Mechanisms of small RNA regulation in early embryonic development

The promise of embryonic stem (ES) cells in regenerative medicine is based on their potential to make every cell in the body, a property coined pluripotency. We still do not fully understand the molecules that underlie pluripotency. It is essential for us to do so, in order to improve on the generation and quality control testing of the embryonic stem cells. Exciting recent work has shown that modifications to the genome that do not change the actual DNA sequence, but do change how that sequence is presented, is a central component of pluripotency. These modifications have been coined epigenetic modifications because they are not altering the underlying genetic code. Specifically, it was recently shown that these epigenetic modifications maintain the stem cell’s capacity to proliferate while poising them to differentiate into all tissues of body. They do so by keeping the programs required for differentiation into adult tissues off, but still accessible to activation. Failure in establishment and/or maintenance of the correct epigenetic program leads to diminished pluripotency and even tumor risk. Unfortunately, very little is known about how the epigenetic program of embryonic stem cells is established and maintained. The purpose of this grant is to understand the role of different classes of small non-coding RNAs in early embryonic development and the establishment and maintenance of the epigenetic program of ES cells. Last year we discovered a diversity of classes of small RNAs in embryonic stem cells including canonical microRNAs, short hairpin RNAs, mirtrons and endogenous siRNAs (endo-siRNAs). The first three appear to be subclasses of microRNAs and likely act a similar fashion to repress the production of proteins that would alter the properties of ES cells. In contrast, how the endo-siRNAs act is unknown. In non-mammalian species they have been showed to regulate the epigenetic status of the cell. To determine their function in ES cells, we are deleting the genetic loci that produce them. Soon we will test what roles they have in ES cell function, the molecular constitution of cells, and the epigenetic modifications of the genome. At the same time as we published our discovery of endo-siRNAs in ES cells, two other groups found a large population of endo-siRNAs in unfertilized eggs (oocytes). To test the role of endo-siRNAs in oocytes, we made two mutants, one that removes all microRNAs and another that removes microRNAs and endo-siRNAs. By comparing these two mutants we can infer the role of endo-siRNAs. To our surprise, endo-siRNAs appear to be the central small RNA players in the oocytes. Even more shocking, the entire microRNA functional pathway appears to be silenced in oocytes and the very early embryo. We predict that this silencing of miRNA function may be central to vast potential of early embryonic cells. We are now working to see how miRNA function is silenced and will then determine how such suppression influences developmental potential.

To fulfill the promise of pluripotent stem cells, both embryonic and induced pluripotent stem cells, it is essential to fully understand their properties and how those properties can be manipulated to make any cell in the human body. The best way to reach that goal is to understand the relationships between these cells that grow in a culture dish in the laboratory and the equivalent cells in the developing embryo. As working with human embryos comes with many ethical concerns, an important alternative is the mouse model. Indeed, much of what we have learned in the mouse model has later been confirmed in human. Therefore, we use a combination of the mouse model and human cells to dissect the molecular basis of stem cell function and differentiation toward adult tissues. In particular, we have been focusing on a class of molecules called small RNAs that were only discovered in the 1990s and became widely appreciated in the past decade. There are several classes of these small RNAs, two of which our lab focuses on, microRNAs and endogenous siRNAs. We have found these small RNAs are essential for normal mammalian development and growth and differentiation of stem cells. In the past year of this grant, we have been expanding on these findings dissecting the relative roles of the two classes of small RNAs, and the individual small RNAs within those classes that are responsible for specific functions. We recently discovered that endogenous siRNAs are absolutely essential for maturation of oocytes and, therefore, are very important in human fertility. However, we do not know how they exactly function and what other molecules they interact with, a new focus we are pursuing. We also discovered that microRNA function is transiently silenced during oocyte maturation and early embryonic development. This finding was surprising and suggests that miRNA suppression almost certainly has an essential role in early development. We have made progress toward determining the mechanism of suppression, which will allow us to understand its role. We are also testing a potential role for microRNA suppression in the efficient production of high quality pluripotent stem cells. Additionally, we have found that microRNAs play important functions in the earliest differentiation events of embryonic stem cells and their embryonic counterpart, the transition of the inner cell mass to the epiblast. We are making progress on which microRNAs are responsible and how they interact with the other molecules in the cell. Finally, we are dissecting for the first time the role of endogenous siRNAs in embryonic stem cells by removing them and determining the cellular consequence. This research is expected to enable to us to more easily manipulate cell fates to produce high quality cells that could be used to study diseases of many types as well as reintroduce healthy tissue into patients with degenerative diseases.

To fulfill the promise of pluripotent stem cells, both embryonic and induced pluripotent stem cells, it is essential to fully understand their properties and how those properties can be manipulated to make any cell in the human body. The best way to reach that goal is to understand the relationships between these cells that grow in a culture dish in the laboratory and the equivalent cells in the developing embryo. As working with human embryos comes with many ethical concerns, an important alternative is the mouse model. Indeed, much of what we have learned in the mouse model has later been confirmed in human. Therefore, we use a combination of the mouse model and human cells to dissect the molecular basis of stem cell function and differentiation toward adult tissues. In particular, we have been focusing on a class of molecules called small RNAs that were only discovered in the 1990s and became widely appreciated in the past decade. There are several classes of these small RNAs, two of which our lab focuses on, microRNAs and endogenous siRNAs. We have found these small RNAs are essential for normal mammalian development and growth and differentiation of stem cells. In the past year, we have been following up exciting roles for endogenous siRNAs in the development of the egg. These small RNAs are essential for the growth of a healthy egg by controlling accurate distribution of DNA. We also discovered that microRNA function is actively suppressed during this important time window. We have made headway into discovering the proteins responsible for this suppression. We believe these proteins will give us hints into how the egg prepares the genome to produce the cells that can replace any tissue in our body. MicroRNA first function at the time an embryo implants into the uterus of the mother. We have found that two groups of microRNAs are acting at this stage. We believe these specific microRNAs play essential roles in the transition from the cells that can make every tissue to a more specialized state where they can only make specific cells. To test this idea, we have made stem cells and mice that are deleted for these miRNAs to see how their loss influences developmental potential of the cell. Similarly, we are determining roles for endogenous siRNAs at these later stages of development by making mice where they have been removed. Together these results are giving new and important insights into the role of small RNAs in early embryonic development. This research is expected to enable to us to more easily manipulate cell fates to produce high quality cells that could be used to study diseases of many types as well as reintroduce healthy tissue into patients with degenerative diseases.

To fulfill the promise of pluripotent stem cells, both embryonic and induced pluripotent stem cells, it is essential to fully understand their properties and how those properties can be manipulated to make any cell in the human body. The best way to reach that goal is to understand the relationships between these cells that grow in a culture dish in the laboratory and the equivalent cells in the developing embryo. As working with human embryos comes with many ethical concerns, an important alternative is the mouse model. Indeed, much of what we have learned in the mouse model has later been confirmed in human. Therefore, we use a combination of the mouse model and human cells to dissect the molecular basis of stem cell function and differentiation toward adult tissues. In particular, we have been focusing on a class of molecules called small RNAs that were only discovered in the 1990s and became widely appreciated in the past decade. There are several classes of these small RNAs, two of which our lab focuses on, microRNAs and endogenous siRNAs. We have found these small RNAs are essential for normal mammalian development and growth and differentiation of stem cells. In the past year, we have been looking more deeply into the mechanism by which the mammalian egg suppresses one of these classes of small RNAs, the microRNAs, but not the other, the endogenous siRNAs. We have also been studying how microRNAs are used shortly after fertilization first to maintain pluripotency (the ability to make all cells of the body) and then to promote differentiation into what eventually will become all the adult tissues. Understanding these mechanisms should enable us to adopt them in order to manipulate many cells to become other types of cells through a process called reprogramming. Reprogramming is the cornerstone of regenerative medicine as it allows one to replace damaged tissues. In other experiments, we have been looking into how microRNAs interact with additional molecular mechanisms in the cells. In particular, we have been studying the association of microRNAs and epigenetic changes in the cells. Understanding how these two mechanisms work together will enhance our ability to reprogram cells. Finally, we continue to tackle the role of the other class of small RNAs, the endogenous siRNAs. We are using reporters, genetic manipulation, and rescue strategies to discover the first examples of endogenous siRNA–gene interactions in mammals, once again focusing on early embryonic development. Together these results are giving new and important insights into the role of small RNAs in early embryonic development. This research is expected to enable to us to more easily manipulate cell fates to produce high quality cells that could be used to study diseases of many types as well as reintroduce healthy tissue into patients with degenerative diseases.

To fulfill the promise of pluripotent stem cells, both embryonic and induced pluripotent stem cells, it is essential to fully understand their properties and how those properties can be manipulated to make any cell in the human body. The best way to reach that goal is to understand the relationships between these cells that grow in a culture dish in the laboratory and the equivalent cells in the developing embryo. As working with human embryos comes with many ethical concerns, an important alternative is the mouse model. Indeed, much of what we have learned in the mouse model has later been confirmed in human. Therefore, we use a combination of the mouse model and human cells to dissect the molecular basis of stem cell function and differentiation toward adult tissues. In particular, we have been focusing on a class of molecules called small RNAs that were only discovered in the 1990s and became widely appreciated in the past decade. There are several classes of these small RNAs, two of which our lab focuses on, microRNAs and endogenous siRNAs. We have found these small RNAs are essential for normal mammalian development and growth and differentiation of stem cells. In the past year, we have made significant achievements in understanding how microRNAs influence what cells become and how microRNAs themselves are regulated during early phases of cell specialization. For example, we have used microRNAs to understand how groups of genes can function together in networks to promote the de-specialization of adult cells back to embryonic stem cells. We have also used the same microRNAs to subdivide the earliest events of embryonic stem cell differentiation allowing to us follow these events during both normal development as well as during the production of induced pluripotent stem cells. Similarly, using genetic tools, we are beginning to understand the function of these microRNAs in the context of the entire organism as well as in the culture dish. Finally, we are using these microRNAs to dissect how changes in structure of DNA are regulated during early differentiation leading to the unique molecular profiles of developing cell types. Together these results are giving new and important insights into the role of small RNAs in early embryonic development. This research is expected to enable to us to more easily manipulate cell fates to produce high quality cells that could be used to study diseases of many types as well as reintroduce healthy tissue into patients with degenerative diseases.

To fulfill the promise of pluripotent stem cells, both embryonic and induced pluripotent stem cells, it is essential to fully understand their properties and how those properties can be manipulated to make any cell in the human body. The best way to reach that goal is to understand the relationships between these cells that grow in a culture dish in the laboratory and the equivalent cells in the developing embryo. As working with human embryos comes with many ethical concerns, an important alternative is the mouse model. Indeed, much of what we have learned in the mouse model has later been confirmed in human. Therefore, we use a combination of the mouse model and human cells to dissect the molecular basis of stem cell function and differentiation toward adult tissues. In particular, we have been focusing on a class of molecules called small RNAs that were only discovered in the 1990s and became widely appreciated in the past decade. There are several classes of these small RNAs, two of which our lab focuses on, microRNAs and endogenous siRNAs. We have found these small RNAs are essential for normal mammalian development and growth and differentiation of stem cells. In the past year, we have made great progress in two major fronts. First we dissected the role of a key family of miRNAs expressed in early mammalian embryos. These miRNAs are expressed from two different locations in the genome, which are expressed at different times and places. Removal of one of these two loci results in a neural tube defect, a common developmental disorder of humans. We have dissected the mechanisms of this defect providing broad insights into how genes and cellular processes function together to form a proper neural tube. These insights provide a major leap forward in our understanding how neural tube defects in humans may occur. Second, by dissecting the regulation of expression of the two miRNA locations and more broadly the cell types that they mark, we have made a major advance in the understanding of how the developmental potential of cells are retained during the early stages of embryonic development. We have learned how epigenetic factors and transcription factors work together to ensure that genes, which must be activated at later developmental stages, are brought to the starting line prior to the opening of the gates. Without such “priming” of the genes, they cannot be activated and thus development cannot precede. It is very likely similar mechanisms are associated with failed pregnancies. This work concludes the funding period of this grant. The many discoveries we have made and published over the length of this grant should have a profound impact on how we can manipulate cells to understand and treat a variety of diseases.