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Fig. 3. SMP-1 expressed on the lumen-facing side of vulva rings guides vulva cell migration. B,D,F,H,J,L are DIC microscopic images of A,C,E,G,I,K, respectively. The SMP-1::GFP translational reporter signal is expressed specifically on the vulva lumen membrane (white arrows in A-J) of vulva cells that have formed rings (shown at different chronological stages of vulva morphogenesis; at the beginning in A,B and nearing completion in I,J). (A,B) The lumen formed by both vulF (small arrow) and vulE (large arrow) rings show the SMP-1::GFP signal. (C-F) Wild-type vulva cells that do not change their shape or migration pattern do not obviously express the SMP-1::GFP signal (red arrows in F), with the exception of possibly one cell just joining the forming vulva proper (blue arrows in E,F). During the later stages of vulva morphogenesis (G-J), the SMP-1::GFP signal highlights cell membrane protrusions radiating out from the lumen (G,I; inset in I shows an enlargement of the region near the asterisk). (K,L) In plx-1(ev724) animals, mutant cells that fail to migrate properly towards the presumptive vulva midline fail to express SMP-1::GFP (red arrows) in comparison with cells that migrate correctly (white arrows). (M,N) Fluorescence confocal microscopy in accordance with the epifluorescence data (see A-J above) shows that the SMP-1::GFP reporter is sequentially turned on during vulva morphogenesis in cells that have finished (or have nearly finished) forming a vulva ring. As vulva morphogenesis progresses, the last formed vulva ring begins to show SMP-1::GFP on its lumen surface. (M) Only two vulva rings express the SMP-1::GFP reporter (white arrows), but later (N) two additional rings express the SMP-1::GFP signal. There is a strong gonadal and uterine SMP-1::GFP staining in A,C,N. M and N are dorsal and dorsolateral projections of 3D confocal images, respectively. D, dorsal; P, posterior; unlabelled arrow indicates left-right depth. Scale bars: in A, 25 µm for A-L; 6.3 µm for inset in I; 1.0 µm in M; 2.7 µm in N.





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