Figure 2.

BMP4 sufficiency and concentration dependence for CPEC differentiation from mouse ESC-derived neural progenitors. A, Schematic of the differentiation method. Neural aggregates are treated for 5–7 DIV with BMP4. B, qRT-PCR of 5-day M1 and M2 aggregates treated with BMP4 (normalized to no BMP4 controls). CPEC markers are strongly induced, while neural progenitor markers Sox1 and Foxg1 are downregulated in a BMP4 concentration-dependent manner. C–G', ISH and ICC of sectioned M1 aggregates. Ttr and Aqp1 expression are detected in peripheral regions of BMP4-treated aggregates (arrowheads; n = 11/13 and 7/7 aggregates, respectively), but not in aggregates without BMP4 (n = 0/6 and 0/3 aggregates). Expression often occurs in vesicles that coexpress Ttr and Aqp1 (adjacent sections in G and G') and localize Aqp1 to the apical (luminal) surface (G', arrowheads) as seen in CPECs in vivo. H–I, ICC of sectioned M2 aggregates. Ttr::RFP (native fluorescence) and Aqp1 expression (arrowheads) are BMP4-dependent (n = 6/8 and 0/6 aggregates with and without BMP4, respectively), and colocalize in cell clusters toward the periphery of aggregates (I). J, K, Electron microscopy of M1-derived BMP4-treated aggregates. Vesicle-lining cells have extensive microvilli (white arrowheads), rare cilia, and juxtalumenal tight junctions (black arrowheads) characteristic of CPECs in vivo. Scale bars: C–I, 50 μm; J, 2 μm; K, 0.5 μm.