Worm Breeder's Gazette 12(3): 37 (June 15, 1992)

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.

lin-31 Acts as a Transcriptional Regulator of Vulval Cell lineages and Encodes A Fork head/HNF.3 DNA Binding Domain

Leilani M. Miller, Maria E. Gallegos, Stuart K. Kim

Stanford University Medical Center, Dept. of Developmental Biology, Stanford, CA 94305

In vulval development the vulval precursor cells (P3-8.p) express one of three potential cell fates (1°, 2°, or 3°) in response to extracellular signals originating from the anchor cell, other Pn.p cells and probably the hyp-7 syncytium. Mutations in lin-31 cause vulval precursor cell fates to become deregulated; that is, any vulval precursor cell can express any one of the three cell fates. Thus, lin-31 is required for the Foper specification of all three cell fates. Our previous genetic studies (see WBG 12-2, p. 97) revealed that lin-31 acts at a late step in the vulval development pathway. In double mutant combinations, the lin-31 mutant phenotype is epistatic to the vulvaless mutant phenotypes of lin-2 , lin-7 , lin-10 , let-23 and let-60 .

To clone lin-31 ,we initially showed that a 190 kb YAC clone could rescue lin-31 mutant worms. We then used recombination in yeast to rapidly narrow the lin-31 rescuing activity to a 9 kb region of genomic DNA (see WBG 12-2, p.38). Sequence analysis, Northern blotting and cDNA cloning experiments indicate that lin-31 is the only gene within this region. Iin-31 has compelling similarity (65% amino acid identity over 105 amino acids) to the DNA binding domains of the Drosophila Fork head protein and the rat liver transcriptional activator HNF-3 .This result suggests that lin-31 acts as a transcriptional regulator of vulval cell lineages. The regulatory activity of lin-31 is likely to be under the control of vulval signalling genes such as lin-2 , lin-7 lin-10 , let-23 and let-60 .

Four lin-31 alleles display allele-specific polymorphisms in the 9 kb rescuing region. Three insertion alleles map about 1 kb upstream of the DNA binding domain and one deletion allele maps 2 kb downstream of the DNA binding domain.

Using lin-31 oligonucleotides derived from the DNA binding domain, we have used anchored PCR to obtain several lin-31 cDNAs. Preliminary characterization of these cDNAs suggests that lin-31 may undergo differential RNA splicing. Two types of cDNAs have been obtained and these cDNAs differ by the presence or absence of a 2 kb intron. We also observe two RNA bands (3.4 kb and 1.5 kb) in Northern blotting experiments, using one of the cDNAs as a probe. Both transcripts are expressed at all developmental stages and are altered or missing in lin-31 insertion or deletion mutants. Since the two lin-31 transcripts differ by about 2 kb, it is possible that the alternative splice can explain both classes of transcripts.

Our current efforts are focused on understanding how lin-31 can alternatively specify the 1°, 2°, and 3° cell fates. One possibility is that alternative splicing patterns allow lin-31 to regulate different sets of genes in vulval cells undergoing different cell lineages. Another possibility is that the same lin-31 gene product associates with different regulatory factors to specify alternative cell fates.

Literature Cited:

WBG 12-2, p. 97

WBG 12-2, p.38