Worm Breeder's Gazette 13(3): 91 (June 1, 1994)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

The expression pattern of a C dc42 -lacZfusion

Rob van Weeghel[1], Ming-shiu Hung[2], Jin-Quan Run[2], Joe Culotti[1], Jeff Way[2]

[1]Samuel S. Lunenfeld Research Institute, 600 University Ave. Toronto, Ontario MSG 1X5

[2]Biology Dept., Nelson Labs, Busch Campus, Rutgers University, Piscataway NJ 08855.

The yeast CDC42 gene encodes a small, membrane-anchored G-protein that controls yeast cell polarity. In Saccharomyces cerevisiae, C dc42 pis localized to sites on the plasma membrane where secretory vesicles fuse to cause membrane growth, namely, the bud and tips of shmoos. In a cdc42 mutant, a bud will not form, and instead secretory vesicles fuse all over the mother cell membrane and the cell just gets bigger. C dc42 is activated by the guanine nucleotide exchange factor encoded by CDC24 ,which has a region of similarity to the unc-73 gene, which functions in direction-dependent processes including axon guidance/outgrowth, cell migration, and asymmetric cell division (WBG 12, #4, 29).

We cloned a C. elegans C dc42 homologue, generated a C dc42 -lacZfusion, coinjected it with the Roller plasmid pRF4 ,and generated an integrated line. In this line, we see two phases of expression (WBG 12, #4, 40).

Early embryonic expression.

C dc42 -lacZexpression can be first detected at the ~80 cell stage (120 min. of development). By 140 minutes of embryonic development, staining is intense throughout the embryo, quenching the DAPI counterstain. Staining remains strong until later in embryonic development (2-fold/3-fold stage) when a worm can be seen in the egg; at this point, the pattern begins to resemble the larval/adult pattern. In larva and adults, staining is extremely variable.

Larva/adult expression.

Expression is seen in the following cells, in approximate order of intensity:

Epithelial cells of the pharynx: these form long, slender projections within the pharynx and have three-fold symmetry (see Worm Book, p. 101).

m7 cells of the pharynx (these form the grinder) and intestinal cells.

The CAN neurons, the excretory canal, the ventral nerve cord neurons, the HSNs, the ALMs, and the PVDs, the distal tip cell, and a subset of unidentified neurons in the head (10-50) and tail (4-10). The CAN staining is very strong, and it is usually possible to seen its neuronal processes extending along the lateral lines.

Staining is not seen in the hypodermis, germ-line, young fertilized eggs, or gonad epithelium (except distal tip cell). Very rarely, a body-wall muscle cell will stain. It is important to remember that the staining in some cells is so strong that staining in others may be unobservable by comparison, so we cannot say that C dc42 is not expressed in cells like the hypodermis.

From the nature of yeast C dc42 and its possible relationship to unc-73 ,this pattern could be interpreted along several lines: polarized secretion, axon guidance, cell migration, etc. One working hypothesis to explain the above pattern is to say that C dc42 expression correlates with the level of polarized secretion that a cell is carrying out. The intestinal cells (and possibly the m7 cell) presumably secrete large amounts of digestive enzymes. The pharynx epithelial cells are, unfortunately, among the least-studied cells in the animal, but these cells might secrete a worm equivalent of saliva to lubricate the passage of bacteria through the pharynx (if anybody has thoughts or info on this idea, please tell us). Among neurons, the staining intensity could simply correspond with their level of metabolic activity, or it could depend on whether the neurons undergo long migrations to reach their final position. To test between these ideas, we examined staining in a mec-4 ( u253 null) strain, which should reduce the metabolic activity of ALMs, but not the PVDs. In C dc42 -lacZ; mec-4 (+)gravid adults, 50% of the ALMs and 42% of the PVDs were stained, and in a mec-4 (-)strain, 10% of the ALMs and 52% of the PVDs were stained, supporting the polarized secretion/metabolic activity idea (It seems unlikely that the expression level is simply a function of metabolic activity unrelated to secretion, because expression in muscle cells is so low.)