RNA-seq timecourse analysis during heart looping morphogenesis. (A) Schematic of zebrafish heart (green) looping during the time period covered by the timecourse. Although several overlapping morphogenetic events are occurring, including cardiomyocyte maturation, initial trabeculation and sino-atrial (SA) node, atrioventricular canal (AVC) and valve formation, animals were staged based on heart looping. (B) Multidimensional scaling (MDS) of the RNA-seq samples. Relative distances between samples indicate their relative similarity (closer indicates more similar). Letters indicate the replicate (A, B or C); numbers (and colors) indicate the hours post-fertilization that the sample was collected. Each sample contained pooled hearts from ∼200 embryos. Outliers A30 and B42 were excluded from subsequent analysis. (C) Volcano plot showing the maximum log₂-scaled fold change of any time point compared with 30 hpf on the x-axis and the Phred-scaled P-value of the negative binomial likelihood ratio test, which tests for differential expression anywhere in the timecourse, on the y-axis. The blue horizontal line shows a Phred-scaled P-value cutoff of 13 (equivalent to a P-value of 0.05). The green vertical lines indicate a log₂ fold change cutoff of 1 and −1. Further analyses were conducted only on the differentially expressed genes in the upper left and upper right quadrants. (D) Heat map and hierarchical clustering of genes showing statistically significant changes in gene expression over the timecourse. Red indicates high expression and blue indicates low expression. Replicates were first averaged to create one column per time point.