Spotlight on Isl1: A Key Player in Cardiovascular Development and Diseases

Cardiac transcription factors orchestrate a regulatory network controlling cardiovascular development. Isl1, a LIM-homeodomain transcription factor, acts as a key player in multiple organs during embryonic development. Its crucial roles in cardiovascular development have been elucidated by extensive studies, especially as a marker gene for the second heart field progenitors. Here, we summarize the roles of Isl1 in cardiovascular development and function, and outline its cellular and molecular modes of action, thus providing insights for the molecular basis of cardiovascular diseases.

Lamb1a regulates atrial growth by limiting second heart field addition during zebrafish heart development

During early vertebrate heart development the heart transitions from a linear tube to a complex asymmetric structure, a morphogenetic process which occurs simultaneously with growth of the heart. Cardiac growth during early heart morphogenesis is driven by deployment of cells from the Second Heart Field (SHF) into both poles of the heart. Laminin is a core component of the extracellular matrix (ECM), and although mutations in laminin subunits are linked with cardiac abnormalities, no role for laminin has been identified in early vertebrate heart morphogenesis. We identified tissue-specific expression of laminin genes in the developing zebrafish heart, supporting a role for laminins in heart morphogenesis. Analysis of heart development in lamb1a zebrafish mutant embryos reveals mild morphogenetic defects and progressive cardiomegaly, and that Lamb1a functions to limit heart size during cardiac development by restricting SHF addition. lamb1a mutants exhibit hallmarks of altered haemodynamics, and blocking cardiac contractility in lamb1a mutants rescues heart size and atrial SHF addition. Together this suggests that laminin mediates interactions between SHF deployment and cardiac biomechanics during heart development and growth in the developing embryo.

Abstract MP245: Tet Proteins Regulate Second Heart Field Multipotent Progenitors Differentiating To Myocytes

DNA methylation and demethylation play an important role in shaping the epigenetic landscape and chromatin accessibility to control gene expression during development in mammals. Ten-eleven Translocation (Tet1, Tet2 and Tet3) is a family of dioxygenases that catalyze DNA methylation oxidation with ultimate DNA demethylation. Our previous study showed that cardiac-specific deletion of Tet2 and Tet3 could disrupt YY1-mediated long range chromatin interactions during heart development and lead to ventricular non-compaction cardiomyopathy. However, it is still unclear whether and how Tet protein mediated epigenetic modifications contribute to cardiac lineage specification during embryonic development. In this study, we generated cardiac specific Tet1-3 triple deficient (Tet-TKO) mouse lines using various cardiac specific Cres to evaluate the function of Tet protein in regulating cardiac lineage specification. We observed developmental defects at outflow tract (OFT) in Tet-TKO embryos, suggesting that Tet deficiency affects the second heart field (SHF) development. Single cell RNA-seq analysis further revealed the accumulation of multipotent SHF progenitors and subsequent halt of myocyte differentiation upon Tet depletion. At the molecular level, we found that Tet ablation perturbs the transcriptional network of Islet1, a transcription factor that is crucial for cardiac development in embryos. Overall, our study demonstrates a critical role of Tet-mediated epigenetic regulation for embryonic cardiac development.

Maternal hyperglycemia impedes second heart field-derived cardiomyocyte differentiation to elevate the risk of congenital heart defects

Congenital heart disease (CHD) is the most frequently occurring structural malformations of the heart affecting ~1% of live births. Besides genetic predisposition, embryonic exposure to teratogens during pregnancy increases the risk of CHD. However, the dose and cell-type-specific responses to an adverse maternal environment remain poorly defined. Here, we report a dose-response relationship between maternal glucose levels and phenotypic severity of CHD in offspring, using a chemically-induced pregestational diabetes mellitus (PGDM) mouse model. Embryos from dams with low-level maternal hyperglycemia (matHG) displayed trabeculation defects, ventricular wall thinning, and ventricular septal defects (VSD). On the other hand, embryos from dams with high-level matHG display outflow tract malformations, ventricular wall thinning and an increased rate of VSD. Our findings show that increasing levels of matHG exacerbates CHD occurrence and severity in offspring compared to control embryos. We applied single-cell RNA- sequencing to define matHG-related transcriptional differences in E9.5 and E11.5 hearts as comparing to controls. Disease-dependent gene-expression changes were observed in Isl1+ second heart field (SHF) and Tnnt2+ cardiomyocyte subpopulations. Lineage tracing studies in Isl1-Cre; RosamTmG embryonic hearts showed Isl1+-SHF-derived cardiomyocyte differentiation was impaired with matHG. This study highlights the influence of matHG-dosage on cardiac morphogenesis and identifies perturbations in the Isl1-dependent gene-regulatory network that affect SHF-derived cardiomyocyte differentiation contributing to matPGDM-induced CHD.

Persistent Ventricle Partitioning in the Adult Zebrafish Heart

The vertebrate heart integrates cells from the early-differentiating first heart field (FHF) and the later-differentiating second heart field (SHF), both emerging from the lateral plate mesoderm. In mammals, this process forms the basis for the development of the left and right ventricle chambers and subsequent chamber septation. The single ventricle-forming zebrafish heart also integrates FHF and SHF lineages during embryogenesis, yet the contributions of these two myocardial lineages to the adult zebrafish heart remain incompletely understood. Here, we characterize the myocardial labeling of FHF descendants in both the developing and adult zebrafish ventricle. Expanding previous findings, late gastrulation-stage labeling using drl-driven CreERT2 recombinase with a myocardium-specific, myl7-controlled, loxP reporter results in the predominant labeling of FHF-derived outer curvature and the right side of the embryonic ventricle. Raised to adulthood, such lineage-labeled hearts retain broad areas of FHF cardiomyocytes in a region of the ventricle that is positioned at the opposite side to the atrium and encompasses the apex. Our data add to the increasing evidence for a persisting cell-based compartmentalization of the adult zebrafish ventricle even in the absence of any physical boundary.

Persistent ventricle partitioning in the adult zebrafish heart

The vertebrate heart integrates cells from the early-differentiating first heart field (FHF) and the later-differentiating second heart field (SHF) emerging from the lateral plate mesoderm. In mammals, this process forms the basis for the development of the left and right ventricle chambers and subsequent chamber septation. The single ventricle-forming zebrafish heart also integrates FHF and SHF lineages during embryogenesis, yet the contributions of these two myocardial lineages to the adult zebrafish heart remain incompletely understood. Here, we characterize the myocardial labeling of FHF descendants in both the developing and adult zebrafish ventricle. Expanding previous findings, late gastrulation-stage labeling using drl-driven CreERT2 recombinase with a myocardium-specific, myl7-controlled loxP reporter results in predominant labeling of FHF-derived outer curvature and the right side of the embryonic ventricle. Raised to adulthood, such lineage-labeled hearts retain broad areas of FHF cardiomyocytes in a region of the ventricle that is positioned at the opposite side to the atrium and encompasses the apex. Our data add to the increasing evidence for a persisting cell-based compartmentalization of the adult zebrafish ventricle even in the absence of any physical boundary.

Genetic and Cellular Interaction During Cardiovascular Development Implicated in Congenital Heart Diseases

Congenital heart disease (CHD) is the most common life-threatening congenital anomaly. CHD occurs due to defects in cardiovascular development, and the majority of CHDs are caused by a multifactorial inheritance mechanism, which refers to the interaction between genetic and environmental factors. During embryogenesis, the cardiovascular system is derived from at least four distinct cell lineages: the first heart field, second heart field, cardiac neural crest, and proepicardial organ. Understanding the genes involved in each lineage is essential to uncover the genomic architecture of CHD. Therefore, we provide an overview of recent research progress using animal models and mutation analyses to better understand the molecular mechanisms and pathways linking cardiovascular development and CHD. For example, we highlight our recent work on genes encoding three isoforms of inositol 1,4,5-trisphosphate receptors (IP3R1, 2, and 3) that regulate various vital and developmental processes, which have genetic redundancy during cardiovascular development. Specifically, IP3R1 and 2 have redundant roles in the atrioventricular cushion derived from the first heart field lineage, whereas IP3R1 and 3 exhibit redundancy in the right ventricle and the outflow tract derived from the second heart field lineage, respectively. Moreover, 22q11.2 deletion syndrome (22q11DS) is highly associated with CHD involving the outflow tract, characterized by defects of the cardiac neural crest lineage. However, our studies have shown that TBX1, a major genetic determinant of 22q11DS, was not expressed in the cardiac neural crest but rather in the second heart field, suggesting the importance of the cellular interaction between the cardiac neural crest and the second heart field. Comprehensive genetic analysis using the Japanese genome bank of CHD and mouse models revealed that a molecular regulatory network involving GATA6, FOXC1/2, TBX1, SEMA3C, and FGF8 was essential for reciprocal signaling between the cardiac neural crest and the second heart field during cardiovascular development. Elucidation of the genomic architecture of CHD using induced pluripotent stem cells and next-generation sequencing technology, in addition to genetically modified animal models and human mutation analyses, would facilitate the development of regenerative medicine and/or preventive medicine for CHD in the near future.

Lamb1a regulates atrial growth by limiting excessive, contractility-dependent second heart field addition during zebrafish heart development

During early vertebrate heart development, the heart transitions from a linear tube to a complex asymmetric structure. This process includes looping of the tube and ballooning of the emerging cardiac chambers, which occur simultaneously with growth of the heart. A key driver of cardiac growth is deployment of cells from the Second Heart Field (SHF) into both poles of the heart, with cardiac morphogenesis and growth intimately linked in heart development. Laminin is a core component of extracellular matrix (ECM) basement membranes, and although mutations in specific laminin subunits are linked with a variety of cardiac abnormalities, including congenital heart disease and dilated cardiomyopathy, no role for laminin has been identified in early vertebrate heart morphogenesis. We identified dynamic, tissue-specific expression of laminin subunit genes in the developing zebrafish heart, supporting a role for laminins in heart morphogenesis.lamb1amutants exhibit cardiomegaly from 2dpf onwards, with subsequent progressive defects in cardiac morphogenesis characterised by a failure of the chambers to compact around the developing atrioventricular canal. We show that loss oflamb1aresults in excess addition of SHF cells to the atrium, revealing that Lamb1a functions to limit heart size during cardiac development by restricting SHF addition to the venous pole.lamb1amutants exhibit hallmarks of altered haemodynamics, and specifically blocking cardiac contractility inlamb1amutants rescues heart size and atrial SHF addition. Furthermore, we identify that FGF and RA signalling, two conserved pathways promoting SHF addition, are regulated by heart contractility and are dysregulated inlamb1amutants, suggesting that laminin mediates interactions between SHF deployment, heart biomechanics, and biochemical signalling during heart development. Together, this describes the first requirement for laminins in early vertebrate heart morphogenesis, reinforcing the importance of specialised ECM composition in cardiac development.