Fertilization is a complex process involving several steps, including sperm activation, oocyte maturation, chemotaxis, gamete recognition, and cell fusion. Many of these essential steps are controlled and regulated by intercellular communication between gametes. This thesis work examines the communication events that occur between sperm and oocytes cells that facilitate fertilization. Oocyte meiotic maturation is one example of a step in fertilization in which intercellular signaling between gametes is required. In many species, sperm prepare the oocyte for fertilization by providing signals for meiotic maturation. Oocyte meiotic maturation is defined by the transition between diakinesis and metaphase I and is accompanied by MAP kinase activation, nuclear envelope breakdown, and meiotic spindle assembly. C. elegans sperm signal oocyte meiotic maturation using the major sperm protein (MSP) as a hormone. Interestingly, the MSP also functions as the central cytoskeletal protein required for the amoeboid motility of nematode sperm. The discovery of MSPs signaling role raised the question of how sperm export MSP to signal oocytes at a distance. MSP lacks a hydrophobic leader sequence and C. elegans sperm lack many standard secretory components, such as ribosomes, ER, or Golgi. Using light and electron microscopy we analyzed the mechanism of MSP release from sperm. We demonstrate that sperm bud novel MSP vesicles to signal distant oocytes. These 150-300 nm MSP vesicles contain both an inner and an outer membrane, with MSP sandwiched in between. Budding protrusions from the cell body contain MSP, but not the MSD proteins, which counteract MSP filament assembly, suggesting that MSP may generate the protrusive force for its own vesicular export. MSP vesicles are labile structures that generate long-range MSP gradients for signaling at oocyte and sheath cell surfaces. Both spermatozoa and non-motile spermatids bud MSP vesicles, but their stability and signaling properties differ. Spermatozoa generate a long-range, short-acting signal, whereas spermatids generate a long-acting signal. EM results suggest that differential vesicle stability affects the physical and temporal range of signaling. We hypothesize that the MSP vesicle release mechanism is in itself signal dependent and signals derived from the female animal initiate MSP vesicle release.

The transcription factor Pancreatic and duodenal homeobox 1 (Pdx1) is essential for formation of the pancreas and for glucose-responsive insulin secretion from mature pancreatic beta cells. In humans and mice, heterozygosity for inactivating mutations in the Pdx1 gene confers a high risk of early-onset type II diabetes. Additional genes that have been linked to heritable type II diabetes encode transactivating factors that bind to the upstream promoter region of Pdx1, suggesting that Pdx1 may play a central role in the maintenance of mature beta cell function. These findings prompted close scrutiny of cis-regulatory elements in the Pdx1 promoter, leading to the identification of proximal and distal regions of phylogenetically conserved sequences with enhancer-like activity. In the first part of my dissertation, I present experimental data employing a transgene-based genetic complementation strategy to demonstrate that the distal conserved region of the Pdx1 promoter is required for high-level expression in postnatal duodenal and stomach mucosal epithelia of mice. Reduced expression of Pdx1 in these tissues leads to decreased numbers of specific enteroendocrine cell types. The second part of the dissertation presents evidence that reduced expression of Pdx1 in pancreatic endocrine progenitor cells during embryogenesis results in decreased production of pancreatic beta cells and increased numbers of glucagon- and pancreatic-polypeptide-expressing cells. The third experimental chapter of the dissertation provides evidence that heterozygosity for Pancreatic transcription factor 1a (Ptf1a), which encodes a critical regulator of pancreatic gene expression, alters the timing of endocrine and exocrine pancreatic differentiation during early stages of pancreatic organogenesis in mice. Collectively, these experiments demonstrate that the discrete developmental transitions and specific cell fates that arise during pancreatic organogenesis require different levels of key transcription factors. This information is useful for understanding the regulation of differentiation of diverse daughter cells from common progenitor cell populations in the embryonic pancreas. Future experiments will apply this knowledge to improve techniques for promoting differentiation of cultured stem cells to produce mature pancreatic beta cells.

Kidney development in mammals is characterized by reciprocal tissue interaction between the ureteric bud and the metanephric mesenchyme. The outgrowing epithelial bud invades the overlying mesenchyme, inducing epithelial differentiation of these renal progenitor cells. In turn, signals from the metanephric mesenchyme allow for continued growth and branching of the ureteric bud, which will form the collecting system. This program is regulated largely at the transcriptional level and the co-factor Cited1 has been implicated in regulation of metanephric mesenchyme differentiation. Here we investigate the expression and function of Cited1 in the developing mouse kidney. Cited1 is expressed in the metanephric mesenchyme subsequent to ureteric bud invasion and is restricted to a subset of cells that aggregate tightly around the bud tip. This structure, known as the cap mesenchyme, contains renal progenitor cells and will undergo epithelial differentiation in response to signals from the ureteric bud. As this program is initiated, Cited1 is down regulated, and is absent in early epithelial structures. Despite its unique expression pattern, deletion of Cited1 does not disrupt kidney development. We hypothesized that this was due to redundancy through Cited2 and Cited4, other members of this gene family expressed in the developing kidney. Deletion of either Cited2 or Cited4 alone or in combination with Cited1 did not disrupt the ability of the metanephric mesenchyme to differentiate or the ureteric bud to branch. Though we have a general appreciation that the cap mesenchyme gives rise to nephrons, careful lineage analysis of this progenitor population has not been previously reported. By exploiting the unique expression domain of Cited1, we have generated a transgenic mouse that expresses an inducible Cre recombinase, allowing us to tag cap mesenchyme cells and follow their fate potential. These studies address the fate of the cap mesenchyme in vivo for the first time using a genetic system and define which cell types do and do not arise from this population. In addition, this line will be useful in a broader sense as a powerful tool for conditional gene deletion within the cap mesenchyme.

Each time budding yeast divide they ensure that both the mother and daughter cell inherit a vacuole. Because budding yeast divide asymmetrically by budding, the vacuole must be actively transported into the bud. As the mother cell begins budding, a tubular and vesicular segregation structure forms which is transported into the bud by the myosin V motor, Myo2, bound to the vacuole-specific myosin receptor Vac17. Upon arriving in the bud the segregation structure is resolved to found the daughter vacuole. The mechanism that regulates segregation structure resolution in a spatially dependent manner is unknown. Directionality in vacuole transport is ensured by the bud-specific degradation of Vac17. It has been proposed that bud-specific degradation of Vac17 is promoted by proteins localized to, or activated solely in, the bud. The p21-activated kinases (PAK) Cla4 and Ste20 are localized to and activated in the bud. Here I report that Cla4 localized to the segregation structure just prior to segregation structure resolution. Cells lacking PAK function failed to resolve the segregation structure. Overexpression of either Cla4 or Ste20 inhibited vacuole inheritance and this inhibition was suppressed by the expression of non-degradable VAC17. Finally, PAK activity was required for Vac17 degradation in late-M and CLA4 overexpression promoted Vac17 degradation. I propose that Cla4 and Ste20 are bud-specific proteins that promote segregation structure resolution and degradation of Vac17.

Bves is a transmembrane protein that influences cell migration, motility, and epithelial integrity through previously undefined mechanisms. In this dissertation, I present evidence that identifies the molecular basis for Bves function. Bves interacts with VAMP3, a SNARE protein that facilitates vesicular transport and specifically recycles β-1 integrin. Here, we demonstrate that Bves is important for VAMP3-mediated receptor recycling that underlies cell adhesion and migration both in vitro and in vivo. Thus, Bves plays a regulatory role in governing SNARE protein function. Similarly, Bves interacts with GEFT, a guanine nucleotide exchange factor that modulates Rho GTPase activity in a broad range of cellular processes including cell migration and protrusion formation. As detailed in this work, Bves regulates cell motility and morphology through regulation of Rho GTPases Rac1 and Cdc42. Thus, through interaction with GEFT, Bves influences Rho GTPase signaling cascades. Taken together, these studies explain the previously reported phenotypes upon Bves depletion at the molecular level and provide a basis to further examine the function of Bves in normal and possibly diseased states. Thus, the significance of this work lies in the identification and characterization of the molecular mechanism underlying Bves function. Overall, this dissertation summarizes our current understanding of Bves function at the molecular level in regulating diverse signal cascades.

This project characterizes CENP-F protein function at a cellular and organ level. First, I outline our plan used to identify novel binding partners. The first binding partner I describe is syntaxin 4 and the interaction between CENP-F and this SNARE protein regulates vesicular transport along the microtubule network. Second, I discuss the binding of CENP-F and Hook2, a centrosomal protein, and the role of CENP-F in centrosomal microtubule nucleation. Additionally, novel immunohistochemical and gene ablation reagents were developed to complete these studies. These two binding partners both illustrate a vital role of CENP-F with the microtubule network throughout the cell. To introduce our model of organogenesis, I outline the process of heart development, with emphasis on the avian model. Given the dynamic and high expression of CENP-F in this vital organ, it is an excellent model in which to study the role of CENP-F in vivo. Finally, I report our initial findings with the heart-specific deletion of CENP-F in the mouse. The ablation of CENP-F in developing cardiomyocytes leads to a small heart with thinner walls that develops progressive dilated cardiomyopathy and irregular conduction patterns. Investigations into the underlying function that is disrupted the CENP-F-/- hearts resulting in these phenotypes are ongoing. Overall, this thesis characterizes the function of CENP-F via binding partner identification and disruption of protein function using cell biological methodologies and genetic deletion. These approaches elucidate function with the microtubule network in vesicular transport and centrosomal microtubule nucleation and show an important role in normal murine heart development.

Export of mature, translationally competent messenger ribonucleoprotein particles (mRNPs) from the nucleus to the cytoplasm is a highly orchestrated process that includes transcription, capping, splicing, and 3' end formation. During these steps, the mRNP protein composition is continually altered and such remodeling is critical for proper gene expression. A striking example of mRNP remodeling occurs during mRNA export through the nuclear pore complex (NPC), the only portal for trafficking across the nuclear envelope. This study aims at deciphering the localized remodeling and release of mRNA at the cytoplasmic side of the NPC in the budding yeast Saccharomyces cerevisiae. I demonstrate that the conserved mRNA export factor Gle1 and IP6 physically interact and stimulate the RNA-dependent ATPase activity of Dbp5, a DEAD-box protein predicted to provide directionality in the mRNA export mechanism. Furthermore, I generated gle1 mutants defective in IP6 binding. In vivo and in vitro analyses of these mutants indicate that Gle1 binds IP6 in vivo, and that this binding is essential for proper mRNA export. I propose that IP6 regulates mRNA export by binding conserved basic residues within the C-terminal domain of Gle1 resembling other highly phosphorylated inositol-binding domains. Dbp5 has been shown to be recruited to mRNA as early as transcription and might accompany the mRNP through the NPC. With juxtaposed Gle1 and Dbp5 docking sites on the NPC cytoplasmic face, I propose that Gle1-IP6 activation of Dbp5 spatially controls RNP remodeling during a late mRNA export step. The GTPase activity of Ran, the regulator of protein export, is also stimulated by compartmentalized factors. Thus, localized NTPase activation may be a global mechanism for controlling the directionality of nucleocytoplasmic transport.

<p> The sheer complexity of evolutionary systems biology requires us to develop more sophisticated tools for analysis, as well as more probing and biologically relevant representations of the data. My research has focused on three aspects of evolutionary systems biology. I ask whether a gene&rsquo;s position in the human metabolic network affects the degree to which natural selection prunes variation in that gene. Using a novel orthology inference tool that uses both sequence similarity and gene synteny, I inferred orthologous groups of genes for the full genomes of 8 mammals. With these orthologs, I estimated the selective constraint (the ratio of non-synonymous to synonymous nucleotide substitutions) on 1190 (or 80.2%) of the genes in the metabolic network using a maximum likelihood model of codon evolution and compared this value to the betweenness centrality of each enzyme (a measure of that enzyme&rsquo;s relative global position in the network). Second, I have focused on the evolution of metabolic systems in the presence of gene and genome duplication. I show that increases in a particular gene&rsquo;s copy number are correlated with limiting metabolic flux in the reaction associated with that gene. Finally, I have investigated the proliferative cell programs present in 6 different cancers (breast, colorectal, gastrointestinal, lung, oral squamous and prostate cancers). I found an overabundance of genes that share expression between cancer and embryonic tissue and that these genes form modular units within regulatory, proteininteraction, and metabolic networks. This despite the fact that these genes, as well as the proteins they encode and reactions they catalyze show little overlap among cancers, suggesting parallel independent reversion to an embryonic pattern of gene expression.</p>

<p> Plumage color evolution in birds has been the focus of theoretical and empirical research on sexual selection since Darwin. Many of the yellow, orange, and red hues seen in bird plumage are the result of carotenoid pigmentation. While a great number of recent studies have examined the functions of carotenoid-based plumage coloration in a single species, few have examined the evolutionary history of this trait in a comparative phylogenetic context. Using the New World blackbirds as a model clade, I focus on two questions that a comparative phylogenetic approach can uniquely address. First, what is the history of evolutionary change in carotenoid color that led to the colors seen in extant blackbird taxa? Second, by what proximate mechanisms have carotenoid pigments evolved? In Chapter 1, I present an ancestral state reconstruction of carotenoid-based plumage coloration across the Icterid phylogeny, based on reflectance measurements of museum skins. My results show robust evidence that red coloration was gained repeatedly from a yellow common ancestor. In Chapter 2, I used pigment biochemistry of meadowlark (<i>Sturnella</i>) and Cacique (<i>Cacicus</i>) feathers to test whether independent gains of red coloration are the result of parallel or convergent metabolic mechanisms. Meadowlarks have evolved red coloration using a different set of carotenoids than caciques, but the caciques have evolved the same set of carotenoids twice. This suggests that red coloration evolved by convergent evolution among different blackbird clades, but evolved by parallel evolution within the caciques. Lastly, in Chapter 3 I examine the relationship between color and carotenoid pigmentation in orioles, a blackbird clade in which orange has been gained at least twice independently from a yellow common ancestor. I found red-producing keto-carotenoids only in orange species and never in yellow species. This result is a striking contrast to our expectation for a continuous gradient of a carotenoid pigment concentration. These results suggest that repeated gains of C4-oxygenation ability best explain evolutionary changes in orange coloration in orioles. To summarize, I showed using phylogenetic comparative methods that blackbirds have repeatedly evolved towards redder carotenoid coloration. Using HPLC biochemistry, I showed that each of these gains of orange and red coloration is likely the result of a gain of C4-oxygenation ability. The prevalence of gains of orange and red coloration suggests that there may be a directional bias towards evolving longer-wavelength carotenoid plumage. The research presented in these chapters provides the phylogenetic framework necessary for future studies to examine the functional causes underlying the repeated evolution of carotenoid-based coloration.</p>

<p> Key to understanding the transition that vertebrates made from water to land is determining the developmental and genomic bases for the changes. New bioinformatic tools provide an opportunity to automate the discovery, broaden the number of, and provide an evidence-based ranking for potential candidate genes. I sought to explore this potential for the fin/limb transition, using the substantial genetic and phenotypic data available in model organism databases. Model organism data was used to hypothesize candidate genes for the fin/limb transition. In addition, 131 fin/limb candidate genes from the literature were extracted and used as a basis for comparison with candidates from the model organism databases. Additionally, seven genes specific to limb and 24 genes specific to fin were identified as future fin/limb transition candidates.</p>