So, now we are going to go into more detail about trans-generational epigenetic inheritance via the gametes in terms of mouse models. So, in this first lecture, we are going to talk about the Agouti viable yellow allele. So, we will consider both this allele in terms of the trans-generational effects, but also in the next lecture we will think about environmental effects on these allele because, it's such a good model and the one that's best studied. And then in the subsequent lecture, we will talk about axin fused, another allele which displays transgenerational epigenetic inheritance in mice. And then towards the end of this week, we are going to start to think about the mechanism using both of these models, but mainly Avy as the example. And we will come back to the end thinking about whats happening in humans. So, in this picture what you can see are three genetically identical mice sitting on my hand. So, these mice, despite the fact that one's completely yellow, one's completely brown, it's called agouti or it basically looks like a normal wild-type mouse. And then one is mottled, it has patches of brown and patches of yellow. So, despite the fact I have these three distinctly different coat colours, they are genetically identical at every locus that you can test for in the mouse. So, these alleles, these mice are part of a strain called the agouti viable yellow strain, and they carry the agouti viable yellow allele. So, how is it they can be so different phenotypically, and yet genetically identical? It's similar to if you knew monozygotic twins. Identical twins that had different hair colour, that were born with different hair colour, or different eye colour. Something distinctly different about them that wasn't due to one of them dying their hair, but rather, this is the way they were born. So, in this case we know how this works. For the agouti viable yellow allele, we have the agouti gene drawn here with the yellow exons. But upstream of the agouti gene, about 100 kilobases upstream, there's an IAP, an intracisternal A-particle that we've spoken about before. It's a repetitive element that has jumped in upstream. These IAPs, or intracisternal A-particles, this is actually acting in this direction. So, it's direction of transcription for itself is away from the agouti gene. However, it has a very very strong promoter within its long terminal repeat that also acts in the other direction. This direction here. So, and this can drive expression of the agouti gene that's 100 kilobases away. So, the agouti gene normally only comes on for a very short window during the hair cycle. And it means that you get a long hair that is mostly brown or black, but has a small fleck of yellow. Because what the agouti protein does is that it's a singly molecule that causes a switch of this dark pigment production to a yellow pigment production. However, if you have this constitutive promoter that's happening, this very strong strong promoter in the IAP, long term not repeat. That these driving expression of agouti all the time constitutive expression of agouti, then you end up with an animal that has a completely yellow coat. It's because there's, agouti is on all the time, and therefore you always switch to making a yellow pigment. It, actually this animal that has a yellow coat also has other issues, because agouti is not only involved in pigment production, but is also involved in other aspects of normal biology. And so, if you have agouti on all the time, this animal ends up also being obese and diabetic. So, what happens stochastically on occasion, is that this long terminal repeat of the intracisternal A-particle will be silenced. And so then, what happens is you get normal expression of the Agouti gene. So, if we silence it here, and this is associated with DNA methylation, which is shown here by these nice lollipop structures that I've shown you before. We now also know that when it's silenced, it's also, this stochastic silencing is also associated with changes to the chromatin packaging. So, we have more tightly compacted chromatin, and the appropriate histone modifications that are made associated with silence chromatin. And if this happens, it means that the agouti gene can go about its normal functioning. It's still just turned on for that short window in the hair cycle, and so, you get an animal that is called pseudoagouti. It's not like a normal agouti coloured mouse, because it's still has this possession of these Avy allele, and so, that's why it's pseudoagouti. It's pretending a normal wild type mouse. So, it will have this normal wild type coat colour, but it will also be normal weight and will not suffer from type II diabetes. So, the difference between these two animals that are shown here is an epigenetic difference, and only an epigenetic difference. They are genetically identical but epigenetically different. So, it's these epigenetic differences that exist at the long terminal repeat of the intercisternal A-particle which is driving expression of the agouti gene and the agouti viable yellow allele. That is defining the differences in their coat colour in their layered obesity, or lack of obesity. So, the fact that you've got these two very different phenotypes, despite genetic identity is termed variable expressivity. So, this gene, the Agouti gene in the Avy allele is variably expressed between genetically identical individuals. Variable expressivity is a term that's been used often in human genetics to describe this same sort of thing which is a variable outcome. But in that case in human genetics, we don't necessarily know that the underlying genetics are identical. It'll be a variable outcome despite perhaps two people having the same mutation in a disease gene. but we don't know necessarily know that the rest of their genome is identical. In the case of mice, we do know the rest of the genome is identical. So, it's a slight variance in this definition. So, other than these perfectly yellow and perfectly agouti mice, there are also the mice that live in the middle. So, they're the ones that have got the patches of agouti coloured fur, and the patches of yellow fur. So, these are called mottled mice, and you can really compare them to when we've looked at the calico cat. We have this variegated expression of the Avy allele. A variegated constitutive expression of the agouti gene. So, these mosaics, these that are, these mottled mice, mean that obviously in some cells you have active expression of agouti. And that's because you've got an unmethylated and open common structure at the controlling IAP. But in contrast, you have other cells, other clones of cells where agouti is only expressed in the normal hair cycle when it should do. And this is associated with a silenced IAP because of DNA methylation and other chromatin marks. So, the reason why you have these clonal patches, these patches that you can actually see, patches of rather than just being a mess throughout the whole of the mouse, where you have one cell that's yellow, neighbouring another that's pseudoagouti. The reason you have these visible patches, just like in the calico cat, is because this epigenetic state, this stochastic epigenetic state at the IAP is established at gastrulation, when there are very small numbers of cells. This epigenetic state, just like for an X inactivation is then mitotically heritable. So, you get this spectrum of phenotypes with the Avy allele mice from purely yellow all the way through to purely pseudoagouti, and a range in between of mottled animals. So, this allele shows variegation, because you can get these animals that display this mottled coat colour, and variable expressivity between these genetically identical animals. We know that the epigenetic state is established in early development, and it's heritable for the lifetime of this organism. So, alleles that display these effects of variegation and variable expressivity, particularly in the context of genetic identity, in this case are termed metastable epialleles. And this is a term which is used to describe them because they are epialleles. They differ in the state, their epigenetic state. And they're metastable because, while they can be switched between generations, which I'll show you in the coming slides, they are stable for the lifetime of that organism. So, an animal with a yellow coat will always have a yellow coat. They're not going to become mottled later in life. Okay. So, now I want to think about this switching between generations, and come back to the idea of trans-generational epigenetic inheritance. So, I'm going to go through a number of pedigrees so that we can think about how the agouti viable yellow allele phenotype or their methylations take these inherited. So, here at the top, we're thinking about a male that carries the agouti viable yellow allele who has an active allele. He has a yellow coat. And he is mated with a female that doesn't carry the Avy allele, and she has a black coat. So, she carries a different allele. She doesn't carry an agouti allele. She carries a non-agouti allele. And basically with, from what it means is that you can ignore the contribution of her alleles. So, while I've drawn her here, we're not considering the pups that didn't inherit the agouti viable yellow alleles. So, basically, you can ignore the contribution from the black animals in these pedigrees. What we're then looking at is in the offspring looking at their coat colours. So, a yellow male, if you collect enough offspring, you'll find that 40% of his offspring will have a yellow coat, 40% of his offspring will be mottled, and 40% will be pseudo-agouti. So in other, I'm sorry 20% will be pseudoagouti, I can't add up. So, the 20% that are pseudoagouti will be methylated at that IAP in the Avy allele. And the 40% for the yellow will be unmethylated at that IAP. If you take a male that differs in his coat colour phenotype, here he is pseudoagouti. So, he has a silent agouti viable yellow allele, and it's heavily methylated. And you again look at proportions of these different phenotypes in the offspring, the spectrum of phenotypes. You find the same thing, 40% yellow, 40% mottled, 20% pseudoagouti. So, despite the fact that these two males had, themselves had a different expression of the Avy allele, their offspring show the same spectrum of phenotypes. So, this suggests that there's clearing and resetting between generations, because there's no memory of the phenotype of the father. We don't have a skewing based on the DNA methylation state and the expression state seen in the dad in this case. But something quite different occurs when you look following maternal inheritance of the Avy allele. So, now, on this right hand panel, we are looking at a female that carries the Avy allele and it has a yellow coat which has unmethylated allele mated with a black male. Again, he's not contributing the coat colour alleles we are interested in. so, we don't show the pups that inherit only his, that don't inherit the Avy allele. Here, what we see is when the mother has an active Avy allele which is hypomethylated. And you find she has 50% of her pups have a yellow coat, 50% are mottled, and she doesn't have any that are pseudoagouti. There are none that inherit the silent allele. Whereas, if we look at a female that is Pseudoagouti, so, has a silent methylated allele, she'll have 40% yellow, 40% Mottled, and 20% Pseudoagouti. So, the difference between these two spectrums coat colours in the offspring, there's a difference because of the epigenetic state in the mother. So, this is what would appear to be some sort of memory of the phenotype of the mother. Not the genotype, these two animals are genetically identical, the yellow mother, and the pseudoagouti mother. But they are epigenetically different. So, this memory of the phenotype of mother raises the possibility that this trans-generaltional epigenetic inheritance through the gametes. But we need to do further experiments to show this is true, and I'll explain what's being done in this case. But this says that potentially, there's some of the epigenetic marks which are found on one of these alleles which are passed through to the next generation. Because of the difference in the phenotypes in the off spring. So, what are the possibilities here if we think about those controls and the questions that you could say. In the mouse, because we know and we can prove that these animals are genetically identical, we actually don't need to worry so much about genetic alterations. However, what we do know is that this female will go on to be an obese, diabetic adult. And when she's conceived and had babies, she's certainly at least overweight, if not already obese. And so, could it be that the development in the environment of an obese mother is what changes this spectrum of phenotypes in the babies. So, could it be that it's the intra-uterine environment that was experienced? So, we need to think about this, and we also need to think about when is the effect established? So, when is this coat colour effect established? And is there any different behaviour of a mother, this mother compared with that mother. Okay. So, first of all we'll think about the intra-uterine environment. The experiments that were done is they took this same sort of breeding. They had a yellow female breeding with a black male. But instead of allowing that mother to go on and litter, they took the zygotes so, the fertilised eggs and they transferred them into a mother that is not obese. She in fact is just a normal mouse in that strain that doesn't carry the Avy allele, the black female. So, she's normal weight, and she's not, she's not obese. And if you take these eggs and transfer them over just after fertilisation, you still get 40%, sorry 50% yellow, 50% mottled, the same as if you let the the breeding happen naturally. So, we don't all of a sudden by changing around the intrauterine environment in this case, cause some sort of shift in the spectrum of phenotypes, or shift in the silencing of the Agouti viable yellow allele. And this tells you that this effect is via the gametes. We took the gametes that were from here, the fertilised egg, transfer them over, and already, this was already predetermined back at those, in those gametes, in the fertilised egg at least. And so, this rules out the contribution of the intrauterine environment in this case. What it doesn't rule out is that oogenesis happens differently in this obese female or overweight female. This was ruled out by separate experiments that were done through breeding that I won't go through in detail in these experiments, but you'll just have to believe me that they ruled out these differences as well. So, in the case of the agouti viable yellow allele, we know that this is set up in utero, and it's set up shortly after gestation. We know this through detail of molecular analysis, but also because of those patchy the size of the patches on that mottled mouse, just like with the calico cat. So, we can now say that this is the example, this is an example, at least in mice, where we do indeed see trans-generational epigenetic inheritance through the gametes. All of the controls have been performed. And it's because of this difference in the phenotypes on this spectrum of phenotypes following female transmission that we can see where we can see there's a memory of the phenotype of the mother. A memory of the epigenotype of the mother. So, in the next lecture what we're going to do is consider how the environment can alter the epigenetic state of the agouti viable yellow allele. And we'll think about this being a very epigenetically sensitive allele and really a model to work with in terms of this, because first of all, we know a lot about it. But secondly, coat colour’s very easy to examine and so, it really gives us a tractable system to look at these sorts of questions.
