During development of the Caenorhabditis elegans gonad, the gonadal leader cells, called distal tip cells (DTCs), migrate in a U-shaped pattern to form the U-shaped gonad arms. The ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family metalloproteases MIG-17 and GON-1 are required for correct DTC migration. Mutations in mig-17 result in misshapen gonads due to the misdirected DTC migration, and mutations in gon-1 result in shortened and swollen gonads due to the premature termination of DTC migration. Although the phenotypes shown by mig-17 and gon-1 mutants are very different from one another, mutations that result in amino acid substitutions in the same basement membrane protein genes, emb-9/collagen IV a1, let-2/collagen IV a2 and fbl-1/fibulin-1, were identified as genetic suppressors of mig-17 and gon-1 mutants. To understand the roles shared by these two proteases, we examined the effects of the mig-17 suppressors on gon-1 and the effects of the gon-1 suppressors and enhancers on mig-17 gonadal defects. Some of the emb-9, let-2 and fbl-1 mutations suppressed both mig-17 and gon-1, whereas others acted only on mig-17 or gon-1. These results suggest that mig-17 and gon-1 have their specific functions as well as functions commonly shared between them for gonad formation. The levels of collagen IV accumulation in the DTC basement membrane were significantly higher in the gon-1 mutants as compared with wild type and were reduced to the wild-type levels when combined with suppressor mutations, but not with enhancer mutations, suggesting that the ability to reduce collagen IV levels is important for gon-1 suppression.

-Topical drug treatment of the eye exposes ocular tissues to a high drug concentration. Genotoxicity assessment in ocular tissues has not been established to date. Therefore, we investigated the in vitro comet assay by incubating cultured human corneal epithelial (HCE-T) cells with known mutagens. The alkaline comet assay was conducted to measure the DNA strand breakage yield. When the cells were incubated with methyl methanesulfonate (MMS) for 1 hr, hydrogen peroxide (H 2 O 2 ) for 15 min and actinomycin D (AMD) for 1 hr, statistically significant increases of percentage (%) DNA in the tail were noted in MMS-, H 2 O 2 -, and AMD-treated cells at 100, 10, and > 10 μM, respectively. When the cells were treated with mitomycin C (MMC) or 5-bromouracil (5-BrU), %DNA in the tail was unchanged even at the highest concentration. Hedgehog cells were found in MMS-and H 2 O 2 -treated cells at 1000 and > 100 μM, respectively. The response to each compound was consistent with results previously reported in other cells. In conclusion, the in vitro comet assay using HCE-T cells can detect DNA strand breakage induced by mutagens. This method has a possibility to become a conventional screening tool to assess the genotoxicity of drugs applied to ocular surface.

During development of the Caenorhabditis elegans gonad, the gonadal leader cells, called distal tip cells (DTCs), migrate in a U-shaped pattern to form the U-shaped gonad arms. The ADAMTS ( a d isintegrin a nd m etalloprotease with t hrombo s pondin motifs) family metalloproteases MIG-17 and GON-1 are required for correct DTC migration. Mutations in mig-17 result in misshapen gonads due to the misdirected DTC migration, and mutations in gon-1 result in shortened and swollen gonads due to the premature termination of DTC migration. Although the phenotypes shown by mig-17 and gon-1 mutants are very different from one another, mutations that result in amino acid substitutions in the same basement membrane protein genes, emb-9/collagen IV a1 , let-2 / collagen IV a2 and fbl-1/fibulin-1 , were identified as genetic suppressors of mig-17 and gon-1 mutants. To understand the roles shared by these two proteases, we examined the effects of the mig-17 suppressors on gon-1 and the effects of the gon-1 suppressors and enhancers on mig-17 gonadal defects. Some of the emb-9 , let-2 and fbl-1 mutations suppressed both mig-17 and gon-1 , whereas others acted only on mig-17 or gon-1 . These results suggest that mig-17 and gon-1 have their specific functions as well as functions commonly shared between them for gonad formation. The levels of collagen IV accumulation in the DTC basement membrane were significantly higher in the gon-1 mutants as compared with wild type and were reduced to the wild-type levels when combined with suppressor mutations, but not with enhancer mutations, suggesting that the ability to reduce collagen IV levels is important for gon-1 suppression.

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