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-to-adult molt. The seam cells exit the cell cycle, fuse, and contribute to the synthesis of an adult-type cuticle. 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 loss-of-function (lf)
lin-14 and
lin-28 mutants, the L/A switch occurs abnormally early, at the L3-to-L4 molt. In contrast,
lin-4(lf) and
lin-29(lf) mutants never develop an adult cuticle; instead, they continue to synthesize a larval-type cuticle and undergo extra molts. Of these four genes,
lin-29 is the most downstream and direct regulator of the L/A switch1. Activities of
lin-4,
lin-14 and
lin-28 are required for the proper timing of
lin-29 activity. However, we think there are additional genes involved in this process. Our approach towards identifying additional heterochronic genes is to express green fluorescent protein (GFP) under the control of a
lin-29-dependent promoter and to screen for mutants that temporally mis-express GFP. We have constructed a
col-19::GFP fusion for this purpose.
col-19 is one target of the LIN-29 zinc finger transcription factor2,3. The
col-19 gene is normally activated in adult hypodermal cells, but is never activated in
lin-29 mutants. Wild-type worms begin expressing the
col-19::GFP fusion from an integrated array shortly after the L4-to-adult molt. We found that
col-19::GFP expression is dependent on the heterochronic gene pathway: In
lin-14 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; 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. We mutagenized RG240 animals and screened for mutants with restored expression of
col-19::GFP. We have screened a total of 46,800 haploid genomes we have obtained 52 mutants that now express
col-19::GFP. Some of the mutants we have isolated suppress additional
lin-4 defects including lack of adult alae synthesis and the vulval defect. We are in the process of isolating the new mutations away from
lin-4 to see if they have phenotypes alone. We have isolated new alleles of
lin-14 in these screens, thereby validating this approach for identifying heterochronic genes. In addition, we have identified mutants whose phenotypic characteristics are distinct from
lin-14 and
lin-28, and thus likely represent new heterochronic genes. We will present a summary of our screens including phenotypic and genetic analysis of the mutants we have isolated. 1. Ambros, V. (1989). Cell 57:49-57 2. Liu, Z., Kirch, S., and Ambros, V. (1995). Development 121:2471-2478 3 Rougvie, A. and Ambros, V. (1995). Development 121:2491-2500