Worm Breeder's Gazette 11(3): 47
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 lin-14 gene produces two transcripts that apparently encode proteins that differ from each other at the N-terminus (see Burglin, et al., WBG, this issue). Transcript A splices exon 4 to exon 5, while transcript B splices exons 1, 2, and 3 directly to exon 5. We used RNA probes specific for exons 3, 4, and 5 in order to analyze by RNAase protection the regulation of these two transcripts in staged N2 animals and various mutants. In addition, we used anti-lin-14 antibody staining to assess whether the product of the lin-14A transcript is expressed in distinct cell lineages or cell types, or at distinct times during development. Transcript B is present in about three-fold greater abundance than transcript A, consistent with their representation in the cDNA library. This ratio does not change detectably at any post-embryonic stage (the jury is still out on relative embryonic expression). During post-embryonic development, both transcripts are expressed at highest levels (about 1/50 myo-1 ) at L1. Both transcripts coordinately decrease in abundance from L2 to adult stages when the level is about 10% L1 abundance, even though no protein is detectable at these stages. One recessive mutant, lin-14( n360), specifically affects transcript B only. Transcript B is almost entirely absent in this mutant, while transcript A is unaffected. This result is consistent with the observation that n360 is associated with an allele-specific RFLP in the genomic fragment containing exon Since n360 animals express almost no transcript B, we could observe the temporal and spatial regulation of lin-14 protein accumulations from transcript A. n360 L1 larvae were stained with a polyclonal anti- lin-14 antibody. All hypodermal seam cell and hyp5 nuclei stain intensely, while hyp7, P cells, neurons, body muscle, and intestinal nuclei stain very faintly. All these nuclei stain intensely at this stage in wild-type. Since most of the lin-14 protein present must be produced from transcript A, most of the observed staining must be lin- 14A product. Therefore, we propose that lin-14A product is expressed predominantly in the hypodermal seam, while lin-14B product is expressed in a variety of cell types. We are attempting to make antibodies which are specific to the N-terminus of each of the two proteins to confirm these observations. n360 is a lin-14 a+b- mutant as determined genetically by Ambros and Horvitz (1987): seam cells execute normal L1 hypodermal cell fates, but execute precocious fates at L2 and later stages, while lin-14 a-b- alleles execute precocious fates at all postembryonic stages. Our molecular analysis of n360 indicates that lin-14b genetic function is encoded by the lin-14B transcript (what a coincidence). The antibody staining results suggest that L1 seam cell fates are normal in n360 because lin-14A protein accumulates at normal levels in the hypodermal seam. Later fates in the seam are precocious in n360 because either they require non-autonomous interactions dependent on lin-14B expression in other tissues, or because they are dependent on lin-14B expression in the seam, or even more complicated schemes. Because the lin-14A protein accumulates in the same set of cell nuclei whose fates are controlled by the lin-14a gene activity, it is likely that the lin-14a gene activity is cell autonomous. Although hypodermal cell fates are normal at L1 in n360 animals, these mutants form precocious L1 dauers like other lin-14 mutants (Liu and Ambros, G&D 3:2039). Our results indicate that while accumulation of lin-14A protein in the seam is sufficient to specify L1 cell lineages in those cells, it is not sufficient to block L1 dauer formation. This suggests that temporal control of dauer formation is dependent on the level of lin-14B protein in cells other than the hypodermal seam (neurons?). While lin- 14A and lin-14B transcripts have similar temporal expression profiles, their products differ in their cellular pattern of expression. It is likely that the two transcripts are derived from separate promoters, so cell type-specific cis-acting regulatory sites may be present in the two promoters. In addition, we expect that the distinct lin-14 proteins produced in each of these cell types will specify different cell fates.