Mutant <i>Zic2</i> Founder line characterization.

<p>Mutant <i>Zic2</i> Founder line characterization.</p

Microinjection Results.

<p>Microinjection Results.</p

Additional file 1: Figure S1. of Pcsk5 is required in the early cranio-cardiac mesoderm for heart development

Hoxa3Cre-driven deletion of Pcsk5. Ethidium bromide stained agarose gel showing multiplex polymerase chain reaction products of allele-specific genotyping from embryonic hearts. Primer details are as in Fig. 5. Five hearts of the Pcsk5 Δ1/flox ; Hoxa3Cre + embryos were analysed. The floxed allele is almost completely absent in this hearts indicating a loss of Pcsk5 floxed allele. (TIF 477 kb

<i>Zic2</i> genomic structure and TALEN binding sites.

<p>A) Genomic structure of the murine <i>Zic2</i> (upper panel) with an enlargement of exon 2 (lower panel), showing the binding sites of the two monomers for TALEN-A and TALEN-B together with the binding sites of the PCR primers, Zic2-F and Zic2-R used to genotype the mutant alleles. B) Example of the Cel1 endonuclease assay showing cleavage of the PCR amplicon from example heterozygous mutant CD1 embryos, injected with TALEN-A mRNAs, TALEN-B mRNAs and control eGFP mRNA. The fragments obtained correspond to the predicted cleavage of the 346 bp amplicon within the spacer region of the TALEN-A and TALEN-B recognition sites as expected.</p

Examples of the curly tail phenotype seen in some of the mutant founder mice.

<p>A) TALEN-A C3H founder 10. B) TALEN-B C57BL/6 founder 1.</p

Evi1 regulates the expression of other CHD genes during embryonic heart development.

<p>(A) The number of CHD genes represented in Evi1 ChIP-Seq data (Evi1 bound genes) or in the list of genes regulated by Mecom. An enriched number of CHD genes were found bound or regulated by Mecom (50 out of 143 genes), p = 0.0453 and p = 0.0276, respectively. These genes represent potential Mecom target genes in heart development. (B) Mecom regulates the expression of 23 CHD genes, which contain Evi1-binding sites specifically in heart. Heart and head (neural crest) tissues were harvested from WT and Evi1^δex3/δex3 embryos of somite number 9 to 18. RT-qPCR assays were performed. Genes considered to be mis-regulated in Evi1^δex3/δex3 hearts were increased or decreased in expression by at least three fold in average for all samples of the same time-point. These graphs are representative of two to five independent experiments.</p

Expression of Mecom mRNA in cardiac structures of wild type embryos.

<p>(A–D) Whole mount mRNA <i>in situ</i> hybridization to show Mecom expression. A–C) Expression during subsequent stages of heart tube formation E8.5 (black brackets). D) At E9.5 Evi1 is expressed in the endothelial cells and in the endocardium of the heart and in the mesenchyme of the aortic arches. Expression also includes a population of migrating neural crest cells (white arrowhead). E–J) E10.5 Sagittal sections (from right to left) showing Evi1 in the aortic arches (a), mesenchyme of the secondary heart field (black arrowheads), outflow and atrio-ventricular canal endocardium including the cushions.</p

Cardiac malformations and failure in Evi1^δex3/δex3 mice.

<p>(A) Transverse sections and (B) 3D reconstruction (left-ventral oblique view) of hearts from Evi1^δex3/δex3 or wild type littermate (+/+) E15.5 embryos analyzed by magnetic resonance imaging (MRI). The aorta (Ao), right ventricle (RV), left ventricle (LV), ventricular septum (VS), trachea (Tr), aortic arch (AoA) and ductus arteriosus (DA) are indicated. Ventricular septal defect (VSD), interrupted aortic arch (IAA) and common arterial trunk (CAT) were observed in Evi1^δex3/δex3 hearts. (C) List of the congenital heart defects identified in fifteen E15.5 embryos of various different genotypes by MRI and 3D reconstruction. (D) Hematoxylin and eosin staining of 5 µm sections of a sick Evi1^δex3/δex3 pup. Subcutaneous and other tissue edema (white spaces) was present, consistent with heart failure.</p

Spontaneous lethal bone marrow depletion in mice harboring an Evi1 exon3 deletion in the hematopoietic system.

<p>(A) Histology was performed on sick Vav-iCre; Evi1^fl3/fl3 and littermate control mice. Bone marrow depletion was observed in the mutant mice. Adipose tissue replaced the hematopoietic cells in the bone marrow. (B) Increased erythropoiesis in the spleen of Vav-iCre; Evi1^fl3/fl3 mice. No visible border was found between the red pulp and white pulp. Erythroid cells are shown by the arrows. Excess erythropoiesis in spleen likely happens to compensate for bone marrow loss. (C) H&E stained sections of the brain of a dying Vav-iCre; Evi1^fl3/fl3 mouse. Hemorrhages (red areas) were visible at several locations (also see Fig. S3E in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089397#pone.0089397.s001" target="_blank">File S1</a>). (D) Histological sections of tissues from dying Vav-iCre; Evi1^fl3/fl3 animals showing bacteremia. Red arrows indicate the presence of bacteria in alveolar capillaries. Giemsa stains reveal the presence of cocci or small rods within glomerular capillaries. No sign of immune system defense (inflammatory cells) was observed despite the infection.</p

Deletion of Evi1 exon3 generates a hypomorphic allele.

<p>(A) Sequenced products obtained after 5′RACE from wild type or Evi1^δex3/δex3 mutant embryos. (B) Table showing the fraction of embryos of each genotype detected at different stages of embryonic development. The Mendelian ratios were not affected by the Evi1 exon3 deletion. (C) Pictures of 28 hr-old littermates highlight the poor health of dying Evi1^δex3/δex3 pups. (D) Kaplan-Meyer curves for wild type, Evi1^δex3/+ and Evi1^δex3/δex3 progeny indicate lethality of all Evi1^δex3/δex3 pups by three days after birth (n = 5 to 16 per genotype). Log rank test, Chi square p value <0.0001. (E) RT-qPCR from cDNA of E14.5 embryos. The primers used amplified the regions between Evi1 exons 2 and 3, 3 and 4 or 13 and 14. Mean of three different samples per condition. The standard deviation is shown. (F) Expression of Evi1 and γ-tubulin protein products in E14.5 wild type or E17.5 Evi1^δex3/+ and Evi1^δex3/δex3 mutant embryos (100 µg protein/lane). (G) Nucleotide sequence of Evi1 cDNA in the exon 3 and 4 genomic region. Two ATG sites are present in exon 3 and one in exon 4. All ATGs are conserved in higher vertebrates.</p