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Patterning Development in the Early Embryo: The Role of Bicoid

Transcript of Part 3: Evolution of Bicoid-based Patterning in the Diptera

00:00:04.17		My name is Eric Wieschaus and I'm a HHMI investigator and professor
00:00:09.08		at Princeton University. For the previous two parts of this lecture we've
00:00:15.20		been talking about how pattern is established in the early Drosophila
00:00:18.22		embryo, and we focused on a molecule called bicoid that is deposited as an RNA
00:00:25.12		at the anterior end of the Drosophila egg during oogenesis in the mother.
00:00:30.25		And then is translated into a protein and forms a protein gradient in
00:00:36.19		the early embryo. And the model and then the reason why this protein,
00:00:43.11		this bicoid gradient is important is that it is thought to be the major
00:00:50.11		determinant in establishing the pattern of gene transcription in the embryo
00:00:55.03		such that different concentrations of the bicoid protein at different points
00:00:59.29		along the anterior posterior axis activate expression of particular genes
00:01:06.25		like the hunchback gene in green here, or the Krüppel gene in red.
00:01:11.18		Now, we've seen that if you examine this gradient in Drosophila embryos
00:01:17.12		or if we examine the expression of the downstream targets, we see that their
00:01:21.12		extraordinarily constant from one embryo to the next. And this is probably
00:01:26.24		what you want if you want to have a system which is establishing pattern
00:01:30.00		and controlling the behaviors of individual cells in the embryo. Now
00:01:35.10		in the last lecture we talked about some of the biophysical parameters
00:01:40.22		and cell biological parameters that might give rise to these constant
00:01:45.05		distributions of bicoid or constant transcription patterns.
00:01:51.05		Now one of the underlying assumptions for all of that work
00:01:59.07		is based on actually a fact. If you look at fly eggs, not only are the expression
00:02:06.01		patterns constant, but the actual sizes of the eggs are constant. And that's
00:02:10.29		an important idea because if you think about any of the mechanisms
00:02:13.29		that we think about when we talk about how one would establish a gradient,
00:02:17.12		they are very sensitive to the size of the egg. If you want to establish
00:02:25.02		hunchback expression up to 48% egg length, and you are doing that
00:02:30.12		by having a gradient of a molecule that diffuses with a particular
00:02:35.24		diffusive constant and establishes a particular constant shape
00:02:39.21		in its distribution as bicoid appears to do, then that mechanism for
00:02:45.13		patterning would be very sensitive to variation in egg size.
00:02:52.19		And if you look at Drosophila eggs in the particular way you raise them
00:02:55.21		in the laboratory, in the way that we measure them, in the stocks
00:02:59.17		we've measured them to establish these gradients and look at them.
00:03:01.29		What we see is that actually individual fly eggs are remarkably similar,
00:03:08.05		wild-type normal eggs are remarkably similar in their size, and so it's basically
00:03:12.09		consistent that such a mechanism could function to pattern
00:03:19.01		embryonic development in Drosophila melanogaster. The problem though
00:03:24.02		with that model arises if you go outside of Drosophila. If you go outside of
00:03:29.12		fruit flies and extend your observations to other kinds of embryos
00:03:35.26		from even other embryos of other insect species, even other fly species.
00:03:39.28		As we all know, from our own personal experience, flies come in different sizes.
00:03:46.13		There are the nice little small Drosophila fruit flies with red eyes
00:03:52.01		that we kind of raise in the lab and have such affectionate feelings for
00:03:56.05		and there are also disgusting flies like house flies and blow flies that
00:03:59.21		kind of fly around and invade our picnics and are less attractive.
00:04:07.06		The bigger flies are the uglier ones, the smaller flies are nicer and sweeter, generally.
00:04:13.29		Now what is also true though, not only is the adult flies that we see
00:04:19.25		are of different sizes, but also if you look at the eggs in the embryos
00:04:23.27		that these different fly species make, they are also different sizes.
00:04:29.16		So Drosophila melanogaster for example makes eggs that are about
00:04:34.04		500 microns long. There are even Drosophila species like
00:04:37.16		Drosophila busckii that make eggs that are even smaller than Drosophila,
00:04:43.20		but the big flies like Musca domestica, the house fly,
00:04:49.01		or Calliphora, blow flies or green bottle flies, Lucilia, that are big
00:04:55.29		obviously as adults much bigger than Drosophila, and the eggs
00:05:00.01		that they make are substantially bigger. Now all of these flies
00:05:08.15		these higher Diptera are closely related. And even though
00:05:14.01		bicoid as an RNA or a gene product was a newly evolved solution
00:05:19.25		to the problem of patterning in the embryo and arose during the evolution
00:05:23.17		of the Diptera, all of these insects here share the common feature
00:05:29.13		that their anterior/posterior patterning depends on bicoid.
00:05:33.20		And yet the bicoid gradients that are forming in these eggs are forming
00:05:39.18		in eggs which are very large or very small. Now, one of the interesting
00:05:47.08		features of all these eggs though is even though they are different sizes
00:05:51.12		if you actually look at the development of these embryos
00:05:54.08		you can see that the early development is remarkably similar in that if you
00:05:58.24		go back and remember how early development in Diptera starts with
00:06:02.18		fertilization followed by these nuclear cleavages in the syncytial embryo
00:06:06.21		and then a pause at 2.5 hours to form the syncytial blastoderm
00:06:14.25		that then transforms itself by the formation of cell membranes between
00:06:19.01		nuclei and into a cellular blastoderm and a gastrula, that process
00:06:22.17		that takes about 2-3 hours in Drosophila melanogaster
00:06:26.11		is also observed in all of these other insects and it also is observed
00:06:31.03		with exactly the same time, 2.5 hours, the same kinetics
00:06:35.17		and if you look at divisions of the nuclei, if you look at individual,
00:06:41.04		say this is an embryo, at the syncytial Drosophila embryo from
00:06:46.14		the big green bottle fly, Lucilia versus Drosophila melanogaster
00:06:52.08		or Drosophila busckii, the eggs have the same shape
00:06:56.14		and although you can probably barely make it out, you can barely see the nuclei
00:07:02.28		in the Lucilia and the nuclei in Drosophila melanogaster or busckii
00:07:07.04		are somewhat smaller, if you blow up the pictures of the individual eggs
00:07:12.00		and look at the nuclear distributions you can see that all these eggs
00:07:16.09		at 2.5 hours have the same number of nuclei. They all have about 100 nuclei
00:07:21.00		going from the anterior to the posterior end of the egg.
00:07:23.23		They're all being patterned over the same time constraints
00:07:27.15		and they're all being patterned by bicoid. If you look at transcription
00:07:35.13		the other remarkable similar thing between all these insects that
00:07:38.25		are all so closely related even though their sizes are different, is that
00:07:42.15		all of them activate transcription at this critical 2 hour period in response to bicoid
00:07:50.19		and if you look at the patterns of gene expression
00:07:52.23		if you look at say hunchback or giant, the two different gap genes
00:07:56.02		in Drosophila or in Musca or pair-rule genes like
00:08:02.23		paired or evenskipped, they show exactly comparable scaled patterns.
00:08:09.00		Even though the eggs are bigger and even though the cells are bigger
00:08:13.13		the patterns per cell are exactly the same. Now these are transcriptional
00:08:18.20		responses, they're genes which are transcribed at the blastoderm stage
00:08:24.05		directly or indirectly in response to the bicoid gradient. And so the question
00:08:29.17		that you'd like to ask, is how is it during the course of evolution,
00:08:34.03		as egg size changes, how does the embryo or the species adjust to using
00:08:43.03		a bicoid gradient to establish pattern. There are really two simple ways
00:08:48.01		that you can think about it. One way is that each of these genes,
00:08:52.05		like hunchback and any of the targets of bicoid activation
00:08:56.14		is going to have a control region which will respond to bicoid concentration.
00:09:03.04		And as you change the length of the egg, one strategy
00:09:08.04		would be to keep the bicoid gradient the same shape
00:09:11.20		and the concentration distribution the same, and yet change
00:09:16.20		the cis-acting control regions of each of these genes. Adjust them
00:09:19.28		during evolution and we know that that's generally what happens
00:09:22.18		during the course of evolution. Alternatively, during evolution
00:09:30.05		you could adjust with the size of the egg not by changing
00:09:35.15		the control responses of individual genes, but by somehow
00:09:39.03		changing the manner or changing the physical properties
00:09:43.07		that establish the bicoid gradient itself such that in bigger eggs
00:09:50.10		the gradient extends longer and in smaller eggs it is shorter.
00:09:57.15		This seemed to us initially a less likely alternative, partially because
00:10:03.19		many of the cases in evolution that we know about involve
00:10:06.26		changes in cis-acting control regions. But when we actually looked
00:10:10.16		at the bicoid distribution in these other insect species, what we observed
00:10:15.14		was that not only does bicoid form gradients in big eggs and the small eggs
00:10:24.14		but if you look at the big egg, if you compared the distribution to say
00:10:29.14		in melanogaster. In melanogaster the gradient falls exponentially
00:10:34.10		over an area like this, and if you look in Lucilia the distribution
00:10:41.11		of bicoid protein extends much longer, much farther
00:10:46.17		into the length and into the egg. In terms of microns, that is the bicoid gradient
00:10:53.13		in a bigger egg, is bigger, proportionately bigger, because if we then
00:10:59.03		replot the data, not in terms of absolute lengths as here
00:11:02.29		but in terms of relative lengths along the eggs you can see that
00:11:07.01		the bicoid gradients in the big eggs and the small eggs are
00:11:10.08		exactly equivalent scaled to the size of the egg. What that means then
00:11:16.14		in turn, is that somehow during the course of evolution of these insects
00:11:23.03		the bicoid gradient has changed. The properties that establish the gradient
00:11:30.18		have changed to allow this gradient to now span
00:11:35.03		a bigger or smaller egg and can provide positional information along
00:11:44.05		the whole length of the egg. So how does this happen? One simple strategy
00:11:52.15		would be to change bicoid as the species evolve they change bicoid,
00:12:00.07		they change the properties of the bicoid protein such that it moves faster,
00:12:03.13		such that it is degraded less rapidly for example, such that it ultimately
00:12:07.19		the gradient that you get out of this bicoid would extend farther
00:12:15.23		and thus establish gradients of comparable shape when
00:12:21.05		scaled back to the actual shape of the egg. To begin to test or
00:12:27.22		think about those models we've cloned the bicoid genes from these
00:12:33.12		different species and compared their structure to
00:12:40.06		that of Drosophila melanogaster bicoid, and if you look at that
00:12:43.06		bicoid is reasonably well conserved particularly in regions of the protein
00:12:48.28		which are functionally well defined. The homeodomain that binds DNA
00:12:52.23		or other regions that have been implicated at least suggestively
00:12:56.11		as being involved in protein stability. Most of the sequences are the same
00:13:06.04		but not surprisingly when you look at any particular sequence,
00:13:10.09		any particular region of these proteins, there are amino acids differences.
00:13:14.25		And so one possibility is that these different species specific bicoids
00:13:20.02		have evolved and the changes in their sequence, either the ones
00:13:24.08		that I've indicated here or changes in other regions of the protein
00:13:28.15		are actually responsible for adjusting the shape of the gradient
00:13:35.10		such that it can now function in larger eggs or smaller eggs.
00:13:40.10		So to test that possibility, you need to begin to hope to identify
00:13:45.04		the regions that have changed in the bicoid protein.
00:13:48.08		What Thomas Gregor and Alistair McGregor in the lab did was to
00:13:53.00		take these cloned bicoid genes from the other species,
00:13:58.03		tag them with EGFP and put them back into melanogaster to ask
00:14:04.07		what type of gradients that they make. And the surprising result here,
00:14:08.19		one that we hadn't anticipated was that if you compare,
00:14:13.29		if you take a bicoid protein from Calliphora for example
00:14:17.14		that will make a large gradient that extends more than a
00:14:23.05		millimeter through the entire Calliphora egg which is
00:14:27.19		one and a half millimeters long, and you put it into a Drosophila egg
00:14:33.01		which is only five hundred microns long, one possible result would have been
00:14:38.25		that this bicoid protein because of its changed properties, the protein
00:14:43.00		that it moves faster or that it degrades less, would make a Calliphora
00:14:47.10		sized gradient in a Drosophila egg and you can imagine
00:14:52.07		that would result in a catastrophe for development
00:14:56.16		for the Drosophila embryo that was depending on that gradient.
00:14:59.09		But what you actually see, the amazing thing is that these bicoid gradients
00:15:03.12		that are established in these eggs, actually using the Calliphora protein
00:15:09.20		are identical, surprisingly identical not to the gradients
00:15:16.15		that those same proteins would have made in Calliphora
00:15:18.13		but to the gradient that is made in Drosophila melanogaster.
00:15:22.27		So, for example in this figure here we can see the Calliphora bicoid
00:15:28.25		extending out in a visible sense to about 48% where we'd be activating
00:15:33.27		hunchback, and that's very similar to the distribution that you'd see
00:15:40.02		when you took the Drosophila EGFP and put it in the same egg.
00:15:45.28		And you can measure that and show that these distributions overlapped
00:15:50.10		each bicoid that regardless of the source of the bicoid protein put into a
00:15:58.24		Drosophila melanogaster egg that protein will make a gradient
00:16:03.06		of the same shape, the same size, as the melanogaster bicoid.
00:16:08.20		So we know that what that's telling us is that the fact that Dipteran bicoids
00:16:18.07		expressed in Drosophila make Drosophila sized gradients is that during
00:16:24.23		the course of evolution it's not bicoid that has changed to allow for the
00:16:31.23		adjustment of the egg shape and the egg size, but some other property
00:16:36.29		of the egg. So these proteins put in a melanogaster egg will produce
00:16:44.10		melanogaster type gradients. Now we've done the same kinds of experiments
00:16:51.02		using other tagged proteins, EGFP, we've altered the bicoid area,
00:16:56.00		just put straight, if you can imagine just taking EGFP or EGFP
00:16:59.18		with an NLS, a nuclear localization sequence, and localizing the RNA
00:17:05.10		to the anterior end of the egg and ask will any protein,
00:17:08.10		any GFP-tagged protein put into a Drosophila egg make a gradient
00:17:13.04		the shape of bicoid. And what we found is that's not true.
00:17:15.17		That each individual protein put into the egg makes a gradient of a particular
00:17:21.21		size and a particular distribution. But all of the bicoid proteins
00:17:25.17		put into the Drosophila egg at the anterior end make gradients
00:17:30.17		of a particular size. So the interesting conclusion then from these
00:17:35.03		experiments is that during the course of evolution, it's not that bicoid
00:17:42.17		has changed to adjust for the size of the eggs, but actually
00:17:46.23		bicoid has been conserved. What's been conserved in bicoid is
00:17:51.00		the property of the protein that allows it to build gradients of a
00:17:55.28		particular size when put into Drosophila eggs. And all the bicoid molecules,
00:18:04.06		but other proteins do not have that property.
00:18:09.04		What's actually diverged during evolution has been not the protein itself
00:18:18.27		but the environment that we put the protein in
00:18:24.10		and then that raises the kinds of questions that you'd like to answer now.
00:18:28.15		What are those properties? What could influence bicoid? What's the
00:18:37.19		features of bicoid, the bicoid protein itself, that allows it
00:18:42.11		to respond and make gradients and there are obvious experiments
00:18:45.09		that we're in the process of doing where you can identify the regions
00:18:50.28		of the bicoid protein that are essential for it to make
00:18:55.25		a gradient of a particular size and shape that's characteristic of bicoid.
00:19:00.24		And then the other question is what are the features that change
00:19:07.02		as you change egg size that change those distributions.
00:19:11.26		How is it that you are able to maintain the property of the protein
00:19:15.26		on the one hand and then change the size of the egg
00:19:18.14		and change the movement of the proteins. So those are
00:19:24.28		really essential questions for understanding how during the course
00:19:34.07		of evolution you are able to use the same system, the same protein,
00:19:39.23		over and over again. They will require different kinds of measurements
00:19:47.21		and being able to work with multiple species and multiple variants
00:19:52.13		both of being able to put bicoid proteins into melanogaster
00:19:57.25		but able to also put variant proteins into the bigger and smaller eggs.
00:20:01.26		But what they'll also require is to distinguish between these different models
00:20:06.20		to give it the underlying mechanisms that are controlling the distribution
00:20:11.16		is again the kind of quantitation that we talked about in the second lecture
00:20:16.22		and it's my own belief that the future of developmental biology
00:20:21.16		in general and the refined understanding of problems
00:20:28.23		in development will depend heavily on our ability to combine
00:20:34.01		both those quantitative visual techniques for analyzing distributions
00:20:40.21		of molecules with the powerful techniques of molecular biology
00:20:45.00		that allow you to manipulate the sequences and structures
00:20:50.03		of those proteins and also the other features of the egg.
00:20:56.12		So I'll stop there and thank you for your attendance.

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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