1 00:00:15 --> 00:00:21 What we talked about last time was formation of different types of 2 00:00:21 --> 00:00:27 cells in the embryo, different positions, different 3 00:00:27 --> 00:00:33 shapes. Am I on? And we talked about one of the 4 00:00:33 --> 00:00:39 overriding principles of all of this being the control of gene expression. 5 00:00:39 --> 00:00:45 I'll explore this a little more with a couple of slides in a moment. 6 00:00:45 --> 00:00:51 Let me just write some points. So we talked about the control of gene 7 00:00:51 --> 00:00:57 expression as being an overriding principle of how cells 8 00:00:57 --> 00:01:03 types are formed. We talked about the fact that cell 9 00:01:03 --> 00:01:08 types were formed in a stepwise fashion. And different parts of the 10 00:01:08 --> 00:01:13 embryo indeed in a stepwise fashion. Not all at once. And we talked 11 00:01:13 --> 00:01:23 about a combinatorial code -- 12 00:01:23 --> 00:01:27 -- of regulators that, together, specify different types of 13 00:01:27 --> 00:01:32 cells. So here we are to remind you talking 14 00:01:32 --> 00:01:37 about cell type in the formation module stepping through our board of 15 00:01:37 --> 00:01:42 life. And here are a couple of slides to remind you what we talked 16 00:01:42 --> 00:01:47 about in last lecture to bring you up to speed. You should look at the 17 00:01:47 --> 00:01:52 PowerPoint on the Web if you have not already. Cell type is defined 18 00:01:52 --> 00:01:58 by the proteins a particular cell makes. 19 00:01:58 --> 00:02:03 For example, red blood cells carry oxygen around the body because they 20 00:02:03 --> 00:02:08 make globin which is able to carry oxygen. The set of proteins that 21 00:02:08 --> 00:02:13 are made are defined by which genes in a cell are active, 22 00:02:13 --> 00:02:18 or expressed is the term you should know. And, therefore, 23 00:02:18 --> 00:02:23 which genes are active, also called the control of gene 24 00:02:23 --> 00:02:29 expression, controls which cell type forms. 25 00:02:29 --> 00:02:33 This triad is essential for you to understand what we're going to be 26 00:02:33 --> 00:02:37 talking about now and really for the rest of the course. 27 00:02:37 --> 00:02:41 So you should really get it. If you don't see me, go back and 28 00:02:41 --> 00:02:46 look at the previous lecture. And this lecture is going to also 29 00:02:46 --> 00:02:50 reinforce this triad. I pointed out last time that gene 30 00:02:50 --> 00:02:55 expression can be controlled at many levels. 31 00:02:55 --> 00:02:59 In fact, at any point from transcription, 32 00:02:59 --> 00:03:04 even from replication actually of the DNA through transcription, 33 00:03:04 --> 00:03:09 splicing, translation, export of the RNA to the cytoplasm, 34 00:03:09 --> 00:03:13 I haven't written up here, messenger RNA stability, protein 35 00:03:13 --> 00:03:18 modification, protein export. Any of these steps is along the way 36 00:03:18 --> 00:03:23 to getting the final gene product. And any of these steps can be 37 00:03:23 --> 00:03:28 controlled to allow the readout of the final gene product or not. 38 00:03:28 --> 00:03:31 I told you that along the way to becoming the final type of cell that 39 00:03:31 --> 00:03:35 a cell is going to become, it goes through a couple of steps. 40 00:03:35 --> 00:03:39 Or it can go through many steps, and we'll talk about that 41 00:03:39 --> 00:03:43 extensively today. Cells begin as uncommitted or naive. 42 00:03:43 --> 00:03:46 They don't know what they're going to become. They then become 43 00:03:46 --> 00:03:50 committed or determined to a particular fate, 44 00:03:50 --> 00:03:54 a position or a structure, but they may not look very different 45 00:03:54 --> 00:03:58 from that first set of uncommitted cells. 46 00:03:58 --> 00:04:03 And then they go on eventually to form so-called differentiated cells 47 00:04:03 --> 00:04:09 or the final cell type which has the function of that particular cell 48 00:04:09 --> 00:04:14 type. And so the point is if there is a stepwise formation of the final 49 00:04:14 --> 00:04:20 cell type. And that final cell type is directed through the action of 50 00:04:20 --> 00:04:26 regulatory factors that we termed inducers or determinants. 51 00:04:26 --> 00:04:31 There is nothing magic about these. Inducers referred to factors that 52 00:04:31 --> 00:04:35 were involved in cell-cell signaling. Determinants referred to regulatory 53 00:04:35 --> 00:04:40 factors that acted within a cell and were inherited by it. 54 00:04:40 --> 00:04:44 You shouldn't be trying to write all this down now actually because 55 00:04:44 --> 00:04:49 this is really review. And if you weren't at the last 56 00:04:49 --> 00:04:53 lecture, try to get some of it to wash over you and then go back and 57 00:04:53 --> 00:04:58 make sure you really know this stuff. 58 00:04:58 --> 00:05:02 And then as the differentiated cells form there is activation of a set of 59 00:05:02 --> 00:05:06 genes called differentiation genes which are the things that carry out 60 00:05:06 --> 00:05:11 the final cell function, the globin protein in red blood 61 00:05:11 --> 00:05:15 cells, the neurofilaments and neurotransmitters in neurons and so 62 00:05:15 --> 00:05:20 on. And we'll talk about this again as we go through today. 63 00:05:20 --> 00:05:24 OK. So this is not unrelated to what we are going to 64 00:05:24 --> 00:05:29 talk about today. Because what I want to do today is 65 00:05:29 --> 00:05:35 to explore with you how you get a particular kind of cell. 66 00:05:35 --> 00:05:41 And I want to use that exploration to underline some of the principles 67 00:05:41 --> 00:05:46 that I haven't yet covered as to how cell type is determined in the 68 00:05:46 --> 00:05:52 embryo. And the cell type I'm going to talk to you about is skeletal 69 00:05:52 --> 00:05:58 muscle. No, this is not the [UNINTELLIGIBLE]. 70 00:05:58 --> 00:06:20 OK. So the point today is to look 71 00:06:20 --> 00:06:26 at a particular cell type and how it forms. The type we've chosen is 72 00:06:26 --> 00:06:32 skeletal muscle. Skeletal muscle is one of three 73 00:06:32 --> 00:06:38 kinds of muscle in your body. The other two being cardiac in your 74 00:06:38 --> 00:06:43 heart and smooth muscle around your internal organs. 75 00:06:43 --> 00:06:48 Skeletal muscle acts in a voluntary fashion to allow movement. 76 00:06:48 --> 00:06:54 It's an extremely complex structure. The final form of skeletal muscle 77 00:06:54 --> 00:06:59 includes 200 proteins which have the extraordinary property of 78 00:06:59 --> 00:07:05 contractility in response to nerve input. 79 00:07:05 --> 00:07:09 I'm not going to talk about the contractile process, 80 00:07:09 --> 00:07:13 although it's fascinating. And you're not going to be tested 81 00:07:13 --> 00:07:17 on this for the physiology of contractility either. 82 00:07:17 --> 00:07:21 But if you're interested in this, I would suggest you look at Chapter 83 00:07:21 --> 00:07:25 47 in your book which talks about this quite nicely. 84 00:07:25 --> 00:07:31 Skeletal muscles are arranged in large groups of fibers. 85 00:07:31 --> 00:07:37 And each muscle fiber is a multinucleate cell, 86 00:07:37 --> 00:07:43 is a giant cell that consists of many myofibrils or myofibrils that 87 00:07:43 --> 00:07:49 are together in a sheath and are surrounded by many nuclei. 88 00:07:49 --> 00:07:55 So skeletal muscle forms from the fusion of many mononucleate cells to 89 00:07:55 --> 00:08:00 give a multinucleate syncytium. And during the process of skeletal 90 00:08:00 --> 00:08:05 muscle formation there are a number of steps that one can delineate. 91 00:08:05 --> 00:08:11 One can talk about a kind of cell called a myoblast which is a single 92 00:08:11 --> 00:08:20 mononucleate cell -- 93 00:08:20 --> 00:08:30 -- that then fuses to form, through many steps, something called 94 00:08:30 --> 00:08:37 a myotube which is multinucleate. And these various myotubes come 95 00:08:37 --> 00:08:43 together to eventually form a muscle fiber. 96 00:08:43 --> 00:08:48 Muscle is obviously an extremely 97 00:08:48 --> 00:08:52 important cell type for movement. All of the meat that you eat, if 98 00:08:52 --> 00:08:56 you're a meat eater, is muscle, essentially a skeletal 99 00:08:56 --> 00:09:00 muscle. And there are many diseases that are associated with abnormal 100 00:09:00 --> 00:09:05 skeletal muscle. Muscular dystrophy, 101 00:09:05 --> 00:09:11 for example, is a deficit of one particular protein in the muscle, 102 00:09:11 --> 00:09:17 affects one in about 3,000 boys. OK. So the myofibril is a very 103 00:09:17 --> 00:09:23 beautiful construction of many proteins organized in a very ordered 104 00:09:23 --> 00:09:29 structure. And the deal is that these proteins slide relative to one 105 00:09:29 --> 00:09:34 another during contractility. And all of these bands of proteins 106 00:09:34 --> 00:09:40 slide in an ordered fashion relative to one another as the muscle 107 00:09:40 --> 00:09:45 contracts. It's an extraordinary story in physiology itself. 108 00:09:45 --> 00:09:51 And I regret I don't have time to go through it with you. 109 00:09:51 --> 00:09:56 But I want to use this rather as an example of a cell type and to 110 00:09:56 --> 00:10:02 explore with you how the cell type forms during development. 111 00:10:02 --> 00:10:08 So the story I'm going to tell you has to do with a particular 112 00:10:08 --> 00:10:14 transcription factor called MyoD. MyoD, shown in green here, no, not 113 00:10:14 --> 00:10:20 shown in green here. That's the DNA double helix. 114 00:10:20 --> 00:10:26 Shown in red here binds to a particular sequence on 115 00:10:26 --> 00:10:33 DNA, its target site. It binds as a dimer. 116 00:10:33 --> 00:10:41 And it acts to activate the transcription of genes required for 117 00:10:41 --> 00:10:49 muscle formation. OK. So MyoD is a transcription 118 00:10:49 --> 00:10:57 factor. And, as I'll tell you a moment, it gives rise to the 119 00:10:57 --> 00:11:05 determination of skeletal muscle. Now, I'm going to tell you a few 120 00:11:05 --> 00:11:11 things about this that are general principles of how this transcription 121 00:11:11 --> 00:11:17 factor works and then I'll go through some slides with you. 122 00:11:17 --> 00:11:23 MyoD is both necessary, although I'm going to clarify that in a 123 00:11:23 --> 00:11:30 moment, and sufficient for muscle determination. 124 00:11:30 --> 00:11:34 This is a dyad of words that is very important, necessary: required for, 125 00:11:34 --> 00:11:39 sufficient: enough to. And whenever you're thinking about gene function, 126 00:11:39 --> 00:11:43 this is the dyad you want to think about. It may not be a gene, 127 00:11:43 --> 00:11:48 and then I'll tell you in a moment. I'm going to put a caveat here with 128 00:11:48 --> 00:11:53 respect to the necessary, OK? But necessary and sufficient is 129 00:11:53 --> 00:11:57 one of these litanies that you want to ask yourself when you're thinking 130 00:11:57 --> 00:12:02 about regulatory factors. The other thing that I want to tell 131 00:12:02 --> 00:12:08 you is that MyoD is a member of a gene family. What's a gene family? 132 00:12:08 --> 00:12:13 A gene family is a group of genes with similar structure and often 133 00:12:13 --> 00:12:19 similar function. This gene family that MyoD belongs 134 00:12:19 --> 00:12:24 to is called the MRF gene family, which stands for muscle regulatory 135 00:12:24 --> 00:12:30 family. And there are four members. 136 00:12:30 --> 00:12:38 And these members share some 137 00:12:38 --> 00:12:45 function with one another. And that leads us to another term 138 00:12:45 --> 00:12:52 which is the term redundant. MyoD is an extremely important gene. 139 00:12:52 --> 00:12:59 And if you make knockouts in mice there is some phenotype, 140 00:12:59 --> 00:13:06 but they have muscle. And it's not until you make a gene 141 00:13:06 --> 00:13:13 mutation in another member of the MRF family that you see mice that 142 00:13:13 --> 00:13:20 don't have any skeletal muscle. So redundant in this case refers to, 143 00:13:20 --> 00:13:27 or in all cases refers to shared function. It can also refer to, 144 00:13:27 --> 00:13:35 and does in this case, it can also refer to alternate pathways. 145 00:13:35 --> 00:13:39 So the notion that we have is that we're starting here as an 146 00:13:39 --> 00:13:43 uncommitted cell and we're going to end up over there as a 147 00:13:43 --> 00:13:47 differentiated muscle cell. And we can get there by walking 148 00:13:47 --> 00:13:51 straight this way, but we could also get there by 149 00:13:51 --> 00:13:55 walking up and out the back and all the way around and to that same spot 150 00:13:55 --> 00:13:59 in front. And both of those would be bona fide pathways that would get 151 00:13:59 --> 00:14:04 you from point A to point B. So sometimes there can be alternate 152 00:14:04 --> 00:14:11 pathways that will get you along the path of forming particular cell 153 00:14:11 --> 00:14:18 types or even carrying out some kind of biochemical function. 154 00:14:18 --> 00:14:25 OK? This is a general principle of biology. So let's look at this in a 155 00:14:25 --> 00:14:31 bit more detail with some slides. The notion is that MyoD binds to the 156 00:14:31 --> 00:14:35 promoters of many different genes expressed in skeletal muscle, 157 00:14:35 --> 00:14:39 together with other transcription factors, some of which may be common 158 00:14:39 --> 00:14:43 to all of these genes and some of which may be different. 159 00:14:43 --> 00:14:47 And it activates their transcription. 160 00:14:47 --> 00:14:51 And you can see I haven't drawn the genes in a really accurate way. 161 00:14:51 --> 00:14:55 These are just representations. There's no double-stranded DNA 162 00:14:55 --> 00:15:00 meant to be implied or not implied there. 163 00:15:00 --> 00:15:04 MyoD is sufficient to activate the skeletal muscle program. 164 00:15:04 --> 00:15:09 And this is a very exciting finding about 15 years ago when it was shown 165 00:15:09 --> 00:15:14 that one could take the MyoD gene, and you actually put it in a virus, 166 00:15:14 --> 00:15:19 and then you can infect a group of cells that normally wouldn't become 167 00:15:19 --> 00:15:24 muscle with this virus. And lo and behold the virus that is 168 00:15:24 --> 00:15:29 expressing the MyoD protein will turn these cells into 169 00:15:29 --> 00:15:34 skeletal muscle. And this was the first example of a 170 00:15:34 --> 00:15:38 regulatory protein that could take one cell type and turn it into 171 00:15:38 --> 00:15:42 another cell type. And MyoD and the other MRFs are 172 00:15:42 --> 00:15:47 very good at doing this. They can, for example, take brain 173 00:15:47 --> 00:15:51 cells and turn those into muscle. They can really take most cell 174 00:15:51 --> 00:15:55 types and turn them into muscle cells. So MyoD is sufficient to 175 00:15:55 --> 00:16:00 convert non-muscle to muscle cells. And this is actually particularly 176 00:16:00 --> 00:16:05 extraordinary because, as I showed you in a one line 177 00:16:05 --> 00:16:09 schematic on this board, forming skeletal muscle is a 178 00:16:09 --> 00:16:14 multi-step program. You start off with these cells 179 00:16:14 --> 00:16:19 called, they call them myotome cells, and then you get these things called 180 00:16:19 --> 00:16:23 myoblasts which are dividing. The myoblasts start to align in 181 00:16:23 --> 00:16:28 arrays. And then the cells fuse with one another, 182 00:16:28 --> 00:16:33 which is really unusual for cells. I mean it's a really kind of weird 183 00:16:33 --> 00:16:37 cell type. And you get these long tubes that have got thousands of 184 00:16:37 --> 00:16:42 nuclei in them. And then the tubes start to make 185 00:16:42 --> 00:16:46 proteins that align themselves in these very ordered ways that allow 186 00:16:46 --> 00:16:51 them to slide relative to one another, and you get this 187 00:16:51 --> 00:16:56 multinucleate myotube that has contractile function. 188 00:16:56 --> 00:17:00 And what's really cool about MyoD is that it can start this whole process 189 00:17:00 --> 00:17:05 which then follows one step after another. So it's been termed a 190 00:17:05 --> 00:17:09 master regulatory switch because it can activate a whole cell 191 00:17:09 --> 00:17:17 type specific program. 192 00:17:17 --> 00:17:21 Now, in contrast, and this is what I was alluding to 193 00:17:21 --> 00:17:25 on the board, knockout or removal, a genetic mutation of MyoD does not 194 00:17:25 --> 00:17:30 remove skeletal muscle from mice. So this is a micrograph of muscle. 195 00:17:30 --> 00:17:34 You can see the ordered myotubes from a wild type mouse. 196 00:17:34 --> 00:17:38 This is a MyoD null mouse. And there's perfectly fine skeletal 197 00:17:38 --> 00:17:42 muscle there. It's a little, there's a little less of it, but 198 00:17:42 --> 00:17:47 it's still there and the mouse is viable. However, 199 00:17:47 --> 00:17:51 if you go in and you make a double mutation in the MyoD gene and 200 00:17:51 --> 00:17:55 another member of the family called Mif5, there is no muscle at all in 201 00:17:55 --> 00:18:00 any region, or no skeletal muscle at all in any region of the body. 202 00:18:00 --> 00:18:03 The other muscle, the heart muscle and the muscle 203 00:18:03 --> 00:18:07 around the intestine, the smooth muscle is fine, 204 00:18:07 --> 00:18:11 but the skeletal muscle is completely absent. 205 00:18:11 --> 00:18:15 So we can say that the MRFs are necessary for muscle formation but 206 00:18:15 --> 00:18:18 that MyoD is a member of a redundant gene family. And that seems to be 207 00:18:18 --> 00:18:22 the rule in biology, that genes come in families, 208 00:18:22 --> 00:18:26 and very often one can take over the function of another when one of them 209 00:18:26 --> 00:18:30 is mutated. And that serves as a kind of fail-safe for making sure 210 00:18:30 --> 00:18:37 that development works properly. 211 00:18:37 --> 00:18:42 OK. So here comes something to think about that I alluded to before 212 00:18:42 --> 00:18:48 spring break. I remember this. Don't know if you do. MyoD is, 213 00:18:48 --> 00:18:53 in the entire embryo the MyoD gene is only expressed in the future 214 00:18:53 --> 00:18:59 skeletal muscle. This is a picture of a frog embryo. 215 00:18:59 --> 00:19:03 And you can see these stripes along the back of the frog. 216 00:19:03 --> 00:19:07 These are regions of the embryo called the myotones. 217 00:19:07 --> 00:19:11 They're part of the somites that I'll talk about. 218 00:19:11 --> 00:19:15 And this is what's going to become the future skeletal muscle. 219 00:19:15 --> 00:19:19 And this is where MyoD is expressed. And, brilliant, 220 00:19:19 --> 00:19:23 only there. And now this is a chicken embryo and you see the same 221 00:19:23 --> 00:19:27 thing. MyoD is expressed in these stripes along the back in the future 222 00:19:27 --> 00:19:31 myotones of the somites. It's also expressed in the future 223 00:19:31 --> 00:19:35 limbs as the muscle of the limbs is starting to form. 224 00:19:35 --> 00:19:39 And so you're going to ask me, or you should, how does MyoD get 225 00:19:39 --> 00:19:43 activated just in the future skeletal muscle? 226 00:19:43 --> 00:19:47 Because in a way this is a copout. I've told you MyoD activates the 227 00:19:47 --> 00:19:51 skeletal muscle program, and it's only expressed in cells 228 00:19:51 --> 00:19:55 that are future skeletal muscle. Well, how does it know to be 229 00:19:55 --> 00:20:00 expressed just in this one region of the embryo? 230 00:20:00 --> 00:20:05 And to answer that question we have to actually go back to the dawn of 231 00:20:05 --> 00:20:10 time to fertilization. And I'm going to take you through 232 00:20:10 --> 00:20:16 some of the steps that will get MyoD expression into this particular 233 00:20:16 --> 00:20:21 pattern that will allow the skeletal muscle to grow out in just the right 234 00:20:21 --> 00:20:27 places of the embryo to give just the right amount and the right 235 00:20:27 --> 00:20:32 position of skeletal muscle. OK. So. And to do that we are 236 00:20:32 --> 00:20:37 going to pose some questions. And I'm going to start by telling 237 00:20:37 --> 00:20:42 you something and then pose a question. If you look at an embryo, 238 00:20:42 --> 00:20:48 you could look back at that chick one if you wanted to do or you can 239 00:20:48 --> 00:20:53 look at this fish. Here is your fish fillet, 240 00:20:53 --> 00:20:58 your skeletal muscle, and it is on the so-called dorsal or backside 241 00:20:58 --> 00:21:03 of the embryo. So last time we talked about dorsal 242 00:21:03 --> 00:21:08 and ventral as two of the so-called axis or positional coordinates in 243 00:21:08 --> 00:21:13 the embryo. And the muscle arises from a region that is termed dorsal 244 00:21:13 --> 00:21:18 which is the back. OK? So you might ask me, 245 00:21:18 --> 00:21:23 how do you know this? And I'll tell you how I know this. 246 00:21:23 --> 00:21:28 You should ask me how do I know this. OK, so I'm going to. 247 00:21:28 --> 00:21:33 Having put questions into your mouths, I'm going to tell you how 248 00:21:33 --> 00:21:39 you know during the development of an organism where a particular cell 249 00:21:39 --> 00:21:45 type is going to arise from. And the technique I'm going to tell 250 00:21:45 --> 00:21:51 you about is something called fate mapping. So let's begin with the 251 00:21:51 --> 00:21:57 sentence muscle, and I'm talking about skeletal 252 00:21:57 --> 00:22:03 muscle when I write muscle, arises from the dorsal or the back 253 00:22:03 --> 00:22:08 of the embryo. OK? And so how do you know this? 254 00:22:08 --> 00:22:14 How is this known? And it's known through use of a technique called 255 00:22:14 --> 00:22:20 fate mapping. What is fate mapping? So this is very interesting. The 256 00:22:20 --> 00:22:25 idea is to take a very early embryo, I've shown you here a fish embryo, 257 00:22:25 --> 00:22:31 and to inject one or a few of its cells that you can distinguish by 258 00:22:31 --> 00:22:37 some kind of morphological, obvious indicator from the rest of 259 00:22:37 --> 00:22:42 the embryo. And to inject those cells with a dye. 260 00:22:42 --> 00:22:47 So here we've used a fluorescent dye. And we've put that dye near 261 00:22:47 --> 00:22:52 the site of the embryo that's got this bump. OK? 262 00:22:52 --> 00:22:57 And as the embryo develops the cells will inherit that dye because 263 00:22:57 --> 00:23:02 it's a non-diffusible dye. And that dye will be inherited. 264 00:23:02 --> 00:23:07 And in the later fish, we're looking down now onto its brain, 265 00:23:07 --> 00:23:12 you can see that the whole front of the brain is outlined with these 266 00:23:12 --> 00:23:16 green fluorescent cells. And what this tells you is that the 267 00:23:16 --> 00:23:21 cell you injected initially has gone on to give rise to this whole front 268 00:23:21 --> 00:23:26 region of the brain. OK? So fate mapping tells you what 269 00:23:26 --> 00:23:37 cells will become. 270 00:23:37 --> 00:23:41 All right. I know it's on the slide, but it's important that you 271 00:23:41 --> 00:23:45 understand this because it is distinguished from something I'm 272 00:23:45 --> 00:23:49 going to tell you later. So you can do this for skeletal 273 00:23:49 --> 00:23:53 muscle. And I'm going to talk about frog development because so much is 274 00:23:53 --> 00:23:57 known. So here is a frog at the 32 cell stage. And one can see, 275 00:23:57 --> 00:24:01 because of the size of the cells and because of pigmentation differences 276 00:24:01 --> 00:24:05 I'll show you in a moment, which side of the embryo is what. 277 00:24:05 --> 00:24:09 And I've injected a cell here that's called the C3 cell, 278 00:24:09 --> 00:24:13 all these cells have got different names, with dye. 279 00:24:13 --> 00:24:17 And later on you can see, in this thought experiment, 280 00:24:17 --> 00:24:21 that the somites are labeled with the blue dye. So the C3 cell, 281 00:24:21 --> 00:24:25 and actually the one on the other side of embryo, 282 00:24:25 --> 00:24:30 are going to give rise to skeletal muscle. OK. 283 00:24:30 --> 00:24:34 So the reason that I wrote on the board what was on the slide already 284 00:24:34 --> 00:24:39 is that it's really important to distinguish what cells are going to 285 00:24:39 --> 00:24:43 become from when they've made the decision to become it. 286 00:24:43 --> 00:24:48 So the next thing we want to know is when cells decide what they're 287 00:24:48 --> 00:24:52 going to become. And I can rephrase that by saying 288 00:24:52 --> 00:25:01 when is the fate decision made? 289 00:25:01 --> 00:25:06 And fate mapping doesn't tell you that. All it tells you is what a 290 00:25:06 --> 00:25:12 cell is going to become, not when the cell has decided to 291 00:25:12 --> 00:25:18 become that thing. For this you have to do some other 292 00:25:18 --> 00:25:24 assay, and you do assays that are generally called determination 293 00:25:24 --> 00:25:30 assays. And I'll show you an example of one such assay. 294 00:25:30 --> 00:25:34 So here's our 32 cell stage frog embryo again with the C3 cell 295 00:25:34 --> 00:25:38 labeled. And one can go into the embryo and dissect out that cell. 296 00:25:38 --> 00:25:42 Frog embryos are about a millimeter in diameter. And you can use very 297 00:25:42 --> 00:25:47 fine glass needles to tease out that cell from the embryo. 298 00:25:47 --> 00:25:51 And you can grow it in a very simple culture method with just some 299 00:25:51 --> 00:25:55 saline solution and the cell will grow fine and divide. 300 00:25:55 --> 00:26:00 And you can ask whether that cell goes on to make skeletal muscle. 301 00:26:00 --> 00:26:04 OK? Does it know that it's going to be skeletal muscle? 302 00:26:04 --> 00:26:08 And the answer at this stage of development is no, 303 00:26:08 --> 00:26:13 it doesn't. So if you remove the cell, grow it in culture, 304 00:26:13 --> 00:26:17 it goes on to become these things called fibroblasts which are cells 305 00:26:17 --> 00:26:21 that spread out in the dish. They don't fuse. They don't make 306 00:26:21 --> 00:26:26 all these muscle proteins. And so the cell, although it's 307 00:26:26 --> 00:26:30 destined to become muscle, has not yet made the decision to 308 00:26:30 --> 00:26:35 take that leap of fate. However, if you wait a few hours, 309 00:26:35 --> 00:26:40 and we're actually on your handouts now. If you wait a few hours and 310 00:26:40 --> 00:26:45 you go and you remove the cells that have arisen from C3, 311 00:26:45 --> 00:26:50 which are divided now. They're still labeled and you can 312 00:26:50 --> 00:26:55 distinguish them because it's a very powerful dye and doesn't diffuse. 313 00:26:55 --> 00:27:01 So you can remove that same group of cells a bit later. 314 00:27:01 --> 00:27:06 And you can then grow those cells in a culture, in a simple saline 315 00:27:06 --> 00:27:11 solution. And lo and behold they go on, after some hours, 316 00:27:11 --> 00:27:16 and form multinucleate myotubes. They go on to form muscle. So 317 00:27:16 --> 00:27:22 somewhere between the 32 cell stage and the stage of a much older embryo 318 00:27:22 --> 00:27:27 of many hundreds of cells, the cells have made the decision to 319 00:27:27 --> 00:27:32 go onto their particular fate. So something has changed in terms of 320 00:27:32 --> 00:27:36 the regulatory genes that they're expressing, presumably, 321 00:27:36 --> 00:27:40 that allow them to go and become muscle. OK. 322 00:27:40 --> 00:27:56 So let's break this 323 00:27:56 --> 00:28:02 down a bit more. And let's ask the question, 324 00:28:02 --> 00:28:07 as to what inputs are required to turning on MyoD activation in a more 325 00:28:07 --> 00:28:11 defined sense? And the first thing I'm going to 326 00:28:11 --> 00:28:16 talk about is how the embryo knows where dorsal is. OK? So 327 00:28:16 --> 00:28:25 dorsal determination. 328 00:28:25 --> 00:28:29 And what I'm going to tell you is a very interesting tale of 329 00:28:29 --> 00:28:34 determinants, of cell autonomous regulatory factors that are 330 00:28:34 --> 00:28:38 specifically required to tell the embryo where the future dorsal or 331 00:28:38 --> 00:28:43 backside of the embryo is going to be. 332 00:28:43 --> 00:28:48 And I'll tell you that these determinants act to stabilize a 333 00:28:48 --> 00:28:54 particular protein that is a transcription factor, 334 00:28:54 --> 00:29:00 and this protein is called beta-catenin. 335 00:29:00 --> 00:29:06 And beta-catenin, once it's stabilized, 336 00:29:06 --> 00:29:13 so it is a transcription factor, goes on to activate a set of genes 337 00:29:13 --> 00:29:20 that are dorsal specific genes, but not MyoD. We're not there yet. 338 00:29:20 --> 00:29:37 Gene. G. I cannot spell, can I? 339 00:29:37 --> 00:29:41 OK. So let's discuss this using your handouts. 340 00:29:41 --> 00:29:46 This is a movie that I was showing you at the beginning. 341 00:29:46 --> 00:29:50 Woops. Let's show it to you again because I want to point 342 00:29:50 --> 00:29:57 something out. 343 00:29:57 --> 00:30:00 All right. This is a four cell embryo. Maybe it's going to be an 344 00:30:00 --> 00:30:04 eight cell embryo. Do you see how these embryos have 345 00:30:04 --> 00:30:08 got some, two cells that are kind of darkly pigmented and two cells that 346 00:30:08 --> 00:30:12 are lightly pigmented? Yeah? The lightly pigmented cells 347 00:30:12 --> 00:30:15 are the ones that are going to be the future dorsal site of the embryo. 348 00:30:15 --> 00:30:19 OK? That's where the muscle is going to come from. 349 00:30:19 --> 00:30:23 And these cells on the other side, on the ventral side of the embryo, 350 00:30:23 --> 00:30:27 that's where the belly is going to come from. 351 00:30:27 --> 00:30:32 Now, how does this happen? Well, it happens, and I've 352 00:30:32 --> 00:30:38 augmented the diagram on one of your handouts a bit so you'll have to add 353 00:30:38 --> 00:30:44 a circle. It happens because of movement of these purple dorsal 354 00:30:44 --> 00:30:50 determinants. And these purple dorsal determinants, 355 00:30:50 --> 00:30:56 as I'll show you, move within the newly fertilized embryo. 356 00:30:56 --> 00:31:01 In fact, within about 16 minutes after fertilization these dorsal 357 00:31:01 --> 00:31:06 determinants have moved in the embryo and have told the embryo 358 00:31:06 --> 00:31:11 where the future dorsal side is going to be. And the way they move 359 00:31:11 --> 00:31:16 is that, in fact, the early egg and the zygote consist 360 00:31:16 --> 00:31:21 of two kinds of cytoplasm, an outer sphere of cytoplasm that's 361 00:31:21 --> 00:31:26 kind of gelatinous and an inner sphere of cytoplasm that 362 00:31:26 --> 00:31:31 is more liquid. And in this outer gelatinous 363 00:31:31 --> 00:31:35 cytoplasm sit these purple determinants. And when 364 00:31:35 --> 00:31:40 fertilization takes place these move by a good angle, 365 00:31:40 --> 00:31:44 at least 30 degrees relative to where they were, 366 00:31:44 --> 00:31:48 and relative to this inner liquid cytoplasm, which is why I've got 367 00:31:48 --> 00:31:53 this doted equator there to indicate that there is a relative movement of 368 00:31:53 --> 00:31:57 these determinants. OK? It's not the whole embryo 369 00:31:57 --> 00:32:02 that's moving. It's just part of the embryo that's 370 00:32:02 --> 00:32:06 moving relative to the other part. And this movement of determinants 371 00:32:06 --> 00:32:10 defines the dorsal and the ventral side of the embryo. 372 00:32:10 --> 00:32:15 OK. Here's a movie to show you that. 373 00:32:15 --> 00:32:22 OK. So this is a movie that is 374 00:32:22 --> 00:32:27 about the first 20 minutes of fertilization. 375 00:32:27 --> 00:32:32 And you can see here pigment moving from one region to another region. 376 00:32:32 --> 00:32:35 Now, the pigment is not the determinants. It just moves with 377 00:32:35 --> 00:32:39 the determinants. OK? So this is an indication that 378 00:32:39 --> 00:32:43 there is stuff moving, actually, from the vegetal pole 379 00:32:43 --> 00:32:47 region up towards this so-called animal pole region. 380 00:32:47 --> 00:32:51 So the terms animal and vegetal pole indicate two different sides of 381 00:32:51 --> 00:32:55 the radially symmetric egg. The animal pole is pigmented. 382 00:32:55 --> 00:32:59 The vegetal pole is not. And they're just useful landmarks. 383 00:32:59 --> 00:33:03 And, again, this is a frog embryo. Things may be similar in humans, 384 00:33:03 --> 00:33:07 but we understand much more about frogs. So that's why I'm going to 385 00:33:07 --> 00:33:11 tell you how things work in frogs. OK. So there is a manifestation of 386 00:33:11 --> 00:33:15 these dorsal determinants moving. These dorsal determinants move by a 387 00:33:15 --> 00:33:19 really fantastic process. They move on microtubules. 388 00:33:19 --> 00:33:23 Remember microtubules? What part of the embryo, 389 00:33:23 --> 00:33:27 what part of the cell were microtubules part of? 390 00:33:27 --> 00:33:33 Anyone remember? Dredge back in your memories. 391 00:33:33 --> 00:33:39 I heard something. Yes? Thank you. Cytoskeleton. 392 00:33:39 --> 00:33:45 Yes. See me for a frog afterward. So the microtubules are part of the 393 00:33:45 --> 00:33:51 so-called cytoskeleton. They are large rods of proteins, 394 00:33:51 --> 00:33:57 polymers of proteins. And when the sperm enters the egg it brings with 395 00:33:57 --> 00:34:03 it a centriole that is a microtubular organization center. 396 00:34:03 --> 00:34:07 And what it does, in this series of three pictures, 397 00:34:07 --> 00:34:12 is to take this mass of red microtubules that are oriented in 398 00:34:12 --> 00:34:17 every which way, and it organizes them so they form 399 00:34:17 --> 00:34:21 these beautiful parallel tracks of microtubules in the outer cytoplasm 400 00:34:21 --> 00:34:26 or between the outer and inner cytoplasm of the egg or of the 401 00:34:26 --> 00:34:31 zygote, of the fertilized egg. So the sperm nucleus or the sperm, 402 00:34:31 --> 00:34:36 excuse me, the sperm centriole is organizing this microtubular array. 403 00:34:36 --> 00:34:41 And it's on these tracks of microtubules that these determinants 404 00:34:41 --> 00:34:45 move, kind of like train tracks. And I'll tell you in a moment, it 405 00:34:45 --> 00:34:50 really is that way. So, oh, it didn't quite work. 406 00:34:50 --> 00:34:55 But OK. So here is a zygote and here is this protein I've told you 407 00:34:55 --> 00:35:00 about, beta-catenin. Now, in the zygote beta-catenin is 408 00:35:00 --> 00:35:06 phosphorylated. And that renders it unstable for 409 00:35:06 --> 00:35:11 various reasons. And it's also cytoplasmic. 410 00:35:11 --> 00:35:17 OK? With the action of the determinants I've been telling you 411 00:35:17 --> 00:35:22 about, by about the two to four cell stage, so very soon after 412 00:35:22 --> 00:35:28 fertilization the beta-catenin on one side of the embryo, 413 00:35:28 --> 00:35:34 the side where these determinants are has been dephosphorylated. 414 00:35:34 --> 00:35:38 So there is a post-translational modification. Dephosphorylated. 415 00:35:38 --> 00:35:43 And that renders it stable for various reasons. 416 00:35:43 --> 00:35:47 It's no longer subject to protein degradation. It also allows it to 417 00:35:47 --> 00:35:52 enter the nucleus. It allows it to act as a 418 00:35:52 --> 00:35:57 transcription factor. So these determinants are changing 419 00:35:57 --> 00:36:01 the stability and the protein structure of this beta-catenin 420 00:36:01 --> 00:36:06 transcription factor. Beta-catenin is really an important 421 00:36:06 --> 00:36:10 protein. This is a wild type frog embryo. Two wild type frog embryos. 422 00:36:10 --> 00:36:14 The head, tail, here's the eye forming. In embryos that have been 423 00:36:14 --> 00:36:19 depleted of beta-catenin you get these kinds of blobs that have no 424 00:36:19 --> 00:36:23 dorsal structures at all. They've got no muscle. They've got 425 00:36:23 --> 00:36:27 no nervous system. Nothing that would normally come 426 00:36:27 --> 00:36:32 from the dorsal side of the embryo. So what is the identity of these 427 00:36:32 --> 00:36:37 dorsal determinants? And, again, this you have partly on 428 00:36:37 --> 00:36:42 your handouts. And the identity of the dorsal 429 00:36:42 --> 00:36:47 determinants ergo is something that stabilizes beta-catenin and 430 00:36:47 --> 00:36:52 dephosphorylates it. And what these are believed to be 431 00:36:52 --> 00:36:57 presently are two proteins called GBP and dsh. OK? 432 00:36:57 --> 00:37:02 You don't need to know the names. It's the principle that's important 433 00:37:02 --> 00:37:08 here. But what's very interesting is that GBP and dsh proteins bind to 434 00:37:08 --> 00:37:13 another protein called kinesin, and kinesin attaches or is part of 435 00:37:13 --> 00:37:18 the microtubule system. So you get these determinants 436 00:37:18 --> 00:37:24 attaching to the microtubules, and as these microtubules polymerize 437 00:37:24 --> 00:37:29 and have a rotation associated with them, it's not quite clear they 438 00:37:29 --> 00:37:35 rotate at this point, this GBP and dsh are associated with 439 00:37:35 --> 00:37:40 the microtubules. And then these two proteins, 440 00:37:40 --> 00:37:44 GBP and dsh, also called disheveled, inhibit another protein called GSK3 441 00:37:44 --> 00:37:48 which is a kinase that phosphorylates beta-catenin and 442 00:37:48 --> 00:37:52 renders it unstable. So you're getting here dorsal 443 00:37:52 --> 00:37:56 determination. You can go think about this more 444 00:37:56 --> 00:38:00 clearly. Dorsal determination because you are inhibiting 445 00:38:00 --> 00:38:05 an inhibitor. All right? Complicated? 446 00:38:05 --> 00:38:09 Yes. Beautifully understood? More or less. This is really the 447 00:38:09 --> 00:38:14 most beautiful example that we know of how you get determination of a 448 00:38:14 --> 00:38:18 particular region of the embryo. OK. So one step. We probably have 449 00:38:18 --> 00:38:23 about 20 steps to go. And clearly we're not going to 450 00:38:23 --> 00:38:27 cover them all so I'm giving you a little meander through the number of 451 00:38:27 --> 00:38:33 steps that are involved. So another step informing muscle, 452 00:38:33 --> 00:38:39 that I want to tell you about, is forming a particular kind of cell 453 00:38:39 --> 00:38:46 from which the muscle will arise. So not only does muscle arise from 454 00:38:46 --> 00:38:52 cells that are dorsally located. Muscle also arises from a cell type 455 00:38:52 --> 00:39:01 called the mesoderm. 456 00:39:01 --> 00:39:06 The mesoderm is part of a system of cells that are the earliest known 457 00:39:06 --> 00:39:12 cell types in the embryo. And these are the germ layers. 458 00:39:12 --> 00:39:18 And the germ layers consist of mesoderm, and also of two other 459 00:39:18 --> 00:39:24 cells types called ectoderm and endoderm. OK. 460 00:39:24 --> 00:39:30 And these cell types were discovered a lot time ago. 461 00:39:30 --> 00:39:33 They're not differentiated cell types but they do have different 462 00:39:33 --> 00:39:37 properties. They are stepwise along the way to becoming different things. 463 00:39:37 --> 00:39:41 The mesoderm, shown in red here, 464 00:39:41 --> 00:39:44 gives rise to the muscle, blood, kidney, heart and various 465 00:39:44 --> 00:39:48 other things. OK? The ectoderm, for example, 466 00:39:48 --> 00:39:52 gives rise to all of the nervous system, and the endoderm to the gut 467 00:39:52 --> 00:39:56 and the lung. And they get their names from their position 468 00:39:56 --> 00:40:00 in the early embryo. The endoderm is on the inside, 469 00:40:00 --> 00:40:06 the ectoderm on the outside, and the mesoderm between the two. 470 00:40:06 --> 00:40:11 OK. How does the mesoderm form? The mesoderm forms through another 471 00:40:11 --> 00:40:16 complicated system. It forms through the action of a 472 00:40:16 --> 00:40:22 transcription factor called VegT. And this is a maternally expressed 473 00:40:22 --> 00:40:27 transcription factor. So we can distinguish maternal and 474 00:40:27 --> 00:40:33 zygotic effects, genetically and in development. 475 00:40:33 --> 00:40:41 VegT is maternally expressed. That means it is present in the egg. 476 00:40:41 --> 00:40:50 VegT in term activates in just one region of the embryo another gene 477 00:40:50 --> 00:40:59 called nodal. It's actually a set of genes. Nodal is 478 00:40:59 --> 00:41:08 a secreted factor. It is actually a ligand and, 479 00:41:08 --> 00:41:16 therefore, an inducer. And it is zygotically expressed. 480 00:41:16 --> 00:41:24 So another principle, "zygotically X" for expressed, 481 00:41:24 --> 00:41:33 and "maternally X" for expressed. OK. 482 00:41:33 --> 00:41:37 This is one your handouts. So we're talking now about the same 483 00:41:37 --> 00:41:42 time of development that we've been talking about but a different system 484 00:41:42 --> 00:41:47 that is working in parallel. Here is an embryo. And I've shown 485 00:41:47 --> 00:41:51 you between the 200 and the 500 cell stage that has got this VegT 486 00:41:51 --> 00:41:56 transcription factor present in the nucleus. It's a transcription 487 00:41:56 --> 00:42:01 factor. And you can find a concentration 488 00:42:01 --> 00:42:05 gradient of this thing. In one part of the embryo at this 489 00:42:05 --> 00:42:09 equatorial region there are low amounts of it. 490 00:42:09 --> 00:42:14 Towards the vegetal pole there are high amounts. And where you have 491 00:42:14 --> 00:42:18 this low amount of VegT you get a band of cells that's going to become 492 00:42:18 --> 00:42:23 mesoderm. This mesoderm then goes on to express this nodal gene which 493 00:42:23 --> 00:42:27 is a secreted ligand. And, for those of you who are 494 00:42:27 --> 00:42:31 familiar with this, it's a member of something called 495 00:42:31 --> 00:42:37 the TGF-beta family. So this gives you another set of 496 00:42:37 --> 00:42:43 cells to deal with. And what I'm going to tell you now 497 00:42:43 --> 00:42:49 is that to think about where the skeletal muscle comes from we have 498 00:42:49 --> 00:42:56 to superimpose the two things I've told you about becoming dorsal and 499 00:42:56 --> 00:43:02 becoming mesodermal. So the next thing we need to do is 500 00:43:02 --> 00:43:09 to talk about the dorsal mesoderm. And we can write an equation here 501 00:43:09 --> 00:43:16 where beta-catenin plus nodal makes dorsal mesoderm. 502 00:43:16 --> 00:43:23 And I'll tell you that this dorsal mesoderm has a very special property 503 00:43:23 --> 00:43:31 and a special name. It's called the organizer. 504 00:43:31 --> 00:43:36 Is the dorsal mesoderm the future skeletal muscle? 505 00:43:36 --> 00:43:42 I'm sorry to tell you it's not really. There's another step 506 00:43:42 --> 00:43:48 involved. And I will tell you in a moment that the organizer is going 507 00:43:48 --> 00:43:54 to induce the precursors of the skeletal muscle. 508 00:43:54 --> 00:44:00 So let me write this down just to give you a sense of where we are. 509 00:44:00 --> 00:44:04 OK. So let's move on here. And let's define dorsal mesoderm by 510 00:44:04 --> 00:44:09 an equation. And, in fact, you can define this whole 511 00:44:09 --> 00:44:13 process by an equation. But, of course, the genes that have 512 00:44:13 --> 00:44:18 to be expressed and so on have to be expressed in a specific temporal 513 00:44:18 --> 00:44:23 order. So it's not a simple linear equation. It's got a time component, 514 00:44:23 --> 00:44:28 too. OK. So here we have beta-catenin on the dorsal side. 515 00:44:28 --> 00:44:33 And we have mesoderm defined by nodal signaling in a band across the 516 00:44:33 --> 00:44:38 equator. And if we superimpose them we get a region where both of these 517 00:44:38 --> 00:44:43 factors are in the same group of cells. And where both of these 518 00:44:43 --> 00:44:49 factors are in the same group of cells we get a special type of 519 00:44:49 --> 00:44:54 mesoderm formed called the dorsal mesoderm or the organizer. 520 00:44:54 --> 00:45:00 The organizer is a very famous piece of mesoderm. 521 00:45:00 --> 00:45:05 And it is famous for this reason. This is a very old experiment that 522 00:45:05 --> 00:45:10 was done in the 1920s where one could take a piece of donor tissue 523 00:45:10 --> 00:45:16 from a donor embryo, that is the future organizer, 524 00:45:16 --> 00:45:21 and move it to a host embryo, and put it on the side of the embryo 525 00:45:21 --> 00:45:27 where the organizer wasn't. OK? So to put it on the ventral 526 00:45:27 --> 00:45:31 side of the host embryo. And when this was done, 527 00:45:31 --> 00:45:35 to the surprise of the investigators, because they really didn't do the 528 00:45:35 --> 00:45:39 experiment for this reason, they found that they got these 529 00:45:39 --> 00:45:43 conjoined twins. Conjoined twin embryos. 530 00:45:43 --> 00:45:46 Here's your host embryo. And this is a second embryo that's 531 00:45:46 --> 00:45:50 formed. And you could look at see where that piece of transplanted 532 00:45:50 --> 00:45:54 tissue went. And when you did you found it was in a strip along the 533 00:45:54 --> 00:45:58 back of the embryo, but most of the second embryo, 534 00:45:58 --> 00:46:02 the conjoined embryo came from tissue that was the host 535 00:46:02 --> 00:46:06 embryo tissue. In other words, 536 00:46:06 --> 00:46:10 this piece of transplanted tissue had organized or induced a second 537 00:46:10 --> 00:46:15 embryo, including all the skeletal muscle. OK? And including a head 538 00:46:15 --> 00:46:19 and a brain and so on. This is a very famous experiment, 539 00:46:19 --> 00:46:24 very famous piece of tissue. And for this experiment the Nobel Prize 540 00:46:24 --> 00:46:29 was awarded to Hans Spemann in 1935. 541 00:46:29 --> 00:46:33 Here are conjoined frog embryos made by transplanting organizers. 542 00:46:33 --> 00:46:38 You can see they have two heads and they're joined at their tail region. 543 00:46:38 --> 00:46:43 What about humans? Well, the same thing is true in human embryos. 544 00:46:43 --> 00:46:48 Usually identical twin embryos will split somewhere between the two cell 545 00:46:48 --> 00:46:52 stage and a very early stage where there is something called the inner 546 00:46:52 --> 00:46:57 cell mass. And you get two identical embryos made and you get 547 00:46:57 --> 00:47:02 completely separate embryos made. Sometimes, very rarely, 548 00:47:02 --> 00:47:06 you get incomplete splitting of the embryo. And the organizer is split 549 00:47:06 --> 00:47:10 in two in a human embryo, and you land up with human conjoined 550 00:47:10 --> 00:47:14 twins. This is very rare. The survival of conjoined twins is 551 00:47:14 --> 00:47:19 very rare. There are some instances, and I've put on your handout a 552 00:47:19 --> 00:47:23 diagram of, these are actual girls who had been diagramed that were 553 00:47:23 --> 00:47:27 just like the frogs, with shared heads, with a shared 554 00:47:27 --> 00:47:32 trunk and separate heads. So the processes in frogs, 555 00:47:32 --> 00:47:37 as far as we know, are very similar to those in humans. 556 00:47:37 --> 00:47:42 Now, what I'm going to do because I have very interesting stuff I'd like 557 00:47:42 --> 00:47:47 to finish this off, finish off here, but I see time is 558 00:47:47 --> 00:47:52 creeping on, is to end off with a teaser. And to tell you that the 559 00:47:52 --> 00:47:57 final step in getting MyoD on is to activate these things 560 00:47:57 --> 00:48:02 called somites. And I'm going to take five minutes 561 00:48:02 --> 00:48:06 at the beginning of next lecture to complete this and then we will move 562 00:48:06 --> 00:48:09 onto our next topic. So thank you.