1. Temporal Regulation of Cell Development: The Heterochronic Gene Pathway in Caenorhabditis elegans David Cooper, Department of Biology, York College of Pennsylvania Methods (1) http://lc-molecular.wikispaces.com/ http://neurodojo.blogspot.com Two separate experiments were conducted: Two crosses to examine the impact on Seam Cells and expression of the hbl-1 gene of a lin-42;lin-14 double mutant An RNAi experiment to look for altered phenotypes when lin-42 and lin-14 mutants are grown under other mutant backgrounds Literature Cited AMBROS, V., 1989, A hierarchy of regulatory genes controls a larva-to-adult developmental switch in C. elegans. Cell 57: 49-57. AMBROS, V., HORVITZ, H.R., 1984, Heterochronic mutants of the nematode Caenorhabditis elegans. Science 226: 409-416. MOSS, E.G., 2007, Heterochronic genes and the nature of developmental times. Current Biology 17: R425- R434. Crosses were started with a control N2 strain which, by definition, has no mutations. Worm Strain RNAi Wild TypeLin-42Lin-42;Lin-14Lin-28 Vector (BVB8) Alae: L4 SCN a : 16.75 (n=20) Alae: L3 Normal Shape Alae: L3 BLP Alae: L3 SCN: 11.15 (n=20) Hbl-1 (BVB3) Alae: L4 SCN: 10.91 (n=22) Alae: L3 DPY b Alae: L3 STRL Alae: L3 SCN: 9.7 (n=20) Lin-41 (I4J11) Alae: L4 SCN: 15.39 (n=18) DPY STRL c Alae: L3 DPY BLP Alae: L3 SCN: 10.75 (n=24) Lin-42 (pkk220*) Alae: L4 SCN: 15.85 (n=20) No apparent effects Alae: L3 BLP Alae: L3 SCN: 10.68 (n=25) Hbl-1::GFP; Lin-42; Lin-14: Cross appeared unsuccessful Rolling phenotype observed in males Rolling males did not seem to be able to successfully mate SCM::GFP; Lin-42; Lin-14: Cross was successful. Hermaphrodites produced that had: Seam cell marker, blipped Able to produce offspring with same phenotype Average Seam Cell Number: 11.4 (n=5) Results (1) What is RNAi? Utilizes a viral defense mechanism against single-stranded RNA Tricks organism into shutting off its own gene Can be done fairly efficiently and quickly E. coli induced to separately produce four different types of RNAi: Vector, lin-42, lin-41, hbl-1 Four different strains plated onto each RNAi. Wild type, lin-42, lin-42; lin-14, lin-28 lin-28 mutants have the same phenotype as lin-42 Methods (2) Results (2) Results: Lin-42 on Lin-41 RNAi: Animals appeared sterile and were highly deformed Lin-28 on Hbl-1 RNAi: Average Seam Cell number was highly unusual Table 1: Results of the RNAi experiments. Each strain was plated onto each of the four RNAi types. a Seam Cell Number b Dumpy Phenotype c Unconfirmed Sterility Lin-42; lin-14 Seam Cells Average = 11.4 Adults should have 16 Previous reports: no symmetric double division Lin-42; lin-14 hbl-1 expression Phenotype does not match expected. Results inconclusive. lin-42 has been shown in the past to suppress hbl-1 phenotype RNAi lin-42 animals on lin-41 RNAi have a very severe phenotype lin-28 animals on hbl-1 RNAi have an unusual seam cell count Corresponds with previously established reports on hbl-1 RNAi Introduction Objectives The specific objectives of this study were: 1)Determine if a Lin-42;Lin-14 double mutant alters the seam cell count or the expression of the hbl-1 gene 2)Compare and contrast four different strains grown under four different mutant backgrounds I would like to thank the University of Medicine and Dentistry of New Jersey – Graduate School of Biomedical Science for hosting the SURE Program. I would also like to thank Dr. Eric Moss, Dr. Bhaskar Vadla, Jennifer Alaimo, and Kevin Kemper for helping me to conduct this research and interpret my data. Lastly I would like to thank Dr. Kaltreider for being my mentor and helping me refine my written report and poster. Acknowledgements Results (Summary): In Caenorhabditis elegans, a complex set of genes have been discovered, collectively known as the heterochronic gene pathway. These genes control the timing of cell development, ensuring that structures develop at the proper time during maturation. Some genes are understood, while others have remained difficult to characterize. The heterochronic genes Lin- 42 and Lin-14 were the focus of this study. The protein lin-42 is known to be related to the family of proteins responsible for circadian rhythms in mammals. Lin-14 produces a nuclear protein believed to be critical to early larval development. These affect development of Seam Cells as well as the expression of the hbl-1 gene Conclusions Analysis of the results imply that lin-14 is epistatic to lin-42. Further investigation into the two unusual RNAi strain phenotypes may help reveal more about the different genes relationships to one another.