429B Poster - 05. Reproduction and gametogenesis
Friday April 08, 2:00 PM - 4:00 PM

Authors:
Savannah Muron ^1,2; Leif Benner ^1,3; Brian Oliver ¹

Affiliations:
1) National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD; 2) Department of Biology, University of Tampa, Tampa, FL; 3) Department of Biology, Johns Hopkins University, Baltimore, MD

Keywords:
b. oogenesis; h. translational regulation

An important component in the continuity of life is sexual reproduction, yet the genetic determinants of germline differentiation to create a sex-specfic gamete remain largely uncovered. Exploring the activity of transcription factors essential to these processes give needed insight to this concept. In Drosophila, OVO is required for female germ cell viability. ovo is a self-regulating gene that encodes two antagonistic isoforms, OVO-A and OVO-B, with OVO-A acting as a repressor and OVO-B acting as an activator. To reveal the behavior of OVO, we chose to visualize its activation during germline development and oogenesis. A sequence containing a 3x-FLAG-HA tag was placed at 4 locations in the open reading frame of the ovo locus, allowing us to tag different isoforms and visualize its expression and cellular localization. This tag was placed at the N-terminus of the OVO-B transcript (ovo^ovo-Nterm, tagging both OVO isoforms), the N-terminus of the OVO-A transcript (ovo^ovo-A, tagging OVO-A), the C-terminus of all transcripts (ovo^ovo-Cterm, tagging both OVO isoforms), and the N-terminus of an OVO-B transcript in which the promoter of OVO-A was deleted (ovo^ovo-ΔAP, tagging OVO-B). Staining in adult ovaries of these different alleles revealed the presence of nuclear OVO in the germarium and differentiating germ cells of the adult ovaries. ovo^ovo-Cterm had a persistent staining pattern from germline stem cells throughout developing egg chambers, while ovo^ovo-Nterm showed a weaker staining intensity in the germline stem cells but stronger in region 2a of the germarium and stage 4 egg chambers. ovo^ovo-ΔAP replicated staining patterns of ovo^ovo-Nterm. ovo^ovo-A appears to have a decreased staining intensity than the other alleles but follow the same staining pattern as ovo^ovo-Nterm. We also looked at the expression of OVO in the adult male germline and were able to detect the presence of OVO in ovo^ovo-Nterm and ovo^ovo-Cterm in germ cells near the hub, but staining was lost in differentiating spermatogonia, while ovo^ovo-A staining was not detectable. Immunoblotting analysis of the alleles show that ovo^ovo-Nterm and ovo^ovo-ΔAP have identical banding patterns, but the ovo^ovo-Cterm had different protein bands, while ovo^ovo-A protein was not detectable. The localization and banding patterns between ovo^ovo-Cterm and ovo^ovo-Nterm suggest that OVO may be regulated at the protein level. We are continuing to determine the expression pattern of these alleles during embryogenesis and larval development in males and females to gain a better insight into ovo expression and regulation.