Wnt signaling in Pristionchus pacificus gonadal arm extension and the evolution of organ shape

Abstract

Changes in organ morphology have been essential to the evolution of novel body forms and in permitting organisms to invade new ecological niches. Changes in the arrangement of cells and tissues and in the regulation of morphological movements are fundamental to evolutionary transitions of organ shape and function. However, little is known about the genetic and developmental control of these changes. We use interspecific differences in the migration and extension of the nematode hermaphrodite gonadal arms to study the generation of morphological novelty. We show that the extending Pristionchus pacificus gonadal arms display a ventral migration that is unique to the Diplogastridae in comparison to the Rhabditidae, including Caenorhabditis elegans, and other nematodes. This results in the distal gonad residing along the ventral side of the body in P. pacificus in contrast to lying on the dorsal side of the body as in C. elegans. We show that at the cellular level this morphogenetic movement is regulated by signals from the developing vulva and the sister gonadal arm. We further show that in P. pacificus Wnt signaling is essential for this regulation. We show genetic and molecular evidence that suggest the Wnt ligands Ppa-mom-2 and Ppa-cwn-2 are components of the signaling mechanism. Supporting these findings, the hermaphrodite gonad of Ppa-bar-1 mutant animals mimics the shape of the C. elegans hermaphrodite gonad; the arms fail to extend ventrally. Thus, this genetic analysis of gonad migration provides insight into the mechanisms underlying the generation of morphological novelty and organ shape.

Fig. 1.

The gonadal arms of P. pacificus hermaphrodites have a novel ventral arm extension in comparison to C. elegans. (A) Cladogram of nematode species (34). The asterisk indicates data taken for O. tipulae CEW1 from ref. , for Mesorhabditis sp. PS1179 from ref. , and for P. redivivus PS1163 from ref. . Line diagrams indicate the path of hermaphrodite gonadal arm extension, and the solid dot indicates the position of the vulva. The ventral gonadal arm extension is unique to the Diplogastridae. Light gray shading indicates the Rhabditidae family. Medium gray shading indicates the Diplogastridae family. With the exception of Mesorhabditis sp. and P. redivivus, which have monodelphic gonads, all strains are didelphic. However, even in Mesorhabditis sp. and P. redivivus the gonadal arm passes the position of the vulva and does not make a ventral gonadal arm extension. (B–D Upper) Nomarski photographs of animals. (B–D Lower) Color-coded cartoons of the photographs to identify individual tissues: The somatic gonad and germ line are outlined and shaded in tones of purple and red. Purple, one gonadal arm in focus from uterus to distal tip; light red, the other gonadal arm that is mostly out of the plane of focus; red, embryos; small dark red amyboid-like shapes, sperm; solid dark red ovals, representative germ cell nuclei. The digestive tract is outlined and shaded in blue. Dark blue, pharynx; blue, gut; light blue, the lumen of the intestine; navy blue ovals, representative gut nuclei. Solid white triangles denote the distal tip of the gonadal arms. Solid open triangles denote the position of the vulva, i.e., the ventral side of the animal. Dashed open triangles represent the homologous position where the vulva would reside in a wild-type animal; i.e., there is no vulva. Small arrows indicate the position of the anus, i.e., the ventral side of the animal. Line diagrams represent the path of gonadal arm elongation. (B) Wild-type fully adult C. elegans N2 hermaphrodite. (C) Wild-type fully adult P. pacificus PS312 hermaphrodite. (D) P. pacificus PS312 young adult in which the entire vulva has been laser-ablated; i.e., P(5–8).p were killed immediately after hatching. Animals without a vulva must be scored at young adult stage before they fill with embryos and the internal morphology is disturbed.

Fig. 2.

The ventral gonadal arm extension is regulated by the Wnt signaling pathway. (A) A Ppa-bar-1 homozygous mutant animal. For a description of cartoon color coding and line diagram see the Fig. 1 legend. Homozygous Ppa-bar-1 animals grow more slowly, they are smaller, their intestines are not as darkly colored, and their intestines do not fluoresce as their wild-type siblings. (B and C) Photographs of J4-stage wild-type P. pacificus PS312 animals for Wnt ligand RNA in situ hybridizations. Note the dark staining in the vulva, indicated by a solid triangle. (B) Ppa-mom-2 is expressed in a subset of the descendants of the secondary cells P5.p and P7.p (C) Ppa-lin-44 is expressed in multiple vulval cells. (D) Putative model for the cellular mechanism for signaling ventral gonadal arm extension. Color coding and symbols are the same as for the other cartoons with the following exceptions: Solid dark red circles at the tip of each gonadal arm represent the DTC nuclei. Vulval tissue is outlined and shaded in green; solid blue circles represent the nuclei of the descendants of P6.p that form the center of the vulva; solid red circles represent the nuclei of the descendants of P5.p and P7.p, respectively, that form the periphery of the vulva; solid green ovals represent vulval nuclei at a later stage in development. The black/white gradient within the body of the animal represents a proposed gradient in Ppa-UNC-6/Netrin along the dorsal–ventral body wall. (Upper) Late J3-stage/early J4-stage P. pacificus hermaphrodite. Both gonadal arms are shown; the arm normally hidden behind the gut is partly transparent. Green arrows indicate the production of a signal, putatively including Ppa-MOM-2 and Ppa-CWN-2, plus additional Wnt ligands. Red inhibitory arrows indicate the production of a signal from the extending gonadal arms. These signals are likely to be processed by the DTCs to carry out the novel P. pacificus ventral migration. By analogy to C. elegans, this is likely done by expressing Netrin receptors in the DTCs that can interpret the Netrin gradient. (Lower) Late J4-stage/young-adult-stage P. pacificus hermaphrodite. Note the resulting pretzel-like gonad morphology of the adult as a result of novel developmental signaling in P. pacificus in comparison to C. elegans.