Worm Breeder's Gazette 11(3): 46
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.
Anti-lin-14 antibodies detect a nuclear protein in specific somatic cells of late embryos and early to mid-L1 animals that quickly disappears during late L1 and later stages. Two major proteins of 76 and 67 kD are detected in immunoblots of protein extracts only from embryos and L1 larvae. These proteins do not correspond to the lin- 14a and lin-14b gene activities (proposed by Ambros & Horvitz, 1987) nor to the lin-14A and lin-14B transcripts (see Burglin et. al., WBG this issue) because both proteins are detected in various lin-14a-b+ and a+b- mutants. We have analyzed the temporal and cellular expression patterns of lin-14 protein accumulation in various heterochronic mutants, i.e. lin-4(e912), lin-28(n719), lin-29(n333), and the double mutants lin-4( e912);lin-28(n719) and lin-14(n536sd);lin-28(n719). lin-4, like lin- 14 semidominant mutants, exhibits a retarded phenotype, displays inappropriately high levels of the 76 and 67 kD lin-14 proteins at all developmental stages, and shows inappropriate lin-14 staining in nuclei at L2, L3, L4 and adult stages. Thus, the lin-4 gene negatively regulates lin-14 protein levels after larval stage 1. Northern blots and RNase protections suggest that this lin-4 regulation of lin-14 is post-transcriptional, as with the lin-14 gain- of-function (gof) mutants; the temporal regulation of both lin-14 transcripts is normal in a lin-4 mutant (Wightman & Ruvkun, unpublished results). In contrast, lin-28 animals have a steeper lin- 14 temporal gradient compared to wild-type; little to no lin-14 protein is visible in the hypodermal and intestinal nuclei of early to mid-L1 animals while the ventral cord neurons, body wall muscle and nerve ring nuclei are comparable in intensity to wild-type. Thus, the lin-28 gene activity positively regulates lin-14 protein levels in specific cell lineages in L1 animals. We do not yet know if this effect is transcriptional or post-transcriptional. These data suggest that at least in the hypodermal and intestinal lineages, lin-14 does not act downstream of lin-28 but instead either up-regulates lin-14 gene expression or controls the stability/activity of the lin-14 protein. Because of the opposing effects of lin-4/lin-14gof and lin- 28 gene activities on lin-14, we examined the temporal regulation of lin-14 expression in double mutants to characterize their epistasis relationship in molecular terms. lin-4;lin-28 and lin-14gof;lin-28 mutants show lin-14 staining in late embryos to mid-L1 stages which fades to undetectable in late L1 and later stage animals. Therefore, while both lin-4/lin-14gof and lin-28 mutations affect lin-14 protein levels, the lin-28 mutation is epistatic to the effects of the lin-4 and lin-14gof mutations on the pattern of lin-14 protein accumulation. This molecular epistasis is consistent with the genetic epistasis of lin-28 to lin-4 and lin-14gof mutations. lin-29 animals show no variation from the wild-type pattern of lin-14 protein confirming that it is indeed downstream of lin-14 in the epistasis pathway. We have also found that in wild-type animals, the down-regulation of lin-14 protein levels is linked to actual developmental time, (not to GMT!). We have investigated this by allowing eggs to hatch and suspending animals at the L1 stage by starvation for up to 125 hours. There is no decline in lin-14 protein levels in these arrested L1s by immunoblot or immunostaining analyses. Northern blots showed that lin- 14 transcript levels are similar in starved L1s compared to newly hatched L1s. However, in the presence of cycloheximide, starved L1s show a steep decline in lin-14 staining at 24 to 48 hours. These data suggest that rather than being stable, lin-14 protein is continually translated in these arrested L1s. If starved L1s are then fed and develop into L2s, the lin-14 protein levels fade as occurs during normal development so that upon feeding, a signal to resume postembryonic development causes a decrease in lin-14 protein levels. This decrease in lin-14 is probably attained by negative post- transcriptional regulation of the lin-14 3'UTR (see Burglin et. al., WBG this issue) The lin-4 gene product is a good candidate for this negative regulatory activity. Our data predicts that the lin-4 gene may become active after this L1 feeding switch. In starved L1s of lin- 28 and lin-14(n179ts25C) mutants, the lin-14 protein staining fades faster than in wild-type suggesting that either a functional lin-14 protein positively autoregulates lin-14 gene expression or protein stability, and that the lin-28 activity is necessary for this effect.