Heart morphogenesis gene regulatory networks revealed by temporal expression analysis

Abstract

During embryogenesis the heart forms as a linear tube that then undergoes multiple simultaneous morphogenetic events to obtain its mature shape. To understand the gene regulatory networks (GRNs) driving this phase of heart development, during which many congenital heart disease malformations likely arise, we conducted an RNA-seq timecourse in zebrafish from 30 hpf to 72 hpf and identified 5861 genes with altered expression. We clustered the genes by temporal expression pattern, identified transcription factor binding motifs enriched in each cluster, and generated a model GRN for the major gene batteries in heart morphogenesis. This approach predicted hundreds of regulatory interactions and found batteries enriched in specific cell and tissue types, indicating that the approach can be used to narrow the search for novel genetic markers and regulatory interactions. Subsequent analyses confirmed the GRN using two mutants, and, and identified sets of duplicated zebrafish genes that do not show temporal subfunctionalization. This dataset provides an essential resource for future studies on the genetic/epigenetic pathways implicated in congenital heart defects and the mechanisms of cardiac transcriptional regulation.

Dynamics of cardiomyocyte transcriptome and chromatin landscape demarcates key events of heart development

“…4C,D; Supplemental Table 6). We identified sox10, klf6a, and irf2bp2a, which were previously linked to zebrafish heart morphogenesis (Hill et al 2017), as hub genes linked to their effector genes containing corresponding binding motifs in NFRs within their proximal promoter regions. Because the vast majority of genes within the turquoise module exhibited significant increase in expression and chromatin accessibility within associated NFRs between 24 and 48 hpf, it suggests the presence of a metabolic switch that takes place in CM between those developmental stages.…”

Dynamics of Cardiomyocyte Transcriptome and Chromatin Landscape Demarcates Key Events of Heart Development

“…Supplement Table 6). We have identified sox10, 294 klf6a and irf2bp2a, which were previously linked to zebrafish heart morphogenesis (Hill et al 2017), 295 as hub genes linked to their effector genes containing corresponding binding motifs in NFR localized 296 in proximal promoter regions. As the vast majority of genes within the turquoise module exhibited 297 significant increase in gene expression and chromatin accessibility within associated NFRs between 298 24 and 48 hpf, it suggests the presence of a metabolic switch that takes place in CM between those 299 developmental stages.…”

SLALOM: A Simple and Rapid Method for Enzymatic Synthesis of CRISPR-Cas9 sgRNA Libraries

“…Similar to the E. coli genome library, sequencing validation of the zebrafish heart library found spacers that were predominately 18 or 19 bp long ( Figure 6C). As sgRNAs in this library were expected to only target expressed genes, we compared the library to a set of genes detected in an existing zebrafish heart RNA-seq dataset 25 . These data showed the library targets at least 11,217 of the genes expressed in the heart ( Figure 6D).…”