Why are urban forests not replacing themselves?  For example, recent surveys demonstrate that adult trees are dying faster than new saplings are recruiting in Seattle (Washington), implying that the health of local coniferous forests is in decline.  Extensive resources are spent planting trees to combat this problem, but the underlying causes of this "lost generation" of trees remains unknown.  We investigate factors limiting seedling establishment of native conifers (Thuja plicata, Tsuga heterophylla) in Seattle, and use results to suggest strategies resource managers can use to address the "lost generation" problem.  We examine the effect of removing non-native English Ivy (Hedera helix) and adding mulch to understand what is limiting conifer regeneration, and to explore possible management techniques for improving growth and survival of transplanted seedlings.  We measured survival, general health (visually assessed), and height as response variables to the two treatments.  As of yet, no studies have empirically tested both of these factors.  We observed the highest transplant survival rates in areas where mulch was added, for both focal species.  Surviving transplants in the mulch treatment were also healthier than the transplants with no mulch added.  Ivy presence did not affect health or survival of either species, but it did affect growth of Tsuga heterophylla.  Treatments affected growth of the two species differently.  Growth of Thuja plicata was not affected by either treatment.  For Tsuga heterophylla, however, growth was highest in the ivy-removed treatment, but was not significantly affected by the presence of mulch.  Our results suggest that seedling survival is limited by a lack of mulch and that competition with invasive understory plants may limit growth for some species.  Our recommendations for park management are therefore that restoration transplants should be paired with mulch addition, as well as Hedera helix removal for Tsuga heterophylla, to maximize survival and growth.

The geographic ranges of species are strongly influenced by climate. As temperatures increase, species are expected to shift poleward in latitude and upward in elevation, a prediction supported by observations of range shifts in recent decades. Species interactions also play a crucial role in setting geographic ranges of global biomes, and could be critical in determining the response of species to climate change. Due to limited data on interspecific competition in the boreal biome, Earth's largest land biome, it is largely unclear how climate change will affect the range limits of the individual tree species that occupy it. In recent decades, spruce trees (Picea engelmannii) have increased in abundance at high elevation locations formerly dominated by larch trees (Larix lyallii). This change is thought to result from improved climatic conditions for spruce establishment but has not been tested. I hypothesize that spruce have higher establishment rates than larch where the two co-occur when temperatures are high. To test this hypothesis, I will harvest 120 stems of each species at locations in the North Cascade Mountains in the summer of 2012. I will then count the number of annual growth rings for each sapling to determine establishment year. Next, I will model the relationship between establishment rates and climate in different decades of the last ~100 years for each species. Finally, I will compare the establishment-climate relationships of the two species to infer how the relative abundances of the species might shift with warming. These results have large-scale implications for changes we should expect to see in global biomes as warming continues. In particular, this hypothesis suggests that spruce will replace larch as climate change progresses.

Cell migration and cell adhesion are crucial processes for embryonic development, homeostasis and cancer progression. Regulated changes in adhesion are coordinated with cytoskeletal change, which result in morphogenesis. Conversely, the loss of cell to cell adhesion can promote tumor cell metastasis and change in cell motility. Thus, examining the specific regulator of the adhesion and cytoskeleton is important in terms of furthering our understanding of the development and disease progression. We specifically study an important protein regulator called p120 catenin and its involvement in cell migration in the zebrafish embryo. An extensive literature implies that p120 catenin both positively and negatively regulates cell adhesiveness through phosphorylation. Cell cultures show that phosphorylation of serine residues strengthens the adhesion and that phosphorylation of tyrosine residues increases the cell mobility. After the morpholino knockdown of normal p120 catenin, I will co-inject the mutant Y228F p120 catenin. Then I will observe whether the mutant without the tryrosine phosphorylation rescued normal development. For this I will use in situ staining of the somites with myoD at the 3 to 15 somite stage. To observe migration and protein localization, we will use a confocal microscope with the embryos' cells labeled with cherry fluorescent p120 catenin, green fluorescent Rho GTPase, or a green fluorescent plasma membrane. If mutant p120 catenin, which cannot be phosphorylated, can rescue successfully, we conclude the phosphorylation site does not play an important role in cell-cell adhesion or cell migration. However, if it does not rescue the morpholino knockdown, tyrosine phosphorylation at the 228 site is important for p120 catenin’s modulation of cell–cell adhesion and cell migration.

Gastrulation is a crucial step in embryogenesis, creating differentiation between ectoderm, mesoderm, and endoderm, as well as forming necessary organs. Transmembrane and juxtamembrane proteins located at the adherens junction, such as cadherins and p120 catenin, work together to either promote cell motility or adhesion. Using Danio rerio embryos as a model organism, we hypothesized that when p120 catenin is at the plasma membrane it binds and stabilizes the cadherins, causing cell clustering and increased adhesion. When p120 catenin is in the cytoplasm it activates Rac1 and Cdc42 (two members of the Rho GTPase family), which causes cell motility. By using a “depletion-rescue” experimental approach, p120 catenin levels are depleted by injecting a splice-morpholino, then “rescued” by coinjecting wild-type Cdc42 or Rac1. This indicates that p120 catenin in healthy embryos works to activate Cdc42 and Rac1 during gastrulation, resulting in cellular motility. When dominant-negative and constitutively-active mutants of Cdc42 or Rac1 are injected in combination with morpholino, the embryos do not “rescue” well, if at all. We postulate that wild type GTPases “rescue” well because the can receive a signal and activate migration directionally while the constitutively-active GTPases activate migration in all directions. These studies on the regulation of cell motility can help to elucidate larger signaling pathways, leading to additional effects on cell survival, growth, and invasiveness, all of which may be caused by the upstream p120 catenin.

Gastrulation is the process during which cells migrate and tissue layers differentiate in early embryo development. This process requires a balance between cell-cell adhesion mediated by cadherin-catenin interactions and stimulation of cell migration. Our research focuses on p120 catenin (p120ctn) dependent cell migration during gastrulation in zebrafish. P120ctn stabilizes cadherins at the plasma membrane and interacts with Rac1 and Cdc42 GTPases to stimulate cell migration, and with RhoA GTPase to regulate cell-cell adhesion. P120 binds and translocates RhoA to the plasma membrane where a RhoA gunanine nucleotide exchange factor stimulates release of GDP and binding of GTP. GTP-RhoA activates RhoA kinase which activates myosin light chains, inducing cross links to stabilize actin filaments, thus increasing the strength of cell-cell adhesion. In this project we aim to address what the role of RhoA GTPase is in the p120ctn signaling pathway as well as how phosphorylation of p120ctn affects the RhoA-p120ctn binding interaction. By knocking down p120ctn and attempting to rescue developmental deficiencies, we can elucidate how p120ctn interacts with RhoA to regulate cell-cell adhesion. We hypothesize that injection of GDP-bound (DN) RhoA will rescue a splice-morpholino p120ctn knockdown allowing low concentrations of p120ctn to activate Rac1 and Cdc42 GTPases that promote cell migration. Our microinjections of DN-RhoA mRNA into one-cell zebrafish embryos have shown that DN-RhoA, and not WT-RhoA or CA-(GTP-bound) RhoA, is able to rescue p120ctn knockdown. Additionally, p120ctn is regulated by phosphorylation and we are currently interested in how tyrosine phosphorylation of p120ctn affects its ability to bind and regulate RhoA. We hypothesize that tyrosine phosphorylation site mutants will be unable to rescue splice-morpholino p120ctn knockdown, and therefore be essential regulation sites. Using confocal fluorescence microscopy and a binding assay to measure GTP-RhoA, we hope to elucidate whether these sites are directly involved in the p120ctn-RhoA binding interaction.