Using RNA-Mediated Interference to Investigate the Role of Novel Proteins in Caenorhabditis elegans Gonadogenesis

During development, cells divide, differentiate into various cell types, and organize into functional tissues and organs. We are interested in understanding the cellular and molecular basis of organ formation in the nematode Caenorhabditis elegans. To this end, we are investigating two novel proteins that have a role in gonadogenesis, SYS-1 and NUD-1. SYS-1 was identified via a genetic screen for mutants with defective gonads. The role of SYS-1 at the cellular level has been characterized (1), but its molecular role is unclear. NUD-1 is homologous to a protein in fungi responsible for mediating nuclear migration (2). Using reverse genetics to “knock-down” the protein levels, C. elegans NUD-1 was shown to have a role in gonadogenesis, but the specific phenotoype has not been described in detail. We are using RNA mediated interference (RNAi) to investigate both of these proteins. RNAi is a method by which adding an excess of mRNA corresponding to a specific gene actually represses the production of that protein. Information learned about gonad formation in worms is likely to advance our understanding of organ development in more complex organisms. INTRODUCTION The mechanisms by which cells divide, differentiate, migrate, and die during development are very important and interesting, but they are not fully understood. A complex series of processes is required for proper development, and it is essential that they be precisely coordinated to form a normal functioning organism. Likewise, a combination of multiple cellular processes is needed for proper formation of an organ. The molecular basis of organ formation is the basis for this study. A model system for studying organ development: the Caenorhabditis elegans gonad In order to study organ development, Caenorhabditis elegans, a 1mm long non-parasitic soil nematode, was employed as a model organism. C. elegans proves to be a good model system for investigating these highly conserved processes due to its genetic and molecular similarity to higher organisms, such as humans. In addition, these worms are easy to grow, they have a short lifespan, and their genome has been entirely sequenced. They are small and transparent which allows them to be examined at the level of individual cells, and the development of all of their somatic cells is essentially constant from animal to animal. C. elegans has often been used to study organ development, and this study focuses specifically on the role of two proteins, NUD-1 and SYS-1, in the development of the hermaphrodite gonad. The C. elegans hermaphrodite gonad is a complex organ with distinct cell-types and functional tissues (3). There are four larval stages of development (L1-L4) of a worm before it becomes an adult. When a worm hatches as an L1, the football-shaped gonad consists of only four cells referred to as the gonad primordium. These are the cells that will ultimately form the adult gonad. Of these four cells, two are somatic precursors (Z1 and Z4) and two are germ cells (Z2 and Z3). The somatic precursors will form the various cells/tissues that house the germ cells and support their development. The somatic gonadal cells develop in a nearly identical manner in all hermaphrodites allowing the processes to be studied at the cellular level. From L1-L4, the gonad adopts the U-shaped morphology of the adult organ and gives rise to the mature gonad tissues such as the spermathecae, uterus, and sheath. It has been shown that the SYS-1 and NUD-1 proteins are necessary for gonad formation to occur properly (1,2). The proteins of interest: SYS-1 and NUD-1 SYS-1 and NUD-1 proteins were identified using distinct approaches, but were both found to be involved in gonad formation. SYS-1 was discovered using a forward genetics approach. Forward genetics is a process by