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Abstract

Regenerative organs maintain a dynamic equilibrium throughout the life of the organism by resisting and repairing damage from both internal and external threats. This balance is achieved by replacing lost or damaged cells through a process primarily mediated by stem cells, which are capable of self-renewal to maintain their pool and differentiate to form specialized cell types. The decision to either divide or differentiate is tightly regulated by a genetic network, disruption of which can trigger uncontrolled stem cell proliferation and differentiation, leading to the formation of large tumors. While several studies have used Drosophila melanogaster to explore the roles of Notch, JNK, EGFR, and Wnt signaling pathways in regulating stem cell function,many genes that interact with these pathways remain unidentified. In this study, I generated tumor models of the Drosophila midgut and malpighian tubules to identify and characterize novel genes involved in stem cell regulation.

Using the CRISPR/Cas9 system, I developed a method to introduce mutations in the progenitor cells of the midgut. With this approach, I generated a Notch loss-of-function midgut tumor model, characterized by increased stem cell proliferation and secretory enteroendocrine cells. Through single-cell RNA sequencing (scRNA seq), I identified several differentially expressed genes, from which I found 28 genes whose knockdown reduced tumor growth and proliferation. I further showed that one of the candidate genes, nerfin-1, is required for the proliferation of stem cells during ApcRNAi;RasV12-mediated midgut tumorigenesis, infection-induced regeneration, and homeostasis. From the scRNA seq dataset, I also observed that Notch loss-of-function tumors activated the Toll signaling pathway in the midgut. I demonstrated that active Toll signaling is required for promoting the proliferation of both the Notch loss-of-function and ApcRNAi;RasV12 tumors in the midgut. Additionally, I showed that genetic activation of the Toll signaling pathway in progenitor cells was sufficient to trigger the rapid proliferation of stem cells via the JNK signaling pathway. I also demonstrated that the activation of the Toll signaling pathway in enterocytes serves as a niche signal, that drives stem cell proliferation in the midgut.

Lastly, I generated a renal cell carcinoma fly tumor model by introducing ApcRNAi;RasV12 in renal stem cells of the malpighian tubules. I showed that tumor-bearing malpighian tubules activate the Wnt, Pi3K/Akt/mTor, and Raf/MEK/ERK signaling pathways, all of which are essential for tumor proliferation. Additionally, I identified five genes whose knockdown reduced tumor proliferation in the malpighian tubules, the human homologs of which are upregulated in renal cell carcinoma patients.

In summary, my research demonstrates that Drosophila melanogaster is a valuable model, which can be used to decode the genetic networks that regulate stem cell function. By generating tumor models in the midgut and malpighian tubules, identifying transcriptomic changes, and characterizing gene function, my work lays the groundwork for further research in flies and higher model organisms, including humans.