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Spatially resolved RNA-sequencing of the embryonic zebrafish heart

ABSTRACT: Development of specialized cell types and structures in the vertebrate heart is regulated by spatially-restricted molecular pathways. Disruptions in these pathways can cause severe congenital cardiac malformations or functional defects. To better understand these pathways and how they regulate cardiac development and function we used tomo-seq, combining high-throughput RNA sequencing with tissue sectioning, to establish a genome-wide expression dataset with high spatial resolution for the developing zebrafish heart. Analysis of the dataset revealed over 1100 genes differentially expressed in sub-compartments. Pacemaker cells in the sinoatrial region induce heart contractions, but little is known about the mechanisms underlying their development and function. Using our transcriptome map, we identified spatially restricted Wnt/β-catenin signaling activity in pacemaker cells, which was controlled by Islet-1 activity. Moreover, Wnt/β-catenin signaling at a specific developmental stage in the myocardium controls heart rate by regulating pacemaker cellular response to parasympathetic stimuli. Thus, this high-resolution transcriptome map incorporating all cell types in the embryonic heart can expose spatially-restricted molecular pathways critical for specific cardiac functions.

ORGANISM(S): Danio rerio

PROVIDER: GSE104057 | GEO | 2018/02/06

REPOSITORIES: GEO

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Project description:Summary: Activation of the evolutionarily conserved, developmental Wnt pathway has been reported during maladaptive cardiac remodeling. Although the function of Wnt-transcriptional activation in development is well described, the consequences of Wnt pathway activation, as well as its cardiac-specific regulatory role in the adult heart, is largely unknown. We show that β-catenin and Transcription factor 7-like 2 (TCF7L2), the main nuclear components of the Wnt-transcriptional cascade, and their transcriptional activity are increased upon pathological remodeling in both murine and human hearts. To understand the consequences of increased Wnt signaling pathway activity, we utilized an in vivo mouse model in which β-catenin is acutely stabilized in adult cardiomyocytes (CM), leading to increased ventricular TCF7L2 expression and activation of its target genes. Mice with stabilized β-catenin displayed cardiac hypertrophy, increased mortality, reduced cardiac function and altered calcium homeostasis, similar to experimentally induced hypertrophy. Moreover, we observed a re-activation of Wnt-dependent developmental gene programs including activation of the Wnt/β-catenin-independent pathway, increased CM cell cycling with poly-nucleation and cytoskeletal disorganization, underscoring a central role in adult tissue remodeling. By integrating transcriptome analyses and genome-wide occupancy (ChIP-seq) of the endogenous ventricular TCF7L2, we show that upon aberrant Wnt activation, TCF7L2 induces context and Wnt-specific gene regulation in pathological remodeling. Interestingly, β-catenin stabilized ventricles showed increased histone H3 lysine 27 acetylation (H3K27ac) and TCF7L2 recruitment to novel disease-associated gene-specific enhancers. Importantly, using integrative motif analyses and experimental evidences, our data uncovered a role for GATA4 as a cardiogenic regulator of TCF7L2/β-catenin complex and established a paradigm for cell-specific effects of Wnt signaling. Altogether, our studies unraveled the nuclear Wnt-TCF7L2-associated chromatin landscape and its role in adult tissue remodeling leading to heart failure. Purpose: The aim of this study was to compare transcriptome profiles (RNA-seq) of normal (containing a Cre recombinase positive locus- Cre "positive" control with a WT β-catenin locus; to eliminate effects of Cre-mediated cardiac toxicity) and β-catenin stabilized murine adult cardiac ventricles. Methods: Adult cardiac tissue mRNA profiles for normal and Wnt-activated mice were obtained using deep sequencing, in triplicates, using Illumina HiSeq2000. The sequence reads that passed quality filters were analyzed at the transcript isoform level with TopHat followed by DESeq2. qPCR validation was performed using TaqMan and SYBR Green assays Conclusions: Our study represents the first detailed analysis of the processes triggered upon Wnt activation in the adult heart, which was so far, not investigated. We report that this Wnt activation in the adult heart maintains its developmental function; however due to the lack of adequate developmental plasticity in the adult heart, culminates in pathological remodeling.

Project description:Activation of the evolutionarily conserved, developmental Wnt pathway has been reported during maladaptive cardiac remodeling. Although the function of Wnt-transcriptional activation in development is well described, the consequences of Wnt pathway activation, as well as its cardiac-specific regulatory role in the adult heart, is largely unknown. We show that β-catenin and Transcription factor 7-like 2 (TCF7L2), the main nuclear components of the Wnt-transcriptional cascade, and their transcriptional activity are increased upon pathological remodeling in both murine and human hearts. To understand the consequences of increased Wnt signaling pathway activity, we utilized an in vivo mouse model in which β-catenin is acutely stabilized in adult cardiomyocytes (CM), leading to increased ventricular TCF7L2 expression and activation of its target genes. Mice with stabilized β-catenin displayed cardiac hypertrophy, increased mortality, reduced cardiac function and altered calcium homeostasis, similar to experimentally induced hypertrophy. Moreover, we observed a re-activation of Wnt-dependent developmental gene programs including activation of the Wnt/β-catenin-independent pathway, increased CM cell cycling with poly-nucleation and cytoskeletal disorganization, underscoring a central role in adult tissue remodeling. By integrating transcriptome analyses and genome-wide occupancy (ChIP-seq) of the endogenous ventricular TCF7L2, we show that upon aberrant Wnt activation, TCF7L2 induces context and Wnt-specific gene regulation in pathological remodeling. Interestingly, β-catenin stabilized ventricles showed increased histone H3 lysine 27 acetylation (H3K27ac) and TCF7L2 recruitment to novel disease-associated gene-specific enhancers. Importantly, using integrative motif analyses and experimental evidences, our data uncovered a role for GATA4 as a cardiogenic regulator of TCF7L2/β-catenin complex and established a paradigm for cell-specific effects of Wnt signaling. Altogether, our studies unraveled the nuclear Wnt-TCF7L2-associated chromatin landscape and its role in adult tissue remodeling leading to heart failure.

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Spatially resolved RNA-sequencing of the embryonic zebrafish heart

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