microRNAs (miRNAs) are short non-coding RNAs that down-regulate gene expression by pairing with sequences in the 3UTR of target mRNAs. They play critical roles in diverse developmental and physiological events but the exact function of most miRNAs remains to be elucidated. Identification of bona fide targets is challenging due to partial base pairing between miRNAs and target 3UTRs. To understand miRNA function during early vertebrate development, I first analyzed the general rules for miRNA:mRNA base pairing. By comparing the silencing ability of different miRNA recognition sites, I show that combinations of weak binding sites are just as effective as perfect sites for miRNA function. In addition, I uncovered a novel tissue-inductive role for miR-92 during early zebrafish development. Most previous work has focused on control of a number of cancer-related genes by miR-92, but here, using gain-of-function, loss-of-function, target identification, and genetic epistasis experiments, I demonstrate that miR-92 also regulates transcription factor gata5 and that such regulation is essential for proper endoderm formation and left-right patterning in zebrafish.

Sex is a major challenge to evolutionary theory, because of the apparent paradox of its ubiquity among multicellular eukaryotes with the substantial evolutionary costs associated with it. The past four decades have seen the development of a large and robust collection of hypotheses purporting to explain the benefits of sex and reconcile this paradox, while empirical research testing the predictions of these hypotheses in nature has only begun to gather momentum more recently. Neochlamisus leaf beetles are one such natural system with great potential for study of the evolutionary tradeoffs of sexuality and asexuality thanks to the prevalence of gynogenetic asexuality within the genus. Gynogenesis is a form of asexual reproduction in which females require insemination to stimulate the development of unreduced eggs into clonal offspring with no paternal genetic contribution. Gynogenesis requires the coexistence of asexual females with females and donor males of the same nominal species, allowing highly controlled comparisons of ecologically and biologically similar sexual and asexual animals. In this thesis, I present a new mitochondrial DNA dataset consisting of diverged sexual and asexual lineages of Neochlamisus and describe the phylogenetics and patterns of molecular evolution observed in each lineage, with explicit regard to reproductive mode. I then test two a priori predictions of differences in molecular evolution between the sexual and asexual lineages related to reproductive mode. My results are consistent with a higher rate of accumulation of putatively harmful nonsynonymous mutations in the asexual lineage, and with a selective sweep of the mitochondrial genome in the sexual lineage driven by cytoplasmic incompatibility-inducing strains of the intracellular bacterial parasite Wolbachia.

A systematic Drosophila forward genetic screen for photoreceptor synaptic transmission mutants identified no-on-and-no-off transient C (nonC) based on loss of retinal synaptic responses to light stimulation. The cloned gene encodes phosphatidylinositol 3-kinase-like kinase (PIKK) Smg1, a regulatory kinase of the nonsense-mediated decay (NMD) pathway. The Smg proteins act in an mRNA quality control surveillance mechanism to selectively degrade transcripts containing premature stop codons, thereby preventing the translation of truncated proteins with dominant negative or deleterious gain of function activities. At the neuromuscular junction (NMJ) synapse, an extended allelic series of Smg1 mutants show impaired structural architecture, with decreased terminal arbor size, branching and synaptic bouton number. Functionally, loss of Smg1 results in a ~50% reduction in basal neurotransmission strength, as well as progressive transmission fatigue and greatly impaired synaptic vesicle recycling during high-frequency stimulation. Mutation of other NMD pathways genes (Upf2 and Smg6) similarly impairs neurotransmission and synaptic vesicle cycling. These findings suggest that the NMD pathway acts to regulate proper mRNA translation to safeguard synapse morphology and maintain synaptic functional efficacy.

The adult vertebrate forebrain is responsible for a diverse set of behaviors, and as such exhibits complex anatomy. This complexity is generated during embryogenesis whereby a specific spatiotemporal sequence of transcriptional and signaling programs promotes specification of different cell types based on the location and developmental potential of progenitors. Six3 is one such transcription factor that exerts multiple functions in the development of anterior neural tissue of vertebrate embryos. Whereas complete loss of Six3 function in the mouse results in failure of forebrain formation, its hypomorphic mutations in human and mouse can promote holoprosencephaly, a forebrain malformation resulting, at least in part, from abnormal telencephalon development. However, Six3s roles in telencephalon patterning and differentiation are not well understood. The zebrafish genome contains three Six3-related genes facilitating analysis of different partial loss-of-function combinations. I analyzed zebrafish embryos deficient in two of three Six3-related genes, six3b and six7, representing a partial loss of Six3 function. Telencephalon forms in six3b;six7-deficient embryos, however ventral telencephalic domains are reduced and dorsal domains are expanded. Decreased cell proliferation or excess apoptosis cannot account for the ventral deficiency. Instead, six3b and six7 are required during early segmentation for specification of ventral progenitors, similar to the role of Hedgehog signaling in telencephalon development. Unlike in mice, Hedgehog signaling is not disrupted in embryos with reduced Six3 function. Furthermore, six3b overexpression is sufficient to compensate for loss of Hedgehog signaling in isl1- but not nkx2.1b-positive cells, suggesting a novel Hedgehog-independent role for Six3 in telencephalon patterning. Additional investigations into the interactions between Six3 and known telencepnalon patterning genes showed that Six3 promotes ventral telencephalic fates through transient regulation of foxg1a expression and repression of Wnt/β-catenin pathway. As Six3-related genes are expressed broadly in prechordal mesoderm and anterior neuroectoderm, transgenic zebrafish were generated to identify the spatial requirement for Six3 function in telencephalon patterning. My studies help define the cellular mechanisms of Six3-mediated dorsoventral patterning in telencephalon and present a novel genetic mechanism by which Six3 regulates this process.

Human Potassium channels

certain Indian coccids

Recent empirical studies conducted in disparate ecosystems have shown that greater species diversity has positive effects on ecosystem functioning; however, other studies have found neutral or sometimes negative results. It is still unclear why the relationship between biodiversity and functioning varies among studies, but perhaps, investigating this relationship across spatial and temporal scales will lead to further understanding. One theory predicts that local niche complementarity among species (the partitioning of species based upon niche differentiation) is predicted to positively affect local ecosystem functioning at the local spatial scale. However, more recent theory predicts that greater local diversity may hinder local ecosystem functioning when diversity is enhanced through regional processes. I suggest community assembly as a way to incorporate both the local and regional processes that determine biodiversity and its consequent effects on ecosystem functioning. From this, I propose a hump-shaped relationship between diversity and ecosystem functioning at local spatial scales, but a linear increase of functioning with diversity at regional spatial scales. Thus, species diversity may have different effects on ecosystem functioning across different spatial scales. Species diversity may affect ecosystem functioning differently across time as environmental conditions shift. Through integrating recent theoretical models in ecosystem ecology and empirical examples of food-webs in community ecology, the effects of herbivore diversity on ecosystem functioning (grazing of primary producers) were examined under unchanged (no nutrients added) and changed (nutrients added) environmental conditions. I found that communities with higher species richness and diversity did not significantly differ from lower diversity communities in grazing intensity in the unchanged environments. However, higher diversity communities did have a significant effect on the biomass of primary producers in the nutrient enriched environments, while lower diversity communities did not. This empirical study showed that the functioning of local communities is dependent on the environmental conditions present in the habitat. Overall, this investigation found that the relationship between species diversity and ecosystem functioning may be dependent on spatial scale and environmental changes over time.

The effects of nutrient input into aquatic systems has been studied frequently; typically, these studies report an increase in algal biomass and a decrease in species diversity in response to an increase of nutrients. However, it is not clear why similar aquatic communities will respond differently to nutrient additions of similar magnitudes, resulting in alternative communities. Because variance in natural ecosystems is pervasive, perhaps it is this variability that helps determine the final community. I proposed that the total amount of nutrient input and the variability of nutrient input would affect the abundances and composition of species. A natural survey was conducted to measure the variable levels of nutrients in several aquatic systems. Experimental ponds were used to test the effects of variable rates and timing of nutrient inputs upon an aquatic community; experimental treatments manipulated the total amount of nutrient input (high v. low), the rate of nutrient input (annually, monthly or weekly), the timing of the nutrient input (early v. mid- season), and the trophic status at which these treatments were imposed (mesotrophic v. eutrophic). The effects of the variability of nutrient input was at least as important as the total amount of the nutrient input. There were large impacts upon species diversity, abundances and composition. Although these effects were manifested in many trophic groups, the response to the variability was most strikingly found within the primary producers, which showed large shifts in abundance and composition.

Mitotic segregation defects such as multipolar spindles, anaphase bridges, and micronuclei have long been observed in cancer cells, but it is not known whether these defects lead to aneuploidy or even contribute to tumorigenesis. We visualize living oral squamous carcinoma cells with stable expression of GFP-histone H2B fusion. Expression of this fusion protein labels chromosomes clearly and does not disrupt the cell cycle, alter the doubling time or produce any defects previously unseen in fixed cells. These carcinoma cells survive the formation of anaphase bridges and micronuclei and complete a second cell division in the same amount of time as unaffected cells. Micronuclei were formed after every division that contained an anaphase bridge in cells we examined. Most often, each daughter cell contained a micronucleus. These results suggest that the chromosome breaks at multiple points along its length and breaking may not be due to a "tug of war" between spindle poles. The movement of micronuclei was very dynamic compared to the nuclei during interphase and micronuclei do not appear to be transcriptionally active. Using long-term live cell imaging we were also able to observe the fate of these cells through two divisions and have determined the length of each phase of mitosis. Anaphase bridges and lagging metaphase chromosomes both lengthen mitosis, suggesting that the mitotic spindle checkpoints are at least partially active in cells. The mitotic delays occurred during metaphase in these defective cells. We have also analyzed centrosomal components including the mitotic apparatus protein, NUMA. No correlations were found between protein expression of NuMA and gene amplification or segregation defects. In summary, we have shown that cells continue to proliferate after the occurrence of mitotic defects and these defects contribute to chromosomal instability.

Control of directionality in integrase-mediated site-specific recombination reactions is achieved by an architectural change fashioned by a class of accessory proteins know as recombination directionality factors (RDFs). In the mycobacteriophage L5 system, no RDF had been previously identified. In the course of this work, the gene, 36, was identified using in vivo screens and shown to play this role. The protein was over expressed using an E. coli expression system and then used to create an in vitro excision reaction assay. Initial work was done in characterizing the excision reaction including supercoiling and host factor requirements. Further analysis has shown that the protein binds specifically to a region within the left side of attP and attR. Once bound, complexes are formed that inhibit integration in the case of attP and stimulate excision when attR is present. Additional work was done to increase the utility of the L5 based integrating vector system, which has been commonly used to create stably integrated single copy transformants. The system lacked an effective means to recover DNA for high copy replication or curing of strains containing inserted DNA. With the identification of the L5 xis gene, we have been able to develop systems that allow the recovery of DNA into E. coli and curing strains of the integrated DNA. The third part of this work involved the characterization of the RDF class of proteins. Since this class of proteins is composed of a diverse group of small proteins, no previous attempt had been made to characterize them. Extensive data mining yielded a collection of 63 putative or known RDFs. Further analysis of sequence data, chemical characteristics and other known properties argues that this class of proteins has evolved from multiple ancestral origins.