Investigating how Notch, and JAK/STAT Signaling Synergistically Regulate Intestinal Stem Cell Homeostasis Using Engineered Optogenetic Alleles

Abstract

Stem cells are a class of undifferentiated cells that have the unique ability to give rise to a variety of specialized cell types during tissue development and growth. These cells communicate amongst one another by sending and receiving signals from multiple pathways that regulate cell fate decisions by promoting either self-renewal or differentiation. The Notch, and JAK/STAT signaling pathways are central regulators of multicellular development and are vital for tissue maintenance. The interplay between the Notch, and JAK/STAT pathways required for tissue homeostasis has not been fully elucidated, particularly as it relates to the intestinal epithelium. I utilize the Drosophila melanogaster midgut as a model system to study stem cell dynamics and more specifically, how Notch and JAK/STAT signaling cooperatively regulate intestinal stem cell turnover. In order to accomplish this, I employed both traditional and optogenetic methods to elicit Notch blockade and ectopic activation of JAK/STAT signaling in the midgut. First, I demonstrated that ectopic JAK/STAT signaling paired with Notch knockdown causes substantial ISC overproliferation, leading to the formation of large ISC tumors spanning the entire midgut. Quantitative assessment of ISC pools confirmed that Notch and JAK/STAT signaling work in a synergistic manner, rather than an additive manner, to regulate ISC homeostasis. I also utilized two optogenetic alleles, Opto-Delta and Opto-Domeless, to recapitulate Notch blockade and JAK/STAT hyperactivation using light. Opto-Delta was tested in the intestinal epithelium and demonstrated the ability to block Notch signaling in the ISCs, resulting in the formation of stem cell clusters. Subsequently, an Opto-Domeless construct was created and expressed in the nervous system during embryogenesis to assess its efficacy to activate JAK/STAT signaling. The embryos that expressed Opto-Domeless exhibited a significant decrease in viability when subjected to the light condition, suggesting possible light-responsiveness. Expression of Opto-Domeless in the midgut was also achieved, however, ISCs expressing Opto-Domeless appeared to have JAK/STAT activity in the absence of light exposure, suggesting that Opto-Domeless is not behaving in a binary fashion and will require further validation. Altogether, these findings reveal great potential for light-gated proteins, as they provide a powerful approach to disentangle dynamic cellular signals by using light.