The Vertebrate Retina, Photoreceptors, and Color Vision
Transcript of Part 4: The Evolution of Trichromatic Color Vision
00:00:02.06 I'm Jeremy Nathans. I'm a professor at the Johns Hopkins Medical School 00:00:05.28 and an investigator of the Howard Hughes Medical Institute. 00:00:09.09 This is the third of three lectures on the vertebrate retina 00:00:12.29 on its structure, function, and evolution. 00:00:16.12 And in this third part, we're going to focus on the evolution of trichromatic color vision. 00:00:21.02 Now, if we just consider the requirements that an organism needs to fulfill, 00:00:28.07 to evolve a new dimension of color vision... 00:00:31.21 And here, I'll use the word dimension in the way Maxwell used it. 00:00:35.22 We saw in the last lecture that Maxwell envisioned a 3D space - a color space... 00:00:41.22 a perceptual space, where each dimension corresponded to the degree of excitation 00:00:48.09 of a single type of photoreceptor cell. 00:00:51.06 If we ask what one needs to do to add an additional dimension to that color space... 00:00:57.27 And that's exactly the evolutionary step that we're going to consider in this lecture, 00:01:02.17 this transition from a simple or simpler dichromatic color vision 00:01:08.25 to the trichromatic color vision that we humans enjoy today. 00:01:12.01 If we ask what's essential to make that evolutionary step, 00:01:16.24 I think we can divide it into three parts. 00:01:20.12 First, the organism needs to evolve a new light-sensing visual pigment. 00:01:26.23 And this would be presumably encoded by a distinct gene. 00:01:30.12 The gene would encode a protein whose sequence differs from those of the pre-existing visual pigment proteins. 00:01:36.10 And then, that gene needs to be expressed in a second requirement. 00:01:43.29 It needs to be expressed in a distinctive set of photoreceptor cells. 00:01:47.17 Distinct from the visual pigment expression that was pre-existing in that organism. 00:01:53.14 And finally, if those two steps are fulfilled, the organism still needs one thing more, 00:01:58.27 and that is it needs the neural circuitry to extract the information from that new receptor 00:02:04.09 and compare it to the information that it's receiving from the pre-existing receptors. 00:02:08.28 As we proceed in this lecture, we'll consider each of these three steps, 00:02:13.13 and what we know about them, and how we think they might work. 00:02:16.15 Now, how do we know that an organism has color vision? 00:02:22.01 How do we know that an animal has color vision? 00:02:24.04 At some level, you have to ask the animal. 00:02:28.03 Of course, if the animal is a human, that's simple. 00:02:31.23 You can set up a color vision test that Newton set up, as we saw in the last lecture, 00:02:36.10 in which, for example, two spectrally pure lights are superimposed, 00:02:41.15 and they would match in appearance a third light. 00:02:44.26 And we can ask the person, do they match or do they not match? 00:02:48.02 And if they don't match, we can, for instance, give the person a dial and let them adjust the intensity 00:02:53.10 of one or another of those lights until a match is made. 00:02:56.21 That's the classical human color vision testing. 00:02:59.14 For a non-human animal, where one doesn't have the advantage of verbal communication, 00:03:04.24 we have to set up a test that will determine whether the animal can make those kinds of discriminations... 00:03:10.29 chromatic discriminations. And the test is of the sort shown here. 00:03:16.00 Now, this is a little monkey who has the task of deciding which one of these three images 00:03:23.00 looks different from the other two. 00:03:24.19 I should say this particular slide is a lab joke 00:03:27.23 We don't usually show them pictures of people... we show them just a blank panel of one or another color 00:03:34.06 or one or another intensity, and the animal decides which of the three panels looks different. 00:03:39.02 But here, this little lady is going to decide that this set of three panels has one that stands out, 00:03:47.23 (it's the one on the right, obviously - it's in color and the other two are in black and white), 00:03:51.20 and she'll press a little lever underneath the bowl that corresponds to that panel, 00:03:57.06 and she'll get a little candy reward in that bowl for figuring out the right answer. 00:04:00.26 And this sort of test will be repeated on many trials - 00:04:05.04 the picture that is different would be changed in its location 00:04:09.28 among the three different locations, and in particular, one would vary the intensity. 00:04:16.22 Now, remember, in the real world we don't use photos like this - portraits like this. 00:04:22.00 We're just using blank panels of a solid color. 00:04:25.11 But, we would vary the intensities of the colors at random. 00:04:28.29 And that's a very important point to make, because we want to make sure 00:04:32.27 that the animal is not cueing on intensity, 00:04:34.27 but is cueing strictly on color. 00:04:37.22 And the animal has to be smart enough and also have the color vision ability 00:04:43.01 to realize that the test can be figured out by determining which color looks different 00:04:50.08 in dependent of intensity differences. 00:04:52.12 Now, we're going to focus on evolution in the primate lineage, 00:04:58.17 and I want to just remind you that primates are quite diverse, although we think of them 00:05:02.19 as just one little subset of the mammals. 00:05:06.03 Here we have just three representative primates - gibbons on the left, a gorilla in the middle, 00:05:10.20 and a bushbaby on the right. 00:05:11.24 I want to just remind you that this diversity is really enormous. 00:05:17.23 The bushbaby on the right could easily fit into the palm of the hand of the gorilla in the middle. 00:05:22.18 And, within the primate lineage, color vision is also diverse. 00:05:27.24 The diversity correlates in an interesting way with the habitats of the primates. 00:05:34.26 Now, recall from plate tectonics that the continents were not separated for all time, as they are now. 00:05:44.05 But, South America and Africa used to be contiguous, and they've been moving steadily apart. 00:05:50.11 That's been going on for about 150 million years. 00:05:54.07 About 50 or 75 million years ago, there was a complete separation of all of the animal species, 00:06:02.03 in the sense that there was no communication whatsoever, 00:06:04.23 and the new and old world primates in particular were free to go their separate evolutionary ways. 00:06:10.09 And, as you'll see in a minute, they have, with respect to their color vision. 00:06:13.26 Now, we could ask, in the case of the evolution of trichromacy within the primate lineage, 00:06:22.17 what advantage is there? Why not stick with the dichromacy 00:06:26.16 which characterizes all of the non-primate mammals... all of the lower mammals. 00:06:30.28 And, although I don't think we really know the answer to this question, 00:06:34.24 there are some ideas which are quite plausible. 00:06:37.06 One of the most plausible is that it is easier to detect fruit among foliage, 00:06:44.02 and in particular, the ripeness of fruit, if one has trichromatic color vision. 00:06:47.22 Now, we've got a sense of that in the preceding lecture, when we looked at an image 00:06:52.22 of a fruit stand as seen by a normal trichromat, and various of the types of dichromats 00:06:59.02 in the human population. 00:07:00.09 Dichromats clearly have a more difficult time judging the ripeness of fruit, 00:07:04.26 and for animals that get a substantial fraction of their food from fruit, as many primates do, 00:07:11.10 this could be a significant selective pressure. 00:07:14.19 It's also possible that there are other visual tasks 00:07:19.01 which would be better performed with trichromatic color vision. 00:07:21.28 Some animals - some primates - are brightly colored, and it's a general pattern 00:07:28.07 that those animals which have bright coloration, be they birds or 00:07:31.16 fish - tropical fish, for example - also have excellent color vision to see that coloration. 00:07:37.26 So, the answer is not in, in terms of what the real selective pressures are for trichromatic color vision, 00:07:45.06 but it is widespread among primates, and we presume there is some positive selective value to it. 00:07:51.00 Now let's revisit a slide that we saw on the previous lecture, just briefly, 00:07:54.27 and this is just to remind ourselves that the basic mammalian arrangement, 00:07:59.07 as shown by this dendrogram in the center, for the mouse, 00:08:02.15 is one in which there are just two cone pigment genes. 00:08:06.20 There's a longer wave one (an M pigment in the mouse) 00:08:09.17 and a shorter wave one (it happens to be UV-sensitive in the mouse). 00:08:12.22 And, in the case of humans and other old-world primates, like gorillas and macaques, and so on, 00:08:19.03 we have seen a split in the longer wave pigment lineage - a very recent split - 00:08:24.27 so that now we have both L and M pigments in place of just a single pigment. 00:08:29.27 But those pigments are still very similar in their sequences. 00:08:33.05 Let's examine one of the earliest clues to the origins of primate trichromacy, 00:08:41.09 and that was gleaned by looking at the way in which color signals are analyzed in the retina. 00:08:48.21 If we look at the basic dichromat arrangement, 00:08:52.08 (this is the arrangement typical of non-primate mammals) 00:08:55.10 it's quite simple. They have a shorter wave cone (an S cone) 00:08:59.05 and they have a longer wave one, here I just call it M/L - the precursor of our M and L pigments. 00:09:04.27 And, these two cone types feed in an antagonistic way into a circuit which ultimately 00:09:15.20 is essentially a blue vs. yellow circuit - a shorter wave vs. a longer wave light circuit 00:09:23.06 which then goes to the brain. And, this might be, for example, an excitatory input 00:09:29.19 from the longer wave side and an inhibitory input from the shorter wave side, 00:09:33.11 and the output (the ganglion cell that is the output of this little circuit) 00:09:39.00 is essentially a differential analyzer for long vs. shorter wave lights. 00:09:43.07 That's a basic circuit that is present in all mammals. 00:09:46.04 We have essentially that same circuit in our retinas still, 00:09:49.05 and, as I'll show you in a minute, it has not changed very much. 00:09:52.05 But, if we look at the primate retina, at the old-world primate retina, 00:09:58.23 a retina like ours, we see that the circuitry has changed remarkably little. 00:10:06.18 So, for example, that circuit that I showed you on the previous slide has now just been 00:10:10.26 enhanced by adding the M and L cone inputs to the limb 00:10:16.24 that previously received the ancestor of those cones. 00:10:20.07 But, it makes no distinction between these two new cone types. 00:10:24.00 M and L cones are treated equivalently, as regards this short vs. longer wave circuit. 00:10:31.14 Ultimately, after summing them at the first stage, 00:10:34.27 they are compared in an antagonistic fashion (+ vs. -) in a second stage 00:10:40.09 and one gets essentially the same sort of output. 00:10:43.07 (A blue vs. yellow type output.) 00:10:46.22 There is a second circuit, though, which is involved in comparing M vs. L signals. 00:10:55.06 This circuit is again an antagonistic one - one of the cone signals is for example excitatory, 00:11:04.12 that's shown here as the L signal, the other inhibitory (the M signal). 00:11:07.13 But, there's a curious feature of this circuit which would appear to be a new circuit added on 00:11:14.11 and not present in the more primitive mammals. 00:11:18.03 And that is actually... it's not a new circuit... this circuit pre-exists 00:11:21.28 as simply a circuit involved in spatial vision, and it uses, in our ancestors, 00:11:30.09 a single cone type - the precursor of the M and L cones 00:11:33.25 and simply compares a signal, for example, in the center of a receptive field 00:11:39.10 to the signal in the surround - a signal that's completely achromatic 00:11:43.19 (a non-color signal) that tells the organism something about intensity differences 00:11:48.03 in one place vs. another place. 00:11:50.00 It appears that the same circuitry, essentially unchanged as far as we can tell, 00:11:55.08 has now been coopted to tell us something about color, as well as spatial intensity. 00:12:01.20 In fact, the two pieces of information - the two kinds of information - are now convoluted 00:12:08.05 within the signal. And, it's not entirely clear exactly where and exactly how 00:12:13.25 those signals would be deconvoluted. 00:12:15.20 But this observation - this finding that the circuitry within the retina appears to have changed 00:12:25.00 little if at all, in the transition from dichromat to trichromat vision is a striking one. 00:12:32.23 And it argues that the principal evolutionary event, and perhaps the only evolutionary event, 00:12:38.25 in making this transition was the acquisition of a new kind of cone. 00:12:43.12 Now, I want to just mention one thing about this peculiar circuitry. 00:12:50.15 I think you can appreciate there's an asymmetry here, in the sense that the 00:12:54.14 circuit does not compare S vs. M and S vs. L and L vs. M in a symmetric way 00:13:03.03 the way it could have. 00:13:04.21 And, because of this sort of peculiar pairwise comparison of S vs. M+L 00:13:11.23 and M vs. L, we have essentially a pair of axes for color comparisons. 00:13:18.22 One can think of them as sort of an X and Y axis space, 00:13:23.12 which has resulted in a peculiar psychophysical phenomenon, in which, when we view 00:13:31.15 the wavelengths of light which one can splay out in order of their wavelengths, 00:13:37.28 from short wavelengths at the ultraviolet end through the blues, the greens, 00:13:41.25 the yellows, the oranges, the reds, to the longer wavelengths, 00:13:45.10 essentially a linear physical stimulus where we go from short wave to long wave... 00:13:53.02 The visual system - our visual system - actually converts this linear scale into a circular scale. 00:14:02.00 And, I think we've all appreciated this from kindergarten, when we first started playing with crayons, 00:14:06.29 that if you draw the colors in what seems to be a natural order - natural in the sense 00:14:13.06 that the gradations between colors is minimal, 00:14:15.04 we naturally arrange them in the order red, orange, yellow, green, blue, violet, but then 00:14:25.29 we think violet and red are actually rather similar - there's sort of a purple transition between them, 00:14:31.23 but we naturally connect it into a circular pattern. Why do we do that? 00:14:35.19 Why do we make a circle out of what is physically a linear input? 00:14:40.11 And the answer is, because we're using this pair of axes to do our color comparisons 00:14:46.05 in the visual system, we are basically looking at red vs. green - that is the L vs. M dimension 00:14:56.20 in one comparison, and we're looking at the short wave (the blue) vs. the sum of red and green 00:15:02.28 (blue vs. yellow) in the other dimension. 00:15:06.02 And simply by dividing it in these two dimensions - red/green and yellow/blue, 00:15:11.08 we naturally create a two-dimensional system in which we psychologically 00:15:17.16 complete the circle. That's sort of an aside, but it's a natural outcome 00:15:21.27 of that way in which we analyze color. 00:15:24.16 Now let's talk a bit about the details of visual pigment sequence 00:15:29.29 and the implications of those sequences for the evolution of primate trichromacy. 00:15:35.20 In the previous lecture, we looked a little bit at the sequence differences that distinguish 00:15:42.05 the different human pigments, one from the next, 00:15:44.22 and we remarked on the striking similarity between 00:15:48.18 long and middle wave pigments (the L and M pigments). 00:15:51.20 And if we look at those comparisons in a little greater detail, 00:15:56.10 and then ask functionally, what's the significance of the various amino acids 00:16:00.19 that differ between L and M pigments, 00:16:02.14 what we see is that there are really only three major players in terms of the spectral sensitivity - 00:16:09.14 the spectral tuning, especially the differential tuning of L and M pigments. 00:16:14.04 That is, there's an amino acid at 277, which can either be phenylalanine or tyrosine, 00:16:19.15 at 285, which can be either alanine or threonine, 00:16:22.27 or at 180 which can be either alanine or serine - in fact, this is the one that's polymorphic 00:16:28.00 in the gene pool in the L pigment. 00:16:30.10 And those three together are responsible for virtually all of the spectral difference 00:16:38.12 between the L and M pigments. 00:16:40.21 If phenylalanine is present at 277, if alanine is present at 285, and alanine at 380, 00:16:47.23 the pigment will absorb at 530nm maximally. 00:16:51.24 If tyrosine is present at 277, threonine at 285, and serine at 180, 00:16:57.12 the pigment will absorb at maximally at 560nm. It'll be an L pigment. 00:17:01.17 And those three players are of interest 00:17:07.06 because they have been seen in both new and old world primates, 00:17:11.07 as we'll discuss in just a minute. 00:17:14.05 But at this point, I need to take a slight interlude and say a few words about the X chromosome 00:17:21.07 and the significance of X-linkage, because X-linkage turns out to loom very large in this story. 00:17:27.17 In all mammals, males differ from females in their chromosomal constitution. 00:17:33.07 Males have an X chromosome and a Y chromosome and females have two X chromosomes. 00:17:40.02 The genes on the X chromosome, of which there are many thousands, 00:17:45.12 must be expressed in both males and females at the appropriate level. 00:17:49.07 Yet, females are endowed with twice the number of copies of those genes as males have. 00:17:56.00 So, how does one avoid expressing twice as many copies of the corresponding mRNAs 00:18:03.19 in a female, compared to a male? If one did, that would be disastrous. 00:18:08.12 It would wreak havoc with development. 00:18:10.07 Mammals have hit upon a peculiar solution to this problem. 00:18:15.13 There is a selective inactivation of one of the two X-chromosomes 00:18:20.18 in each cell in the female body. Now, just for completeness, let me just say 00:18:25.22 it's not every gene on the X chromosome that obeys this rule, but most of them do. 00:18:29.16 And, what we see is that, at an early point in development, 00:18:35.12 when the embryo consists of on the order of a few thousand cells, 00:18:38.14 each of those cells decides with essentially a molecular coin toss, 00:18:42.18 which of the two X chromosomes will be inactive and which will remain active. 00:18:47.00 And the result is that all female mammals are mosaics, 00:18:50.01 as shown here for this cat - this female cat has an X-linked coat color variation. 00:18:58.04 She's a heterozygote. And, I think you can appreciate that there are patches of her coat 00:19:03.02 that are one color and other patches that are another color 00:19:05.16 that are sort of haphazardly mixed to some extent. 00:19:07.20 But, there are other parts where there are little chunks that are solidly one or the other. 00:19:13.14 And, this sort of mosaicism then can play itself out in the context of any number of X-linked genes, 00:19:22.09 and it turns out that it plays itself out in the context of the visual pigment genes 00:19:27.04 in new world primates in an especially interesting way. 00:19:30.16 New world primates, as it turns out, have trichromatic color vision 00:19:37.09 only for females. Males are dichromats among the new world monkeys. 00:19:42.27 And among the females, in those populations, only a fraction are trichromats. 00:19:48.17 (Roughly two-thirds.) 00:19:50.06 And, the way this works is that those new world monkeys 00:19:56.24 have a single X-linked visual pigment gene. 00:20:00.22 They don't have the gene duplication that we have. 00:20:03.17 But that gene is polymorphic in the population. 00:20:06.19 And, the different alleles encode different spectrally-sensitive variants of that pigment. 00:20:12.27 So, for example, for one typical new world monkey species, 00:20:18.17 we see that there are three alleles - one maximally sensitive at 535nm 00:20:24.10 (rather like our M pigment), another at 550nm (sort of midway between the M and L pigments), 00:20:31.03 and a third at 563nm (quite close to our L pigment). 00:20:36.01 By comparison, our old world arrangement involves two genes (two or more in some cases), 00:20:43.19 in tandem on the X chromosome, and they encode pigments of 530nm at peak absorption 00:20:50.26 and 560nm. 00:20:52.08 Now the striking finding, when one looks at the sequences 00:20:55.23 of new and old world monkey pigment genes, 00:20:59.20 is that the new world monkeys are using exactly the same three amino acid differences 00:21:05.29 to differentially tune the spectral sensitivities as we use 00:21:10.23 (we, old world primates use) to differentially tune the spectral sensitivities of our pigments. 00:21:15.19 So, just to recap, we use at these three critical sites, alanine, phenylalanine, and alanine 00:21:23.17 in our M pigment and serine, tyrosine, and threonine in our L pigment, 00:21:30.12 and the new world monkeys do exactly the same thing. 00:21:34.01 And the pigment that's halfway in between has an alanine and a phenylalanine and a threonine. 00:21:40.14 That is, it's sort of a hybrid between M and L, just as you might have expected. 00:21:45.20 And, this is quite a striking finding, because we know from work 00:21:50.03 both by site-directed mutagenesis and by sequencing the genes from different mammals, 00:21:55.00 that this is not the only way to tune these pigments. 00:21:58.05 There are other ways (there are other amino acids, other positions) that can vary 00:22:02.29 that can give rise to spectral tuning to either shorter or longer wavelengths. 00:22:08.07 And the fact that new world and old world primates use exactly the same amino acid variations 00:22:15.27 to tune their spectral sensitivities argues that this is not likely a coincidence - 00:22:21.19 not likely a convergence on the same solution, but rather that they arose from a common ancestor. 00:22:26.27 And we suspect that the common ancestor is basically the new world arrangement. 00:22:33.19 That is, this allelic variation pre-existed, we imagine, 00:22:38.16 in the earliest primates that pre-dated the separation of new and old worlds. 00:22:44.27 And, as shown schematically on this next slide, 00:22:47.25 we can envision, therefore, a timeline (time now going from left to right) 00:22:52.03 in which non-primate mammals, shown at the very top, 00:22:56.12 have gone their separate route and remain dichromatic, 00:22:59.22 and along the primate lineage (or the proto-primate lineage), 00:23:04.25 we see, at some point before the split of new and old world, 00:23:09.15 this polymorphism appearing in the X-linked pigment genes. 00:23:13.03 And then, with the split of new world (who have maintained that system) 00:23:17.23 and old world primates, there has been the acquisition at some point 00:23:24.24 in the old world primate lineage, of this gene duplication that we now enjoy. 00:23:30.01 This harks back to the previous lecture when I mentioned that we believe that the duplication 00:23:36.15 occurred between variant sequences that were already different. 00:23:42.08 That is, between a pre-existing L-like pigment and a pre-existing M-like pigment. 00:23:47.14 And, that's in a sense a bit of an exception to the way that people think 00:23:54.08 that gene evolution usually occurs. 00:23:55.12 The classic mode of gene evolution involves the duplication of a gene 00:24:00.15 to make an identical copy and then, over time, the acquisition of mutations 00:24:05.13 within that copy or perhaps as well the parental copy, to change their sequence and their function. 00:24:11.16 So that's essentially duplication followed by divergence - 00:24:14.26 that's certainly the classic mode of gene evolution, 00:24:19.01 and it's been documented in many, many instances. 00:24:22.12 This is really the reverse. This is divergence followed by duplication. 00:24:27.20 And, it begs the question of whether there might be other examples of this sort as well. 00:24:33.14 Now, this really, I think addresses the first of the three challenges 00:24:42.23 for evolving a new dimension of color vision, 00:24:44.27 that is, the production of a pigment that has a novel absorbance spectrum. 00:24:49.21 So, this has happened initially by allelic variation, 00:24:54.07 and now it's been cemented in the old world lineage by gene duplication, 00:24:57.04 and now, let's consider the second of the three requirements. 00:25:01.23 And that is a way in which the novel pigment gene could be expressed in a class 00:25:07.29 of photoreceptor cells distinctive from the class that is expressing the pre-existing pigments. 00:25:12.23 The first clue to how this might work for the L and M pigments 00:25:19.19 came from a study of those rare humans 00:25:23.15 who are missing the expression of both M and L sensitivities. 00:25:29.28 These individuals have what's called blue cone monochromacy. 00:25:33.07 They have normal blue cones with which they can see the world, and they have normal rods. 00:25:37.28 But, I think you can appreciate, since the rod system doesn't contribute to color vision, 00:25:43.13 and the blue cones are the only cone system that they possess, 00:25:46.14 there is no ability to compare the blue cone output to any other cone type. 00:25:51.11 And therefore, these individuals are monochromats - they have a fully monochromatic 00:25:57.05 color world - that is, just a one-dimensional world, in which there is essentially only intensity 00:26:02.00 and they have no sense of color at all. 00:26:05.01 So, this is true color blindness. 00:26:06.21 Now, it turns out that blue cone monochromacy is X-linked, 00:26:09.19 as are the common dichromacies and anomolous trichromacies. 00:26:13.09 And, in fact, blue cone monochromacy arises from sequence variation at the L and M gene locus. 00:26:20.18 And there are two basic mechanisms for blue cone monochromacy. 00:26:24.28 The first, which I'll describe here just for completeness, 00:26:27.04 which is really not that relevant to our story, 00:26:29.22 is the one that you might have guessed, knowing what you know thus far 00:26:33.24 about how this came about. 00:26:35.25 And that is that the wild type array (just one example is shown here at the top - 00:26:41.12 one L and two M pigment genes), can by the sort of homologous recombination 00:26:45.13 that we considered in the previous lecture, be reduced to a single gene within the array. 00:26:50.21 And that can either be an L pigment gene or an L/M hybrid gene. 00:26:55.07 This is no news at this point. The individual who carries those genes would be a dichromat. 00:27:01.24 But then, that gene could suffer an additional mutational event. 00:27:06.12 It could, like any gene, suffer some sort of mutation which disrupts protein function or structure, 00:27:12.08 and as a result, this individual would now, by virtue of these two mutational steps, 00:27:18.11 be missing all functional long and medium wave pigment genes. 00:27:24.05 And this is, in fact, seen in about half of blue cone monochromats. 00:27:27.28 Blue cone monochromacy occurs at a frequency of about 1 person in 100,000, 00:27:33.01 and about half of blue monochromats have the sort of mutational event I've illustrated here. 00:27:38.23 The other half, though, are more germane to the lecture today. 00:27:44.19 And that is, these individuals have, in one step, suffered a mutational event 00:27:51.15 which has eliminated the expression of the L and M pigment gene array. 00:27:56.00 And interestingly, in many of them, that mutational event, in fact, 00:28:00.28 has not affected the coding regions of those genes at all. 00:28:04.05 So, here's an example of a normal array - one L and 2 M pigment genes (this is a typical array). 00:28:10.09 And here, below, is the array with a deletion of sequences upstream. 00:28:18.02 That deletion, as far as we can tell from analyzing those individuals, 00:28:24.22 has completely eliminated the expression of all the genes in the array. 00:28:28.14 So there's some sequence - there's some critical sequence upstream - 00:28:31.05 which is involved in their expression. 00:28:33.06 This is not completely surprising - in the mammalian gene expression business, 00:28:38.13 this would be called an enhancer (a sequence at some distance from the genes that it controls) 00:28:45.29 and presumably it is involved in some sort of chromatin assembly 00:28:51.09 of promoter and enhancer sequences, 00:28:54.02 and their associated proteins, which control transcription. 00:28:57.12 Now, we know that this sequence is important in the control of gene expression 00:29:05.11 because if we produce a transgenic mouse which carries the human upstream sequences - 00:29:11.02 the promoter region of the L pigment gene, for example plus this upstream region, 00:29:16.22 which this enhancer-like region which we infer is important 00:29:20.18 from the analysis of blue cone monochromats, 00:29:22.23 we can see that combination of sequences can drive the expression of a reporter, 00:29:28.02 (in this case, the lacZ... the beta-galactosidase gene from E. coli) 00:29:32.05 in cone photoreceptor cells in the mouse retina - that's what these blue cells are. 00:29:37.18 These are now cells that have been stained with the beta galactosidase substrate, 00:29:43.19 X-gal, and in this transgenic mouse retina, that substrate is then converted to a blue precipitate, 00:29:51.02 indicating the location of cells expressing the lacZ or beta galactosidase transgene. 00:29:57.17 If exactly the same sort of mouse is produced with sequences 00:30:02.12 that are missing that upstream segment, 00:30:04.16 just the kind of deletion that a blue cone monochromat might have, 00:30:08.16 there's no expression whatsoever in the retina. 00:30:12.22 So, this sequence is involved in transcriptional control, 00:30:16.07 and the mouse experiment as well as the sequences of many mammals, 00:30:20.01 reveal that, in fact, this upstream region (which we're now going to call a locus control region 00:30:26.00 because it's controlling the locus of L and M pigment gene) 00:30:30.25 that this upstream sequence, in fact, is highly conserved among mammals. 00:30:34.16 It's present in dogs, cows, cats, whatever... 00:30:39.23 And, it's also present in us. 00:30:43.17 So, here's this sequence, just shown schematically as a little purple ball, 00:30:46.14 and, although we don't know the details, we presume that in the ancestral mammals, 00:30:51.23 the ones with just a single visual pigment gene, 00:30:55.04 or in new world primates, where they have a single gene, but the gene is polymorphic, 00:31:00.07 somehow it acts on or with the adjacent promoter to affect transcription in cone photoreceptors. 00:31:09.01 Now, this locus control region turns out to be outside 00:31:16.11 of the region which was duplicated in evolution to create the L and M pigment genes. 00:31:21.17 We have only (we, that is, old world primates) 00:31:27.27 have only a single copy, as do the lower, non-primate mammals. 00:31:31.29 And therefore, one presumes that this locus control region acts 00:31:37.15 on the L pigment promoter in L cones and then on the M pigment promoter in M cones. 00:31:45.06 That's a reasonable assumption, although I should say it's really more of a model 00:31:50.15 than a summary of data. 00:31:52.09 This model makes a simple prediction. 00:31:55.27 And that is, that the pairing of the locus control region either with the L pigment gene 00:32:02.00 in L cones or the M pigment gene in M cones 00:32:04.29 is a critical event in the development of those cones - 00:32:08.00 in the decision to become either an L or an M cone. 00:32:11.28 And, it's also the case that there's no other molecular difference 00:32:18.07 that we know about between those two cell types. 00:32:20.18 So that the possibility exists that that's the only decision that distinguishes them from each other. 00:32:27.11 And so, with that sort of idea in mind, let's just consider for a moment, 00:32:32.24 how that decision might be made and now we're really considering models 00:32:38.24 for which we don't have firm data, but I think they constrain the way 00:32:43.19 in which we would think about the process. 00:32:45.15 We can imagine that, in an ancestral dichromatic mammal, 00:32:53.08 for example, in the new world primates of today, 00:32:55.25 there's a single transcription factor, shown by this black ball - this ancestral transcription factor, 00:33:03.00 which mediates the pairing of the locus control region with the 00:33:07.27 single nearby promoter. There's really no decision to be made here... 00:33:11.20 There's only one gene that's available for expression in any given cell. 00:33:16.14 And, that pairing then would facilitate the expression of that one gene 00:33:23.09 in the one kind of longer wave cone cells that that organism had. 00:33:26.22 Now, in primates like us - old-world primate - where there are two different genes, 00:33:33.20 an L and an M pigment gene, we could, in the context of this... what I will call the standard model, 00:33:39.02 envision that there are transcription factors specific for either the L or the M cones, 00:33:44.25 and these are shown as either red or green balls, and that those orchestrate the pairing 00:33:49.25 of the locus control region with the appropriate promoter. 00:33:54.07 And that would be, I think, the sort of standard model that one thinks about 00:33:59.01 in the expression of genes in any particular set of distinctive cells, 00:34:04.06 say kidney vs. liver... that would be orchestrated 00:34:07.01 by cell-type specific transcription factors in those cases. 00:34:11.05 But, let's just consider a, perhaps simpler model, 00:34:15.04 and one that has a different set of predictions - 00:34:20.07 that's a stochastic model of cell type-specific gene expression. 00:34:23.17 And in this model, we can imagine that, in fact, 00:34:28.07 the ancient transcription factor - the ancestral transcription factor... 00:34:31.16 this black ball, which mediates the pairing in dichromat mammals, 00:34:36.24 and presumably in new world monkeys, in fact is the same transcription factor that we have. 00:34:41.09 Then, the pairing in L cones or M cones is simply determined by a molecular coin toss. 00:34:46.20 It can either involve pairing to the L pigment promoter or the M pigment promoter. 00:34:52.11 Once the pairing has occurred, presumably it's stable. 00:34:55.16 There are various molecular mechanisms one can envision for stabilizing it... 00:34:59.16 DNA methylation, for example, and other modifications. 00:35:05.09 But, that that initial choice would be made without reference to any other decision-making process, 00:35:12.02 and that once the choice is made, that is what has set the destiny of that photoreceptor 00:35:16.25 as being either an L or an M cone... 00:35:19.03 Now that's a very simple model. It makes a number of predictions. 00:35:26.14 And one prediction it makes is that the decision to express a gene in one cone or another - 00:35:34.29 either L or M - is actually a decision process that any mammal can effect. 00:35:40.25 Any mammal can do this decision-making. It doesn't need a special transcription factor 00:35:48.02 family that's specific to L or M cone types. 00:35:52.18 And so that has been tested - that idea has been tested 00:35:55.02 by placing a version of the human L and M pigment gene array, with the locus control region 00:36:03.01 into the mouse genome. So, here's construct that was inserted into the mouse genome. 00:36:08.07 There's a locus control region at the left end, 00:36:11.29 followed by an L pigment promoter, shown in red, 00:36:14.07 followed by one enzymatic reporter, alkaline phosphatase, 00:36:18.03 which produces a brown histochemical product in those cells in which it's expressed, 00:36:23.26 followed by an M pigment promoter and the beta galactosidase, or lacZ 00:36:29.23 reporter that we've seen before which gives a blue product in those cells in which it's expressed. 00:36:35.03 And this was inserted into the mouse genome at exactly one copy. 00:36:38.20 And then the retina was examined, and lo and behold, 00:36:43.11 it was observed that, not only was the expression not only exclusively in cone photoreceptors, 00:36:48.19 (that was expected), but that cones were either brown or blue, 00:36:54.12 and only very rarely were they both. 00:36:56.26 That is, the mouse appears to be quite capable of doing this molecular coin toss 00:37:03.16 and deciding which of the two genes to express 00:37:06.20 and expressing them in a mutually exclusive fashion. 00:37:10.08 The fact that new world monkeys have, what we know is a stochastic mechanism for doing this, 00:37:16.12 argues that stochastic mechanism in general are plausible mechanisms 00:37:21.15 for generating an L/M mosaic, 00:37:24.02 and extracting information, again in ways we don't fully understand, 00:37:29.04 but extracting information to generate trichromacy. 00:37:32.14 Now, let's take this one step further. 00:37:38.02 If it's possible for a mammal which has never experienced trichromacy to produce a mosaic 00:37:46.05 of L and M type cones in its retina simply by being presented with those genes in its genome, 00:37:52.11 let's ask, could that mammal, if given a new visual pigment gene, immediately use it for color vision. 00:38:01.02 And that experiment was done also in the mouse. 00:38:04.28 So, here is a mouse who has had in its genome, 00:38:11.20 a human visual pigment gene inserted - a long wave pigment gene inserted. 00:38:15.27 She's a female and she has on one of her X chromosomes a normal mouse pigment gene, 00:38:22.10 an M pigment gene which absorbs maximally at about 510 nm, 00:38:25.20 and she has on her other X chromosome a human L pigment gene which encodes a pigment 00:38:31.20 that absorbs maximally at about 560 nm. 00:38:33.29 And she is being given the mouse version of that task that we saw earlier on 00:38:38.29 for our little monkey friend. In this case, she has to decide which of these three panels 00:38:45.21 shown here has a light that is different from the other two. 00:38:49.16 And if she decides correctly, she gets a little drop of soymilk, 00:38:53.11 which she's drinking right now from above the panel which is different. 00:38:56.11 So there's a little tube here, which will produce a drop of soymilk 00:39:01.06 when she guesses the correct panel. 00:39:02.22 And, of course, the test is repeated many times over... 00:39:06.11 the locations of the different color lights are varied randomly among the panels, 00:39:12.03 and the mouse is asked in this sense, by measuring how many times it gets a drop of soymilk, 00:39:19.19 (which is measured electronically)... The mouse is asked, can you see the difference between 00:39:26.08 the colors even if we randomize intensity? 00:39:28.19 Well, when this test was done, in an initial test, the mouse was simply asked 00:39:34.20 whether it could see longer wavelength lights more efficiently 00:39:39.06 now that it had a human L pigment gene, than could a normal mouse. 00:39:43.15 And the answer is yes. 00:39:45.10 A mouse - the heterozygous mouse, with both M and L pigments, required less long wave light 00:39:52.25 compared to a control mouse with only M pigment. Not surprising. 00:39:56.22 But it just shows that the mouse L pigment works and can transduce signals 00:40:01.01 and tell the brain that it has captured light. 00:40:03.16 But the more telling experiment is here, where we're looking at 00:40:07.14 discrimination between lights of different wavelength. 00:40:12.15 So here we have a three-way forced choice task of just the sort we saw the mouse performing 00:40:16.25 and because there are three panels, and one of the three is correct, 00:40:21.08 an animal that cannot pass this test 00:40:24.13 will simply guess on every round and get it right, as shown by these data points down here, 00:40:30.13 33% of the time. So these are mice that cannot pass the test, 00:40:34.20 but they realize quite quickly that it's better to guess than to do nothing 00:40:38.21 because even if they guess, they'll get a drop of soymilk a third of the time. 00:40:42.27 But a mouse that has the engineered human pigment, as well as the normal mouse pigment, 00:40:48.06 (and these, of course, are females because it's on the X chromosome) 00:40:51.14 and which by virtue of X-inactivation has created a mosaic 00:40:55.00 of the different cone types within its retina 00:40:57.00 (a rather fine-grained mosaic, we now know)... 00:41:02.14 That kind of mouse can discriminate a test wavelength (shown here at variable wavelengths 00:41:07.19 from 500-600 nm) from a control, standard wavelength of 600nm 00:41:14.14 quite substantially better than chance. 00:41:17.08 So, roughly 70 or 75% of the time, when that test wavelength is 500 or 520 or 540 or 580 or 560 nm, 00:41:28.24 that mouse can tell the difference between the test wavelength 00:41:32.10 and the standard control wavelength. 00:41:35.14 And again, intensities have been varied so that the animal cannot cue on intensity. 00:41:41.05 It must be seeing the color aspect of the stimulus. 00:41:47.09 Of course, when the test wavelength gets very close to the standard... 00:41:51.06 when it's at say, 590nm, or its exactly the standard, 600nm, 00:41:56.17 then the mouse begins to fail the test. 00:41:59.08 And of course, when it's exactly the same wavelength, it fails completely, 00:42:01.07 and it's just guessing at this point 00:42:03.02 what the right answer might be. 00:42:04.19 But this experiment has a number of interesting implications for color vision evolution 00:42:11.03 and perhaps brain evolution, in general. 00:42:13.12 In the context of the evolution of trichromatic color vision, what it says is that 00:42:18.15 there is a plasticity that is built in naturally, in the mammalian brain, 00:42:23.18 that allows it to take an input - a novel sensory input, 00:42:28.04 and make sense of it... and make sense of it in the context of pre-existing sensory inputs. 00:42:33.05 And from the point of view of brain evolution in general, 00:42:37.15 I think this points to what is probably the winning strategy for brain evolution. 00:42:41.09 That is, to have the system built with a degree of plasticity that allows it to take advantage, 00:42:47.24 perhaps immediately advantage, of genetic changes that affect parts of the system, 00:42:52.25 say the front end system - the receptors themselves. 00:42:57.02 and to extract information that is useful, even though the only genetic change 00:43:03.17 has been in the receptor and not in the neural wiring. 00:43:06.03 And, of course, specifically, from the point of view of the evolution of trichromatic color vision, 00:43:11.12 this would argue that the first primate who acquired an additional genetic change 00:43:17.11 to create a novel pigment immediately saw a world of color that no primate had ever seen before.