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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.
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