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First published online 16 February 2005
doi: 10.1242/dev.01694

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Vulva morphogenesis involves attraction of plexin 1-expressing primordial vulva cells to semaphorin 1a sequentially expressed at the vulva midline

¹ Samuel Lunenfeld Research Institute of Mount Sinai Hospital, 600 University Avenue, Toronto M5G 1X5, Canada
² Department of Molecular and Medical Genetics, University of Toronto, Toronto M5S 1A8, Canada

View larger version (85K): [in a new window]   Fig. 1. Vulva ring formation defects in plexin 1 (plx-1) and semaphorin 1a (smp-1) mutants characterized by DIC and the AJM-1::GFP reporter (see Materials and methods). (A) Dorsolateral perspective; (B-F) ventral views; (G-J) dorsolateral projections of 3D confocal images. Arrowheads indicate the vulva midline (innermost position of the vulva primordium and site of the presumptive vulva lumen). (A) Schematic showing an intermediate step in vulva morphogenesis (cell fate identities are indicated by letters). (B-F) Focal plane of F and other cells that have formed rings are out of focus. (B) At the beginning of larval stage 3, primordial vulva cells are linearly arranged in wild-type animals. Starting from the midline of the primordium, wild-type vulva cells sequentially modify their shape and generate processes (white arrows) that migrate around and under their inner neighbors. (C) In smp-1(ev715) and plx-1(ev724) single mutants and smp-1(ev715); plx-1(ev724) double mutants at the same stage, vulva cells are misguided (detached cells indicated by red arrow and mis-positioned cells indicated by white dots). (D) In wild-type adult hermaphrodites, the vulva appears as seven stacked concentric rings. However, in plx-1(ev724) mutants (E,F), the ring structure is disorganized: some cells are detached from the primordium (red arrows) and vulva cell processes do not migrate correctly around their inner neighbors (white arrows). (G-J) Fluorescence confocal microscopy and software-assisted 3D image reconstruction was also used to characterize vulva cell extensions and ring morphogenesis (see Materials and methods). Migrating cells send out processes ventrally around their inner neighbors within the vulva primordium in a sequential manner. Intermediate steps show the lumen-facing membrane of the migrating processes of the first (centrally positioned) cells during early stages (arrows in G) and, at a later stage, the processes of distally positioned cells (arrows in H). Seven stacked vulva rings (white arrows in I) signal the end of morphogenesis. In plx-1(ev724) mutants vulva (J), cells may fail to migrate towards the midline (shown by a gap) and frequently fail to extend processes around their inner neighbors (arrows showing the lumen-facing membrane). Mutant cells can adopt an abnormal crescent shape, which causes secondary invaginations (asterisks). (A,G-J) The perspective orientation is indicated with the triple arrows (D, dorsal; P, posterior; unlabelled arrow indicates left-right depth). Scale bars: 25 µm in B,C; 25 µm in D-F; 3.8 µm for G; 2.3 µm for H; 3 µm for I; 5.3 µm for J.

View larger version (109K): [in a new window]   Fig. 2. The plx-1::gfp transcriptional reporter and the plx-1p::PLX-1::GFP translational reporter are expressed during vulva morphogenesis. In wild type (A-F), expression is predominant in P5.p and P7.p descendants and the reporters are widely expressed in most cells undergoing ring formation. The signal increases during later phases of ring formation. (A,C) Transcriptional reporter. (B,D) Translational reporter. The GFP signal of the plx-1p::PLX-1::GFP translational reporter is first (B) distributed on the whole cell membrane of early expressing cells and highlights filopodia-like extensions but later (D) begins to concentrate at the cell surface zone contacting neighboring cells (arrowheads). As vulva morphogenesis progresses (E,F), the PLX-1::GFP signal is widely expressed at the cell membrane of ring forming cells (arrowheads). The PLX-1::GFP signal is localized predominantly on the lumen side of vulva ring forming cells of wild-type animals (arrows in E,F). In smp-1(ev715) mutants (G,H), vulval cells that do not display a migration phenotype show the same pattern of expression as wild type. By contrast, cells displaying an aberrant migration phenotype in smp-1(ev715) and unc-73(rh40) mutants (arrowheads in I-L) often lose the predominant localization of the PLX-1::GFP signal at the lumen-facing membrane. F,H,J,L are DIC microscopic images of E,G,I,K, respectively. (M) Fluorescence confocal 3D microscopy revealed the PLX-1::GFP signal highlighting the lumen-facing surface of crescent-shaped cells forming vulva rings (M is a 3D confocal projection, N is a sketch of the staining in M with PLX-1 highlighted in green). The signal is maintained for a while in the vulva cells that have completed their migration and formed a ring (white arrows in M). However, as externally positioned cells are recruited to initiate their migration towards the presumptive vulva midline, the PLX-1::GFP signal becomes concentrated in cell surface membrane that is just entering the vulva proper (red arrow in M). D, dorsal; P, posterior; unlabelled arrow indicates left-right depth. Scale bars: 25 µm in A-L; 1.8 µm in M.

View larger version (115K): [in a new window]   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.

View larger version (85K): [in a new window]   Fig. 4. Vulva cell migration defects are caused by ectopic expression of smp-1. Observations use DIC microscopy (A,B,E) and the AJM-1::GFP reporter (C,D) (see Materials and methods). (A,B) Lateral views with anterior towards the left; (C) top-down projection from a 3D confocal image. D, dorsal; P, posterior; unlabelled arrow indicates left-right depth. (D,E) Ventral views, anterior towards the left. (A,B) Animals carrying the plx-1p::SMP-1 transgene display vulva morphology defects (B) when compared with wild-type animals (A). In plx-1p::SMP-1 transgenic animals, vulva cells from each side frequently (see Table 1) fail to migrate towards the presumptive vulva midline, forming two invaginations (arrowheads in B) as seen in smp-1 and plx-1 mutants. (C) Three dimensional image reconstructions of the GFP signal from the AJM-1::GFP reporter in animals carrying the plx-1p::SMP-1 transgene (see Materials and methods) reveal vulva cells from the anterior and posterior sides that do not complete their migration to form a vulva ring (arrows). (D,E) In smp-1(ev715) animals carrying the plx-1p::SMP-1 transgene, frequent widely separated multiple invaginations are observed on both anterior and posterior sides of the presumptive vulva (arrows). Scale bars: in A, 25 µm for A-B, D-E; in C, 8 µm for C.

View larger version (33K): [in a new window]   Fig. 5. A SMP-1 and PLX-1 based model of cell migration during vulva morphogenesis. (A) Lateral perspective; D, dorsal; P, posterior; unlabelled arrow indicates left-right depth. A series of short-range migrations is repeated for the formation of vulva rings. (B) Ventral perspective of the boxed area in A. Intermediate steps of the proposed model for vulva cell migration showing the PLX-1-expressing leading edge as a blue band on the future lumen side of cells poised to enter the forming vulva. The leading edge spreads ventrally under forming vulva rings expressing SMP-1 on their lumen surface (green spiked ring). SMP-1 expression increases dramatically in the newly formed ring, while the next external vulva half ring (not yet expressing SMP-1) initiates its migration by extending processes underneath it. (C) A genetically derived model of the molecular cascade regulating vulva morphogenesis. SMP-1, PLX-1 and CED-10 function in the same pathway when guiding vulva cells (attractive guidance to the vulva midline). UNC-73 and MIG-2 act in one or more parallel pathways whose function is similar to that of the SMP-1/PLX-1 signaling pathway. It is possible that MIG-2 and CED-10 also function downstream of PLX-1 (broken arrows from UNC-73 and MIG-2), as suggested by the literature, indicating that UNC-73 can activate MIG-2 and CED-10. Additional guidance mechanisms may function in parallel with PLX-1 and MIG-2 (broken arrows with question mark on the right).

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