Worm Breeder's Gazette 13(3): 62 (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 Effects of Homeotic Genes on vit-lacZ Expression

Peg MacMorris, Tom Blumenthal

Biology Dept., Indiana Univ., Bloomington IN 47405

In the course of our studies of vitellogenin gene regulation, we have found highly stereotyped, reproducible, temporal patterns of expression of vit/lacZ fusion genes within the adult hermaphrodite intestine. We examined synchronized populations Or adult worms of two strains at regular intervals after the L4 -adultmolt by X-gal staining. These strains contain independently integrated arrays of a gene in which the lacZ coding region is fused to a 3.9 kb upstream fragment of vit-2 and a 923 bp downstream fragment of vit-6 .In each strain, instead of ß-gal activity appearing approximately simultaneously in all intestinal cells, certain cells express the transgene well before others, and some never express it at all. Since all intestinal cells normally express copious amounts of yolk protein, we assume that the fusion is debilitated in some way that magnifies differences between the different cells, and thus provides a sensitive assay for factors influencing the onset of vit gene expression.

Two distinct regions turn the fusion on first, int5 and 6 nuclei in the middle of the animal, and int8 nuclei near the posterior. In some individuals only the posterior nuclei stain and in others only the mid-region nuclei stain, demonstrating that expression in the two foci initiates independently. The int7 nuclei between the two early regions rarely stain in young adults. Later, a third focus near the anterior end, int2 ,develops. Areas of expression extend from each focus after their initial appearance to include surrounding cells in a graded maner, suggesting a gradient of influence. The end result is that in older adults it is common for all nuclei from int2 - int8 to stain, yet int1 and int9 nuclei rarely show expression. The staining pattern is often asymmetric, confined to one side of the animal in each region; it is imprecise (the mid-region focus can be either in int5 or int6 cells); and the three foci are clearly independent (examples abound in which expression occurs in one region in the absence of the others). Frequently, the two nuclei within a single intestinal cell are either both stained, although not always with the same intensity, or they are both unstained.

This spatial pattern gives us an opportunity to explore the effects of the C. elegans homeotic genes on endodermal tissue. The HOM-C genes, lin-39 , mab-5 and egl-5 ,have previously been shown to contribute to spatial patterning of ectoderm and mesoderm, namely epidermal and neuronal cells of the P lineage, and some sex-specific and body wall muscle cells. They act combinatorially to give cells in each region of the body spatial information that regulates their subsequent development. They are known to act in the L1 and later and to have their greatest effects on postembryonic development.(1) If we align the known regions of the HOM-C genes' influence with adjacent regions of the intestine at the adult stage, we would expect the domains of influence for the three genes to be: lin-39 ,anterior to mid-body ( int3 -6); mab-5 ,mid-posterior ( int5 -9)and egl-5 ,posterior ( int9 ).To determine whether mutations in these 3 genes influence the pattern of vit/lacZ expression, we crossed a vit/lacZ integrated strain to the HOM-C mutants lin-39 , mab-5 and egl-5 and analyzed them for alterations in the timing and pattern of ß-gal activity by X-gal staining.

The data clearly show that each of these mutations alters the pattern of lacZ expression, and the staining patterns are consistent with the predicted actions of the mutations. In lin-39 (lf) mutants, expression of anterior and middle region ( int2 -6)cells is delayed and reduced; in mab-5 ,the predominant early peak of int8 expression is eliminated and some early expression occurs in int7 ,which is rarely seen in wild-type backgrounds. Egl-5 (lf)mutants have a less dramatic effect, merely reducing int8 expression. These results are generally consistent with the expected overlapping domains of the HOM-C genes. It should be noted that the data do not discriminate between direct effects on the intestinal cells and indirect effects on cells in each region that affect intestinal expression. Nevertheless, it is clear that the intestine, an endodermal tissue, experiences region-specific influences from the homeotic genes.

Literature Cited:

(1) Wang et al. (1993), Cell 74: 29-42