Worm Breeder's Gazette 11(4): 61
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.
To develop tools which will allow a detailed genetic and molecular study of how heterochronic genes control stage-specific gene expression, we have constructed reporter genes in which the - galactosidase gene is driven by the promoters of stage-specific collagen genes. Collagens are major components of C. elegans cuticle. col-7 and col-19 genes are adult-specific, accumulating their mRNA only at the L4 to adult molt (Cox and Hirsh, 1985; Cox et al., 1989). In contrast, the col-17 gene is larval-specific with its mRNA present at larval molts and absent at the L4 to adult molt (See Liu and Ambros, WBG 11-2,99). We fused 2.7 kb 5' sequences of col-19, including 7 aa coding sequence, to the -galactosidase gene in vector 22.04 (kindly provided by Andy Fire). This vector contains a SV40 nuclear localization signal, which targets fusion proteins to the nuclei (Fire et al., 1990) . We also fused the col-7 gene (780bp upstream and approximately 200 aa. coding sequences) to the -galactosidase gene in Fire's 35.41 vector, which contains a synthetic transmembrane domain that prevents secreted proteins from being transported out (Fire et al., 1990). Finally, we constructed a fusion between the col-17 gene (800bp 5' and 94 aa coding sequences) to the -gal gene of 35.41. All these constructs were then micro-injected along with rol-6 (su1006) PRF4 plasmid (Mello et al., WBG 11-1, 18). Stable (but extra-chromosomally inherited) roller lines were established and stained with X-gal. All three constructs showed consistent stage-specific reporter -gal expression. Both col-19/ -gal and col-7/ -gal are expressed beginning at the L4 to adult molt and beyond. However, it appears that the two fusion genes are expressed in different sets of hypodermal cells: col- 19/ -gal staining first appears in vulva cells of L4 to adult molt animals and subsequently in seam cells of young adults and finally in syncitial cells and Pnp cells but not vulva cells; col-7/ -gal staining seems restricted to lateral hypodermal seam cells. This apparent difference could reflect the fact that these two reporters use two different fusion vehicles. We plan to construct col-19 fusions using the same vector used for col-7/ -gal in order to compare the expression of these two genes directly. In contrast, the larva- specific col-17 promoter directs -gal expression only at the larva molts including late embryo, L1, L2, L3 molts but not at the L4 to adult molt. The col-17/ -gal expression seems to restricted to cells in the head, vulva and tail of larvae. We have not examined in detail the identities of these cells. The adult-specific col-19/ -gal construct was introduced into heterochronic mutants by microinjection together with PRF4 and stable roller lines were examined for -gal activity. The adult-specific col- 19/ -gal expression was completely blocked in lin-29(n546) retarded mutants, and was activated at early (L3) molt in lin-28(n719) and lin- 14(n179ts) precocious mutants. This abnormality in the stage-specific col-19 expression correlates with the abnormally early or late adult cuticle formation in the precocious or retarded mutants respectively. Thus, heterochronic genes regulate the stage-specific transcriptional initiation of, at least partly, collagen genes. This finding is consistent with that lin-29 may encode a Zinc-finger transcription factor (Rougvie et al., WBG 11-2, 39) and that lin-29 has been shown, by genetic epistatic tests, to be a most direct regulator of genes involved in the cell differentiation process including collagen genes. We have not yet examined the expression of col-7/ -gal and col-17/ - gal gene fusions in heterochronic mutant background. We expect that these stage-specific fusion reporters will be useful genetic and biochemical tools for characterizing heterochronic genes and isolating new heterochronic genes.