Strength of interactions in the Notch gene regulatory network determines patterning and fate in the notochord

Development of multicellular organisms requires the generation of gene expression patterns that determines cell fate and organ shape. Groups of genetic interactions known as Gene Regulatory Networks (GRNs) play a key role in the generation of such patterns. However, how the topology and parameters of GRNs determine patterning in vivo remains unclear due to the complexity of most experimental systems. To address this, we use the zebrafish notochord, an organ where coin-shaped precursor cells are initially arranged in a simple unidimensional geometry. These cells then differentiate into vacuolated and sheath cells. Using newly developed transgenic tools together with in vivo imaging, we identify jag1a and her6/her9 as the main components of a Notch GRN that generates a lateral inhibition pattern and determines cell fate. Making use of this experimental system and mathematical modeling we show that lateral inhibition patterning is promoted when ligand-receptor interactions are stronger within the same cell than in neighboring cells. Altogether, we establish the zebrafish notochord as an experimental system to study pattern generation, and identify and characterize how the properties of GRNs determine self-organization of gene patterning and cell fate

Binary recombinase systems for high-resolution conditional mutagenesis

Conditional mutagenesis using Cre recombinase expressed from tissue specific promoters facilitates analyses of gene function and cell lineage tracing. Here, we describe two novel dual-promoter-driven conditional mutagenesis systems designed for greater accuracy and optimal efficiency of recombination. Co-Driver employs a recombinase cascade of Dre and Dre-respondent Cre, which processes loxP-flanked alleles only when both recombinases are expressed in a predetermined temporal sequence. This unique property makes Co-Driver ideal for sequential lineage tracing studies aimed at unraveling the relationships between cellular precursors and mature cell types. Co-InCre was designed for highly efficient intersectional conditional transgenesis. It relies on highly active trans-splicing inteins and promoters with simultaneous transcriptional activity to reconstitute Cre recombinase from two inactive precursor fragments. By generating native Cre, Co-InCre attains recombination rates that exceed all other binary SSR systems evaluated in this study. Both Co-Driver and Co-InCre significantly extend the utility of existing Cre-responsive allele

Fisetin protects against cardiac cell death through reduction of ROS production and caspases activity

Myocardial infarction (MI) is a leading cause of death worldwide. Reperfusion is considered as an optimal therapy following cardiac ischemia. However, the promotion of a rapid elevation of O2 levels in ischemic cells produces high amounts of reactive oxygen species (ROS) leading to myocardial tissue injury. This phenomenon is called ischemia reperfusion injury (IRI). We aimed at identifying new and effective compounds to treat MI and minimize IRI. We previously studied heart regeneration following myocardial injury in zebrafish and described each step of the regeneration process, from the day of injury until complete recovery, in terms of transcriptional responses. Here, we mined the data and performed a deep in silico analysis to identify drugs highly likely to induce cardiac regeneration. Fisetin was identified as the top candidate. We validated its effects in an in vitro model of MI/IRI in mammalian cardiac cells. Fisetin enhances viability of rat cardiomyocytes following hypoxia/starvation - reoxygenation. It inhibits apoptosis, decreases ROS generation and caspase activation and protects from DNA damage. Interestingly, fisetin also activates genes involved in cell proliferation. Fisetin is thus a highly promising candidate drug with clinical potential to protect from ischemic damage following MI and to overcome IRI.This work was supported by FNR, the Luxembourg National Research Fund, FNR-CORE INFUSED project. At the NorLux Laboratory and the Proteome and Genome Research Unit of LIH, it was also supported by funding from Luxembourg’s Ministry of Higher Education and Research (MESR).S

Pluripotency factors regulate the onset of Hox cluster activation in the early embryo

Pluripotent cells are a transient population of the mammalian embryo dependent on transcription factors, such as OCT4 and NANOG, which maintain pluripotency while suppressing lineage specification. However, these factors are also expressed during early phases of differentiation, and their role in the transition from pluripotency to lineage specification is largely unknown. We found that pluripotency factors play a dual role in regulating key lineage specifiers, initially repressing their expression and later being required for their proper activation. We show that Oct4 is necessary for activation of HoxB genes during differentiation of embryonic stem cells and in the embryo. In addition, we show that the HoxB cluster is coordinately regulated by OCT4 binding sites located at the 3′ end of the cluster. Our results show that core pluripotency factors are not limited to maintaining the precommitted epiblast but are also necessary for the proper deployment of subsequent developmental programs.This work was funded by the Spanish government (grants BFU2017-84914-P and PID2020-115755GB-I00 to M.M.; BFU2016-74961-P and BFU2016-81887-REDT to J.L.G.-S.), the Andalusian government (grant BIO-396 to J.L.G.-S.), and the European Research Council (ERC; grant agreement 740041 to J.L.G.-S.). M.T. held Juan de la Cierva fellowships from the Spanish government (FJCI-2017-31791 and IJC2019-038897-I), R.R. and R.D.A. held FPU fellowships from the government, and J.V. was the recipient of a “La Caixa” fellowship. Work in the laboratory of J.L.G.-S. was supported by a María de Maetzu Unit of Excellence Grant (MDM-2016-0687) to the Department of Gene Regulation and Morphogenesis of the CABD. The CBMSO is supported by an institutional grant from the Fundación Ramon Areces, and the CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN), and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MICIN/AEI/10.13039/501100011033). : With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2020-001041-S)

Transient fibrosis resolves via fibroblast inactivation in the regenerating zebrafish heart

After myocardial infarction in the mammalian heart, millions of cardiomyocytes are lost and replaced by fibrotic scar tissue. While fibrosis is persistent in adult mammals, there are some vertebrates, including zebrafish, with the capacity for regeneration. This process does not occur in the absence of fibrosis. Here we studied subpopulations of collagen-producing cells and analyzed their fate after complete regeneration of the zebrafish myocardium. Our data show that fibroblasts persisted in the regenerated heart but shut down the profibrotic program. While fibrosis could be considered as detrimental to the regeneration process, our study reveals a positive effect on cardiomyocyte proliferation. Accordingly, a fibrotic response can be beneficial for heart regeneration. In the zebrafish (Danio rerio), regeneration and fibrosis after cardiac injury are not mutually exclusive responses. Upon cardiac cryoinjury, collagen and other extracellular matrix (ECM) proteins accumulate at the injury site. However, in contrast to the situation in mammals, fibrosis is transient in zebrafish and its regression is concomitant with regrowth of the myocardial wall. Little is known about the cells producing this fibrotic tissue or how it resolves. Using novel genetic tools to mark periostin b - and collagen 1alpha2 (col1a2)-expressing cells in combination with transcriptome analysis, we explored the sources of activated fibroblasts and traced their fate. We describe that during fibrosis regression, fibroblasts are not fully eliminated but become inactivated. Unexpectedly, limiting the fibrotic response by genetic ablation of col1a2 -expressing cells impaired cardiomyocyte proliferation. We conclude that ECM-producing cells are key players in the regenerative process and suggest that antifibrotic therapies might be less efficient than strategies targeting fibroblast inactivation

Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma

The mesothelium lines body cavities and surrounds internal organs, widely contributing to homeostasis and regeneration. Mesothelium disruptions cause visceral anomalies and mesothelioma tumors. Nonetheless, the embryonic emergence of mesothelia remains incompletely understood. Here, we track mesothelial origins in the lateral plate mesoderm (LPM) using zebrafish. Single-cell transcriptomics uncovers a post-gastrulation gene expression signature centered on hand2 in distinct LPM progenitor cells. We map mesothelial progenitors to lateral-most, hand2-expressing LPM and confirm conservation in mouse. Time-lapse imaging of zebrafish hand2 reporter embryos captures mesothelium formation including pericardium, visceral, and parietal peritoneum. We find primordial germ cells migrate with the forming mesothelium as ventral migration boundary. Functionally, hand2 loss disrupts mesothelium formation with reduced progenitor cells and perturbed migration. In mouse and human mesothelioma, we document expression of LPM-associated transcription factors including Hand2, suggesting re-initiation of a developmental program. Our data connects mesothelium development to Hand2, expanding our understanding of mesothelial pathologies

Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma.

The mesothelium lines body cavities and surrounds internal organs, widely contributing to homeostasis and regeneration. Mesothelium disruptions cause visceral anomalies and mesothelioma tumors. Nonetheless, the embryonic emergence of mesothelia remains incompletely understood. Here, we track mesothelial origins in the lateral plate mesoderm (LPM) using zebrafish. Single-cell transcriptomics uncovers a post-gastrulation gene expression signature centered on hand2 in distinct LPM progenitor cells. We map mesothelial progenitors to lateral-most, hand2-expressing LPM and confirm conservation in mouse. Time-lapse imaging of zebrafish hand2 reporter embryos captures mesothelium formation including pericardium, visceral, and parietal peritoneum. We find primordial germ cells migrate with the forming mesothelium as ventral migration boundary. Functionally, hand2 loss disrupts mesothelium formation with reduced progenitor cells and perturbed migration. In mouse and human mesothelioma, we document expression of LPM-associated transcription factors including Hand2, suggesting re-initiation of a developmental program. Our data connects mesothelium development to Hand2, expanding our understanding of mesothelial pathologies

Characterisation of cardiomyocyte plasticity and the role of fibroblasts during zebrafish heart regeneration

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 12-01-2018El pez cebra es un organismo modelo ampliamente usado para estudiar la regeneración de corazón, en el que los cardiomiocitos preexistentes proliferan y reemplazan el miocardio perdido. Durante el desarrollo, los progenitores mesodérmicos del campo cardiaco primario forman un tubo cardiaco, al cual se añaden las células del campo cardiaco secundario. Aquí investigamos si los derivados de ambos campos en el pez dan lugar a distintas poblaciones de cardiomiocitos, y el grado de plasticidad durante la regeneración. El trazado de linaje de las células tbx5a-positivas también nos permitió investigar el destino de los cardiomiocitos durante la regeneración en adulto. Mientras que los análisis de trazado de linaje previos sugirieron que cada capa de cardiomiocitos es derivada de la misma capa, aquí describimos que los cardiomiocitos de las trabéculas pueden cambiar su especificación y diferenciarse en miocardio cortical. La regeneración del corazón está precedida de una respuesta fibrótica. Por lo tanto, fibrosis y regeneración no son respuestas mutuamente excluyentes. Tras una criolesión, colágeno y otras proteínas de matriz extracelular se acumulan en el lugar del daño. A diferencia de lo que ocurre en mamíferos, la fibrosis es una respuesta transitoria y simultánea a la regeneración de la nueva pared miocárdica. Aquí describimos que durante la regresión de la fibrosis, los fibroblastos no son completamente eliminados, sino que se inactivan. Sorprendentemente, limitar la respuesta fibrótica por ablación de las células que expresan col1a2 no estimuló la regeneración, sino que disminuyó la proliferación de cardiomiocitos. Concluimos que la regeneración del corazón de pez cebra es un proceso en el que hay una gran plasticidad de cardiomiocitos, y las células que producen matriz extracelular y se inactivan, promueven la proliferación de cardiomiocitos.The zebrafish is an established model organism to study heart regeneration, in which pre-existing cardiomyocytes (CMs) proliferate to replace the lost myocardium. During development, mesodermal progenitors from the first heart field (FHF) form a primitive cardiac tube, to which cells from the second heart field (SHF) are added. Here we investigated whether FHF and SHF derivatives in the zebrafish give rise to distinct CM populations, and examined the degree of cell fate plasticity of SHF derivatives during heart regeneration. Using tbx5a-lineage tracing we found that the adult zebrafish heart is also composed of CM populations from the FHF and SHF. Furthermore, ablation of FHF-derived CMs in the embryo is compensated by expansion of SHFderived cells. tbx5a lineage-tracing was also employed to investigate the fate of trabecular CMs during adult heart regeneration. While previous clonal analysis suggested that the different myocardial layers are rebuilt by CMs within each layers, we describe that trabecular CMs can switch their fate and differentiate into cortical myocardium. Heart regeneration is preceded by a fibrotic response. Thus, fibrosis and regeneration are not mutually exclusive responses. Upon cardiac cryoinjury, collagen and other extracellular matrix (ECM) components accumulate at the injury site. Unlike the situation in mammals, fibrosis in zebrafish is transient and its regression is concomitant with regrowth of the myocardial wall. We describe that during fibrosis regression, fibroblasts are not fully eliminated and become inactivated. Unexpectedly, limiting the fibrotic response by genetic ablation of col1a2-expressing cells not only failed to enhance regeneration but also impaired CMs proliferation. We conclude that zebrafish regeneration is a process that requires CM plasticity, and involves ECM-producing cells that become inactive and promote CMs proliferation

Use of echocardiography reveals reestablishment of ventricular pumping efficiency and partial ventricular wall motion recovery upon ventricular cryoinjury in the zebrafish.

While zebrafish embryos are amenable to in vivo imaging, allowing the study of morphogenetic processes during development, intravital imaging of adults is hampered by their small size and loss of transparency. The use of adult zebrafish as a vertebrate model of cardiac disease and regeneration is increasing at high speed. It is therefore of great importance to establish appropriate and robust methods to measure cardiac function parameters.Here we describe the use of 2D-echocardiography to study the fractional volume shortening and segmental wall motion of the ventricle. Our data show that 2D-echocardiography can be used to evaluate cardiac injury and also to study recovery of cardiac function. Interestingly, our results show that while global systolic function recovered following cardiac cryoinjury, ventricular wall motion was only partially restored.Cryoinjury leads to long-lasting impairment of cardiac contraction, partially mimicking the consequences of myocardial infarction in humans. Functional assessment of heart regeneration by echocardiography allows a deeper understanding of the mechanisms of cardiac regeneration and has the advantage of being easily transferable to other cardiovascular zebrafish disease models

Binary recombinase systems for high-resolution conditional mutagenesis

Conditional mutagenesis using Cre recombinase expressed from tissue specific promoters facilitates analyses of gene function and cell lineage tracing. Here, we describe two novel dual-promoter-driven conditional mutagenesis systems designed for greater accuracy and optimal efficiency of recombination. Co-Driver employs a recombinase cascade of Dre and Dre-respondent Cre, which processes loxP-flanked alleles only when both recombinases are expressed in a predetermined temporal sequence. This unique property makes Co-Driver ideal for sequential lineage tracing studies aimed at unraveling the relationships between cellular precursors and mature cell types. Co-InCre was designed for highly efficient intersectional conditional transgenesis. It relies on highly active trans-splicing inteins and promoters with simultaneous transcriptional activity to reconstitute Cre recombinase from two inactive precursor fragments. By generating native Cre, Co-InCre attains recombination rates that exceed all other binary SSR systems evaluated in this study. Both Co-Driver and Co-InCre significantly extend the utility of existing Cre-responsive alleles