Fig. 4

MZepcam mutants display secondary increase in proliferation and apoptosis of EVL cells and basal keratinocytes.

(A) Confocal images of maternal/zygotic epcam (MZ-/-) mutant and wild type (WT) embryos at indicated stages. EVL cells and their nuclei are in green (anti-GFP immunostaining of tg(cytokeratin8:GFP) product; row 1), nuclei of basal keratinocytes in red (anti-p63 immunostaining; row 2), and BrdU-positive nuclei in blue (row 3). Row 4 shows merged images; row 5 magnified views of regions boxed in row 4 in red. They show examples of BrdU-positive EVL (in light blue; green+blue) and basal cell nuclei (in purple; red+blue). (B) Percentages of BrdU-positive EVL (in blue) and basal layer (in red) nuclei in WT and MZ-/- embryos at the indicated stages of development. (C) Confocal image of aggregate of p63-positive basal cells (in red) at 48 hpf; the aggregate is devoid of BrdU incorporation (in green), whereas BrdU-positive nuclei are present outside the aggregate, indicating that the assay worked. (D) Analysis of apoptosis levels within epidermal aggregates of MZ-/- embryos. At 48 hpf, aggregates contain very few or no TUNEL-positive cells (green), while more apoptotic cells are found in aggregates at 96 hpf.