smarce1 mutants have a defective endocardium and an increased expression of cardiac transcription factors in zebrafish

Abstract

SWI/SNF or BAF chromatin-remodeling complexes are polymorphic assemblies of homologous subunit families that remodel nucleosomes and facilitate tissue-specific gene regulation during development. BAF57/SMARCE1 is a BAF complex subunit encoded in animals by a single gene and is a component of all mammalian BAF complexes. In vivo, the loss of SMARCE1 would lead to the formation of deficient combinations of the complex which might present limited remodeling activities. To address the specific contribution of SMARCE1 to the function of the BAF complex, we generated CRISPR/Cas9 mutations of smarce1 in zebrafish. Smarce1 mutants showed visible defects at 72 hpf, including smaller eyes, abnormal body curvature and heart abnormalities. Gene expression analysis revealed that the mutant embryos displayed defects in endocardial development since early stages, which led to the formation of a misshapen heart tube. The severe morphological and functional cardiac problems observed at 4 dpf were correlated with the substantially increased expression of different cardiac transcription factors. Additionally, we showed that Smarce1 binds to cis-regulatory regions of the gata5 gene and is necessary for the recruitment of the BAF complex to these regions.

Figure 1

(A) Schematic diagram of smarce1 gene. The DNA-binding high mobility group is shown. CRISPR/Cas9 mediated-deletions were produced in exon IV. The nucleotide sequence of the RNA-guide targeted region is shown in gray with the PAM in red and the deletions are indicated in lines below. (B) The body curvature, smaller eyes and cardiac edema are shown. (C) Graphs showing rescue experiments with injected smarce1 mRNA. A significant lower number of edematous embryos (left) and a higher survival rate (right) were determined in groups of the mRNA-injected relative to buffer-injected embryos. Values represent the average of three independent injections of 100 ± 10 embryos per experiment. ***P < 0.001.

Figure 2

Morphological defects in smarce1 mutants. (A) The heart morphological defects of mutated embryo can be appreciated in comparison with a heart from a normal sibling (arrow) at 72 hpf. B-C. Sagittal sections stained with hematoxylin-eosin from 96 hpf normal (B) and mutant (C) embryos. (D–F) Confocal Z-projections of cmlc2:GFP/smarce1 normal (D) and defective hearts (E,F) at 72 hpf. In F nuclei were stained with DAPI. A: atria, AVC: atrio-ventricular canal, EC: endocardium, Ht: heart, V: ventricle. Scale bars: 25 μm.

Figure 3

Rescue of the heart looping by smarce1 mRNA injection in smarce1 mutant embryos. Whole-mount in situ hybridization detects transcripts for the cardiomyocyte marker cmlc2. A-B. Embryos at 48 hpf showing a normal heart (A) and a smarce1^−/− heart with a defective looping (B). (C–H) Analysis of heart looping at 72 hpf. Buffer injected embryos present a normal heart in control siblings (C) and a non-looped heart in all the mutants (D). (E–H) smarce1 mRNA injected embryos showing the rescue of the heart looping in the mutant embryos. Complete rescue is shown in (F); a partial rescue in (G); and the failing of rescue in (H). Scale bars: 50 μm.

Figure 4

Patterns of expression of atria and ventricle markers in smarce1 mutants. Chamber expression in smarce1 heterozygous (upper panel) and homozygous (lower panel) embryos. Left to right: 48 hpf embryos showing the expression of the ventricular cardiomyocyte marker vmhc, embryos showing the expression of the atria cardiomyocyte marker amhc, 72 hpf embryos stained with MF20 and S46 antibodies to detect the ventricle (red) and atrium (green) in wildtype and mutant embryos. Scale bar: 25 μm.

Figure 5

Defects in the endocardium in smarce1 mutants. Embryos at 9- (A,B), 14- (C,D) and 20- (E,F) somite stage showing the expression the endothelial marker flk1. While a normal ALPM pattern is observed in mutant embryos up to 14 somites, at 20 somite stage mutant embryos present an abnormal distribution of endocardial cells. Expression of nfatc1 in embryos at 26 (G-I), 30 (J–L) and 35 hpf (M,N) reveals a disorganized pattern of the endocardium. Embryos at 24 hpf showing expression of the endocardial marker gata5 (O–Q) and the myocardial marker cmlc2 (T–V). Expression of gata5 in the endocardium of the mutant embryos is abnormal and has a lower signal. A dysmorphic pattern for cmlc2 is detected in one out of six embryos (V). R,S. Expression of nppa in 48 hpf embryos. An arrow shows the absence of nppa at the atrioventricular junction. (W, X) Expression of versican in embryos at 4 dpf. Scale bars: 50 μm.

Figure 6

(A) Quantitation by QPCR of gene expression of cardiac factors. 2 dpf and 4dpf embryos were genotyped and evaluated. For all genes n ≥ 3. *P < 0.05, **P < 0.01. (B) In vivo Smarce1-binding sites were detected by chromatin immunoprecipitation using a SMARCE1 antibody. Cropped gel images representing typical results from ChIP assays in: the regulatory module located from the transcription initiation site +1 to −1415, which includes the basic promoter (gata5-Bp), the module located from −1469 to −1892 (gata5-A), the module located from −7823 to −8213 (gata5-B) and the module located from −16805 to −17147 (gata5-C). IgG, Smarce1, Smarca4, Arid1a and Pbrm indicate the PCR products from genomic DNA precipitated using the corresponding antibody. WT and Mut indicate the source of genomic DNA. The input is the PCR product from the total input genomic DNA. Full-length gels are presented in Supplementary Figure 3.