Browsing by Subject "Biology, General"

A fundamental goal in the diverse field of evolutionary biology is reconstructing the historical processes that facilitated lineage diversification and the current geographic distribution of species diversity. Oceanic islands provide a view of evolutionary processes that may otherwise be obscured by the complex biogeographic histories of continental systems, and have thus provided evolutionary biology with some of its most lasting and significant theories. The Indian Ocean island of Madagascar is home to an extraordinarily diverse and endemic biota, and reconstructing the historical processes responsible for this diversity has consumed countless academic careers. While the flowering plant genus Coffea is but one lineage contributing to Madagascar's staggering floral diversity, it is representative of the common evolutionary theme of adaptive radiation and local endemism on the island. In this dissertation, I employ the genus Coffea as a model for understanding historical biogeographic processes in the Indian Ocean using methods of molecular phylogenetics and population genetics. In the molecular phylogenetic study of Coffea presented in chapter 2, I show that Madagascan Coffea diversity is likely the product of at least two independent colonization events from Africa, a result that contradicts current hypotheses for the single origin of this group.

Species of Coffea are known to exhibit self-incompatibly, which can have a dramatic affect on the geographic distribution of plant genetic diversity. In chapter 3, I identify the genetic mechanism of self-incompatibility in Coffea as homologous to the canonical eudicot S-RNase system. Baker's Rule suggests that self-incompatible lineages are very unlikely to colonize oceanic islands, and in chapter 4, I test this hypothesis by characterizing the strength of self-incompatibility and comparing S-RNase polymorphism in Coffea populations endemic to isolated Indian Ocean islands (Grande Comore and Mauritius) with that of Madagascan/African species. My findings suggest that while island populations show little evidence for genetic bottleneck in S-RNase allelic diversity, Mauritian endemic Coffea may have evolved a type of "leaky" self-incompatibility allowing self-fertilization at some unknown rate. Through the application of traditional phylogenetic methods and novel data from the self-incompatibly locus, my dissertation contributes a wealth of new information regarding the evolutionary and biogeographic history of Coffea in the Indian Ocean.

A long-held assumption in animal behavior is that females and males differ fundamentally in their mating strategies. Females are thought to be more choosy because female reproduction typically is limited by parental investment. Males, on the other hand, are expected to compete among themselves for access to females or resources, since male reproduction is limited primarily by mating access. This dichotomy is challenged by the increasing realization that males can be choosy and females also compete aggressively. It remains unclear, however, if and how selection acts on aggressive behavior in the context of intrasexual competition among females (reviewed in Chapter 1). In this thesis, I use a population of free-living tree swallows (Tachycineta bicolor) to test predictions about the selective pressures shaping aggressive behavior in females. First, using an experimental manipulation of nest site availability, I demonstrate that more aggressive females have a competitive edge in acquiring nestboxes, a critical limiting resource required for breeding (Chapter 2). This result shows that more aggressive females are more likely to breed and, thus, that females experience direct selection to be aggressive in the context of competition for mating opportunities. Next, I demonstrate a fitness cost of female aggression (Chapter 3): high levels of aggression in females are not associated with the quantity of offspring, but instead, more aggressive females had offspring of lower quality (i.e. reduced mass). Using a cross-fostering approach, I explore the causal link between female aggression and offspring mass, and I find that a trade-off between female aggression and maternal care best accounts for this cost of aggression. Site differences may create variation in how selection shapes female aggression, but the overall finding that more aggressive females have lower quality offspring indicates that this cost may work counter to selection favoring aggressive behavior in the context of competition over nestboxes. Understanding the evolution of female aggressiveness in a biparental system is incomplete without examining how males may alter the selective environment shaping female behavior. In Chapter 4, I explore the potential role of a female's mate in offsetting the costs of aggression. Males appear to mitigate these costs for their female partners, but not by compensating for poor parenting by aggressive females. Instead, females invest more heavily in reproduction, laying more and larger eggs, when mated to a male that is more different from her own phenotype. If this differential investment outweighs the cost of aggressiveness in terms of offspring quality, then male phenotype may play a key role in understanding the selective pressures shaping the evolution of aggressive behavior in females. Altogether, this dissertation explores the costs and benefits of female aggressive behavior. The focus on aggressiveness as a sexually selected trait in females provides a much needed parallel to the wealth of information already known about the selective pressures shaping sexually selected traits in males.

Because species respond individually to climate change, understanding community assembly requires examination of multiple species from a diversity of forest niches. I present the post-glacial phylogeographic history of an understory, parasitic herb (Epifagus virginiana, beechdrop) that has an obligate and host specific relationship with a common eastern North American hardwood tree (Fagus grandifolia, American beech). The migration histories of the host and parasite are compared to elucidate potential limits on the parasite's range and to understand their responses to shared climate change. Two chloroplast DNA regions were sequenced and 9 microsatellite loci genotyped from parasite specimens collected throughout the host's range. These data were compared with available cpDNA sequences from the host (McLachlan et al. 2005) and host fossil pollen records from the last 21,000 years (Williams et al. 2004). Analyses of genetic diversity reveal high population differentiation in the parasite's southern range, a possible result of long term isolation within multiple southern glacial refuges. Estimates of migration rates and divergence times using Bayesian coalescent methods show the parasite initiating its post-glacial range expansion by migrating northward into the northeast from southern areas, then westward into the midwest, a pattern consistent with the development of high density beech forests. This result is strongly confirmed through spatial linear regression models, which show host density plays a significant role in structuring parasite populations, while the initial migration routes of the host are irrelevant to parasite colonization patterns. Host density is then used as a proxy for the parasite's habitat quality in an effort to identify the geographic locations of its migration corridors. Habitat cost models are parameterized through use of the parasite's genetic data, and landscape path analyses based on the habitat map show a major migration corridor south of the Great Lakes connecting the northeast and midwest. Host density was the major determinant controlling the parasite's range expansion, suggesting a lag time between host and parasite colonization of new territory. Parasites and other highly specialized species may generally migrate slower due to their complex landscape requirements, resulting in disassociation of forest assemblages during these times. From these results, the low migration capacities of highly specialized species may be insufficient to outrun extirpation from their current ranges.

The chemical cues that provide an olfactory portrait of mammalian individuals are in part detected by chemosensory receptors in the vomeronasal organ (VNO). By and large, the pertinent receptor-cue combinations used for olfactory communication are unidentified. Here we identify members of the formyl peptide receptor (FPR) family of G protein coupled receptors as candidate chemosensory receptors in the VNO of mice. We demonstrate that N-formylated mitochondrially encoded peptides presented by the major histocompatibility complex (MHC) molecule H2-M3 stimulate a subset of the VNO sensory neurons (VSNs). We show that one VNO localized FPR, Fpr-rs1, is differentially activated by strain specific variants of N-formylated peptides. We show that N-formylated peptides can function as chemosignals in a strain selective pregnancy block. We propose that this link between self-recognition peptides of the immune system and chemosensory pathways provides a possible molecular means to communicate the nature of an individual's maternal lineage or strain.

The teleost zebrafish (danio rerio) has a highly elevated regenerative capacity compared to mammals, with the ability to quickly and correctly regenerate complex organs such as the fin and the heart following amputation. Studies in other highly regenerative systems suggest that regenerative capacity is directly related to the homeostatic demands of a given tissue, such as high basal levels of cell turnover or the ability to modify tissue size in response to homeostatic changes. However, it is not known if this relationship is present in vertebrate tissues with blastema-based regeneration. To test this idea, we investigated whether markers associated with regeneration are expressed in uninjured zebrafish tissues, and if treatments that block regeneration also lead to homeostatic defects over long periods.

We found that regenerative capacity is generally required for homeostasis in the fin, as multiple genetic treatments that block regeneration also led to a degenerative loss of distal fin tissue in uninjured animals. In addition, we found that there is extensive cell turnover in the distal fin tissues, accompanied by expression of critical effectors of blastemal regeneration. Both cell proliferation and gene expression were sensitive to changes in Fgf signaling, a factor that is critical for fin regeneration.

In the heart, we found that although there is little cell turnover in uninjured adult animals, the zebrafish heart can undergo rapid, dramatic cardiogenesis in response to animal growth. These growth conditions induce cardiomyocyte hyperplasia similar to regeneration, and induce gene expression changes in the epicardium, a tissue that is critical for cardiac regeneration. We find that the epicardium continually contributes cells to the uninjured heart, even in the absence of cardiac growth. If this contribution is prevented via a long-term block of Fgf signals, scarring can result, indicating that continual activity of epicardium derived cells (EPDCs) is critical for cardiac homeostasis. We have generated reagents that allow us to visualize EPDCs, and find that they contribute cardiac fibroblasts and perivascular cells during rapid cardiac growth. Uncovering the fate of EPDCs during cardiac homeostasis and regeneration will allow us to better understand their function, and may lead to the development of regenerative therapies for human cardiovascular diseases.

TThe proposed research will provide a set of 16 near complete mtDNA gene orders. The observed gene rearrangements will be used to investigate the phylogeny of the Sub-Class Hydroidolina and the Family Hydractiniidae. All the medusozoan classes contain a linear mitochondrial genome (mtDNA genome), whereas the Class Anthozoa contains a circular mtDNA genome (Bridge et al., 1992). The linear structure of the medusozoan mtDNA genomes is the most likely reason why these genomes are underrepresented, because the most rapid methods of mtDNA genome sequencing take advantage of the circular nature of most animal mtDNA. In a circular genome where mtDNA gene order is unknown prior to isolation, the forward and reverse primer(s) are designed based on the sequences of one or two highly conserved regions. Linear mtDNA requires a more involved approach, making it more difficult to sequence in its entirety than circular mtDNAs. In chapter 1, a novel assay to determine linear mtDNA gene orders is presented. In chapter 2, the near-complete mtDNA genome sequences are presented, as well as 7 near-complete mtDNA gene orders determined by the mtDNA gene order assay. These data are used to investigate relationships in the Family Hydractiniidae. Finally, in chapter 3, an additional 9 near complete mtDNA gene orders are used to explore relationships in the sub-class Hydroidolina. This study significantly increases the number of known near-complete mtDNA genomes, as well as their mtDNA.

A key question in developmental biology is how organisms attain a final size. Deviations in growth patterns can produce different/new phenotypes and these changes can play fundamental roles in ecology and evolution. The size of an organism and of its constitutive organs is determined by the growth rate and the duration of the growing period. In insects, peptide hormones such as insulin-like growth factors have been shown to be involved in determining the growth rates by coordinating metabolism, cell proliferation and cell size. In contrast, steroid hormones, such as ecdysone, are involved in determining life stage transitions, and thus the termination of the growing period. Although it is clear that insulin and steroid hormones are both involved in the regulation of growth, the ways in which these two regulators interact is yet to be determined. Furthermore, it is not clear how organs and body growth are coordinated during development to arrive to their correct proportions. In this study, using the tobacco hornworm Manduca sexta and its wings as a model system, I examine the developmental mechanisms involved in the regulation of organ growth and how developmental processes can drive morphological evolution. First, I examine how the hormonal events that take place during the termination of the body growth period affect wing disk growth. Second, by using gene expression assays and in vitro cultures, I examine the interaction between bombyxin, the Lepidopteran insulin-like growth factor, and ecdysone, the molting hormone, and their contributions to wing imaginal disk growth. Finally, by using three different size strains of M. sexta, I examine the developmental basis of the allometric relationship between the wings and the body. My results show that during the final instar of M. sexta larval development, wing imaginal disks are sensitive to the hormonal events that terminate the growth period. Furthermore, I show that the bombyxin requirement for wing disk growth is restricted to the early days of the final instar unlike the constitutive effects seen in other species. After the larva has passed a particular critical weight, bombyxin is not necessary for wing disk growth, although its absence does decrease the growth rate. In contrast, ecdysone is required for promoting the growth of wing imaginal disks primarily through its stimulation of cell proliferation. Finally, I show how selection on body size has unpredictable consequence for the response of wing size. These results demonstrate how specific allometries have a developmental basis in the cross-talk of the various signals that regulate growth itself. Therefore, direct selection on allometric relationships may not need to be strong in order to hold scaling relationships constant, at least over short evolutionary periods.

Linker for activation of T cells (LAT) is a transmembrane adaptor protein that lacks any intrinsic enzymatic or transcriptional activity. Upon TCR engagement, LAT is phosphorylated at its membrane-distal tyrosine residues, which mediate the binding of Grb2/Sos, PLCγ1, and GADS/SLP-76 complexes. SLP-76 (SH2 domain-containing leukocyte protein of 76kD) is a cytosolic adaptor protein that can interact with a variety of other adaptor proteins and signaling effectors. Through its constitutive binding of GADS, SLP-76 is recruited to the plasma membrane via LAT following TCR stimulation. Together, LAT and SLP-76 nucleate a large multi-molecular signaling complex, which couples TCR proximal signaling to downstream biochemical events, including calcium mobilization and Ras-MAPK pathway activation.

LAT is important in early thymocyte development as LAT-deficient mice have a complete block at the DN3 stage. To study the role of LAT beyond the DN3 stage, we generated mice in which the lat gene could be deleted by Cre recombinase. Deletion of LAT after the DN3 stage allowed largely normal development of DP thymocytes. However, LAT-deficient DP thymocytes were severely defective in responding to stimulation via the TCR and failed to efficiently differentiate into SP thymocytes. Moreover, deletion of LAT in peripheral mature T cells rendered these T cells completely unresponsive to CD3 crosslinking due to abolished calcium mobilization and Ras-ERK activation. Long-term survival and lymphopenia-driven homeostatic proliferation of the LAT-deficient naïve T cells were also severely impaired. Together, these data indicate that, in addition to its role in pre-TCR signaling, LAT also plays an essential role in thymocyte development during the transition from the DP to SP stage, as well as in mature T cell activation and homeostasis.

Similar to LAT, SLP-76 is also critical for T cell function and thymocyte development. While the functions of various SLP-76 domains have been extensively studied, the role of the sterile alpha motif (SAM) domain in SLP 76 function remains unknown. By generating SLP 76 knock in mice with the SAM domain deleted, we showed that the absence of the SAM domain resulted in impaired positive and negative thymic selections, leading to a partial block of thymocyte development at the DP to SP transition. TCR-mediated IP3 production, calcium flux, and ERK activation were all decreased in these ΔSAM-SLP-76 knockin T cells, leading to defective IL 2 production and proliferation. Moreover, despite normal association between GADS and SLP-76, TCR-mediated SLP 76 clustering was inhibited by the deletion of the SAM domain, likely causing the aforementioned TCR signaling defects. These data demonstrated for the first time that the SAM domain is indispensable for optimal SLP-76 signaling.

The role of two cell extrinsic signals, T cell receptor (TCR) ligation and interleukin-7, in promoting glucose uptake and survival of T lymphocytes is examined in this work. Both of these signals are capable of regulating the uptake and fate of glucose, but the requirement of this regulation for T cell homeostasis and functionality remains unclear. To examine the role of TCR mediated increases of glucose metabolism and the signals involved, primary murine T cells were activated in vitro and the role and regulation of glucose uptake was examined. We show that glucose uptake is limiting in T cell activation and that CD28 costimulation is required for maximal glucose uptake following TCR stimulation by upregulating expression and promoting the cell surface trafficking of the glucose transporter Glut1. Regulation of T cell glucose uptake and Glut1 was critical, as low glucose prevented appropriate T cell responses. Additionally, transgenic expression of Glut1 augmented T cell activation, and led to accumulation of readily activated memory-phenotype T cells with signs of autoimmunity in aged mice. To further examine the regulation of glucose uptake, we analyzed CD28 activation of Akt, which appeared necessary for maximal glucose uptake of stimulated cells and which we have shown can promote Glut1 cell surface trafficking. Consistent with a role for Akt in Glut1 trafficking, transgenic expression of constitutively active Akt (mAkt) increased glucose uptake of resting T cells, but did not alter Glut1 protein levels. Therefore, CD28 appeared to promote Akt-independent upregulation of Glut1 protein and Akt-dependent Glut1 cell surface trafficking. In support of this model, co-expression of Glut1 and mAkt transgenes resulted in a synergistic increase in glucose uptake and accumulation of activated T cells in vivo that were largely independent of CD28. Induction of Glut1 protein and Akt regulation of Glut1 trafficking are therefore separable functions of CD28 costimulation that cooperate to promote glucose metabolism necessary for T cell activation and proliferation.

Glucose uptake is dramatically increased in response to TCR and costimulation signaling, however, glucose uptake must be maintained at a low level in naive T cells to promote survival and homeostasis. Interleukin-7 (IL-7) plays a central role in maintaining naive T cell homeostasis, and mediates this effect in vivo at least in part through control of homeostatic proliferation and inhibition of apoptosis. IL-7 can promote glucose uptake and glycolysis in vitro and may also promote glucose metabolism in vivo to maintain T cell survival. To determine if IL-7 regulates T cell metabolism in vivo, we generated a transgenic model for conditional IL-7 receptor (IL 7R) expression on IL-7R-/- T cells. T cells in this model developed normally and, consistent with previous work, deletion of the IL-7R transgene in vivo led to cell death even in an otherwise normal lymphoid compartment. Importantly, in vivo deletion of IL 7R also led to decreased cell size and glycolytic flux. However, glucose uptake was not altered following deletion of the IL-7R indicating that while not essential for glucose uptake, IL-7 is required for maintenance of glycolysis. These data are the first to identify a signal required in vivo to regulate lymphocyte metabolism and demonstrate that in addition to its well-defined roles in homeostatic proliferation and cell survival, IL-7 plays a key and non-redundant role to maintain T cell glycolysis. Together, these data concerning the role of TCR, costimulation, and IL-7 in the regulation of glucose uptake and metabolism exemplify the importance of cell extrinsic signals and the regulation of glucose utilization.