Abstract 1285: Wnt2 Plays a Key Role in Cardiac Development via a Non-canonical Pathway
Embryonic stem (ES) cells are a promising source of cardiomyocytes, but their clinical application has been hindered by the lack of selective differentiation methods. Although several signals are involved in heart development, the precise signals that mediate cardiomyocyte differentiation remain undetermined. Wnt family has a potent effect on the various organ development. To address this issue, we investigated the expression of wnt genes in the embryonic heart. Then we applied these findings to establish an efficient protocol to induce cardiomyocytes in vitro. [Methods and Results]
We analyzed TOP-EGFP mice to clarify whether canonical wnt signal pathway is important in the developing heart. TOP-EGFP mice are transgenic mice in which the EGFP gene is located under the β-catenin binding site so that the EGFP protein expresses when the canonical pathway is activated. They did not reveal any GFP in early stage heart, indicating that the canonical pathway is not involved.
Expression of non-canonical wnt was screened. Whole mount in situ hybridization of wnt2 and nkx2.5 (positive control) was performed at mouse embryonic stages E7.5, E8.0, E8.5 and E9.0. Wnt2 was strongly expressed in the the heart-forming area. It was clearly expressed at the cardiac crescent at E7.5 and at the late crescent stage at E8.0 and in the linear heart tube at E8.5 and it was markedly expressed in both atrium at E9.0 after looping.
We applied this embryonic wnt2 expression pattern to ES cell differentiation. Using siRNA we knocked-down wnt2 protein in various phases, which inhibited formation of beating EB, and decreased cardiac muscle genes only during the primitive stage (day 2–4). Also adding wnt2 protein to embryonic cells in the appropriate phase led to a marked induction of Nkx2.5, GATA4, Tbx5, and MEF2C. And more it rapidly and strongly induced various cardiac proteins. [Conclusions] Wnt2 was strongly expressed in the heart-forming region. We applied this finding to develop an effective protocol for obtaining cardiomyocytes from mouse ES cells by adding wnt2 protein and also to inhibit generation of cardiomyocytes by inhibition of wnt2 signaling. We concluded that wnt2 plays a key role in cardiac development via a non-canonical pathway.