Figure 5.

Gfra1 and Gfra2 Are Essential for In Vitro Cardiomyocyte Differentiation from Mouse ESCs

(A) Flo analyses of TNNT2 at day 10 of differentiation. WT, wild-type; G1-KO, Gfra1-KO; G2-KO, Gfra2-KO; DKO, Gfra1/2-DKO; R-KO, Ret-KO; ISO CTRL, isotype control.

(B) Quantitative analyses of flow cytometry show the severe impairment of cardiomyocyte differentiation in Gfra1/2 DKO ESC lines. ^∗p < 0.05 versus WT in Student’s t test. n = 5.

(C) Immunocytochemical analyses of each KO ESC line 10 days after induction of cardiomyocyte differentiation. Gfra1/2 double-KO (DKO) ESCs exhibited severe defects in TNNT2⁺ cardiomyocyte differentiation. Scale bar, 100 μm.

(D) qPCR analyses for Gfra1 and Gfra2 in Gfra2 KO ESC lines and in Gfra1 KO ESC line at day 7, respectively. Note the elevated expression of Gfra1 in Gfra2 KO ESCs. ^∗p < 0.05 versus WT in Student’s t test. N.S., not significant. Bar graph represents mean ± SEM. n = 3.

(E and F) Flow cytometrical analyses of TNNT2 in differentiation day 10 ESC lines. G&N, Gdnf, and Nrtn. Gdnf- and Nrtn-null ESC lines did not show any statistically significant difference in cardiomyocyte differentiation efficiency compared to WT. N.S., not significant versus WT. Bar graph represents mean ± SEM. n = 3. (F) Immunofluorescent images of TNNT2 in ESC lines at differentiation day 10. Scale bar, 100 μm.

(G) See also Figures S4–S6.