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 "vectors and more vectors."

Harrison SW, Fire A

[International C. elegans Meeting, 1989]

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

Fire A, Harrison SW

[International C. elegans Meeting, 1989]

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 (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]

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 et al. (1991) International C. elegans Meeting "MULTIPLE CIS-ACTING SIGNALS REGULATE MyoD EXPRESSION"

Fire A, Krause MW, Priess JR, Harrison SW, Weintraub H

[International C. elegans Meeting, 1991]

We have previously shown that a myoD::Bgal fusion construct correcdy mimics the myoD expression pattern in body wall muscle cells and their precursors (Krause et al. Cell, 63, 907). We constructed and tested a set of deletions, substitutions and insertions upstream of the myoD coding region in order to map cis-acting control signals which are responsible for the observed expression pattern. The results of this analysis indicate that a 3kb redon upstream of myoD coding sequences contains multiple sites with a variety of roles in regulating myoD expression. Several aspects of this analysis are summarized below l) Different signals for establishment and maintenance of myoD expression: Many of the deletion constructs show differential effects on precursor and mature body wall muscle expression. One upstream segment is required for maximal expression in precursor cells, while a distinct segment is needed for maximal expression in mature muscle. 2) Ectopic expression: All of the deletion mutants show some ectopic expression (expression in tissues not seen with the oridnal construct). This primarily occurs in posterior gut cells or in gland [g2] and mardnal cells in the pharynx. The deletion analysis has not indicated that discrete negative regulatory elements or 'silencers' exist. Instead, at least some of the ectopic expression actually appears to result from a positive interaction caused by close juxtaposition of upstream elements from the myoD gene with specific sequences in the plasmid vector. Therefore, we withhold judgment on whether there are negative control elements suppressing ectopic expression that act in normal regulation of myoD. 3) Lineage specific signals active in muscle precursor cells: The body wall muscle precursors are derived from the C, D, MS, and AB lineages. Several of the deletions show a marked preference for expression in one or more sets of muscle precursor cells. These patterns are consistent with a preference based on lineage. In particular, one set of upstream deletion constructs is active primarily in muscle precursors derived from the C lineage while another set is active preferentially in MS (and possibly D) derived muscle precursors. These lineage specific signals are presumably the interface between the lineage information dving self-identity to cells in the early embryo and factors such as myoD which function subsequently to allow expression of one particular cell fate in several diverse lineages.