Date of Award

6-13-2025

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Biology

First Advisor

Jennifer Mierisch

Second Advisor

Rodney Dale; Terry Grande; Thomas Sanger

Abstract

The Notch signaling pathway plays a significant role in gonad development and spermatogenesis, but little is known about its specific targets in the testis. The Drosophila testis contains two populations of stem cells: germline stem cells and cyst stem cells, which give rise to differentiating germline and somatic cyst cells, respectively. Notch signaling is activated in the somatic cyst cells by the Delta ligand from the germline in a region of the testes coined the “transition zone”, where somatic cyst cells progress from early cyst cells surrounding gonialblasts and 2-cell germline cysts to late cyst cells surrounding 16-cell spermatocyte cysts. We have found that increased Notch signaling in somatic cyst cells prevents their complete transition from early to late cyst cell fate, leading to an arrest in late spermatogenesis and sterility. These results suggest Notch signaling is required to enter the transition state, but too much Notch signaling inhibits a complete transition to the late cyst cell fate. To explore the downstream effectors through which Notch mediates these effects, RNA-sequencing was performed on testes overexpressing activated Notch and 1535 potential Notch targets were identified. Through genetics and gene expression analysis, we identified two members of the Enhancer of Split Complex (E(spl)m3-bHLH and E(spl)m-bHLH) and groucho (gro) as downstream targets of the Notch signaling pathway in spermatogenesis. To investigate the direct effects of gro activation in the testes, we expressed overactive Gro protein in the somatic cyst cells using the Gal4/UAS system. Overexpression of activated Gro resulted in a decrease of cells in the transition state and a delayed onset of the transition zone when compared to the control. These results suggest too much Gro results in continued repression of Notch targets in somatic cells, preventing the transition of somatic cells from early to late cyst cell fate. Knocking down E(spl)m3-bHLH and E(spl) E(spl)m-bHLH also causes a delayed onset of the transition zone, suggesting that Notch activation of these targets is required to promote the transition state. Our results support a model in which Notch signaling is required for the transition state but must be turned off for somatic cells to completely transition to the late cyst cell fate to promote the late stages of sperm development.

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