Epiblastic Cited2 deficiency results in cardiac phenotypic heterogeneity and provides a mechanism for haploinsufficiency

Abstract

AimsDeletion of the transcription factor Cited2 causes penetrant and phenotypically heterogenous cardiovascular and laterality defects and adrenal agenesis. Heterozygous human CITED2 mutation is associated with congenital heart disease, suggesting haploinsufficiency. Cited2 functions partly via a Nodal→Pitx2c pathway controlling left–right patterning. In this present study we investigated the primary site of Cited2 function and mechanisms of haploinsufficiency.Methods and resultsA Cited2 conditional allele enabled its deletion in particular cell lineages in mouse development. A lacZ reporter cassette allowed indication of deletion. Congenic Cited2 heterozygous mice were used to investigate haploinsufficiency. Embryos were examined by magnetic resonance imaging, by sectioning and by quantitative real-time polymerase chain reaction (qRT-PCR). Epiblast-specific deletion of Cited2 using Sox2Cre recapitulated penetrant and phenotypically heterogenous cardiovascular and laterality defects. Neural crest-specific deletion using Wnt1Cre affected cranial ganglia but not cardiac development. Mesodermal deletion with Mesp1Cre resulted in low penetrance of septal defect. Mesodermal deletion with T-Cre resulted in adrenal agenesis, but infrequent cardiac septal and laterality defects. β-Galatactosidase staining and qRT-PCR demonstrated the efficiency and location of Cited2 deletion. Murine Cited2 heterozygosity is itself associated with cardiac malformation, with three of 45 embryos showing ventricular septal defect. Cited2 gene expression in E13.5 hearts was reduced 2.13-fold in Cited2+/− compared with wild-type (P = 2.62 × 10−6). The Cited2 target gene Pitx2c was reduced 1.5-fold in Cited2+/− (P = 0.038) hearts compared with wild-type, and reduced 4.9-fold in Cited2−/− hearts (P = 0.00031). Pitx2c levels were reduced two-fold (P = 0.009) in Cited2+/− embryos, in comparison with wild-type. Cited2 and Pitx2c expression were strongly correlated in wild-type and Cited2+/− hearts (Pearson rank correlation = 0.68, P = 0.0009). Cited2 expression was reduced 7474-fold in Sox2Cre deleted hearts compared with controls (P = 0.00017) and Pitx2c was reduced 3.1-fold (P = 0.013). Deletion of Cited2 with Mesp1Cre resulted in a 130-fold reduction in cardiac Cited2 expression compared with control (P = 0.0002), but Pitx2c expression was not affected.ConclusionThese results indicate that phenotypically heterogenous and penetrant cardiac malformations in Cited2 deficiency arise from a primary requirement in epiblast derivatives for left–right patterning, with a secondary cell-autonomous role in the mesoderm. Cardiac malformation associated with Cited2 haploinsufficiency may occur by reducing expression of key Cited2 targets such as Pitx2c.

Mice Carrying a Hypomorphic Evi1 Allele Are Embryonic Viable but Exhibit Severe Congenital Heart Defects

“…We used magnetic resonance imaging (MRI) to visualize organ formation in six Evi1 δex3/δex3 , three Evi1 δex3/+ and six E15.5 control littermates, as previously described [41]. Structural abnormalities were observed in the hearts of all six Evi1 δex3/δex3 embryos (Fig.…”

Cited2 Is an Essential Regulator of Adult Hematopoietic Stem Cells

“…To investigate a functional requirement for Cited2 in adult hematopoiesis, we generated Cited2 fl/fl Mx1-Cre conditional knockout mice (MacDonald et al., 2008), in which treatment with poly(I)-poly(C) (pIpC) induces efficient gene deletion in hematopoietic cells (Kuhn et al., 1995). We treated Cited2 fl/fl Mx1-Cre and Cited2 fl/fl mice with pIpC (Figure 1B) and refer to these as Cited2 Δ/Δ and Cited2 fl/fl mice, respectively.…”

Cited2 Gene Controls Pluripotency and Cardiomyocyte Differentiation of Murine Embryonic Stem Cells through Oct4 Gene

“…Conditional knock-out of Cited2 in heart and hematopoietic tissues also displayed multiple phenotypes (18, 26 -29). In particular, deletion of Cited2 at the epiblast stage using Sox2-Cre phenocopies cardiac defects in Cited2 total knock-out mouse model (28). Depletion of Cited2 in the hematopoietic tissues via Mx1-Cre affects hematopoietic stem cell quiescence and apoptosis (26,27).…”