Worm Breeder's Gazette 16(4): 34 (October 1, 2000)

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.

Functional divergence of beta-catenins in C. elegans

Lakshmi Natarajan, Nina Witwer, David M. Eisenmann

University of Maryland Baltimore County, Baltimore, MD 21250.

Vulval development in C. elegans is an excellent model system to study cell signaling in development. In a screen for mutants defective in vulval formation, bar-1 was identified. bar-1 was cloned and found to encode a C. elegans homolog of the b-catenin/ Armadillo family of proteins. b-catenin proteins function in two different processes; Wnt signaling and cell adhesion.

Vertebrates have two b-catenin family members, b-catenin and plakoglobin, and Drosophila has one, Armadillo. In contrast, C.elegans has three members of this family (BAR-1, WRM-1 and HMP-2), of which WRM-1 functions in Wnt signaling in the embryo, BAR-1 functions in Wnt signaling in larval life and HMP-2 plays a role in cellular morphogenesis during embryogenesis. Interestingly, the three b-catenin homologs in C. elegans show only about 17-28% amino acid identity to vertebrate b-catenins and to each other, while homologs from other species show between 50-70% identity with vertebrate b-catenins. The question that follows is why does the worm have three highly divergedb-catenin family members? Are they diverged to perform different functions or are their Wnt signaling and adhesion functions still conserved? We addressed this question by taking two approaches.

First, we performed a directed yeast two hybrid assay using four b-catenin proteins (BAR-1, HMP-2, WRM-1 and mouse b-catenin) fused to the Gal4 activation domain. We then tested these fusions for interactions in yeast with several proteins each fused to the Gal4 DNA Binding domain. These proteins were Wnt signaling factors POP-1, APR-1 and LIT-1, the alpha catenin homolog HMP-1 and the histone deacetylase EGL-27.

Our results show that BAR-1 interacts strongly with POP-1 (TCF homolog) and APR-1 (APC homolog) and does not interact with LIT-1, EGL-27 and HMP-1. This suggests that BAR-1 may have a Wnt signaling function and not a cell adhesion function. Consistent with the results that BAR-1 interacts with APR-1 and POP-1, work from Alex Hajnal's lab has shown that apr-1 antisense RNA results in bar-1-like defects in vulval development and our lab has found that pop-1 RNAi on larvae results in a vulval phenotype, suggesting both factors act in this process with BAR-1. WRM-1 on the other hand interacts less strongly with POP-1 and is the only b-catenin tested which interacts with LIT-1. This is consistent with the fact that both POP-1 and LIT-1 are involved in WRM-1 signaling in embryogenesis. However, we did not see an interaction with WRM-1 and APR-1 even though APR-1 functions in embryonic Wnt signaling. Finally, HMP-2 does not interact with any of the Wnt signaling components but strongly interacts with vertebrate and C. elegans a-catenin (HMP-1). These results suggest that HMP-2 may have evolutionarily diverged from an ancestral b-catenin perform different functions. Similar results have recently been reported by Korswagen et al. (1) The vertebrate b-catenin tested in our assay interacts with vertebrate a-catenin, but does not interact with any of the C. elegans proteins.

As another approach to understanding if the threeb-catenins have been conserved in function despite sequence divergence, we are determining if WRM-1 and HMP-2 can substitute for BAR-1 function during vulval development. wrm-1 and hmp-2 driven by the bar-1 promoter have been injected into bar-1 mutant animals and are being analyzed for rescue of the bar-1 vulval phenotype. Preliminary results show that wrm-1 is able to rescue the bar-1 mutant phenotype in the vulva suggesting that wrm-1 and bar-1 might have similar functions but different expression patterns. Taken together, our evidence suggests that C. elegans may have oneb-catenin which functions in cell morphogenesis (HMP-2) and two which function in Wnt signaling (BAR-1 and WRM-1). Therefore, the three C. elegans b-catenins may be less conserved in sequence because they have been free to diverge due to this segregation of function.

(1) Korswagen et al. Nature 406, 527-32 (3rd Aug 2000)