Metabolism, mTOR, and the Mysteries of Diapause

Asis Hussein is a graduate student (Biochemistry) in the Ruohola-Baker Lab and the lead author of a new paper exploring how and why some mammalian species are able to pause their pregnancies under certain conditions.

Life begins in a swirl of science and mystery. We explain what we can with biology and fill in the gaps with a mix of wonder and speculation.  There are some facts we know for sure. Once fertilization occurs, the self-organizing development of a new being unfolds with incredible speed, power, and precision. In the embryo, the tightly packed cells that will give rise to all other cells are ready to carry out their coded instructions – to go forth and multiply.

Only one force is designed by evolution to halt this forward momentum.

Imagine a mouse, sheltered in the nook of a forest, nursing her newborns. Her maternal instincts tell her that the time is not right for more mouths to feed. Another pregnancy so soon could put life-threatening pressure on the mother, the litter, and the embryo. Fortunately, nature has devised an elegant solution. Under the right environmental conditions, a recently fertilized embryo can enter diapause, a reversible state of dormancy that suspends the development of the unborn until the environmental conditions are more favorable for survival for the growing mouse family.

The diapause stage stops the development, for a period of days or months, until the time is right for the developing embryo to awaken, implant to the uterus and resume development into a viable offspring.  This enigmatic dormancy-by-design has drawn widespread interest among scientists and science fiction writers alike. If humans could control cellular time, stop development and aging, could certain diseases, and perhaps even death itself be delayed? To answer these questions, scientists are looking closely at the nature of embryonic development and the mechanisms of dormancy.

Pinpointing the Genes that Put Cells to Sleep

One of those scientists is a University of Washington Biochemistry graduate student named Asis Hussein. “Diapause has been documented in more than 130 mammalian species, but the biological mechanisms that regulate the entry and exit of an embryo into and out of diapause are not well understood,” says Hussein. At the most fundamental level sits an even more tantalizing two-part question. Why is it that some cells involved in early development (or say, cancer) can essentially go to sleep and is it possible to control these processes to improve human health?

This diagram shows the two-pronged sequence of events that leads to the entry of diapause. A key factor is the presence of a protein known as glutamine which plays a central role in triggering diapause.

Answers to these questions are now emerging. Hussein, a PhD student in the Ruohola-Baker Lab in the UW Institute for Stem Cell and Regenerative Medicine (ISCRM), is the lead author of a study published in the journal Developmental Cell. The findings advance current understanding of embryonic development and have important implications for cancer research.

In the investigation, the research team performed a detailed comparison of mouse embryos in diapause to non-diapause mouse embryos.  “One thing we noticed was that the lipid profile was very different,” explains Hussein.  “More fatty acids had accumulated in the diapause embryos, suggesting that this substance, which serves as a nutrient source for cells, does something to trigger diapause in mice.”

The abundance of fatty acids, in turn, drew the researchers to a group of proteins (involved in the NFKB pathway) that play a critical role in embryonic cell survival and noticed that genes associated with these proteins were highly active in the diapause embryos. Following the metabolic and gene expression clues, the investigators examined the function of a protein known to provide cells with glutamine, an amino acid that can regulate the metabolic pathway, mTOR.  Using cutting edge CRISPR gene-editing technology, Hussein and Julie Mathieu, Assistant Professor in Comparative Medicine, showed that cutting off the flow of glutamine caused embryos to change their epigenetic make-up, and exit diapause.

Epigenetics are the changes that occur to our genetic makeup (the chromosomes) during our development and our lives. These changes, which can be caused by environmental or chemical factors, do not alter our DNA sequence, but they do affect the way cells interpret our genetic code and which proteins our cells manufacture. The role of epigenetics in the entry and exit of diapause is a key finding of the paper, specifically, that epigenetic changes governed by metabolism are responsible for the entry and exit of diapause.

Validating a Hunch: Is it Possible to Control Cell Dormancy?

The ISCRM research team genetically edited mouse embryos in vitro (seen here) and observed that the absence of glutamine transporters induced an exit from diapause.

“Epigenetics has brought back Lamarckian vs Darwinian discussions, and proven that dramatic changes in gene expression may happen without any alterations in the genes themselves, but by chemical marks in histones that organize the genes,” says Hannele Ruohola-Baker, Professor of Biochemistry, Associate Director of ISCRM, and the study’s Principal Investigator. “It is this histone-code that controls embryonic progression, and the lack of it leads to the enigmatic diapause.”

Identifying new metabolic and genetic signatures of diapause was just one part of the discovery process for the ISCRM researchers. The next challenge was to validate the hunch. Could understanding which genes are involved in the entry of exit of diapause be put to good use – could scientists learn to lull cells to sleep or wake them up?

For this, the team used mouse embryonic stem cells as a model – an efficient way to mimic diapause without involving a living animal. Using both chemistry (drugs) and gene editing techniques, the team was able to induce a diapause-like state in the embryonic cells. Assistance came from California, where Oliver Fiehn, a Professor in the UC Davis Genome Center, performed highly sensitive mass spectrometry analysis that revealed a close match between the gene expression data in the stem cells and the actual mice.

Implications for Cancer and IVF

At the root of the research is a desire to apply new understandings of cell biology to widespread health concerns. In fact, ISCRM co-founder Tony Blau, a noted cancer researcher was involved with the early stages of the investigations described in the new paper.  In the early phase of the investigation, the researchers gathered detailed genetic information on mouse embryos and began hunting for genes involved in diapause.

Mouse embryos in pre-implantation stage (L) and in diapause (R). Stem cells are shown in magenta.

Carol Ware, Professor of Comparative Medicine and Associate Director of ISCRM, worked with Blau at the inception of the project. Ware explains the enduring interest in diapause for human medicine. “Diapause can be an essential means of survival for some species, and it can be employed under environmental stress in others.  The million dollar question is whether human embryos naturally experience diapause. There are anecdotal reports suggesting this is possible, and we presume that diapause can be artificially induced in humans.  Understanding the mechanism of entry into and release from diapause is a compelling first step in understanding how to control this cellular response for medical therapies.”

Ruohola-Baker elaborates on the implications of the insights revealed in the paper.  “This study explores mechanisms behind the very nature of pregnancy in mammals. What we are learning about diapause could lead us to more effective IVF approaches, for example. And it gives us new information about the behavior of stem cells and cancer cells.”

Hussein summarizes the significance of the findings for cancer research. “We’ve now identified genes that are critical for diapause. We believe this translates directly to cancer cells, which also go in and out of a quiescent state, which explains why some of them survive rounds of chemotherapy for example. It puts us on a pathway to wonder what if the field could design a therapeutic that could wake cancer cells up at the right time so the drugs don’t miss them.”

Acknowledgements:
This work is supported by the ISCRM Fellows Program Award for A.M.H., ISCRM Innovation Pilot Award for J.M. and grants from the National Institutes of Health R01GM097372, R01GM97372-03S1, and R01GM083867 for H.R.-B., 1P01GM081619 for C.B.W. and H.R.-B.