Figure 3

Exogenous BMP4 leads to decreased overall proliferation and increased apoptosis in the neural domain of the cultured otocysts. (A) BMP4, when added at 10 ng/ml or 20 ng/ml to otocysts after three days in culture, significantly inhibits proliferation, tested by an 8 h BrdU pulse 24 h after adding BMP4. (Asterisks indicate p < 0.05 (10 ng/ml) and p < 0.01 (20 ng/ml), unpaired Student's t-test, n = 7; error bars represent standard deviations). (B) Addition of BMP4 after three days in culture leads to an increase in TUNEL-positive cells when analyzed on the seventh day (asterisks indicate p < 0.01 (10 ng/ml) and p < 0.001 (20 ng/ml), unpaired Student's t-test, n = 7; error bars represent standard deviations). (C) In sections of control otocysts analyzed on the seventh day in culture, apoptotic cells, identified by TUNEL staining (red), mostly occurred outside of the Pax-2-positive areas (shown in blue). (D) BMP4 at 10 ng/ml noticeably increased the number of TUNEL-positive cells in the potential neural domain, identified by green islet-1-positive cell nuclei. (E,F) Similar to (C,D), but the neural domain is labeled with antibody to beta-III tubulin (shown in green). Note the increase of TUNEL-positive cell numbers within the neural domain. (G) Noggin does not affect the number of apoptotic cells.