Worm Breeder's Gazette 13(3): 31 (June 1, 1994)
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.
The maternal product of the skn-1 gene is required to specify the fate of the ventral-most 4-cell stage blastomere, called EMS(1). In skn-1 mutant embryos, EMS fails to produce its normal cell types and instead adopts a fate similar to its sister, P2 .Sequence analysis suggests that skn-1 encodes a transcription factor, SKN-1 ,and biochemical studies indicate that SKN-1 is a sequence specific DNA binding protein that is capable of acting as a transcriptional activator(2). Antibody staining has been used to show that SKN-1 accumulates to peak levels at the 4-cell stage in the nuclei of EMS and P2 ,is present at lower levels in the 8-cell stage daughters of EMS and P2 ,and fades to undetectable levels by the 12-cell stage(3). Thus skn-1 may specify blastomere fate by regulating the expression of downstream target genes in descendants of EMS. To date, however, no downstream targets of skn-1 have been identified.
To define the genetic pathway that controls the development of EMS and its descendants, we want to identify zygotically expressed genes that are downstream targets of SKN-1 transcriptional regulation. Specifically, we are using differential display techniques(4) to identify mRNAs that are expressed in descendants of EMS between the 4-cell and 28-cell stages during wild-type embryogenesis. To identify such messages, we are comparing the pattern of mRNA expression in 4-cell to 28-cell stage skn-1 mutant embryos to the pattern seen in early mex-1 mutant embryos. We expect zygotic genes that normally are activated by SKN-1 regulation to be off in skn-1 mutant embryos. However, in mex-1 mutant embryos, skn-1 functions not only in EMS but also ectopically in other blastomeres that consequently adopt EMS-related fates(5). Thus we would expect genes that are activated by skn-1 to be expressed at abnormally high levels in 4-cell to 28-cell stage mex-1 mutant embryos. Using the differential display technique, we are collecting cDNA clones of messages that may be absent in early skn-1 mutant embryos but expressed ectopically in early mex-1 mutant embryos. We will screen these cDNA clones first by using RNAse protection assays to determine if the corresponding messages are differentially expressed in skn-1 and mex-1 mutant embryos, relative to an actin control. Differentially expressed cDNA clones will then be analyzed by in situ hybridization to determine the expression patterns of their corresponding messages in early embryos. We hope to identify messages that (1) are expressed specifically in EMS descendants between the 4-cell and 28-cell stages in wild-type embryos; (2) fail to be expressed in skn-1 mutant embryos; and (3) are expressed ectopically in mex-1 mutant embryos. cDNA clones that meet these criteria will be studied further to see if they are targets of SKN-1 regulation, and if they are necessary for proper EMS development.
2. Blackwell, T.K., Bowerman, B., Priess, J.R. and Weintraub, H., unpublished results.
3. Bowerman, B., Draper, B.W., Mello, C. and Priess, J. (1993) Cell, 74, 443-452.
4. Liang, P. and Pardee, A.B. (1992) Science, 257, 967-971.
5. Mello, C., Draper, B.W., Krause, M., Weintraub, H. and Priess, J.R. (1992) Cell, 70, 163-176.