Physical forces greatly influence the growth and function of an organism. Altered gravity can perturb normal development and induce corresponding changes in gene expression. Understanding this relationship between the physical and biological realms is important for NASA s space travel goals. We use combined RNA-Seq and qRT-PCR to profile changes in early Drosophila melanogaster pupae exposed to chronic hypergravity (3 g three times Earth s gravity) to highlight gravity-dependent pathways and gene products. Robust transcriptional response was evident among the pupae developed in a hypergravity environment compared to control. 1,513 genes showed significantly (p < 0.05) altered gene expression in the 3 g samples. These findings were supported with qRT-PCR data. Major biological processes affected include ion transport redox homeostasis immune and humoral stress response proteolysis and cuticle development.

Genome-wide transcriptional profiling shows that reducing gravity levels in the International Space Station (ISS) causes important alterations in Drosophila gene expression. However simulation experiments on ground without space constraints show weaker effects than space environment. A global and integrative analysis using the gene expression dynamics inspector (GEDI) self-organizing maps reveals a subtle response of the transcriptome using different populations and microgravity and hypergravity simulation devices. These results suggest that in addition to behavioural responses that can be detected also at the gene expression level the transcriptome is finely tuned to normal gravity. The alteration of this constant parameter on Earth can have effects on gene expression that depends both on the environmental conditions and the ground based facility used to compensate the gravity vector. Alternative and commons effects of mechanical facilities like the Random Positioning Machine and a centrifuge and strong magnetic field ones like a cryogenically cooled superconductive magnet are discussed. We compare the effects over the gene expression profile of different gender/age Drosophila imagoes in 3-4 days-long experiments under altered gravity conditions into three GBF (Ground Based Facilities for micro/hyper- gravity simulation) using whole genome microarray platforms. Descriptions of different GBFs (treatments): LDC means Large Diameter Centrifuge. Samples can be placed under three conditions: inside LDC (at certain g level) at the LDC rotational control and at external 1g control (outside the LDC). RPM means Random Positioning Machine. Samples can be placed under two conditions: inside RPM (at nearly 0g Microgravity level) and at external 1g control (outside the RPM). At the magnet means INSIDE the Magnetic levitator (another GBF). Samples can be placed under four conditions: inside Magnet 0g* (at microgravity with magnetic field) inside Magnet at 1g* (internal control with magnetic field) or inside the magnet 2g* (at hypergravity with magnetic field) and at external 1g control (outside the magnet)

In prospective human exploration of outer space the need to maintain a species over several generations under changed gravity conditions may arise. This paper reports the analysis of the third generation of fruit fly Drosophila melanogaster obtained during the 44.5-day space flight (Foton-M4 satellite 2014 Russia) followed by the fourth generation on Earth and the fifth generation under conditions of a 12-day space flight (2014 in the Russian Segment of the ISS). The obtained results show that it is possible to obtain the third-fifth generations of a complex multicellular Earth organism under changed gravity conditions (in the cycle weightlessness - Earth - weightlessness) which preserves fertility and normal development. However there were a number of changes in the expression levels and content of cytoskeletal proteins that are the key components of the spindle apparatus and the contractile ring of cells.

Gene expression levels were determined in 3rd instar and adult Drosophila melanogaster reared during spaceflight to elucidate the genetic and molecular mechanisms underpinning the effects of microgravity on the immune system. The goal was to validate the Drosophila model for understanding alterations of innate immune responses in humans due to spaceflight. Five containers of flies with ten female and five male fruit flies in each container were housed and bred on the space shuttle (average orbit altitude of 330.35 km) for 12 days and 18.5 hours with a new generation reared in microgravity. RNA was extracted on the day of shuttle landing from whole body animals (3rd instar larvae and adults) hybridized to Drosophila 2.0 Affymetrix genome arrays and the expression level of all genes was normalized against the gene expression level from the corresponding developmental stage animals raised on ground. Spaceflight altered the expression of larval genes involved in the maturation of plasmatocytes (macrophages) and their phagocytic response as well as the level of constitutive expression of pattern recognition receptors and opsonins that specifically recognize bacteria and of lysozymes antimicrobial peptide pathway and immune stress genes hallmarks of humoral immunity. Larval microarrays (FL 6 samples) are based on RNA extracted from 6 independent sets of 50 mid 3rd instar larvae reared in microgravity and collected on the day of landing after 12 days and 18.5 hours on the space shuttle and the same number of control larvae raised on ground (GL 6 samples). Adults microarrays (F1 3 samples) are based on RNA from 3 sets of 20 adult females each that emerged during spaceflight and within 4 hours of landing and the same number of adult females from the corresponding ground control containers (G1 3 samples).

The project focuses on understanding the transcriptional and post-transcriptional mechanisms that regulate early seedling development in spaceflight and microgravity. One of the goals of the PRR experiment was to study the role of small regulatory RNAs in plant response to the space environment using Arabidopsis thaliana.

Transcriptional crosstalk between mammary gland liver and adipose tissue Experiment Overall Design: Pregnant and Lactating rats exposed to 3 gravity conditions

Experimentation on the International Space Station has reached the stage where repeated and nuanced transcriptome studies are beginning to illuminate the structural and metabolic differences between plants grown in space compared to plants on the Earth. Genes that are important in setting up the spaceflight responses are being identified; their role in spaceflight physiological adaptation are increasingly understood and the fact that different genotypes adapt differently is recognized. However the basic question of whether these spaceflight responses are required for survival has yet to be posed and the fundamental notion that spaceflight responses may be non-adaptive has yet to be explored. Therefore the experiments presented here were designed to ask if portions of the plant spaceflight response can be genetically removed without causing loss of spaceflight survival and without causing increased stress responses. The CARA experiment compared the spaceflight transcriptome responses of two Arabidopsis ecotypes Col-0 and WS as well as that of a PhyD mutant of Col-0. When grown with the ambient light of the ISS phyD displayed a significantly reduced spaceflight transcriptome response compared to Col-0 suggesting that altering the activity of a single gene can actually improve spaceflight adaptation by reducing the transcriptome cost of physiological adaptation. The WS genotype showed an even simpler spaceflight transcriptome response in the ambient light of the ISS more broadly indicating that the plant genotype can be manipulated to reduce the transcriptome cost of plant physiological adaptation to spaceflight and suggesting that genetic manipulation might further reduce or perhaps eliminate the metabolic cost of spaceflight adaptation. When plants were germinated and then left in the dark on the ISS the WS genotype actually mounted a larger transcriptome response than Col-0 suggesting that the in-space light environment affects physiological adaptation which further implies that manipulating the local habitat can also substantially impact the metabolic cost of spaceflight adaptation.

Transcriptional crosstalk between mammary gland liver and adipose tissue Experiment Overall Design: Pregnant and Lactating rats exposed to 3 gravity conditions

In this study transcriptome profiling was used to gain insight into the spaceflight adaptation role of Altered response to gravity-1 (Arg1) a gene known to affect gravity responses in plants on Earth. The study compared expression profiles of cultured lines of Arabidopsis thaliana derived from wild type (WT) cultivar Col-0 to profiles from a knock-out line deficient in the gene encoding (ARG1 KO) both on the ground and in space. The cell lines were launched on SpaceX CRS-2 as part of the Cellular Expression Logic (CEL) experiment of the BRIC17 spaceflight mission. The cultured cell lines were grown within 60mm Petri plates in Petri Dish Fixation Units (PDFUs) that were housed within the Biological Research In Canisters (BRIC) hardware. Spaceflight samples were fixed on orbit. Differentially expressed genes were identified between the two environments (spaceflight and comparable ground controls) and the two genotypes (WT and ARG1 KO). Each genotype engaged unique genes during physiological adaptation to the spaceflight environment with little overlap. Most of the genes altered in expression in spaceflight in WT cells were found to be Arg1-dependent suggesting a major role for that gene in the physiological adaptation of undifferentiated cells to spaceflight.

Spaceflight poses risks to the central nervous system (CNS), and understanding neurological responses is important for future missions. We report CNS changes in Drosophila aboard the International Space Station in response to microgravity (SFμg) and artificially simulated Earth-gravity (SF1g) via inflight centrifugation as a countermeasure. While inflight behavioral analyses of SFμg exhibit increased activity, postflight analysis displays significant climbing defects, highlighting the sensitivity of behavior to altered gravity. Multi-omics analysis shows alterations in metabolic, oxidative stress, and synaptic transmission pathways in both SFμg and SF1g; however, neurological changes immediately postflight, including neuronal loss, glial cell count alterations, oxidative damage, and apoptosis, are seen only in SFμg. Additionally, progressive neuronal loss and a glial phenotype in SF1g and SFμg brains, with pronounced phenotypes in SFμg, are seen upon acclimation to Earth conditions. Overall, our results indicate that artificial gravity partially protects the CNS from the adverse effects of spaceflight.