Investigating the coordination of germline stem cell fate by ecdysone signaling and BMP signaling in the Drosophila ovary

Abstract

Germline stem cells (GSCs) receive multiple secreted signals that regulate their fate. How these signals are integrated to balance self-renewal with differentiation are not well understood. Here, we use the Drosophila ovary as a model to understand how signal convergence is achieved. Ecdysone signaling and Bone Morphogenetic Protein (BMP) signaling are well-known signals that promote GSC self-renewal. Over-expression of Ecdysone Receptor (EcR), knock-down of bag of marbles (bam), or over-expression of constitutively active thickveins (tkv) in all germ cells results in germ cell tumors at similar stages of differentiation. This suggests that ecdysone signaling and BMP signaling converge on a common point of regulation. We perfomed single-cell RNA sequencing (scRNAseq) to assess the potential transcriptomic differences between the three tumor genotypes. UMAP distribution and differential gene expression show that the over-expression of EcR.B1 and constitutively active tkv (TkvACT) yield undifferentiated cells that are more similar to each other than they are to the bam-depleted cells. Knock-down of BMP signaling in the EcR.B1 over-expression or knock-down of ecdysone signaling in TkvACT over-expression did not rescue differentiation. This data suggests that a direct interaction between ecdysone signaling and BMP signaling is unlikely, and that the two pathways likely function indirectly by converging on common transcriptional targets to regulate GSC maintenance and differentiation. We identified a subset of putative common targets, including ecdysone inducible protein 63E (Eip63E or E63E), because their expression is upregulated in response to EcR.B1 or TkvACT over-expression. We performed other bioinformatic analyses, such as Single Cell rEgulatory Network Inference and Cluster (SCENIC), pseudotime analysis, and single cell velocity (scVelo) to help identify other potential common targets of ecdysone signaling and BMP signaling. Our results suggest that ecdysone signaling and BMP signaling converge on a common point of regulation to coordinate GSC fate.