So, in mammals so far, we've thought about random X inactivation. The fact that you could have either the maternal or paternal X chromosome inactivated within the female nucleus. However, this isn't the only form of X inactivation that occurs. So, we know that random X inactivation occurs around gastrulation. The choice is heritable. But a second type of X inactivation can occur that is studied predominantly in mice, and also in marsupials. called imprinted X inactivation. In this case, there isn't a random choice about which X chromosome is selected, but rather it's dictated that it will be the paternally derived, the X chromosome derived from the father, that will be chosen to be inactive. So, we know that this form of imprinted X inactivation, at least in mice, occurs in pre-implantation development. So that's before the embryo has made a placenta. And then occurs in the placenta itself. It also, it's interesting to note that, while it doesn't seem to occur particularly frequently in human cells, in marsupials, in the it occurs in all tissues. So marsupials, you might know non-placental mammals that are, indigenous to Australia and South America. And in these marsupials, these non-placental mammals imprint X inactivation is occurring in all tissues. So including in the adult marsupial females. And so because of this difference between having it occasionally in mice, and having it all the time in marsupials. And very rarely, or never in adult, in female humans. It argued that maybe this imprinted form of X inactivation, the case where the choice is removed, and it's no longer random It's perhaps an archaeic mechanism. So lets think about, when each of these types of imprinting occurs, in the mouse. And, for most of the X inactivation lectures we're going to be predominantly thinking about what occurs in the mouse. Because we can learn a lot about the molecular mechanisms in a model system like the mouse that and these studies, are much more challenging, in humans. Although some of these studies have now been performed in humans, they've been much more recent, because it's only been very recently we've had the ability to perform these studies in humans. So here I'm showing you a picture of kind of the circle of life if you like. So we start out and we have. A sperm and an oocyte. They fuse to form a zygote. And then go through the preimplantation phase of development. Going through from the two cell stage, the four cell stage, the marula, and ending up at the blastocyst that I mentioned. So somewhere between the two and the four cell stage, this is where imprinted X activation first occurs in the mouse. And so, this is where the paternal X chromosome is silenced. So we're not exactly sure and it probably depends on which locus you look at on the X chromosome, exactly when this first occurs, but it is certainly between this two and four cell stage. So in this, for this pre-implantation period, it's only the maternally derived X chromosome which is active. So, we then form the blastocyst. And the blastocyst is a ball of cells, that has on the outside a layer of cells that will go on to form, which is called trophectoderm, which will go on to form the placenta. And within that ball of cells, is the inner cell mass. This inner cell mass here, depicted here will go on to form the embryo itself and therefore, also, go on to form the adult. So, these two different parts of the blastocyst, are actually treated differently, in terms of X inactivation. So, the trophectoderm, that will go on to form the placenta maintains the imprinted X inactivation that was found in that pre-implantation period. So the X inactivation that was first established between the two and four cell stage, is then continuing to be maintained in these extra embryonic tissues in the placenta. That will support the growth of the embryo. However the inner cell mass of the blastocyst which will go on to become the embryo does something different. So a blastocyst is present at around e3.5 so embryonic day 3.5 to embryonic day 4.5. of just gestation in the mouse and embryonic 5.5 we know that random X inactivation takes place. This is when you have this gastrulus stage of development that's drawn here. At this gastrulus stage of development in the the epiblast. The epiblast is what the portion of the embryo that will actually go on make the embryo itself and the adult being in the end. And the rest of it are really, membranes that are associated, are going to become the placenta. So what we now know is that, within the cells of the inner-cell mass, these cells will have to, remove the imprinted X inactivation, remove that paternal imprint, the silencing of the paternal X chromosome and then start random X inactivation of fresh at embryonic day 5.5. From this point onwards, this random X inactivation is maintained, so it's maintained through the somatic cells of the mid-gestation embryo shown here, so it's maintained through to the somatic cells of an adult female. So, seeing as there has to be a switch from imprinted X inactivation to random X inactivation from the inner cell mass to the epiblast. something must be happening there. And indeed, what we actually find is that the cells of inner cell mass contain two active X chromosomes. And this is extremely unusual. It happens just twice in the development of a female mammal. First in the inner cell mass, and this is because they need to clear that imprint in X inactivation that was found earlier in development in that pre-implantation phase. And secondly, it happens again in the primordial germ cells. So, the primordial germ cells are during the development of the of the eggs. Then what will happen, or sperm, but in this case the eggs, you find that X inactivation is again cleared or removed. So, here you will again find two active X chromosomes. So, these two exceptions to the rule are interestingly both pluripotent cell types. Meaning that they are able to give rise to all the lineages in the embryo there seems to be some link between pluripotency and X inactivation status in this case that you don't have X inactivation in these cell types. So what we also know is that cells in the inner cell mass. Can be used to derive embryonic stem cells. So, you may have heard something about embryonic stem cells but, these cells can be used again to derive any of the embryonic lineages but, not the placenta. The placenta as I mentioned comes from the trophectoderm and so the embryonic stem cells can't make the trophectoderm, can't make the placenta. And these embryonic stem cells have been extremely useful for studying X inactivation because when we can drive embryonic stem cells from the inner cell mass they can be grown indefinitely in culture. And when we do that we can therefore have many, many more cells than can be derived from the inner cell mass itself and of a small embryo. But, additionally if we take these embryonic stem cells and stimulate them to differentiate. Then they'll undergo X inactivation, random X inactivation, just as it occurs during normal development from E5.5 onwards. And so much of what we've learned about X inactivation at least in the mouse has come from studies of embryonic stem cells that have been derived and differentiate them, differentiating them in culture. And so over the subsequent lectures that as we go through each of the different mechanisms involved in X inactivation and the stages of X inactivation much of what we've learned has been coming from those embryonic stem cells with a smaller number of studies that are actually done in embryos that are freshly taken.
