Germ stem cells self-renew, differentiate, and secure genome transmission to the next generation. Germ stem cells depend on niches, which are specialized microenvironments that instruct stem cell self-renewal. Niche extension is a phenomenon in which the niche cells extend elaborate cellular protrusions that surround stem cells. While niche extension is conserved across organisms, the function and regulators of niche extension remain unknown. An essential component of niches is the basement membrane. Basement membranes are secreted, cell-associated, thin sheets of extracellular matrix that structurally support niches, and can regulate stemness and cell proliferation. Type IV collagen is a major component of basement membranes. Discoidin domain receptors (DDRs), are a subset of receptor tyrosine kinases that are specifically activated by collagen, and often signal through mitogen-activated protein kinase (MAPK) cascades. Misregulation of Type IV collagen and mutations in mammalian DDRs have both been linked to infertility, however, their role in promoting fecundity is unknown. The Caenorhabditis elegans (C. elegans) germline niche is composed of only a single cell, the distal tip cell (DTC), which extends long processes that enwrap and maintain germ stem cells by activating Notch signaling, thus sustaining germ stem cells and fertility. The genetic and visual experimental tractability of C. elegans allows for rigorous identification of mediators of niche extension and their role in fertility. Through an expression and DTC-specific RNAi screen, I found that the C. elegans discoidin domain receptor DDR-2 promotes the formation of type IV collagen aggregates/puncta along DTC niche processes. In the absence of DDR-2, type IV collagen accumulates internally in the DTC niche and niche process extension is dramatically reduced. Based on strong preliminary data, my central hypothesis is that DDR-2 and type IV collagen function in a positive feedback loop to direct the secretion of type IV collagen anchoring puncta, which drives niche extension and expands the germ stem cell pool.
In Aim 1, I will determine how DDR-2 and type IV collagen interact in the DTC to drive niche extension and germline maintenance through site of action studies, genetic analysis, live cell imaging of vesicular trafficking, and fertility assays.
In Aim 2, I will determine the downstream effectors of DDR-2 in DTC extension using genetic epistasis analysis and cell biological studies. Through preliminary findings of a large scale RNAi screen, I will focus on the role of MAPK/ extracellular signal-regulated kinases (ERK) signaling and vesicular trafficking regulators. Ultimately I expect my studies will establish a new role for discoidin domain receptor function in germ line niche formation and fertility, which will help in our understanding and treatment of fertility disorders in humans. !
Misregulation of germ stem cells is one of the main causes of infertility. The proposed studies will determine the role of a conserved cell-matrix receptor, the discoidin domain receptor, in forming the germ stem cell niche, a protective specialized environment that maintains germ stem cells. Elucidating how germ stem cells are sustained and sheltered, will be crucial in our increasing our understanding reproduction and developing more effective treatments for human fertility. !
