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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.

This material is based upon work supported by the National Science Foundation and the National Institute of General Medical Sciences under Grant No. 2122350 and 1 R25 GM139147. Any opinion, finding, conclusion, or recommendation expressed in these videos are solely those of the speakers and do not necessarily represent the views of the Science Communication Lab/iBiology, the National Science Foundation, the National Institutes of Health, or other Science Communication Lab funders.

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