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.

Unlike mammals, western clawed frog (Xenopus tropicalis) tadpoles are able to completely regenerate their spinal cord after tail amputation. This complete spinal cord regeneration is due to the ability of their neural progenitor cells (NPCs) to differentiate into neurons successfully. Our research focuses on two transcription factors—Meis1 and Pbx3– that are upregulated by regenerating neurons and are necessary for successful regeneration. We aim to elucidate how these two proteins are working together to guide successful spinal cord regeneration in X. tropicalis tadpoles. I am specifically investigating Meis1 and Pbx3 splice variant expression during neural regeneration. Previous work in mice found that different known splice variants of Pbx3 have different expression patterns. While X. tropicalis has two predicted splice variants each of Meis1 and Pbx3, nothing is known about their individual expression or function. I sought to fill in this gap by looking at Meis1 and Pbx3 splice variant expression in different tissues and over regenerative time. Based on previous research in mice, I hypothesize that both splice variants of Meis1 and Pbx3 have different gene expression patterns in different cell types over regenerative time. I aimed to investigate this hypothesis by doing two experiments. My first experiment was to study the expression of each splice variant over regenerative time by performing qPCRs in order to look at the presence of splice variant mRNA in uninjured, 24, and 72 hours post-amputation (hpa). For my second experiment, I am making in situ hybridization probes specific for each splice variant to identify their tissue-specific expression patterns. The experiment is being performed over regenerative time to observe how expression in tissues changes at the same time points hpa used in the first experiment.

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.