Chronic obstructive pulmonary disease (COPD) is a debilitating lung disease that is currently the third leading cause of death worldwide. Recent reports have indicated that dysfunctional iron handling in the lungs of COPD patients may be one contributing factor. However, a number of these studies have been limited to the qualitative assessment of iron levels through histochemical staining or to the expression levels of iron-carrier proteins in cells or bronchoalveolar lavage fluid. In this study, we have used time of flight secondary ion mass spectrometry (ToF-SIMS) to visualize and relatively quantify iron accumulation in lung tissue sections of healthy donors versus severe COPD patients. An IONTOF 5 instrument was used to perform the analysis, and further multivariate analysis was used to analyze the data. An orthogonal partial least squares discriminant analysis (OPLS-DA) score plot revealed good separation between the two groups. This separation was primarily attributed to differences in iron content, as well as differences in other chemical signals possibly associated with lipid species. Further, relative quantitative analysis revealed twelve times higher iron levels in lung tissue sections of COPD patients when compared to healthy donors. In addition, iron accumulation observed within the cells was heterogeneously distributed, indicating cellular compartmentalization. With increasing global burden, COPD is currently the third leading cause of death worldwide3. Cigarette smoke exposure remains one of the main etiological factors for COPD3. Tobacco smoke contains 440–1150 µg/g of iron, of which 0.06% is transferred to mainstream cigarette smoke. Lung exposure to iron increases upon regular cigarette smoking2, potentially as a result of direct supply of iron through cigarette smoke, or as a result of complexation of host iron by oxygen donor ligands found on cigarette smoke particulate matter. Abnormally increased iron deposition has been reported in the lungs of both current and former smokers4. Furthermore, levels of non-heme iron and of iron-binding proteins including ferritin, lipocalin-2 and lactoferrin, are increased in lung tissue, sputum, BALF, and cells of COPD patients, relative to non-smokers15. Although cigarette smoking is the major cause of COPD, occupational exposures, including those to dust, fumes and diesel exhaust, have emerged as important additional risk factors for COPD, especially in nonsmokers16. Iron has also been shown to accumulate in lung tissue of people exposed to particulate air pollutions17, suggesting this may be another source of iron linked to an increased risk of developing COPD.Chronic obstructive pulmonary disease (COPD) is a debilitating lung disease encompassing airway inflammation (chronic bronchitis), destruction of lung tissue (emphysema) and remodeling of the small airwaysviz oxygen transport and energy production, increased free iron could be detrimental due to its potential to generate free radicals18. In addition to environmental risk factors such as cigarette smoke, genetic factors have also been reported to influence COPD susceptibility1. Genome wide association studies have identified iron responsive element binding protein 2 (IREB2) as an important candidate for a COPD susceptibility gene, suggesting a functional disease link between abnormal iron metabolism and COPD3. IREB2 is a gene on human chromosome 15, coding for iron regulatory protein 2 (IRP2), which plays a vital role in maintaining cellular iron homeostasis18.Although iron is an essential trace element required for many vital biological processes 19. In this study, we have employed time of flight secondary ion mass spectrometry (ToF-SIMS) to visualize and relatively quantify iron accumulation in human lung tissue sections of COPD (GOLD IV) patients versus (vs.) healthy donors. Previous studies have employed manual methods for iron quantification in diseased vs. healthy human or mouse lung tissue sections7, which inevitably introduces risks of biased data interpretation and variability between analyses. ToF-SIMS is a versatile technique that has been used in different areas of research, including analysis of metals20, polymers21 and complex biological samples. Over the recent years this technique has been introduced for biomedical studies on human tissue samples such as liver22, kidney23, skeletal muscle24 and various forms of cancer such as prostate cancer25 and breast cancer26.According to the Global initiative for chronic Obstructive Lung Disease (GOLD) criteria, COPD patients are classified into GOLD stage I (Mild), Stage II (Moderate), Stage III (Severe) and Stage IV (Very severe) based on the severity of airflow limitation28 and matrix application29. However, with imaging mass spectrometry it is still challenging to detect a wide range of ion species with sufficient spatial resolution. To overcome this limitation, the delayed extraction mode has been developed as an alternative method. In this mode, the extracted secondary ions from the sample surface are collected with a certain delay after the arrival of primary ions30, which makes it possible to record secondary ion images of ion species with very high spatial resolution and a mass resolution of several thousands in a single run.In this technique, a microfocused beam is impinged on discrete points of a sample surface, generating secondary ions that are analyzed to generate a mass spectrum at each pixel point from the sample. When it comes to chemical analysis of biological samples, ToF-SIMS is a powerful technique in comparison to other single methods, as this technique in parallel can provide identification and localization of substances at high spatial resolution (<400 nm) without the need for labelling7, is heterogeneous. Thus, ToF-SIMS appears as a powerful tool to image and relatively quantify lung tissue iron overload, allowing clear differentiation between healthy vs. COPD donors based on their lung tissue iron content. In addition, the technique can distinguish other chemical differences such as alterations in lung tissue lipid composition between the two groups.In the present study, we successfully demonstrate that ToF-SIMS can be employed for visualization and relative quantification of iron in human lung tissue sections from both healthy and diseased donors (COPD (GOLD IV)). Further, upon application of the delayed extraction mode where spatial resolution down to 400 nm is attainable, we can show that the intracellular iron distribution within cells tentatively identified as macrophages, emerging as the major cell type responsible for iron accumulation in lung tissue3++ primary ions using the high current bunched mode were used to visualize the total iron signal  showing the signal in contrast to protein fragments  in tissue from a healthy donor . H&E staining was performed on healthy donors Fig.  and COPDnor Fig.  and a COnor Fig. , respectnor Fig. , S1a, whnor Fig. , S1b.FigNext, iron ion imaging of a COPD (GOLD IV) patient’s lung tissue was compared to standard histochemical iron (Perls’ Prussian blue) staining performed on a consecutive lung tissue section. Correlations regarding lung tissue regions of iron overloading was observed between the two methods Fig. , howeverIt is worth noting though, that for both conventional histochemical iron staining and ToF-SIMS analysis, the paraffin fixed lung tissue sections were de-paraffinized using xylene. Since xylene is an aggressive chemical, the risk for iron delocalization and leaching needs to be considered. Still, the good correlation between iron staining and ToF-SIMS imaging performed on consecutive serial sections indicates that such delocalization is limited. Another potential issue is that since the sampling area is relatively big (approximately 15*22 mm), charging and topographic effects can be present. Normalisation of the iron signal to the total ion signal did however, not significantly alter the ion image for iron. Also, the quality of the obtained spectra based on achieved mass resolution and lack of peak tailing  of lung tissue sections. In this study, we used ToF-SIMS for relative quantification of iron in lung tissue sections of healthy donors vs. COPD (GOLD IV) patients. For this purpose, three sections from three different donors in each group were chosen, five areas per section were randomly selected for iron imaging, and the ion signal for iron was analyzed and averaged. The relative quantification revealed approximately twelve times higher levels of iron in COPD (GOLD IV) patients’ lung tissue sections compared to that of healthy donors  patients´ lung tissue, we decided to investigate whether the pattern of iron distribution could be assessed down to the cellular or the subcellular level. To achieve this, we used the ToF-SIMS delayed extraction mode, allowing lateral resolution down to 400 nm while maintaining a high mass resolution. Applying this technique is an efficient way to overcome the issue of compromising between a high mass resolution and a sub-µm lateral resolution in conventional ToF-SIMS.7 and by analyzing consecutive sections with Perls’ Prussian blue/ nuclear fast red as seen in Fig. 1. Mitochondria are the main consumers of intracellular iron, and iron regulation by mitochondria may play a vital role in many cellular processes1. Similarly, lysosomal iron turnover is also an important component of intracellular iron regulation31 and the contribution of lysosomal iron to these subcellular regions should also be considered. The exact subcellular localization of iron inside these macrophages remains to be determined and is currently the subject of further investigation.The heterogeneous nature of iron distribution was revealed by overlaying microscopic total ion and iron signal images Fig.  of COPD Following the identification of heterogeneous distribution of iron within the iron positive cells, we performed depth profiling on these cells to confirm that the measured iron signal was from intracellular deposits. In this mode, the ion intensity in the z-depth, as sputter time (s), of the tissue section can be monitored. The results clearly demonstrated that when entering the cells, the signal from iron increased whereas the signal from plasma membrane lipids including phosphatidylethanolamine (PE) and phosphatidylcholine (PC) decreased Fig. , indicatNext, multivariate data analysis was performed to explore whether the lung tissue ToF-SIMS spectra could differentiate and distinguish healthy control subjects from COPD patients based on the chemical composition of the tissue.Principle component analysis (PCA) was carried out on the collected and normalized spectra obtained from ToF-SIMS analysis of three different lung tissue samples from each group ) with five regions for analysis randomly chosen on each sample. In the PCA analysis, the two groups could be discerned using the first (PC1: t[1]) vs. the third (PC2: t[3]) principal component Fig. . However32. With the same dataset, OPLS-DA analysis revealed an excellent separation between healthy donors and COPD (GOLD IV) patients  and thus a major driver of the separation between the two groups  in the COPD (GOLD IV) samples. These peaks are likely to be emanating from different lipids, as it has been previously reported that more than 210 lipids were differentially expressed in COPD, including a large portion of sphingolipids that were significantly upregulated in smokers with COPD35. The lung lipidome and lipid signaling have been implicated in regulating many of the biological processes associated with COPD. Several inflammatory cytokines upregulated in COPD have the potential to modulate the activity of lipid-modifying enzymes such as phospholipases, sphingomyelinases and sphingosine kinases, which might consequently result in an altered lipid composition that could affect cellular integrity. For instance, it has been shown that dysregulation of the sphingolipid metabolism may be an important driver of progressive alveolar destruction and remodeling in COPD36. Thus, analysis of the COPD lung lipidome may provide novel approaches to biomarker development and identification of potential therapeutic targets33. However, this is not in the scope of this paper and further investigation is required to better understand the effect of COPD on lipid expression.The loading plot corresponding to the OPLS-DA score plot revealed that iron was one of the most prominent biological masses amongst the highest loading peaks 38 and silicon dioxide (SiO2)39) in rat lung tissue sections and in aerosolization research to study the migration of excipients onto the surface of inhaled drugs42. ToF-SIMS has also been employed in lipidomics profiling of lung tissue from Pseudomonas aeruginosa infected Cystic Fibrosis Transmembrane Conductance Regulator deficient (CFTR−/−) mice. This study reported a significant increase in cholesteryl sulfate (m/z 465.4) in lung epithelium from CFTR−/− mice in response to bacterial infection43. ToF-SIMS has also been routinely used in studying the lateral organization of lipids and proteins in pulmonary surfactant systems45.ToF-SIMS is a novel analytical technique gaining increasing importance in life sciences due to its relatively high spatial resolution and sensitivity combined with its scope for deep chemical profiling analysis. Thus, mass spectrometry imaging provides an excellent opportunity to obtain an unprecedented amount of information on the distribution of elements and molecules in biological samples46, nor dependent on histopathologic scoring, which is at best semi-quantitative. Relative quantification of iron load by ToF-SIMS revealed approximately twelve times higher levels of iron in lung tissue sections from severe COPD patients as compared to healthy control donors. This agrees with a previous qualitative assessment showing increased iron deposition in very severe COPD patients’ lung tissue using the Perls’-DAB histochemical iron staining protocol7. The same study7 identifies alveolar macrophages as the predominant iron positive cell population. Despite clear evidence of iron accumulation in lung tissue obtained from COPD patients, the source of iron remains obscure. Increased iron content in COPD lungs could possibly result from cigarette smoke exposure4, genetic predisposition e.g. IREB2 polymorphism3, alveolar microhemorrhage47, systemic inflammation47, air pollutants or other unknown factors. Iron laden alveolar macrophages have also been reported in Idiopathic Pulmonary Fibrosis47 and elevated iron levels have been found in the respiratory tracts of cystic fibrosis patients48. In addition to respiratory diseases, dysregulated iron metabolism and iron accumulation have been observed in neurodegenerative disease conditions such as Alzheimer’s disease, Parkinson’s disease, Multiple sclerosis and Amyotropic Lateral Sclerosis50.Our study has several important strengths. To the best of our knowledge, we are the first to successfully use ToF-SIMS to image iron in human lung tissue sections at a spatial resolution of 400 nm and to perform relative iron quantification. This application of ToF-SIMS is highly valuable as it is more precise in quantification and less error prone compared to conventional histochemical methods. For example, unlike common histochemical methods, ToF-SIMS detection of iron is not altered by anthracotic material (accumulation of carbon in the lungs due to repeated exposure to particles)3+) iron and soluble ferrous (Fe2+) iron. At physiological pH, iron pre-dominantly exists as insoluble ferric iron, bound to carrier proteins such as transferrin51, ferritin9, lactoferrin52, ceruloplasmin53 and/or lipocalin 28, or unbound in the form of cell free hemoglobin/heme or non-transferrin bound iron (citrate or acetate bound iron), all of which are found in the BALF. To be absorbed, iron must be in the ferrous (Fe2+) state. Intracellular Fe2+ is then either stored  or utilized for essential biologic processes including heme synthesis and Fe-S cluster synthesis. Under conditions of excessive intracellular iron, surplus iron may be deposited as insoluble polymeric iron(III) with oxo-bridges termed hemosiderin54. In this study we are unable to determine if the iron detected by ToF-SIMS is ferric, ferrous, insoluble or soluble or if it is bound to a specific protein. Further studies are required to determine the nature of this accumulated iron in COPD.Our study also has limitations. ToF-SIMS does not discriminate between oxidized, ferric  patients as compared to healthy donors. Most of this iron was shown to be heterogeneously distributed within the iron-positive cells, consistent with accumulation within discrete cellular organelles such as mitochondria or lysosomes. Using multivariate analysis OPLS-DA, we show that COPD patients and healthy controls can be clearly separated on basis of the chemical composition of their lung tissue. The iron signal was found to be a major driver causing this separation. However, other species, Transplanted lung tissue from three end stage COPD (GOLD IV) patients and three healthy donors age-matched to the COPD group were employed for this study. The studies were approved by the Swedish Research Ethical Committee . All research was performed in accordance with relevant guidelines and regulations. Signed informed consent forms were received from the study subjects or their closest relatives.57. 3 µm thick tissue sections were mounted on microscopic glass slides and used for ToF-SIMS imaging and Perls’ Prussian blue iron staining.The lung tissue was dissected into small pieces and fixed with 4% buffered formalin over-night, followed by dehydration and paraffin embedding process as routinely performed for biobank storage and described previously*3++ primary ions at 25 keV energy were applied to record ToF-SIMS spectra of positive ions. Both the high current bunched mode with a mass resolution of m/∆m = 6000 fwhm at m/z 100, as well as the delayed extraction mode, with a mass resolution of m/∆m = 4000 fwhm at m/z 100 were used. The delayed extraction was set to an extraction voltage of 2 kV, the rise time to 40 ns and the delay was adjusted to 5 µs. The pulsed primary ion current was 0.3 pA and 0.25 pA for high current bunched mode and delayed extraction mode respectively. The maximum ion dose density of Bi3++ was kept below 4 × 1011 cm−2. To avoid charging effects, electron flooding was used. In total three sections obtained from three different donors in each group  with five random areas from each section were analyzed. Depth profile analysis was performed using pulsed Bi3++ gun (0.26 pA) while sputtering was carried out with a C60++ beam with a current of 0.78 nA.A ToF-SIMS 5 instrument  equipped with a liquid metal primary ion source was used for all ToF-SIMS experiments. Bi+, [CH]+, [CH2]+, [CH3]+, and [C5H15PNO4]+ were used. Spectra obtained from individual samples were analyzed using the search peaks function in the Surface Lab software. Parameters employed to select peaks were as followed: counts >100, S/N > 3, width 0.8 Da. In the following step, the complete data set consisting of 804 peaks were normalized to the primary ion dose of the respective spectra and then subjected to principle component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) data analysis using SIMCA . Low mass peaks, m/z below 30, were excluded from the analysis as they were related to redundant and not chemically specific peaks such as hydrocarbon clusters. The scores and loading plots revealed which ions  were responsible for most of the variation between the two groups, healthy and COPD.The Surface Lab software  was used to analyze all ToF-SIMS data. For mass calibration, signals of [C]Formalin-fixed, paraffin embedded lung tissue sections were deparaffinized and rehydrated using xylene and decreasing concentrations of alcohols and washed in de-ionized water. Tissue sections were then stained with Hematoxylin  for 8 minutes and kept under running tap water for 5 minutes to allow stain to develop. The sections were then stained with eosin  for 5 minutes, after which they were dehydrated through treatment with increasing concentrations of alcohols and xylene. Finally, the tissue sections were mounted and scanned using Aperio CS2 scanner , and analyzed using Aperio ImageScope .Formalin-fixed, paraffin embedded lung tissue sections were deparaffinized and rehydrated using xylene and decreasing concentrations of alcohols and washed in de-ionized water. Slides were then incubated in Perls’ stain  as per the manufacturer’s protocol for 30 minutes at room temperature (RT) and then washed with distilled water. For nuclear staining, slides were incubated with nuclear fast-red solution and incubated for 5 minutes at RT. Following, the slides were washed with distilled water for 5 minutes, dehydrated and mounted. Slides were scanned using Aperio CS2 scanner  and analyzed using Aperio ImageScope .Supplementary information


Lower vertebrate and neonatal mammalian hearts exhibit the remarkable capacity to regenerate through the reprogramming of pre-existing cardiomyocytes. However, how cardiac injury initiates signaling pathways controlling this regenerative reprogramming remains to be defined. Here, we utilize in vivo biophysical and genetic fate mapping zebrafish studies to reveal that altered hemodynamic forces due to cardiac injury activate a sequential endocardial-myocardial signaling cascade to direct cardiomyocyte reprogramming and heart regeneration. Specifically, these altered forces are sensed by the endocardium through the mechanosensitive channel Trpv4 to control Klf2a transcription factor expression. Consequently, Klf2a then activates endocardial Notch signaling which results in the non-cell autonomous initiation of myocardial Erbb2 and BMP signaling to promote cardiomyocyte reprogramming and heart regeneration. Overall, these findings not only reveal how the heart senses and adaptively responds to environmental changes due to cardiac injury, but also provide insight into how flow-mediated mechanisms may regulate cardiomyocyte reprogramming and heart regeneration. When confronted with pathological or physiological stress, tissues and organs can respond through the adaptive reprogramming of differentiated cell-types, which leads to repair in some instances and disease in others . In the Recent studies have revealed that biomechanical forces generated by blood flow can contribute significantly to heart development through modulating Notch, BMP and Erbb2 signaling pathways . During Tg(Tp1:eGFP) Notch reporter line, we initially examined the activation of Notch signaling in vmhc:mCherry-nitroreductase (NTR) hearts that were ventricle-ablated with metronidazole (MTZ) treatment at 5 dpf. Consistent with previous studies  Notch reporter line, which expresses a destabilized d2GFP as previously reported ; Tg(vmhc:mCherry-NTR) ventricle ablated hearts inhibits not only the reactivation of key early cardiac transcriptional regulators such as gata4, hand2 and nkx2.5 (+) using a combination of Tg(amhc:CreERT2) and Tg(cmlc2:LoxP-AmCyan-STOP-LoxP-zsYellow) [Tg(myl7:CSY)] ‘switch’ reporter lines, we additionally discovered that this heat-shock hsp70l:dnMAML-GFP Notch inhibition also prevents the ability of c-aYFP+ atrial CMs to reprogram and transform into ventricle CMs (Tg(hsp70l:LoxP-mKate2-Stop-LoxP-dnMAML-GFP) [Tg(hsp70l:RS-dnM)] and Tg(kdrl:Cre) lines, which together allow for heat-shock induction of dnMAML-GFP specifically in the endocardium  after vmhc:mCherry-NTR ventricle MTZ-ablation decreases the activation of gata4, hand2 and nkx2.5 expression  transgenic tools to genetically inhibit Notch signaling during cardiac injury at 5–6 dpf. Heat shock induction of d nkx2.5  but alsod nkx2.5 , thus led nkx2.5 . Throughicle CMs . Becauseicle CMs   line, which labels red blood cells in dsRed, we visualized and measured in vivo blood flow dynamics by confocal microscopy and particle image velocimetry (PIV) analyses in uninjured (control) and ventricle-injured hearts at six dpf    of blooded flow) .klf2a expression, which primarily is located in specific regions of the AVC of uninjured hearts (Tg(klf2a:H2B-GFP) line, which expresses nuclear-GFP in response to changes in hemodynamic flow/stress  hearts  hearts , suggest) hearts , we furtreporter . Overallgata2a mutant, which exhibits decreased blood cells  and Tg(β-actin2:loxP-DsRed-STOP-loxP-eGFP) [or Tg(β-actin2:RSG)] lines to genetically label atrial CMs (c-aGFP+), we also discovered that reducing blood flow and viscosity under these conditions significantly reduces the contribution of c-aGFP+ labeled atrial CMs to the ventricle after ventricle ablation  but also ventricle-ablated hearts  and ventricle-ablated hearts  BMP reporter line, which has been previously used to detect BMP signaling in the myocardium of zebrafish hearts  (mss5Tg(cmlc2:GFP) (Tg(β-actin2:loxP-DsRed-STOP-loxP-eGFP)s928, abbreviated Tg(β-actin2:RSG) (fb2Tg(myl7:loxP-AmCyan-STOP-loxP-ZsYellow), abbreviated Tg(myl7:CSY) , abbreviated Tg(hsp70l:dnM) , abbreviated Tg(Tp1:d2GFP)  abbreviated Tg(Tp1:nls-mCherry) (Tg(kdrl:ras-mCherry)s896 (Tg(kdrl:Cre)s898 (Tg(gata1:DsRed) (mw30and Tg(BRE-AAVmlp:eGFP), abbreviated Tg(BRE:d2GFP)  was generated as previously described . We used the following transgenic lines: NTR)s957 ; Tg(cmlcGFP)mss5 ; Tg(β-acin2:RSG) ; Tg(myl7yl7:CSY) ; Tg(hsp770l:dnM) ; Tg(cmlcERT2)pd10; Tg(EPV.1:d2GFP) ; Tg(T2KTmCherry) ; Tg(kdrlrry)s896 ; Tg(kdrl:Cre)s898; Tg(klf2H2B-GFP) ; Tg(gata1:DsRed) ; and Tg(E:d2GFP) . We utilv4sa1671 , klf2aiglf2aig4, , gata2a a2a um27  and erbbrbb2st50 . The hspescribed  with theE-hsp70l  was usedsa1671Trpv4, ig4klf2a and um27gata2 mutants were genotyped by PCR as described (https://zfin.org/zf_info/zfbook/stages/index.html), were genotyped by fin clip as described (st50erbb2 homozygous mutant embryos were identified by the previously characterized aberrant cardiac morphology (escribed . Adult fescribed . The mutescribed . The erbrphology .Tg(vmhc:mCherry–NTR) zebrafish were treated with 10 mM of Metronidazole (MTZ)  in egg water with 0.2% DMSO (dimethylsulphoxide)  for 2 hr at 28°C in the dark at 3, 4 or 5 dpf as previously described . Briefly, 24 hpt zebrafish were fixed overnight in 4% PFA at 4°C, washed in Phosphate Buffered Saline with 0.1% Tween 20 (PBT) and dehydrated through a graded methanol series, and then incubated in 100% methonol at −20°C for at least 2 hr before graded rehydration to PBT. Samples were then incubated in 10 μg/mL proteinase K for 1 hr at room temperature, washed three times with PBT and refixed in 4% PFA for 20 min. After washing three times in PBT, samples were incubated with TUNEL staining solution overnight at 37°C and then washed five times with PBT before acquiring images using a Nikon C2 confocal microscope.Live zebrafish were embedded in 1% low melting agarose in a glass-bottom culture dish (MatTek), and the heart contraction was recorded by an Andor iXon EMCCD camera at a frame rate of 40 ms/ frame. The fractional area change was calculated as FAC = (End diastolic area-End systolic area)/End diastolic area x 100%.Tg(vmhc:mCherry-NTR) fish hearts were imaged and analyzed at 0 hpt (before ablations), 24 hpt and 96 hpt using a Nikon C2 confocal microscope  where an average of 25 stacks every 5 μm were acquired. To specifically determine the ventricular area, 3D reconstructions of these fish hearts were analyzed to calculate the total area covered by vmhc:mCherry-NTR cells using ImageJ imaging analyses . Total ventricular area was normalized to the average 0 hpt non-ablated heart ventricular area.To determine the ventricular area and the subsequent tissue recovery in ablated and DMSO control fish, ablated and control bmp10, gata4, klf2a, hand2, nkx2.5, notch1b and nrg1. Fish were analyzed under a Leica M205 FA stereo microscope. Magnifications of bright field images of the area containing the fish heart are shown. Blue corresponds with riboprobe staining. Heart shape was outlined based on anatomic observation under the microscope. Quantitation of the number of hearts showing increased staining of the corresponding marker as compared to non-ablated controls  vs total number of observed hearts is provided in each corresponding figure legend  (n/d).Whole-mount in situ hybridizations were performed as previously described , using thsp70l:dnM or the hsp70l:RS-dnM transgenes were treated with DMSO (control) or MTZ (ablated) as described. Immediately after DMSO/MTZ treatment, fish were heat-shocked for 5 min at 42°C followed by 1 hr at 37°C. Heat-shock was repeated every 12 hr until corresponding analysis was performed. Expression of dnMAML-GFP was detected under fluorescence microscopy.Fish containing the vmhc:mCherry-NTR transgene was lost after fixation treatment; thus anti-MF20/anti-MHC was used to detect myocardium for these studies. The primary antibodies used in this study include: anti-GFP ; anti-MF20/anti-MHC  (+/MF20+ double positive or EdU+/cmlc2:GFP+ double postive cells within the ventricle and the atrium of control and ablated fish.Immunofluorescence staining was performed as previously described for anti-phospho-histone H3 studies . Red fluma Bank) ; and antma Bank) . DetectiTg or Tg zebrafish were treated with a 10 mM 4-hydroxytamoxifen (4-OHT) solution (Sigma) or with 0.1% ethanol (unlabeled control) at 3, 4 or 5 dpf for 4 hr at 28°C and subsequently washed with fresh egg water several times. Hearts with GFP/YFP genetically labeled atrial CMs were treated with DMSO (control) or MTZ (ablated) at 12 hr post 4-OHT labeling  and analyzed under a fluorescent scope every 12 hr up to 4 days. To identify ventricular GFP-positive CMs, hearts from Tg were imaged with a Nikon C2 confocal at 60, 72 or 96 hpt as previously described  or Tg hearts, zebrafish were imaged with a Nikon C2 spectral detector  and a 488 laser. YFP signals were obtained by extracting the minimum matching YFP wavelength from spectral data after calibration with YFP only positive controls as previously described (+) in the ventricle, maximum intensity projections of at least 25 serial 5 μm z-stacks were used to create 3D reconstructions. The ventricles were outlined based on the morphology observed in the bright-field channel. Similarly, the surface area covered by c-aGFP+ or c-aYFP+ CMs was outlined based on presence of GFP or YFP fluorescence. The total surface area of the outlined ventricles and the outlined c-aGFP+ and c-aYFP+ CMs was calculated by ImageJ . The percentage of the ventricle covered by c-aGFP or c-aYFP was obtained by dividing the c-aGFP or c-aYFP area by the total surface area in the ventricle.Lineage tracing was performed as previously described . Tg(vmhcescribed . To idenescribed . To deteTg(Tp1:d2GFP) or Tg(klf2a:H2B:GFP) hearts were imaged with a Nikon C2 confocal microscopy at the indicated time points. An average of 25 serial 5 μm z-stacks were acquired. Mean fluorescence levels were calculated using methods previously described  control and ablated fish hearts were imaged with a Nikon C2 confocal microscopy at the indicated time points. An average of 25 serial 5 μm z-stacks were taken, and then images were analyzed with ImageJ . Individual stacks were analyzed to determine the presence of Tp1:nls-mCherry and/or klf2a:H2B-GFP positive nuclei in the atria and the AVC. In the ventricle, Tp1:nls-mCherry positive cells were identified as those nuclei that were anatomically located beneath the myocardial layer. The myocardial layer was identified as vmhc:mCherry-NTR positive/klf2a:H2B-GFP negative.To determine the number of Tg fish at six dpf (24 hpt). Fish were imaged with a Leica Sp5  resonance scanner at 50–100 frames per second, at 512 × 128 pixels of resolution. 10 z-stacks were obtained, each separated by 10 μm for a total of 2 min. Bright field blood flow videos for two dpf wild-type control and trpv4 -/- mutant were recorded by an Andor iXon EMCCD camera at a frame rate of 40 ms/ frame.Blood flow videos were obtained by imaging control and ablated To determine the velocity of the blood inside the ventricle from obtained microscopy videos, an in-house multigrid ensemble particle image velocimetry (PIV) software was developed, as previously described . PIV detFlow rates through different sections of the cardiac chambers were calculated from the PIV measurements using1/Q0Q between the amplitudes of the fundamental frequency flow harmonic  and the time-averaged flow   . These aTo decrease cardiac contractility after MTZ (ablated) or DMSO (control) treatments, fish were immediately incubated in egg water with 1 mg/ml of tricaine  or 10 μM of blebbistatin  for 12 hr and then washed three times for analyses. To inhibit BMP or Erbb2 signaling after MTZ (ablated) or DMSO (control) treatments, fish were immediately treated with 10 μM Dorsomorphin (DM)  or 5 μM AG1478 , respectively. Fish were treated for 24, 48, or 72 hr as indicated. To inhibit BMP, Erbb2 or Notch signaling after MTZ (ablated) or DMSO (control) treatments, fish were immediately treated with 10 μM Dorsomorphin (DM) , 5 μM AG1478 , or 10 mM DAPT  until indicated time for each experiment.Tg fish were treated with DMSO (control) or MTZ (ablated) as described. Fish were subsequently treated with DMSO, DAPT or blebbistatin as described above. To determine the presence of BRE:d2GFP, hearts were imaged with a Nikon C2 confocal microscopy at 48 hpt. An average of 25 serial 5 μm stacks was acquired for each heart. To identify BRE:d2GFP/vmhc:mCherry-NTR double positive co-expressing cells, individual stacks were analyzed with ImageJ .To study the myocardial activation of BMP signaling, 5 dpf t or Binomial tests were used as indicated in the corresponding figure legends.Box plots and statistical analysis were performed using GraphPad Prism 7. ANOVA, un-paired Student’s Animal experimentation: All procedures involving animals were in accordance with the National Institutes of Health Guide for the care and use of Laboratory Animals, and approved by the University of California at San Diego Institutional Animal Care and Use Committee (IACUC) (Protocol number S08316). In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.in vivo myocardial reprogramming" for peer review at eLife. Your article is being evaluated by Marianne Bronner as the Senior Editor, a Reviewing Editor, and three reviewers.Thank you for sending your article entitled "Hemodynamic-mediated endocardial signaling controls eLife. Given the list of essential revisions, including new experiments, the editors and reviewers invite you to respond within the next two weeks with an action plan and timetable for the completion of the additional work. We plan to share your responses with the reviewers and then issue a binding recommendation. The reviewers' comments are detailed below.All of the reviewers see merit in the manuscript but also raise issues that will require extensive revisions. Our concern is that these revisions may take a considerable time beyond the two to three months required for a revision in Reviewer #1:In this manuscript, Chi and colleagues describe an endocardial to myocardial signalling pathway that mediates cardiomyocyte (CM) reprogramming and proliferation in the embryonic zebrafish ventricular ablation model. Increased oscillatory flow, sensed in part by the Trpv4 mechanosensory channel, is required to activate endocardial Klf2 and Notch activity, which in turn influences Bmp and Erbb2 activity in CMs. While these components have all been shown to influence cardiac development, and in many cases adult cardiac regeneration, this work provides a holistic analysis of this pathway in the embryonic setting.Overall, the manuscript is clearly written, with well presented and supported experimental results.Comments:1) The data for the Notch response in Figure 1—figure supplement 2 is somewhat confusing. Heightened Notch reporter activity in the ventricle is shown/discussed (panels A and B), yet Notch1b expression in panel F is clearly atrial, yet called "throughout the heart" in the text. This should be clarified.nkx2.5 appears to be induced in the atrium, but not ventricle, following Notch inhibition. Does this imply incomplete reprogramming of atrial CMs? Again, this should be discussed. Are these representative images?2) In Figure 1—figure supplement 3 panel L, klf2/tp1 (Notch) double-positive cell, especially given the red colour of the NTR transgene as well.3) In Figure 2—figure supplement 1, it is unclear what is being counted as a cmlc2:NTR-based) CM ablation has been reported , where presumable blood flow is minimal.4) The authors present and support a model where alterations/increases in oscillatory flow trigger an endocardial to myocardial cascade that initiates cardiac reprogramming/proliferation. However, regeneration of the embryonic heart following full  In this study, the assessment of cardiac regeneration is primarily based on pH3 staining (~4 cells per heart). More robust assays for cardiomyocyte proliferation and cardiac function will strengthen the data.2) A recent study shows that the embryonic heart cannot fully recover from the ablation of a small population of cardiomyocytes. The injured hearts develop trabeculation defects which ultimately lead to heart failure. In this study, the ablation covers the entire ventricle, yet the majority of ablated embryos display recovered ventricular morphology and function at 9 dpf. To clarify the discrepancy, it would be helpful if the authors could elaborate on how they assess the efficiency of ablation and the recovery of function and morphology.Reviewer #3:erbb2 signalling.The authors report the consequences of heart injury in the adult zebrafish, after metronidazole mediated ablation of ventricular cardiomyocytes. Using a range of genetically modified fishes and drug treatments, they identify a hemodynamic sensitive repair mechanism, mediated by endocardial Klf2a and Notch signalling, promoting cardiomyocyte proliferation in both the ventricle and atrium, as well as atrial-to-ventricle cardiomyocyte contribution. This response reactivates a pathway involved in myocardium trabeculation, in which endocardial Notch promotes cardiomyocyte proliferation via Bmp and The topic of heart repair is of high interest because of the major medical issue, but also the complexity emerging from the growing number of pathways involved in experimental models. By combining sophisticated genetics and hemodynamic measures with standard molecular and cellular analyses, the article identifies a novel repair mechanism. However, there are some important issues to address and the writing of the manuscript requires clarifications.vmhc. Is there a precursor state that cardiomyocytes need to revert to, to elicit a response? To which stage in normal development is this precursor state similar? What are the changes at the level of the genome? The observations that gata4, nkx2.5 and hand2 are activated is not convincing, since it is unclear whether this activation is in the endocardium or myocardium and whether it is required for the repair response. Hand2 is known to be activated in the endocardium downstream of Notch during trabeculation (PMID 25497097), gata4 is known to be expressed in endocardial cells (ex PMID 16914500).It is unclear why the response to injury is qualified as reprogramming. Reprogramming is used in the context of conversion of cardiac fibroblasts into cardiomyocytes, or of cardiomyocytes into pluripotent stem cells, and is associated with an erasure of epigenetic marks in the genome. What is the rationale here to refer to reprogramming and not just activation of proliferation? For example, atrial cardiomyocytes may be "pushed" from a crowded chamber to the necrotic ventricular region, where they could start to express vmhc:mCherry after 24hpt in Figure 1—figure supplement 3, Figure 3J-M, Figure 5F-H seems less dramatic compared to previous Zhang et al., 2013 publication. So how variable is the injury? Does this affect the valves, which would in itself promote retrograde flow?The conclusion on "regeneration" is overstated. The quantification shown as "percentage of fish" presumably corresponds to the survival rates, whereas a conclusion on regeneration would require the recovery of the tissue to be quantified. How many cardiomyocytes are lost after metronidazole treatment? How many are recovered at 72hpt, and which percentage derives from atrial cells? These quantifications are important to discuss the relative importance of the growing number of repair mechanisms. The effect of ventricular necrosis seen with klf2a, notch reporter, nrg1 activation. Thus, is the hemodynamic response confined to the boundary zone? Or to the valve region which is competent to activate klf2a? Please explain the discrepancy between the Tp1 Notch reporter, activated in the ventricle, and notch1 expression, more striking in the atrium.The localisation of the response is always described as "throughout the heart". However, images show some degree of regionalisation, which is not discussed. The boundary of the injured area, which is also the valve region, is more affected by gata2 mutants? Is blood flow affected in trpv4 mutants?The interpretation of blebbistatin as a perturbation of hemodynamics is questionable, given that this drug also impacts proliferation (decreased in control hearts) and cell migration. Is the flow oscillatory in The use of drugs requires control experiments to show the impact on targeted signalling pathways in this context .erbb2 signalling is not demonstrated in this context. Whether Bmp and erbb2 signalling are confined to, or required, in the myocardium is not shown in this context, but only based on literature in the context of trabeculation.The conclusion that endocardial Notch promotes myocardial Bmp and The Discussion section should be extended to comment the limitations/open questions of the study and the impact of the hemodynamic response for heart regeneration compared to alternative pathways, in terms of quantitative contribution, kinetics or regionalisation. The level of similarities between fish and mouse heart repair can also be discussed, rather than assumed. The last sentence is overstated. [Editors' note: the authors’ plan for revisions was approved and the authors made a formal revised submission.]Reviewer #1:In this manuscript, Chi and colleagues describe an endocardial to myocardial signalling pathway that mediates cardiomyocyte (CM) reprogramming and proliferation in the embryonic zebrafish ventricular ablation model. Increased oscillatory flow, sensed in part by the Trpv4 mechanosensory channel, is required to activate endocardial Klf2 and Notch activity, which in turn influences Bmp and Erbb2 activity in CMs. While these components have all been shown to influence cardiac development, and in many cases adult cardiac regeneration, this work provides a holistic analysis of this pathway in the embryonic setting.Overall, the manuscript is clearly written, with well presented and supported experimental results.We thank reviewer 1 for her/his positive and thoughtful evaluation of the manuscript and her/his helpful suggestions to strengthen and deepen the conclusions of the manuscript.Comments:1) The data for the Notch response in Figure 1—figure supplement 2 is somewhat confusing. Heightened Notch reporter activity in the ventricle is shown/discussed (panels A and B), yet Notch1b expression in panel F is clearly atrial, yet called "throughout the heart" in the text. This should be clarified.notch1b expression in new Figure 1—figure supplement 3  panel F and dynamic Notch reporter activity shown in new Figure 1—figure supplement 3 panel D is particularly increased in the atrium and atrioventricular canal region; however, both notch1b expression and Notch reporter activity is weakly present in the ventricle. These findings correlate closely to the levels of oscillatory flow after cardiac injury as shown in Figure 2. We have clarified these findings in the Result section as suggested by reviewer 1. Please see subsection “Endocardial Notch signaling controls myocardial reprogramming”.We appreciate reviewer 1’s concern regarding the data for the Notch response to cardiac ventricle injury. Reviewer 1 is correct that 2) In Figure 1—figure supplement 3 panel L, nkx2.5 appears to be induced in the atrium, but not ventricle, following Notch inhibition. Does this imply incomplete reprogramming of atrial CMs? Again, this should be discussed. Are these representative images?nkx2.5 expression and reprogramming after Notch inhibition.We thank reviewer 1 for noting this issue. This data is due to incomplete inhibition of reprogramming after heat-shock induction of dnMAML Notch inhibition. Reviewer 1 is correct that new Figure 1—figure supplement 4 panel L  is meant to be a representative image. We have provided a better representative image for Figure 1—figure supplement 4 panel L, which shows more clearly the inhibition of 3) In Figure 2—figure supplement 1, it is unclear what is being counted as a klf2/tp1 (Notch) double-positive cell, especially given the red colour of the NTR transgene as well.klf2a:H2B-GFP is primarily activated in endothelial/endocardial cells in response to hemodynamic flow changes, we are counting endocardial cells that are co-expressing both klf2a:H2B-GFP and Tp1:nls-mCherry. To clarify these findings, we have provided better images including planar images and separation of fluorescence channels in new Figure 2—figure supplement 2 as suggested by reviewer 3. As a result, the endocardial cells can be better distinguished from the cardiomyocytes because Tp1:nls-mCherry appears to be more nuclear in the endocardial cells and more cytoplasmic in cardiomyocytes, and the endocardial and myocardial layers are also better separated in these planar images. In addition to these changes, we have also included new data/analyses and text to support our findings/interpretations  The authors present and support a model where alterations/increases in oscillatory flow trigger an endocardial to myocardial cascade that initiates cardiac reprogramming/proliferation. However, regeneration of the embryonic heart following full (cmlc2:NTR-based) CM ablation has been reported , where presumable blood flow is minimal.cmlc2:NTR-based hearts, which may mediate myocardial reprogramming and regeneration. Finally, the level of recovery after cmlc2:NTR ablation in the Curado et al., studies is lower than the level of recovery after vmhc:NTR ablation. This difference in recovery could be due to weaker beating and reduced oscillatory flow in the cmlc2:NTR ablated hearts. To address reviewer 1’s comment, we have discussed some of these possibilities in the discussion to account for the differences in findings between studies. Please see the Discussion section.We appreciate reviewer 1’s comment. There are experimental differences between our studies and the Curado et al., 2007 that may make it more challenging to directly compare findings. For example, ablations were performed at 48 hpf in the Curado et al. studies whereas we performed our ablations later after the heart has completed cardiac looping and formed cardiac trabeculae. Furthermore, the heart does not appear to be completely ablated in the Curado et al., studies , suggesting that the hearts are beating (presumably more weakly). Although blood flow could be weaker in the Curado ablated hearts, the actual hemodynamic flow dynamics  is unclear in these hearts since they were not examined. It is quite possible that flow is oscillatory in various regions of these Reviewer #2:In this manuscript, Gálvez-Santisteban et al. use a nice set of biophysical and genetic assays to characterize injury-induced cellular responses in embryonic hearts. The main conclusion is that cardiac injury alters oscillatory blood flow and induces Trpv4-mediated Klf2-Notch signaling in endocardial cells to regulate cardiomyocyte reprogramming and regeneration in erbb2 and bmp dependent manner. While this manuscript is clearly written and the data are nicely presented, a better characterization of the injury model and more robust assays for regeneration are needed before publication.We thank reviewer 2 for her/his concerns and appreciate her/his critical evaluation of our studies. We appreciate the suggestions to clarify our data and improve our manuscript.1) In this study, the assessment of cardiac regeneration is primarily based on pH3 staining (~4 cells per heart). More robust assays for cardiomyocyte proliferation and cardiac function will strengthen the data.vmhc:NTR ventricular cardiomyocyte ablation model. Please see new Figure 1—figure supplement 1 and subsection “Endocardial Notch signaling controls myocardial reprogramming”.We thank reviewer 2 for her/his comment. Our assessment of cardiomyocyte proliferation by phospho-Histone H3 (pH3) staining was based on established standards as previously published . However, to further validate this approach and strengthen the data, we have also performed EdU cardiomyocyte proliferation studies in our cardiac injury and regeneration model. Additionally, functional contractile and ventricular tissue recovery studies have been included to complement the cardiomyocyte proliferation experiments and show that zebrafish hearts are able to regenerate and functionally recover from cardiac injury in the 2) A recent study shows that the embryonic heart cannot fully recover from the ablation of a small population of cardiomyocytes. The injured hearts develop trabeculation defects which ultimately lead to heart failure. In this study, the ablation covers the entire ventricle, yet the majority of ablated embryos display recovered ventricular morphology and function at 9 dpf. To clarify the discrepancy, it would be helpful if the authors could elaborate on how they assess the efficiency of ablation and the recovery of function and morphology.vmhc. As a result, our vmhc:cherry-NTR lines may not be able to ablate these neural crest-derived cardiomyocytes, thus potentially accounting for differences in outcomes between these studies. However, we do interestingly observe in our studies that not all hearts recover from ventricular injury, suggesting the possibility that in some of our vmhc:cherry-NTR ablations, we are ablating these neural crest-derived cardiomyocytes. To address reviewer 2’s comment, we have included these explanations in our discussion to account for the discrepancies in findings between these studies. Please see the Discussion section.We appreciate reviewer 2’s comment and the comparison of our findings to those from the Abdul-Wajid et al., (2018). The discrepancy in findings between these studies may be due to the differences in the cardiomyocytes that are ablated. Since different cardiomyocyte ablation lines are used between the studies, we may not be ablating the same cardiomyocytes as in the Abdul-Wajid et al., studies, which are neural crest-derived and may not necessarily express Reviewer #3:The authors report the consequences of heart injury in the adult zebrafish, after metronidazole mediated ablation of ventricular cardiomyocytes. Using a range of genetically modified fishes and drug treatments, they identify a hemodynamic sensitive repair mechanism, mediated by endocardial Klf2a and Notch signalling, promoting cardiomyocyte proliferation in both the ventricle and atrium, as well as atrial-to-ventricle cardiomyocyte contribution. This response reactivates a pathway involved in myocardium trabeculation, in which endocardial Notch promotes cardiomyocyte proliferation via Bmp and erbb2 signalling.The topic of heart repair is of high interest because of the major medical issue, but also the complexity emerging from the growing number of pathways involved in experimental models. By combining sophisticated genetics and hemodynamic measures with standard molecular and cellular analyses, the article identifies a novel repair mechanism. However, there are some important issues to address and the writing of the manuscript requires clarifications.We appreciate reviewer 3’s thorough review of our manuscript and her/his interest in our studies. We have carefully reviewed her/his comments and provided response and plan to address concerns.It is unclear why the response to injury is qualified as reprogramming. Reprogramming is used in the context of conversion of cardiac fibroblasts into cardiomyocytes, or of cardiomyocytes into pluripotent stem cells, and is associated with an erasure of epigenetic marks in the genome. What is the rationale here to refer to reprogramming and not just activation of proliferation? For example, atrial cardiomyocytes may be "pushed" from a crowded chamber to the necrotic ventricular region, where they could start to express vmhc. Is there a precursor state that cardiomyocytes need to revert to, to elicit a response? To which stage in normal development is this precursor state similar? What are the changes at the level of the genome? The observations that gata4, nkx2.5 and hand2 are activated is not convincing, since it is unclear whether this activation is in the endocardium or myocardium and whether it is required for the repair response. Hand2 is known to be activated in the endocardium downstream of Notch during trabeculation (PMID 25497097), gata4 is known to be expressed in endocardial cells (ex PMID 16914500).We appreciate reviewer 3’s query and agree that reprogramming can be used to describe the conversion of fibroblasts into another differentiated cell-type, or of a differentiated cell-type into pluripotent stem cells. However, conversion of one differentiated cell type/fate to another differentiated cell-type/fate, such as α to β cell conversion in the pancreas, is also noted to be a reprogramming process as described by several groups . Thus, we have used the term reprogramming to also describe the conversion of atrial cardiomyocytes into ventricular cardiomyocyte to repair the injured ventricle as observed in our genetic-labelling lineage tracing studies . Furthermore, we have provided data to show cardiomyocytes may revert back to a precursor state likely through activating early cardiogenesis transcription factors as others have previously shown in cardiac regeneration studies . To confirm these findings, we have used three different cardiac transcription factors involved in cardiomyocyte development rather than just relying on one. Thus, the confluence of the three transcription factor data along with our cell fate conversion/lineage tracing studies is the basis for why we use the term reprogramming in our manuscript; however, we are amenable to using other similar terms as reprogramming to describe the process including conversion and trans-differentiation when appropriate. Finally, we agree that it would be interesting to examine the extent of reprogramming of cardiomyocytes in our studies and in general, particularly from a genome/epigenome level as suggested by reviewer 3; however, such studies are beyond the scope of our current work to understand the role of hemodynamic forces during cardiac injury and repair.The conclusion on "regeneration" is overstated. The quantification shown as "percentage of fish" presumably corresponds to the survival rates, whereas a conclusion on regeneration would require the recovery of the tissue to be quantified. How many cardiomyocytes are lost after metronidazole treatment? How many are recovered at 72hpt, and which percentage derives from atrial cells? These quantifications are important to discuss the relative importance of the growing number of repair mechanisms. The effect of ventricular necrosis seen with vmhc:mCherry after 24hpt in Figure 1—figure supplement 3, Figure 3J-M, Figure 5F-H seems less dramatic compared to previous Zhang et al., 2013 publication. So how variable is the injury? Does this affect the valves, which would in itself promote retrograde flow?vhmc is specifically expressed in ventricular cardiomyocytes and thus the vmhc:cherry-nitroreductase line specifically ablates ventricular cardiomyocytes and not valve endocardial cells.We understand reviewer 3’s concerns regarding the regeneration of the heart after ventricular injury. Our ventricular injury and regeneration studies were performed in a similar manner as we previously described in Zhang et al., 2013, which addresses issues raised by reviewer 3 including recovery of tissue, percentage of atrial cardiomyocytes that regenerate the ventricle, etc. Thus, we have cited these studies in our manuscript. Moreover, we have clarified our definition of the quantification shown as "percentage of recovered fish” in Figure 1F,1Q, Figure 4F, Figure 5Q and Figure 6K. These recovered fish represent fish that have recovered ventricular tissue and contractility at 96 hpt as previously described . Please see subsection “Ventricular ablations”. Furthermore, to support these findings, we have quantified the area of the ventricular tissue at 24 hours and 96 hours post metronidazole treatment . To address how many cardiomyocyte are lost after metronidazole treatment, we have performed TUNEL cell death assays as we previously described in Zhang et al., 2013 and provided quantitative/statistical data of these studies to address how variable our ablations/injuries are . To complement TUNEL cell death assays, we have also provided how much ventricular tissue is lost at 24 hours post metronidazole treatment as we previously described in Zhang et al., 2013 . With regards to the percentage of the regenerated ventricle that derives from genetically labeled atrial cardiomyocytes, we have included this data in our manuscript . Finally, The localisation of the response is always described as "throughout the heart". However, images show some degree of regionalisation, which is not discussed. The boundary of the injured area, which is also the valve region, is more affected by klf2a, notch reporter, nrg1 activation. Thus, is the hemodynamic response confined to the boundary zone? Or to the valve region which is competent to activate klf2a? Please explain the discrepancy between the Tp1 Notch reporter, activated in the ventricle, and notch1 expression, more striking in the atrium.Tp1:eGFP notch reporter and notch1b expression is because Tp1:eGFP does not report dynamic Notch activity due to eGFP perdurance as recently reported by Han et al., (PMID: 27357797). Thus, we have used a Tp1:d2GFP line to track dynamic Notch reported activity, which corresponds more closely to notch1b expression in injured hearts. We have clarified this point in the text . Additionally, we have also revised the text as suggested by reviewer 1  as well as provided additional analyses  and revisions in the text as suggested by reviewer 3 .) to address reviewer 3’s other concerns regarding regionalization of injury response.We agree with reviewer 3 that there is some degree of regionalization of injury response. This regionalization is closely associated with hemodynamic changes during ventricular injury as we discussed in response to reviewer 1, comment 1. The discrepancy between The interpretation of blebbistatin as a perturbation of hemodynamics is questionable, given that this drug also impacts proliferation (decreased in control hearts) and cell migration. Is the flow oscillatory in gata2 mutants? Is blood flow affected in trpv4 mutants?gata2 and trpv4 mutants; PMID: 19924233, PMID: 27221222, PMID: 25959969) to not only confirm these hemodynamic findings but also further examine potential mechanisms. Thus, to address this particular concern, we have provided more details/explanations in the text on the different approaches that were used to confirm findings as well as provide better referencing of work that has used these hemodynamic approaches before. Please see subsection “Reducing intra-cardiac hemodynamic forces impairs myocardial reprogramming”. Finally, we have provided particle image velocimetry (PIV) analyses to show that blood flow is similar between wild-type and trpv4 -/- mutant hearts .-/-erbb2 mutant to confirm the AG1478 studies. Thus, we have explained that DMSO is the control in these studies and also referenced Han et al., PMID: 27357797 to clarify that the drugs used in these studies were previously verified to block respective signaling pathways. Please see subsection “Hemodynamic-mediated endocardial signaling pathways activate BMP and Erbb2 signaling to regulate myocardial reprogramming and regeneration”.We appreciate reviewer 3’s comment. We have used DMSO as a control for the drugs used in these particular studies. Furthermore, we are using specific drugs to perturb Notch, BMP and Erbb2 signaling in a similar manner as we have published and validated previously . Additionally, we have included an The conclusion that endocardial Notch promotes myocardial Bmp and erbb2 signalling is not demonstrated in this context. Whether Bmp and erbb2 signalling are confined to, or required, in the myocardium is not shown in this context, but only based on literature in the context of trabeculation.BRE:d2GFP data in Figure 7A-F does show that endocardial Notch signaling mediates myocardial Bmp signaling during ventricular injury. Thus, we have revised our text to reflect that the Neuregulin data suggests that endocardial Notch signaling may regulate Erbb2 signaling during myocardial regeneration, which has been implicated in cardiac regeneration previously . Additionally, we have not only referenced previous work that reveals that BMP signaling is present in the myocardium of zebrafish hearts but also provided better images to show that BRE:d2GFP Bmp reporter activity is present in cardiomyocytes of injured hearts but is altered after blocking Notch signaling .We appreciate reviewer 3’s interpretation of the data in Figure 7 and agree that our Neuregulin data in Figure 7M-R is suggestive that endocardial Notch signaling during cardiac injury promotes Erbb2 signaling to regulate myocardial regeneration. However, our The discussion should be extended to comment the limitations/open questions of the study and the impact of the hemodynamic response for heart regeneration compared to alternative pathways, in terms of quantitative contribution, kinetics or regionalisation. The level of similarities between fish and mouse heart repair can also be discussed, rather than assumed. The last sentence is overstated.We thank reviewer 3 for her/his suggestion. We have revised our discussion to include her/his suggestion. Please see Discussion section.


During erythropoiesis, hematopoietic stem and progenitor cells transition to erythroblasts en route to terminal differentiation into enucleated red blood cells. Transcriptome-wide changes underlie distinct morphological and functional characteristics at each cell division during this process. Many studies of gene expression have historically been carried out in erythroblasts, and the biogenesis of β-globin mRNA—the most highly expressed transcript in erythroblasts—was the focus of many seminal studies on the mechanisms of pre-mRNA splicing. We now understand that pre-mRNA splicing plays an important role in shaping the transcriptome of developing erythroblasts. Recent advances have provided insight into the role of alternative splicing and intron retention as important regulatory mechanisms of erythropoiesis. However, dysregulation of splicing during erythropoiesis is also a cause of several hematological diseases, including β-thalassemia and myelodysplastic syndromes. With a growing understanding of the role that splicing plays in these diseases, we are well poised to develop gene-editing treatments. In this review, we focus on changes in the developing erythroblast transcriptome caused by alternative splicing, the molecular basis of splicing-related blood diseases, and therapeutic advances in disease treatment using CRISPR/Cas9 gene editing. This system of transcriptional regulation is currently being exploited to discover how chromosomal regions interact and how chromatin looping might be a therapeutic target in diseases of RBCs3.Erythropoiesis is the developmental pathway by which red blood cells (RBCs)—specialized hemoglobin-containing cells that deliver oxygen throughout the body—are produced from hematopoietic stem and progenitor cells (HSPCs). Morphologically, this includes the loss of the cell nucleus and acquisition of a characteristic disk-like shape . Early m4. Globin pre-mRNA was an early model substrate for the investigation of splicing mechanisms6, and mutations in globin genes at the 5′ and 3′ boundaries of the introns—termed splice sites (5′ and 3′ SSs)—proved to be the cause of various forms of thalassemia7. Thalassemias are hemoglobin deficiencies resulting from aberrant globin expression. Some thalassemia mutations cause intron retention (IR) and lead to nonsense-mediated decay (NMD), a major gene regulatory mechanism that degrades mRNA transcripts containing premature termination codons (PTCs) present in retained introns9. Finally, other thalassemia mutations disrupt the nucleotide sequence that signals 3′ end cleavage and polyadenylation of β-globin mRNA, showing the importance of this RNA processing mechanism in health and disease. This review will focus on the common roles of splicing in diseases of erythropoiesis with an emphasis on recent insights into the transcriptomes of developing RBCs, the effects of splicing factor mutations that drive myelodysplastic syndromes (MDSs), and current efforts to restore normal globin expression in thalassemias using CRISPR/Cas-mediated genome editing.RNA biology is an area in which erythropoiesis, globin gene regulation, and disease mutations have led to fundamental discoveries. The globin genes contain two non-coding intervening sequences (introns), which are removed by the spliceosome in a process termed pre-mRNA splicing10. Transcriptome-wide profiling using RNA sequencing (RNA-seq) has allowed unbiased dissection of changes that occur along this developmental pathway11. The greatest number of changes in gene expression in human erythroblasts—either upregulation or downregulation—occurred between the late basophilic to polychromatic and polychromatic to orthochromatic stages, where roughly equal numbers of genes are upregulated as are downregulated. These transcripts were enriched for differently annotated functions reflecting cellular events in the differentiation process , which are RNA-binding proteins that regulate how the spliceosome assembles on pre-mRNA and how different 5′ and 3′ SSs are chosen13. This suggested a regulatory feedback loop, whereby changes in splicing factors could affect the splicing of many downstream genes necessary for development. Subsequent work has focused on identifying stage-specific changes in splicing transcriptome-wide using RNA-seq, a less biased approach that does not rely on known intron–exon boundaries10. In addition, mapping the gene expression networks governed by splicing in erythroid differentiation has aided in identifying the functional significance of splicing regulation14.Splicing can contribute to the regulation of transcript levels by activating cellular programs, such as NMD, to reduce transcript levels. Moreover, alternative splicing leads to the expression of different transcripts and protein products from the same gene4.1R gene, which encodes a protein that is crucial for erythrocyte membrane integrity15. Changes in expression levels and specific binding of the hnRNP A/B protein affect this developmental switch15. Since then, alternative splicing has emerged as a more widespread phenomenon18. The muscleblind-like protein 1 (MBNL1) is a sequence-specific splicing factor that undergoes extensive alternative splicing during differentiation17. Chenget al. showed that a specific Mbnl1 isoform which includes the alternative exon 5 accumulates in the nucleus in later stages of erythroid differentiation17. The inclusion Mbnl1 isoform is responsible for regulating the splicing of downstream genes important for erythroid differentiation, as knockdown of the Mbnl1 inclusion isoform alone blocked differentiation and caused defects in proliferation. Mirroring the previous findings observed by microarrays, Pimentelet al. report a program of highly dynamic alternative isoform switching in late-stage human erythroblasts using RNA-seq19. An increase in steady-state levels of transcripts containing PTCs, which likely trigger NMD of these transcripts, was observed in the later stages of differentiation, suggesting that alternative splicing coupled to NMD may be a novel, stage-specific gene regulatory mechanism.One of the first and most well-characterized examples of alternative splicing in erythropoiesis is the stage-specific inclusion of exon 16 of the23. IR was only recently recognized as a widespread occurrence25, and developing erythroid cells exhibit robust IR. Pimentelet al. showed that late human erythroblasts accumulate hundreds of transcripts containing retained introns and that these IR transcripts are enriched for splicing factors and iron-homeostasis factors26. These results were corroborated at the single-cell level in human immortalized myelogenous leukemia K562 cells27. The top three categories of nuclear IR transcripts by gene ontology analysis were RNA metabolism, RNA splicing, and the C complex spliceosome. The retained introns detected in late human erythroblasts were more likely to be found next to alternative exons that contained PTCs26, in line with previous studies suggesting that IR followed by NMD is an important mechanism that regulates levels of splicing factors28.IR is a class of alternative splicing wherein an intron is not removed by the spliceosome, potentially introducing PTCs and targeting the transcript for NMD. Alternatively, it is possible that certain intron-retained transcripts remain in the nucleus and undergo splicing with delayed kinetics26. Indeed, a series of highly conserved cryptic SSs were identified for their activity in promoting IR in SF3B1 transcripts29. The identified intronic sequences are sufficient to promote IR in SF3B1 and can also promote retention when inserted into other introns. The cryptic exons generated by these SSs are proposed to act as splicing decoys, sequestering components of the spliceosome and ultimately preventing productive splicing by blocking the appropriate cross-intron interactions needed to define the intron for splicing. Alternatively, reduced levels of SF3B1 might preferentially affect splicing efficiencies or the half-lives of (pre-)mRNAs encoding splicing factors or both. Although we presume that most of these instances reflect the downregulation of IR transcripts, the possibility that certain splicing events are delayed remains. Interestingly, delaying gene expression through IR is physiologically relevant in other cell types, including developing spermatocytes, neuronal cells, platelets, granulocytes, and stimulated macrophages33. In the case of erythroid differentiation, how introns are retained in a seemingly stage-specific and cell type-specific way remains to be fully understood.How is IR triggered during erythropoiesis? Key insights are emerging from studies of transcripts encoding the important core splicing factor SF3B1. SF3B1 expression is also subject to IR during erythroid differentiation suggesting that SF3B1 regulation by IR may constitute a regulatory hub leading to the downregulation of transcripts encoding other splicing factors37. Generally, mutations in either cis or trans can affect splicing outcomes. Cis mutations may disrupt the intrinsic sequences that demarcate SSs in a transcript (5′ and 3′ SSs). In contrast, mutations in any number of the core spliceosome machinery can produce splicing defects in trans, which causes deleterious effects for a large number of downstream splicing substrates. Both types of splicing defects have been characterized in the erythroid lineage38.Misregulation of splicing underlies a growing number of human diseases39. β-thalassemia is one of the most prevalent diseases caused by somatic mutations worldwide, yet currently the only available curative treatment is an allogenic transplant of HSPCs from a matched donor. This option is unavailable for many patients because of the cost of treatment and limited availability of matched donors. Although we possess a quite-thorough understanding of the molecular basis and pathophysiology of this disease, better treatments are still sorely needed. Many β-thalassemia patients are dependent on transfusions from blood donors to maintain proper levels of healthy, circulating RBCs. However, this therapy often leads to complications related to iron overload, including organ damage. The majority of β-globin alleles that cause thalassemia contain point mutations  during spliceosome assembly and helps to identify SSs that are used in catalysis. SF3B1 mutations are generally associated with disease, and the most common point mutant found in subtypes of MDS is70. SRSF2 is essential for hematopoiesis, andSrsf2 gene knockout in mice is embryonic lethal71. In one study, the P95H mutant was found to disrupt the recognition of exonic splicing enhancer regions, and in the case of the oncogeneEzh2, this resulted in the inclusion of a toxic exon and a decrease inEzh2 transcript levels72. Similarly, the P95H mutant expressed in an MDS cell line was shown to specifically affect alternative splicing of genes that are important for cancer development and apoptosis71. These results were generally corroborated with a conditional knock-in mouse model to look at steady-state effects rather than in the regenerative context of lethally irradiated mice as in previous mouse models73. Konet al. found increased occurrences of alternative splicing in about 20% of transcripts73. However, in these mice, there were only a few differentially spliced genes that were targets for NMD, and no change in the expression ofEzh2, suggesting that the context of regenerative stress may have an impact on the role of SRSF2 in promoting aberrant alternative splicing. Using a technique that measure protein–RNA interactions genome-wide, SRSF2 P95H was found to differentially bind alternative exons, but interestingly this does not predict the outcome of alternative splicing74. The genes most affected were RNA processing factors, suggesting a cascade effect, where mutant SRSF2 promotes misregulation of splicing factors and thereby causes aberrant splicing and downregulation of a host of downstream targets.SRSF2 is a member of the serine/arginine (SR)-rich family of proteins, which are involved in regulating SS selection for both constitutive and alternative splicing by binding exonic sequence enhancer. Multiple lines of evidence support a role for mutant SRSF2 in driving myelodysplasias by promoting changes in alternative splicing. In a K562 cell line expressing the most common P95H mutation, alternative splicing events were correlated directly with the altered ability of the P95H mutant to bind a specific RNA motifde novo MDS, and this mutation promoted exon skipping in a minigene reporter75. RNA-seq data from patients with this same mutation showed again a small increase in exon skipping, and the nucleotide just upstream of the 3′ SS was a predictor of exon usage for mutant but not wild-type splicing76. These results were verified by using RNA-seq data from 167 patients with acute myeloid leukemia, confirming that U2AF1 S34F/Y mutants show a preference for CAG over UAG 3′ SSs, causing downstream aberrant splicing events77. However, in both of these cases, the downstream targets with altered splicing were variable and did not contain much overlap.U2AF1  binds to most 3′SSs, assisting the assembling spliceosome in identification of the 3′SS to be used during splicing catalysis. Unbiased whole-genome sequencing found the S34F mutation in around 9% of 150 patients withet al. developed a doxycycline-inducible transgenic mouse carrying the allele in the myeloid, lymphoid, stem, and progenitor cell lineages78. After transplant of the transgenic bone marrow to lethally irradiated mice, peripheral blood total white blood cell counts were decreased, as was the case for B cells and monocytes78. However, there was no change in RBC or platelet counts, and the mice showed no evidence of bone marrow dysplasia and did not develop MDS or acute myeloid leukemia after one year of transgenicU2af1 S34F expression. By analyzing transcriptome sequences of myeloid progenitors from the induced transgenic mice, they identified 742 cases of alternate splicing junctions, and these were mostly alternate 3′ SS usage with an enrichment for CAG 3′ SS sequence over UAG in wild-type controls. Finally, in an attempt to link altered splicing patterns to the cause of myeloid disease, Parket al. derived cell lines with the S34F mutation and showed that, in addition to aberrant alternative splicing, many transcripts are alternately processed at the 3′ end, specifically through use of a distal cleavage and polyadenylation site79. They show that one such alternatively processed transcript,Atg7, causes an autophagy defect that may explain how changes in splicing and RNA processing can drive oncogenic transformation. MDS patients harboring the S34F mutation had increased levels ofAtg7 mRNA with the distal cleavage and polyadenylation site. Importantly, the authors show that use of the distal site results in translation repression and that the corresponding decrease in ATG7 protein levels is sufficient to transform cells.To investigate the effects of expressing the S34F mutant of U2AF1 in an animal model, Shirai52. One unifying mechanism proposed by Chenet al. is that while mutations in splicing factors cause distinct splicing defects, they cause a common replication stress by triggering elevated R-loop formation80. The authors show that increased R-loops compromise hematopoiesis and that resolution of R-loops by overexpression of RNase H partially rescues proliferation. This is an intriguing mechanism by which splicing factors may play auxiliary roles in transcriptional regulation. They propose that this insult to genomic stability may be a common mechanism which makes progression to more severe disease phenotypes more likely when other mutations are coexisting. Retained introns have been proposed to act as substrates for R-loop formation, contributing to genomic instability and DNA damage in the case of severe spinal muscular atrophy81. More work will need to be done to understand the contribution of both the splicing and the genomic stability defects of mutant splicing factor alleles on MDS.The recent discovery of splicing factors as commonly mutated proteins in MDS was unexpected. Although these mutations affect proteins in a common pathway, the outcome in each case is a distinct pattern of altered splicing. Additionally, these mutations are associated with variable prognoses and subclasses of MDS. SF3B1 is strongly associated with a ringed sideroblast phenotype and a relatively positive prognosis, whereas U2AF1 and SRSF2 are associated with more advanced myelomonocytic leukemia and with worse outcomes4.1R transcript82, a signature alternative splicing event in RBC development (see above). Naturally occurring human variants in RBM38 expression were discovered through a high-throughput genome-wide association study screen and validated to show changes in a subset of genes that are alternatively spliced during terminal erythropoiesis83. Interestingly, RBM38 was additionally shown to interact with eIF4G in the cytoplasm to enhance the translation of a subset of mRNAs in terminal differentiation84. Moreover, an alternatively spliced isoform of the transcriptional repressor GFI1B produced by a natural variant causes defects in megakaryopoiesis but not erythropoiesis, suggesting that variation among individuals can play a role in developmental dynamics85. Future investigations should illuminate further connections among alternative splicing, transcription, and translation during hematopoiesis.Mutations in several other RNA-binding proteins have been proposed to contribute to misregulation of splicing during erythropoiesis. For example, RBM38 has been shown to regulate alternative splicing in terminal erythropoiesis, specifically activating inclusion of exon 16 of the19 and IR29 as major regulatory mechanisms associated with differentiation will no doubt yield insight into the complexity of how tissue-specific changes in splicing are relayed to affect gene expression. Mutations affecting core spliceosomal proteins  as well as genes important for mature RBC function  have revealed aberrant splicing which leads to hindered erythropoiesis, often in unexpected ways. Promising treatments for MDS, in particular, rely on modulating splicing as a mechanism to specifically target defective splicing in the myeloid lineage88. Many questions remain as to how mutations in ubiquitous splicing factors specifically cause defects in the myeloid lineage and what the molecular mechanism is for how downstream targets of these alternatively spliced transcripts contribute to disease progression.Erythropoiesis provides an excellent model in which to study RNA splicing in both healthy and diseased states. Recent work identifying alternative splicingBPS, branch point sequence; HSPC, hematopoietic stem and progenitor cell; IR, intron retention; MBNL1, muscleblind-like protein 1; MDS, myelodysplastic syndrome; NMD, nonsense-mediated decay; PTC, premature termination codon; RBC, red blood cell; RNA-seq, RNA sequencing; SS, splice site