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Patterning Development in the Early Embryo: The Role of Bicoid

Transcript of Part 1: Patterning Development in the Embryo

00:00:05.18		I'm Eric Wieschaus and I'm a HHMI investigator and a
00:00:11.22		professor at Princeton University. My laboratory works on the way
00:00:15.24		embryos develop and I'm particularly interested in the gene activities
00:00:20.28		and the cell biologically processes that operate in the very early embryo
00:00:26.00		to transform what seems to us like a simple freshly fertilized egg into
00:00:31.25		a complex organism with cells with particular morphologies and
00:00:39.17		particular organs all in the right place at the right time. Now these processes
00:00:45.01		are interesting to all of us I think especially with respect to human development,
00:00:50.21		because we are all interested in where it is that we come from, how is it that
00:00:54.09		a single cell is able to give rise to something as complex as you or I.
00:00:59.12		But over the past 20 years we have also learned that all these process
00:01:06.12		that occur in embryonic development and all the gene activities
00:01:08.28		and all the cell shape changes and the controls of cell adhesion
00:01:13.04		and cytoskeleton cell structure that one sees happening and can follow
00:01:20.02		and study in embryos are the same kinds of processes that involve
00:01:23.10		the same kind of molecules that operate throughout the life of almost all organisms.
00:01:29.26		So one of the goals of our work is not just to understand how embryos develop,
00:01:35.14		but to understand how cells and living organisms control their gene activity,
00:01:41.01		how they control their cell shape, how they control all these processes
00:01:45.03		that are essential for life. Not only of the embryo and for embryonic
00:01:48.29		development but for everything that we see today. So what I'm going
00:01:53.03		to talk to you about in my lectures, is my basic plan, or what I'd like to do is to
00:01:57.08		first describe to you a little bit about how the fruit fly embryo,
00:02:01.23		which is the embryo that we work on in the lab develops, and point out to you
00:02:05.24		some of the really interesting features that have made this embryo
00:02:09.12		really helpful, a really powerful tool for understanding development.
00:02:14.12		And then in the second part of my talk, what I'll do is focus in on one particular question
00:02:23.17		which is how is it that in an embryo cells come to achieve different fates,
00:02:30.14		different patterns of gene activity. How do cells come to be different from
00:02:33.25		each other, where does that pattern arise? And as you'll see, we'll start
00:02:37.24		through a basic description of the process and then work our way through
00:02:43.02		gene activities and basic cellular functions but what we'd really like
00:02:49.13		to end up with is a deeper almost biophysical understanding of these
00:02:55.15		processes, and that's my goal. And then what I'd like to do is also expand back
00:02:59.05		out and talk about embryonic development, talk about it in the context
00:03:03.05		of not just fruit fly embryos but more broadly of all organisms
00:03:08.13		on this earth and how processes evolve and have changed during
00:03:12.09		the history of life on this earth. Okay, so the very first slide though and to begin
00:03:16.09		what I wanted to do was just to show you an image of a Drosophila embryo.
00:03:20.29		This is a scanning EM, it's a fruit fly egg, or a Drosophila egg
00:03:25.04		almost immediately after fertilization. It's still a single cell. It's about
00:03:30.08		maybe half a millimeter or 500 microns long. If you look at the surface you
00:03:36.10		don't see anything very much. If you were to look inside you'd see that
00:03:39.28		in this zone here there's the nucleus of the single cell, which is the product
00:03:45.19		of the fusion of the female pronucleus and the male pronucleus. If you look
00:03:51.05		at it and it doesn't really look very interesting. Amazing thing though
00:03:56.05		is what this embryo has to do to become interesting is to convert from being
00:04:01.21		a single cell into a multicellular organism where individual cells
00:04:08.01		can assume different fates and begin to do different things.
00:04:11.03		Now one of the interesting things about fly embryonic development in
00:04:15.16		Drosophila is that the embryo is able to do this extraordinarily rapidly.
00:04:20.09		Such that after two and a half hours this single cell has now transformed itself
00:04:25.16		into a multicellular organism with now 6,000 cells, about a hundred cells
00:04:31.26		along the anterior with the future head and future tail anterior/posterior axis
00:04:36.25		of the embryo. And the way that its able to do this transformation so rapidly
00:04:42.17		is that what it's done is that unlike most other organisms where when
00:04:48.00		cells divide and replicate you have a DNA replication and then a mitosis and
00:04:52.01		that's followed by cell division. In Drosophila during the early stages
00:04:56.23		of development these mitotic divisions, these replications of nuclei
00:05:02.17		occur without cell division such that an individual fertilized egg which starts
00:05:08.00		out with a fused of a single nucleus goes after one mitotic cycle goes from
00:05:12.17		one nuclei to two nuclei, the cycles are synchronous so the nuclei divide
00:05:16.10		again without cell division so that you have from two nuclei to four nuclei, eight nuclei.
00:05:22.05		And what happens after about an hour and a half to two hours is that
00:05:27.00		through a sequence of thirteen of these synchronous rounds of nuclear replication
00:05:32.20		the embryo is now still a single cell, but it's a single cell with 6,000 nuclei on
00:05:40.10		the surface. And amazingly then, at that point these mitotic divisions
00:05:45.01		temporarily stop and it's only at that point that the cytoskeleton
00:05:51.02		and the membrane synthesis is reorganized in this embryo to now make
00:05:56.02		new membrane such that membrane can be pulled down, plasma membrane
00:06:00.16		can be pulled down between individual nuclei to separate them
00:06:03.20		or partition them into individual cells. And it's after that process that's called
00:06:08.09		cellularization that the embryo has now converted itself from one cell
00:06:12.27		into an embryo with multiple cells, 6,000 cells. And it's only at that
00:06:19.17		point that those cells can begin, that when you have individual cells, that those cells
00:06:24.23		can begin to become different from each other and show distinct behaviors
00:06:28.14		that are ultimately related to their fates as skin or muscle. Now
00:06:33.02		there's one other thing that's really interesting about this phase
00:06:37.04		and that's that if you look at the early stages when the embryo is undergoing
00:06:42.13		these rapid mitotic divisions all the gene products that it needs, all the proteins
00:06:47.06		and all the RNAs are supplied by the embryo's mother, put into
00:06:52.14		the unfertilized egg before fertilization and what the embryo
00:06:56.28		is doing is just going through a cyclic pattern of DNA replication and mitosis
00:07:02.16		doing the same thing over and over again using the same gene products,
00:07:07.00		these gene products that are supplied by the mother. What happens
00:07:12.05		once those repetitive cycles are done and the embryo wants to do
00:07:17.15		something new or different, is that it begins to transcribe its own genome.
00:07:22.29		So it begins to make what we call zygotic RNAs and zygotic proteins. And so at
00:07:27.27		this point, it becomes a very interesting point for us in development,
00:07:32.29		because initially for this first two and a half hours, the embryo has been
00:07:37.19		doing something repetitive over and over using only maternally supplied gene
00:07:42.25		products and then the sense is that's when it begins to transcribe its own
00:07:47.14		genes and transcribe specific genes that are required ultimately to do new
00:07:53.18		things, to go on to the next step in development. So the sense is this stage
00:07:59.10		becomes really important for us to look at because it marks a stage
00:08:04.26		not only where something new begins to happen, you stop mitosis,
00:08:10.18		you begin to change and cells begin to become distinct from each other
00:08:14.01		but it's also associated with adding new gene products. Now I'd like to
00:08:20.11		though just continue on with the description of development,
00:08:23.09		for you to keep in mind though that now what we're going to be looking at
00:08:26.20		begins to reflect the active contribution of genes in the embryo itself.
00:08:33.03		What happens between these two stages you go from uniform behaviors
00:08:37.03		to distinct cellular behaviors you can see that again looking at the scanning EMs
00:08:41.11		of the egg. Here, this is again the embryo that  I showed you before
00:08:44.24		with 6,000 cells, 100 cells all arranged along the anterior/posterior axis
00:08:50.11		all the cells look pretty much the same. If I'd fixed this embryo for scanning
00:08:54.18		EM about five minutes later, what you would have seen is that now all of the
00:09:00.00		cells in the embryo are no longer the same shape. Their clearly distinct
00:09:04.28		things happening, there's an area here that's ultimately going to form the
00:09:09.06		head of the embryo that's marked off by this fold, it's called the cephalic or
00:09:15.00		head fold from the rest of the embryo. There are other things beginning
00:09:18.06		to happen in the embryo clearly by this stage at this point, 5-10 minutes
00:09:23.08		later, the cells are marking, they're showing distinct behaviors
00:09:29.15		and showing how different they are. We can actually watch these behaviors
00:09:33.03		in living embryos by tagging the surfaces of cells with fusion proteins between
00:09:41.05		GFP and various membrane proteins so we can follow individual cell shapes.
00:09:45.12		And when you watch this movie again what you can see, this is again
00:09:47.29		the blastoderm stage that we talked about and watch in this area here
00:09:51.21		all the cells are pretty much the same, but right about there you can begin
00:09:54.15		to see this fold happening and then you begin to see remarkable changes
00:09:58.28		in the behaviors of cells, you can see, this movement of cells as they sweep
00:10:03.28		and move around the end of the embryo. Obviously as you watch
00:10:10.18		the process, we'll watch it one more time, all the cells are the same,
00:10:14.08		individual cells begin to become distinct, and with the extraordinary
00:10:20.12		reproducible pattern, so embryos always make a head and they always
00:10:24.11		make a head right here, they're always separated by folds. This behavior
00:10:29.07		here, these cells here that are moving into this invagination
00:10:31.29		here are ultimately going to form the endodermal or gut regions
00:10:36.07		of the embryo. We can roll this embryo over and now we can watch from
00:10:40.09		the ventral side, and we can see that other things are going on. You can
00:10:43.23		actually see a little bit of this head fold here but what's more striking
00:10:48.11		is this movement of cells, and watch right here you'll see a fold forming.
00:10:53.11		These are future muscle cells that are going to be brought into the interior
00:10:57.13		of the embryo, because obviously that's where the embryo wants to have muscles.
00:11:01.25		Now, by looking at embryos and characterizing the behaviors of individual cells
00:11:09.09		and the overall changes in morphology in the behaviors of individual cells.
00:11:13.18		What we've learned, is that all major morphological changes, all growth,
00:11:20.13		all changes in the visible appearance of the embryos involve local changes in cell
00:11:27.14		shape. The initial changes occur without any cell division. There is
00:11:31.05		no growth. There's no mitosis anymore after these first initial cell divisions.
00:11:42.22		The embryo will begin mitosis later, but at this stage these major changes
00:11:48.09		that we saw in the movie are all happening because individual cells in specific
00:11:52.19		places change their shape say from being long and square, to being rounded up.
00:11:58.19		And it's that kind of cell shape change that produces ultimately
00:12:03.12		the changes that we see here that will say separate the future head
00:12:07.07		from the tail of the embryo. So what we want to know is why is it
00:12:10.27		that certain cells, and only certain cells in certain places change their shape,
00:12:16.20		and others not? We know that those cell shape changes correlate
00:12:26.16		with patterns of gene expression. So that if we go back to this head fold
00:12:31.22		here that separates the head from the rest of the embryo, we can look
00:12:38.16		and see there is actually a single row of cells that are making the fold and
00:12:44.04		there are genes that are expressed exactly in stripes in this embryo that have
00:12:49.01		just begun to be transcribed at this stage that we talked about before.
00:12:53.05		The stage right when the embryo has completed the mitotic divisions that precede
00:12:59.21		the cell behaviors and mark the infolding of these cells such that
00:13:06.01		the cells that change their shape right here are cells for example that
00:13:09.27		are not expressing the green gene here which is a gene called paired, or
00:13:19.16		the orange gene which is a gene called runt. And so there are specific patterns
00:13:24.04		of gene expression, of transcription, that have arisen at this stage, 20 minutes
00:13:31.10		before the cells have begun to change shape that direct the cells
00:13:35.14		and control their cell behaviors. But all that does is of course is just push
00:13:42.13		the question back. We want to know why it is that cells in a given region
00:13:47.26		of an embryo show particular patterns of cell behavior, particular shapes
00:13:52.05		and now I tell you well that's because their expressing different gene
00:13:54.13		products that doesn't really answer the question because if what you
00:13:58.03		really want to know is how is it that spatial and temporal patterns of
00:14:01.09		gene expression are established at the blastoderm stage. And go back again
00:14:08.11		to this central idea that it's at this stage the onset, right before gastrulation,
00:14:15.08		right before these cell shape changes begin to occur that individual genes
00:14:22.13		come to be expressed in the embryo in specific patterns. I indicated that before
00:14:28.25		that stage cell behaviors were uniform and maternal RNAs and proteins,
00:14:33.25		and they depend on maternal RNAs and most of those maternal RNAs
00:14:39.14		and proteins, and actually for a long time we thought all of them were
00:14:43.08		uniformly distributed throughout the egg, but what we've learned now is that
00:14:48.13		we have to put the emphasis on mostly these maternal RNAs and proteins
00:14:54.02		that are supplied and necessary during these early stages are mostly
00:14:57.17		uniform but there's a very small number of proteins and RNAs that are put
00:15:02.05		into the egg by the mother and show distinct patterns of distribution.
00:15:06.09		And one of the most important of these and this will be important
00:15:08.27		for the remainder of my talk is a protein called bicoid. It's a transcription factor
00:15:13.15		supplied by the mother, it's present during these early stages and if you look
00:15:18.24		at its distribution in the early embryo you can see that this protein is localized
00:15:24.14		at high concentrations at the anterior end of the egg, the future head region
00:15:28.24		of the egg, and then grades off in cells as we move more and more posterior
00:15:38.20		in the embryo. And one of the things we've learned and that I'll tell you more
00:15:43.04		about is the controlling role for this protein distribution in establishing
00:15:49.04		the patterns of gene expression and transcription that occur at these stages
00:15:54.10		at this process right before gastrulation and that are responsible for the cell
00:15:59.08		shape changes. Now what's really going to be essential to the problem
00:16:11.07		is that if we have a graded distribution for protein, a maternal protein
00:16:17.13		in the egg. A protein that the mother has directly put into the egg.
00:16:23.11		How does that distribution of maternal bicoid protein established?
00:16:28.19		How is it formed? And from wonderful experiments by
00:16:31.21		Christiane Nüsslein-Volhard and Wolfgang Driever and a number of other laboratories
00:16:35.08		we've learned that what's central here is that this protein gradient that we can
00:16:42.06		see in this embryo here, it's about two hours old, arises not because
00:16:51.03		the mother puts the protein into the egg, and not because she puts
00:16:54.04		the protein in a graded fashion but instead what she does is when she's
00:16:59.18		making the egg back in the ovary back long before fertilization, when she was
00:17:03.28		making the egg, she deposits the RNA that encodes this protein and anchors
00:17:10.21		it to the cytoplasm in the anterior end of the egg. This RNA is not translated
00:17:16.14		during oogenesis, as long as she's holding this egg, as long as egg
00:17:21.01		is not fertilized the RNA sits there in an inactive form. When the egg is
00:17:27.15		fertilized, a consequence of fertilization or activation of this egg is that this
00:17:35.17		RNA is released from its anchor and begins to be translated because
00:17:42.16		the protein is not anchored, the protein is thought to diffuse from this source of
00:17:46.26		synthesis, continue to make protein constantly here from the RNA,
00:17:52.16		that's localized here, but the protein diffuses and what's established over time
00:17:59.12		in these first two hours, is a gradient of this transcription factor bicoid.
00:18:05.13		What then happens is that ultimately, and this is a little cartoon diagram
00:18:15.08		that the highest concentrations of the bicoid protein will be at the anterior
00:18:21.27		end of the egg the concentration will fall, this is a transcription factor
00:18:26.27		and there are genes in the embryo that are going to become
00:18:31.23		transcriptionally activate at this time this is the stage where
00:18:36.05		major transcriptional activation occurs in the embryo but
00:18:40.09		those genes are activated by bicoid as a transcription factor
00:18:48.04		in a concentration dependent way. So there are certain genes for example like
00:18:52.00		the hunchback gene that are activated by relatively higher concentrations
00:18:57.03		of bicoid protein and so show expression only in the anterior most
00:19:03.02		48% of the egg. So we can see here, that other genes that can be activated by
00:19:08.04		lower thresholds for example, the Krüppel gene shown here, and that
00:19:12.14		Krüppel gene then and the hunchback gene define domains
00:19:20.24		of gene expression, are the genes in fact that are expressed
00:19:25.07		in the embryo and are involved in establishing those spatial patterns.
00:19:30.09		Now, that understanding of development was quite remarkable,
00:19:39.16		something that the role of maternal RNAs and maternal proteins
00:19:43.24		bicoid was the first such maternal RNA which was functionally demonstrated
00:19:49.21		to provide this gradient like form of information across the whole embryo.
00:19:55.29		Discovered by Christine Nusslein-Volhard and Wolfgang Driever more than
00:19:59.04		15 years ago had an extraordinary impact in developmental biology
00:20:04.27		and our understanding of the processes of embryonic development.
00:20:09.13		This created a great deal of excitement cause you always ask in science
00:20:14.10		why was that result so exciting, what was so important about it.
00:20:17.26		And so I think there are a couple of interesting things that happen.
00:20:22.13		If you look at the model that you have a graded maternal protein that
00:20:29.09		controls transcription defined by having downstream targets, genes activated
00:20:39.15		in a concentration dependent way, a specific threshold. You establish a pattern
00:20:44.17		of gene expression, but what it's really telling us is that in biology information
00:20:50.25		is quantitative. People had even speculated that mothers may
00:20:56.05		put gene products in the egg to establish pattern. No one knew what the nature of
00:21:01.10		that maternal information was. What these experiments are telling us is that
00:21:05.11		information in biology is largely quantitative. The cells make choices
00:21:11.12		based on levels or concentrations of bicoid protein. And what that does
00:21:19.23		is it also tells us that the choice process depends not just
00:21:26.00		on concentration levels, but on the ability of nuclei, or cells,
00:21:32.10		to measure concentration and make permanent cell choices in response
00:21:39.15		to those measured concentrations. So one of the things we'll talk about is
00:21:43.02		how is it? What do we know about cells? What are these measurements?
00:21:46.24		How do they actually work? Is this the right way of thinking about the process?
00:21:51.02		What are the problems thinking about the process, but what the
00:21:53.25		experiments did is that by emphasizing the quantitative nature
00:21:57.15		of the information they changed how a developmental biologist
00:22:01.14		thought about the process of development. Another interesting thing
00:22:05.17		if you go back and look at the process and think about how it is that
00:22:09.05		the bicoid gradient is itself established. How is it you provide information
00:22:16.19		to an embryo. The initial localization, the initial pattern is a localized RNA
00:22:24.04		which is very finely localized to the anterior end. There's not a lot of
00:22:34.00		information in a simple localization of a single molecule in a single place.
00:22:39.04		What's important is that the final, that is we'll say the information rich
00:22:44.05		distribution that the cells are going to use to make their developmental
00:22:47.05		traces is not that initial distribution, but the final distribution is achieved by
00:22:57.06		simple physical parameters. You localize an RNA and then when that RNA
00:23:04.03		begins to make a protein, the protein diffuses. And if you think in terms
00:23:08.18		of what kinds of things can impact on that, things like how fast
00:23:12.18		molecules move, how fast are they degraded, all those things will ultimately
00:23:19.13		set up and define what the pattern of distribution is. All those things
00:23:26.21		are potentially measureable and so what's exciting about having a cartoon
00:23:31.18		picture of a localized RNA and a protein gradient, is that we believe that
00:23:38.20		once you have that cartoon in your head it directs you to say,
00:23:44.00		well what is interesting if we want to test this. Can we actually measure
00:23:47.11		diffusion constants? Can we actually measure degradation? Can we
00:23:52.23		actually test the model, can we as a biologist test the model by defining
00:23:59.09		the biophysical parameters associated with the generation
00:24:02.27		of biological information? And lastly, there is one other interesting point
00:24:11.03		that it took me a very long time to appreciate about this model,
00:24:15.15		but I think is really really essential is that after bicoid was discovered
00:24:19.29		and those remarkable initial characterizations, the expectation was,
00:24:25.15		it was at a time where we were coming to realize as molecular biologists that
00:24:30.15		most of the genes and most of the processes that one sees say in
00:24:35.04		one species or in one organism, most of those same genes are also found
00:24:39.22		in other organisms and they probably also function in much the same way.
00:24:42.29		So there was initially an expectation that the bicoid protein given
00:24:48.18		its predominate and central role, the central role that it plays in
00:24:54.14		governing embryonic development in Drosophila given its importance,
00:25:00.02		that one would quickly identify similar proteins the bicoid gene in the frog
00:25:07.02		or the bicoid gene in humans maybe even. And what's remarkable
00:25:12.09		and what was unexpected at the time was that in contrast to many genes
00:25:17.01		that are conserved to a hide degree of fidelity across all species, bicoid,
00:25:22.05		the protein of the bicoid gene, is a fairly new invention. Meaning that
00:25:29.06		even if you look in other insects, even if you look in other Diptera or other flies,
00:25:33.28		you don't find the bicoid protein. Bicoid evolved at a point in the history
00:25:42.12		or the evolution of the higher flies as a new gene, a new solution
00:25:49.18		to a problem that must have been old and has always existed for all embryos,
00:25:52.28		because all embryos have to be able to establish pattern. But what's interesting
00:25:57.06		then about this particular problem about bicoid, is that it's not conserved.
00:26:03.06		It's a new solution to a fundamental developmental problem.
00:26:07.24		And so on the one hand that's interesting because you can say well how is it
00:26:11.05		that organisms in the course of their evolution establish new problems
00:26:15.26		and new solutions. Why do they do that, and what's the nature of those
00:26:19.23		new solutions. The other interesting thing, the question that then though
00:26:26.13		arises from the evolution of bicoid is that although we've said that bicoid
00:26:33.18		is a fairly recent evolution among the Diptera, there are a number of flies
00:26:38.05		which we'll see have from the point where bicoid was established as a
00:26:43.15		patterning mechanism have a number of fly species that have continued
00:26:50.22		to use bicoid and those species have evolved in other ways. As you'll see
00:26:55.18		they make big eggs and small eggs and so what becomes really interesting
00:26:59.06		about bicoid is how is it, during the course of evolution, when a new solution
00:27:04.24		arises. How is it after an organism or group of organisms chooses
00:27:13.02		that solution. How is it that during the course of further evolution
00:27:17.29		that solution is modified or changed or adapted to make it suitable as the
00:27:24.03		individual organism evolve and radiate and establish themselves into different niches.
00:27:29.26		And so bicoid, I believe, ultimately from the standpoint of evolution really
00:27:33.28		provides an interesting opportunity to study how evolutionary pathways
00:27:39.17		are modified. So basically for the remainder two parts of this lecture what I'd
00:27:47.15		like to do is focus on those questions. I'd like to talk a little bit about
00:27:51.03		how it is that this bicoid gradient is established. What have we been able
00:27:57.24		to learn by applying biophysical techniques to the establishment,
00:28:01.12		to fly embryos to figure how molecules move and how stable are the actual patterns
00:28:08.10		of bicoid in the embryo. Is it stable enough to provide information
00:28:14.22		that cells can make choices on? And then in the very last part,
00:28:17.23		I'd like to speculate a little bit and tell you a little bit
00:28:20.10		more about our experiments on evolution. Thank you.

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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