Fig. 4

Leader cells drive tissue morphogenesis in hydrogels. a Expression of SLUG and SOX9 in leader cells. Fluorescence image of actin-rich terminal ductal lobular unit (TDLU) protrusions stained with phalloidin (purple), α-SLUG (green), and α-SOX9 (red). Nuclei stained with 4′,6-diamidino-2-phenylindole (DAPI; blue) and quantification of double-positive leader cells (yellow) indicated below. Scale bars represent 50 μm. b Expression of cytokeratin 14 (CK14) and CK8/18 in TDLU protrusions. Fluorescence images of actin-rich protrusions stained with phalloidin (purple), α-CK14 (green), and α-CK8/18 (red). Nuclei stained with DAPI (blue) and quantification of CK14 positivity in leader cells indicated below. Scale bars represent 50 μm. c Bright-field images of TDLU protrusions containing multiple divergent leader cells (green arrowheads). d Time-lapse microscopy of a day 8 TDLU showing a single leader cell protruding at the leading ductal edge (red line), followed by ductal elongation (blue line) and the eventual appearance of another leader cell at the new leading edge. See Additional file 6: Movie S4 and Additional file 7: Movie S5. Time denotes hours:minutes following onset of recording. e Schematic depiction of the single duct elongating in (d), highlighting in red the protruding leader cell that precedes ductal elongation. Arrows indicate the direction of invasion and elongation. f Time-lapse analysis of an 8-day TDLU that changed its direction during ductal elongation. A single leader cell (black arrowhead) appears to reorient the direction of ductal growth (arrows). See Additional file 8: Movie S6. Scale bars represent 50 μm