Feb. 27 Determination of Cell Fate How does a cell know what to become when it grows up?

As a cell matures, ultimately the mature body as about 220 cell types. How do you know which of these to become? We are looking at two main ways that cells determine what to become. Two Simple Models: 1) External/Surrounding Environment and Associated Signals Extrinsic Factors 2) What is inside of you (i.e. internal makeup) Intrinsic Factors Both of these can potentially instruct cells in following a particular developmental pathway to a particular destiny to specialize into specific cells tissues organs This section is a bit different overall, goal to understand how cells know what to become as they grow. We are going to primarily be looking at experimental evidence and see what we can deduce from it. Most of what we learn is just blind faith We are going to learn science based on the raw data. Each experiment will involve learning who/when/what/why. So we need to know the person, procedure, and concluded. The date is just for referencehow long have we known this information? Experiment #1: by Driesch (1892). He was working with sea urchins. He performed a very simple experiment in which he took a sea urchin embryo at the four-cell stage (morula/preblastula) and he separated these four cells from each other four independent cells. He cultured these four cells in seawater. He asked will of embryo give rise to a normal animal? So instead of all four giving rise to one larva (that undergoes metamorphosis and becomes one urchin), what if we just take one of these cells. What would happen? He found that indeed one of these cells alone can give rise to all of the body parts of what would is expected. The resulting larvae were a little smaller (obviously, because we are stating with the biomass), but everything was there (three germ layers, organs, etc.). He concluded that these cells alone have the ability to drive the entire body plan. This ability is known as totipotency To be totipotent (total power) means to have the ability to produce the entire animal. Thus, it is the capability of the nucleus to create the development of an entire, intake animal. There was nothing lost by separating the cells. Experiment #1 Implications: The data suggests that, early in development, cells and their nuclei are totipotent. They then have the ability and the necessary genetic information to drive the development of the entire animal. So if these cells are totipotent in the beginning, what changes during development? Is this a trait of every cell of our body? Probably not But we need to explore the question We are now moving to the 1950s and examine a technique called Nuclear Transplantation (NT). NT is a technique of transplanting a nucleus into an egg, once you have removed the eggs original nucleus. This was originally performed in frogs because the amphibian egg is very large and easy to manipulate. It can handle a fair bit of abuse. So, in this primitive method, they go in with a glass needle and remove the eggs nucleus. The intermediate is an enucleated egg. They then inject

(through microinjection) some nucleus from a different cell. The first experiments were done were just to take frog embryo cells, suck out the nucleus, and inject it into a donor somatic cell nucleus. This injection mimics what a sperm would do in activating the egg. So now, this newly crafted egg starts dividing. When the procedure works, an adult frog is eventually formed. Freddy was the first cloned animal, a leopard frog that was cloned from an early embryonicstaged nucleus introduced into a new egg and given a chance to direct the building of the frog. We would call this nucleus totipotent because it has the ability to direct the entire developmental process. This is just rehashing what we already knew. But How is this lost? Experiment #2: Briggs and King (1952) applied NT techniques in transplanting nuclei of different ages into this enucleated eggs. The graph shows the ages of the frogs: Late Blastula, Early Gastrula, Late Gastrula, Neurula, Tailbud tadpoles, and Heartbeat tadpoles. You can notice that as the use a nucleus from a late blastula, only 80% of the eggs will develop normally. Notice the trend of the graph. As the source of the nucleus that is introduced ages, there is a dramatic drop until you hit zero. When you use a fairly advanced cell (80 hours old), totipotency is essentially lost. Experiment #2 Implications: As cells differentiate they lose their ability to direct development. We can conclude that this totipotent ability is lost as cells age and differentiate. So in experiment #1 we concluded that early cells are totipotent, but experiment #2 reveals that this is not a permanent condition. The question becomes, is it ever possible to get an embryo and normal tadpole from a differentiated cell nucleus? Experiment #3: Diberardino (1983) addressed the question above. She worked with a terminally differentiated cell (a frog erythrocyte, which has a nucleus). This is a known terminally differentiated cell type. It is not intermediate and it is not a stem cell. She does NT. She transplants it into an enucleated egg and 7% of these develop into blastulas and then abort/they stop (no where near a tadpole). No surprise here, Briggs and King suggest that we cant find totipotency within a differentiated cell. Then she does something interesting, she does another transfer: Serial Nuclear Transplantation. She uses these blastulas and the source for nuclei and these are injected into a second generation of enucleated eggs. They are given another chance. First generation only got so far But if we take that nucleus and inject it into another egg, can it drive the development of this new egg even farther than the first attempt. Experiment #3 Implications: Yes, almost half (49%) of these serial transfer products become blastulas. Some (7%) actually moved into the gastrulation phase and started to organize germ layers. Unfortunately, none of the embryos progressed past these stages. But indeed the serial transfer made a big difference. But why did this work? Experiment #3 Parallel Experiment: She also did a parallel experiment where instead of working with a mature egg (the type that would be ovulated and out in the pond water), she harvested immature oocytes and injected them. She then treated these with progesterone to induce oocyte maturation. She then removed the oocyte nucleus. Low and behold, she observed much greater success: 16% blastulas, 4% gastrulas, and 4% neurulas! So there is something about passage of this erythrocyte nucleus through the maturation process that unmasks some developmental potential that was not manifested otherwise. So she did her serial transplantation from her first experimental again, and she observed much greater success. None of them ever metamorphasized so no adult frogs were produced. Because she never found adult frogs, it was believed that these nuclei could not achieve the required level of totipotency required to built an entire animal.

So the dogma moving through the 1980s was that nuclei, once they leave totipotency state, they are unable to return: totipotency is lost and restricted. You can develop partial power pluripotency where you can develop organ systems and such and tissue types. If you can only build different tissues, you are pluripotent. Only are you totipotent if you can drive the development of an entire animal. How does the Serial NT improve the success rate? How come these nuclei in round one didnt do so well, but you can give them another chance and they do better? This is describing the embryos in round two (the red comparison on the slide). Apparently, totipotent nuclei are present in embryos with limited development. Maybe these totipotent nuclei are prevented form directing development due to the presence of non-totipotent nuclei, which cause partial cleavage. So at this point, they are suggesting that not all the cells within a developing embryo are on the same footing in terms of their ability to direct development. Some have this totipotency and others dont. Problem is, everybody has to work together if you are part of the same embryo. So even though you have the ability, if your neighbors cant hold up their end of the bargain of sorts, you are not going to get a normal animal developing, because they fail to follow through with their restricted ability. How does this ability improve through Serial NT? The best explanation for this apparent improvement in nuclear transfer success is the following. It is believed that nuclei from slow-dividing somatic cells cannot complete their chromosome replication in time for the first cleavage of a recipient egg, which always takes place according to the time schedule of the egg So here we are, we have taken a somatic cell nucleus (and it has its own cell cycle) and we are injecting into an entirely new cell with its own schedule. And if the two are not in sync, something is not going to work correctly. For example, the egg will always divide 1.5 h after fertilization. Somatic cells though can take up to six hours to complete their chromosomal replication. So if this egg starts to divide and the nucleus (which is slower) isnt up to speed, the egg is going to divide and the daughter cells arent going to have the correct number of chromosomes because the newly introduced somatic nucleus isnt on the same time schedule as the egg. So, when these transplanted nuclei are forced into early mitosis with incompletely replicated chromosomes, they are likely to suffer chromosomal damage and generate chromosomal defective embryos This is why only 7% progressed to blastula stage. However, sometimes it happens that a transplant nucleus fails to undergo chromosome segregation when the egg divides into two cells and this whole nucleus moves into one of the two cells. So now it has a second chance to replicate in the next round of mitosis. As a result, partial blastulas are obtained because one of these two cells undergoes cleavage and the other dies (lacks a nucleus). So these nucleus you can recover, give them a second chance, and are now more synced with the cell cycle rhythm that is expected and they can help promote longer development. So what we are saying is, as nuclei work and start off some of them are better off in terms of developing into the right timeframe than others. If you get them another chance, they will get into the same schedule as the egg and drive development even farther. Nuclear Transplants allow you to test the nuclear potency via multiple chances/series.

Experiment #4: Wilmut used NT with mammals (specifically sheep). He works for a biotech company who wants to clone sheep to produce pharmaceutical products in their body secretions (milk). So you could engineer a sheep to give it a human gene that produces some product like blood clotting factors and this is expressed in the milk of the sheep. But when this one sheep dies, youve lost the golden goose. Youve lost the source of golden eggs. But Wilmut wanted to clone these sheep so they can mass-produce whole flocks of these sheep. These sheep do exist today! He was also granted a license to do this with humans. What he tried to do was get the egg and the somatic cell together. He worked with a sheep system: an enucleated oocyte that he fused with a quiescent mammary gland cell. This mammary gland cell was starved so instead of it dividing normally it was quiescent. We think that this allowed the cell cycles to synchronize with the oocyte. The two were fused and the resulting product was transferred to a surrogate mother and after 200+ tries, one of the worked and Dolly was born. Notice: The face fur color was their marker to show that indeed dolly came from the nucleus and not the oocyte. The oocyte came from a black-faced sheep and the nucleus from a white-faced sheep. So white-faced offspring indicated that the oocyte was derived from the injected nucleus. Maybe her genetics failed prematurely? She didnt die from a natural death (she was put down). But maybe her aged genome was pre-destined for early death. Work was done on her telomeres though. Telomeres are a reflection of age. Her telomeres were indeed a little shorter than what you would expect. The cloning of Dolly has led to a whole revolution. There is a whole industry of cloning pets, panda bears (and other endangered species), etc. What about cloning human tissue? This is somewhat controversial. Experiment #4 Implications: Wilmut suggests that indeed, if the conditions are right, you can restore totipotency. This is what the whole industry of biotech cloning relies on and attempts to optimize. Experiment #5: Blau (1985) performed a somatic cell hybridization experiment. She fused two somatic cells and asking questions about gene expression. The two cells she is fusing are a liver cell and a muscle cell (two very different tissue types). So when they come together into one fused cell, it forms a novel cell that doesnt exist in nature Hetereokaryon Notice, she used two different sources: human liver cell and mouse muscle cell. So this is a cross species hetereokaryon. We see the two nuclei. She then asked, can we detect human muscle proteins in this novel cell? She added antibodies for human muscle proteins and they bound Indeed, the hetereokaryon was producing human muscle protein But this was not a human muscle cell, it was a liver cell. So the only way to get human muscle protein from a human liver cell, would be to awaken the muscle genes of the liver cell to make muscle products. Experiment #5 Implications: She concluded that indeed you could awaken genes if the context is right for these dormant genes to come out of inactivity. Apparently, the context was getting exposed to some cytoplasm of a muscle cell, even though it was from a different species Thus, there must have been some factors in there that were recognized by the human genome and turned on the muscle genes in the human nucleus. >> Cytoplasm can activate gene expression! << So can these factors reestablish totipotency? Yes!

March 4 Today, we are wrapping up the list of experiments that help us understand cell fate determination The data suggests that over time, as cells specialize that totipotency is restricted and is lost as cells are dedicated towards a final destiny/job. However, we can reverse this specialization if we know the right genetic cocktail (combination of genetic factors) to introduce into the cell to bring it back to a quasi-embryonic state. Experiment #6: Jaenisch (2007) started with fibroblasts, which is a standard/easily-obtainable somatic cell, and they introduce various transcription factors to get this induced pluripotent stem cell. Which, then typically, in vitro, is treated under standard cell culture conditions to direct it towards a particular cell fate. These conditions weve known about for quite a bit because work has been done on natural embryonic stem cells. Now, instead of using an embryonic cell we are using this iPS (induced Pluripotent Stem Cell). Experiment #6 Implications: Clinical applications are currently happening right now using this technology. This cycle cell story here is done in an animal model and hypothetically could work in humans. There have, however, been a few applications in the field of medicine to come about through this technology. One is developing cells for screening drugs for effectiveness. So as a pharmaceutical company, you come out with a new product and you want to test it to make sure that it is biologically active and doesnt have adverse side effects. Typically, you dont want to move right into humans for testing. You start with animals and then move into human-like models, and only then do you move into human clinical trials. They have taken human iPS cells and differentiated heart cells from them iCell Cardiomyocytes! These are available commercially for companies to test their drugs on to see how these drugs interface with human heart muscle cells. This serves as a screening application for this iPS technology. In Japan, there have been clinical trials for individuals with macular degeneration (degeneration of the retina resulting in blurry and altered vision). They have taken skin cells from people with the condition, derived iPS cells from them, and performed cell differentiation in a dish to produce retina cells. They then implant these retina cells into the retina of the patient to see if these will help restore proper function of the retina. This outcome has yet to be fully realized and understood. There are two dangers of concern when it comes to this standard protocol. One of these is cancer. The way this first generation of iPS were generated was using an oncogene (c-Myc) This was a big red flag that as you introduce this gene into cells that might transform this into cancer cells in the future. Well, they have counted this problem by determining a cocktail that omits c-Myc. However, another problem (two) is that these cells are reprogrammed by introducing these transcription factors in the form of a viral infection. So the virus is the vector that through infection introduces into the skin cell these transcription factors. As these viruses integrate into the skin cell genome, supposedly randomly, they could potentially interrupt some sequence that is suppressing cancer (i.e. a suppressor gene) and illicit cancer development down the line.

Therefore, the use of viral vectors has been frowned on because of the potential to inactivating genes involved in suppressing cancer. Again, this has been solved by using ways of introducing factors that do not require viral integration. So these two problems have been overcome from the traditional work that Jaenisch started. A third problem is the teratoma problem. Some of these cells, being pluripotent (ability to give rise to any type of tissue), even though they have been passed through differentiation, there may be a few of these cells that havent differentiated. So when these are introduced into the human body, they could chose to go rogue and develop into something other than what is intended. This becomes the Teratoma. This is a growth that is derived from a stem-like cell. Example: foot tissue developing inside the brain of a baby. The teratoma then is evidence that these cells have pluripotency, unfortunately in its clinical manifestation it is a unintended entity. The question was asked last year, can we do this in vitro cell differentiation in vivo (in the living body). Diagram: They produced a transgenic mouse where they put the four transcription factor genes (these factors that have the power to take a cell and transform it into the iPS stage). They took these four factors and introduced them into the genome of the mouse in an inducible form. Now, these genes are already in the genome of the mouse, but they are tightly regulated. So what we are doing is adding a gene cassette where we can control at will these genes and turn them on and off. The way they control these genes is by inserting them under a promoter that is inducible by an antibiotic (in this case, Doxycycline). So whenever the antibiotic is present in the mouse, it activates these genes. Transgenic Mouse No Antibiotic Life is normal Transgenic Mouse Ingest Antibiotic Teratoma Formation The antibiotic surges through the body and activates the gene cassette which then causes a systemic, in-vivo production of these transforming factors What happens? Now that we have given it this transformative cock tail inside of every cell, what happens? Well, these animals quickly developed teratomas. This is no surprise because we said that these teratomas were attribute of producing iPS cells. So these masses that developed where traced to the action of these four factors producing iPS cells inside of the body. So these differentiated cells came to a stem-like cells and these stem-like cells started to develop these teratomas. What is unusual, some of these teratomas started to organize themselves into little baby mice what??? So these are male mice that have baby mice growing inside of them. This shows that not only do these stem-like cells have the ability to form different tissues, but they seem to have the ability to build an entire new individual. So There is something about the in vivo production of these cells that gives them an even broader ability of driving development. In fact, when they drew blood from these mice, they could find circulating in the bloodstream iPS cells which had a broader ability of developmental potential than those derived in vitro. Therefore, they described these cells as being totipotent-like. Because they have the ability to drive the development of structures normally not seen in iPS cells derived in the dish. This is just another example of iPS cells and the potential that they can have on driving development.

Direct Reprogramming [In vivo reprogramming of adult pancreatic exocrine cells to beta cells] Can we ever bypass this intermediate stage (this stem-like cell stage which has the teratoma/cancer problem). Can we ever go from one cell type to another? The answer is yes! This is a very powerful technique called direct reprogramming. First performed at Harvard by researchers interested in studying the pancreas. They have discovered the cocktail of factors that when injected into the pancreas, builds new insulin producing (beta) cells. The pancreas has a few important roles. It is not only an endocrine organ (generating insulin for regulating blood sugar), but it is also an exocrine organ producing secretory enzymes involved in digestion. So what they did, they discovered this cock tail that when injected into the pancreas of a mouse, transforms the exocrine cells into beta cells. This happens within a day or two. So we have an adult mouse that has trouble producing insulin. They simple injected these factors, and now these factors transform exocrine cells into insulin secreting cells. The diagrams show the pancreas of a WT mice (with an islet that produces insulin). The next diagram is showing an injected mouse, where you not only have the islets but you also have exocrine cells that are now producing insulin (red is indicative of insulin production). They asked, now that we can transform cells into a totally new job. We go from exocrine to endocrine. Does this reverse diabetes? They have an animal model where you treat it with an antibiotic (streptozotocin, STZ) and it disrupts normal pancreatic function (no insulin production). The graph shows fasting blood sugar, normal blood sugar is <100. After a day of being on STZ, you can see blood sugar goes up to 400+ (this is the diabetic model). On day one, some of the mice where injected with these transcription factors. Within a day, blood sugar dropped by almost 50% and stayed significantly lower than those mice that didnt receive the cocktail. It is not quite the same as the baseline but it is a lot better than being 500+. One reason they think it isnt back to the baseline has to do with the delivery of the insulin. If you study the pancreas and its anatomy, these islets are designed with blood vessels closely surrounding them to maximize export of insulin into the blood stream. In this model, exocrine cells are randomly turned on and there is no organized delivery system for insulin to be exported from the tissue. Direct reprogramming has been discovered to work in many different types of tissues and organs. We can now take a differentiated cell, introduce transcription factors, and transform these into a new cell type. Direct Reprogramming in a Living Heart This has been applied to the heart. This is a model where they cause a heart attack in a mouse. The way they do that is by tying off one of the coronary arteries (Notice the X). So the blood supply to the ventricle wall is gone and you have in infarction (which is where the cell wall is starved for blood dies). Instead of having nice tissue, you are left with a scar of fibrous, non-contractile tissue. The heart dilates but doesnt contract like it should. What they did was inject a cocktail. Discovering these cocktails is the hard part. What is the cocktail that will take a heart fibroblast and transform it into a heart contracting muscle cell. Well, they discovered the cocktail. GHMT is the four-part cocktail that spells muscle cell. They injected it into the heart. They watched to see what would change in this mouse. This heart attack was not

fatal, but it was enough to cripple and damage the heart but not kill the animal. So they let the heart heal with this injection and then they took cross sections at different points in time to look at the anatomy of the heart and how it has changed. GFP = a control injection GHMT = the transcription cocktail Out of 9 mice, 5 of them have this severe phenotype: Extreme thinning and fibrous scar of the ventricle wall. Only 2 of the 9 mice, had the nice thick, fleshy ventricle. The majority of the mice werent doing very well. We then compare these ten mice with ten that were injected with the cocktail. Only 1 of the 10 mice, has this thinning of the ventricle wall. Most of them, 7 of 10, had the nice thick wall. Furthermore, when the heart function was analyzed (ejection fraction and stroke volume). Those that received the cocktail were functioning significantly better than the control. Some even better than mice that didnt have the induced heart attack Functionally and anatomically, we can now directly reprogram the heart in a living animal. They have actual done this with the SA node. They have figured out the cocktail that produces SA node cells and they can create an SA node by injecting cells of the heart. So people that need pacemakers, we can now rebuild in vivo SA nodes at least in the animal model. We havent tried this in humans yet. So there is a race to discover these cocktails to build specific tissues in vivo. This whole field is exploding And it revolves around the question, what determines the fate of the cell? We have actually figured out genetically, at least the transcription factors, which allow us to reprogram cells in many cases. So these are the experiments We are now moving into the biology behind the experiments. Mosaic vs. Regulative Development These experiments have allowed us to classify animals in two broad categories: This table is an attempt to categorize all animals into two camps. Realize that this is a human approach and some animals resist nice compartmentalization but as broad strokes we can categorize animals in these two categories: We are Deuterostomes Are vertebrates (chordates) and echinoderms (sea urchins). These animals establish their cell fate somewhat late in development and these are base on extrinsic factors (things outside of the cell that impinge on the cells fate, regulative development or indeterminate development). This was supported by the work of Driesch. Remember, he separated the sea urchin cells at the four-cell stage and each built a new individual. This was regulative and these had not yet committed to some specialization, at least at the four-cell stage. What are these extrinsic factors? Other animals use intrinsic factors; these are things inside of the cell that establish cell fate quite early. So from the egg you inherit some sort of factor that from day one, that factor is going to direct you towards a particular destiny. These are found in Protostomes Mollusks, Annelids, Arthropods. This is known as mosaic development which means that every cell has its own unique package of determinates and you put them all together to get the entire embryo Just like a mosaic is made up of smaller individual pieces that makes the overall picture collectively.

So what this means is if you lose a cell from the embryo in a protostome, because it has its own unique contribution, you will only have a partial embryo. Because there is nobody else who can makeup for that lose of material. Rouxs work in 1888 demonstrated this. He went into a 2 cell stage frog and killed one of the cells with a hot needle. Notice, he only obtained half an embryo. So losing half of the embryo was catastrophic there is detrimental lose that cant be made up by the one living cell. This is unlike the sea urchin model in which you could lose of the cells and still obtain a complete individual. But frogs are deuterostomes!!! What??? But this data is used for supporting the mosaic/protostome model. How? His conclusion was some sort of artifact because of this dead tissue that most likely held back the development of the living tissue. So if he could have separated the dead cell from the living cell, he might have seen an intact embryo and a normal individual. So what are the intrinsic factors that cells can inherit Using the insect model and tunicates. Finally, we will use mammals and urchins to analyze what are the extrinsic factors that drive their development and cell fate determination. We will also study frogs in this regard. Ooplasmic Determinants Ooplasmic Determinants These are the intrinsic factors that are inherited by cells that undergo cleavage by that fertilized egg. These originate in the oocyte. And they are passed on to the daughter cells as they divide. This then serves as a wonderful example of cytoplasmic localization. Because what we are suggesting is that the cytoplasm is not uniform and as it is partitioned through division into daughter cells, each daughter cell is different because it gains slightly different types of cytoplasm. So the model system was studied by Okada in 1974. He was studying one particular cell fate known as the pole cell. The fate he was studying was the fate of pole cells, which develop at the posterior end of the early embryo. About 40 pole cells form at the posterior end. It had been known that at this end where the pole cells develop, before fertilization if you look at the egg at that end, you would find polar granules (colored in red). They are complexes of RNA and protein that are only found at the posterior end of the egg. As that cytoplasm is divided up through cytokinesis, these granules are inherited by the pole cells. And the pole cells are the future germ line of the animal. So when you study pole cell determination, you are studying germ cell determination. So, Okada wanted to test whether or not there is causality between pole cell development and bearing the polar granules. Because it could be just an association or just a coincidence. They might not actually be instructive of the pole cells to develop. He tries to demonstrate that these polar granules do indeed cause the pole cells to form. So this is the experiment. He performs a microinjection experiment. First of all, he showed that indeed you could sterilize a fly by shinning UV light on the posterior end of the egg. When that UV light shines on the egg, the idea is that it damages the RNA and protein of the polar granules so that they are no longer functional and pole cells do not form. So by inactivating the polar granules action, you stop pole cell formation. So it is a type of loss of function experiment. We can also do gain of function experiments. Could we force pole cells to develop where they normally dont form? Yes! He took some of the cytoplasm from either the anterior end or the posterior end and injected it into the irradiated eggs (which would normally not make pole cells). Pole cells develop after

injection with posterior but not anterior cytoplasm. The two ends are different! Poleplasm This suggests that there is indeed something in the cytoplasm of the insect egg that is transferable and drives pole cell development. When you move it around, you can move around pole cell development. They asked what is in the polar granule? What are the specific components that drive pole cell determination? They performed a chemical analysis and discovered: mtlrRNA (mitochondrial large ribosomal RNA) and Oskar protein. This protein is produced and localized in the polar granule under the influence of other proteins (tudor, aubergine, and vasa). Some of the roles of these are still unclear. Somehow they regulate transcription of Oskar gene. Oskar protein seems to be a pivotal player in the whole polar granule story. [tudor, aubergine, vasa] Translation Initiation Oskar Protein They have tested this in a couple of ways. If you over produce Oskar proteins (upregulate expression), it ups the number of pole cells. There is a dosage effect of Oskar protein. It has also been shown that you can mis-localize Oskar protein. Instead of having at the posterior end, you can force it form at the anterior end. When the protein moves, pole cell formation also moves. You can thus get pole cells forming at the wrong end. Indeed, Oskar seems to be an important determinate in the number and location of pole cells. Ooplasmic Determinants Tunicates Sea Squirts Aquatic, sessile organism. They anchor on a rock or substrate in the marine environment and pump water through their body. They have a muscular wall and move water through and filter feed. Whittaker was studying tunicates and he was looking at what determines muscle development in the animal. Now it had been known for quite awhile that when you look at the 8-cell stage of the tunicate embryo, that you can study in a dish (much like sea urchin), that two of the cells (designated B4.1) give rise to muscle tissue. So part of the way they do this cell fate mapping is that they just dissect the embryo and separate these pairs of cells. These pairs alone when cultured will give rise to certain structures. Of all of the pairs, only one of them (B4.1) gives rise to muscle. What is interesting; when you look physically at the embryo, the B4.1 cells literally have a different color to them. This is called the yellow crescent. It was hypothesized that maybe this yellow crescent contains some type of determinate that tells cells to become muscle (myoplasm = muscle determining cytoplasm). Well, this again could just be a coincidence. This was tested. What happens if we manipulate the yellow crescent? Can we manipulate muscle formation by disrupting the yellow crescent? Whittaker (1973) found an enzyme abundant in muscle called Acetylcholinesterase. He found a stain that reveals the presence of this enzyme, this is showing an early embryo, and you can see all of the darkness and cells expressing AChE. This serves as a histological marker for muscle development. So whatever turns black in the embryo eventually becomes muscle. So he uses this as a tracer to follow muscle development in the embryo. Whittakers Experiments with Tunicates He does two experiments. One he just pulls out the B4.1 pair of cells and does the staining technique. This is the mass of cells and they turn intensely blackconsistent with muscle. The rest of the embryo remained unstained.

Can you force cells that normally dont become muscle into becoming muscle by forcing them to have the yellow crescent? They way he did this was by altering cell division. So this is the normal progression of cell divisions of this animal. You can see the normal egg and the first division that splits it into two. Now we have two cells that have the yellow crescent. These then divide again (meridional) and two cells have the crescent. Then, you have an equatorial cleavage and you end up with eight cells and only two cells have the crescent. When this stage is stained, only two cells reveal staining. Between the equatorial cleavage stage (4 to 8 cell), he compressed the embryo, which displaces the mitotic spindle so it wouldnt split equatorially but instead it would split meridionally. So in this case, each of these two cells that have the yellow crescent would split in half and the two daughter cells would inherit pieces of yellow crescent. So in the end, half of the embryo has the yellow crescent, instead of just of the embryo. Then he asked, now that we have these two extra cells that have the yellow crescent, do they indicate muscle development? Indeed, yes. 4 of the eight cells stained black and thus indicate that as the yellow crescent is partitioned into new cells, so does muscle cell development. Macho-1: A Myoplasmic Determinant But what is in that yellow crescent that drives muscle development? What is the molecular basis for myoplasm? Well it took over 20 years, Nishida (2001) discovered Macho-1 This is believed to be a key determinate in the myoplasm. If you look in the unfertilized egg and stain to localize the Macho-1 RNA, you will find it localized in the yellow crescent. At the eight-cell stage, it is only found in the B4.1 pair which is what we would expect! We then can inject Macho-1 RNA into cells that normally dont have it and look to see if they start to express markers for muscle development. So in this case, he is injecting these pairs of cells with Macho-1 (pink and blue, wild type of different amounts). These pink bars represent the expression of AChE, which is the muscle marker, and myosin. Wherever wild type Macho-1 RNA was injected, AChE and Myosin expression increased! However, they mutated this RNA so that the product that it normally encodes would not be active. As you can see, when the mutant was injected (yellow), you dont see any marker (AChE or Myosin) expression. It doesnt matter in the B4.1 cells because it already has the endogenous Macho-1. Macho-1 encodes for a transcription factor, which is no surprise, because we have been talking about the power of transcription factors to direct and govern cell specialization. So Macho-1 contains a zinc finger-based motif (sequence of amino acids that allows it to bind to DNA and regulate transcription). It also is localized to myoplasm. If you express it cells that normally wouldnt otherwise give rise to muscle, these cells start to develop into muscle. When Macho-1 is suppressed, you lose muscle marker development (green and gold bars) and you gain endoderm markers (Alkaline Phosphatase). So this is lose of function tests Macho-1 is very important in getting muscle to form in this animal. What is Macho-1 doing? Well, it regulates genes but what is its target? What is Macho-1 doing? The current data suggests that Macho-1 targets Tbx genes. Particularly, Macho-1 targets Tbx6b. This is the experimental data. These tunicate embryos and we are staining and the dark, brown color is representative of AChE. So you can see that muscle development is found in much of the

animal. If you overexpress Macho-1, you get excessive muscle development (lots of marker). If you knock it down, you get a reduction in darkness. You can compliment this by overexpressing Tbx6b even though you have under expressed Macho1. If you allow the downstream gene to be active, you can restore this overexpression phenotype of muscle. Macho-1 Tbx6b Muscle Development So Macho-1 is inherited from the egg and as cells gain this factor, it turns on genes that turn on muscle development (the Tbx6b gene). How does an Ooplasmic Determinant Work? Intrinsic Factors! The study of bicoid in the fruit fly. Nusslein-Volhard, she won the Nobel Prize for her work with fruit fly genetics. In 1986, she helped pinned down the contribution for this particular gene (biocoid) in the anterior (head) region of the fly development. Being a protostome, we would predict that in order to get a head, that particular cells fate would be governed by intrinsic factors inside of the egg that are inherited by cells at that end of the embryo. So here is the egg, we find bicoid localized at the anterior end. The cells that develop from this end are destined to become head. If you knock out the bicoid gene, bicoid is no longer presence. You not only lack a head, but you get a tail that develops instead of a head. You get a two-tailed embryo. This never develops into an adult fly. So this illustrates the principle of the default pathway. Remember, the default pathway there is a certain developmental goal that body parts and tissues are moving towards and it will move there unless something intervenes and pushes it off to a new direction. So apparently, at the anterior end, the default pathway is tail unless you have bicoid present to transform the tail default destiny into the head anatomy. We would never have known this unless we removed bicoid to reveal the default programming. So what is the data? We have lost function and we have lost the head. We can see bicoid gene product localized at the anterior end. The Bicoid gene product is a transcription factor and we find it in the nucleus. In the wild type, we find a gradient of the bicoid protein product and it diffuses as you move towards the posterior end. Bicoid alone is sufficient for anterior development Can we restore head development by simply adding back Bicoid mRNA? Yes. Here we have various embryos. This would be the mutant that normally doesnt make a head. If we inject bicoid mRNA into the anterior end you get a anteriorly localized head. If you inject it into the same bcdmutant,but into the middle, you get head developing in the middle and the default pathway of tails at each end. Or you can produce a two headed fly by injecting bicoid mRNA into the posterior end of a wild-type embryo. Bicoid = Ooplasmic Determinate that encodes for head. March 6 So weve been looking at how cells know what to become as they specialize and what determines the fate of the cells. Typically, it is something inside (intrinsic factors) the cell and is inherited by the cells by the

egg. So you can envision that if you have an egg and these determinates are localized throughout the cytoplasm, as the cytoplasm is cleaved, as certain cell will inherit a certain cocktail of determinates. Thus, these cells develop differently Requires all of the cells to account for all body structures Mosaic Theory. If we remove a cell, we remove a determinate and there is no cell that can replace the deficient. Insects, Tunicates, Snails, etc. Protostomes Follow the Mosaic Model We are not talking about extrinsic factors (things outside of the cell). These are signals that tell the cell what to become. We are going to look at a number of experiments. First starting off generally and then moving in to try to understand more of the biochemical basis and how this cell to cell signally works. The Role of Cell Position in Cell Fate Determination This experiment was performed using mouse embryos in 1977. Kelly performed a cell rearrangement experiment with mouse embryos. In this case, you can fertilize mouse eggs in vitro and allow them to develop. He worked with the eight-cell stage embryos. At this stage, there are basically two types of territories of cells that can be distinguished. You have the four inner cells that give rise to inner cell mass and the remaining cells give rise to trophoblast. The first experiment he performed wasnt depicted on the slide. He took these eight cells, pulled them all apart and randomly reassembled them. So what use to be on the inside is now on the outside and vice versa and he would still get a viable mouse from this scrambled embryo. So apparently, at the eight-cell stage, a cell is flexible in what it can become. Because in this experiment, it could adopt a new location and still give rise to proper development. So if you are an inner cell, but in the new scrambled embryo you find yourself on the outside, you no longer move towards being ICM. But you adopt the trophoblast destiny. This suggests that location determines your fate! In this case, he performed cell rearrangements and recombined them with different cells of different embryos that based on lines of mice with different fur colors. The colors of these cells are indicative of future mouse fur color. So we have an albino mouse embryo (white) and we have a black mouse embryo (black). In this case, he would take a cell from any location from the albino mouse embryo. And surround it by four black mouse embryo. He would incubate this and implant the blastocyst into a surrogate mother. Based on this, what color of the mouse was born? The mouse was albino! Because we said the ICM gives rise to the animal. The inside determines the ICM. This in indeed what they found. So even though this cell could come from the outside, once it is placed in the inside, it inherited a new developmental goal towards ICM. The other thing he noticed is that you can obtain a complete mouse from just five cells instead of the original eight cells. This is very similar to the sea urchin story with Driesch where you could get a complete urchin from just of the original embryo. In this case we have 5/8th of the original embryo. So in this case, not every cell is needed like it would be in the mosaic model because we can pull off three of these cells and not use them and we still get an entire mouse. Regulative Model for development as cell fate has not been locked in yet. This has practical implication for humans too! Because there is a test that can done the lab called pre-implantation genetic diagnosis. Where they can perform genetic testing on embryos

produced in vitro (IVF). Some of these embryos, people want to learn a bit about them before they are implanted. So they can take a cell from the eight-cell stage human embryo and remove it and test that one cell using PCR to determine whether it has certain mutations, sex, tissue types, etc. So there are a number of genetic tests that can be performed in which we can learn about the embryo from that one cell. The remaining seven cells could then be used in implantation and produce a viable baby! Humans too have expendable cells in the early embryo. So Kellys work suggests that indeed the neighborhood in which you live in determines your fate. Inner neighborhood ICM Animal Outer neighborhood Trophoblast Placenta The experiments we are going to be looking at when it comes to extrinsic factors are referred to as whos your neighbor experiments. We are going to look at a number of experiments in which the neighbors were changed and we ask the question does cell fate change too. How Cell-Cell Interactions Influence Cell Fate Horstadius did more cell recombination experiments. He moved cells around and put them into novel arrangements and asked questions. What do you get? He worked with 64-cell stage sea urchin embryo. In this case, the colors represent the germ line fates. So the top half (animal hemisphere) of the normal sea urchin embryo is destined to become ectoderm. So the gut is depicted in gold and it coms from the macromere region of the vegetal hemisphere and then you have micromeres that give rise to skeleton. He performed a number of experiments. He took partial embryos and observed development. First, he took just the animal half alone. This half only derives a partial embryo and only gives rise to derivatives of the animal hemisphere (ectoderm). So this is definitely an altered embryo. So abnormal development here occurs if you only have part of the embryo. Then he started to add back pieces of the original embryo. He adds back the micromeres of the vegetal pole. When he adds these back, he observed an almost recognizable larva. He also got a gut developing, even in the absence of any cells that normally give rise to gut. This guy was derived from the animal hemisphere. Cells that normally give rise to ectoderm, changed their fate and produced endoderm derivatives. So apparently their neighbors could influence these cells. In the absence of this vegetalizing factor or signal from the micromeres, the animal hemisphere just followed their normal state of progression of becoming ectoderm. However, under the influence of the micromeres (usually not neighbors), all of a sudden the animal hemisphere behaves differently and starts to build body parts that are normally built from the neighboring macromeres of the vegetal hemisphere. Horstadius hypothesized that there must be some factors Vegetalizing Factors. These are emitted from the micromeres and whoever is neighboring was influenced to build gut. So we see endoderm derived from cells that are pretty close to the micromeres under this factor influence. So in this case, part C, the animal hemisphere cells happen to be under the influence of these factors from the micromeres and they themselves behave like the macromeres would have normally donebuild gut. Thus, this work hypothesized that there are chemical signals that are released from cells that are sensed from other cells and these cells react in response and change fate. This is the principle of induction. We are about to unpack the principle of induction.

How Cell-Cell Interactions Influence Cell Fate I Frogs have been the model system for understanding induction. Nieuwkoop worked with whos your neighbor experiments, recombining cells who are normally not neighbors, and asking the question due to these new signally systems and conditions, how does this affect cell fate? Mesoderm Induction How? Normally, mesoderm develops from the marginal zone region of the early frog embryo. The top roof is ectoderm, followed by mesoderm, and endoderm on the bottom. Nieuwkoop dissected these early embryos (blastulas) and took the roof and recombined it with novel neighbors. In this case, he removes the marginal zone cells and places the roof (the animal cap) and places it right next to the vegetal cells. The animal cap then instead of giving rise to ectoderm like it normally would, it gave rise to mesoderm derivatives. Obviously, at this stage, the animal cap cells were not fixed in their fate to become ectoderm because they can be redirected to become something else (if they have new neighbors). So what are these mysterious factors (red arrows in part B) released during chemical induction that apparently instructs cells to become mesoderm. Normally, the marginal zone cells receive that directive But now, because there are new neighbors, the animal cap receives those directives and becomes mesoderm. Question: How do scientists determine/describe what is mesoderm, endoderm, ectoderm? Answer: Before genetic markers, scientists looked for anatomical/histological changes. Now, they look for genetic markers and through PCR/reverse-transcriptase. What are these factors? Mediators of induction What are these factors that are instructive of mesoderm development. How Cell-Cell Interactions influence Cell Fate II Mesoderm is not just one class There are many subcategories. Paraxial, Intermediate, and Lateral Plate. So there is regionality in mesoderm development. Therefore, are their regionality to these signals? Is the dorsal signal than the ventral signal verses the lateral signal? Could this explain why there are different kinds of mesoderm? Another experiment was done by placing the animal cap over different regions of the vegetal cells. We can see the animal cap (A1-A4). The vegetal cells are divided into D1 to D4. D1 is future dorsal side and D4 is future ventral side. The dorsal side is where the organizer develops (thus represents a key type of mesoderm) He did see the regionality reflected When D1 is placed underneath the animal cap, you primarily get dorsal type of mesoderm. When you place D4 underneath the animal cap, you get primarily ventral (with some intermediate).

Therefore, the signals are not the same. There must be specific regional signals that induce the development of specific types of mesoderm. How Cell-Cell Interactions Influence Cell Fate III This launches us into a bunch of cell signaling We are hypothesizing that the vegetal cells give rise to this mesoderm-inducing suite of signals. They come in a variety of flavors. There are special signals on the dorsal side that gives rise to the organizer (the dorsal lip of the blastopore @ the marginal zone/blastopore). These signals in particular are important to keep track of because they are very important in specifying an important part of the embryo. This organizer is often called Spemanns Organizer (named after Hans Spemann). Nieuwkoop has also been recognized The Nieuwkoop Center is the source of these organizeinducing signals (D1). Proteins can be secreted from cells. There must be signaling pathways that moderate the action of the signal once it gets inside the target cell. How beta-Catenin is stabilized dorsally: the Wnt Pathway. First/Main Pathway: Wnt Pathway. This is a pathway that involves extracellular signaling (Wnt Proteins) binding to a surface receptor a transduction of information into the cell. The receptor is called Frizzled and it interacts with a protein called Disheveled. These odd names describe features in fruit-fly knockouts phenotypes. Wnt Frizzled (Receptor) Disheveled Disheveled is an inhibitor. Disheveled inhibits Glycogen Synthase Kinase-3 (GSK-3) Being a kinase, GSK-3 phosphorylates B-Catenin. Phosphorylated b-catenin is marked for degradation. So, B-Catenin is one of the linker proteins that anchor cadherins to the cytoskeleton. This is a structural role of catenin (thus moderating cell adhesion). B-Catenin can also play a genetic/transcriptional role. So B-catenin must get inside the nucleus So thus far Wnt Frizzeled (Receptor) Disheveled / GSK-3 -/ B-Catenin. It binds to a transcription factor called LEF/TCF (T-Cell Factor). This a transcription factor that with the help of B-catenin, binds to target genes and activates them. This pathway is also driven in cancer So people interested in cancer have really been studying the Wnt pathway Some evidence that some forms of cancer are traced to when cell adhesion is altered where b-catenin is normally working outside of the nucleus (anchoring things), when this role is disrupted, b-catenin breaks free and triggers gene transcription. This pathway, however, is critical for developing the dorsal side of the embryo. B-catenin is thus localized dorsally. Why is it there and why do we care?

We care because b-catenin can activate unique genes on the dorsal side which would be absent from the ventral side of the embryo Dorsal Mesoderm = organizer So, the Wnt Pathway: Allows genes to be activated through extracellular signals. It is regulated by a trio of proteins. So if Wnt wasnt around, GSK-3 would not be inhibited. And GSK3 along with axin and APC form a three-part complex that binds to b-catenin and leads to its degradation.

GSK-3 phosphorylates b-catenin and marks it for degradation by the proteasome with the help of a protein called B-TRcp breakdown of b-catenin. So when GSK-3 is activated, b-catenin is not active This is what happens on the ventral side (b-catenin is degraded). On the dorsal side, there are inhibitors for this three-part complex. Disheveled is one of these. Another one is GBP (GSK-3 Binding Protein). GBP binds GSK-3 and prevents it from doing its work. And without GSK-3 activate, b-catenin can survive

So we have two ways of blocking GSK-3: Disheveled and GBP. The third player that aids in the survival of b-catenin is Casein Kinase Ie This inhibits the axin component (one of the three members of the complex responsible for breaking down catenin).

The bottom line: All of this signaling is going to be uniquely occurring in the dorsal side and leads to the survival of b-catenin dorsally Thus acting like a regional marker for where the dorsal mesoderm is going to form. How the Cortical Rotation differentially stabilizes -catenin Why is the dorsal side so different in this way? What governs the activities of these various proteins? The gray crescent This is a marker on one side (opposite of the sperm entry). The cortical rotation causes morphological differences in the early frog. There are also biochemical differences too! When the shift occurs (30 degrees), it trans-locates the inhibitors of GSK-3 to the dorsal side (disheveled (normally located in the vegetal pole), and GBP). These things block GSK-3 and thus b-catenin is stabilized dorsally. Where on the ventral side, GSK-3 is active So we end up with a gradient (lots of b-catenin in the dorsal side and few in the ventral side). So the reason we have an enrichment of b-catenin dorsally is because we have a regional enrichment of these factors (GBP and DSH) that aid in the survival of b-catenin by inhibiting GSK3. We have set in place b-catenin in the dorsal side were the organizer/dorsal mesoderm is going to develop Locating the Cortical Dorsal Determinant I This work was done in Japan and was painstakingly performed. This is one of the few articles that only has one author on it. Crazy! What he did was go into a frog egg and divide it into five different regions. He sucked out using a micropipette cytoplasm from these different regions (this is cortical cytoplasm). So we have animal hemisphere and vegetal hemisphere (which we can determine via color in the frog model). These were injected into a recipient embryo and they looked for this twinning effect. Recall, this is the phenotype that you obtain when you graft in the dorsal lip (when you add another organizer). We realized that indeed you could find the same activity in an unfertilized egg (the dorsal lip activity as well as in an embryo that has a dorsal lip). So he is getting twining just by inserting cytoplasm from an egg but only when it came from the vegetal hemisphere (96% twinning success). He also did the same experiment with a number of different ages of embryos. This next frame is using a 32-cell stage embryo that he partitioned into these quadrants. So he has these four tiers. He does the same microinjections. He discovers the greatest success of twinning to come from the dorsal mid-zone or below (C1 and D1). This is exactly where we would predict b-catenin to be stabilized. So what was being translocated here from the vegetal to dorsal side? The twinning was caused by the abnormal survival of b-catenin, stimulating a secondary organizer and giving rise to another body axis. So far we have a dorsal determinate called b-catenin It survives there do to the inhibition of its antagonist.

It specifies dorsal development What is the actual action? It stimulates gene expression How b-catenin induces the Organizer

The Gene Cascade: Know the activation pathway. These genes are the products of b-catenin As b-catenin moves into the nucleus, it binds to transcription factors (Tcf3) and activates twin/siamois genes. These encode proteins that are regulatory factors that activate other genes (Chordin, Noggin, Goosecoid). These three genes are markers for the organizer. We think these three mediate some of the actions of the organizer and its ability to craft the body axis. These three genes are the workman of what is stimulated by b-catenin. B-catenin doesnt actually do the work It simply is a trigger that trips the switch for these genes to be activated that actually do the work of organizing. Know what keeps b-catenin alive! How Mesoderm is induced: An Overall Picture This is the model for the quiz and the answer to a final test question. Xnr = xenopus nodal related gene

The above diagram tries to portray which genes are acting where to specify mesoderm regionality. We have already talked about dorsal side But what about ventral and intermediate mesoderms? The idea is that we start off with: b-catenin stabilized dorsally and in the vegetal hemisphere we have VegT and Vg1 factors. These are transforming growth factor family members. These are colored in blue and represent regional gene action. This is the same signal seen Gene Cascade (TGF-B Family). This family specifically are found in the vegetal hemisphere VegT and Vg1 affect Xnr Where we have no b-catenin, VegT and Vg1 turn on Xnr in low amounts. Therefore, ventrally, where there is no b-catenin, we get little amounts of Xnr expression. As we move dorsally, as we get into the area where b-catenin is present, in the presence of b-catenin plus these Veg factors, we get elevated Xnr. This is all in the vegetal cells that signal the marginal zone to form the mesoderm. Low levels of Xnr found ventrally translates into ventral mesoderm. As we move dorsally, more Xnr is expressed (due to b-catenin) and that specifies organizer So we end up with organizer dorsally and ventral mesoderm (two broad classes) However, There is one more player What defines intermediate mesoderm? How ventral mesoderm is induced: antagonism between inducers BMP (Bone Morphogen Protein) and a specific Wnt Protin called XWnt8. Wherever b-catenin activity is low, BMP activity is high. The reason for this is the products of the organizer (Chordin, Noggin, Follistatin etc.) bind and inactivate BMPs. So when you look at the

marginal zone of the frog, we are asking the question what patterns the intermediate mesoderm? Well, dorsally we have the organizer which has its molecules (Chordin, Noggin, Follistatin, etc.) These are BMP inhibitors. Organizer Molecules (Chordin, Noggin, Follistatin, etc.) Bind and Block BMPs As we move away from this inhibitory action, BMP activity increases as we move ventrally. BMP is only allowed to be active in the ventral side. Same thing happens with Xwnt8 and its inhibitor Frzb. Frzb is blocker of Frizzeled. Frizzeled is the receptor for the Xwnt8 protein. Frzb contains the same site for binding Xwnt8, so in the presence of Frzb, Xwnt8 binds to Frzb site instead of the Frizzeled site and the signal is not transduced inside the cell. Frzb is a dorsal related organizer molecule. Xwnt8 is expressed throughout the ventral and intermediate mesoderm. Xwnt8 is highest ventrally.

Ventral Mesoderm is going to develop where Xwnt8 and BMP is the highest. Dorsal Mesoderm is going to develop where they are lowest Intermediate Mesoderm forms where there is intermediate. Story: Gene Expression. March 10 We are wrapping up the induction story.

Induction and the CNS We are looking at experimental evidence, conclusions, and associated models. So this is the work of Spemann, he was the guy who did the dorsal lip grafting and won the Nobel Prize for discovering the primary organizer in amphibians. This is another grafting he did with ectoderm. He was studying newtsa type salamander. He took ectoderm from a donor (pink color embryo). In this case, based on its location this should become neural tissue. So this is neural ectoderm. He takes this and transplants it into this yellow recipient embryo in a whole new location. This new location is where epidermis usually develops. So he moves the natively occurring ectoderm and replaces it with this new graft. Low and behold, even though what he transferred was derived from neural ectoderm, adopted a new cell fate and become epidermis. So what should have become neural became epidermal. This suggests that indeed that at least at this point at development the newt embryo ectoderm is not fixed in terms of neural vs. epidermal fate. In this case, we can easily switch the fate just by changing the neighborhood in which this tissue exists. He repeated the same experiment with an older embryo set. However, he obtained different results. In fact the transplanted ectoderm did not change fate into epidermal tissue. Even though it is now in the epidermal neighborhood, it sticks to its presumed origin of being neural ectoderm. This ultimately develops a secondary neural plate and a secondary body axis. What happened between the early gastrula and late gastrula stage to fix the fate of this neural ectoderm? Neural Ectoderm gives rise to brain and spinal cord and is found dorsally. What happens during gastrulation to fix these fates? We know that underneath, at least at this later point in development, there is a whole involuted layer of future mesoderm. This involuted layer is absent in the early gastrula. So in this particular involuted tissue, at least in this region it becomes part of the notochord, was suspected to be the culprit for what fixed the overlying ectoderm to its particular fate. For years, it was suggested that this underlying tissue would tell the overlying tissue to become neural ectoderm. And when this isnt present (i.e. in the early gastrula), that message hasnt been sent yet and these cells are still flexible (not fixed). But once it receives this message, this tissue becomes fixed. This is not quite true, but this underlying tissue is still very important. Regionality in Neural Induction This underlying tissue is called chordamesoderm. When we study the central nervous system, we know that there is regionality to the development of that system. The brain develops anteriorly and caudally we have spinal chord. So if you remember, mesoderm also develops regionally. Example, dorsal is very different than ventral and intermediate is unique too. So maybe, the anterior chordamesoderm is behaving differently than the posterior. So the overlying ectoderm becomes brain at one location and spinal cord at another. If this is the case, there must be some different interactions and signals going on. Now they did an experiment to demonstrate the regional components of the influence of the underlying chordamesoderm to the overlying ectoderm.

So this story is where the performed tissue transplanting of the dorsal lip of different ages into the inside of a developing amphibian embryo. This little lip was placed underneath the ectoderm. What they found was that depending on the age of the lip, the influence the type of neural tissue that would form from ectoderm. Young Gastrula Dorsal Lip (barely starting involution process) Overlying Ectoderm develops into a brain. However, Older Gastrula Dorsal Lip Overlying Ectoderm develops into spinal cord. So there is something to do with the age of the lip and its ability to drive central nervous system development. This chordamesoderm seems to vary in its power based on its age. The idea here was that this future chordamesoderm that is about to involute and when it does its work, since it is going in first, it is going to move and adopt an anterior region and thus the overlying ectoderm is going to brain (anterior). Later dorsal lip isnt going to involute as far. It is going to underlie the posterior ectoderm and instruct it to become posterior neural tissue (spinal chord). So just like in mesoderm induction, there is regonality in the influence of ectodermal differentiation. So this process of building the ectoderm into neural tissue is called Neural Induction. This is not the correct term now The current term is Neuralization. Thus, Neuralization is what the ectoderm experiences to become neural tissue. Neural Development as the Default Pathway There were some experiments done about 25 years ago, where they were able to shift the ectodermal fate independently of any underlying chordamesoderm. What these experiments revealed was that neural development is actually a default pathway. The ectoderm is preprogrammed to become brain/spinal cord unless it is told to do something else (i.e. become epidermal). Neural vs Epidermal. That something else is epidermis. So as the ectoderm is developing, it faces two main pathways: neural or epidermal. Now we believe the neural pathway is what all ectoderm will become unless it is told to do otherwise. BMP4 Signaling The same BMP4 that patterns ventral mesoderm formation. So signals that are operating in the marginal zone to tell those cells to become ventral mesoderm are also spilling out into the ectoderm and telling the ectoderm not to become neural but to become epidermis. BMP4 Epidermal Fate These arrows are the BMP4 signals and the blue color is the epidermal fate. But the chordamesoderm underlying the ectoderm modifies the BMP4 signaling and now block it, revealing the default pathway to become neural. These experiments are some that reveal this tendency. You can take some ectoderm based on its location you know should become epidermal (blue). If you simply separate the cells, this dilutes the BMP4 signal and you neural pathway kicks in. You dont get a functional brain/spinal cord but it starts to show gene expression consistent with neural ectoderm development.

You can also take this same epidermal fated ectoderm and genetically knock down the BMP4 receptor and reveal neural fate. Now in frongs we dont have a knockout frog for this but we can do morpholinos and other microinjections that knockdown particular targeted genes and look for changes in phenotypes. If we do that with BMP4 receptors, it transforms the tissue into a neural fate. Likewise, you can also get similar results by treating them with molecules secreted by the organizer that block BMP4 signaling. Those same molecules bind and block BMP4 and influence ventral mesoderm events they are also influencing the ectoderm. Organizer Molecules (Chordin, Noggin, Follistatin, etc.) Bind and Block BMP4s BMP4 signaling promotes ventral mesoderm but it is antagonized by organizer molecules. These same molecules spill over into the ectoderm and thus neutralize the dorsal ectoderm. Where they are not influenced, BMP4 signaling drives the epidermal fates. Development of the ectoderm and mesoderm are married together via the organizer molecules that share in common the ability to block the development of certain fates. A 2-step Model for Neuralization So here is the in vivo model for what occurs. This underlying tissue is making BMP4 inhibitors and thus unveils the neural development of what is overlying it. Neuralization is not the same is Neurulation. Neurulation is the building of neural folds and the neural tube of the central nervous system. Neuralization is the signals that kickstart Neurulation. Neuralization Neurulation Neuralizing Factors (Chordin, Noggin, Follistatin) Suppress BM4 These factors can be seen being excreted by chordamesoderm into the overlying ectoderm. Focus on Steps 1 and 3. Step 2 involves head formation and we are not following that story. The initial signal blocks BMP4 activity. Now, we have to explain anterior and posterior differences. Posteriorizing Factors These are molecules that form a gradient (purple) that patterns the ectoderm so that the anteriorly you have brain and posteriorly you have spinal chord. Involves Wnt Signaling, FGF (Fibroblast Growth Factor), and Retinoic Acid gradients These are the signals that tell the ectoderm to be brain verses spinal chord. These all work together to build the central nervous system We have already seen the evidence for blocking BMP4 and how it unveils neural development. What helps posterior development of spinal chord though?

Testing how FGF is involved in Neuralization This is one experiment where they are working with frogs and they took an early frog embryo and they dissect out the dorsal marginal zone (where the organizer is going to form, future dorsal lip). They take these cells (called DMZ cells) and they dissociate them in a suspension. They mix these cells with future ectoderm cells (derived from roof of blastocoel of stage 10 gastrula). Thus, the DMZ cells serve as the potential signal from the future mesoderm to the ectoderm. What kind of ectoderm will these cells be signaled to become? So we take these ectoderm cells and we mix them with varying levels of DMZ cells. Which is a way of changing the dosage of the factors produced by the mesoderm. We follow two genes: a marker for posterior neural development (a spinal chord specific gene) and a marker for epidermal development (keratin). The more signaling (the more DMZ cells) ,the stronger the response is for becoming neural tissue. So at maximal DMZ cells, you see a 100% expression of the spinal chord gene. Inversely, at low levels of DMZ, you see 100% expression of keratin. High DMZ Posterior Neural Development They performed the same experiment in which the blocked FGF signaling pathways. In the absence of FGF signaling, you get no posterior neural gene marker expression. This shows the absolute requirement of FGF signaling to achieve posterior neural development. Next Slide: There is also Wnt Signaling and RA involved in instructing posterior neural development. This work is looking specifically at Wnt signaling and b-catenin. There are three experiments show here. One of these is from the textbook. The first experiment they did was to take the roof of the blastula; these are the cells that are supposed to become ectoderm. They treated these with Xwnt8 protein (this is the signal that operates in the marginal zone to promote ventral mesoderm). Does it have anything do with ectoderm, particularly with posterior development? They used different doses of Xwnt8 (0, 2, and 20 [nM]) and then they asked the question: do these cells express gees that are consistent with certain types of neural development? RT-PCR allows them to determine this. The intensity of the bands in the gel are indicative of the activity of that particular gene. If we see a white band, we know the gene was expressed. If there is no band, the gene was not expressed. What we are looking at specifically are these last three rows of varying XWnt8 concentrations. You can see that the marker for anterior (Otx2) is shutting down as you increase the amount of XWnt8. But the Krox20 marker (posterior) is turned on as the amount of XWnt8 is increased. So we would predict that as you move more posteriorly along the body axis towards the tail where the spinal cord is developing, the ectoderm should have more Wnt signaling as you head in that direction. They can test this directly by inserting a gene recorder. This is a luciferase gene that produces light under the control of Wnt signaling. So they injected it into the developing neural ectoderm (so they have all of these cells as the develop dorsally), and they can monitor the light production, which is reflective of Wnt signaling. Notice as you move from brain to spinal chord, the white bar

increases. Thus, showing that you have an increase in Wnt signaling as you move from head to toe. Which is what we predicted from the first experiment: the more Wnt signaling protein the more posterior development you have. This is experiment #2. Thus, showing that Wnt signaling is elevated in the more posterior portions of the body. We can also stain this ectoderm for b-catenin protein inside of the cells. So they did this, sliced up the developing backside into eight regions, stain them under the microscope with antibodies for bcatenin and look for the intensity of the staining. You can see the greatest staining was found posteriorly and as you move towards the brain (#8), the amount of b-catenin drops off. Thus, b-catenin parallels Wnt signaling. The bottom line is that b-catenin and Wnt signaling are also very important in pattering neural development in the developing frog. Thus ends the data that supports the two-step model. Principle of Overlapping Mechanisms: Neuralization DeRobertis, who came up with the model of mesoderm induction, developed a frog that failed to produce mesoderm. So obviously this is a pretty altered frog that lacks mesoderm. What was curious about this frog is that it still developed a brain and a spinal chord. Now this is a little troublesome because we are saying that this mesoderm here is full of signals talking to the overlying ectoderm and instructing it to become neural. So without, mesoderm, you dont have the inhibitors of BMP and thus epidermal fates are promoted. So the big question became, what is going on in these frogs that lack mesoderm that allows them to build brain/spinal cord? This led to the discovery of a whole new region of the embryo BCNE Center Colored in the diagram in green. This unveiled a neat example of the principle of overlapping mechanisms in the story of neuralization. Meaning, there is more than one way of building a brain and spinal chord in a frog. The first way was that Spamanns organizer releases these BMP4 inhibitors that blocks the BMP4 and thus unveils the neural fate. But without these signals, there is another center that somehow makes up for the deficit. Blastula Chordin Noggin Expressing Center (BCNE) This is occurring before gastrulation occurs. It occurs in the blastula stage. There is a region right above the future dorsal lip (where the organizer will form), that expresses chordin and noggin (the same inhibitors of BMP4). Thus, these cells are pre-patterned to become neural. Work has shown that this center serves as the future brain of the frog. The first thing they did was sandwiching experiments. They took embryonic tissue pieces and sandwiched them together. There is something about taking an explant from an embryo and putting it against a partner piece that allows the two to talk to each other and promote some type of development that they alone could not accomplish.

So they took a sandwich from the BCNE (colored in red, DSW) and one from the ventral portion (colored in blue, VSW). When these grew in a dish, they obtained brain tissue from the BCNE sandwich. Likewise, they obtained epidermis from the ventral sandwich. Furthermore, if they excised the BCNE (top right experiment), which is seen in the part B, the developing tadpole lacked a brain. You could compliment this by grafting in a BCNE from another embryo. So here is the lacking of the BCNE and now we graft in a novel BCNE. This compliments and you form a brain. But if you graft it in from the ventral potion, you dont get complementation and thus no brain. The bottom left experiment, they marked the BCNE with a Lac-Z gene so they could trace it through the b-galactosidase production through development. So they graft it in and the BCNE now has a nice color marker. They followed this and the tadpole had a blue brain! This tissue, in normal development, specifies the brain. If something fails for the BCNE to work, there is a backup system to Spemanns organizer that secretes the BMP4 inhibitors and allows neural development to continue. So this is the model we believe operates. Currently, to build a brain in a frog incorporates two pathways that compliment each other in case one fails. March 11 Weve been talking about this phenomenon of how cells know what to become through the neighborhood in which they live. This is referring to those extrinsic factors, often chemicals, which are emitted by neighbors that then impinge and instruct other cells. Also known as Instructive Induction. Instructive Induction Two examples. These are whos your neighbor experiments where they co-cultured tissues of various combinations and asked the question, what happens? The first experiment was done with chickens. They took the tissue that underlies the developing skin of a chicken (dermal mesenchyme, colored pink). Then they took wing epidermal epithelium (ectoderm) as the target tissue and combined them. Some of the mesoderm would come from the wing, thigh, and foot. They would underlie the ectoderm with mesoderm of various sources: wing, thigh, and foot. They followed what types of feathers would develop on the ectoderm as a way to try to represent regionality of the fate of that ectoderm. We know that the feathers of a chicken (or any bird) are not uniform across the body. As you would expect, wing mesoderm + wing epidermis = wing feathers However, you could shift the feather identity by simply placing under it a different source of mesoderm. So with the neighborhood changing, apparently this ectoderm responds and shifts its own developmental progression and progresses thigh feathers consistent with thigh signals and scales/claws when foot mesoderm is used.

This is an example of instructive induction, where some cells are waiting to get the go ahead and information from surrounding cells in knowing what to become. There is one dramatic example of this has to do with tooth development. They tested the idea of whether or not birds have the capability of producing teeth. There is a chicken mutant in which they actually produce teeth. There is a mutation that creates a novel structure in the mouth of the bird. These teeth are like crocodile teeth (reptilian in nature). This shows that birds do have some ability to produce teeth. This second experiment, they try to awaken this ability to produce teeth in a chicken. This is done in vitro. They take oral chicken ectoderm (chick jaw epithelia) and underlie it with mouse molar mesenchyme (mesoderm). They co-cultured these. They found that they could grow teeth in the chicken. This picture is a tooth growing. March 11 Pattern Formation Pattern formation is going to be illustrated with five stories. Pattern Formation in Hydra Hydra is a small, aquatic organism. They have a very simple body plan. We are going to use hydra as our simplest story to try to illustrate the broad principles of pattern formation. Pattern Formation = how to achieve an ordered, spatial arrangement of cells. The process that builds a spatially organized arrangement. This is typically some part of our body. For example, how do we build the pattern of forming five digits and not six digits? How come our thumb is morphologically different than the other digits? Actually, all of the digits are slightly different in morphology. How is this pattern devised? The cells must know where they are and thus develop accordingly. When we imagine the analogy between these skydivers and cells, they have to know where they are spatially. The model we are going to look at first is the French Flag Model. This is going to be the fundamental model for driving all of our discussions on pattern formation. All of the five stories can be explained using this model. It was devised in Europe (hence French Flag) in the 1960s to try to explain embryonic pattern formation. It originally was birthed out of studies with chickens and we will study those experiments later but we can now apply it to many different context of how patterns develop in the human and animal world. So what is the model? Well first of all, we have to understand what the French Flag looks like. We have three stripes: blue, white, and red. Each of these taking up a third of the flag. What the model suggests is that each cell within a given area of a developing embryo knows its position by sensing a chemical called a morphogen. This chemical morphogen is found in a gradient that cells monitor and pick up their position by figuring out how much morphogen is present where they currently exist.

The flag represents a developmental field. This is a discrete area of an embryo with boundaries that is developing. For example, our hands, liver, etc. are specific developmental fields. These have discrete structure and boundaries. Every cell of the hand or developing liver knows where its at by sensing some kind of chemical informationthe morphogen. Just like each thread of the flag knows where its at based on its chemical morphogen concentration: blue vs white vs red. If we were to look across the flag, if we moved from left to right, we would notice that the morphogen concentration changes. The graph to the right represents this hypothetical morphogen. As you move from left to right, you move from high levels of morphogen to low levels of morphogen. Where you are on this continuum of morphogen concentration, communicates which color you should be. If you have high levels, you are instructed to be a blue thread. But if you drop below a certain threshold, you are not going to be blue. You have to be something else because you no longer qualify to be a blue thread. But you havent dropped low enough to drop into the red zone, so you become the intermediate phenotype of white thread. Then if you have very low levels, below the red threshold, you are instructed to be red. So as cells sense where they are and adopt appropriate colors, we build a flag. So every cell knows what they should become and as long as they are following the same guideline (the same morphogen gradient), they are then ordered accordingly towards the correct color to build the intended pattern. So morphogen gradients are going to be the key to pattern formation. We are going to unpack this throughout this packet. The Regenerative Powers of Hydra It was named after its regenerative capabilities. The capability of regeneration is illustrated in the hydra. This animal can regenerate any missing body part. If you chop off its head, it will regrow a head. If you chop off its foot, it will regrow its foot. Actually, you can throw this entire animal into a blender, chop it up, and take the mass of mixed up cells, and it will rebuild the entire animal again! The progression on the left shows this. You see 0.5 hour after blending. The cells are just clumped together and allowed to grow. 144 hours later, we have regenerated the head, body column, and a foot. Amazing For this to be pulled off, by the way there is no other animal that can do this, those cells must have positional information and must known how to reconstruct the entire body plan from scratch. Pattern Formation in Hydra Hydra follows a pretty basic body plan: head, body column, and foot. The body column is full of stem cells. It is only two cell layers thick. It has ectoderm and endoderm-like layers and no mesoderm. These cells never stop dividing as long as the animal is fed. As these cells divide they migrate away from their home in the body column and the move up or down. They either move into the head region (which consists of a mouth/hypostome and tentacles) or they move down into the bottom half where they form part of the foot or where they build a baby hydra (a form of asexual reproduction). This process NEVER STOPS!

This animal does not have a defined life span. Therefore, it is immortal in the sense that if you fed it, it will continue to survive. You can kill it, but it never experiences old age (senescence). It is perpetually producing cells. These cells arent always retained by the animal. Otherwise, the animal wouldnt stop growing and it would get bigger and bigger! It always stops at a certain mature size, but never stops dividing and migrating cells. How does this animal that is always producing new cells never get to big? Well, the cells that it doesnt need it either a) builds a baby hydra and are lost in the form of offspring or b) they just sluff off. When a cell ends up in the tentacle, it only stays there for four days and then it is lost. It is pushed off the end. Therefore, there is a conveyor belt of cells that are produced through mitosis, that are translocated to he tentacles, and eventually lost. Residence Time in Tentacle = 4 days before it is lost This raises the question, if we start off as a column cell through mitotic divisions and migrate to the head, how do these cells know they are know part of the tentacle? As the cells migrate upwards and enters the head, it must know its position and thus adopts a new pattern (in this case building a tentacle or building a hypostome). Or if you move downwards, how does it know it is now part of the foot/basal disk/etc. The question is, if this is the pattern and cells are always on the move figuring out where they are, how do they actually know where they are at? The simplest answer is a morphogen gradient. There is a morphogen gradient that goes head to toe and that cells pick up on and thus specialize and differentiate accordingly to that. The story we are about to unfold we have a fair amount of information but there are some big gaps in the data. So you might not be satisfied with the storyline At least this serves as an illustration to the point that somehow cells can pick up on positional signals by morphogen gradients. Pattern Formation in Hydra [French Flag Model Slide] Lets apply the French Flag model to the Hydra pattern formation. How does this model help us explain the ability to regenerate missing body parts? So we take a hydra and we chop it in half. So we have only half a hydra. Instead of the animal, we are going to envision the flag representing the hydra. Just like the hydra has an axis (head to toe axis), the flag also has an axis (blue to red). Obviously, after the bisection, whatever morphogen gradient was there, we now only have half of the gradient. The new wedge shows the partial gradient (partial flag). This animal now can reconstruct what is missing. This process is known as morphallaxis. Because it does not require any new cell division. So we are not growing a new head, we are taking the cells left behind and rearranging them to restore the head that is gone (with no increase in cell number). You can chop a hydra in half and irradiate it with x-rays which block mitosis and you will still grow back a head, even though there is no cell division. You end up with a smaller animal because you are starting with a smaller number of cells, but all of the body parts are intact. So we go back to a complete flag, albeit it is now a smaller flag than the original. How does this connect to the morphogen gradient? If we only have half a gradient, how come we can pull off a full gradient now where there is the blue and red threshold from just the partial gradient?

Gradient in Head Regeneration We now invoke a concept called positional value. This can be hard to understand but positional value is a state of being and it varies across the morphogen gradient. So the positional value of this thread here that made the white part of the flag exists in a different part of the gradient than a cell that encodes the red part of the flag. So they have different colors and thus different positions. This positional value is a pliable thing, we can change it as needed. So what happens is these cells that were only half way up the gradient, when you chop away part of the flag/animal, these cells are now relatively at the top. There positional value has therefore shifted because they are the highest point. Being the highest point, they shift their positional value to be that of blue. So what use to be down here, shifts and we create a new gradient and instructs all of the cells that are left behind, to adopt a new state. We can recreate blue, even though we didnt have the information for that by simply changing the positional value of this third of the left behind cells. Morphallaxis allows for positional information and where you are to be shifted around a bit. If you think about, it has to be that way because if you can blend up an animal and allow these cells to come together, they must have the ability to be flexible and figure out a new way to grow from their prior life! This is the idea, as you move through the head to the toe of the animal, there is this gradient of positional value that is colored in red. This is just a parameter trying tot describe your relative position in the whole scheme of things and it varies (as expected). It is influenced by a morphogen gradient (colored in green). In this case, there is a hypothesized chemical called a head inhibitor it is produced in the head (thus why it is highest in the head region). As you move down the body, it is found in a gradient with decreasing levels towards the foot. This head inhibitor prevents heads from forming. So it is normal for the animal to only have one head. As this head forms, it produces this inhibitor to make sure no other heads that choose to supplement the intended body plan and form a multi-headed animal. This is a control mechanism to control how many heads we have. Produce a Head Suppress other heads As you move down the body, if a head were to form, it would form farthest from the head and the head inhibitor. This would probably form a new baby hydra. They always form far from the parent head of the animal So lets say we decapitate our hydra. This is similar to cutting the flag in half. So we have a headless hydra and the source of the head inhibitor is now gone. So in order for this to work, the head is always producing this mysterious head inhibitor. This is always happening and this inhibitor breaks down and must be replenished by the presence of the head. So this chemical thus starts to decline and the decline is going to be greatest where the head use to be, because this is where the source was. The greatest change is going to be near the head. As the inhibitor levels drop, cells in this region sense that and use the head inhibitor as a cue for their positional value. As the inhibitor levels change, they adopt a new positional value. Furthermore, there is no head inhibitor so they commit to becoming new head (change their positional value). So the positional value near this end adopts head-ness and begins producing

head inhibitor again. And we have now restored the gradients. However, the animal is 2/3 the size since we didnt undergo mitosis. We have just taken these remaining cells and rearranged them to reproduce the missing head. Based on relative concentrations of morphogens. In this case, it is hypothesized that this inhibitor is a head inhibitor in the hydra. We are predicting that there is some type of gradient that exist across the body of Hyrda and it originates from the head. Morphogens are chemicals that communicate positional information. Thus, if you sense the head inhibitor, you are not going to become a head. But if you dont have much head inhibitor, then maybe you will become head Pattern Formation in Hydra There are actually two gradients of two different chemicals morphogens. Lets so a little example where we do a grafting experiment. They chop off part of the body column (cells waiting to get instruction on what to become: head or foot). These are in that stem-like cell part of the animal where they are produced but not fated towards head or foot, yet. You can graft this into a host hydra. You can also color these cells to track them. What happens to the grafted hydra? Will it build a head? 20% in this experiment Head forms 80% in this experiment No head forms

However, if you removed the host head, the majority of these grafts would indeed form a new secondary head. So with head loss of the host, it promotes over 4 times the frequency of building a secondary head. So this suggests that indeed the head is producing some chemical that is suppressing additional head. If you remove the head, you get an additional head. However, is this found in a gradient? Because we are saying that this head inhibitor is positional information in an unequal concentration down the body axis. So we perform the same experiment, and we leave the head on, but we graft the colored piece of donor hydra at different positions along the body column. Some of the grafts are close to the head, some of are midway, and some are close to the foot. The data shows that the farther you are away from the head, the better your chances are as a graft at forming a new secondary head. This thus reflects the head inhibitor gradient. As you move from the head to the base, the amount of inhibitor decreases and success increases! They showed if you inhibit gap junction function, the gradient no longer functions. Gap junctions form conduits for cell-to-cell movement of material. In this case, whatever this inhibitor is, it uses gap junctions to move along the body axis. It is hypothesized to be a small peptide. This seems to be chemically what the inhibitor is. This hasnt been pinned down tightly in hydra They also discovered, it also matters where you took the donor graft in the success of forming a secondary head later. So in this case, we always took the donor piece from the midsection. What if we took the donor from near the head or near the foot?

The graph on the right shows this data. The closer the donor grafts from the head, the higher percentage of head formation, and the greater the success. This seems counter intuitive because we are thinking what if I take part of the this piece close to the donor head, so it should be loaded with head inhibitor, but it somehow does a really good job at producing head when grafted into the same spot in a host hydra. Well, two assumptions: 1) The head inhibitor is short-lived. When grafts are removed from the inhibitor source, it breaks down Thus it has a very short half-life. So just because it started from a high inhibitor region, it doesnt keep that head inhibitor region. So it is now under the mercy now of some other kind of influence which we are calling a head activator. This is also produced by the head, but is much more long lived than the head inhibitor! So if you are going to become a head, particularly an extra head, the two informational signals are morphogens called head inhibitor and head activator. They are both found in gradients with large amounts at the head. Depending on where you are in the body column, the amounts of these chemicals vary. This determines whether or not you are producing a head or not. We will revisit this in more detail. Hydra self-organization: Using aggregates to show organizer action Does Hyrda have an organizer? Remember, the dorsal lip in the frog can organize the entire body plan? Hensen node is similar in chickens. Does Hydra have an organizer? Yes it does! They did an experiment in which they did the blended hydra protocol. In this case they took a red hydra and blended it up and obtained a cluster of cells. They implant in this cluster pieces of the head, specifically the hypostome (the oral disc region right in the middle). They watched to see what happens! In this particular example, the mass of red cells have three hypostomes and every hypostome organizes its own body axis. So you end up with three hydras all joined together, because they came from the same mass of cells. Each body was formed from its own hypostome. So the organizer is the hypostome. Functionally, it is the equivalent of the dorsal lip in the frog. Now you had to have a certain amount of cells of that hypostome in order to get the organizing effect. This is what the graph shows. It had to be around 5-15 cells (60m) needed for head activation. This is an example of a two headed, aggregate hydra. You can see the two body axis and the hypostome cells that were creating a body axis surrounded by little tentacles. Indeeed, hydra has an organizing region The Hypostome When they stained it for gene expression, they found that these regions express Wnt gene expression. Just like Wnts are important, b-catenin, etc. in organizing frog mesoderm. B-catenin and Wnt signaling we would predict to play important roles in the hydra. Head Organizer and Wnt Signaling

We can actually do experiments surrounding b-catenin and Wnt signaling. We can chemically inhibit GSK-3 (that target of degrading b-catenin) and watch what happens in the hydra. So these are hydras that are bathed in a chemical (ALP) that inhibits GSK-3. These animals develop a bottle brush phenotype instead of having only tentacles up at the top, you get dozens of them lining the body column. At the base of each little tentacle, is Wnt gene expression. Its almost like they are trying to build little heads. If this is true, if by blocking GSK-3, we are getting all of these Wnts, we would expect b-catenin levels to be elevated. Because this is an outcome of preserving Wnt signaling and blocking GSK-3. So this shows the staining of b-catenin under the influence of inhibiting GSK-3. So they used different levels of ALP (inhibits GSK-3) and the more ALP the more b-catenin survived (stained green). These are also associated with organizer development. The bottom figure shows a grafting experiment that shows its association with organizer development. They treated the hyrda with ALP and cut out some of the column and grafted it into a headless host hydra that was colored black. If they just didnt use a bottlebrush hydra, they never found a secondary axis (CONTROL). However, if they used the bottle brush with the elevated b-catenin, 100% of the grafts formed a secondary body head. This shows the organizer ability. Where that when that little piece is grafted on doesnt make the head, instead it directs the formation of the head from the black surrounding column cells. So the little graft is white, corresponding to the donor tissue, and the rest of the developing head is black and derived from the host body column. This is just evidence that as we build a head, b-catenin and Wnt signaling are part of that organizing process. So this helps generate these gradients of head activator and head inhibitor. Transgenic Hydra The ultimate of this is to make a transgenic hydra in which almost every cell produce b-catenin. This case they put b-catenin coding region under the promoter for b-actin. So wherever a cell made actin, the cells also made b-catenin. So we asked, what happens when you have systemic bcatenin? Well, since b-catenin functions in organizing, you end up with a big jumbled mess. You have all sorts of organizing activity within the animal, each trying to build its own body. Chaos! All of this tissue loaded with b-catenin, when grafted into a host hydra, acts as an organizer and you get lots of secondary body axes forming. March 13 Just to recap what weve been studying, Hyrda is an animal that has no defined lifespan. It seems to live until killed or starvation. The reason for this is due to stem-like cells in the body column. As these cells divide, they migrate and populate the body. As cells die, cells are constantly being replenished. We have upward and downward movement of cells. The challenge is that these cells start out undifferentiated and as they change their location, they need positional information to realize their position along the body axis. The answer to this is due to morphogens which are found in gradients along the body.

As a cell, you sense a chemical compound at a certain concentration and you translate that into positional and output of what you are going to become. The story we are talking about is that at the head end. A similar story does occur at the foot end but we know more about the head. So, weve looked at data where we graft body parts and we noticed that head formation is influenced by both an activator and an inhibitor. These both come from the head and establish a gradient with lowest levels near the foot. So where and what a cell becomes is influenced by these two chemicals. We think at least one of them is a small peptide sequence that is sensed. Otherwise, we have no clue what the chemicals actually are. When we look at the head, as a cell moves up there is actually two options that it has in terms of body parts. It can become a tentacle or part of the hypostome. If you remember, the hypostome is of particular importance because it has the ability to function as the organizer! So it is extra special in the sense that it has the ability to organize the entire body plane if given the cells to direct. So when we analyze head development, we are going to separate hypostome and tentacle development. So if we look at head activator which is HA, we can break that down into Hypostome Activator (because if that cell is going to be part of the hypostome it needs a different signal than one destined for tentacle). We also have a tentacle activator (a different chemical). These two cannot coexist in the same location because they are physically different locations. The hypostome activator actually has a suppressive action on tentacle activator in the model. There are also inhibitory influence interplaying on cells. So if a cell passes up into the head region, it not only senses these activators but also senses the inhibitors. There is a corresponding hypostome inhibitor and tentacle inhibitor. So depending on which is strongest determines which fate these head-migrating cells become. Pattern Formation in Hydra They have discovered a gene that seems to represent this. The gene is HyAlx and was discovered at the University of California (2000). It was a gene that seems to represent tentacle activation. As cells move into the tentacle and become part of the tentacle, you would expect tentacle activator to be doing its work. This is by definition. This gene is an aristaless-related gene. However, there is a gene in fruit flies called Aristaless, this is how the gene above was named. So we call it the Hydra Aristaless Gene (HyAlx). This gene is what we are going to be looking at and as we go through the data, equate this gene with an indicator of tentacle activation. So as cells are moving up the body column, they pass through what is called the tentacle zone/tentacle border. Once they pass this border, they are now committed to becoming part of the tentacle. So as cells move through this passageway, they commit to becoming tentacle and this is where the gene is expressed. The image on the left is a hydra body and a gene probe is used for identifying locations of HyAlx. Notice, the based of every tentacle shows gene expression. So cells that are about to enter the tentacle turn this gene on. We think this is a signature for the tentacle formation.

Now if this were true, we should be able to test this. This could just be a coincidence that if the gene is turned on in this spot. So loss of function experiments were performed and this showed that when this gene was suppressed, tentacle formation was also suppressed. So, lost of HyAlx compromises tentacle formation. Therefore, indicative of a causality between HyAlx being active at the base of each tentacle. Pattern Formation in Hydra [regeneration] They also studied this gene during regeneration. So they chopped off the head of the hydra. Notice the stump of the hydra at 20 hours after decapitation. You can see the HyAlx gene expression all throughout the stump. As the tentacles start to form, they are preceded by a concentration/localization of HyAlx which ultimately becomes ring-like as the tentacle emerges from the ring-like area of expression. Notice, two days after decapitation, the head is back to normal. So the time course of gene expression is consistent with a contributing with tentacle formation. In sequencing this gene, it turns out to contain a homeodomain which is a portion of a protein that allows it to bind to DNA and regulate genes. These are signatures of proteins that regulate other genes. Thus, we expect the HyAlx gene to controls a whole network of tentacle specifying genes. So the data suggests that this gene is important in the formation of tentacles, and thus is an indicator of tentacle activation. Pattern Formation in Hydra [Reaction Diffusion Model] Now, a model has been put together that tries to explain the interplay of all of these players. This model is based on a general model called the Reaction Diffusion Model. This model applies to many different situations in developing animals. It has been used to try to explain zebra stripes and stripes on fish and some of the intricate patterns on peacock feathers. The question becomes, how do these patterns emerge? The model tries to explain the interplay of two substances: One is inhibitory and one is stimulatory. So we are about to look at this model as it applies to hydra because it accounts for the two players that play a role in hydra development. Realize that this model can be applied in many different ways. It is a mathematical model and the graphs we are looking at are computer generated using modeling software. It helps to try to show that what we see in nature can be replicated using a mathematical model. So in order for this model to work, there must be some parameters. These are underlying assumptions as to how things interact and behave. The green represents the activator and we can equate it to the head activator in the hydra application. Remember: the head activator in hydra has two manifestations: hypostome and tentacle. Likewise, there is an inhibitor colored in red. Being a head inhibitor, it is also going to describe tentacle and hypostome inhibition. Each of these rectangles represents a field of cells in the body. In this case, pretend the hydra that just lost its head, we are looking at the stump. So this table represents the cut end of the stump where the head use to be. On this stump, are all sorts of cells that are spread out in 2-D. These cells now are going to interface with each other as they produce the activator and inhibitor. So the

center of the stump is going to behave a bit differently than then surrounding area based on the modeling. Assumptions: 1. This activator has the ability to produce more activator through autocatalysis. What this means is that when cells produce activator, the product of that can cause the same cell to make activator. This is an example of positive feedback. So here in the middle, if we have cells that are making activator, more activator is going to be made in the middle and it piles up. The amount of activator being made is represented by the height of these surfaces. So in the middle, we have a hotspot of activator production. 2. This activator doesnt diffuse very fast. So even though it is being made here, it has the opportunity to move down a concentration gradient, it doesnt do that very quickly. So it tends to pile up. This is why over time, you see this peak getting higher and higher 3. This activator actually stimulates the inhibitor to be produced by cells. This inhibitor quickly moves away from its site of production and moves down its concentration gradient. So we would expect the inhibitor under the influence of the activator is also being made in the same spot, but it doesnt stay there Thus the height of the inhibitor peak isnt as tall as the activator (green peak). 4. The inhibitor diffuses very quickly. So we have a green cone representing the influence of activator on the cells and surrounding it is more of a foothills of inhibitor, which based on its name, inhibits the activator. So even though the activator is slow in diffusing, what little does escape the center zone and out on the periphery, is quickly overwhelmed by the inhibitor. So what this means is that you follow it over time and the activator peak gets taller and taller and the sharper because of the interplay of these two factors. Pattern Formation in Hydra [Reaction Diffusion Model Part 2] Now we are going to apply this to the hydra more specifically. It shows the interplay of the activators. This playing field of sorts represents the table, stump, or region of hyrda body. And through time, we can see the building up of these peaks. So in the end, you end up with these peaks representing localized regions of activator. There is another factor called Source Density. This is a third component that interplays with the web of influences. By definition, it is the capability to produce a head. At the head end of the Hydra, the Source Density would be the highest. So SD since promotes head, it is a positive influence in terms of wanting to make a tentacle or hypostome. SD Positive Influence So we have a SDH that influences the Hypostome and a SDT that influences the Tentacles The influences can be separated based on the regions of the head. Just like we separated the inhibitor. So we have a SD that influences the hypostome and we have a SD that influences the tentacle. 4 Elements of the Model: 1) All four elements explain the observed gene expression and 2) HyAlx and these seem to explain how the head grows.

1. Hypostome activation (HA) increases when SDH is greater than Hypostome inhibition (HI). So if the positive influence outweighs the negative influence, this activator is going to be promoted to do its job. If this is the case, if the hypostome activator is allowed to work we build a hypostome. So if we look at the hypostome activator table, wherever HA is active we are going to build a hypostome. We can follow this through the on the purple table.

2. What about the tentacle activator? There are two actual criteria for tentacle activation to do its job. The SD has to override the inhibitory action of TI. In addition, there has to be a threshold. There has to be an absolute amount of SD that has to be high enough to kick this whole thing off. Not only does SDT > TI, but this has to be above a certain threshold. 3. HA inhibits TA 4. TI is less stable than hypostome inhibition. When you cut off the head, the inhibitor is broken down, it has a short half life and doesnt stick around very long. In fact when we compare the two inhibitors, the tentacle inhibitor is less table than the HI. So when you lose the head, this inhibitory influence is lost first and later the HI influence is lost. Okay, lets imagine we chop off the head of a hydra. We are asking what happens in this stump that is left behind? Well since it is high up in the body, this is a region of high source density (the ability to produce head). This is represented by the hill. So instead of being just flat, this region of the body has a fair amount of SD. This SD is greater than the threshold required to build a tentacle. So one of the two criteria is already met for restoring tentacle. That is we have gone above the threshold needed to make a tentacle for the source density.

We also need to make sure the SD is greater than the inhibitor (TI). Luckily, since we chopped off the head, the source of the TI is gone. Therefore, TI is lost quite quickly. So now the TI is lower than the SD. When SD > TI, you have now meet the requirements for building a tentacle and TA launches. We said that TA is linked with activation of HyAlx gene expression. So we predict that based on these two criteria that this gene is going to be expressed right at the cut edge of our stump and this is exactly what we see. So this gene is faithful to the model. In

reality, the model was built to explain the data. Notice what happens as we follow it over time, there is a restriction in the region of the future hypostome and it is restricted to where the tentacles are found. Notice that over time, as the head is gone, the hypostome inhibitor drops, because it has a limited lifespan and it becomes low enough at a point where SDH > HI. Thus, this causes a rise in HA. Now we said the hypostome is an organizer. The organizer has the ability to form the body axis; in this case the head is what we are focusing on. As HA increases and the hypostome starts to be replaced, SDH goes up because of the organizer activity of the hypostome that is being restored. Unfortunately, wherever hypostome activation (HA) is tentacles cannot form! So even though this starts off broadly, it begins to suppress tentacle activation in the middle and in the end, you end up with surrounding peaks of TA (each one for a tentacle) surrounding the central peak of hypostome activation (HA). Overtime, we go from a broad expression to a restricted tentacle activator expression. This is showing in a model what this would look like. As you look at the models, each color represents a different influence in the story. The rectangles try to represent the cells that are going to experience the particular influence (HA, TA, SD). Hypostome forms in the middle of the stump and tentacles form around the central hypostome. Pattern Formation in Hydra [Reaction Diffusion Model Part 3]

Lets now apply the model to the other end of the animal. What we just looked at was chopping the head off, so we were high in the body and had high SD (the ability to form a head). What if we move down towards the foot. We said down here in the bottom fourth of the animal, little buds develop that grow into baby hydras. This area is just the opposite in terms of SD in respect to the head region. It turns out, by definition, the budding zone has a SD that is higher than the HI. What this means is that the criteria for element #1 of the model are already met! So even though SD is low (the peak in green is not as big), but because the conditions for HA are met, we see the hypostome activation starts. Nothing happens with the tentacles, because the tentacle criteria have not been met yet. As the hypostome grows, it elevates the SD (remember, the hypostome is an organizer and it has the ability change SD). As SD goes up, the conditions for building tentacles kick in and grow around the hypostome. And you end up with the same results you saw above. In this case, hypostome development preceeded tentacle development. This is exactly opposite to the first case. It all has to do with the interplay of source density with the model.

So in the end you end up with one hypostome and four tentacles surrounding it and a nice SD in the head Makes sense ??? So the HyAlx gene has given us a clue in trying to understand the interplay of all of the forces that are involved in influencing how the head forms.

Hyrda species differ in the order that tentacles arise on a new bud: The HyAlx gene has been looked at in a totally different context and reinforces the role of this gene in tentacle formation. This is a study done in Europe where they studied different species of Hydra. Hydra is very interesting. Some of them contain photosynthetic algae in the cells. Thus, it is a green animal. It is driven by photosynthesis! But there are different species of Hydra and some species grow their tentacles in a different timing pattern than others. Some species have what is called synchronous tentacle formation (H. vulgaris AEP). These tentacles grow all at the same time. So every tentacle is the same length because they all emerge at the same time and grow in unison. However, H. oligactis shows two tentacles get a head start. So two emerge and then two more start to grow. So you end up with two long ones and two short ones. This is asynchronous. They asked a question (which btw, this tentacle pattern timing is a species characteristic), what was the basis for this. They did a big screening of the genome of Hydra and they did gene searches. They found a unique gene called HYM301. They took this gene from Oligactis and inserted it into Vulgaris. So you can see wild-type vulgaris (synchronous) and we take the gene and express it transgenically and we can see the it transforms it into the asynchronous phenotype of Oligactis.

So this HYM301 gene has the power to drive the timing of tentacle emergence and it seems to be the basis for species differentiation in these two Hydra. So this HYM301 gene must have some influence on tentacle formation (a timing influence). If this is the case, if HyAlx is important in tentacle formation, does HyAlx expression change when you alter the timing of tentacle development through this introduction of the HYM301 gene. The answer is yes! Here we are with a couple of decapitated Hydras. They are needing to regrow their tentacles. We are noticing the wild-type (synchronous) and we have uniform expression. We also see the transgenic one which is expressing asynchronous formation. You can see an increase in HyAlx gene expression So changing tentacle timing and its formation also changes HyAlx gene expression. Thus, HyAlx and tentacle formation go hand-in-hand. This is the story of Hydra, morphogens and how their concentrations vary across the body of the animal, giving positional information, and we have at least the story for Hydra, a marker that helps us follow these morphogens.