corast^l325/stl325 embryos display reduced survival and heart failure. (A) Light microscopy showing the emergence of pericardial edema between 72 and 120 h post fertilization (hpf). P, pericardium; H, heart. Phenotype ratios are presented on the right. (number of trials=4; number of samples per trial=48) (B) Survival curve of cora^stl325 embryos relative to their unaffected clutchmates (number of trials=4; number of samples per trial=48). (C) Quantification of light microscopy images measuring area of the pericardium (number of trials=4; number of samples per trial=48). (D) Quantification of light microscopy images measuring ejection fraction (number of trials=4; number of samples per trial=48). (E) Lightsheet microscopy generated images of 96 hpf embryos harboring the cmlc::GFP transgenic reporter. Hearts are shown in systole and diastole. A, atrium; V, ventricle. Quantification of ejection fraction (n=4). (F) Quantification of changes in cell dimension using lightsheet-generated videos (n=4).

cora^stl325 hearts display hypoplasia and reduced proliferation. (A) Fluorescent microscopy images of 96 hpf embryos (left, cmlc2::GFP; n=8) and H&E staining (right; n=4). (B) Fluorescent microscopy images of 96 hpf embryos stained with the SF46 (orange) and MF20 (green) showing normal atrioventricular patterning (left). Electron microscopy showing intact sarcomeres in control and cora^stl325 cardiomyocytes (right; n=4). (C) Brightfield and fluorescence microscopy time-course of cmlc2::GFP expression between 48 and 120 hpf (Control (n=12) and cora^stl325 (n=8). (D) Quantification of atrial and ventricular cross-sectional areas obtained from C [Control (n=12) and cora^stl325 (n=8)]. (E) BrdU immunostaining (red) of 96 hpf embryos embedded in paraffin [Control (n=6) and cora^stl325 (n=12)].

cora^stl325 embryos have craniofacial deformities and neuroanatomical defects. (A) Head morphology of control and cora^stl325 embryos at 96 hpf. (B) Head-to-body and head-to-eye ratios. Control (n=13), cora^stl325 (n=9). (C) Alcian Blue staining of control and cora^stl325 embryos at 96 hpf (left), with quantification of major jaw elements (right). EP, Ethmoid plate; MC, Meckel's Cartilage; T, Trabeculae. n=9 per experimental group. (D) H&E staining of the jaw of control and cora^stl325 embryos at 96 hpf. (E) Fluorescence microscopy images of Tg(crestin::GFP) and Tg(sox10::RFP) control and cora^stl325 embryos. (F) X-ray microscopy-generated images of 96 hpf embryos (left) and quantification of tissue volume (right). Br, brain Control (n=4), cora^stl325 (n=4).

cora^stl325 encodes a nonsense mutation in taf5. (A) Workflow for mapping cora^stl325 using whole genome next generation sequencing. (B) Manhattan plot displaying homozygosity ratio for the AB* background (y-axis) as a function of chromosome location (x-axis). A value of 2.0 indicates 100% homozygosity for the AB* background. Chr. 1: chromosome 1. (C) Fine chromosomal mapping identified a 24 cM region (red box) that was linked with the cora^stl325 phenotype (top). Genotyping to identify the point mutation leading to a nonsense mutation in taf5. The nonsense mutation was linked to the cora^stl325 phenotype (bottom). (D) Graphical representation of the taf5 genomic locus and TAF5 protein including major functional domains. Red box indicates the mutated base and resulting nonsense mutation.

cora^stl325 is a null allele of taf5. (A) Brightfield images of control, cora^stl325, taf5^stl852, and cora^stl325/ taf5^stl852 embryos at 96 hpf. (B) Measurement of pericardial area and ejection fraction in control, cora^stl325, taf5^stl852, and cora^stl325/ taf5^stl852 embryos at 96 hpf. n=22. (C) Measurement of head-to-body and head-to-eye ratios in control, cora^stl325, taf5^stl852, and cora^stl325/ taf5^stl852 embryos at 96 hpf. n=22. (D) Survival of control, taf5^stl852, and cora^stl325/taf5 CRISPR embryos. n=42. (E) RTPCR for taf5 mRNA in control, cora^stl325, taf5^stl852, and cora^stl325/ taf5^stl852 embryos at 96 hpf. n=7. (F) Time course of taf5 mRNA expression by whole-mount in situ hybridization (top) and taf5 mRNA expression in cora^stl325 and taf5^stl852 embryos at 96 hpf. Note: all brackets marked with ‘*’ represent P<1E-4.

taf1 deletion recapitulates the corazoncito phenotype. (A) Brightfield images of control and taf1^{stl456/stl456} embryos at 96 hpf. (B) taf1 mRNA expression measured by in situ hybridization (left) and RT-PCR (right). N=6 per experimental group. (C) Quantification of pericardial area (left) and ejection fraction (right) in control (n=30) and taf1^{stl456/stl456} (n=12) embryos at 96 hpf. (D) Quantification of head-to-body ratio (left) and head-to-eye ratio (right) in control (n=21) and taf1^{stl456/stl456} (n=20) embryos at 96 hpf. (E) Alcian Blue staining of control and taf1 knockout (KO) embryos. (F) X-ray microscopy-generated images of control and taf1 KO embryos at 96 hpf (left). Quantification of brain volume (right). N=4 per experimental group. Br, brain (red), heart (blue). (G) Whole-mount in situ hybridization of crestin and sox10 mRNA expression in control and taf1 KO embryos at 96 hpf. (H) Whole-mount in situ hybridization of neuronal (neurog1) and brain region markers (six3b, Adcyap1b, zic2a).

RNA sequencing of control and cora^stl325 identifies derangements metabolic gene expression. (A) Principal component analysis (PCA) plot of pooled sequencing libraries generated from control and cora^stl325 embryos at 96 hpf. Each dot represents a pool of 100 embryonic hearts. (B) Bar graph of the top Gene Ontology (GO) pathways associated with genes differentially expressed between control and cora^stl325 hearts. (C) Heat map of metabolic genes significant with significantly reduced expressed in cora^stl325 hearts. (D) Graphical representation of the number of differentially expressed metabolic genes associated with the TCA cycle (left) and the electron transport chain (right). Made with BioRender.

Defective oxidative metabolism in cora^stl325 and taf1 knockout embryos. (A) RTPCR showing the reduced expression of genes involved in the TCA cycle (Asdph, Suclg2) and the electron transport chain (Aifdm5, Cox4il) in cora^stl325 and taf1^stl456 embryos at 96 hpf. Each data point represents a pool of 20 embryos. (B) Reduced basal oxygen consumption rates (OCR) in cora^stl325 (n=12) and taf1^stl456 (n=12) embryos compared to controls at 96 hpf (left). Preserved basal OCR in ttn^{stl853/stl853} (n=20) compared to controls (n=18) embryos at 96 hpf (right). Data were obtained from a Seahorse XF24 Bioanalyzer. (C) Targeted metabolite assays measuring NAD/NADH (left) and ADP/ATP (right) in control, cora^stl325 taf1^stl456, and ttn^{stl853/stl853} embryos at 96 hpf. n=4 pools of 100 embryos per genotype. (D) Oxygen consumption rates of isolated mitochondria for fatty acid metabolism. n=4 pools of 100 embryos per genotype

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