Complex Interdependence Regulates Heterotypic Transcription Factor Distribution and Coordinates Cardiogenesis.

Transcription factors (TFs) are thought to function with partners to achieve specificity and precise quantitative outputs. In the developing heart, heterotypic TF interactions, such as between the T-box TF TBX5 and the homeodomain TF NKX2-5, have been proposed as a mechanism for human congenital heart defects. We report extensive and complex interdependent genomic occupancy of TBX5, NKX2-5, and the zinc finger TF GATA4 coordinately controlling cardiac gene expression, differentiation, and morphogenesis. Interdependent binding serves not only to co-regulate gene expression but also to prevent TFs from distributing to ectopic loci and activate lineage-inappropriate genes. We define preferential motif arrangements for TBX5 and NKX2-5 cooperative binding sites, supported at the atomic level by their co-crystal structure bound to DNA, revealing a direct interaction between the two factors and induced DNA bending. Complex interdependent binding mechanisms reveal tightly regulated TF genomic distribution and define a combinatorial logic for heterotypic TF regulation of differentiation.

A novel dual reporter embryonic stem cell line for toxicological assessment of teratogen-induced perturbation of anterior-posterior patterning of the heart.

Reliable in vitro models to assess developmental toxicity of drugs and chemicals would lead to improvement in fetal safety and a reduced cost of drug development. The validated embryonic stem cell test (EST) uses cardiac differentiation of mouse embryonic stem cells (mESCs) to predict in vivo developmental toxicity, but does not take into account the stage-specific patterning of progenitor populations into anterior (ventricular) and posterior (atrial) compartments. In this study, we generated a novel dual reporter mESC line with fluorescent reporters under the control of anterior and posterior cardiac promoters. Reporter expression was observed in nascent compartments in transgenic mouse embryos, and mESCs were used to develop differentiation assays in which chemical modulators of Wnt (XAV939: 3, 10 µM), retinoic acid (all-trans retinoic acid: 0.1, 1, 10 µM; 9-cis retinoic acid: 0.1, 1, 10 µM; bexarotene 0.1, 1, 10 µM), and Tgf-ß (SB431542: 3, 10 µM) pathways were tested for stage- and dose-dependent effects on in vitro anterior-posterior patterning. Our results suggest that with further development, the inclusion of anterior-posterior reporter expression could be part of a battery of high-throughput tests used to identify and characterize teratogens.

Fibronectin mediates mesendodermal cell fate decisions.

Non-cell-autonomous signals often play crucial roles in cell fate decisions during animal development. Reciprocal signaling between endoderm and mesoderm is vital for embryonic development, yet the key signals and mechanisms remain unclear. Here, we show that endodermal cells efficiently promote the emergence of mesodermal cells in the neighboring population through signals containing an essential short-range component. The endoderm-mesoderm interaction promoted precardiac mesoderm formation in mouse embryonic stem cells and involved endodermal production of fibronectin. In vivo, fibronectin deficiency resulted in a dramatic reduction of mesoderm accompanied by endodermal expansion in zebrafish embryos. This event was mediated by regulation of Wnt signaling in mesodermal cells through activation of integrin-ß1. Our findings highlight the importance of the extracellular matrix in mediating short-range signals and reveal a novel function of endoderm, involving fibronectin and its downstream signaling cascades, in promoting the emergence of mesoderm.

Reptilian heart development and the molecular basis of cardiac chamber evolution.

The emergence of terrestrial life witnessed the need for more sophisticated circulatory systems. This has evolved in birds, mammals and crocodilians into complete septation of the heart into left and right sides, allowing separate pulmonary and systemic circulatory systems, a key requirement for the evolution of endothermy. However, the evolution of the amniote heart is poorly understood. Reptilian hearts have been the subject of debate in the context of the evolution of cardiac septation: do they possess a single ventricular chamber or two incompletely septated ventricles? Here we examine heart development in the red-eared slider turtle, Trachemys scripta elegans (a chelonian), and the green anole, Anolis carolinensis (a squamate), focusing on gene expression in the developing ventricles. Both reptiles initially form a ventricular chamber that homogenously expresses the T-box transcription factor gene Tbx5. In contrast, in birds and mammals, Tbx5 is restricted to left ventricle precursors. In later stages, Tbx5 expression in the turtle (but not anole) heart is gradually restricted to a distinct left ventricle, forming a left-right gradient. This suggests that Tbx5 expression was refined during evolution to pattern the ventricles. In support of this hypothesis, we show that loss of Tbx5 in the mouse ventricle results in a single chamber lacking distinct identity, indicating a requirement for Tbx5 in septation. Importantly, misexpression of Tbx5 throughout the developing myocardium to mimic the reptilian expression pattern also results in a single mispatterned ventricular chamber lacking septation. Thus ventricular septation is established by a steep and correctly positioned Tbx5 gradient. Our findings provide a molecular mechanism for the evolution of the amniote ventricle, and support the concept that altered expression of developmental regulators is a key mechanism of vertebrate evolution.

TAF1 bromodomain inhibition as a candidate epigenetic driver of congenital heart disease.

Heart formation requires transcriptional regulators that underlie congenital anomalies and the fetal gene program activated during heart failure. Attributing the effects of congenital heart disease (CHD) missense variants to disruption of specific protein domains allows for a mechanistic understanding of CHDs and improved diagnostics. A combined chemical and genetic approach was employed to identify novel CHD drivers, consisting of chemical screening during pluripotent stem cell (PSC) differentiation, gene expression analyses of native tissues and primary cell culture models, and the in vitro study of damaging missense variants from CHD patients. An epigenetic inhibitor of the TATA-Box Binding Protein Associated Factor 1 (TAF1) bromodomain was uncovered in an unbiased chemical screen for activators of atrial and ventricular fetal myosins in differentiating PSCs, leading to the development of a high affinity inhibitor (5.1 nM) of the TAF1 bromodomain, a component of the TFIID complex. TAF1 bromodomain inhibitors were tested for their effects on stem cell viability and cardiomyocyte differentiation, implicating a role for TAF1 in cardiogenesis. Damaging TAF1 missense variants from CHD patients were studied by mutational analysis of the TAF1 bromodomain, demonstrating a repressive role of TAF1 that can be abrogated by the introduction of damaging bromodomain variants or chemical TAF1 bromodomain inhibition. These results indicate that targeting the TAF1/TFIID complex with chemical compounds modulates cardiac transcription and identify an epigenetically-driven CHD mechanism due to damaging variants within the TAF1 bromodomain.

Cholecystokinin peptide signaling is regulated by a TBX5-MEF2 axis in the heart.

The procholecystokinin (proCCK) gene encodes a secreted peptide known to regulate the digestive, endocrine, and nervous systems. Though recently proposed as a biomarker for heart dysfunction, its physiological role in both the embryonic and adult heart is poorly understood, and there are no reports of tissue-specific regulators of cholecystokinin signaling in the heart or other tissues. In the present study, mRNA of proCCK was observed in cardiac tissues during mouse embryonic development, establishing proCCK as an early marker of differentiated cardiomyocytes which is later restricted to anatomical subdomains of the neonatal heart. Three-dimensional analysis of the expression of proCCK and CCKAR/CCKBR receptors was performed using in situ hybridization and optical projection tomography, illustrating chamber-specific expression patterns in the postnatal heart. Transcription factor motif analyses indicated developmental cardiac transcription factors TBX5 and MEF2C as upstream regulators of proCCK, and this regulatory activity was confirmed in reporter gene assays. proCCK mRNA levels were also measured in the infarcted heart and in response to cyclic mechanical stretch and endothelin-1, indicating dynamic transcriptional regulation which might be leveraged for improved biomarker development. Functional analyses of exogenous cholecystokinin octapeptide (CCK-8) administration were performed in differentiating mouse embryonic stem cells (mESCs), and the results suggest that CCK-8 does not act as a differentiation modulator of cardiomyocyte subtypes. Collectively, these findings indicate that proCCK is regulated at the transcriptional level by TBX5-MEF2 and neurohormonal signaling, informing use of proCCK as a biomarker and future strategies for upstream manipulation of cholecystokinin signaling in the heart and other tissues.

GATA-targeted compounds modulate cardiac subtype cell differentiation in dual reporter stem cell line.

BACKGROUND: Pharmacological modulation of cell fate decisions and developmental gene regulatory networks holds promise for the treatment of heart failure. Compounds that target tissue-specific transcription factors could overcome non-specific effects of small molecules and lead to the regeneration of heart muscle following myocardial infarction. Due to cellular heterogeneity in the heart, the activation of gene programs representing specific atrial and ventricular cardiomyocyte subtypes would be highly desirable. Chemical compounds that modulate atrial and ventricular cell fate could be used to improve subtype-specific differentiation of endogenous or exogenously delivered progenitor cells in order to promote cardiac regeneration. METHODS: Transcription factor GATA4-targeted compounds that have previously shown in vivo efficacy in cardiac injury models were tested for stage-specific activation of atrial and ventricular reporter genes in differentiating pluripotent stem cells using a dual reporter assay. Chemically induced gene expression changes were characterized by qRT-PCR, global run-on sequencing (GRO-seq) and immunoblotting, and the network of cooperative proteins of GATA4 and NKX2-5 were further explored by the examination of the GATA4 and NKX2-5 interactome by BioID. Reporter gene assays were conducted to examine combinatorial effects of GATA-targeted compounds and bromodomain and extraterminal domain (BET) inhibition on chamber-specific gene expression. RESULTS: GATA4-targeted compounds 3i-1000 and 3i-1103 were identified as differential modulators of atrial and ventricular gene expression. More detailed structure-function analysis revealed a distinct subclass of GATA4/NKX2-5 inhibitory compounds with an acetyl lysine-like domain that contributed to ventricular cells (%Myl2-eGFP+). Additionally, BioID analysis indicated broad interaction between GATA4 and BET family of proteins, such as BRD4. This indicated the involvement of epigenetic modulators in the regulation of GATA-dependent transcription. In this line, reporter gene assays with combinatorial treatment of 3i-1000 and the BET bromodomain inhibitor (+)-JQ1 demonstrated the cooperative role of GATA4 and BRD4 in the modulation of chamber-specific cardiac gene expression. CONCLUSIONS: Collectively, these results indicate the potential for therapeutic alteration of cell fate decisions and pathological gene regulatory networks by GATA4-targeted compounds modulating chamber-specific transcriptional programs in multipotent cardiac progenitor cells and cardiomyocytes. The compound scaffolds described within this study could be used to develop regenerative strategies for myocardial regeneration.