Early Endocardial Morphogenesis Requires Scl/Tal1

The primitive heart tube is composed of an outer myocardial and an inner endocardial layer that will give rise to the cardiac valves and septa. Specification and differentiation of these two cell layers are among the earliest events in heart development, but the embryonic origins and genetic regulation of early endocardial development remain largely undefined. We have analyzed early endocardial development in the zebrafish using time-lapse confocal microscopy and show that the endocardium seems to originate from a region in the lateral plate mesoderm that will give rise to hematopoietic cells of the primitive myeloid lineage. Endocardial precursors appear to rapidly migrate to the site of heart tube formation, where they arrive prior to the bilateral myocardial primordia. Analysis of a newly discovered zebrafish Scl/Tal1 mutant showed an additional and previously undescribed role of this transcription factor during the development of the endocardium. In Scl/Tal1 mutant embryos, endocardial precursors are specified, but migration is severely defective and endocardial cells aggregate at the ventricular pole of the heart. We further show that the initial fusion of the bilateral myocardial precursor populations occurs independently of the endocardium and tal1 function. Our results suggest early separation of the two components of the primitive heart tube and imply Scl/Tal1 as an indispensable component of the molecular hierarchy that controls endocardium morphogenesis

αE-catenin-dependent mechanotransduction is essential for proper convergent extension in zebrafish

Cadherin complexes mediate cell-cell adhesion and are crucial for embryonic development. Besides their structural function, cadherin complexes also transduce tension across the junction-actomyosin axis into proportional biochemical responses. Central to this mechanotransduction is the stretching of the cadherin-F-actin-linker α-catenin, which opens its central domain for binding to effectors such as vinculin. Mechanical unfolding of α-catenin leads to force-dependent reinforcement of cadherin-based junctions as studied in cell culture. The importance of cadherin mechanotransduction for embryonic development has not been studied yet. Here we used TALEN-mediated gene disruption to perturb endogenous αE-catenin in zebrafish development. Zygotic α-catenin mutants fail to maintain their epithelial barrier, resulting in tissue rupturing. We then specifically disrupted mechanotransduction, while maintaining cadherin adhesion, by expressing an αE-catenin construct in which the mechanosensitive domain was perturbed. Expression of either wild-type or mechano-defective α-catenin fully rescues barrier function in α-catenin mutants; however, expression of mechano-defective α-catenin also induces convergence and extension defects. Specifically, the polarization of cadherin-dependent, lamellipodia-driven cell migration of the lateral mesoderm was lost. These results indicate that cadherin mechanotransduction is crucial for proper zebrafish morphogenesis, and uncover one of the essential processes affected by its perturbatio

Cardiac regenerative capacity: an evolutionary afterthought?

Cardiac regeneration is the outcome of the highly regulated interplay of multiple processes, including the inflammatory response, cardiomyocyte dedifferentiation and proliferation, neovascularization and extracellular matrix turnover. Species-specific traits affect these injury-induced processes, resulting in a wide variety of cardiac regenerative potential between species. Indeed, while mammals are generally considered poor regenerators, certain amphibian and fish species like the zebrafish display robust regenerative capacity post heart injury. The species-specific traits underlying these differential injury responses are poorly understood. In this review, we will compare the injury induced processes of the mammalian and zebrafish heart, describing where these processes overlap and diverge. Additionally, by examining multiple species across the animal kingdom, we will highlight particular traits that either positively or negatively affect heart regeneration. Last, we will discuss the possibility of overcoming regeneration-limiting traits to induce heart regeneration in mammals

A heterozygous mutation in cardiac troponin t promotes ca2+ dysregulation and adult cardiomyopathy in zebrafish

Cardiomyopathies are a group of heterogeneous diseases that affect the muscles of the heart, leading to early morbidity and mortality in young and adults. Genetic forms of cardiomyopathy are caused predominantly by mutations in structural components of the cardiomyocyte sarcomeres, the contractile units of the heart, which includes cardiac Troponin T (TnT). Here, we generated mutations with CRISPR/Cas9 technology in the zebrafish tnnt2a gene, encoding cardiac TnT, at a mutational “hotspot” site to establish a zebrafish model for genetic cardiomyopathies. We found that a heterozygous tnnt2a mutation deleting Arginine at position 94 and Lysine at position 95 of TnT causes progressive cardiac structural changes resulting in heart failure. The cardiac remodeling is presented by an enlarged atrium, decreased ventricle size, increased myocardial stress as well as increased fibrosis. As early as five days post fertilization, larvae carrying the TnT RK94del mutation display diastolic dysfunction and impaired calcium dynamics related to increased Ca2+ sensitivity. In conclusion, adult zebrafish with a heterozygous TnT-RK94del mutation develop cardiomyopathy as seen in patients with TnT mutations and therefore represent a promising model to study disease mechanisms and to screen for putative therapeutic compounds

Notch and Bmp signaling pathways act coordinately during the formation of the proepicardium.

The epicardium is the outer mesothelial layer of the heart. It encloses the myocardium and plays key roles in heart development and regeneration. It derives from the proepicardium (PE), cell clusters that appear in the dorsal pericardium (DP) close to the atrioventricular canal and the venous pole of the heart, and are released into the pericardial cavity. PE cells are advected around the beating heart until they attach to the myocardium. Bmp and Notch signaling influence PE formation, but it is unclear how both signaling pathways interact during this process in the zebrafish. Here, we show that the developing PE is influenced by Notch signaling derived from the endothelium. Overexpression of the intracellular receptor of notch in the endothelium enhances bmp expression, increases the number of pSmad1/5 positive cells in the DP and PE, and enhances PE formation. On the contrary, pharmacological inhibition of Notch1 impairs PE formation. bmp2b overexpression can rescue loss of PE formation in the presence of a Notch1 inhibitor, but Notch gain-of-function could not recover PE formation in the absence of Bmp signaling. Endothelial Notch signaling activates bmp expression in the heart tube, which in turn induces PE cluster formation from the DP layer.Nadia Mercader was funded by the Spanish Ministry of Economy and Competitiveness through grant BFU2014-56970-P (Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016. Programa Estatal de I+D+i Orientada a los Retos de la Sociedad Retos Investigación: Proyectos I+D +i 2016, del Ministerio de Economía competitividad e Industria), and cofunding by Fondo Europeo de Desarrollo Regional (FEDER). Nadia Mercader is also supported by the European Industrial Doctorate Program EID 722427. Nadia Mercader and Julien Vermot are supported by the Swiss National Science Foundation grant ANR-SNF 310030L_182575. This project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No. 708312 (MP) and from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme: GA Nº682938. Laura Andrés-Delgado was funded (2014-16) through the postdoctoral fellowship Ayudas Postdoctorales 2013. José Luis de la Pompa was supported by grants SAF2016-78370-R, CB16/11/00399 (CIBER CV) and RD16/0011/0021 (TERCEL) from the Spanish Ministry of Science and Innovation. The CNIC is supported by the Ministry of Science and Innovation and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505).S

Epigenetic state changes underlie metabolic switch in mouse post-infarction border zone cardiomyocytes

Myocardial infarction causes ventricular muscle loss and formation of scar tissue. The surviving myocardium in the border zone, located adjacent to the infarct, undergoes profound changes in function, structure and composition. How and to what extent these changes of border zone cardiomyocytes are regulated epigenetically is not fully understood. Here, we obtained transcriptomes of PCM-1-sorted mouse cardiomyocyte nuclei of healthy left ventricle and 7 days post myocardial infarction border zone tissue. We validated previously observed downregulation of genes involved in fatty acid metabolism, oxidative phosphorylation and mitochondrial function in border zone-derived cardiomyocytes, and observed a modest induction of genes involved in glycolysis, including Slc2a1 (Glut1) and Pfkp. To gain insight into the underlying epigenetic regulatory mechanisms, we performed H3K27ac profiling of healthy and border zone cardiomyocyte nuclei. We confirmed the switch from Mef2-to AP-1 chromatin association in border zone cardiomyocytes, and observed, in addition, an enrichment of PPAR/RXR binding motifs in the sites with reduced H3K27ac signal. We detected downregulation and accompanying epigenetic state changes at several key PPAR target genes including Ppargc1a (PGC-1α), Cpt2, Ech1, Fabpc3 and Vldrl in border zone cardiomyocytes. These data indicate that changes in epigenetic state and gene regulation underlie the maintained metabolic switch in border zone cardiomyocytes

Inflammatory response in hematopoietic stem and progenitor cells triggered by activating SHP2 mutations evokes blood defects

Gain-of-function mutations in the protein-tyrosine phosphatase SHP2 are the most frequently occurring mutations in sporadic juvenile myelomonocytic leukemia (JMML) and JMML-like myeloproliferative neoplasm (MPN) associated with Noonan syndrome (NS). Hematopoietic stem and progenitor cells (HSPCs) are the disease propagating cells of JMML. Here, we explored transcriptomes of HSPCs with SHP2 mutations derived from JMML patients and a novel NS zebrafish model. In addition to major NS traits, CRISPR/Cas9 knock-in Shp2 D61G mutant zebrafish recapitulated a JMML-like MPN phenotype, including myeloid lineage hyperproliferation, ex vivo growth of myeloid colonies, and in vivo transplantability of HSPCs. Single-cell mRNA sequencing of HSPCs from Shp2 D61G zebrafish embryos and bulk sequencing of HSPCs from JMML patients revealed an overlapping inflammatory gene expression pattern. Strikingly, an anti-inflammatory agent rescued JMML-like MPN in Shp2 D61G zebrafish embryos. Our results indicate that a common inflammatory response was triggered in the HSPCs from sporadic JMML patients and syndromic NS zebrafish, which potentiated MPN and may represent a future target for JMML therapies

The centriolar satellite protein Cfap53 facilitates formation of the zygotic microtubule organizing center in the zebrafish embryo

In embryos of most animal species, the zygotic centrosome is assembled by the centriole derived from the sperm cell and pericentriolar proteins present in the oocyte. This zygotic centrosome acts as a microtubule organizing center (MTOC) to assemble the sperm aster and mitotic spindle. As MTOC formation has been studied mainly in adult cells, very little is known about the formation of the zygotic MTOC. Here, we show that zebrafish (Danio rerio) embryos lacking either maternal or paternal Cfap53, a centriolar satellite protein, arrest during the first cell cycle. Although Cfap53 is dispensable for sperm aster function, it aids proper formation of the mitotic spindle. During cell division, Cfap53 colocalizes with γ-tubulin and with other centrosomal and centriolar satellite proteins at the MTOC. Furthermore, we find that γ-tubulin localization at the MTOC is impaired in the absence of Cfap53. Based on these results, we propose a model in which Cfap53 deposited in the oocyte and the sperm participates in the organization of the zygotic MTOC to allow mitotic spindle formation