Degree

Doctor of Philosophy (PhD)

Department

Biological Sciences

Document Type

Dissertation

Abstract

This dissertation describes the use of Drosophila neuroblasts (NBs) to model human ribosomopathies; the overall goal is to understand why specific stem cell and progenitor cell populations are the primary targets in nucleolar stress as seen in the ribosomopathies. Chapter 1 provides an overview of relevant literature. Chapter 2 describes nucleolar stress in Drosophila neuroblasts as a model for human ribosomopathies. For this, we induce nucleolar stress by using the UAS-GAL4 system to express RNAi that depletes Nopp140 transcripts, and we also employ homozygous, CRISPR-Cas9-generated Nopp140 gene disruptions with a systemic null phenotype (Nopp140-/-). Embryonic lethality was observed under RNAi depletion of Nopp140 as well as homozygous and heterozygous Nopp140 disruption. Larval lethality occurred at the second instar stage in Nopp140-/- line, similar to the previously generated complete Nopp140 deletion line, KO121. Larval brain development was severely impaired in Nopp140-/- larvae and in larvae that expressed neuron-specific RNAi that depletes Nopp140. The hypoplastic brain phenotype was due to reduction in NB populations as well as the proliferative capacity of the dividing NBs. While the majority of NB lineages in wild-type brains are at S-phase and proliferative at day 3 after larval hatching as indicated by EdU labeling assay, only the Mushroom Body (MB) NBs are at S-phase and proliferative in the Nopp140-/- larval brain. Furthermore, these MB NBs retained fibrillarin within their nucleoli, while fibrillarin redistributed to the nucleoplasm in the surrounding cells. Hence, we conclude that MB NBs are more resilient to nucleolar stress induced by the loss of Nopp140 compared to other neuroblast lineages. This finding strengthens the use of Drosophila neuroblasts as a model for the human ribosomopathies, and we hypothesize that different neuroblast lineages respond variably to nucleolar stress. Chapter 3 describes repeat polymorphisms in the Nopp140 gene that result in two Nopp140 alleles, Nopp140-Long and Nopp140-Short, that differ by exactly 96 bps within the central domain. We provide evidence showing preferential amplification of the Nopp140-Long allele compared to that of the Nopp140-Short allele, which we determined to be a PCR artefact, for reasons that remain unknown. Chapter 4 closes with conclusions and future studies.

Date

3-18-2019

Committee Chair

DiMario, Patrick

Available for download on Thursday, March 26, 2020

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