During the larva to adult transition the seam cells undergo terminal differentiation. The seam cells stop dividing, fuse with each other and synthesize the adult cuticle. AFF-1 is a fusogen in C. elegans required for the fusion of the lateral seam cells at the L4-adult transition, generating the lateral epithelial syncytium on each side of the animal. The seam cells terminal differentiation is controlled by the heterochronic pathway. The zinc finger transcription factor LIN-29 is the most downstream regulator of the seam cells terminal differentiation.
lin-29 regulates the collagen gene
col-19 which is one of the genes involved in the seam cells terminal differentiation.
lin-29 is a good candidate to be a positive regulator of AFF-1 expression in the seam cells. To determine whether
lin-29 controls
aff-1 through transcriptional regulation we examined
lin-29(
n482) loss-of-function mutant worms which also express
aff-1p::GFP. While wild-type worms express
aff-1 in the seam cells starting from the L4 stage, we found that some of the
lin-29 mutant worms had a reduced or no expression of
aff-1 in the seam cells at the L4 stage. These results suggest that
lin-29 may activate
aff-1 transcriptionally. However, the mutant allele that was chosen has partial penetrance and may not be a complete null allele. Therefore, we chose different
lin-29 mutant alleles that are complete null alleles for further examination of
aff-1 regulation. We are currently generating strains of either
lin-29(
n333) or
lin-29(
n546) which also express
aff-1p::GFP. Next, we will also test the influence of ectopic expression of
lin-29 on
aff-1 expression. It was shown that
lin-29 binds to specific sites of
col-19 promoter (1). In order to determine if
lin-29 regulates
aff-1 directly, we will examine whether similar sites exist in
aff-1 promoter using comparison of the
col-19 and
aff-1 promoter sequences. We will also examine whether
lin-29 binds to sites in
aff-1 promoter in vitro, and whether these sites are indeed necessary for in vivo activation of
aff-1. In case our results indicate that
lin-29 activates
aff-1 indirectly we will search for other factors that are involved in this regulatory pathway. An alternative hypothesis is that
lin-29 regulation of
aff-1 activity is posttranscriptional. To investigate this possibility we have generated the translational reporter AFF-1::mCherry. We found that in Sf9 heterologous insect cells AFF-1::mCherry induces cell-cell fusion. We will show expression of AFF-1::mCherry in the seam cells and explore
lin-29 potential regulation of AFF-1 localization and activities in the terminal differentiation of the seam cells. (1) Rougvie, A. E. and Ambros, V. (1995) Development 121, 2491-2500.