Morphological markers of life-stage and fertility in <i>C. elegans</i>.

<p>Detailed descriptions of <i>P. pacificus</i> vulva and gonad development are available [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077079#B38" target="_blank">38</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077079#B39" target="_blank">39</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077079#B42" target="_blank">42</a>]. Panels include a DIC micrograph on top and a cartoon of the micrograph below. For body orientation, anterior is to the left and dorsal to the top. Black arrows mark the position of the vulva, the egg- laying organ. Asterisks in the cartoons denote developing embryos <i>in </i><i>utero</i> and are not shown in the micrograph for clarity. White triangles denote the position of oocytes. Tissues are denoted by color: gut, blue; uterus and spermatheca, purple; germ line (including the germ cells, oocytes, and developing embryos), red. Ovals and circles depict easily seen nuclei within tissues. (A) Young adult hermaphrodite. The entire posterior gonad arm is shown. The red arrow outlines the path of gonad arm extension starting proximal to the vulva and terminating with the arrowhead at the distal tip. As an adult, this animal has made sperm, oocytes, and contains embryos, has two fully reflexed and inflated gonadal arms, and a fully everted vulva with a slit like morphology. The black asterisk denotes an egg in the spermatheca that has just been fertilized but has not developed an eggshell yet. In contrast, L4 larvae never contain embryos as they only begin to produce gametes late in the L4-stage. The L4 gonad arms are smaller and not as inflated, and early in L4 have only reached the dorsal side of the animal, but do not reach the center above the vulva until late in L4. (B) Wild type early L4-larva hermaphrodite detail of uterus and vulva. The vulva has a characteristic “Christmas tree” like shape, it is not everted. The uterus is empty and un-inflated. Gametes have not been produced, the gonad arms are skinny and contain relatively few germ cells making the gonad arms difficult to capture in the same focal plane as the vulva. (C) Wild type adult hermaphrodite detail of uterus and vulva. The uterus is full of multicellular embryos. The gold asterisk denotes an embryo with a clearly visible eggshell. The eggshell is present as an oval around the ball of cells. The embryo and shell are separated by a slim cleared liquid-filled space. (D) Adult fog-<i>2</i> female detail of uterus and vulva. <i>fog-2</i> females do not produce sperm and contain no embryos. Hence the uterus and spermatheca remain unexpanded. Unfertilized oocytes stack up in the gonad arms and become compressed, giving a “piano key” phenotype. Eventually pressure may push an oocyte into the uterus and the oocyte will be laid, but without an eggshell. Laid oocytes have a “mushy” appearance and remain single celled until decomposition. The edge of a laid embryo has a refractory appearance due to the eggshell.