Emilie A. Bard-Chapeau1, Dorota Szumska2, Bindya Jacob3, Belinda Q. L. Chua1, Gouri C. Chatterjee4, Yi Zhang5, Jerrold M. Ward1, Fatma Urun6, Emi Kinameri6, Stéphane D. Vincent7, Sayadi Ahmed1, Shoumo Bhattacharya2, Motomi Osato3, Archibald S. Perkins5, Adrian W. Moore6, Nancy A. Jenkins1¤, Neal G. Copeland1*¤
1 Institute of Molecular and Cell Biology, Singapore, Singapore
2 Welcome Trust Centre for Human Genetics, Oxford, United Kingdom
3 Cancer Science Institute, Singapore, Singapore
4 MYSM School of Medicine, Yale University School of Medicine, New Haven, Connecticut, USA
5 Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, Rochester, New York, USA
6 RIKEN Brain Science Institute, 2-1 Hirosawa, Wako-shi, Saitama, Japan
7 Department of Development and Stem Cells, Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS UMR 7104, Inserm U964, Université de Strasbourg, Illkirch, France
¤ Current address: Methodist Hospital Research Institute, Houston, Texas, USA
* E-mail: firstname.lastname@example.org
Published in PLOS ONE in February 2014.
The ecotropic viral integration site 1 (Evi1) oncogenic transcription factor is one of a number of alternative transcripts encoded by the Mds1 and Evi1 complex locus (Mecom). Overexpression of Evi1 has been observed in a number of myeloid disorders and is associated with poor patient survival. It is also amplified and/or overexpressed in many epithelial cancers including nasopharyngeal carcinoma, ovarian carcinoma, ependymomas, and lung and colorectal cancers. Two murine knockout models have also demonstrated Evi1’s critical role in the maintenance of hematopoietic stem cell renewal with its absence resulting in the death of mutant embryos due to hematopoietic failure. Here we characterize a novel mouse model (designated Evi1fl3) in which Evi1 exon 3, which carries the ATG start, is flanked by loxP sites. Unexpectedly, we found that
germline deletion of exon3 produces a hypomorphic allele due to the use of an alternative ATG start site located in exon 4, resulting in a minor Evi1 N-terminal truncation and a block in expression of the Mds1-Evi1 fusion transcript. Evi1dex3/dex3 mutant embryos showed only a mild non-lethal hematopoietic phenotype and bone marrow failure was only observed in adult Vav-iCre/+, Evi1fl3/fl3 mice in which exon 3 was specifically deleted in the hematopoietic system. Evi1dex3/dex3 knockout pups are born in normal numbers but die during the perinatal period from congenital heart defects. Database searches identified 143 genes with similar mutant heart phenotypes as those observed in Evi1dex3/dex3 mutant pups. Interestingly, 42 of these congenital heart defect genes contain known Evi1-binding sites, and expression of 18 of these genes are also effected by Evi1 siRNA knockdown. These results show a potential functional involvement of Evi1 target genes in heart development and indicate that Evi1 is part of a transcriptional program that regulates cardiac development in addition to the development of blood.
Figure Legend: (A-D) Deletion of Evi1 exon3 generates a hypomorphic allele. Evi1Δex3/Δex3 animals do not die in utero like previously published phenotypes for Evi1Δex4/Δex4 and Evi1Δex7/Δex7. Moreover, Evi1Δex3/Δex3 display a different phenotype compared to exon 4 and 7 knockout mice. (A) Kaplan-Meyer curves for wild type, Evi1Δex3/+ and Evi1Δex3/Δex3 progeny indicate lethality of all Evi1Δex3/Δex3 pups by three days after birth (n=5 to 16 per genotype), but not during embryonic development. Log rank test, Chi square p value <0.0001. (B) Flow cytometric profiles of wild type, Evi1Δex3/+ and Evi1Δex3/Δex3 littermate fetal livers at E14.5. HSC and progenitor cell subpopulations were detected by a combination of markers (KSL: c-Kit+, S: Sca-1+, L: lineage-, or KL-CD34+). We found a significant reduction, but not total depletion of cells in the Evi1-deleted samples; p values are from an unpaired t-test between +/+ and Evi1Δex3/Δex3 fetal livers. (C) Colony forming counts from cells of 3 fetal livers of each genotype at E14.5. We observed a significant reduction, but not depletion, in colony formation between +/+ and Evi1ex3/Δex3 fetal livers, p=0.0057 (unpaired t-test). These results suggest that the perinatal lethality observed in Evi1Δex3/Δex3 mice was not caused by hematopoietic defects. (D) 3D reconstruction (left-ventral oblique view) of hearts from Evi1ex3/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 Evi1ex3/Δex3 hearts. These types of congenital heart defects (CHD) are known to be viable in utero but lethal during the neonatal phase of life for other mouse knockouts, and thus likely represent the major cause of perinatal lethality seen in Evi1ex3/Δex3 pups. (E) Evi1 controls the expression of genes that regulate heart development.This diagram represents the number of CHD (congenital heart defect) 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.(F-I) Profound depletion of hematopoietic cells in adult mice carrying an Evi1 exon3 deletion. (F) Kaplan-Meyer survival curves indicate significant lethality in Vav-iCre; Evi1fl3/fl3 mice, with a median survival of 7.7 weeks (Vav-iCre targets the hematopoietic compartment). These adult mice also displayed leucopenia, severe anemia and thrombocytopenia. (G) Flow cytometric profiles of bone marrow cells from Vav-iCre/+;Evi1fl3/fl3 and littermate control mice (Evi1fl3/+ or Evi1fl3/fl3). HSC and progenitor cell subpopulations were detected by a combination of markers (KSL: c-Kit+, S: Sca-1+, L: lineage-). We found a depletion of cells in Evi1-deleted samples, p=0.00011 and p=0.0024 (unpaired t-test). (H) Colony forming counts for cells from bone marrow of young Vav-iCre;Evi1fl3/fl3 and littermate control mice (Evi1fl3/+ or Evi1fl3/fl3). N=3 for each group, p=0.0019 (unpaired t-test). No colony could be formed in the mutants. (I) Histology was performed on sick Vav-iCre; Evi1fl3/fl3 and littermate control mice. Bone marrow depletion was observed in the mutant mice. Adipose tissue replaced the hematopoietic cells in the bone marrow. Additional histolopathology experiments showed that these animals died of hemorrhages in vital organs or severe bacterial infections.