Cell competition (CC) is a universal, quality control process found across the taxonomic spectrum in which neighboring or interacting cells compare their relative fitness (e.g., growth rates.) In this process the “winner” cells contribute to the further development of the organism or tissue, while suboptimal cells, or “losers,” are outcompeted. This process has been studied in organisms ranging from fungi to humans, and has both developmental and disease relevance. For example, during cancer development, neoplastic cells can function as super-competitors in a tissue, outcompeting wild-type cells and driving tumor growth.

Although there is intense interest, the molecular mechanisms that underlie this process are not well understood. One potential candidate pathway shown to be involved in tumorigenesis is the Notch Pathway. Recent studies have shown that differentially elevated expression of the Notch ligand Jagged is associated with heightened invasiveness and motility of cancer cells as well as increased tumor development. Here we utilize a novel in vivo system that allows us to study individual stem cells during a migration and niche lodgment process. The unique biology of the colonial tunicate, Botryllus schlosseri, has germline stem cells (GSCs) with genetically determined competitive phenotypes; when GSCs of two individuals are mixed, one genotype will outcompete the other, solely contributing to the germline of subsequent generations. Termed Germ Cell Parasitism (GCP) this clonal dominance is heritable, stable, and reproducible, providing a tractable system in which to study cell competition.

Here we show a potential mechanism for GCP and cell competition in which GCP is partially due to differential homing of winners and losers to the germline niche and partially due to direct interaction between winner/loser cells and with the niche itself. We show that up-regulation of Delta and Jagged occurs in the winner GSCs, providing further support for the role of the Notch pathway in the process. Moreover, live-imaging of transplanted GSCs from winner/loser genotypes reveal that winner and loser GSCs both arrive in the developing germline niche, further suggesting a role for direct interaction between winner/loser GSCs within the niche. Based on these results, we propose that the basis of GSC competition resides in a combination of variation in homing ability and cell competition for niche occupancy controlled by differential expression of the Notch Pathway components.

Background

Results

Conclusions

Examples of metazoan regeneration can be seen within all animal phyla; however, the ability to restore lost tissues upon injury varies greatly. Some species can only partially replace tissues and organs, while others can repeatedly regenerate entire limbs, severed spinal cords, and portions of their brain. There are also extraordinary cases of whole-body regeneration (WBR) where animals can produce entire bodies from small fragments of leftover tissue after injury. A few species of botryllids can even utilize circulating blood borne stem cells for WBR. These animals are in the subphylum tunicata, and evolutionarily classified alongside vertebrata in the phylum Chordata. Botryllids are marine invertebrates that grow by repeated rounds of asexual reproduction to form a colony of clonally derived individuals called zooids. Under normal conditions, zooids are regenerated de novo on a weekly basis, and a colony increases in size by expanding the number of individuals. Two distinct processes that differ by the source of new bodies are responsible for zooid regeneration in the botryllids. In the first, called blastogenesis, new zooids arise from a multipotent epithelium of a pre-existing zooid. In the second, termed vascular budding (VB) or WBR, mobile cells in the vasculature are the source of the new zooid. In some botryllid species, blastogenesis and VB occur concurrently. In others, blastogenesis is exclusively used for expansion while WBR only occurs following injury or exit from dormancy. We studied WBR in two related species: Botryllus schlosseri and Botrylloides diegensis. Both have an extracorporeal vasculature that develops outside the bodies to connect all individuals within a colony. We isolated blood vessels to induce WBR by removing all zooids, primary buds, and secondary buds. This surgery was performed under a dissecting microscope with scalpels and extra-fine tip forceps to prevent damage to the vasculature. Regeneration was then analyzed using time-lapse microscopy with the animal being maintained in a temperature-controlled seawater recirculation basin. We compared and characterized early stages of WBR utilizing these days-long videos. Our data suggest that unlike other botryllid species, new zooid growth following injury in B. schlosseri is not due to WBR, but instead through ectopic development of tissues leftover from the blastogenic process. Despite these differences, we did find a common theme in the two species: developmental competition results in only a single zooid reaching maturity. We utilized this phenomenon to control the number of buds and their spatial orientation in B. schlosseri and found that competition is reversible and mediated by circulating factors and/or cells. This represents a new model to study resource allocation and competition within an individual.

Age-related macular degeneration (AMD) is the leading cause of blindness in people over the age of 50 in the western world. Nearly 2 million people in the U.S. were affected in 2010, and as life expectancy increases, the number of people with AMD will also increase. By the year 2030, it is estimated that over 3 million people in the U.S. alone will be diagnosed with AMD. Although early signs of AMD can be identified easily by a fundus photograph taken by an ophthalmologist, little can be done to cure the disease. Once AMD progresses to advanced stages, irreversible central vision loss can occur, eventually resulting in blindness. In addition to the effect on quality of life, the economic burden of AMD is estimated to be over $340 billion globally. Early AMD can progress into two advanced stages termed wet or dry AMD based on the presence of choroidal neovascularization. Although wet and dry AMD are clinically distinct, they both are triggered by the degeneration of the retinal pigmented epithelium in the macula, the area responsible for central vision. Retinal pigmented epithelial (RPE) cells are a cuboidal, polarized, and highly pigmented monolayer located between the visual photoreceptors and the vascular choroid. They are an essential cell type in the retina as they provide nutrients, support the visual cycle, and remove waste and fluids from the retina, among other functions. Degeneration and the failure of this single layer of cells to repair can have devastating impacts on retinal function and can ultimately result in the death of the overlaying photoreceptors. The pathogenesis driving AMD is not entirely clear, but numerous risk factors have been identified including genetic variants and environmental stressors, but the number one risk factor is age. The lack of treatments for early and advanced stages of AMD can in part be explained by the lack of appropriate cell culture and animal models which recapitulate the progression of the disease. My Ph.D. research focuses on the development of novel in vitro wounding platforms to analyze the innate ability of human RPE to repair. In chapter II, we discuss the development of a platform to induce a state of chronic wounding in RPE monolayers. Chronic wounding resulted in several AMD phenotypes including enlarged cell size and multinucleation, along with an increased inflammatory response. In chapter III, we examine a large macula-sized wounding platform. In this system, RPE cells failed to adequately repair, resulting in loss of cuboidal morphology, loss of pigmentation, and regions of RPE atrophy. Finally, in chapter IV, we use a transcriptomic approach to analyze age-related changes in RPE and how it may affect the ability of RPE to repair wounds in the monolayer. Taken together, the work presented here further our knowledge of RPE wound repair and may also allow for the identification of therapeutics which can improve the ability of RPE to repair.

Background

Results

Conclusions