Browsing by Author "Fox, Donald T"

In order to maintain genome integrity cells employ a set of well conserved DNA damage checkpoints. DNA damage checkpoints are active during interphase and serve to prevent mitosis with broken DNA. Mitosis with broken DNA is associated with DNA segregation errors, genome instability and even cell death in resulting daughter cells. It has recently has been appreciated that cells can compensate for damaged DNA during mitosis. However, little is known about this mitotic DNA damage response.

In this work, I have utilized a genetically tractable system to study mitotic DNA damage responses in Drosophila. During development, Drosophila rectal papillar cells undergo developmentally programmed inactivation of DNA damage responses. Following inactivation, papillar cells undergo two rounds of mitosis. We find that papillar cells fail to undergo cell death or high-fidelity DNA repair prior to mitosis and instead enter mitosis with DNA double stranded breaks (DSBs). Remarkably, papillar cells segregate acentric DNA fragments into daughter cells during mitosis resulting in viable daughter cells, normal organ development and function. Proper segregation and organ formation is dependent on the FANCONI Anemia gene FANCD2. Loss of FANCD2 results in unaligned acentric fragments and mis-segregation of broken DNA resulting acentric micronuclei formation. Mis-segregation of acentric DNA results in cell death and failure to form a developmentally normal and functional organ. Thus, we have uncovered a role for FANCD2 in mitotic DNA damage responses.

Additionally, we find that single-stranded DNA (ssDNA) is present during papillar cell mitosis following DNA DSB induction. ssDNA is present on both the edge of segregating and lagging DNA as well as spanning short regions between fragments of lagging DNA. The observation that ssDNA is present suggests that while papillar cells do not initiate complete repair, some level of DNA resection must occur following DNA DSB induction. In line with this reasoning, we find a role for the DNA damage sensor complex, the MRN complex, in papillar cell survival following I-Cre induction. The MRN complex consists of three components, Mre11, Rad50 and NBS1. Loss of Mre11 or NBS1 results in reduced papillar cell survival following I-Cre induction. Furthermore, Mre11 is a nuclease. Thus, we propose that MRE11 acts at sites of DNA DSBs in papillar cells to create ssDNA. We hypothesize that formation of ssDNA is sufficient to form a DNA/protein bridge between segregating and lagging DNA to enable proper DNA segregation. Interestingly, resistance to DNA damage is also observed in many cancers. We speculate that such DNA damage resistant cancer cells may utilize similar mechanisms to compensate for DNA breaks during mitosis.

Endopolyploid cells (hereafter - polyploid cells), which contain whole genome duplications in an otherwise diploid organism, play vital roles in development and physiology of diverse organs such as our heart and liver. Polyploidy is also observed with high frequency in many tumors, and division of such cells frequently creates aneuploidy (chromosomal imbalances), a hallmark of cancer. Despite its frequent occurrence and association with aneuploidy, little is known about the specific role that polyploidy plays in diverse contexts. Using a new model tissue, the Drosophila rectal papilla, we sought to uncover connections between polyploidy and aneuploidy during organ development. Our lab previously discovered that the papillar cells of the Drosophila hindgut undergo developmentally programmed polyploid cell divisions, and that these polyploid cell divisions are highly error-prone. Time-lapse studies of polyploid mitosis revealed that the papillar cells undergo a high percentage of tripolar anaphase, which causes extreme aneuploidy. Despite this massive chromosome imbalance, we found the tripolar daughter cells are viable and support normal organ development and function, suggesting acquiring extra genome sets enables a cell to tolerate the genomic alterations incurred by aneuploidy. We further extended these findings by seeking mechanisms by which the papillar cells tolerated this resultant aneuploidy.

Codon usage bias is a fundamental aspect of the genetic code. For many years synonymous mutations to a coding sequence were considered to be functionally “silent.” We now appreciate that is not the case and that synonymous codon choice can have drastic implications for gene expression and protein production. A major debate in the field remains whether codon usage bias is evolutionarily selected for to drive efficient translation in a tissue-specific manner. Here we perform an organism wide screen in Drosophila using codon modified reporters to reveal tissue-specific responses to codon usage bias. We uncover a strict limit on rare codon usage for protein expression, and this limit coincides with the rareness limit of endogenous genes in Drosophila. We find that rare codon usage near the edge of this limit is sufficient to impart tissue-specific gene expression, notably in the testis and brain. We define a new codon usage metric, the tissue-apparent Codon Adaptation Index (taCAI) to reveal a conserved enrichment in rare codons in endogenous testis genes of both flies and humans. We further demonstrate that rare codons in the evolutionarily young gene, RpL10Aa, are required for female fertility.

As development progresses, many tissues lose their ability to regenerate via cellular proliferation. In some tissues, including the human heart and kidneys, injury activates a non-proliferative cellular response known as hypertrophy- an increase in cell size to restore lost organ mass. Although the connection between development and injury response is often observed, little is known about the developmental signals that terminate and switch injury responses. Moreover, the role of non-proliferative injury responses in restoring function to recovering tissues remains unexplored.

In this dissertation, I identify the Drosophila hindgut pylorus, an intestinal valve, as a new model to study the developmental regulation of injury responses. By using Drosophila genetics and developing a new method for site-specific genetic ablation, I discovered that the Drosophila pylorus switches its injury responses during tissue development. Injury to the larval pylorus results in accelerated mitotic cycles while injury to the adult pylorus leads to endocycles (DNA replication without division) and hypertrophy. My work identifies developmental hormones and transcription factors that act to regulate the injury response switch through control of fizzy-related, an evolutionary-conserved mitotic inhibitor. Last, I found that under chronic growth conditions, endocycles and hypertrophy protect the pyloric epithelial barrier function. Together my work explores both regulation and function of a developmental injury response switch.

Mitosis involves the faithful segregation of two identical copies of chromosomes into two daughter cells. This process is highly regulated to maintain genome integrity, as mis-segregation of partial or whole chromosomes can lead to genomic instability. Cells are constantly exposed to both endogenous and exogenous forms of DNA damage, which if left unattended to, can contribute to mitotic errors. Cells therefore possess DNA damage responses (DDRs) which involves enacting cell cycle checkpoints, DNA damage repair, and in cases of extreme damage – cell death or senescence.While several lines of investigation have identified key mechanisms of the DDR during interphase of the cell cycle, there are several key questions that remain with regards to how cells deal with damage that persists into mitosis. Further, there is currently a gap in knowledge on the mechanisms, timing, and conditions in which different aspects of the DDR are active and coordinated. In this dissertation, I will demonstrate how I implemented genetic and imaging tools using our laboratory’s previously established model system, Drosophila rectal papillar cells [hereafter papillar cells]. Using this model, I studied (1) mechanisms of the DDR during mitosis, (2) mechanisms that act in the absence of key DDR components, and (3) novel regulators and protein-protein interactions of the mitotic DDR. This body of work contributes to the growing knowledge of how cells tolerate DNA damage that persists into mitosis.

A fundamental question of biology is how tissues are organized. Tissues can be composed of many small cells or comparatively fewer large cells that add nuclear content to facilitate tissue growth. The cells can be separate, discrete units or interconnected collectives. The nuclear composition of a tissue has functional consequences from the tissue physiology to likelihood of cancers and hyperproliferation to the response to stress and tissue damage. These two decisions, to be small or large and to be distinct (mononucleated) or joined (multinucleated), and, especially, the interaction between these choices are poorly understood. In this dissertation, I identify the Drosophila rectal papillae as a new model to study tissue interconnectivity, multinuclearity, and the interaction between nuclear content and cytoplasm-sharing. I played a major role in the discovery that the adult Drosophila rectal papillae share cytoplasm and proteins up to at least 62 kDa. This sharing is developmentally regulated and requires membrane trafficking and gap junction genes instead of canonical cell-cell fusion or incomplete cytokinesis factors. This mechanism of sharing does not appear to involve plasma membrane breaches, a novel way for tissues to share contents. Additionally, I advance the Drosophila larval heart as a model to study nuclear content (ploidy) in heart development and physiology. Together, my work explores how tissues use mononucleate and multinucleate ploidy in development and physiology.

Polyploid cells are generated through a cell cycle variant termed the endocycle. Endocycling cells undergo multiple rounds of genome duplication without an intervening mitosis. Endocycling is known to lead to alterations in chromosomes structure that make mitosis “ill advised”, in the words of one review. However, many polyploid cells retain mitotic capacity, both when polyploidy is induced pathologically, and in some developmental contexts. Using two mitotic polyploid cell types in Drosophila melanogaster, I investigated how chromosomes structure is regulated in pathological and developmental endocycles. By combining genetics, live imaging and chromosome cytology I have discovered two phases of chromosome regulation that, together, ensure accurate mitosis in polyploid cells. The first of these occurs during the endocycle when removal of sister chromatid cohesin by pds5, without mitosis, allows for the formation of paired chromatids. We named this process “cohesin disestablishment”. Secondly, during cell division, mad2 controls the length of mitosis which allows time for sister chromatids to separate into pairs. We named this process “Separation Into Recent Sisters” (SIRS). Together, cohesin disestablishment and SIRS, allow the accurate segregation of chromosomes in polyploid mitotic cells.