Looking on the horizon; potential and unique approaches to developing radiation countermeasures for deep space travel.

Future lunar missions and beyond will require new and innovative approaches to radiation countermeasures. The Translational Research Institute for Space Health (TRISH) is focused on identifying and supporting unique approaches to reduce risks to human health and performance on future missions beyond low Earth orbit. This paper will describe three funded and complementary avenues for reducing the risk to humans from radiation exposure experienced in deep space. The first focus is on identifying new therapeutic targets to reduce the damaging effects of radiation by focusing on high throughput genetic screens in accessible, sometimes called lower, organism models. The second focus is to design innovative approaches for countermeasure development with special attention to nucleotide-based methodologies that may constitute a more agile way to design therapeutics. The final focus is to develop new and innovative ways to test radiation countermeasures in a human model system. While animal studies continue to be beneficial in the study of space radiation, they can have imperfect translation to humans. The use of three-dimensional (3D) complex in vitro models is a promising approach to aid the development of new countermeasures and personalized assessments of radiation risks. These three distinct and unique approaches complement traditional space radiation efforts and should provide future space explorers with more options to safeguard their short and long-term health.

Conserved function of Drosophila Fancd2 monoubiquitination in response to double-strand DNA breaks.

Fanconi anemia genes play key roles in metazoan DNA damage responses, and human FA mutations cause numerous disease phenotypes. In human cells, activating monoubiquitination of the Fanconi anemia protein Fancd2 occurs following diverse DNA damage stimuli. Monoubiquitinated Fancd2 forms nuclear foci to recruit additional repair factors. Fancd2 animal models to date have focused on molecular nulls or whole gene knockdown, leaving the specific in vivo role of monoubiquitination unclear. Using a point mutant in a conserved residue, we recently linked Drosophila Fancd2 monoubiquitination to a mitosis-specific DNA double-strand break response. In this context, we used CRISPR/Cas9 to generate the first animal model of an endogenous mutation in the conserved monoubiquitination site (fancd2K595R). Here, we expand upon our characterization of fancd2K595R. We also introduce and characterize additional Drosophila tools to study fancd2, including new mutant alleles and GFP-tagged rescue transgenes. Using these new reagents, we show the impact of Drosophila Fancd2 on organismal and cell viability, as well as on repair protein localization, in the presence or absence of double-strand breaks. These findings expand our understanding of Fanconi anemia gene function in vivo and provide useful reagents for DNA repair research.

Exploiting codon usage identifies intensity-specific modifiers of Ras/MAPK signaling in vivo.

Signal transduction pathways are intricately fine-tuned to accomplish diverse biological processes. An example is the conserved Ras/mitogen-activated-protein-kinase (MAPK) pathway, which exhibits context-dependent signaling output dynamics and regulation. Here, by altering codon usage as a novel platform to control signaling output, we screened the Drosophila genome for modifiers specific to either weak or strong Ras-driven eye phenotypes. Our screen enriched for regions of the genome not previously connected with Ras phenotypic modification. We mapped the underlying gene from one modifier to the ribosomal gene RpS21. In multiple contexts, we show that RpS21 preferentially influences weak Ras/MAPK signaling outputs. These data show that codon usage manipulation can identify new, output-specific signaling regulators, and identify RpS21 as an in vivo Ras/MAPK phenotypic regulator.

Indispensable pre-mitotic endocycles promote aneuploidy in the Drosophila rectum.

The endocycle is a modified cell cycle that lacks M phase. Endocycles are well known for enabling continued growth of post-mitotic tissues. By contrast, we discovered pre-mitotic endocycles in precursors of Drosophila rectal papillae (papillar cells). Unlike all known proliferative Drosophila adult precursors, papillar cells endocycle before dividing. Furthermore, unlike diploid mitotic divisions, these polyploid papillar divisions are frequently error prone, suggesting papillar structures may accumulate long-term aneuploidy. Here, we demonstrate an indispensable requirement for pre-mitotic endocycles during papillar development and also demonstrate that such cycles seed papillar aneuploidy. We find blocking pre-mitotic endocycles disrupts papillar morphogenesis and causes organismal lethality under high-salt dietary stress. We further show that pre-mitotic endocycles differ from post-mitotic endocycles, as we find only the M-phase-capable polyploid cells of the papillae and female germline can retain centrioles. In papillae, this centriole retention contributes to aneuploidy, as centrioles amplify during papillar endocycles, causing multipolar anaphase. Such aneuploidy is well tolerated in papillae, as it does not significantly impair cell viability, organ formation or organ function. Together, our results demonstrate that pre-mitotic endocycles can enable specific organ construction and are a mechanism that promotes highly tolerated aneuploidy.

Nonmuscle myosin II is required for cell proliferation, cell sheet adhesion and wing hair morphology during wing morphogenesis.

Metazoan development involves a myriad of dynamic cellular processes that require cytoskeletal function. Nonmuscle myosin II plays essential roles in embryonic development; however, knowledge of its role in post-embryonic development, even in model organisms such as Drosophila melanogaster, is only recently being revealed. In this study, truncation alleles were generated and enable the conditional perturbation, in a graded fashion, of nonmuscle myosin II function. During wing development they demonstrate novel roles for nonmuscle myosin II, including in adhesion between the dorsal and ventral wing epithelial sheets; in the formation of a single actin-based wing hair from the distal vertex of each cell; in forming unbranched wing hairs; and in the correct positioning of veins and crossveins. Many of these phenotypes overlap with those observed when clonal mosaic analysis was performed in the wing using loss of function alleles. Additional requirements for nonmuscle myosin II are in the correct formation of other actin-based cellular protrusions (microchaetae and macrochaetae). We confirm and extend genetic interaction studies to show that nonmuscle myosin II and an unconventional myosin, encoded by crinkled (ck/MyoVIIA), act antagonistically in multiple processes necessary for wing development. Lastly, we demonstrate that truncation alleles can perturb nonmuscle myosin II function via two distinct mechanisms--by titrating light chains away from endogenous heavy chains or by recruiting endogenous heavy chains into intracellular aggregates. By allowing myosin II function to be perturbed in a controlled manner, these novel tools enable the elucidation of post-embryonic roles for nonmuscle myosin II during targeted stages of fly development.

An MYH9 human disease model in flies: site-directed mutagenesis of the Drosophila non-muscle myosin II results in hypomorphic alleles with dominant character.

We investigated whether or not human disease-causing, amino acid substitutions in MYH9 could cause dominant phenotypes when introduced into the sole non-muscle myosin II heavy chain in Drosophila melanogaster (zip/MyoII). We characterized in vivo the effects of four MYH9-like mutations in the myosin rod-R1171C, D1430N, D1847K and R1939X-which occur at highly conserved residues. These engineered mutant heavy chains resulted in D. melanogaster non-muscle myosin II with partial wild-type function. In a wild-type genetic background, mutant heavy chains were overtly recessive and hypomorphic: each was able to substitute partially for endogenous non-muscle myosin II heavy chain in animals lacking zygotically produced heavy chain (but the penetrance of rescue was below Mendelian expectation). Moreover, each of the four mutant heavy chains exhibits dominant characteristics when expressed in a sensitized genetic background (flies heterozygous for RhoA mutations). Thus, these zip/MyoII(MYH9) alleles function, like certain other hypomorphic alleles, as excellent bait in screens for genetic interactors. Our conjecture is that these mutations in D. melanogaster behave comparably to their parent mutations in humans. We further characterized these zip/MyoII(MYH9) alleles, and found that all were capable of correct spatial and temporal localization in animals lacking zygotic expression of wild-type zip/MyoII. In vitro, we demonstrate that mutant heavy chains can dimerize with endogenous, wild-type heavy chains, fold into coiled-coil structures and assemble into higher-order structures. Our work further supports D. melanogaster as a model system for investigating the basis of human disease.

Collection, dechorionation, and preparation of Drosophila embryos for quantitative microinjection.

INTRODUCTIONThe quantitative microinjection of drugs, antibodies, toxins, and manipulated RNAs and proteins into Drosophila embryos--the "pharmacological approach"--provides a unique opportunity to analyze cellular functions in the developing embryo, and provides spatial and temporal resolution that is not readily available through genetic studies. These studies require that the observed effects reflect a dose-response relationship so that the data can be accurately interpreted. Thus, these microinjection approaches require a more refined strategy for handling embryos, and the use of appropriately designed chambers to mount and observe the embryos greatly facilitates analysis of the biological response to a given injected material. This protocol outlines the procedures for collection and preparation of Drosophila embryos for quantitative microinjection.

Quantitative microinjection of Drosophila embryos.

INTRODUCTIONThe quantitative microinjection of drugs, antibodies, toxins, and manipulated RNAs and proteins into Drosophila embryos--the "pharmacological approach"--provides a unique opportunity to analyze cellular functions in the developing embryo, and provides spatial and temporal resolution that is not readily available through genetic studies. These studies require that the observed effects reflect a dose-response relationship so that the data can be accurately interpreted. Quantitative microinjections can be readily achieved with the addition of a fluorescent tracer to the solution to be injected. Analysis of the resulting integrated fluorescent intensity following injection can then be used to determine the volume and hence the concentration of the solution injected. This protocol outlines the procedures for the microinjection and quantification of aqueous solutions during high-resolution observation of early development in the Drosophila embryo.

Quantitative microinjection of Drosophila embryos: general strategy.

INTRODUCTIONMicroinjection of Drosophila embryos is a common technique used by a wide range of investigators, but some applications require a refined strategy for handling embryos. This article outlines the general procedures for microinjection and quantification of aqueous solutions during high-resolution observation of early development in the fly embryo. It also describes the design of suitable support slides for the manipulation of Drosophila embryos under upright and inverted microscopes.

Nonmuscle myosin II generates forces that transmit tension and drive contraction in multiple tissues during dorsal closure.

BACKGROUND: The morphogenic movements that characterize embryonic development require the precise temporal and spatial control of cell-shape changes. Drosophila dorsal closure is a well-established model for epithelial sheet morphogenesis, and mutations in more than 60 genes cause defects in closure. Closure requires that four forces, derived from distinct tissues, be precisely balanced. The proteins responsible for generating each of the forces have not been determined. RESULTS: We document dorsal closure in living embryos to show that mutations in nonmuscle myosin II (encoded by zipper; zip/MyoII) disrupt the integrity of multiple tissues during closure. We demonstrate that MyoII localization is distinct from, but overlaps, F-actin in the supracellular purse string, whereas in the amnioserosa and lateral epidermis each has similar, cortical distributions. In zip/MyoII mutant embryos, we restore MyoII function either ubiquitously or specifically in the leading edge, amnioserosa, or lateral epidermis and find that zip/MyoII function in any one tissue can rescue closure. Using a novel, transgenic mosaic approach, we establish that contractility of the supracellular purse string in leading-edge cells requires zip/MyoII-generated forces; that zip/MyoII function is responsible for the apical contraction of amnioserosa cells; that zip/MyoII is important for zipping; and that defects in zip/MyoII contractility cause the misalignment of the lateral-epidermal sheets during seam formation. CONCLUSIONS: We establish that zip/MyoII is responsible for generating the forces that drive cell-shape changes in each of the force-generating tissues that contribute to closure. This highly conserved contractile protein likely drives cell-sheet movements throughout phylogeny.