Link to full list of manuscripts
2024
Goumenaki, Pinelopi; Günther, Stefan; Kikhi, Khrievono; Looso, Mario; Marín-Juez, Rubén; Stainier, Didier Y. R.
The innate immune regulator MyD88 dampens fibrosis during zebrafish heart regeneration Journal Article
In: Nature Cardiovascular Research, vol. 3, iss. 9, pp. 1158–1176, 2024.
@article{nokeyp,
title = {The innate immune regulator MyD88 dampens fibrosis during zebrafish heart regeneration},
author = {Pinelopi Goumenaki and Stefan Günther and Khrievono Kikhi and Mario Looso and Rubén Marín-Juez and Didier Y. R. Stainier},
doi = { 10.1038/s44161-024-00538-5},
year = {2024},
date = {2024-09-13},
urldate = {2024-09-13},
journal = {Nature Cardiovascular Research},
volume = {3},
issue = {9},
pages = {1158–1176},
abstract = {The innate immune response is triggered rapidly after injury and its spatiotemporal dynamics are critical for regeneration; however, many questions remain about its exact role. Here we show that MyD88, a key component of the innate immune response, controls not only the inflammatory but also the fibrotic response during zebrafish cardiac regeneration. We find in cryoinjured myd88−/− ventricles a significant reduction in neutrophil and macrophage numbers and the expansion of a collagen-rich endocardial population. Further analyses reveal compromised PI3K/AKT pathway activation in the myd88−/− endocardium and increased myofibroblasts and scarring. Notably, endothelial-specific overexpression of myd88 reverses these neutrophil, fibrotic and scarring phenotypes. Mechanistically, we identify the endocardial-derived chemokine gene cxcl18b as a target of the MyD88 signaling pathway, and using loss-of-function and gain-of-function tools, we show that it controls neutrophil recruitment. Altogether, these findings shed light on the pivotal role of MyD88 in modulating inflammation and fibrosis during tissue regeneration.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The innate immune response is triggered rapidly after injury and its spatiotemporal dynamics are critical for regeneration; however, many questions remain about its exact role. Here we show that MyD88, a key component of the innate immune response, controls not only the inflammatory but also the fibrotic response during zebrafish cardiac regeneration. We find in cryoinjured myd88−/− ventricles a significant reduction in neutrophil and macrophage numbers and the expansion of a collagen-rich endocardial population. Further analyses reveal compromised PI3K/AKT pathway activation in the myd88−/− endocardium and increased myofibroblasts and scarring. Notably, endothelial-specific overexpression of myd88 reverses these neutrophil, fibrotic and scarring phenotypes. Mechanistically, we identify the endocardial-derived chemokine gene cxcl18b as a target of the MyD88 signaling pathway, and using loss-of-function and gain-of-function tools, we show that it controls neutrophil recruitment. Altogether, these findings shed light on the pivotal role of MyD88 in modulating inflammation and fibrosis during tissue regeneration.
Wang, Zhen-Yu; Mehra, Armaan; Wang, Qian-Chen; Gupta, Savita; da Silva, Agatha Ribeiro; Juan, Thomas; Günther, Stefan; Looso, Mario; Detleffsen, Jan; Stainier, Didier Y. R.; Marín-Juez, Rubén
flt1 inactivation promotes zebrafish cardiac regeneration by enhancing endothelial activity and limiting the fibrotic response Journal Article
In: bioRxiv, 2024.
@article{nokeyq,
title = {flt1 inactivation promotes zebrafish cardiac regeneration by enhancing endothelial activity and limiting the fibrotic response},
author = {Zhen-Yu Wang and Armaan Mehra and Qian-Chen Wang and Savita Gupta and Agatha Ribeiro da Silva and Thomas Juan and Stefan Günther and Mario Looso and Jan Detleffsen and Didier Y.R. Stainier and Rubén Marín-Juez},
doi = {10.1101/2024.09.11.612516},
year = {2024},
date = {2024-09-11},
journal = {bioRxiv},
abstract = {VEGFA administration has been explored as a pro-angiogenic therapy for cardiovascular diseases including heart failure for several years, but with little success. Here we investigate a different approach to augment VEGFA bioavailability: by deleting the VEGFA decoy receptor VEGFR1/FLT1, one can achieve more physiological VEGFA concentrations. We find that following cryoinjury, zebrafish flt1 mutant hearts display enhanced coronary revascularization and endocardial expansion, increased cardiomyocyte dedifferentiation and proliferation, and decreased scarring. Suppressing Vegfa signaling in flt1 mutants abrogates these beneficial effects of flt1 deletion. Transcriptomic analyses of cryoinjured flt1 mutant hearts reveal enhanced endothelial MAPK/ERK signaling and downregulation of the transcription factor gene egr3. Using newly generated genetic tools, we observe egr3 upregulation in the regenerating endocardium, and find that Egr3 promotes myofibroblast differentiation. These data indicate that with enhanced Vegfa bioavailability, the endocardium limits myofibroblast differentiation via egr3 downregulation, thereby providing a more permissive microenvironment for cardiomyocyte replenishment after injury.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
VEGFA administration has been explored as a pro-angiogenic therapy for cardiovascular diseases including heart failure for several years, but with little success. Here we investigate a different approach to augment VEGFA bioavailability: by deleting the VEGFA decoy receptor VEGFR1/FLT1, one can achieve more physiological VEGFA concentrations. We find that following cryoinjury, zebrafish flt1 mutant hearts display enhanced coronary revascularization and endocardial expansion, increased cardiomyocyte dedifferentiation and proliferation, and decreased scarring. Suppressing Vegfa signaling in flt1 mutants abrogates these beneficial effects of flt1 deletion. Transcriptomic analyses of cryoinjured flt1 mutant hearts reveal enhanced endothelial MAPK/ERK signaling and downregulation of the transcription factor gene egr3. Using newly generated genetic tools, we observe egr3 upregulation in the regenerating endocardium, and find that Egr3 promotes myofibroblast differentiation. These data indicate that with enhanced Vegfa bioavailability, the endocardium limits myofibroblast differentiation via egr3 downregulation, thereby providing a more permissive microenvironment for cardiomyocyte replenishment after injury.
Gupta, Savita; Bajwa, Gursimran Kaur; El-Sammak, Hadil; Mattonet, Kenny; Günther, Stefan; Looso, Mario; Stainier, Didier Y. R.; Marín-Juez, Rubén
The transmembrane glycoprotein Gpnmb is required for the immune and fibrotic responses during zebrafish heart regeneration Journal Article
In: bioRxiv, 2024.
@article{nokeyr,
title = {The transmembrane glycoprotein Gpnmb is required for the immune and fibrotic responses during zebrafish heart regeneration},
author = {Savita Gupta and Gursimran Kaur Bajwa and Hadil El-Sammak and Kenny Mattonet and Stefan Günther and Mario Looso and Didier Y. R. Stainier and Rubén Marín-Juez},
doi = {10.1101/2024.09.11.612527},
year = {2024},
date = {2024-09-11},
journal = {bioRxiv},
abstract = {Myocardial infarction occurs when coronary supply of oxygen and nutrients to part of the heart is interrupted. In contrast to adult mammals, adult zebrafish have a unique ability to regenerate their heart after cardiac injury. Several processes are involved in this regenerative response including inflammation, coronary endothelial cell proliferation and revascularization, endocardial expansion, cardiomyocyte repopulation, and transient scar formation. To identify potential regulators of zebrafish cardiac regeneration, we profiled the transcriptome of regenerating coronary endothelial cells at 7 days post cryoinjury (dpci) and observed the significant upregulation of dozens of genes including gpnmb. Gpnmb (glycoprotein non-metastatic melanoma protein B) is a transmembrane glycoprotein implicated in inflammation resolution and tissue regeneration. Transcriptomic profiling data of cryoinjured zebrafish hearts reveal that gpnmb is mostly expressed by macrophages. To investigate gpnmb function during zebrafish cardiac regeneration, we generated a full locus deletion (FLD) allele. We find that after cardiac cryoinjury, animals lacking gpnmb exhibit neutrophil retention and decreased macrophage recruitment as well as reduced myofibroblast numbers. Moreover, loss of gpnmb impairs coronary endothelial cell regeneration and cardiomyocyte dedifferentiation. Transcriptomic analyses of cryoinjured gpnmb mutant hearts identified enhanced collagen gene expression and the activation of extracellular matrix (ECM) related pathways. Furthermore, gpnmb mutant hearts exhibit larger fibrotic scars revealing additional defects in cardiac regeneration. Altogether, these data indicate that gpnmb expressing macrophages modulate inflammation and ECM deposition after cardiac cryoinjury in zebrafish and further highlight the importance of this subset of immune cells to support a regenerative response.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Myocardial infarction occurs when coronary supply of oxygen and nutrients to part of the heart is interrupted. In contrast to adult mammals, adult zebrafish have a unique ability to regenerate their heart after cardiac injury. Several processes are involved in this regenerative response including inflammation, coronary endothelial cell proliferation and revascularization, endocardial expansion, cardiomyocyte repopulation, and transient scar formation. To identify potential regulators of zebrafish cardiac regeneration, we profiled the transcriptome of regenerating coronary endothelial cells at 7 days post cryoinjury (dpci) and observed the significant upregulation of dozens of genes including gpnmb. Gpnmb (glycoprotein non-metastatic melanoma protein B) is a transmembrane glycoprotein implicated in inflammation resolution and tissue regeneration. Transcriptomic profiling data of cryoinjured zebrafish hearts reveal that gpnmb is mostly expressed by macrophages. To investigate gpnmb function during zebrafish cardiac regeneration, we generated a full locus deletion (FLD) allele. We find that after cardiac cryoinjury, animals lacking gpnmb exhibit neutrophil retention and decreased macrophage recruitment as well as reduced myofibroblast numbers. Moreover, loss of gpnmb impairs coronary endothelial cell regeneration and cardiomyocyte dedifferentiation. Transcriptomic analyses of cryoinjured gpnmb mutant hearts identified enhanced collagen gene expression and the activation of extracellular matrix (ECM) related pathways. Furthermore, gpnmb mutant hearts exhibit larger fibrotic scars revealing additional defects in cardiac regeneration. Altogether, these data indicate that gpnmb expressing macrophages modulate inflammation and ECM deposition after cardiac cryoinjury in zebrafish and further highlight the importance of this subset of immune cells to support a regenerative response.
2022
Chowdhury, Kaushik; Lai, Shih-Lei; Marín-Juez, Rubén
Modulation of VEGFA Signaling During Heart Regeneration in Zebrafish Book Chapter
In: in Molecular Biology, Methods; Fiedler, Lorna R.; Pellet-Many, Caroline (Ed.): vol. 2475, pp. 297-312, Humana New York, NY, 2, 2022.
@inbook{Chowdhury2022,
title = {Modulation of VEGFA Signaling During Heart Regeneration in Zebrafish},
author = {Kaushik Chowdhury and Shih-Lei Lai and Rubén Marín-Juez},
editor = {Methods in Molecular Biology and Lorna R. Fiedler and Caroline Pellet-Many},
doi = {10.1007/978-1-0716-2217-9_22},
year = {2022},
date = {2022-04-21},
urldate = {2022-04-21},
volume = {2475},
pages = {297-312},
publisher = {Humana New York},
address = {NY},
edition = {2},
series = {Methods in Molecular Biology},
abstract = {Over the last decades, myocardial infarction and heart failure have accounted every year for millions of deaths worldwide. After a coronary occlusion, the lack of blood supply to downstream muscle leads to cell death and scarring. To date, several pro-angiogenic factors have been tested to stimulate reperfusion of the affected myocardium, VEGFA being one of the most extensively studied. Given the unsuccessful outcomes of clinical trials, understanding how cardiac revascularization takes place in models with endogenous regenerative capacity holds the key to devising more efficient therapies. Here, we summarize the main findings on VEGFA's role during cardiac repair and regeneration, with a particular focus on zebrafish as a regenerative model. Moreover, we provide a comprehensive overview of available tools to modulate Vegfa expression and action in zebrafish regeneration studies. Understanding the role of Vegfa during zebrafish heart regeneration may help devise efficient therapies and circumvent current limitations in using VEGFA for therapeutic angiogenesis approaches.},
keywords = {},
pubstate = {published},
tppubtype = {inbook}
}
Over the last decades, myocardial infarction and heart failure have accounted every year for millions of deaths worldwide. After a coronary occlusion, the lack of blood supply to downstream muscle leads to cell death and scarring. To date, several pro-angiogenic factors have been tested to stimulate reperfusion of the affected myocardium, VEGFA being one of the most extensively studied. Given the unsuccessful outcomes of clinical trials, understanding how cardiac revascularization takes place in models with endogenous regenerative capacity holds the key to devising more efficient therapies. Here, we summarize the main findings on VEGFA's role during cardiac repair and regeneration, with a particular focus on zebrafish as a regenerative model. Moreover, we provide a comprehensive overview of available tools to modulate Vegfa expression and action in zebrafish regeneration studies. Understanding the role of Vegfa during zebrafish heart regeneration may help devise efficient therapies and circumvent current limitations in using VEGFA for therapeutic angiogenesis approaches.
El-Sammak, Hadil; Yang, Bingyuan; Guenther, Stefan; Chen, Wenbiao; Marín-Juez, Rubén; Stainier, Didier Y R
A Vegfc-Emilin2a-Cxcl8a Signaling Axis Required for Zebrafish Cardiac Regeneration Journal Article
In: Circulation Research, vol. 130, iss. 7, pp. 1014-1029, 2022.
@article{nokey,
title = {A Vegfc-Emilin2a-Cxcl8a Signaling Axis Required for Zebrafish Cardiac Regeneration},
author = {Hadil El-Sammak and Bingyuan Yang and Stefan Guenther and Wenbiao Chen and Rubén Marín-Juez and Didier Y R Stainier},
doi = { 10.1161/CIRCRESAHA.121.319929},
year = {2022},
date = {2022-04-01},
urldate = {2022-04-01},
journal = {Circulation Research},
volume = {130},
issue = {7},
pages = {1014-1029},
abstract = {Background: Ischemic heart disease following the obstruction of coronary vessels leads to the death of cardiac tissue and the formation of a fibrotic scar. In contrast to adult mammals, zebrafish can regenerate their heart after injury, enabling the study of the underlying mechanisms. One of the earliest responses following cardiac injury in adult zebrafish is coronary revascularization. Defects in this process lead to impaired cardiomyocyte repopulation and scarring. Hence, identifying and investigating factors that promote coronary revascularization holds great therapeutic potential.
Methods: We used wholemount imaging, immunohistochemistry and histology to assess various aspects of zebrafish cardiac regeneration. Deep transcriptomic analysis allowed us to identify targets and potential effectors of Vegfc (vascular endothelial growth factor C) signaling. We used newly generated loss- and gain-of-function genetic tools to investigate the role of Emilin2a (elastin microfibril interfacer 2a) and Cxcl8a (chemokine (C-X-C) motif ligand 8a)-Cxcr1 (chemokine (C-X-C) motif receptor 1) signaling in cardiac regeneration.
Results: We first show that regenerating coronary endothelial cells upregulate vegfc upon cardiac injury in adult zebrafish and that Vegfc signaling is required for their proliferation during regeneration. Notably, blocking Vegfc signaling also significantly reduces cardiomyocyte dedifferentiation and proliferation. Using transcriptomic analyses, we identified emilin2a as a target of Vegfc signaling and found that manipulation of emilin2a expression can modulate coronary revascularization as well as cardiomyocyte proliferation. Mechanistically, Emilin2a induces the expression of the chemokine gene cxcl8a in epicardium-derived cells, while cxcr1, the Cxcl8a receptor gene, is expressed in coronary endothelial cells. We further show that Cxcl8a-Cxcr1 signaling is also required for coronary endothelial cell proliferation during cardiac regeneration.
Conclusions: These data show that after cardiac injury, coronary endothelial cells upregulate vegfc to promote coronary network reestablishment and cardiac regeneration. Mechanistically, Vegfc signaling upregulates epicardial emilin2a and cxcl8a expression to promote cardiac regeneration. These studies aid in understanding the mechanisms underlying coronary revascularization in zebrafish, with potential therapeutic implications to enhance revascularization and regeneration in injured human hearts.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Background: Ischemic heart disease following the obstruction of coronary vessels leads to the death of cardiac tissue and the formation of a fibrotic scar. In contrast to adult mammals, zebrafish can regenerate their heart after injury, enabling the study of the underlying mechanisms. One of the earliest responses following cardiac injury in adult zebrafish is coronary revascularization. Defects in this process lead to impaired cardiomyocyte repopulation and scarring. Hence, identifying and investigating factors that promote coronary revascularization holds great therapeutic potential.
Methods: We used wholemount imaging, immunohistochemistry and histology to assess various aspects of zebrafish cardiac regeneration. Deep transcriptomic analysis allowed us to identify targets and potential effectors of Vegfc (vascular endothelial growth factor C) signaling. We used newly generated loss- and gain-of-function genetic tools to investigate the role of Emilin2a (elastin microfibril interfacer 2a) and Cxcl8a (chemokine (C-X-C) motif ligand 8a)-Cxcr1 (chemokine (C-X-C) motif receptor 1) signaling in cardiac regeneration.
Results: We first show that regenerating coronary endothelial cells upregulate vegfc upon cardiac injury in adult zebrafish and that Vegfc signaling is required for their proliferation during regeneration. Notably, blocking Vegfc signaling also significantly reduces cardiomyocyte dedifferentiation and proliferation. Using transcriptomic analyses, we identified emilin2a as a target of Vegfc signaling and found that manipulation of emilin2a expression can modulate coronary revascularization as well as cardiomyocyte proliferation. Mechanistically, Emilin2a induces the expression of the chemokine gene cxcl8a in epicardium-derived cells, while cxcr1, the Cxcl8a receptor gene, is expressed in coronary endothelial cells. We further show that Cxcl8a-Cxcr1 signaling is also required for coronary endothelial cell proliferation during cardiac regeneration.
Conclusions: These data show that after cardiac injury, coronary endothelial cells upregulate vegfc to promote coronary network reestablishment and cardiac regeneration. Mechanistically, Vegfc signaling upregulates epicardial emilin2a and cxcl8a expression to promote cardiac regeneration. These studies aid in understanding the mechanisms underlying coronary revascularization in zebrafish, with potential therapeutic implications to enhance revascularization and regeneration in injured human hearts.
Methods: We used wholemount imaging, immunohistochemistry and histology to assess various aspects of zebrafish cardiac regeneration. Deep transcriptomic analysis allowed us to identify targets and potential effectors of Vegfc (vascular endothelial growth factor C) signaling. We used newly generated loss- and gain-of-function genetic tools to investigate the role of Emilin2a (elastin microfibril interfacer 2a) and Cxcl8a (chemokine (C-X-C) motif ligand 8a)-Cxcr1 (chemokine (C-X-C) motif receptor 1) signaling in cardiac regeneration.
Results: We first show that regenerating coronary endothelial cells upregulate vegfc upon cardiac injury in adult zebrafish and that Vegfc signaling is required for their proliferation during regeneration. Notably, blocking Vegfc signaling also significantly reduces cardiomyocyte dedifferentiation and proliferation. Using transcriptomic analyses, we identified emilin2a as a target of Vegfc signaling and found that manipulation of emilin2a expression can modulate coronary revascularization as well as cardiomyocyte proliferation. Mechanistically, Emilin2a induces the expression of the chemokine gene cxcl8a in epicardium-derived cells, while cxcr1, the Cxcl8a receptor gene, is expressed in coronary endothelial cells. We further show that Cxcl8a-Cxcr1 signaling is also required for coronary endothelial cell proliferation during cardiac regeneration.
Conclusions: These data show that after cardiac injury, coronary endothelial cells upregulate vegfc to promote coronary network reestablishment and cardiac regeneration. Mechanistically, Vegfc signaling upregulates epicardial emilin2a and cxcl8a expression to promote cardiac regeneration. These studies aid in understanding the mechanisms underlying coronary revascularization in zebrafish, with potential therapeutic implications to enhance revascularization and regeneration in injured human hearts.
2021
Boskovic, Srdjan; Juez, Rubén Marín; Stamenkovic, Nemanja; Radojkovic, Dragica; Stainier, Didier YR; Kojic, Snezana
In: Gene, vol. 792, pp. 145725, 2021.
@article{nokey,
title = {The stress responsive gene ankrd1a is dynamically regulated during skeletal muscle development and upregulated following cardiac injury in border zone cardiomyocytes in adult zebrafish},
author = {Srdjan Boskovic and Rubén Marín Juez and Nemanja Stamenkovic and Dragica Radojkovic and Didier YR Stainier and Snezana Kojic},
doi = {10.1016/j.gene.2021.145725},
year = {2021},
date = {2021-08-05},
journal = {Gene},
volume = {792},
pages = {145725},
abstract = {Ankyrin repeat domain 1 (ANKRD1) is a functionally pleiotropic protein found in the nuclei and sarcomeres of cardiac and skeletal muscles, with a proposed role in linking myofibrilar stress and transcriptional regulation. Rapid upregulation of its expression in response to both physiological and pathological stress supports the involvement of ANKRD1 in muscle tissue adaptation and remodeling. However, the exact role of ANKRD1 remains poorly understood. To begin to investigate its function at higher resolution, we have generated and characterized a TgBAC(ankrd1a:EGFP) zebrafish line. This reporter line displays transgene expression in slow skeletal muscle fibers during development and exercise responsiveness in adult cardiac muscle. To better understand the role of Ankrd1a in pathological conditions in adult zebrafish, we assessed ankrd1a expression after cardiac ventricle cryoinjury and observed localized upregulation in cardiomyocytes in the border zone. We show that this expression in injured hearts is recapitulated by the TgBAC(ankrd1a:EGFP) reporter. Our results identify novel expression domains of ankrd1a and suggest an important role for Ankrd1a in the early stress response and regeneration of cardiac tissue. This new reporter line will help decipher the role of Ankrd1a in striated muscle stress response, including after cardiac injury.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Ankyrin repeat domain 1 (ANKRD1) is a functionally pleiotropic protein found in the nuclei and sarcomeres of cardiac and skeletal muscles, with a proposed role in linking myofibrilar stress and transcriptional regulation. Rapid upregulation of its expression in response to both physiological and pathological stress supports the involvement of ANKRD1 in muscle tissue adaptation and remodeling. However, the exact role of ANKRD1 remains poorly understood. To begin to investigate its function at higher resolution, we have generated and characterized a TgBAC(ankrd1a:EGFP) zebrafish line. This reporter line displays transgene expression in slow skeletal muscle fibers during development and exercise responsiveness in adult cardiac muscle. To better understand the role of Ankrd1a in pathological conditions in adult zebrafish, we assessed ankrd1a expression after cardiac ventricle cryoinjury and observed localized upregulation in cardiomyocytes in the border zone. We show that this expression in injured hearts is recapitulated by the TgBAC(ankrd1a:EGFP) reporter. Our results identify novel expression domains of ankrd1a and suggest an important role for Ankrd1a in the early stress response and regeneration of cardiac tissue. This new reporter line will help decipher the role of Ankrd1a in striated muscle stress response, including after cardiac injury.
Tsedeke, Ayele Taddese; Allanki, Srinivas; Gentile, Alessandra; Jimenez-Amilburu, Vanesa; Rasouli, Seyed Javad; Guenther, Stefan; Lai, Shih-Lei; Stainier, Didier Y R; Marín-Juez, Rubén
Cardiomyocyte heterogeneity during zebrafish development and regeneration Journal Article
In: Developmental Biology, vol. 476, pp. 259-271, 2021.
@article{nokey,
title = {Cardiomyocyte heterogeneity during zebrafish development and regeneration},
author = {Ayele Taddese Tsedeke and Srinivas Allanki and Alessandra Gentile and Vanesa Jimenez-Amilburu and Seyed Javad Rasouli and Stefan Guenther and Shih-Lei Lai and Didier Y R Stainier and Rubén Marín-Juez},
doi = {10.1016/j.ydbio.2021.03.014},
year = {2021},
date = {2021-08-01},
journal = {Developmental Biology},
volume = {476},
pages = {259-271},
abstract = {Contrary to adult mammals, zebrafish are able to regenerate their heart after cardiac injury. This regenerative response relies, in part, on the endogenous ability of cardiomyocytes (CMs) to dedifferentiate and proliferate to replenish the lost muscle. However, CM heterogeneity and population dynamics during development and regeneration require further investigation. Through comparative transcriptomic analyses of the developing and adult zebrafish heart, we identified tnnc2 and tnni4b.3 expression as markers for CMs at early and late developmental stages, respectively. Using newly developed reporter lines for these genes, we investigated their expression dynamics during heart development and regeneration. tnnc2 reporter lines label most CMs at embryonic stages, and this labeling declines rapidly during larval stages; in adult hearts, tnnc2 reporter expression is only detectable in a small subset of CMs. Conversely, expression of a tnni4b.3 reporter is initially visible in CMs in the outer curvature of the ventricle at larval stages, and it is subsequently present in a vast majority of the CMs in adult hearts. To further characterize the adult CMs labeled by the tnnc2 (i.e., embryonic) reporter, we performed transcriptomic analyses and found that they express markers of immature CMs as well as genes encoding components of the Notch signaling pathway. In support of this finding, we observed, using two different reporters, that these CMs display higher levels of Notch signaling. Moreover, during adult heart regeneration, CMs in the injured area activate the embryonic CM reporter and downregulate the tnni4b.3 reporter, further highlighting the molecular changes in regenerating CMs. Overall, our findings provide additional evidence for CM heterogeneity in adult zebrafish.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Contrary to adult mammals, zebrafish are able to regenerate their heart after cardiac injury. This regenerative response relies, in part, on the endogenous ability of cardiomyocytes (CMs) to dedifferentiate and proliferate to replenish the lost muscle. However, CM heterogeneity and population dynamics during development and regeneration require further investigation. Through comparative transcriptomic analyses of the developing and adult zebrafish heart, we identified tnnc2 and tnni4b.3 expression as markers for CMs at early and late developmental stages, respectively. Using newly developed reporter lines for these genes, we investigated their expression dynamics during heart development and regeneration. tnnc2 reporter lines label most CMs at embryonic stages, and this labeling declines rapidly during larval stages; in adult hearts, tnnc2 reporter expression is only detectable in a small subset of CMs. Conversely, expression of a tnni4b.3 reporter is initially visible in CMs in the outer curvature of the ventricle at larval stages, and it is subsequently present in a vast majority of the CMs in adult hearts. To further characterize the adult CMs labeled by the tnnc2 (i.e., embryonic) reporter, we performed transcriptomic analyses and found that they express markers of immature CMs as well as genes encoding components of the Notch signaling pathway. In support of this finding, we observed, using two different reporters, that these CMs display higher levels of Notch signaling. Moreover, during adult heart regeneration, CMs in the injured area activate the embryonic CM reporter and downregulate the tnni4b.3 reporter, further highlighting the molecular changes in regenerating CMs. Overall, our findings provide additional evidence for CM heterogeneity in adult zebrafish.
2020
Ding, Yi; Raterink, Robert-Jan; Marín-Juez, Rubén; Veneman, Wouter J; Egbers, Koen; van den Eeden, Susan; Haks, Mariëlle C; Joosten, Simone A; Ottenhoff, Tom H M; Harms, Amy C; Alia, A; Hankemeier, Thomas; Spaink, Herman P
Tuberculosis causes highly conserved metabolic changes in human patients, mycobacteria-infected mice and zebrafish larvae Journal Article
In: Scientific Reports, vol. 10, iss. 1, no. 11635, 2020.
@article{nokey,
title = {Tuberculosis causes highly conserved metabolic changes in human patients, mycobacteria-infected mice and zebrafish larvae},
author = {Yi Ding and Robert-Jan Raterink and Rubén Marín-Juez and Wouter J Veneman and Koen Egbers and Susan van den Eeden and Mariëlle C Haks and Simone A Joosten and Tom H M Ottenhoff and Amy C Harms and A Alia and Thomas Hankemeier and Herman P Spaink},
doi = {10.1038/s41598-020-68443-y},
year = {2020},
date = {2020-07-15},
urldate = {2020-07-15},
journal = {Scientific Reports},
volume = {10},
number = {11635},
issue = {1},
abstract = {Tuberculosis is a highly infectious and potentially fatal disease accompanied by wasting symptoms, which cause severe metabolic changes in infected people. In this study we have compared the effect of mycobacteria infection on the level of metabolites in blood of humans and mice and whole zebrafish larvae using one highly standardized mass spectrometry pipeline, ensuring technical comparability of the results. Quantification of a range of circulating small amines showed that the levels of the majority of these compounds were significantly decreased in all three groups of infected organisms. Ten of these metabolites were common between the three different organisms comprising: methionine, asparagine, cysteine, threonine, serine, tryptophan, leucine, citrulline, ethanolamine and phenylalanine. The metabolomic changes of zebrafish larvae after infection were confirmed by nuclear magnetic resonance spectroscopy. Our study identified common biomarkers for tuberculosis disease in humans, mice and zebrafish, showing across species conservation of metabolic reprogramming processes as a result of disease. Apparently, the mechanisms underlying these processes are independent of environmental, developmental and vertebrate evolutionary factors. The zebrafish larval model is highly suited to further investigate the mechanism of metabolic reprogramming and the connection with wasting syndrome due to infection by mycobacteria.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Tuberculosis is a highly infectious and potentially fatal disease accompanied by wasting symptoms, which cause severe metabolic changes in infected people. In this study we have compared the effect of mycobacteria infection on the level of metabolites in blood of humans and mice and whole zebrafish larvae using one highly standardized mass spectrometry pipeline, ensuring technical comparability of the results. Quantification of a range of circulating small amines showed that the levels of the majority of these compounds were significantly decreased in all three groups of infected organisms. Ten of these metabolites were common between the three different organisms comprising: methionine, asparagine, cysteine, threonine, serine, tryptophan, leucine, citrulline, ethanolamine and phenylalanine. The metabolomic changes of zebrafish larvae after infection were confirmed by nuclear magnetic resonance spectroscopy. Our study identified common biomarkers for tuberculosis disease in humans, mice and zebrafish, showing across species conservation of metabolic reprogramming processes as a result of disease. Apparently, the mechanisms underlying these processes are independent of environmental, developmental and vertebrate evolutionary factors. The zebrafish larval model is highly suited to further investigate the mechanism of metabolic reprogramming and the connection with wasting syndrome due to infection by mycobacteria.
Fukuda, Ryuichi; Marín-Juez, Rubén; El-Sammak, Hadil; Beisaw, Arica; Ramadass, Radhan; Kuenne, Carsten; Guenther, Stefan; Konzer, Anne; Bhagwat, Aditya M; Graumann, Johannes; Stainier, Didier YR
Stimulation of glycolysis promotes cardiomyocyte proliferation after injury in adult zebrafish Journal Article
In: EMBO Reports, vol. 21, iss. 8, no. e49752, 2020.
@article{nokey,
title = {Stimulation of glycolysis promotes cardiomyocyte proliferation after injury in adult zebrafish},
author = {Ryuichi Fukuda and Rubén Marín-Juez and Hadil El-Sammak and Arica Beisaw and Radhan Ramadass and Carsten Kuenne and Stefan Guenther and Anne Konzer and Aditya M Bhagwat and Johannes Graumann and Didier YR Stainier},
doi = {10.15252/embr.201949752},
year = {2020},
date = {2020-08-05},
journal = {EMBO Reports},
volume = {21},
number = {e49752},
issue = {8},
abstract = {Cardiac metabolism plays a crucial role in producing sufficient energy to sustain cardiac function. However, the role of metabolism in different aspects of cardiomyocyte regeneration remains unclear. Working with the adult zebrafish heart regeneration model, we first find an increase in the levels of mRNAs encoding enzymes regulating glucose and pyruvate metabolism, including pyruvate kinase M1/2 (Pkm) and pyruvate dehydrogenase kinases (Pdks), especially in tissues bordering the damaged area. We further find that impaired glycolysis decreases the number of proliferating cardiomyocytes following injury. These observations are supported by analyses using loss-of-function models for the metabolic regulators Pkma2 and peroxisome proliferator-activated receptor gamma coactivator 1 alpha. Cardiomyocyte-specific loss- and gain-of-function manipulations of pyruvate metabolism using Pdk3 as well as a catalytic subunit of the pyruvate dehydrogenase complex (PDC) reveal its importance in cardiomyocyte dedifferentiation and proliferation after injury. Furthermore, we find that PDK activity can modulate cell cycle progression and protrusive activity in mammalian cardiomyocytes in culture. Our findings reveal new roles for cardiac metabolism and the PDK-PDC axis in cardiomyocyte behavior following cardiac injury.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Cardiac metabolism plays a crucial role in producing sufficient energy to sustain cardiac function. However, the role of metabolism in different aspects of cardiomyocyte regeneration remains unclear. Working with the adult zebrafish heart regeneration model, we first find an increase in the levels of mRNAs encoding enzymes regulating glucose and pyruvate metabolism, including pyruvate kinase M1/2 (Pkm) and pyruvate dehydrogenase kinases (Pdks), especially in tissues bordering the damaged area. We further find that impaired glycolysis decreases the number of proliferating cardiomyocytes following injury. These observations are supported by analyses using loss-of-function models for the metabolic regulators Pkma2 and peroxisome proliferator-activated receptor gamma coactivator 1 alpha. Cardiomyocyte-specific loss- and gain-of-function manipulations of pyruvate metabolism using Pdk3 as well as a catalytic subunit of the pyruvate dehydrogenase complex (PDC) reveal its importance in cardiomyocyte dedifferentiation and proliferation after injury. Furthermore, we find that PDK activity can modulate cell cycle progression and protrusive activity in mammalian cardiomyocytes in culture. Our findings reveal new roles for cardiac metabolism and the PDK-PDC axis in cardiomyocyte behavior following cardiac injury.
2019
Hu, Wanbin; Yang, Shuxin; Shimada, Yasuhito; Münch, Magnus; Marín-Juez, Rubén; Meijer, Annemarie H; Spaink, Herman P
In: BMC Genomics, vol. 20, iss. 1, no. 878, 2019.
@article{nokey,
title = {Infection and RNA-seq analysis of a zebrafish tlr2 mutant shows a broad function of this toll-like receptor in transcriptional and metabolic control and defense to Mycobacterium marinum infection},
author = {Wanbin Hu and Shuxin Yang and Yasuhito Shimada and Magnus Münch and Rubén Marín-Juez and Annemarie H Meijer and Herman P Spaink},
doi = {10.1186/s12864-019-6265-1},
year = {2019},
date = {2019-11-20},
journal = {BMC Genomics},
volume = {20},
number = {878},
issue = {1},
abstract = {Background: The function of Toll-like receptor 2 (TLR2) in host defense against pathogens, especially Mycobacterium tuberculosis (Mtb) is poorly understood. To investigate the role of TLR2 during mycobacterial infection, we analyzed the response of tlr2 zebrafish mutant larvae to infection with Mycobacterium marinum (Mm), a close relative to Mtb, as a model for tuberculosis. We measured infection phenotypes and transcriptome responses using RNA deep sequencing in mutant and control larvae.
Results: tlr2 mutant embryos at 2 dpf do not show differences in numbers of macrophages and neutrophils compared to control embryos. However, we found substantial changes in gene expression in these mutants, particularly in metabolic pathways, when compared with the heterozygote tlr2+/- control. At 4 days after Mm infection, the total bacterial burden and the presence of extracellular bacteria were higher in tlr2-/- larvae than in tlr2+/-, or tlr2+/+ larvae, whereas granuloma numbers were reduced, showing a function of Tlr2 in zebrafish host defense. RNAseq analysis of infected tlr2-/- versus tlr2+/- shows that the number of up-regulated and down-regulated genes in response to infection was greatly diminished in tlr2 mutants by at least 2 fold and 10 fold, respectively. Analysis of the transcriptome data and qPCR validation shows that Mm infection of tlr2 mutants leads to decreased mRNA levels of genes involved in inflammation and immune responses, including il1b, tnfb, cxcl11aa/ac, fosl1a, and cebpb. Furthermore, RNAseq analyses revealed that the expression of genes for Maf family transcription factors, vitamin D receptors, and Dicps proteins is altered in tlr2 mutants with or without infection. In addition, the data indicate a function of Tlr2 in the control of induction of cytokines and chemokines, such as the CXCR3-CXCL11 signaling axis.
Conclusion: The transcriptome and infection burden analyses show a function of Tlr2 as a protective factor against mycobacteria. Transcriptome analysis revealed tlr2-specific pathways involved in Mm infection, which are related to responses to Mtb infection in human macrophages. Considering its dominant function in control of transcriptional processes that govern defense responses and metabolism, the TLR2 protein can be expected to be also of importance for other infectious diseases and interactions with the microbiome.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Background: The function of Toll-like receptor 2 (TLR2) in host defense against pathogens, especially Mycobacterium tuberculosis (Mtb) is poorly understood. To investigate the role of TLR2 during mycobacterial infection, we analyzed the response of tlr2 zebrafish mutant larvae to infection with Mycobacterium marinum (Mm), a close relative to Mtb, as a model for tuberculosis. We measured infection phenotypes and transcriptome responses using RNA deep sequencing in mutant and control larvae.
Results: tlr2 mutant embryos at 2 dpf do not show differences in numbers of macrophages and neutrophils compared to control embryos. However, we found substantial changes in gene expression in these mutants, particularly in metabolic pathways, when compared with the heterozygote tlr2+/- control. At 4 days after Mm infection, the total bacterial burden and the presence of extracellular bacteria were higher in tlr2-/- larvae than in tlr2+/-, or tlr2+/+ larvae, whereas granuloma numbers were reduced, showing a function of Tlr2 in zebrafish host defense. RNAseq analysis of infected tlr2-/- versus tlr2+/- shows that the number of up-regulated and down-regulated genes in response to infection was greatly diminished in tlr2 mutants by at least 2 fold and 10 fold, respectively. Analysis of the transcriptome data and qPCR validation shows that Mm infection of tlr2 mutants leads to decreased mRNA levels of genes involved in inflammation and immune responses, including il1b, tnfb, cxcl11aa/ac, fosl1a, and cebpb. Furthermore, RNAseq analyses revealed that the expression of genes for Maf family transcription factors, vitamin D receptors, and Dicps proteins is altered in tlr2 mutants with or without infection. In addition, the data indicate a function of Tlr2 in the control of induction of cytokines and chemokines, such as the CXCR3-CXCL11 signaling axis.
Conclusion: The transcriptome and infection burden analyses show a function of Tlr2 as a protective factor against mycobacteria. Transcriptome analysis revealed tlr2-specific pathways involved in Mm infection, which are related to responses to Mtb infection in human macrophages. Considering its dominant function in control of transcriptional processes that govern defense responses and metabolism, the TLR2 protein can be expected to be also of importance for other infectious diseases and interactions with the microbiome.
Results: tlr2 mutant embryos at 2 dpf do not show differences in numbers of macrophages and neutrophils compared to control embryos. However, we found substantial changes in gene expression in these mutants, particularly in metabolic pathways, when compared with the heterozygote tlr2+/- control. At 4 days after Mm infection, the total bacterial burden and the presence of extracellular bacteria were higher in tlr2-/- larvae than in tlr2+/-, or tlr2+/+ larvae, whereas granuloma numbers were reduced, showing a function of Tlr2 in zebrafish host defense. RNAseq analysis of infected tlr2-/- versus tlr2+/- shows that the number of up-regulated and down-regulated genes in response to infection was greatly diminished in tlr2 mutants by at least 2 fold and 10 fold, respectively. Analysis of the transcriptome data and qPCR validation shows that Mm infection of tlr2 mutants leads to decreased mRNA levels of genes involved in inflammation and immune responses, including il1b, tnfb, cxcl11aa/ac, fosl1a, and cebpb. Furthermore, RNAseq analyses revealed that the expression of genes for Maf family transcription factors, vitamin D receptors, and Dicps proteins is altered in tlr2 mutants with or without infection. In addition, the data indicate a function of Tlr2 in the control of induction of cytokines and chemokines, such as the CXCR3-CXCL11 signaling axis.
Conclusion: The transcriptome and infection burden analyses show a function of Tlr2 as a protective factor against mycobacteria. Transcriptome analysis revealed tlr2-specific pathways involved in Mm infection, which are related to responses to Mtb infection in human macrophages. Considering its dominant function in control of transcriptional processes that govern defense responses and metabolism, the TLR2 protein can be expected to be also of importance for other infectious diseases and interactions with the microbiome.
Marín-Juez, Rubén; El-Sammak, Hadil; Helker, Christian S M; Kamezaki, Aosa; Mullapuli, Sri Teja; Bibli, Sofia-Iris; Foglia, Matthew J; Fleming, Ingrid; Poss, Kenneth D; Stainier, Didier YR
In: Developmental Cell, vol. 51, iss. 4, pp. 503-515, 2019.
@article{nokey,
title = {Coronary Revascularization During Heart Regeneration Is Regulated by Epicardial and Endocardial Cues and Forms a Scaffold for Cardiomyocyte Repopulation},
author = {Rubén Marín-Juez and Hadil El-Sammak and Christian S M Helker and Aosa Kamezaki and Sri Teja Mullapuli and Sofia-Iris Bibli and Matthew J Foglia and Ingrid Fleming and Kenneth D Poss and Didier YR Stainier},
doi = {10.1016/j.devcel.2019.10.019},
year = {2019},
date = {2019-11-18},
journal = {Developmental Cell},
volume = {51},
issue = {4},
pages = {503-515},
abstract = {Defective coronary network function and insufficient blood supply are both cause and consequence of myocardial infarction. Efficient revascularization after infarction is essential to support tissue repair and function. Zebrafish hearts exhibit a remarkable ability to regenerate, and coronary revascularization initiates within hours of injury, but how this process is regulated remains unknown. Here, we show that revascularization requires a coordinated multi-tissue response culminating with the formation of a complex vascular network available as a scaffold for cardiomyocyte repopulation. During a process we term "coronary-endocardial anchoring," new coronaries respond by sprouting (1) superficially within the regenerating epicardium and (2) intra-ventricularly toward the activated endocardium. Mechanistically, superficial revascularization is guided by epicardial Cxcl12-Cxcr4 signaling and intra-ventricular sprouting by endocardial Vegfa signaling. Our findings indicate that the injury-activated epicardium and endocardium support cardiomyocyte replenishment initially through the guidance of coronary sprouting. Simulating this process in the injured mammalian heart should help its healing.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Defective coronary network function and insufficient blood supply are both cause and consequence of myocardial infarction. Efficient revascularization after infarction is essential to support tissue repair and function. Zebrafish hearts exhibit a remarkable ability to regenerate, and coronary revascularization initiates within hours of injury, but how this process is regulated remains unknown. Here, we show that revascularization requires a coordinated multi-tissue response culminating with the formation of a complex vascular network available as a scaffold for cardiomyocyte repopulation. During a process we term "coronary-endocardial anchoring," new coronaries respond by sprouting (1) superficially within the regenerating epicardium and (2) intra-ventricularly toward the activated endocardium. Mechanistically, superficial revascularization is guided by epicardial Cxcl12-Cxcr4 signaling and intra-ventricular sprouting by endocardial Vegfa signaling. Our findings indicate that the injury-activated epicardium and endocardium support cardiomyocyte replenishment initially through the guidance of coronary sprouting. Simulating this process in the injured mammalian heart should help its healing.
Collins, Michelle M; Ahlberg, Gustav; Hansen, Camilla Vestergaard; Guenther, Stefan; Marín-Juez, Rubén; Sokol, Anna M; El-Sammak, Hadil; Piesker, Janett; Hellsten, Ylva; Olesen, Morten S; Stainier, Didier Y R; Lundegaard, Pia R
Early sarcomere and metabolic defects in a zebrafish pitx2c cardiac arrhythmia model Journal Article
In: PNAS, vol. 116, iss. 48, pp. 24115-24121, 2019.
@article{nokey,
title = {Early sarcomere and metabolic defects in a zebrafish pitx2c cardiac arrhythmia model},
author = {Michelle M Collins and Gustav Ahlberg and Camilla Vestergaard Hansen and Stefan Guenther and Rubén Marín-Juez and Anna M Sokol and Hadil El-Sammak and Janett Piesker and Ylva Hellsten and Morten S Olesen and Didier Y R Stainier and Pia R Lundegaard},
doi = {10.1073/pnas.1913905116},
year = {2019},
date = {2019-11-26},
journal = {PNAS},
volume = {116},
issue = {48},
pages = {24115-24121},
abstract = {Atrial fibrillation (AF) is the most common type of cardiac arrhythmia. The major AF susceptibility locus 4q25 establishes long-range interactions with the promoter of PITX2, a transcription factor gene with critical functions during cardiac development. While many AF-linked loci have been identified in genome-wide association studies, mechanistic understanding into how genetic variants, including those at the 4q25 locus, increase vulnerability to AF is mostly lacking. Here, we show that loss of pitx2c in zebrafish leads to adult cardiac phenotypes with substantial similarities to pathologies observed in AF patients, including arrhythmia, atrial conduction defects, sarcomere disassembly, and altered cardiac metabolism. These phenotypes are also observed in a subset of pitx2c +/- fish, mimicking the situation in humans. Most notably, the onset of these phenotypes occurs at an early developmental stage. Detailed analyses of pitx2c loss- and gain-of-function embryonic hearts reveal changes in sarcomeric and metabolic gene expression and function that precede the onset of cardiac arrhythmia first observed at larval stages. We further find that antioxidant treatment of pitx2c -/- larvae significantly reduces the incidence and severity of cardiac arrhythmia, suggesting that metabolic dysfunction is an important driver of conduction defects. We propose that these early sarcomere and metabolic defects alter cardiac function and contribute to the electrical instability and structural remodeling observed in adult fish. Overall, these data provide insight into the mechanisms underlying the development and pathophysiology of some cardiac arrhythmias and importantly, increase our understanding of how developmental perturbations can predispose to functional defects in the adult heart.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Atrial fibrillation (AF) is the most common type of cardiac arrhythmia. The major AF susceptibility locus 4q25 establishes long-range interactions with the promoter of PITX2, a transcription factor gene with critical functions during cardiac development. While many AF-linked loci have been identified in genome-wide association studies, mechanistic understanding into how genetic variants, including those at the 4q25 locus, increase vulnerability to AF is mostly lacking. Here, we show that loss of pitx2c in zebrafish leads to adult cardiac phenotypes with substantial similarities to pathologies observed in AF patients, including arrhythmia, atrial conduction defects, sarcomere disassembly, and altered cardiac metabolism. These phenotypes are also observed in a subset of pitx2c +/- fish, mimicking the situation in humans. Most notably, the onset of these phenotypes occurs at an early developmental stage. Detailed analyses of pitx2c loss- and gain-of-function embryonic hearts reveal changes in sarcomeric and metabolic gene expression and function that precede the onset of cardiac arrhythmia first observed at larval stages. We further find that antioxidant treatment of pitx2c -/- larvae significantly reduces the incidence and severity of cardiac arrhythmia, suggesting that metabolic dysfunction is an important driver of conduction defects. We propose that these early sarcomere and metabolic defects alter cardiac function and contribute to the electrical instability and structural remodeling observed in adult fish. Overall, these data provide insight into the mechanisms underlying the development and pathophysiology of some cardiac arrhythmias and importantly, increase our understanding of how developmental perturbations can predispose to functional defects in the adult heart.
Gancz, Dana; Raftrey, Brian C; Perlmoter, Gal; Marín-Juez, Rubén; Semo, Jonathan; Matsuoka, Ryota L; Karra, Ravi; Raviv, Hila; Moshe, Noga; Addadi, Yoseph; Golani, Ofra; Poss, Kenneth D; Red-Horse, Kristy; Stainier, Didier YR; Yaniv, Karina
Distinct origins and molecular mechanisms contribute to lymphatic formation during cardiac growth and regeneration Journal Article
In: Elife, vol. 8, no. e44153, 2019.
@article{nokey,
title = {Distinct origins and molecular mechanisms contribute to lymphatic formation during cardiac growth and regeneration},
author = {Dana Gancz and Brian C Raftrey and Gal Perlmoter and Rubén Marín-Juez and Jonathan Semo and Ryota L Matsuoka and Ravi Karra and Hila Raviv and Noga Moshe and Yoseph Addadi and Ofra Golani and Kenneth D Poss and Kristy Red-Horse and Didier YR Stainier and Karina Yaniv},
doi = {10.7554/eLife.44153},
year = {2019},
date = {2019-11-08},
journal = {Elife},
volume = {8},
number = {e44153},
abstract = {In recent years, there has been increasing interest in the role of lymphatics in organ repair and regeneration, due to their importance in immune surveillance and fluid homeostasis. Experimental approaches aimed at boosting lymphangiogenesis following myocardial infarction in mice, were shown to promote healing of the heart. Yet, the mechanisms governing cardiac lymphatic growth remain unclear. Here, we identify two distinct lymphatic populations in the hearts of zebrafish and mouse, one that forms through sprouting lymphangiogenesis, and the other by coalescence of isolated lymphatic cells. By tracing the development of each subset, we reveal diverse cellular origins and differential response to signaling cues. Finally, we show that lymphatic vessels are required for cardiac regeneration in zebrafish as mutants lacking lymphatics display severely impaired regeneration capabilities. Overall, our results provide novel insight into the mechanisms underlying lymphatic formation during development and regeneration, opening new avenues for interventions targeting specific lymphatic populations.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
In recent years, there has been increasing interest in the role of lymphatics in organ repair and regeneration, due to their importance in immune surveillance and fluid homeostasis. Experimental approaches aimed at boosting lymphangiogenesis following myocardial infarction in mice, were shown to promote healing of the heart. Yet, the mechanisms governing cardiac lymphatic growth remain unclear. Here, we identify two distinct lymphatic populations in the hearts of zebrafish and mouse, one that forms through sprouting lymphangiogenesis, and the other by coalescence of isolated lymphatic cells. By tracing the development of each subset, we reveal diverse cellular origins and differential response to signaling cues. Finally, we show that lymphatic vessels are required for cardiac regeneration in zebrafish as mutants lacking lymphatics display severely impaired regeneration capabilities. Overall, our results provide novel insight into the mechanisms underlying lymphatic formation during development and regeneration, opening new avenues for interventions targeting specific lymphatic populations.
2018
Lai, Shih-Lei; Marín-Juez, Rubén; Stainier, Didier Y R
Immune responses in cardiac repair and regeneration: a comparative point of view Journal Article
In: Cellular and Molecular Life Sciences , vol. 76, iss. 7, pp. 1365–1380, 2018.
@article{nokey,
title = {Immune responses in cardiac repair and regeneration: a comparative point of view},
author = {Shih-Lei Lai and Rubén Marín-Juez and Didier Y R Stainier},
doi = {10.1007/s00018-018-2995-5},
year = {2018},
date = {2018-12-21},
journal = {Cellular and Molecular Life Sciences },
volume = {76},
issue = {7},
pages = {1365–1380},
abstract = {Immediately after cardiac injury, the immune system plays major roles in repair and regeneration as it becomes involved in a number of processes including damage-associated signaling, inflammation, revascularization, cardiomyocyte dedifferentiation and replenishment, and fibrotic scar formation/resolution. Recent studies have revealed that different immune responses occur in the various experimental models capable or incapable of cardiac regeneration, and that harnessing these immune responses might improve cardiac repair. In light of this concept, this review analyzes current knowledge about the immune responses to cardiac injury from a comparative perspective. Insights gained from such comparative analyses may provide ways to modulate the immune response as a potential therapeutic strategy for cardiac disease.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Immediately after cardiac injury, the immune system plays major roles in repair and regeneration as it becomes involved in a number of processes including damage-associated signaling, inflammation, revascularization, cardiomyocyte dedifferentiation and replenishment, and fibrotic scar formation/resolution. Recent studies have revealed that different immune responses occur in the various experimental models capable or incapable of cardiac regeneration, and that harnessing these immune responses might improve cardiac repair. In light of this concept, this review analyzes current knowledge about the immune responses to cardiac injury from a comparative perspective. Insights gained from such comparative analyses may provide ways to modulate the immune response as a potential therapeutic strategy for cardiac disease.
Burg, Leonard; Palmer, Nicholas; Kikhi, Khrievono; Miroshnik, Evgeniya S; Rueckert, Helen; Gaddy, Eleanor; Cunningham, Carlee MacPherson; Mattonet, Kenny; Lai, Shih-Lei; Marín-Juez, Rubén; Waring, Richard B; Stainier, Didier Y R; Balciunas, Darius
Conditional mutagenesis by oligonucleotide-mediated integration of loxP sites in zebrafish Journal Article
In: PLoS Genetics, vol. 14, iss. 11, no. e1007754, 2018.
@article{nokey,
title = {Conditional mutagenesis by oligonucleotide-mediated integration of loxP sites in zebrafish},
author = {Leonard Burg and Nicholas Palmer and Khrievono Kikhi and Evgeniya S Miroshnik and Helen Rueckert and Eleanor Gaddy and Carlee MacPherson Cunningham and Kenny Mattonet and Shih-Lei Lai and Rubén Marín-Juez and Richard B Waring and Didier Y R Stainier and Darius Balciunas},
doi = {10.1371/journal.pgen.1007754},
year = {2018},
date = {2018-11-14},
journal = {PLoS Genetics},
volume = {14},
number = {e1007754},
issue = {11},
abstract = {Many eukaryotic genes play essential roles in multiple biological processes in several different tissues. Conditional mutants are needed to analyze genes with such pleiotropic functions. In vertebrates, conditional gene inactivation has only been feasible in the mouse, leaving other model systems to rely on surrogate experimental approaches such as overexpression of dominant negative proteins and antisense-based tools. Here, we have developed a simple and straightforward method to integrate loxP sequences at specific sites in the zebrafish genome using the CRISPR/Cas9 technology and oligonucleotide templates for homology directed repair. We engineered conditional (floxed) mutants of tbx20 and fleer, and demonstrate excision of exons flanked by loxP sites using tamoxifen-inducible CreERT2 recombinase. To demonstrate broad applicability of our method, we also integrated loxP sites into two additional genes, aldh1a2 and tcf21. The ease of this approach will further expand the use of zebrafish to study various aspects of vertebrate biology, especially post-embryonic processes such as regeneration.
},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Many eukaryotic genes play essential roles in multiple biological processes in several different tissues. Conditional mutants are needed to analyze genes with such pleiotropic functions. In vertebrates, conditional gene inactivation has only been feasible in the mouse, leaving other model systems to rely on surrogate experimental approaches such as overexpression of dominant negative proteins and antisense-based tools. Here, we have developed a simple and straightforward method to integrate loxP sequences at specific sites in the zebrafish genome using the CRISPR/Cas9 technology and oligonucleotide templates for homology directed repair. We engineered conditional (floxed) mutants of tbx20 and fleer, and demonstrate excision of exons flanked by loxP sites using tamoxifen-inducible CreERT2 recombinase. To demonstrate broad applicability of our method, we also integrated loxP sites into two additional genes, aldh1a2 and tcf21. The ease of this approach will further expand the use of zebrafish to study various aspects of vertebrate biology, especially post-embryonic processes such as regeneration.
Boskovic, Srdjan; Marín-Juez, Rubén; Jasnic, Jovana; Reischauer, Sven; Sammak, Hadil El; Kojic, Ana; Faulkner, Georgine; Radojkovic, Dragica; Stainier, Didier Y R; Kojic, Snezana
Characterization of zebrafish (Danio rerio) muscle ankyrin repeat proteins reveals their conserved response to endurance exercise Journal Article
In: PLoS One, vol. 13, iss. 9, no. e0204312, 2018.
@article{nokey,
title = {Characterization of zebrafish (Danio rerio) muscle ankyrin repeat proteins reveals their conserved response to endurance exercise},
author = {Srdjan Boskovic and Rubén Marín-Juez and Jovana Jasnic and Sven Reischauer and Hadil El Sammak and Ana Kojic and Georgine Faulkner and Dragica Radojkovic and Didier Y R Stainier and Snezana Kojic},
doi = {10.1371/journal.pone.0204312},
year = {2018},
date = {2018-09-25},
journal = {PLoS One},
volume = {13},
number = {e0204312},
issue = {9},
abstract = {Muscle proteins with ankyrin repeats (MARPs) ANKRD1 and ANKRD2 are titin-associated proteins with a putative role as transcriptional co-regulators in striated muscle, involved in the cellular response to mechanical, oxidative and metabolic stress. Since many aspects of the biology of MARPs, particularly exact mechanisms of their action, in striated muscle are still elusive, research in this field will benefit from novel animal model system. Here we investigated the MARPs found in zebrafish for protein structure, evolutionary conservation, spatiotemporal expression profiles and response to increased muscle activity. Ankrd1 and Ankrd2 show overall moderate conservation at the protein level, more pronounced in the region of ankyrin repeats, motifs indispensable for their function. The two zebrafish genes, ankrd1a and ankrd1b, counterparts of mammalian ANKRD1/Ankrd1, have different expression profiles during first seven days of development. Mild increase of ankrd1a transcript levels was detected at 72 hpf (1.74±0.24 fold increase relative to 24 hpf time point), while ankrd1b expression was markedly upregulated from 24 hpf onward and peaked at 72 hpf (92.18±36.95 fold increase relative to 24 hpf time point). Spatially, they exhibited non-overlapping expression patterns during skeletal muscle development in trunk (ankrd1a) and tail (ankrd1b) somites. Expression of ankrd2 was barely detectable. Zebrafish MARPs, expressed at a relatively low level in adult striated muscle, were found to be responsive to endurance exercise training consisting of two bouts of 3 hours of forced swimming daily, for five consecutive days. Three hours after the last exercise bout, ankrd1a expression increased in cardiac muscle (6.19±5.05 fold change), while ankrd1b and ankrd2 were upregulated in skeletal muscle (1.97±1.05 and 1.84±0.58 fold change, respectively). This study provides the foundation to establish zebrafish as a novel in vivo model for further investigation of MARPs function in striated muscle.
},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Muscle proteins with ankyrin repeats (MARPs) ANKRD1 and ANKRD2 are titin-associated proteins with a putative role as transcriptional co-regulators in striated muscle, involved in the cellular response to mechanical, oxidative and metabolic stress. Since many aspects of the biology of MARPs, particularly exact mechanisms of their action, in striated muscle are still elusive, research in this field will benefit from novel animal model system. Here we investigated the MARPs found in zebrafish for protein structure, evolutionary conservation, spatiotemporal expression profiles and response to increased muscle activity. Ankrd1 and Ankrd2 show overall moderate conservation at the protein level, more pronounced in the region of ankyrin repeats, motifs indispensable for their function. The two zebrafish genes, ankrd1a and ankrd1b, counterparts of mammalian ANKRD1/Ankrd1, have different expression profiles during first seven days of development. Mild increase of ankrd1a transcript levels was detected at 72 hpf (1.74±0.24 fold increase relative to 24 hpf time point), while ankrd1b expression was markedly upregulated from 24 hpf onward and peaked at 72 hpf (92.18±36.95 fold increase relative to 24 hpf time point). Spatially, they exhibited non-overlapping expression patterns during skeletal muscle development in trunk (ankrd1a) and tail (ankrd1b) somites. Expression of ankrd2 was barely detectable. Zebrafish MARPs, expressed at a relatively low level in adult striated muscle, were found to be responsive to endurance exercise training consisting of two bouts of 3 hours of forced swimming daily, for five consecutive days. Three hours after the last exercise bout, ankrd1a expression increased in cardiac muscle (6.19±5.05 fold change), while ankrd1b and ankrd2 were upregulated in skeletal muscle (1.97±1.05 and 1.84±0.58 fold change, respectively). This study provides the foundation to establish zebrafish as a novel in vivo model for further investigation of MARPs function in striated muscle.
Gauvrit, Sébastien; Villasenor, Alethia; Strilic, Boris; Kitchen, Philip; Collins, Michelle M; Marín-Juez, Rubén; Guenther, Stefan; Maischein, Hans-Martin; Fukuda, Nana; Canham, Maurice A; Brickman, Joshua M; Bogue, Clifford W; Jayaraman, Padma-Sheela; Stainier, Didier Y R
HHEX is a transcriptional regulator of the VEGFC/FLT4/PROX1 signaling axis during vascular development Journal Article
In: Nature Communications, vol. 9, iss. 1, no. 2704, 2018.
@article{nokey,
title = {HHEX is a transcriptional regulator of the VEGFC/FLT4/PROX1 signaling axis during vascular development},
author = {Sébastien Gauvrit and Alethia Villasenor and Boris Strilic and Philip Kitchen and Michelle M Collins and Rubén Marín-Juez and Stefan Guenther and Hans-Martin Maischein and Nana Fukuda and Maurice A Canham and Joshua M Brickman and Clifford W Bogue and Padma-Sheela Jayaraman and Didier Y R Stainier},
doi = {10.1038/s41467-018-05039-1},
year = {2018},
date = {2018-07-13},
journal = {Nature Communications},
volume = {9},
number = {2704},
issue = {1},
abstract = {Formation of the lymphatic system requires the coordinated expression of several key regulators: vascular endothelial growth factor C (VEGFC), its receptor FLT4, and a key transcriptional effector, PROX1. Yet, how expression of these signaling components is regulated remains poorly understood. Here, using a combination of genetic and molecular approaches, we identify the transcription factor hematopoietically expressed homeobox (HHEX) as an upstream regulator of VEGFC, FLT4, and PROX1 during angiogenic sprouting and lymphatic formation in vertebrates. By analyzing zebrafish mutants, we found that hhex is necessary for sprouting angiogenesis from the posterior cardinal vein, a process required for lymphangiogenesis. Furthermore, studies of mammalian HHEX using tissue-specific genetic deletions in mouse and knockdowns in cultured human endothelial cells reveal its highly conserved function during vascular and lymphatic development. Our findings that HHEX is essential for the regulation of the VEGFC/FLT4/PROX1 axis provide insights into the molecular regulation of lymphangiogenesis.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Formation of the lymphatic system requires the coordinated expression of several key regulators: vascular endothelial growth factor C (VEGFC), its receptor FLT4, and a key transcriptional effector, PROX1. Yet, how expression of these signaling components is regulated remains poorly understood. Here, using a combination of genetic and molecular approaches, we identify the transcription factor hematopoietically expressed homeobox (HHEX) as an upstream regulator of VEGFC, FLT4, and PROX1 during angiogenic sprouting and lymphatic formation in vertebrates. By analyzing zebrafish mutants, we found that hhex is necessary for sprouting angiogenesis from the posterior cardinal vein, a process required for lymphangiogenesis. Furthermore, studies of mammalian HHEX using tissue-specific genetic deletions in mouse and knockdowns in cultured human endothelial cells reveal its highly conserved function during vascular and lymphatic development. Our findings that HHEX is essential for the regulation of the VEGFC/FLT4/PROX1 axis provide insights into the molecular regulation of lymphangiogenesis.
2017
Lai, Shih-Lei; Marín-Juez, Rubén; Moura, Pedro Luís; Kuenne, Carsten; Lai, Jason Kuan Han; Tsedeke, Ayele Taddese; Guenther, Stefan; Looso, Mario; Stainier, Didier YR
Reciprocal analyses in zebrafish and medaka reveal that harnessing the immune response promotes cardiac regeneration Journal Article
In: Elife, vol. 6, no. e25605, 2017.
@article{nokey,
title = {Reciprocal analyses in zebrafish and medaka reveal that harnessing the immune response promotes cardiac regeneration},
author = {Shih-Lei Lai and Rubén Marín-Juez and Pedro Luís Moura and Carsten Kuenne and Jason Kuan Han Lai and Ayele Taddese Tsedeke and Stefan Guenther and Mario Looso and Didier YR Stainier},
doi = {10.7554/eLife.25605},
year = {2017},
date = {2017-06-20},
journal = {Elife},
volume = {6},
number = {e25605},
abstract = {Zebrafish display a distinct ability to regenerate their heart following injury. However, this ability is not shared by another teleost, the medaka. In order to identify cellular and molecular bases for this difference, we performed comparative transcriptomic analyses following cardiac cryoinjury. This comparison points to major differences in immune cell dynamics between these models. Upon closer examination, we observed delayed and reduced macrophage recruitment in medaka, along with delayed neutrophil clearance. To investigate the role of immune responses in cardiac regeneration, we delayed macrophage recruitment in zebrafish and observed compromised neovascularization, neutrophil clearance, cardiomyocyte proliferation and scar resolution. In contrast, stimulating Toll-like receptor signaling in medaka enhanced immune cell dynamics and promoted neovascularization, neutrophil clearance, cardiomyocyte proliferation and scar resolution. Altogether, these data provide further insight into the complex role of the immune response during regeneration, and serve as a platform to identify and test additional regulators of cardiac repair.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Zebrafish display a distinct ability to regenerate their heart following injury. However, this ability is not shared by another teleost, the medaka. In order to identify cellular and molecular bases for this difference, we performed comparative transcriptomic analyses following cardiac cryoinjury. This comparison points to major differences in immune cell dynamics between these models. Upon closer examination, we observed delayed and reduced macrophage recruitment in medaka, along with delayed neutrophil clearance. To investigate the role of immune responses in cardiac regeneration, we delayed macrophage recruitment in zebrafish and observed compromised neovascularization, neutrophil clearance, cardiomyocyte proliferation and scar resolution. In contrast, stimulating Toll-like receptor signaling in medaka enhanced immune cell dynamics and promoted neovascularization, neutrophil clearance, cardiomyocyte proliferation and scar resolution. Altogether, these data provide further insight into the complex role of the immune response during regeneration, and serve as a platform to identify and test additional regulators of cardiac repair.
Gerri, Claudia; Marín-Juez, Rubén; Marass, Michele; Marks, Alora; Maischein, Hans-Martin; Stainier, Didier Y R
Hif-1α regulates macrophage-endothelial interactions during blood vessel development in zebrafish Journal Article
In: Nature Communications, vol. 8, no. 15492, 2017.
@article{nokey,
title = {Hif-1α regulates macrophage-endothelial interactions during blood vessel development in zebrafish},
author = {Claudia Gerri and Rubén Marín-Juez and Michele Marass and Alora Marks and Hans-Martin Maischein and Didier Y R Stainier},
doi = {10.1038/ncomms15492},
year = {2017},
date = {2017-05-19},
journal = {Nature Communications},
volume = {8},
number = {15492},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
2016
Marín-Juez, Rubén; Marass, Michele; Gauvrit, Sebastien; Rossi, Andrea; Lai, Shih-Lei; Materna, Stefan C; Black, Brian L; Stainier, Didier Y R
Fast revascularization of the injured area is essential to support zebrafish heart regeneration Journal Article
In: PNAS, vol. 113, no. 40, pp. 11237-11242, 2016.
@article{Marín-Juez2016,
title = {Fast revascularization of the injured area is essential to support zebrafish heart regeneration},
author = {Rubén Marín-Juez and Michele Marass and Sebastien Gauvrit and Andrea Rossi and Shih-Lei Lai and Stefan C Materna and Brian L Black and Didier Y R Stainier},
editor = {-},
doi = {10.1073/pnas.1605431113},
year = {2016},
date = {2016-10-04},
urldate = {2016-10-04},
journal = {PNAS},
volume = {113},
number = {40},
pages = {11237-11242},
abstract = {Zebrafish have a remarkable capacity to regenerate their heart. Efficient replenishment of lost tissues requires the activation of different cell types including the epicardium and endocardium. A complex set of processes is subsequently needed to support cardiomyocyte repopulation. Previous studies have identified important determinants of heart regeneration; however, to date, how revascularization of the damaged area happens remains unknown. Here, we show that angiogenic sprouting into the injured area starts as early as 15 h after injury. To analyze the role of vegfaa in heart regeneration, we used vegfaa mutants rescued to adulthood by vegfaa mRNA injections at the one-cell stage. Surprisingly, vegfaa mutants develop coronaries and revascularize after injury. As a possible explanation for these observations, we find that vegfaa mutant hearts up-regulate the expression of potentially compensating genes. Therefore, to overcome the lack of a revascularization phenotype in vegfaa mutants, we generated fish expressing inducible dominant negative Vegfaa. These fish displayed minimal revascularization of the damaged area. In the absence of fast angiogenic revascularization, cardiomyocyte proliferation did not occur, and the heart failed to regenerate, retaining a fibrotic scar. Hence, our data show that a fast endothelial invasion allows efficient revascularization of the injured area, which is necessary to support replenishment of new tissue and achieve efficient heart regeneration. These findings revisit the model where neovascularization is considered to happen concomitant with the formation of new muscle. Our work also paves the way for future studies designed to understand the molecular mechanisms that regulate fast revascularization.
},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Zebrafish have a remarkable capacity to regenerate their heart. Efficient replenishment of lost tissues requires the activation of different cell types including the epicardium and endocardium. A complex set of processes is subsequently needed to support cardiomyocyte repopulation. Previous studies have identified important determinants of heart regeneration; however, to date, how revascularization of the damaged area happens remains unknown. Here, we show that angiogenic sprouting into the injured area starts as early as 15 h after injury. To analyze the role of vegfaa in heart regeneration, we used vegfaa mutants rescued to adulthood by vegfaa mRNA injections at the one-cell stage. Surprisingly, vegfaa mutants develop coronaries and revascularize after injury. As a possible explanation for these observations, we find that vegfaa mutant hearts up-regulate the expression of potentially compensating genes. Therefore, to overcome the lack of a revascularization phenotype in vegfaa mutants, we generated fish expressing inducible dominant negative Vegfaa. These fish displayed minimal revascularization of the damaged area. In the absence of fast angiogenic revascularization, cardiomyocyte proliferation did not occur, and the heart failed to regenerate, retaining a fibrotic scar. Hence, our data show that a fast endothelial invasion allows efficient revascularization of the injured area, which is necessary to support replenishment of new tissue and achieve efficient heart regeneration. These findings revisit the model where neovascularization is considered to happen concomitant with the formation of new muscle. Our work also paves the way for future studies designed to understand the molecular mechanisms that regulate fast revascularization.