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==Experimental Procedure Summary==
==Experimental Procedure Summary==
We seek to understand how microgravity affects embryonic development in rats and how, if possible, to restore normal growth to embryos while still in microgravity conditions.
We do not currently understand the effect gravity has on cells, but it appears critical to development. Understanding the role gravity plays is an essential step in increasing the human presence in space. Embryonic development has been simulated in microgravity conditions on earth before, and it has been found that the embryos were not able to develop to term. However, already pregnant rats have successfully given birth in space before. It is thought that this developmental failure is a result of the lack of formation of micro tubules at the cellular level, and that this failure happens relatively early in the development process.
According to Kojima et al, the fertilization and implantation of embryos is not senstive to the gravitational vector. However, this study used a series of 1D clinostats. The embryos did perform well after 96 hours, suggesting that five days may be insufficient time to adequetely test the conditions. In a separate experiment, Kojima et al showed that embryos were resorbed if exposed pre-implanatation. Wakayama et al, on the other hand, found a lower fertilization rate in microgravity conditions.
Studies have shown that certain species of fish are capable of mating and producing healthy offspring in space, but that microgravity causes a large degree of morphological variation on terrestial animals. (Crawford) Rats in space have successfully mated and fertilized eggs, but implantation has failed.
Uva et al in 2002 worked with rat cells in a clinostat for 20 hours, and some differences were observed.
It seems that there is a lack of consensus on how exactly embryogenesis is effected by gravity, which makes sense given the gap in knowledge as to the precise way gravtiy affects cells. More current research has suggested that the effects could be due to the cytocskelaton (Crawford-Young).
We hope to precisely identify the time that gravity becomes critical to further development. We will incubate mice embryos under microgravity conditions (actually in an environment with no net gravitational force), and vary the length of time they are allowed to develop both with and without gravity before implantation. It is our hope that we can identify a critical phase of development that requires gravity. In other words, we hope to isolate a (hopefully narrow) window of time that an embryo removed from microgravity before or placed in after will develop normally. We will then examine the embryos at this stage and examine what signal pathways are active and how the cell is interacting with its cytoskelaton support. At this point, we would seek to develop an artificial means of triggering the identified signaling pathways. If time and resources permit, we would also attempt to isolate proteins upstream to begin to understand the mechanism by which gravity acts on it.
Identify the gravity dependent components of embryonic development and develop a signal or environmental based factor capable of restoring development potential. Additionally, by identifying the pathways that gravity affects, we can identify the proteins and perhaps the mechanism by which cells sense the small force.
While the literature does not have consensus on the effectivenss of the clinostat to simulate microgravity or the effects of microgravity on implantation, we believe that microgravity is deterimental to embryonic development and that a 3D clinostat will be sufficient to simulate the conditions as appropriate.
Assuming we see failure of development after a certain point, we expect to find a difference in mRNA expression levels that, when computationally analyzed, will yield potential proteins of interest that can be further examined manually.
We are confident that if we can identify the protein or proteins missing from the microgravity embryo cells, we will be able to infer or deduce the relevant pathways.
If the microgravity fails to affect the cells, then this experiment will demonstrate the inadequacy of the clinostat as a microgravity simulator. In this case, assuming the research budge permits, we can conduct the experiment aboard the ISS.
If there is no detectable difference in mRNA expression levels, then we can attempt to examine the cells before (and after) the critical time for changes. If this is still a failure, then our experiment as stated will fail.
Experimental Procedure Summary
Because a mouse must be implanted within five days of zygote formation, without access to a space laborartoy we are limited to testing in this period of time. We will test whether the embryos still develop after being in microgravity conditions for 0-5 days. We will place 5 mice in on day 1 (group A), and one additional mouse from group B everyday. One mouseher apartment-mate will be a recent WashU graduate studying biology. Our move-in date is July 1st. The apartment is part of a 3 unit house with quiet neighbors on a safe, residential street. It is located will be removed from group A everyday as well. All mice will be implanted on day 5. It is possible that additional mice can not be added to the machine without stopping it. If this is the case, each time period must be run separately to completion.
After data analysis, will repeat microgravity conditions to get the embryos at the critical failure stage, and using RNA-seq sequence both a normal gravity and a microgravity mouse embryo. These steps will repeat until the difference is identified.
Once the mRNA has been isolated, will analyze to determine protein. Literature will be consulted for what role protein plays in pathway. Will attempt to activate protein or anything downstream afterwards. Specifics of this will depend on the pathway identified.
3D Clinostat (for microgravity simulation) or access to ISS
Fertalized mice embryos and media to culture
Materials needed to perform RNA-seq assay
Crawford-Young, Susan J. "Effects of microgravity on cell cytoskeleton and embryogenesis." International journal of developmental biology 50.2 (2006): 183-191.
Kojima, Yoshiyuki, et al. "Effects of simulated microgravity on mammalian fertilization and preimplantation embryonic development in vitro." Fertility and sterility 74.6 (2000): 1142-1147.
Wakayama, Sayaka, et al. "Detrimental effects of microgravity on mouse preimplantation development in vitro." PloS one 4.8 (2009): e6753.