Lateral hypodermal seam cell terminal differentiation (the larval to adult (L/A) switch) involves several coordinate changes in the behavior of seam cells during the L4 molt: cessation of cell division, formation of adult cuticle and cell fusion. The proper timing of the L/A switch requires the normal activity of at least four heterochronic genes:
lin-4,
lin-14,
lin-28 and
lin-29. In
lin-14 (lf) and
lin-28 (lf) mutants, the L/A switch occurs abnormally early, at the L3 molt, and these animals only go through three larval stages. In contrast,
lin-4 (lf) and
lin-29 (lf) mutants never develop an adult cuticle; instead, they continue to synthesize a larval cuticle and undergo extra molts. Of these genes,
lin-29 is the most downstream and direct regulator of the L/A switch1.
lin-14 is a negative regulator of
lin-29 activity, yet LIN-14 protein becomes undetectable by the early L22,3, two stages before LIN-29 is detected in hypodermal cells in the L4 stage (Bettinger & A.R., unpub.). Because of this disparity in the times of action of these gene products, we are searching for other genes that help restrict
lin-29 activity to the L4 molt. Since LIN-29 is a zinc finger protein of the (Cys)2-(His)2 DNA-binding class, it likely activates the L/A switch by controlling transcription of genes involved in hypodermal cell division and differentiation. Among these are cuticle collagen genes that are stage-specifically regulated, such as
col-19 4. Indeed, the
col-19 gene which is normally activated in adults, is never activated in hypodermal cells in
lin-29 mutants, and, in addition, LIN-29 binds to the
col-19 promoter in vitro5. We have constructed a
col-19::GFP fusion for monitoring
lin-29 activity in mutant screens. Wild-type worms begin expressing the
col-19::GFP fusion from an integrated array during the L4 molt. This expression is dependent on the heterochronic gene pathway: In
lin-14 (lf) and
lin-28 mutants GFP expression begins during the L3 molt, and in
lin-4 and
lin-29 mutants GFP expression is not observed. In an effort to identify genes that regulate the L/A switch, we are screening for mutants with altered temporal expression of
col-19::GFP. The strain used for our initial screens, RG240 (
lin-4(
e912); veIs13) contains the integrated
col-19::GFP array in a
lin-4 mutant background. Because the L/A switch is not activated in these worms, the adult cuticles lack alae and GFP expression is not observed. Due to the
lin-4 mutation, these animals also lack a vulva and do not form dauer larvae. Finally, these animals roll due to the presence of
rol-6(
su1006)sd in the array. We mutagenized RG240 animals and screened for the expression of
col-19::GFP. In a screen of 10,000 haploid genomes we obtained three mutants that now express
col-19::GFP. A brief description of two of these mutants is presented here. RG243 (
lin-4(
e912); veIs13;
ve11) mutant animals begin expressing
col-19::GFP as young adults. The adult cuticle contains alae, and approximately 10% of adults have vulval protrusions, indicating that the vulval defect associated with
lin-4 mutants has been partially suppressed. Intriguingly, and in contrast to the parental strain, RG243 mutants appear to roll as larvae, but not as adults, suggesting that the
rol-6 mutation is being stage-specifically suppressed. We have not observed dauers in this strain. RG244 (
lin-4(
e912); veIs13;
ve12) mutant animals express
col-19::GFP as adults, and alae is present on the adult cuticle. Like RG240, RG244 animals lack a vulva, fail to form dauers, and roll during both larva and adult stages. RG244 worms appear larger than the parental strain and have an increased brood size. Our initial results from this screen are encouraging. By screening for restored expression of a
col-19::GFP fusion in a
lin-4 mutant background, we have isolated mutants that suppress additional
lin-4 defects including adult alae synthesis. We are in the process of isolating the new mutations and we are conducting additional screens. 1 Ambros, V. (1989). Cell 57:49-57 2 Ruvkun and Giusto, (1991). Nature 338:313-319 3 Arasu (1991). Genes and Dev. 5:1825-1833 4 Liu, Z., Kirch, S., and Ambros, V. (1995). Development 121:2471-2478 5 Rougvie, A. and Ambros, V. (1995). Development 121:2491-2500