Student Theses and Dissertations

Amma Asare

Date of Award

2017

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Fuchs Laboratory

Abstract

Balanced growth and differentiation is essential to tissue morphogenesis and homeostasis. How imbalances arise in disease states such as cancers is poorly understood. Loss of differentiation is associated with poorer prognosis in human patients and increasing malignancy in animal models. Here we explore this intersection between growth and differentiation in the context of epidermal development, where populations of stem cells are maintained in careful equilibrium and induced to proliferate and differentiate in response to stimuli such as injury or cyclical growth signals. During development, a naïve epidermis undergoes rapid proliferation and differentiation to form skin containing hair follicles, neurons, immune surveillance populations and melanocytes. The epidermis maintains homeostasis through many phases of development including rapid cytoskeletal dynamics and temporally coupled inductions of protein synthesis. However, it is still poorly understood how this maintenance is coordinated and sustained. After creating a quantitative differentiation assay to fractionate proliferative and differentiating cells from the embryonic mouse epidermis, I used transcriptional profiling to gain a deeper understanding of novel aspects of this process in vivo. By profiling time points across development spanning the naïve and fully competent epidermis I dissected mechanisms essential for establishing and maintaining the differentiated interfollicular epidermis. This map of the transcriptional landscape served as a tool for forming hypotheses about the association between a pathway or molecule of interest and epidermal development. Probing these profiles allowed me to directly assess the correlation between expression levels and known key regulators of epidermal differentiation. With this new understanding of signatures associated with key differentiation steps in the normal epidermis, I explored how genes commonly dysregulated in epithelial tumors may be involved in this developmental differentiation process. I mined my transcriptional profiles to identify overlap with genes reported to be dysregulated in a range of epithelial tumors. I then devised in vivo epidermal RNAi screen to identify which of these genes were candidate regulators of normal epidermal development. In utero lentiviral injection allows for direct manipulation of the developing epidermis and continued embryo development. The goal of the screen was to assess whether when a particular gene is lost during early epidermal development, the resulting epidermal clone is formed normally with respect to differentiation. Using a quantitative differentiation assay in combination with barcoded high throughput sequencing, I revealed how each gene altered differentiation. My screen identified a number of novel target genes likely to regulate individual steps of differentiation or differentiation more globally. The use of a tumor prone TGFbRII conditional knockout mouse line allowed for comparison of differentiation behavior in a more disease relevant setting. The use of wild type embryos implicated surprising new genes as potential regulators of differentiation. Focusing on one unexpected hit, peroxisome-associated protein PEX11b, I found that Pex11b-deficient epidermis fails to differentiate and form a barrier essential for life. Further study revealed mitotic changes associated with Pex11b-deficient basal progenitors including a mitotic delay, during which spindles rotate uncontrollably, perturbing polarized divisions and skewing daughter fates. Probing deeper, we discovered that without PEX11b, peroxisomes function, but fail to segregate properly. Intriguingly, peroxisome localization is directly coupled to mitotic progression, and when peroxisomes are ectopically mis-localized, mitotic abnormalities occur. Together, our findings unveil a hitherto unforeseen role for organelle inheritance in mitosis and spindle alignment, in the choice of daughter progenitors to differentiate or remain stem-like, and in maintaining proper tissue architecture.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

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