolif-to show business and promote new cardiac myocytes is high.
Although confocal imaging co-locates the GATA4 to drive EGFP and muscle markers, it is still possible that non-muscular cells induce GATA4 damage after rapid differentiation into proliferative car-diomyocytes. In order to test the extent to which existing cardiomyocytes are regenerated, we have created a strain in which tamoxifen-induces cre by the contraction of the myosin light chain 2 (CMLC2, also known as MYL7) of the TG (CMLC2 regulated sequence drive: Rose Percy) PD10. The measurement of labeling efficiency shows that our 4-HT injection protocol mark, 95% not injured CMLC2: Rose Cooper Cui; B-ACT2:RSG ventricular myocytes and EGFP fluorescence (supplement Figure 4). On the basis of several different indicator line analyses, the CMLC2 drive through the rose crest is specific to cardiac myocytes (supplement Figure 5), and there is no protected research institution by the injury or the vehicle injected into the gating (Figure 2b). Tagged myocardium after five days, we remove the ventricular apex and allow regeneration for 30 days. We found no significant difference in the ratio of myocardial regeneration cmlc2 in EGFP1: Rose Cooper Cui; B-ACT2:RSG tissue compared to 5 or 35 days after injection to collect the non-injured ventricle, the results showed that the majority of new cardiomyo-nuclear cells from the cell expression injury (Figure 2b and 4) before the derivation of CMLC2.

A key aspect of successful regeneration is the newly created cell to the functionality of the existing organization in-company. We mark the whole heart with transmembrane potential-sensitive dye two -4-anepps, and carry out surface flat myocardium including the dense layer and the regenerated optical voltage mapping. In 7 DPA, and less so in the DPA, there is an increased density of the equal-time lines near the apex of the ventricles compared to an unhurt control, indicating a significant slowdown in conduction (Figure 3a). In addition, electrical activity has never been propagated to the regenerated vertex 7 DPA, while the impulse is repeated throughout the ventricles of the ventricle when a large number of new Gata41 myocardium cells appear (Figure 3a). The density of the equal-time line appears normal at the vertex (Figure 3a). These observations directly estimate the conduction velocity, which reveals a slowdown in the distal ventricular conduction at 7 and DPA, while the normal conduction is confirmed in the DPA (Figure 3b). We have also found that at 7 DPA, it is entirely up to the DPA to recover a significantly reduced maximum rate of depolarization evidence, while the action potential duration is the same in each group (figure 3c and supplement Figure 6). Therefore, our imaging data indicate that the electrical coupling of the new apical myocardium begins to occur by, after 2 weeks of injury, with complete coupling in the recovered wall of DPA
the normal regenerative response of a zebra fish's heart injury is considered to be blocking or exceeding the competition for secondary scar Response4. In order to test whether a regenerated scar can occur after a build, we are injured by TG (HSP70:DN-FGFR1) PD1 ventricles and induce expression of dominant negative fibroblast growth factor (FGF) by heat shock receptor for 30 days, resulting in regenerative arrest and scarring19 (Figure 4a,b). Then we enable the FGF signal by removing the heat shock for 14 days or 60 days. Interestingly, 73% of the wounds in the 14-day signaling of the restored FGF increased myocardium, with heat shock protein 70 (supplementary Figure 7): DN-FGFR1; GATA4: Contains GATA4 field EGFP vertex: EGFP1 myocardium (Figure 4c,d). When we restore the FGF signal to the scar heart for a 60-day extension, fibrin cleared from the wound, although we did not observe the loss of scar tissue. It is noteworthy that 90% of the lesions showed histological improvements in the FGF signal after extended recovery, including 60% of the continuous wall-encapsulated scar (figure 4e-h) that has formed muscle. These results suggest that the regenerative signal is maintained in the zebra fish heart with the established damage scar, which in mammals presents an environment for the cell-based therapy of a barrier to myocardial infarction20,21. Therefore, the mechanism behind the regeneration of the zebra fish heart may be associated with myocardial infarction in which the survivors have mature scar tissue and damaged ventricu-lar walls.

Figure 3 | Electrical coupling of regenerated cardiac myocytes. One, the surface of the myocardium adjacent to the unimpaired, 7,14 and the apex of the DPA plant two milliseconds, such as the density map ventricle. Scale 100 mm.
B, the average rate of conduction measured from local velocity vectors indicates that at 7 and DPA speeds are slowed by an average of 6 sem, N5 months from 4th to 7th in the ventricles of the ventricle at each point in time. * P,0.05 (Unidirectional variance analysis (ANOVA)). C, the surface action potential represents the trace, indicating the maximum depolarization rate at 7 DPA slows.

Early studies were evaluated for rapid (EGFP) and slow (nuclear DsRed2) of double-genetically modified TG (CMLC2:EGFP) Folding reporters F1 or twu26; TG (cmlc2:nucdsred2) F2 zebra fish to record evidence of the proliferation of fresh mature cmlc22 progenitor cells into cardiomyo-nucleus cells. Specifically, many of the EGFP1 nucDsRed22 myocardium cells are observed in the developing embryonic heart and in regenerating adult ventricle19,22. We re-examined the cytoplasm of the stituting CMLC2 DsRed2 reporter, removing the cell nucleus localization may reduce the sensitivity of dynamic developmental events to the reporter's element of this development timing analysis (TG (CMLC2: DsRed2) PD15). We observed the embryos in the EGFP1 DsRed22 muscle cells, but were not regenerated in 7 DPA adults, which instead contained EGFP1 DsRed21 muscle cells with each cytoplasmic journalist fluorescence at a lower intensity than in non-regenerative muscles (supplement Figure 8). Plus CMLC2: trace data from the Emerald lineage, these new results argue for a revision of the previous interpretation and indicate that participating in myocardial regeneration possesses or obtains an immature phenotype, reducing CMLC2 expression. This phenotype is observed by myocardial cells in the ultrastructural features of the DPA may reflect regeneration, we did not tricles in the epicardial under the injury of the myocardium cells, including the reduction of muscle structure, irregular Line granule dria, low mitochondrial density (supplement Figure 9).
In short, we have identified a new mechanical aspect of the zeb-rafish heart regeneration that has a close relationship with the origin, function and tolerance of myocardial regeneration. Most importantly, we found that the ventricular wall of the cardiomyocytes Ecotene the subgroup Vates GATA4 regulate the sequence, proliferate, and promote local muscle regeneration of the stantially. The degree to which other cell populations may provide regeneration is subject to further direct linear eage tracking experiments. It is noteworthy that the activation of GATA4: EGFP expressed in the outer membrane myocardium parallel to the epicardial cells in the rapid, cavity-wide damage reaction before they were also included in the regeneration Area19. These muscularizing and vascularization tis-of the similar spa-matching tiotemporal dynamically show important interactions as each of them is activated, multiplied, and integrated into the lesion site.

Figure 4| Recovery GATA4: EGFP expression and a new ventricular wall scar after. A, A, 30 days (D) FGF receptor (FGFR) inhibition after a typical wound resection. HS, heat shock. C,D,GATA4: Induced injury 30 days FGFR inhibition, and 14 days of restoration at room temperature for a regimen of EGFP fluorescence (arrow) (RT; N58). The broken line represents the wound. E,f, normal regeneration (in parentheses) after injury and incubation at room temperature for 60 days. G,h,60 Day room temperature recovery, excision of 30 days after FGFR inhibition, improved wall anatomy (in parentheses) does not remove scars (arrows; N510). Green, egfp-labeled DNFGFR1; through CMLC2 red, muscle cell nuclei: nucDsRed2 in transgenic
A,e and G. Acid Magenta orange g staining (red, serum fibrin; blue, collagen) b,f and H. Animal heat Shock Heart was collected 4 hours before, in E and G, inducing EGFP. Scale 50 mm.

Our findings are intriguing given that the described factors have recently been reported to be introduced to increase the proliferation of differen-tiated cardiomyocytes and improve the function of the injured adult mammal heart23-26. It is possible that the zebra fish heart provides an environment that encourages the optimization of the activation and/or the multiplication of the subgroups of the cardiomyocytes. The CRE-based tools in zebrafish, including those we describe here, can allow precise experiments to manipulate gene expression or function in an attempt to modify a damaged environment or regenerative reaction. With the knowledge of the key sources of new cardiac myocytes, the regulation of the interaction of the cells and molecules mediated by the heart regenera-ash in zebrafish can now be pursued more informatively.

Method Summary

far cross Ekkwill (EK) or ek/ab mixed background zebra fish 6-12 months baby was?? Used in the previously4 described in the ventricular resection procedure. All the genetically modified strains were analyzed and the details of their structure were described in the method. The animal density remained at 4 fish L21 in all experiments. 4-Hydroxy (4-HT) (Sigma Company) soluble in ethanol (5MGML21) is diluted in water to 0.5mgml21 for intra-peritoneal injection. 10% of ethanol is used as a vector control. The quantification of the EGFP tag is described in the method. Heat shock experiment was performed to describe Previously27, using double transgenic hsp70:dnfgfr1; Cmlc2:nucdsred2 or HSP70:DNFGFR1; GATA4:EGFP animals. For BrdU, 2.5 mg of ML21 (Sigma Company) Intraperitoneal injection, 3 days before collection per day. Immunofluorescence, in situ hybridization, and acid magenta orange g stains (detected blood fibrin and collagen) are described previously4. One of the antibodies used in this study was: Anti-MEF2 (rabbit; Santa Cruz Biotechnology Co., Ltd., anti-myosin heavy chain (F59, mouse; Development Studies Hybridoma Library), anti-beta protein (rabbit; Sigma Company), anti-ZF RALDH2 (rabbit: Abmart), anti--brdu (rat; accurate), and anti-GFP (rabbit, only used for co-detection BrdU use; Invitrogen). In this study used in two antibody (Invitrogen) respectively will Alexa Fluor 594 goat anti-Rabbit IgG (h1l) anti-MEF2, will Alexa Fluor 594 Goat Anti-mouse IgG (h1l) for F59, will Alexa Fluor 594 Goat Anti-rat IgG (h1l) for anti-BrdU, and Alexa Fluor 488 goat anti-Rabbit IgG (h1l), for anti-GFP. In situ hybridization and immunofluorescence images are photographed using the Leica DM6000 microscope with a Retiga-exi camera (c-imaging) and confocal images using the Leica SP2 or SP5 confocal microscope. Physiological methods are described in the method.
all methods and any related referenced files are in the Www.nature.com/nature online version.

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Thanks to J. Burris and a. Eastes Zebra fish Care, X. Meng and Development Studies hybridoma Cell Bank antibody, M. Gignac's help, electron microscope, lab members ' comments on manuscripts, and G. Burns P. Shambon and G. The plasmid of the Felsenfeld. This work is supported by the American Heart Association (KK and YF), JDRF (RMA) and JSP (KK) to support the postdoctoral fellowship; NIH training grants HL007208 at the Massachusetts General Hospital (AAW) and HL007101 at the Duke University Medical Center (GFE); from Nhlbi (HL064282 to te,hl054737 to dyrs, and HL081674 to KDP), Nigms (GM075846 to Cam), March corner Money (CAM) grant, funded by the AHA, the Pew Charitable Trust and the Whitehead Foundation (KDP).
Authors and K.D.P. Design experimental strategies, analyze data, and prepare manuscripts. J.e.h Co., Ltd. and Y.F. Generated and characterized as pedigree tracking of genetically modified strains. R.m.a. and D.y.r.s provide undisclosed reagent lineage tracking. J.e.h Co., Ltd. and K.D.P for regeneration experiments. J.e.h. Conducting electron microscopy. A.A.W.,G.F.E and C.A.M. Design physiology experiments and interpreting data. A.A.W. Conducting optical mapping experiments. T.E. Help design strategies and provide key reagents. All authors comment on the manuscript.
For information about the author's reprints and permissions, please visit www.nature.com/reprints. The author claims that there is no financial interest in competition. The letter and required materials should be given to KDP ([email protected]).

Method

construction GATA4: ercreer transgenic animals. ert2-Cre-ert2 Complementary Pcag-ert2creert2 DNA (see 28) clones of the 14.8 thousand base (KB) GATA4 promoter18 downstream. The entire structure is genetically modified using a Web site with a wide range of nuclease method29 i-scei on both sides. The full name of this transgenic line is TG (gata4:ercreer) pd39.
construction CMLC2: Rose Cooper Green Genetically modified animals. The pcre-ert2 of the Cre-ert2 cDNA (see 30) clones the downstream of the CMLC2 Promoter31 of 5.1 KB. A dsred-embodiment cartridge is controlled by lens-specific crystal, α-promoter is also included (CRYAA: Red fluorescent protein), so that by the lens fluorescence32 visual identification of genetically modified animals, and in the upstream sub-clone CMLC2: Rose Percy sequence in the opposite direction. The entire building flank is with the chicken B-bead protein insulator (23-core insulator Element) (by G. Two copies of the core components of the Felsenfeld, and then to the I-SCEI site side. The full name of this GM line is TG (CMLC2: Rose Percy) PD10.
Construction of B-act2: RSG transgenic animals. We sub-clone 9.8 kb of the genotype of the microphone DNA into the modified Pbsk vector immediately after the bactin2 transcription of the starting site of the upstream Polyclonal site by the Ⅰ-sceⅰ restriction enzyme cut-off site side. A loxp red fluorescent protein-STOP-LOXP a EGFP box of sub-clones of bactin2 downstream. The Red fluorescent protein-stop box is used as a marker for transgene, and also prevents EGFP protein from being read through translation. This build was injected into a wild-type embryo using a standard wide-range nuclease transgenic techniques29 single-cell phase. A founder separates and propa-the gate from the injected embryo. The full name of this transgenic line is TG (BACTIN2:LOXP red fluorescent protein-STOP-LOXP EGFP) S928.
Construction of 5-KB b-act2:rng and hsp70:rng transgenic animals. The pbigt carrier is used to enable the inclusion of both sides of the loxp mcherry box with transcription termination of a box, a EGFP box upstream with a labeled nuclear positioning signal three parts (NLS-EGFP) of the second LOXP sequence downstream. The resulting box (Loxp of the Mcherry-stop-loxp NUCEGFP) solution, then the sub-clones downstream, 10-kb the zebra fish bactin2 promoter, or one, 1.5-kb of the hsp70 promoter of the zebra fish, 5.3-kb the fragment. The two copies of the building body flank the chicken globulin insulator (23-core insulator Element) (by G. Felsenfeld), as well as two i-scei-bit sites. The full name of the transgenic strains is TG (Bactin2:loxp MCHERRY-STOP-LOXP-NUCEGFP) PD31 and TG (hsp70l:loxp mcherry-stop-loxp NUCEGFP) PD30. Construction GATA5: rng transgenic animals. GATA5 in the BAC clone dkeyp-73a2 is replaced with a loxp mcherry-stop-loxp NUCEGFP (RNG) by the Red/et recombination Engineering (genebridges) box. In 59 and 39 homologous arms for recombination is a 716 base pair (BP) fragment of the starting codon upstream, while a 517-bp fragment downstream, respectively, and sub-clones to the flank of the Rng box. To avoid potential loss of recombination between the rng box and the BAC vector of the internal source loxp sequence, we replace the vector-derived loxp sequence with the I-SCEI site using the same technique. The final BAC was refined by the Nucleobond BAC 100 kit (Clontech Company), and co-injected with a large-scale nuclease into a single-cell stage of zebra-fish embryos. The full name of this transgenic line is TG (Gata5:loxp's mcherry-stop-loxp NUCEGFP) PD40.
Construction CMLC2: The DsRed2 transgenic animal. The DsRed2 cDNA clones the 5.1-kb cmlc2 the back of the promoter, while the entire box flanks with the I-SCEI site. The full name of this transgenic line is TG (cmlc2: DsRed2) PD15.
4-ht Labeling and EGFP fluorescence CMLC2 quantification: Rose Cooper Cui; B-ACT2:RSG animals. In order to determine the dynamics of the 4-HT tag, we gave a single 20 ml low dose of 4-ht to not be injured CMLC2 (2.5 mg ML21) Intraperitoneal injection: Rose Cooper Cui; B-ACT2:RSG Adult. We found that recombinant egfp fluorescence of the muscle cells in the ventricular portion after 1 days of injection (DPI), and in the increased frequency (, 20% of the ventricular myocytes) and intensity by 2 dpi after injection 3, 4 and 5 days to examine the ventricular portion of the EGFP labeled frequency similar to the 2 dpi of these findings provide evidence that , Intraperitoneal injection of 4-HT can no longer post 2 dpi stimulation of the new recom-bination event in adult zebrafish ventricular myocardium.

in order to quantify the pedigree of EGFP expression and the CMLC2 experiment, the release of CRE recombinant enzyme mediated: Rose Cooper Cui; B-ACT2:RSG animals, three segments including regenerating each heart are selected. For the unhurt control heart, the largest three sections are selected from each heart description previously27. We use the DsRed1 area to reflect the total myocardial portion of the surface area, which is followed by 4-ht injection in the b-act2 of the expression background of the red fluorescent protein sticking to the myocardium: RSG transgenic fish. This may be due to a variety of genetically modified copies, allowing the escape of some side mounted loxp from CRE-mediated recombina-ash (Supplement Figure 2) Red fluorescent protein box. The single-ray image of the ventricular apex adjusts the gain to detect EGFP or red fluorescent protein signals above the background level at 340. (1024 3 1024 pixels). In the area of the injured vertex (504 3 504 pixels) is selected to include most of the regeneration in the image, and
crop using Photoshop software. The vent-ricular vertices of the same area are cropped for the animals that are not injured. The EGFP1 and DsRed1 regions are quantified in the ImageJ software pixels passed, and the percentage of EGFP1 relative DsRed1 area is calculated.
EGFP and GATA4:EGFP fluorescence GATA4 quantification ercreer; B-ACT2:RSG animals. In order to quantify the EGFP1 area, 3 parts, including wound/regeneration, are selected from each heart. Ventricular apex?? The image is used with the Leica DM6000 microscope with a Retiga-exi camera (c-imaging) taken at 320. An area (109 3 109 pixels GATA4:EGFP Animals and 73 3 73 pixels gata4:ercreer; B-ACT2:RSG animals) are designated at each side cut wound edge and trimmed with Photoshop software (see Supplement Figure 2. Cartoons). An area (109 3 218 pixels GATA4:EGFP Animals and 73 3 146 pixels gata4:ercreer; B-ACT2:RSG animals) and transverse areas are specified and clipped at the site of injury. The EGFP1 area is quantified in the ImageJ software pixels passed, and the number of pixels is summed up and averaged, resulting in a EGFP1 area of the edge and a damaged site EGFP1 area for each heart.
transmission electron microscope. The wounded heart was fixed in 2.5% of glutamic acid Raldehyde, in 0.1M of sodium gluconate buffer 2% of formaldehyde. The tissue is then fixed at 1% Oso 4, dehydrated, covered in spurr resin (EMS), and sliced using ultracut or ultracut e ultra thin Slicer (Leica) 70-90 nm thickness. The slices are made of acetic acid oxy-uranium and lead citrate. The microscope was performed in the FEI Tecnai G2 dual transmission electron microscope, and the image was obtained using Hamamatsu ORCA-HR's digital camera and AMT software. Light mapping. The fish was anesthetized with 0.1% of 3-amino-benzoic acid methyl ester, and the heart was isolated within 30 seconds, and placed in the containing Tyrode solution (in mm) NA1 and (136), K 1 (5.4), MG21 (1.0), PO432 (0.3), CA21 (1.8), glucose (5.0) and Hepes (10.0), at ph 7.4. The heart is loaded for 10 minutes and is dissolved in a tyrode solution with transmembrane potential-sensitive dye two -4-anepps (D-1199,invitrogen). After staining, the preparation is transferred to the recording studio (Warner Instrument), which contains a 30 mm (EMD chemical company) containing an excited contraction separator type II myosin in order to suppress the shrinkage of the Tyrode solution. The chamber is mounted in an inverted microscope (TE-2000U, Nikon) equipped with a high speed CCD camera (ccd-heart SMQ, red shirt imaging) with 80 3 80 pixel frame stage. Use the tiny robot in place near the base of the heart for a fine platinum electrode heart on point-stimulation 60 times per minute. The electrode position eliminates the illusion of increasing the conduction speed due to multiple pixels adjacent to the stimulation site while local capture (i.e., virtual electrode effect) 33. The optical action potential is recorded from the epicardial surface of the vertex. Fluorescence is excited to have 120 watts of metal Halide arc lamps (X-lift 122,EXFO) and filtration at 540 6 25 nm. Fluorescence emission is passed through a long-pass emission filter (585 nm) before being focused on the camera. An optical magnification of 32 results in a spatial resolution of 11 mm between the recorded pixels. The camera operates at 2000 frames per second and the signal is digitized to 14-bit accuracy. The signal is digitized at a time (900 khz cutoff), filtered and spatially (weighted average of 4 pixels) in the domain to reduce noise. The time process of recording the change of membrane potential with the action potential of the system is comparable to that of Fidelity recording a patch clamp TECHNIQUE34 action potential.
Optical mapping fluorescence data analysis. The use of fluorescence data obtained from the preparation of custom software for optical action potential analysis from the zebra Fish Heart (matlab,mathworks company) was analyzed. The action potential duration (APD) is defined as 20% depolarization and 80% of the yield or the time interval between the repolarization to the resting potential (APD80). The rise rate of action potentials is the maximum value of the first derivative of the action potential. The activation time is defined as the time of the ascent phase of the action potential in the 50% depolarization process. We have used this standard for local activity because the depolarization of time at half the maximum has been previously indicated in computer simulations close to the maximum cellular sodium influx35 counterpart. The map shown in front of the wave at a constant time interval (2 milliseconds) is composed of this gray fold from the activation of the function provided by MATLAB software using contour drawing. The conduction velocity vector field is used to estimate the activation time of the established algorithm previously36. In short, the local velocity vector, which represents the size and direction of the propagated depolarization wave at each record point, calculates the depolarization time measured by the embedding at each site with a parabolic two-dimensional surface. At each site, the component of the velocity vector calculates the component from the gradient on that surface. The conduction velocity crosses at a site that is located close to each heart vertex (supplement Fig. 10) 200 3 200 square millimeters on average.

[temporary] major contribution of cardiac regeneration through gata41 heart muscle zebra Fish