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Genetic analysis of signal transduction regulation in Drosophila melanogaster

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Proper cell growth and differentiation requires the integration of multiple signaling pathways that are maintained by various post-translational modifications. Many proteins in signal transduction pathways are conserved between humans and model organisms. My dissertation characterizes four previously unknown manners of

Proper cell growth and differentiation requires the integration of multiple signaling pathways that are maintained by various post-translational modifications. Many proteins in signal transduction pathways are conserved between humans and model organisms. My dissertation characterizes four previously unknown manners of regulation in the Drosophila Decapentaplegic (Dpp) pathway, a pathway within TGF-beta family. First, I present data that the Dpp signal transducer, Mothers Against Dpp (Mad), is phosphorylated by Zeste-white 3 (Zw3), a kinase involved in the Wingless pathway. This phosphorylation event occurs independently of canonical phosphorylation of Mad by the Dpp receptor. Using ectopic expression of different alleles of Mad, I show that Zw3 phosphorylation of Mad occurs during the cell cycle in pro-neuronal cells and the loss of phosphorylation of Mad by Zw3 results in ectopic neuronal cells. Thus, Mad phosphorylation by Zw3 is necessary for cell cycle control in pro-neuronal cells. Second, I have shown that the regulator dSno, which has previously been shown to be a TGF-beta antagonist and agonist, is also a Wingless pathway antagonist. Loss of function flip-out clones and ectopic expression of dSno both resulted in changes of Wingless signaling. Further analysis revealed that dSno acts at or below the level of Armadillo (Arm) to inhibit target gene expression. Third, I have demonstrated that the protein Bonus, which is known to be involved in chromatin modification, is required in dorsal-ventral patterning. Further experiments discovered that the chromatin modifier is not only a necessary Dpp agonist, but it is also necessary for nuclear localization of Dorsal during Toll signaling. Last, I showed that longitudinal lacking-like (lola-like) is also required in dorsal-ventral patterning. The loss of maternally expressed lola-like prevents dpp transcription. This shows that lola-like is integral in the Dpp pathway. The study of these four proteins integrates different signaling pathways, demonstrating that the process of development is a web of connections rather than a linear pathway.

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Created

Date Created
2014

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Comparative genomics and novel bioinformatics methodology applied to the green anole reveal unique sex chromosome evolution

Description

In species with highly heteromorphic sex chromosomes, the degradation of one of the sex chromosomes can result in unequal gene expression between the sexes (e.g., between XX females and XY males) and between the sex chromosomes and the autosomes. Dosage

In species with highly heteromorphic sex chromosomes, the degradation of one of the sex chromosomes can result in unequal gene expression between the sexes (e.g., between XX females and XY males) and between the sex chromosomes and the autosomes. Dosage compensation is a process whereby genes on the sex chromosomes achieve equal gene expression which prevents deleterious side effects from having too much or too little expression of genes on sex chromsomes. The green anole is part of a group of species that recently underwent an adaptive radiation. The green anole has XX/XY sex determination, but the content of the X chromosome and its evolution have not been described. Given its status as a model species, better understanding the green anole genome could reveal insights into other species. Genomic analyses are crucial for a comprehensive picture of sex chromosome differentiation and dosage compensation, in addition to understanding speciation.

In order to address this, multiple comparative genomics and bioinformatics analyses were conducted to elucidate patterns of evolution in the green anole and across multiple anole species. Comparative genomics analyses were used to infer additional X-linked loci in the green anole, RNAseq data from male and female samples were anayzed to quantify patterns of sex-biased gene expression across the genome, and the extent of dosage compensation on the anole X chromosome was characterized, providing evidence that the sex chromosomes in the green anole are dosage compensated.

In addition, X-linked genes have a lower ratio of nonsynonymous to synonymous substitution rates than the autosomes when compared to other Anolis species, and pairwise rates of evolution in genes across the anole genome were analyzed. To conduct this analysis a new pipeline was created for filtering alignments and performing batch calculations for whole genome coding sequences. This pipeline has been made publicly available.

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Created

Date Created
2016

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Drosophila CORL phenotypes connect mating, longevity, and insulin signaling

Description

Drosophila CORL (dCORL) is a central nervous system (CNS)-specific gene that is hypothesized to function in Transforming Growth Factor β signaling. It is part of the Corl multigene family that includes mouse and human homologs. dCORL is necessary for Ecdysone

Drosophila CORL (dCORL) is a central nervous system (CNS)-specific gene that is hypothesized to function in Transforming Growth Factor β signaling. It is part of the Corl multigene family that includes mouse and human homologs. dCORL is necessary for Ecdysone Receptor isoform B1 (EcR-B1) protein expression in the mushroom body, a brain region responsible for learning and memory. Beyond this, dCORL function is unknown. As dCORL expression is restricted to the CNS, co-expression experiments were performed to identify dCORL-specific neurons. In these experiments, EcR-B1 protein expression was compared to dCORL mRNA expression revealing that they are not expressed in the same cells. Therefore, EcR-B1 is regulated non-autonomously by dCORL. Co-expression analyses were also conducted utilizing dCORL reporters. For example, the reporter AH-lacZ was co-stained with two pars intercerebralis (PI) markers: Drifter (Dfr; a transcription factor found in the nucleus) and Drosophila insulin-like peptide 2 (dILP2; a peptide present in the neurosecretory cells of the pars intercerebralis [PI].) The results showed that there was complete AH-lacZ co-expression with dILP2 in third instar larval and adult brains. Previous work in our lab on dCORL mutant (Df(4)dCORL) adult longevity showed a connection between mating and increased lifespan; mated mutant females had doubled lifespans compared to virgins. Given the published relationship between insulin and longevity, I hypothesized an association between insulin, dCORL, and mating. Df(4)dCORL mutants were used to analyze the effects of dCORL loss-of-function on dILP2. There was a reduction in the number of dILP2-expressing cells in mutants compared to wild type. In wild type larval and adult PI’s, most dILP2-positive neurons also expressed Dfr. Whereas in adult virgin mutants, all dILP2 neurons were Dfr-positive. Both 3-day and 15-day old mated females showed increased dILP2 cell numbers compared to virgin mutants. In these sets of dILP2 cells only a subset expressed Dfr as in wild type. The mutant phenotypes of mated flies showed partial rescue compared to virgins. This led to the conclusion there were associations between mating, longevity, and insulin signaling through dCORL. Homology between Drosophila and mammalian Corl proteins imply these connections may be seen in mammals.

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Agent

Created

Date Created
2018

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Evolutionary Genetics of CORL Proteins

Description

Transgenic experiments in Drosophila have proven to be a useful tool aiding in the

determination of mammalian protein function. A CNS specific protein, dCORL is a

member of the Sno/Ski family. Sno acts as a switch between Dpp/dActivin signaling.

dCORL is involved in

Transgenic experiments in Drosophila have proven to be a useful tool aiding in the

determination of mammalian protein function. A CNS specific protein, dCORL is a

member of the Sno/Ski family. Sno acts as a switch between Dpp/dActivin signaling.

dCORL is involved in Dpp and dActivin signaling, but the two homologous mCORL

protein functions are unknown. Conducting transgenic experiments in the adult wings,

and third instar larval brains using mCORL1, mCORL2 and dCORL are used to provide

insight into the function of these proteins. These experiments show mCORL1 has a

different function from mCORL2 and dCORL when expressed in Drosophila. mCORL2

and dCORL have functional similarities that are likely conserved. Six amino acid

substitutions between mCORL1 and mCORL2/dCORL may be the reason for the

functional difference. The evolutionary implications of this research suggest the

conservation of a switch between Dpp/dActivin signaling that predates the divergence of

arthropods and vertebrates.

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Agent

Created

Date Created
2019