Worm Breeder's Gazette 14(1): 91 (October 1, 1995)

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.

In Search of novel heterochronic genes with a col-19::GFP fusion

Juan E. Abrahante, Ann Rougvie

Genetics & Cell Biology, University of Minnesota, St Paul MN 55108.

      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