Supplementary Figure 1. Illustrations showing how the anterior primitive streaks were isolated
and cultured. The 2nd sector of the primitive streaks in HH3 chick embryos is regarded as the
anterior primitive streaks, which was dissected, isolated and maintained in cultured for 24 - 48
hours. This applies for Fig. 5 of the main text. For Fig. 6 of the main text, the anterior primitive
streak explants were incubated for 3 - 12 days and then harvested for immunofluorescent staining
with MF20 antibodies.
Supplementary Figure 2. Primer sets used in the RT- PCR study.
Supplementary Figure 3. Ethanol retards development of the gastrulating chick embryos. A-C:
Representative appearance control chick embryos 0 (A), 17 (B) and 38 (C) hours. The embryos
reached stage HH11 with a C-loop heart tube after 38 hours. D-G: Representative appearance of
chick embryos treated with ethanol for 0-38 hours. For ethanol-treated embryos that reached
HH11, some developed abnormal with atypical C-looping heart tube. H: Bar chart showing the
development stages that control and ethanol-treated embryos managed to reach after 38 hour
culture. Scale bars = 600 µm in A-G.
Supplementary Figure 4. Tunicamycin and thapsigargin treatment did not affect heart tube
formation. Chick embryos (HH0) were incubated in the presence of 1μg/ml tunicamycin or
150ng/ml thapsigargin for 45 hours and then immunofluorescently stained with MF20 antibodies.
A-B: Representative bright-field (A) and MF20 immunofluorescent images (B) of tunicamycintreated embryos. B1-B1’: Transverse sections of B (B1) and higher magnification from B1 (B1’).
The sections were counterstained with DAPI. C-D: Representative bright-field (C) and MF20
immunofluorescent images (D) of thapsigargin-treated embryos. B1-B1’: Transverse sections of
D (D1) and higher magnification from D1 (D1’). B1 (B1’). Scale bars = 600 µm in A, C; 200µm
in B, D; 200 µm in B1, D1 and 100 µm in B1’, D1’.
Supplementary movies: Time-lapse microscopic recording of the contracting cardiac progenitor
cells after exposure to RAPA and ethanol. The anterior primitive streak tissues were isolated from
HH3 chick embryos and maintained in culture. Time-lapse recording were made of the cardiac
progenitor cells exposed to saline (control), 40nM RAPA and 342.5mM ethanol. The recordings
revealed RAPA- and ethanol repressed cardiac progenitor cells contract compared with the control.