Fire A et al. (1989) International C. elegans Meeting "vectors and more vectors."

Harrison SW, Fire A

[International C. elegans Meeting, 1989]

Fire A et al. (2000) East Coast Worm Meeting "Postembryonic Muscle Patterning"

Fire A, Kostas SA

[East Coast Worm Meeting, 2000]

We are interested in understanding the genetic and molecular mechanisms that execute muscle patterning programs. The worm has a variety of muscle types that arise through a combination of lineage-specific programs and cell-cell interactions. While most of the muscle cells are born embryonically, a subset of muscles (14 striated muscles, eight uterine muscles and eight vulval muscles) arise postembryonically. All of these are derived from the lone postembryonic mesodermal blast cell, M. Analysis of mutations affecting some of the known developmental control genes has provided an intriguing but still fragmentary picture of pattern regulation in this lineage.1,2,3 Using green fluorescent protein (gfp) reporters that are active in cells derived from M we have screened for new mutants with altered muscle lineages. Our initial analysis has focused on a distinct and easily-scored cell fate decision: the distinction between uterine and vulval muscle. Three mutations that transform presumptive uterine to vulval muscles were isolated from the screen. The mutations are allelic and define a gene (designated mls-1) required for this decision. We found that this gene encodes a member of the T-Box family of transcription factors. Currently efforts are directed toward testing interactions between mls-1 and other regulatory pathways thought to be active within the lineage. Surprisingly, we found that mutations in several of the mog genes result in a similar sex muscle transformation. We are interested in determining if the mog genes interact with a tissue-specific regulatory site downstream of the mls-1 coding region. 1 Corsi, A. et al., Development, in press. 2 Harfe, B. et al., Development 125: 421-429 1998. 3 Greenwald, I. et al., Cell 34: 435-444 1983.

Fire A et al. (1989) International C. elegans Meeting "muscle promoters and enhancers."

Fire A, Harrison SW

[International C. elegans Meeting, 1989]

A Fire et al. (1999) International C. elegans Meeting "Structural requirements for the trigger in RNA-mediated interference"

J Fleenor, S Parrish, A Fire

[International C. elegans Meeting, 1999]

This poster will describe in vivo interference activities for a series of modified double-stranded RNAs that are based on segments of the genes unc-22 and gfp . Double stranded RNAs have been shown to act as potent inducers of gene-specific molecular silencing in C. elegans [a]. This process apparently reflects a well conserved control mechanism: recent reports have confirmed the effectiveness of dsRNA-triggered interference mechanisms in a variety of additional species including plant, insect, and protozoan systems [b-e]. By characterizing structural requirements (on the triggering side) for these two well defined segments in C. elegans , we hope to illuminate general features of the interference mechanism. Our experiments involve a variety of manipulations to produce dsRNAs with sequence or chemical alterations, followed by injection into C. elegans and assays for genetic interference. The manipulations are designed to address the following questions: 1. How precise and extensive are requirements for homology with the target gene? 2. What features distinguish the incoming RNA as "foreign"? 3. What chemical groups on the incoming RNA participate in interference? 4. Do the incoming sense and antisense strands have distinct roles in triggering interference? a. Fire, Xu, Montgomery, Kostas, Driver, Mello. Nature 391, 806 b. Waterhouse, Graham, Wang, PNAS 95, 13959 c. Ngô, Tschudi, Gull, Ullu, PNAS 95, 14687 d. Kennerdell and Carthew, Cell 95, 1017 e. Misquitta and Paterson, PNAS 96, 1451

Fire A et al. (1987) International C. elegans Meeting "expression of reintroduced muscle genes."

Waterston RH, Fire A

[International C. elegans Meeting, 1987]

Fire A et al. (1997) International C. elegans Meeting "SOME PROMOTER ACTIVITY PATTERNS"

Hsieh J, Macarah C, Fire A, Kelly B, Harfe BD, Montgomery MK, Xu SQ

[International C. elegans Meeting, 1997]

We will describe a variety of interesting germline and somatic promoter activity patterns and make some suggestions as to how you might be able to engineer your gene to be expressed in any of all of these patterns.

Fire A et al. (1995) International C. elegans Meeting "EFFICIENT GENE-EXPRESSION: REQUIRES PUNCTUATION!"

Xu SQ, Seydoux GC, Fire A

[International C. elegans Meeting, 1995]

We find that transcripts produced by lacZ fusions and some other heterologous transgenes are predominantly nuclear, frequently in 1-2 spots which may be sites of transcription (Development 120, 2823; P.Okkema, unpublished). In contrast, most endogenous transcripts accumulate in the cytoplasm. One potential difference between lacZ transcripts and endogenous messages is the long intron-less lacZ coding region. Consistent with this idea, we found several years ago (Genetics 135, 385) that a single intron just 5' to lacZ stimulates expression from some promoters. To generate reporter genes which were closer in character to endogenous nematode genes, we have produced vectors with 1-12 artificial introns within the lacZ coding region. For weak promoters, these increase beta-gal expression (by 1-2 orders of magnitude). Stimulation has likewise been seen by making an intron-rich gfp coding region. We've used in situ hybridization to examine RNAs produced from intron-poor and intron-rich unc-54::lacZ fusions. Constructs without introns (or with just one intron) show predominantly nuclear RNA localization, while intronrich constructs accumulate mRNA at high levels in the cytoplasm. The intron-rich reporters provide the potential for more sensitive vectors to assay gene expression. Promoterless and basal-promoter (enhancer-trap) vectors have been constructed with multiple introns in the reporter coding region. These are indeed more sensitive for detecting weak promoter & enhancer activities (higher staining frequency and greater intensity). Zygotic beta-gal expression in very early embryos is dramatically improved, while the germline still refuses to express beta-gal. As the signal increases, background also increases. Injected alone, promoterless intron-rich vectors give some background staining in gut and pharynx. Introns also appear to enhance sporadic background staining in the enhancer assay vectors.

Fire A et al. (2000) East Coast Worm Meeting "Genetic Analysis of Coelomocyte Specification and Patterning"

Fire A, Yanowitz J

[East Coast Worm Meeting, 2000]

We are interested in studying events which are responsible for patterning the late C. elegans embryo. We are using coelomocytes as a model system to study such events since both molecular and functional markers for the coelomocytes are available, since the organism tolerates deviations in the number of coelomocytes, and since the small number of these cell facilitates screening for mutants. Coelomocytes are highly endocytic mesodermal cells that reside in the pseudocoelomic space. Four of the six coelomocytes arise late in embryogenesis from MS progeny; the other two arise from the post-embryonic divisions of M. The lineages and timing of coelomocyte specification present several questions about cell fate decisions. In particular, we are interested in understanding how cells choose between muscle and non-muscle fates, how mesodermal cell fate choices are linked to the cell cycle, and how two unique lineages can give rise to cells with the same terminal phenotypes. To address these questions, we have been screening for changes in the number of coelomocytes by assaying for changes in tissue-specific GFP expression. This screen was faciliated by a strain with extra-bright GFP expression in these cells (provided to us by Dr. J. Fares). Using this strain, we have isolated over 20 mutations. These fall into several phenotypic categories based on (1) which cells are affected, (2) the number of cells, and (3) the timing of changes in cell number. We are currently mapping the mutations and performing more detailed phenotypic characterization.

Fire A et al. (1993) International C. elegans Meeting "Body wall muscles sense anterior-posterior position."

Kostich M, Xu SQ, Krause MW, Fire A

[International C. elegans Meeting, 1993]

Fire A (1985) International C. elegans Meeting "transient expression of microinjected sup-7 DNA and a stable transgenic suppressor line."

Fire A

[International C. elegans Meeting, 1985]