Identifying Stem Cells and their Niche

Niche ModelAlthough adult stem cells are believed to reside in distinct microenvironments, or niches, that function to regulate stem cell behavior, niches have been hard to study because of the difficulty of precisely identifying the stem cells in most tissues.  However, we have developed a set of criteria that facilitates reliable identification of the epithelial follicle stem cells (FSCs) in the Drosophila ovary and we are now mapping their interactions with neighboring cells to better understand the nature of the FSC niche.  Surprisingly, we have found that the FSC niche appears much more dynamic than the few previously characterized niches.  That is, it does not have a fixed position within the tissue and at least some cellular components of the niche turn over regularly during adult life. 

We are also using lineage analysis to follow FSC behavior, track the patterns of FSC daughter cell migration and differentiation, and investigate relevant gene function.  We found that the follicular epithelium surrounding each new follicle is produced by exactly two stem cells that reside in defined positions at the anterior edge of the tissue.  Newly formed germ cells pass by the FSC niches one at a time and FSC divisions are coordinated with this process so that, on average, two FSC daughters contribute to each newly formed follicle.  Interestingly, approximately half of the FSC daughters exhibit a directed migration between FSC niches.  These migrations are a normal part of the process of enveloping incoming germ cells but also serve the important function of providing replacement stem cells when an FSC is lost from the niche.  It appears that interniche migrating stem cells regularly contact the stem cell in the opposite niche and compete with the resident stem cell for niche occupancy.

Epithelial Stem Cell Genetics

Niche ModelThe wnt/wingless, hedgehog, BMP and Notch signaling pathways are all important for FSC function and early epithelial development but little is known about where in the process these signals exert their effects or how they are coordinated to produce a functional, healthy epithelium.  We are investigating the function of key signaling components at specific steps in early follicle formation to map the contributions of these pathways to FSC function and follicle formation. 

In addition, we are interested in the role that misregulation of signaling in the ovarian epithelial cells plays in ovarian cancer.  Oncogenic mutations in p53, K-Ras, and PI3K and wnt signal pathway components are common in ovarian cancer tumors.  Drosophila have homologs of these and other ovarian cancer genes and, like in the mammalian ovary, upregulation of wnt signal in follicle cells leads to hyperplasia.  We are now investigating the interaction between the wnt pathway and other putative ovarian cancer genes in follicle cells as well as screening for markers that identify pre-tumorous cells to better understand the early steps leading to hyperplasia.  Through collaboration with our colleagues in the Center for Reproductive Sciences, we will be able to test whether gene interactions and markers that we identify in the fly ovary will also be present in mammalian model systems and human tissues.

To identify additional genes that are required for proper FSC function, we screened through a collection of over 600 lines bearing lethal mutations.  We have identified several mutants with a follicle stem cell phenotype including ones that accelerate the rate of stem cell loss; confer a “hyper-competitive” stem cell replacement phenotype; and/or cause over-proliferation, perhaps modeling a precancerous state.  

Epithelial Stem Cells and Oogenesis

Oogenesis is well conserved from flies to mammals and studies of the Drosophila ovary have provided valuable insight into common germ cell features such as cyst structure, ring canal and fusome composition, and Balbiani body function.  Somatic cell features have also been modeled in the fly, such as the production of follicle maturation signals by epithelial cells, and the epithelial-to-mesenchymal transition, which is a normal part of follicle development in flies and a characteristic of epithelial ovarian cancer in mammals.  Thus, the fly ovary can be an informative model of both normal and pathological processes in the ovary. 

Our work on the characterization of the follicle stem cells and their associated niche provides an opportunity to use the fly ovary as a model of follicle formation.  By studying the lineage just downstream of the FSCs, we found that FSCs produce “pre-follicle cells” that are developmental intermediates between the FSCs and the polarized epithelium.  Unlike FSCs, they do not remain in the niche and typically do not contribute cells to more than one follicle.  However, the expression status in pre-follicle cells of several markers reflects the FSC state.  That is, they express markers found in the FSCs but not in the polarized epithelial cells, and do not express other markers that are absent in the FSCs and upregulated in the polarized epithelium.  Interestingly, these cells are capable of differentiating into any of the three major follicle cell types in the early FSC lineage through a process that is largely dependent on spatial cues rather than lineage relationships. 

Follicle formation begins when a newly formed germline cyst moves past the FSC niche and contacts these pre-follicle cells.  We found that a Delta signal from the germline activates Notch in some pre-follicle cells, inducing them to migrate along the anterior fact of the cyst toward the opposite niche.  Other pre-follicle cells that do not receive the signal instead migrate away from the niche toward the posterior, directly into the polarized epithelium.  Thus, this signal sets up an asymmetry among pre-follicle cells that is probably important for ensuring proper distribution of pre-follicle cells across the cyst surface and positioning them to receive the appropriate downstream cues for differentiation.  In addition, notch-mediated interniche migration is required for interniche competition and stem cell replacement.