Kristen Tews et al. (2006) Development & Evolution Meeting "Investigating the Role of NUD-1 in C. elegans Gonadogenesis"

Rachel McCabe, Guy Caldwell, Kristen Tews, Jennifer Miskowski, Kim Caldwell

[Development & Evolution Meeting, 2006]

The C. elegans gonad is a complex organ consisting of multiple cell-types that are organized into morphologically and functionally distinct tissues. We are interested in identifying proteins necessary for gonad formation and elucidating the mechanisms by which these proteins act. The C. elegans NUD-1 protein is the structural and functional ortholog of the fungal NudC protein (1). NUD-1/NudC proteins are evolutionarily conserved and have been implicated in diverse processes such as asymmetrical cell division in yeast, nuclear migration in fungi, and neuronal migration in humans. However, the exact function of NUD-1/NudC proteins is unknown. When NUD-1 levels are depleted in hermaphrodites by RNAi, a variety of phenotypes are found, including sterility (1). Observation of nud-1(RNAi) mutants revealed possible defects in both the germ line and somatic gonad. To investigate whether nud-1(RNAi) animals possess somatic gonadal defects, cell-specific GFP reporter constructs have been employed. Most cell types are correctly specified, but there appear to be defects in cellular positioning and tissue organization. Hence, NUD-1 might be required for proper tissue morphology. An antibody generated against a phosphorylated version of mammalian NUDC cross-reacts specifically in C. elegans and is being used in immunofluorescence studies. Robust staining was observed in the germ line of both hermaphrodites and males. Specifically, NUD-1 was found in perinuclear puncta in primary spermatocytes with more diffuse staining in secondary spermatocytes and spermatids. Adult hermaphrodites also revealed staining in the spermathecae, where mature sperm are found, and surrounding the oocytes. These results suggest a role for NUD-1 in spermatogenesis and possibly in oogenesis and are consistent with RNAi results. Double labeling experiments are underway to better define the NUD-1 localization pattern.

Kristen Tews et al. (2005) International Worm Meeting "Investigating the role of NUD-1 in C. elegans hermaphrodite gonadogenesis"

Kim A Caldwell, Guy A Caldwell, Kristen Tews, Jennifer A Miskowski

[International Worm Meeting, 2005]

The C. elegans NUD-1 protein is the structural and functional ortholog of the fungal NudC protein (1). NUD-1/NudC proteins are evolutionarily conserved and have been implicated in diverse processes such as asymmetrical cell division in yeast, nuclear migration in fungi, and neuronal migration in humans. However, the exact function of NUD-1/NudC proteins is unknown. When NUD-1 levels are depleted by RNAi, a variety of phenotypes are found, including sterility (1). Observation of nud-1(RNAi) mutants revealed possible defects in both the germ line and somatic gonad. To investigate whether nud-1(RNAi) animals possess somatic gonadal defects, cell-specific GFP reporter constructs have been employed. To date, the distal tip cells, anchor cell, and uterine pi cells appear to be properly specified and general gonad morphology is correct in the majority of animals. An antibody generated against a phosphorylated version of mammalian NUDC has been found to cross-react in C. elegans and is being used in immunofluorescence studies. We have preliminary evidence that the protein is localized to particular germline cells. Additional experiments to determine the specificity of the staining and to address the significance will be presented. (1) Dawe et al., Dev. Genes Evol. 211, 434 (2001).

Kristen Tews et al. (2003) International Worm Meeting "Investigating the role of NUD-1 in C. elegans hermaphrodite gonadogenesis"

Jennifer A Miskowski, Kim A Caldwell, Guy A Caldwell, Georgette Jones, Kristen Tews

[International Worm Meeting, 2003]

The C. elegans hermaphrodite gonad is a complex organ consisting of multiple cell-types that are organized into morphologically and functionally distinct tissues. We are interested in identifying proteins necessary for gonad formation and elucidating the mechanisms by which these proteins act. The C. elegans NUD-1 protein was identified based on its sequence conservation with the NudC protein of fungi, which has been shown to be involved in nuclear migration (1). However, the exact function of NudC in this process is unknown. NUD-1 plays a role in many aspects of C. elegans development, including gonadogenesis (1). Hermaphrodites where NUD-1 levels have been depleted by RNAi are sterile and have defects in oogenesis and somatic gonad formation. In particular, the proximal region of the gonad is filled with spermathecal tissue with little to no uterine tissue. Interestingly, a NUD-1::GFP reporter protein is expressed in uterine progenitor cells at the somatic gonadal primordium stage in hermaphrodites (SPh). Further characterization of the nud-1 RNAi phenotype and expression pattern is underway. In addition, a genetic screen will be performed that is designed to identify mutations in the nud-1 gene and other loci that function in the same processes as nud-1. A heritable nud-1 mutant will allow for more detailed phenotype characterization that focuses on early stages of gonad development and will permit cell lineage analysis. This work will hopefully shed light on developmental mechanisms needed for organ formation across species, and provide clues about the action and regulation of the NUD-1 protein, in particular. (1) Dawe et al., Dev. Genes Evol. 211, 434 (2001).

Kristen Tews et al. (2004) Mid-west Worm Meeting "Determining the role of NUD-1 in hermaphrodite gonadogenesis by analysis of GFP reporters in nud-1(RNAi) mutants"

Kim A Caldwell, Guy A Caldwell, Kristen Tews, Jennifer A Miskowski

[Mid-west Worm Meeting, 2004]

The C. elegans hermaphrodite gonad is a complex organ consisting of multiple cell-types that are organized into morphologically and functionally distinct tissues. We are interested in identifying proteins necessary for gonad formation and elucidating the mechanisms by which these proteins act. The C. elegans NUD-1 protein was identified based on its sequence conservation with the NudC protein of fungi, which has been shown to be involved in nuclear migration (1). However, the exact function of NudC in this process is unknown. NUD-1 plays a role in many aspects of C. elegans development, including gonadogenesis (1). Hermaphrodites where NUD-1 has been depleted by RNAi are sterile and have defects in oogenesis and somatic gonad formation. In particular, the proximal region of the gonad is filled with spermathecal tissue with little to no uterine tissue. Furthermore, a NUD-1::GFP reporter protein is expressed in uterine progenitor cells at the somatic gonadal primordium stage in hermaphrodites (SPh). To better characterize the gonadal defects in nud-1 (RNAi) animals, cell-specific GFP reporter constructs have been employed. Analysis of GFP expression patterns in nud-1 (RNAi) mutants will hopefully shed light on the particular gonadal cell(s) and/or tissue(s) affected by loss of the NUD-1 protein. (1) Dawe et al., Dev. Genes Evol. 211, 434 (2001).