Fig. 3. (A-P) Phenotypic analysis of embryos stained with phalloidin-rhodamine after injection of CsDelta1, CsDelta2 and CsNotch dsRNA. (A-J) Confocal micrographs of flat preparations of prosomal segments of 180-hour-old embryos; anterior is towards the top. (K-P) Transverse sections through the fourth prosomal segments; medial is towards the left. Prosomal regions of embryos injected with GFP dsRNA as a control (A,F,K), with CsDelta1 dsRNA (B,G,L), CsDelta2 dsRNA (C,H,M) and CsNotch dsRNA (D-P), respectively. (F-J) Higher magnifications of the fourth prosomal hemisegments. (A,F) After injection of GFP dsRNA, the ventral neuroectoderm shows the normal number of invagination sites (about 30 per hemisegment; dots of high phalloidin-rhodamine staining, arrows). (B,G) After injection of CsDelta1 dsRNA, the number of invagination sites is reduced in individual segments (arrows). (C,H) A more severe reduction of invagination sites can be detected after injection of CsDelta2 dsRNA; invagination sites are absent in the whole ventral neuroectoderm. (D,I,) After injection of CsNotch dsRNA, dots of high phalloidin-rhodamine staining can be detected in the positions that correspond to invagination sites in control injected embryos (arrows), although they are much smaller. (E,J) In a more severely affected embryo there is only diffuse phalloidin-rhodamine staining visible in positions that correspond to invagination sites in control injected embryos (arrow). (K) Confocal micrograph of a transverse optical section through an invagination site (arrowheads) of an embryo injected with GFP dsRNA. The cell processes of the basally enlarged cells extend to the apical surface. (L) Transverse optical section through the fourth prosomal hemisegment of an embryo injected with CsDelta1 dsRNA, showing that in a region where invagination sites are missing the neuroectoermal cells form a bulge (arrowhead). (M) Transverse section through the fourth prosomal hemisegment of an embryo injected with CsDelta2 dsRNA. Two bulges of neuroectermal cells are visible (arrowheads). (O,P) The transverse sections through the fourth prosomal hemisegments of embryos injected with CsNotch dsRNA reveal that, although dots of higher phalloidin-rhodamine staining are visible on the apical surface (arrowheads), there are no bottle-like cells visible underneath these dots. The presence of several cell layers suggests that cells that normally invaginate occupy space in the apical layer so that newly formed cells were pushed basally. l1, l2, walking legs 1 to 2 (corresponding to prosomal segments 3 and 4). Scale bars: in A, 150 µm for A-E; in F, 50 µm for F-J; in K, 20 µm for K-P.

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