Project description:Chemical modifications to the tails of histone proteins act as gene regulators that play a pivotal role in adaptive responses to environmental stress. Determining the short and long term kinetics of histone marks is essential for understanding their functions in adaptation. We used Caenorhabditis elegans as a model organism to study the histone modification kinetics in response to environmental stress, taking advantage of their ability to live in both terrestrial and aquatic environments. We investigated the multigenerational genome-wide dynamics of five histone marks (H3K4me3, H3K27me3, H4K20me1, H3K36me1, and H3K9me3) by maintaining P0 animals on terrestrial (agar plates), F1 in aquatic cultures, and F2 back on terrestrial environments. We determined the distributions of histone marks in the gene promoter regions and found that H4K20me1, H3K36me1, and H3K9me3 showed up to eleven-fold differences in density, whereas H3K4me3 and H3K27me3 remained highly constant during adaptation from terrestrial to aquatic environments. Furthermore, we predicted that up to five combinations of histone marks can co-occupy single gene promoters and confirmed the colocalization of these histone marks by structured illumination microscopy. The co-occupancy increases with environment changes and different co-occupancy patterns contribute to variances in gene expressions and thereby presents a supporting evidence for the histone code hypothesis.
Project description:Study abstract: Axolotl salamanders (Ambystoma mexicanum) remain aquatic in their natural state, during which biomechanical forces on their diarthrodial limb joints are likely reduced relative to salamanders living on land. However, even as sexually mature adults, these amphibians can be induced to metamorphose into a weight-bearing terrestrial stage by environmental stress or the exogenous administration of thyroxine hormone. In some respects, this aquatic to terrestrial transition of axolotl salamanders through metamorphosis may model developmental and changing biomechanical skeletal forces in mammals during the prenatal to postnatal transition at birth and in the early postnatal period. To assess differences in the appendicular skeleton as a function of metamorphosis, anatomical and gene expression parameters were compared in skeletal tissues between aquatic and terrestrial axolotls that were the same age and genetically full siblings. The length of long bones and area of cuboidal bones in the appendicular skeleton, as well as the cellularity of cartilaginous and interzone tissues of femorotibial joints were generally higher in aquatic axolotls compared to their metamorphosed terrestrial siblings. A comparison of steady state mRNA transcripts encoding aggrecan core protein (ACAN), type II collagen (COL2A1), and growth and differentiation factor 5 (GDF5) in femorotibial cartilaginous and interzone tissues did not reveal any significant differences between aquatic and terrestrial axolotls. RNAseq samples: Total RNA was isolated from whole body tissue samples of Mexican axolotl salamanders (Ambystoma mexicanum) at the following developmental stages: Embryo at the tail bud stage, newly hatched larva, larva at the limb bud stage, juvenile at 8.5 centimeters, and adult using variations of guanidinium-based protocols. RNA quantity, purity, and integrity of both the individual samples and the resulting pool were determined with an Agilent 2100 Bioanalyzer using the Eukaryotic Total RNA nano series II analysis kit. The pooled RNA sample was poly-A selected and used for Illumina random priming directional library prep. Four lanes were sequenced only on one end providing single end reads and 4 lanes were sequenced at both ends giving paired-end reads. The library was sequenced on an Illumina HiSeq 2000 for 75bp reads producing 147,248,512 single end reads and 2 x 153,254,667 paired-end reads.
Project description:<p>Aquatic insects are well-adapted to freshwater environments, but metabolic mechanisms of such adaptations, particularly to primary environmental factors (e.g., hypoxia, water pressure, dark light and abundant microbes), are poorly known. Most firefly species (Coleoptera: Lampyridae) are terrestrial, but the larvae of a few species are aquatic. We generated 24 global metabolomic profiles of larvae and adults of <em>Aquatica leii</em> (freshwater) and <em>Lychnuris praetexta</em> (terrestrial) to identify freshwater adaptation-related metabolites (AARMs). We identified 110 differentially abundant metabolites (DAMs) in <em>A. leii</em> (adults vs aquatic larvae) and 183 DAMs in <em>L. praetexta</em> (adults vs terrestrial larvae). Furthermore, 100 DAMs specific to aquatic <em>A. leii</em> larvae were screened as AARMs via interspecific comparisons (<em>A. leii</em> vs <em>L. praetexta</em>), which were primarily involved in antioxidant activity, immune response, energy production and metabolism, and chitin biosynthesis. They were assigned to six categories/superclasses (e.g., lipids and lipid-like molecules, organic acids and derivatives, and organoheterocyclic compound). Finally, ten metabolic pathways shared between KEGG terms specific to aquatic fireflies and enriched by AARMs were screened as aquatic adaptation-related pathways (AARPs). These AARPs were primarily involved in energy metabolism, xenobiotic biodegradation, protection of oxidative/immune damage, oxidative stress response and sense function (e.g., glycine, serine and threonine metabolism, drug metabolism-cytochrome P450 and taste transduction), and certain aspects of morphology (e.g., steroid hormone biosynthesis). These results provide evidence suggesting that abundance changes in metabolomes contribute to freshwater adaptation of fireflies. The metabolites identified here may be vital targets for future work to determine the mechanism of freshwater adaptation in insects.</p>
Project description:While considerable progress has been made towards understanding the complex processes and pathways that regulate human wound healing, regenerative medicine has been unable to develop therapies that coax the natural wound environment to heal scar-free. The inability to induce perfect skin regeneration stems partly from our limited understanding of how scar-free healing occurs in a natural setting. Here we have investigated the wound repair process in adult axolotls and demonstrate that they are capable of perfectly repairing full thickness excisional wounds made on the flank. In the context of mammalian wound repair, our findings reveal a substantial reduction in hemostasis, reduced neutrophil infiltration and a relatively long delay in production of new extracellular matrix (ECM) during scar-free healing. Additionally, we test the hypothesis that metamorphosis leads to scarring and instead show that terrestrial axolotls also heal scar-free, albeit at a slower rate. Analysis of newly forming dermal ECM suggests that low levels of fibronectin and high levels of tenascin-C promote regeneration in lieu of scarring. Lastly, a genetic analysis during wound healing comparing epidermis between aquatic and terrestrial axolotls suggests that matrix metalloproteinases may regulate the fibrotic response. Our findings outline a blueprint to understand the cellular and molecular mechanisms coordinating scar-free healing that will be useful towards elucidating new regenerative therapies targeting fibrosis and wound repair. We used microarray analysis to determine the gene expression changes that take place during scar free wound healing in aquatic and terrestrial axolotl salamanders. Epidermal tissue was harvested using a 4mm biopsy punch. Two wounds were made along the flank and posterior to the forelimbs. Harvested epidermis was pooled for each animal. Four biological replicates were collected from uninjured epidermis (D0) and at 1, 3, and 7 days post injury.