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Eugene Opoku-Serebuoh et al. (2002) East Coast Worm Meeting "Functional Studies of the eff-1 Promoter"

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East Coast Worm Meeting,

2002]

Cell fusion is a common phenomenon in multicellular organisms, yet it is poorly understood. In the nematode C. elegans, the developing worm is very selective in choosing which cells and at what times cell fusion occurs. The recently discovered gene eff-1, which stands for epithelial fusion failure, is required for cell fusion in C. elegans.1 Worms that have recessive mutations in eff-1 exhibit a distinct larval phenotype - a constriction of the larval tail whip into a ball - that is a feature of cell fusion failures during morphogenesis. Though the requirement for this gene in cell fusion is known, nothing is known about how it is regulated in development. There are several genes, notably transcription factors, that play crucial roles in the fate decisions of fusing cells, and their expressions are restricted to specific cell types. Thus, it is likely that eff-1 may be regulated by distinct transcription factors and upstream signaling pathways in each of the different cell types. The questions to ask are is eff-1 indeed regulated differently in these cells, what upstream regulatory genes and which cis-regulatory elements in eff-1 may account for specific regulation, and does the differential expression of these transcription factors have any role to play in eff-1 function in these different cells? These questions will be answered using molecular biology, microscopic and transgenic approaches. First, the temporal pattern of expression of eff-1 will be determined. It is expected to be expressed in many tissues in the worm as it is required for cell fusion and roughly, a third of all cells in C. elegans fuse during development. We have begun to observe eff-1 regulation via a transcriptional fusion of the eff-1 promoter to a reporter fluorescent protein. Second, the determination of the length of the minimal promoter fragment that is required for the full expression of the gene will be necessary. This can be tested by observation of a Green Fluorescent Protein (GFP) reporter and by assaying for rescue of cell fusion defects of eff-1 mutants during all stages of development. In addition, this helps in identifying the domains - putative transcription factor binding sites - within the promoter that are responsible for the function of the gene in a limited and well-defined region. The observation of the activity of wild type and mutant eff-1 promoter expression in various transcription factor mutant worms will further aid in explaining how eff-1 is regulated.

E Opoku-Serebuoh et al. (2003) International Worm Meeting "Profiling transcriptional regulation of eff-1 in fusing cell types."

E Opoku-Serebuoh, R Pulak, W Mohler, V Scranton

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International Worm Meeting,

2003]

In C. elegans, cell fusion requires the activity of the eff-1 gene (epithelial fusion failure).*Nematodes that are mutant for this gene fail to fuse their hypodermal, vulval, and pharyngeal cells during development. We find that ectopic expression of eff-1 is lethal to embryos and larvae, suggesting that precise temporal regulation of eff-1 expression in these specific cell types is important for normal development. We have also found that full function of the eff-1 gene in rescuing mutant phenotypes requires ~7.5 of 5'-upstream sequence. Thus we are interested in understanding the transcriptional control of eff-1 expression. A transcriptional fusion of the eff-1 promoter to GFP (eff-1::gfp) reveals a complex spatio-temporal expression pattern. Many cells that are fated to fuse express eff-1::gfp in the moments leading up to cell fusion, suggesting that their fusion fate/competence may be limited specifically by expression of EFF-1 protein. Cell fusions occur during various developmental stages and in various tissues of the animal, and the eff-1promoter appears to be differentially activated during the morphogenetic processes in which cell fusion is involved. We have created a series of transgenic lines that have progressively 5'-truncated versions of eff-1::gfp. We have identified 0.5-kb regions in the promoter that are required for tissue-specific transcriptional activity of the transgene in the pharynx, vulva, and amphid sheath, and a separate region for repression of gut expression. As these upstream domains are likely to contain enhancing and silencing cis regulatory elements, we have now begun site-directed mutagenesis of recognizable transcription-factor binding motifs. Our initial efforts indicate that PHA-4 may act directly upon eff-1 to drive pharyngeal expression. In trying to recognize broad/subtle effects on transcriptional regulation of eff-1, we have begun to analyze post-embryonic developmental expression chronograms (PDECs) of GFP fluorescence in mixed-stage populations from these promoter-deletion strains. PDECs are compilations of 1-dimensional fluorescent intensity measurements along the lengths of hundreds or thousand of worms. Spatio-temporal regulation of a transgene in all larval stages can be recognized in a single 2D image profile. We expect that contributions of upstream regions to quantitative regulation of transcriptional activity will be recognizable by comparisons of PDECs from the various eff-1promoter-deletion strains. Comparison of PDECs represents a unique way of studying the transcription of a developmentally-regulated gene and should be a useful tool for studying other aspects of developmental gene regulation in the worm. *Mohler et al. 2002 Dev Cell 2:355-62.

Eugene Opoku-Serebuoh et al. (2004) East Coast Worm Meeting "Transcriptional regulation and stochasticity of eff-1 in fusing cell types."

William A Mohler, Victoria L Scranton, Eugene Opoku-Serebuoh

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East Coast Worm Meeting,

2004]

In C. elegans, cell fusion requires the activity of the eff-1 gene. Nematodes that are mutant for this gene fail to fuse their hypodermal, vulval, and pharyngeal cells during development. We find that ectopic expression of eff-1 is lethal to embryos and larvae, suggesting that precise temporal regulation of eff-1 expression in these specific cell types is important for normal development. We have also found that full function of the eff-1 gene in rescuing mutant phenotypes requires ~7.5 of 5'-upstream sequence. Thus we are interested in understanding the transcriptional control of eff-1 expression. A transcriptional fusion of the eff-1 promoter to GFP (eff-1p::gfp) reveals a complex spatio-temporal expression pattern. Many cells that are fated to fuse express eff-1p::gfp in the moments leading up to cell fusion, suggesting that their fusion fate/competence may be limited specifically by expression of EFF-1 protein. Cell fusions occur during various developmental stages and in various tissues of the animal, and the eff-1 promoter appears to be differentially activated during the morphogenetic processes in which cell fusion is involved. We have created a series of transgenic lines that harbor progressively 5'-truncated versions of eff-1p::gfp. We have identified 0.5-kb regions in the promoter that are required for tissue-specific transcriptional activity of the transgene in the pharynx, vulva, and amphid sheath, and a separate region for repression of gut expression. Enhancer assay experiments have further been used to confirm the tissue-specific expression patterns conferred by some of these regions in the eff-1 promoter. As these upstream domains are likely to contain enhancing and silencing cis regulatory elements, we have now begun site-directed mutagenesis of recognizable transcription-factor binding motifs. In addition, we are currently performing yeast one-hybrid experiments to identify possible transcription factors that would regulate eff-1 promoter activity in a tissue-specific context. Our initial efforts indicate that PHA-4 may act directly upon eff-1 to drive pharyngeal expression. Interestingly, we observe a stochastic 'all-or-none' effect on pharyngeal expression when two putative PHA-4 binding sites are mutated. The similar expression of eff-1p::gfp by each binucleate cell in the metacorpus of a given animal indicates the existence of a 'noise-damping' signaling mechanism that tightly coordinates gene expression levels among these cells of quite distinct lineage.

Awadzi K et al. (1995) Trop Med Parasitol "The chemotherapy of onchocerciasis XX: ivermectin in combination with albendazole."

Buttner DW, Addy ET, Awadzi K, Opoku NO, Plenge-Bonig A

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Trop Med Parasitol,

1995]

Ivermectin is a potent microfilaricide that also blocks microfilarial release while albendazole is toxic to all intrauterine stages. We investigated whether their combination would permanently sterilize the adult worms. In the first open phase, all 69 patients received 150 micrograms/kg of ivermectin. In the second double-blind phase one week later, 35 patients were randomized to receive 800 mg of albendazole with a fatty breakfast for three consecutive days while 34 patients received matching placebo tablets. Detailed clinical and laboratory examinations were done before treatment and were repeated at intervals over one year. Nodules were excised at three and six months. There was a rapid reduction in skin microfilariae, maximal at four weeks (99.9%). Counts increased subsequently and were between 11 and 18% of initial values at one year. Nodule histology showed no macrofilaricidal activity of the combination. A high proportion of the stretched intrauterine microfilariae were degenerate in both groups. Anterior chamber microfilarial counts were unchanged until day 18 and then fell successively. Low levels persisted in several patients at one year. Dead corneal microfilariae and corneal punctate opacities increased initially, fell with time and then disappeared in most patients. Systemic and ocular reactions were mild to moderate and biochemical abnormalities were minor. A pronounced posttreatment eosinophilia subsided by day 30. There was no significant difference between the two groups in clinical and laboratory tolerance or in alterations in skin and ocular parasites and no important differences in the effect on the adult worms. The combination of ivermectin with albendazole given one week apart is well tolerated but produces no additional effect against Onchocerca volvulus when compared to ivermectin given alone.

Barbara Meyer (2008) Development & Evolution Meeting "Gametogenesis, Meiosis, & Sex Determination"

Barbara Meyer

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