FIG. 1.

Effect of ethylene glycol tetraacetic acid (EGTA) injection into the lateral ventricle of the developing brain. (A) The design of the experiments performed in this study. We performed in utero transplantation with EGTA using neural cells dissociated from developing mouse brains or derived from mouse and human (not shown here) embryonic stem cells (ESCs). Grafted cells and the host brain tissues were characterized. (B) EGTA dissociates apical junctions in the neuroepithelium in a concentration-dependent manner. (a) Low-magnification view of the developing mouse cortex (E14.5). (b) Effects of EGTA on the actin organization (green) of the apical surface assessed from the lateral (left columns) and the apical sides (right columns). Green, actin; white, DNA. (C) Excessive doses of EGTA impair the positioning of mitotic cells in the neuroepithelium. The position of apical mitotic cells [phospho-Histone H3 (PH3)-positive cells, white] was examined 1 day after the injection of EGTA into the E14.5 mouse brain. The population of mitotic cells at the apical surface is essentially the same when 0 and 20 mM EGTA are injected (a, b, and e), while the number of ectopic, non-apical mitotic cells is increased at EGTA concentrations of 30 and 50 mM (c, d, and e). Note that the gross organization of the postmitotic neurons (Tuj1-positive, red) is nearly intact when up to 50 mM EGTA is injected (a–d; right panels). Scale bar in (a)=50 μm. *P<0.05, t-test. Error bars; SEM. (D) The gross brain structures of postnatal (P) mice remain intact after the injection of 20 mM EGTA. (a) Ratio of normal brains at various postnatal stages after the injection of 0, 20, 30, or 50 mM EGTA at E14.5. Postnatal stages were categorized as 0 to 5, 6 to 10, and 11 to 58 days after birth. n, the number of animals tested in each category. (b–e) Brains (left panels) and cortical layers (right panels) at P0 (b, d) and P58 (c, e) after injection of 0 (b, c) or 20 (d, e) mM EGTA at E14.5. Cortical layers were visualized through immunostaining with a VGluT2 antibody [24]. Scale bars for left panels=1 mm, for right panels=200 μm. Color images available online at www.liebertpub.com/scd