Figure 7. NE progenitors transiently express EMT regulator Snail and downregulate adhesion and polarity markers.

(A) Expression of NE, adhesion (adhes), polarity, and EMT markers in NEB progenitors and intermediates (dark green) at steps indicated: before NE selection (pre-selection) and during NE specification (Class 1), migration to form clusters (Classes 2-4), and differentiation (Class 5). Light green cells, maturing NE cell already at target (asterisk); white cells, NE cells before selection and other non-NE epithelial cells. Symbols show high (++), intermediate (+), low (+/−), no (−), or mixed (+ and −) expression. For polarity markers, expression refers to expression restricted to basal (bas), basolateral (bas-lat), or apical domains. See also Figure S7.

(B-E) Confocal micrographs showing dynamic patterns of indicated NE (B), adhesion (C), polarity (D) and EMT (E) markers at indicated steps of NEB formation. Dots, outline of NEB; dashed line, basement membrane. (B) Ascl1 (green) is expressed during migration (left panels; E13) but down-regulated during differentiation (right panels; E18), whereas CGRP (red) is detected only during differentiation. (C) E-cadherin is expressed around entire plasma membrane during NE selection (Ascl1-positive (green) cell at E13; left panels), but is down-regulated in class 3 and 4 intermediates (some of which are marked by ZsGreen in E15 Ascl1^CreER/+;Rosa26^ZsGreen/+ mouse induced with tamoxifen at E12.5) in developing NEB (dotted outline; center panels). E-cadherin is again detected in NE cells (CGRP-positive; green) of mature NEB (PN 2 month; right panels), although expression is excluded from apical cell surface and detected along lateral boundaries in at least some cells. Cells neighboring NEB (not shown) express higher levels of E-cadherin and have larger apical surface that excludes E-cadherin. (D) Before NE selection (E12; left panels) epithelial progenitors are uniformly polarized and tight junction protein ZO-1 (magenta) is detected at apical surface of each cell. ZO-1 is down-regulated and undetected in intermediates (Ascl1^CreER/+;Rosa26^ZsGreen/+ mouse labeled as above) during migration (center panels),when surrounding epithelial cells maintain its expression (inset), but is again detected during differentiation (right panels). (E) Snail proteins (magenta) are detected in some (filled arrowhead) but not other (open arrowhead) early NE progenitors (Ascl1-positive, green) during NE selection (E13; left panels), but all intermediates express Snail during migration (middle panels). Intermediates down-regulate Snail in concert with Ascl1 as clusters form and differentiate (E15; right panels), as seen by declining level of Snail and Ascl1 in some cells (asterisks) within maturing cluster. Snail proteins are induced again later in NE cells (C.K., Joe Ouadah, and M.A.K., unpublished data). Bars, 10 μm.