Estrogens act by binding to estrogen receptors alpha and beta (ERα, ERβ), ligand-dependent transcription factors that play crucial roles in sex differentiation, tumor growth and cardiovascular physiology. Estrogens also activate the G protein-coupled estrogen receptor (GPER), however the function of GPER in vivo is less well understood. Here we find that GPER is required for normal heart rate in zebrafish embryos. Acute exposure to estrogens increased heart rate in wildtype and in ERα and ERβ mutant embryos but not in GPER mutants. GPER mutant embryos exhibited reduced basal heart rate, while heart rate was normal in ERα and ERβ mutants. We detected gper transcript in discrete regions of the brain and pituitary but not in the heart, suggesting that GPER acts centrally to regulate heart rate. In the pituitary, we observed gper expression in cells that regulate levels of thyroid hormone triiodothyronine (T3), a hormone known to increase heart rate. Compared to wild type, GPER mutants had reduced levels of T3 and estrogens, suggesting pituitary abnormalities. Exposure to exogenous T3, but not estradiol, rescued the reduced heart rate phenotype in gper mutant embryos, demonstrating that T3 acts downstream of GPER to regulate heart rate. Using genetic and mass spectrometry approaches, we find that GPER regulates maternal estrogen levels, which are required for normal embryonic heart rate. Our results demonstrate that estradiol plays a previously unappreciated role in the acute modulation of heart rate during zebrafish embryonic development and suggest that GPER regulates embryonic heart rate by altering maternal estrogen levels and embryonic T3 levels.

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Zebrafish are a powerful model system to assess the molecular and cellular effects of exposure to toxic chemicals during embryonic development. To study the effects of environmental endocrine disruptors, embryos and larvae are commonly exposed to supraphysiologic concentrations of these compounds in the water, but their bioavailability in zebrafish is largely unknown. One hypothesis is that supraphysiologic concentrations of estrogens in the water are required to achieve physiologic levels in vivo; however, this has not been directly tested. To test this hypothesis, we developed an assay using radiolabeled estradiol ([3H]E2) to measure uptake from water at multiple concentrations and exposure durations in developing zebrafish from 0 to 5 days postfertilization (dpf). We found that [3H]E2 uptake increased with increasing concentration, duration, and developmental stage. Percent uptake from the total volume of treatment solution increased with increasing exposure duration and developmental stage, but remained constant with increasing concentration. We also found that the chorion, an acellular envelope surrounding embryos through 3 dpf, did not substantially affect [3H]E2 uptake. Finally, we found that at 1 dpf, E2 was preferentially taken up by the yolk at multiple exposure durations, while at 2 dpf E2 was preferentially taken up into the embryonic body. Our results support the hypothesis that exposing zebrafish embryos and larvae to supraphysiologic concentrations of estrogens is required to achieve physiologically relevant doses in vivo. The isotopic assay reported here will provide a foundation for determining the uptake of other compounds for teratogenicity, toxicology and drug discovery studies.

Human immunodeficiency virus (HIV) integrase inhibitors are increasingly being used for antiretroviral therapy (ART), and dolutegravir (DTG/Tivicay) has emerged as a leading core agent. In 2018, the Tsepamo study reported a 6-to 9-fold increase for neural tube defect (NTD) risk among the offspring of mothers receiving DTG during early gestation. Maternal folate (vitamin B9) status is the largest known modifier of NTD risk, so we evaluated folate-related mechanisms of action and the critical period for DTG developmental toxicity. Folate receptor (FOLR1) binding studies indicate DTG is a non-competitive FOLR1 antagonist at therapeutic concentrations. In vitro testing indicates calcium (2mM) increases FOLR1-folate interactions and alters DTG-FOLR1-folate interactions and cytotoxicity. DTG does not inhibit downstream folate metabolism by dihydrofolate reductase (DHFR). Early embryonic exposure toDTG is developmentally toxic in zebrafish, and supplemental folic acid can mitigate DTG developmental toxicity. The results from these studies are expected to inform and guide future animal models and clinical studies of DTG-based ART in women of childbearing age. The human immunodeficiency virus and acquired immune deficiency syndrome (HIV/AIDS) epidemic remains a prominent public health challenge. Globally, there are approximately 36.7 million people living with HIV, with 2.1 million new infections occurring annually, resulting in 1.1 million AIDS-related deaths per year 1 . Current HIV antiretroviral therapy (ART) includes C-C chemokine receptor type 5 (CCR5) antagonists, fusion inhibitors, integrase inhibitors, nucleoside and non-nucleoside reverse transcriptase inhibitors (NRTIs and NNRTIs), and protease inhibitors. Dolutegravir (DTG) is the clinically preferred integrase inhibitor used alone (Tivicay, ViiV Healthcare), or in combination (e.g. Triumeq, ViiV Healthcare) as ART for HIV infection in adults and children. A teratogenic risk for DTG was recently reported after neural tube defects (NTDs) were observed in four infants from mothers who had been taking DTG at the time of conception in Botswana 2 . The World Health Organization (WHO) subsequentlyprovided guidelines that DTG use be avoided by women of childbearing potential unless they used adequate contraception methods 3 . Based on this cohort study, the current guidelines are warranted and prudent, but more evidence is needed before scientific and medical communities can support a causal relationship between DTG and developmental toxicity, including NTDs. Developmental Toxicity of DTG:There is increasing usage of the HIV integrase inhibitors are for ART, and DTG has emerged as the leading compound in this class of medications. The DTG/Tivicay manufacturer reports (US FDA approved Tivicay Prescribing Information, 09/2018) animal reproduction studies showed no evidence of adverse developmental outcomes. Specifically, DTG was administered orally at up to 1,000 mg per kg daily, in rats and rabbits, during the period of organogenesis, days 6-17 and 6-18, respectively...