Embryonic development is a process of regulated growth by which all cells are initially established. Previous studies suggest that there may be differential metabolic regulation in developing embryonic tissues. However, little research has been conducted to determine the specific metabolic factors that are differentially active during development and the key regulatory elements behind these metabolic processes, such as glycolysis. Hypoxy-inducible factor 1-alpha, hif1α, is a stress-induced transcription factor that is known to regulate glycolysis under hypoxic conditions. This project aims to investigate the role of hif1α in regulating the expression of glycolytic genes in the developing axial tissues of the Xenopus tropicalis embryo. X. tropicalis is a unique model for studying embryonic development. Due to their complete early-stage embryo cleavages, inhibitors can be restricted to one side of the embryo allowing for direct comparison with an internal control on the other side of the embryo. Preliminary data from in situ hybridization suggests that the inhibition of hif1α using translation-blocking morpholinos reduces glycolytic gene expression in early-stage X. tropicalis embryos. Based on these results, we plan to further test the regionalization of glycolysis, by determining what causes hif1α to activate a glycolytic gradient in certain tissues in the X. tropicalis embryo. This research implicates hif1α as a potentially important transcriptional regulator of glycolytic gene expression during embryonic development, and can lead to discovering new ways in which differential metabolic regulation can contribute to the form and function of embryos.

While humans cannot regrow a limb after amputation, animals like the Xenopus tropicalis tadpoles can fully regenerate their limbs. How metabolic pathways are reprogrammed to support the demand for anabolic building blocks needed for regeneration is still not understood. Our group has found that while regeneration increases glucose intake, tadpole tail regeneration does not depend on glycolysis for precursors to support proliferation. This result suggests a redirection of glucose flux to pathways other than glycolysis, specifically the pentose phosphate pathway (PPP). Previously, I inhibited glucose-6-phosphate dehydrogenase (G6PD), a key enzyme in the PPP, with the pharmacological antagonists dehydroepiandrosterone (DHEA) and G6PDi and showed that inhibitor-treated tails are much shorter compared to dimethyl sulfoxide (DMSO) controls. From these results, I predicted that the PPP is required throughout regeneration to support increased cell proliferation rates. To investigate if the PPP is required for increased cell proliferation during regeneration, I performed phosphohistone-H3 (pH3) immunohistochemistry to label mitotic cells. Histone-H3 is phosphorylated at the start of mitosis, making pH3 a marker for actively dividing cells. The results showed that amputated tadpoles with inhibited PPP have reduced cell proliferation rates compared to controls, confirming that the PPP is required to support rapid cell proliferation during regeneration. To determine if the PPP has a shorter critical activation window during regeneration, I treated amputated tadpoles with PPP inhibitors while varying the length and start-time of treatments. PPP-inhibited tadpoles have significantly shorter tails as treatment length increases, regardless of the start time for PPP inhibition. This result suggests that PPP activity must be sustained throughout regeneration to fully regrow the tail. My work has therefore identified the PPP as a previously unknown but critical metabolic pathway promoting tadpole tail regeneration. This insight advances our understanding of how metabolic reprogramming provides the carbon building blocks for regeneration.

Chronic pain -- pain that continues beyond expected healing time that may or may not be linked with tissue damage -- is a common condition among older (≥65 years) adults, often caused by osteoarthritis (OA) in weight-bearing joints, such as the knee. Older adults with knee OA often experience movement-evoked pain that is associated with reduced mobility, activity avoidance, and social relationship disruption. Knee OA is also associated with increased falls risk, which is a leading cause of injury and mortality among older adults. However, the role of movement-evoked pain in knee OA and falls risk is unclear. It is conceivable that movement-evoked pain contributes to falls risk via impaired neuromuscular function and knee buckling. Thus, better understanding movement-evoked pain might help identify rehabilitation targets to reduce falls risk. While literature demonstrates differences between movement-evoked pain and pain-at-rest, current assessments cannot accurately discern which surveyed pain levels are movement-evoked. Example methodologies that correlate activity and pain levels include pain diaries, accelerometers, or ecological momentary assessment (EMA) surveys. Such methodologies are often dependent on a participant’s ability to recall and differentiate pain experienced in relation to movement, and do not directly assess what movements evoke specific pain levels. The proposed pilot project: (1) develops a protocol for novel trigger-based smartphone EMAs using the Move 4 accelerometer, (2) evaluates the protocol’s feasibility when implemented in older adults with knee OA, and (3) analyzes relationships between movement-evoked pain, knee buckling, and falls. We anticipate that EMA collection will be acceptable for most participants, but are concerned about the accelerometer. The non-invasive Move 4 is capable of triggering EMAs when specific movement thresholds are achieved in real time. Thus, the proposed project addresses a major shortcoming in the field that currently relies on participant recall and does not capture pain levels immediately following specific movement patterns.