Rapid identification of reproductive toxicants among environmental chemicals using an in vivo evaluation of gametogenesis in budding yeast Saccharomyces cerevisiae.

Infertility affects ∼12 % of couples, with environmental chemical exposure as a potential contributor. Of the chemicals that are actively manufactured, very few are assessed for reproductive health effects. Rodents are commonly used to evaluate reproductive effects, which is both costly and time consuming. Thus, there is a pressing need for rapid methods to test a broader range of chemicals. Here, we developed a strategy to evaluate large numbers of chemicals for reproductive toxicity via a yeast, S. cerevisiae high-throughput assay to assess gametogenesis as a potential new approach method (NAM). By simultaneously assessing chemicals for growth effects, we can distinguish if a chemical affects gametogenesis only, proliferative growth only or both. We identified a well-known mammalian reproductive toxicant, bisphenol A (BPA) and ranked 19 BPA analogs for reproductive harm. By testing mixtures of BPA and its analogs, we found that BPE and 17 ß-estradiol each together with BPA showed synergistic effects that worsened reproductive outcome. We examined an additional 179 environmental chemicals including phthalates, pesticides, quaternary ammonium compounds and per- and polyfluoroalkyl substances and found 57 with reproductive effects. Many of the chemicals were found to be strong reproductive toxicants that have yet to be tested in mammals. Chemicals having affect before meiosis I division vs. meiosis II division were identified for 16 gametogenesis-specific chemicals. Finally, we demonstrate that in general yeast reproductive toxicity correlates well with published reproductive toxicity in mammals illustrating the promise of this NAM to quickly assess chemicals to prioritize the evaluation for human reproductive harm.

A tailed mirtron promotes longevity in Drosophila.

Thousands of atypical microRNAs (miRNAs) have been described in the genomes of animals; however, it is unclear if many of these non-canonical miRNAs can measurably influence phenotypes. Mirtrons are the largest class of non-canonical miRNAs that are produced from hairpins excised by splicing, which after debranching become substrates for Dicer and load into RISC. Most mirtrons require additional processing after splicing to remove 'tail' residues interposed between one of the host intron splice sites and base of the hairpin precursor structure. Despite most mirtrons requiring tail removal no function has been elucidated for a tailed species, indeed for all mirtrons identified function has only been assigned to a single species. Here we study miR-1017, a mirtron with a 3' tail, which is well expressed and conserved in Drosophila species. We found that miR-1017 can extend lifespan when ectopically expressed in the neurons, which seems partly due to this miRNA targeting its host transcript, acetylcholine receptor Dα2. Unexpectedly we found that not only did miR-1017 function in trans but also in cis by affecting splicing of Dα2. This suggests a mechanism for mirtron evolution where initial roles of structural elements in splicing lead to secondary acquisition of trans-regulatory function.

A deficiency screen of the 3rd chromosome for dominant modifiers of the Drosophila ER integral membrane protein, Jagunal.

The mechanism surrounding chromosome inheritance during cell division has been well documented, however, organelle inheritance during mitosis is less understood. Recently, the endoplasmic reticulum (ER) has been shown to reorganize during mitosis, dividing asymmetrically in proneuronal cells prior to cell fate selection, indicating a programmed mechanism of inheritance. ER asymmetric partitioning in proneural cells relies on the highly conserved ER integral membrane protein, Jagunal (Jagn). Knockdown of Jagn in the compound Drosophila eye displays a pleotropic rough eye phenotype in 48% of the progeny. To identify genes involved in Jagn dependent ER partitioning pathway, we performed a dominant modifier screen of the 3rd chromosome for enhancers and suppressors of this Jagn-RNAi-induced rough eye phenotype. We screened through 181 deficiency lines covering the 3L and 3R chromosomes and identified 12 suppressors and 10 enhancers of the Jagn-RNAi phenotype. Based on the functions of the genes covered by the deficiencies, we identified genes that displayed a suppression or enhancement of the Jagn-RNAi phenotype. These include Division Abnormally Delayed (Dally), a heparan sulfate proteoglycan, the γ-secretase subunit Presenilin, and the ER resident protein Sec63. Based on our understanding of the function of these targets, there is a connection between Jagn and the Notch signaling pathway. Further studies will elucidate the role of Jagn and identified interactors within the mechanisms of ER partitioning during mitosis.

Microtubules are necessary for proper Reticulon localization during mitosis.

During mitosis, the structure of the Endoplasmic Reticulum (ER) displays a dramatic reorganization and remodeling, however, the mechanism driving these changes is poorly understood. Hairpin-containing ER transmembrane proteins that stabilize ER tubules have been identified as possible factors to promote these drastic changes in ER morphology. Recently, the Reticulon and REEP family of ER shaping proteins have been shown to heavily influence ER morphology by driving the formation of ER tubules, which are known for their close proximity with microtubules. Here, we examine the role of microtubules and other cytoskeletal factors in the dynamics of a Drosophila Reticulon, Reticulon-like 1 (Rtnl1), localization to spindle poles during mitosis in the early embryo. At prometaphase, Rtnl1 is enriched to spindle poles just prior to the ER retention motif KDEL, suggesting a possible recruitment role for Rtnl1 in the bulk localization of ER to spindle poles. Using image analysis-based methods and precise temporal injections of cytoskeletal inhibitors in the early syncytial Drosophila embryo, we show that microtubules are necessary for proper Rtnl1 localization to spindles during mitosis. Lastly, we show that astral microtubules, not microfilaments, are necessary for proper Rtnl1 localization to spindle poles, and is largely independent of the minus-end directed motor protein dynein. This work highlights the role of the microtubule cytoskeleton in Rtnl1 localization to spindles during mitosis and sheds light on a pathway towards inheritance of this major organelle.

Conserved role for Ataxin-2 in mediating endoplasmic reticulum dynamics.

Ataxin-2, a conserved RNA-binding protein, is implicated in the late-onset neurodegenerative disease Spinocerebellar ataxia type-2 (SCA2). SCA2 is characterized by shrunken dendritic arbors and torpedo-like axons within the Purkinje neurons of the cerebellum. Torpedo-like axons have been described to contain displaced endoplasmic reticulum (ER) in the periphery of the cell; however, the role of Ataxin-2 in mediating ER function in SCA2 is unclear. We utilized the Caenorhabditis elegans and Drosophila homologs of Ataxin-2 (ATX-2 and DAtx2, respectively) to determine the role of Ataxin-2 in ER function and dynamics in embryos and neurons. Loss of ATX-2 and DAtx2 resulted in collapse of the ER in dividing embryonic cells and germline, and ultrastructure analysis revealed unique spherical stacks of ER in mature oocytes and fragmented and truncated ER tubules in the embryo. ATX-2 and DAtx2 reside in puncta adjacent to the ER in both C. elegans and Drosophila embryos. Lastly, depletion of DAtx2 in cultured Drosophila neurons recapitulated the shrunken dendritic arbor phenotype of SCA2. ER morphology and dynamics were severely disrupted in these neurons. Taken together, we provide evidence that Ataxin-2 plays an evolutionary conserved role in ER dynamics and morphology in C. elegans and Drosophila embryos during development and in fly neurons, suggesting a possible SCA2 disease mechanism.

Congenital Zika Virus Infection in Immunocompetent Mice Causes Postnatal Growth Impediment and Neurobehavioral Deficits.

A small percentage of babies born to Zika virus (ZIKV)-infected mothers manifest severe defects at birth, including microcephaly. Among those who appeared healthy at birth, there are increasing reports of postnatal growth or developmental defects. However, the impact of congenital ZIKV infection in postnatal development is poorly understood. Here, we report that a mild congenital ZIKV-infection in pups born to immunocompetent pregnant mice did not display apparent defects at birth, but manifested postnatal growth impediments and neurobehavioral deficits, which include reduced locomotor and cognitive deficits that persisted into adulthood. We found that the brains of these pups were smaller, had a thinner cortical layer 1, displayed increased astrogliosis, decreased expression of microcephaly- and neuron development- related genes, and increased pathology as compared to mock-infected controls. In summary, our results showed that even a mild congenital ZIKV infection in immunocompetent mice could lead to postnatal deficits, providing definitive experimental evidence for a necessity to closely monitor postnatal growth and development of presumably healthy human infants, whose mothers were exposed to ZIKV infection during pregnancy.