Fig. 7. Abnormal marker expression, cell cycle entry and premature apopotosis of amnioserosa cells in Doc mutant embryos. Shown are confocal fluorescent microscopic images with merged amnioserosa scans. (A-D) race in situ hybridization (green) and antibody staining (red) with anti-Hnt (A,B) and anti-C15 (C,D) at stage 12. race expression decreases in the amnioserosa of DocA mutants. Residual expression of race generally correlates with residual Hnt expression (arrow in A) and large C15-stained nuclei (arrow in C; arrowheads, small nuclei). In addition, there are increased levels of race mRNA in the posterior/dorsal head (asterisk) of DocA embryos. (E,F) Stage 10 embryos stained for race mRNA and phospho-Histone H3. Unlike in the wild-type (F), race-stained amnioserosa cells show nuclear phospho-Histone H3 staining in DocA mutants (E, arrow heads). (G,H) Stage 11 embryos after 30 minutes BrdU pulse labeling, double-stained with anti-BrdU (green) and anti-C15 antibodies (red) to visualize amnioserosa nuclei. Overlapping signals appear yellow. While normal amnioserosa cells are arrested in the G2 phase of cell cycle 14 and do not incorporate BrdU (H), BrdU incorporation is detected in a fraction of amnioserosa cells of DocA mutants (arrowheads in G). BrdU incorporation in dorsal ectodermal cells flanking the amnioserosa and other domains is seen in both mutant and wild-type embryos at this stage. (I,J) Stage 11 embryos stained for C15 (red), DNA (Hoechst, blue) and -tubulin (green) after fixation in the presence of taxol. Mitotic spindles in the amnioserosa of DocA mutants (I, arrowheads) indicate dividing amnioserosa cells, which are not seen in wild-type embryos (J). (K,L) Detection of apoptotic cell death in late stage 12 embryos by TUNEL is shown in green and staining with anti-C15 is shown in red. At this stage, there is significant apoptosis within the amnioserosa in DocA mutants (K, arrowheads), but none in wild type (L), although apoptosis can be detected in the head and other regions of wild-type embryos. Most apoptotic amnioserosa cells have already lost C15 expression, but are clearly localized within the amniosera layer. Mutant embryos were identified by triple-staining with anti-ß-galactosidase (A-D; not shown) or by the absence of anti-Doc2 staining performed in parallel to anti-C15 staining (G,I,K). G,H are at 1.75x greater magnification than A-F,K,L; I,J are at 2.5x greater magnification than A-F,K,L.

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