High altitude studies have been insightful in revealing putative hypoxia tolerant genes to improve pathological and physiological conditions that involve hypoxia or ischemia. Whole-genome sequencing of Ethiopian highlanders identified LIPE (Lipase E, Hormone Sensitive Type), as an important gene that allows them to live in chronic low oxygen conditions (Udpa et al., 2014). Using Drosophila melanogaster, ubiquitous knockdown of the ortholog of this gene, hormone-sensitive lipase (Hsl), results in increased tolerance to chronic hypoxia. To elucidate the tissue specificity of the adaptive phenotype, UAS-RNAi(Hsl) line was crossed with different cell- or tissue-specific GAL4 drivers to systematically knockdown this gene, without altering its expression in the rest of the organism. There was a remarkable difference in eclosion rates when Hsl was knocked down in eclosion hormone-expressing neurons, third instar larval fat body, oenocytes, and hemocytes, constituting a four-fold increase over controls (all knockdown crosses were >87%, P<0.005). Since Hsl plays a prominent role in lipid metabolism, its inhibition in the cell(s) or tissue(s) that produced the most profound response to hypoxia tolerance were quantified to provide evidence of any changes in triglyceride levels. An augmented triglyceride concentration was observed in Hsl knockdown crosses, specifically in, UAS-RNAi(Hsl) x da-GAL4 and UAS-RNAi(Hsl) x Eh-GAL4 with a 1.5- to 2-fold increase in hypoxia when compared with controls. This data suggests that lipid accumulation as a result of Hsl knockdown may be biochemically significant in promoting hypoxia tolerance.

Methadone is a synthetic opioid analgesic and a common pharmacological treatment for opioid use disorder (OUD) in pregnant women. Its clinical application blocks the effects of other opioids and attenuates withdrawal symptoms or behaviors leading to relapse or overdose. However, methadone’s ability to readily enter fetal circulation, accumulate in neural tissue, and cause long-term neurocognitive sequelae, has led to concerns regarding its effect on fetal brain development.

In the present research, human induced pluripotent stem cell-derived cortical organoid (hCO) technology was leveraged to probe the etiology of these deficits by understanding how methadone impacts the earliest mechanisms of cortical development.

Bulk mRNA sequencing of 2-month-old hCOs chronically treated with a clinically relevant dose of 1μM methadone revealed a robust transcriptional response to methadone associated with functional components of the synapse, including pre-synaptic vesicular trafficking/release and post-synaptic signal reception machinery, as well as the underlying extracellular matrix (ECM), and cilia. Co-expression network and predictive protein-protein interaction analyses demonstrated that these changes occurred in concert, centered around a regulatory axis of growth factors, developmental signaling pathways, and matricellular proteins (MCPs). Of the central MCPs, thrombospondin 1 (TSP1), which is necessary for synapse formation and maturation, was most prominently downregulated, with dose-dependent reductions in its protein levels. These results indicated that methadone exposure during cortical development alters transcriptional programs associated with synapse biology by modulating ECM and ciliary molecular mechanisms.

To further dissect methadone’s impact at the synapse, ultrastructural imaging was performed to visualize and quantify changes in synaptic features. Improving upon the limitations of prior ultrastructural studies of synapses in brain organoids, culture, imaging, and segmentation methodologies were modified to optimize the volumetric capture of synaptic features via electron microscopy. These techniques revealed that 10μM methadone reduces synaptic vesicle volumes as well as the range of vesicle morphologies in 3-month-old hCOs, which may have implications for the reductions in neurotransmission observed following methadone exposure.

Cumulatively, the findings presented in this dissertation provide novel insight into the molecular underpinnings of methadone’s effect on neurocognitive development, novel methods of studying these effects in brain organoids, and a basis for improving interventions for maternal OUD.

With congenital heart disease as the most common birth defect in the world, investigating the genetic basis of heart development is an important endeavor that can be greatly advanced by studying the model organism Drosophila melanogaster. At the end of embryonic fly heart development, there are three types of pericardial cells (PCs) that are distinguished by their gene expression profiles. Among these PCs, those that specifically express the cardiac master regulator tinman (tin) are not well characterized (Tin-PCs). In this study, we use single-cell RNA sequencing, fluorescent staining, and mutational analysis to characterize the role of Tin-PCs in embryonic fly heart development. We have identified and confirmed the expression of several genes—CrzR, Neurotactin, Lachesin, coracle, sinuous, Wnt4, and cut—that were either distinctly expressed or highly enriched in Tin-PCs, but not in other types of PCs. Here, we present that knockdown of sinuous delayed dorsal closure of the heart and epidermis, knockdown of tin reduced expression of the cell adhesion protein Neurotactin, and loss of cut caused abnormal closure of Tin-PCs while ectopic Cut expression in cardioblasts reduced expression of the PC marker even-skipped. From these findings, we conclude that Tin-PCs play some role in dorsal closure, cardiac cell adhesion, and even-skipped expression in PCs of the Drosophila embryo. By elucidating the role of Tin-PCs in Drosophila embryogenesis, we hope to highlight the great potential of single-cell RNA sequencing in identifying novel cell types and provide further understanding of the cellular and genetic mechanisms that underlie Drosophila heart development.

Heart disease has been the leading cause of death in the U.S. over the past century despite its decreasing rate over the last 50 years. Understanding the function of new genes and pathways in a simple model system may provide novel insights and lead to new therapeutic targets for heart disease in humans. In mammalian systems, calcineurin-NFAT signaling has been implicated in the regulation of the cardiac hypertrophic response. However, how this gene interacts with other molecular pathways in the heart is unknown. In order to better understand this signaling process, the singular function of calmodulin (CaM), calcineurin (Pp2B-14D), and Nuclear Factor of Activated T-Cells (NFAT), in the heart was investigated, using the animal model system Drosophila melanogaster. Loss-of-function and gain-of-function studies were conducted in a tissue-specific manner using the GAL4/UAS system by means of RNAi and overexpression lines, respectively. The flies were dissected to expose the beating heart tube; hearts were filmed and analyzed using Semi-Automated Optical Heartbeat Analysis (SOHA) and key cardiac performance indicators were measured. Slightly significant differences between NFAT gene knockdown and respective controls using two RNAi lines were observed, yet there were no obvious changes observed in cardiac size. However, this may be due to insufficient KD or due to compensation by changes in expression of other genes. Overall, cardiac size was reduced in Pp2B-14D KD and increased in Pp2B-14D OE. Finally, CaM KD resulted in significant contractile defects when compared to their respective controls.

Cardiomyopathy is a feature of many hypoxia-induced diseases, and affects millions of people worldwide suffering conditions including pulmonary disease, inflammation, and high altitude. Interestingly, highlanders with beneficial genetic adaptations to high altitude have remarkably low incidence of cardiomyopathies. In contrast, pathological cardiac hypertrophy is the hallmark feature of disease in other, poorly adapted highland populations. Detailed mechanisms of these cardiac responses remain largely unknown, yet examination of populations selected for survival in hypoxic environments provides a means to unravel genetic contributions to hypoxia-related disease, even for lowland populations.

Drosophila, with its short lifespan and extensive genetic toolbox, is an excellent organism to investigate conserved and novel pathways underlying cardiac hypoxia responses, particularly through use of a unique population with multi-generational adaptation to hypoxia (‘hypoxia-selected’). We advance Drosophila models of cardiac hypoxia and show the fly heart responds differentially to acute, sustained, chronic and multi-generational hypoxia, and these responses are partly mediated by the HIF1α homolog, sima.

We further explore hypoxia-selected fly cardiac physiology, and find effects of an acute hypoxia stress are particularly pronounced in hypoxia-selected fly hearts, even after removal of immediate environmental selection pressure. Most notably, we find persistent reduction in cardiac size in hypoxia-selected flies, but not in control flies exposed to chronic hypoxia, suggesting underlying genetic changes.

We used transcriptome analyses of hypoxia-selected and chronically hypoxic fly hearts to explore contribution of calcineurin to the persistent changes observed in hypoxia-selected fly hearts. Using a heart-specific GAL4 system to modulate expression, we find knockdown of the primary calcineurin A homologues, CanA14F or Pp2B, cause cardiac restriction which phenocopies effects found in hypoxia-selected flies. We propose the calcineurin pathway as uniquely altered in the hypoxia-selected fly heart, and provide insight on mechanisms underlying cardiac adaptation to high altitude and development of cardiac disease.

In summary, genes identified from hypoxia-selected populations, human or fly, can alter responses to normal cardioprotective mechanisms in the fly, as in HIFα mutants (first identified in well-adapted humans) and calcineurin-deficiency (identified in hypoxia-selected flies). We provide new insight into the physiologic mechanisms of cardiac remodeling during various hypoxia exposures, evidence for the genetic basis of cardiac adaptations, and establish markers of cardiac disease states.