DEV02625 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Normal heart development in moderate-mild Hand1 over-expression embryos at E12.5 and E14.5. (A-D) Sagittal sections of control or Hand1 over-expressing embryos at E12.5. (E,F) Frontal sections through isolated hearts from control or Hand1 over-expressing embryos at E14.5. Abbreviations: at, atrium; avc, atrio-ventricular canal; co, control; lv, left ventricle; oe, over-expression; rv, right ventricle. Scale bars: in A, 200 μm for A-D; in E, 50 μm for E, F.

• Supplemental Figure 2 -

  Fig. S2. Generation of embryoid bods precociously over-expressing Hand1. (A) ES cells precociously over-expressing Hand1 were generated by incorporating a tTA transactivator under the control of a TK promoter into the transgenic line containing the Tre2-Hand1 responder. (B) Over-expressing ES cells express Hand1 at day 0 of differentiation, 4 days prior to the detection of endogenous Hand1 by northern blot. This was confirmed by RT-PCR for Hand1 expression following 0, 2 and 4 days of differentiation (inset i). After 14 days of differentiation (asterix), levels of Hand1 are 5- to 6-fold higher in over-expressing EBs compared to controls. This was confirmed by qRT-PCR for Hand1 expression in control versus over-expression day 14 EBs (inset ii). (C) Single cells were isolated from contractile foci derived from the Hand1-null, wild type and control cell lines at day 14 and identified as cardiomyocytes by immunostaining for MF20 (sarcomeric myosin). Abbreviations: co, control; −ve, no RT template negative control; oe, over-expression.