 Hello everyone. I think it's about time to get going. Today we have with us Dr. Furman, originally from Germany, but now here with us today. And she's going to be talking to us about extracellular signals regulating eye development. Thank you very much for having me here today. I gave my last Grand Rounds four years ago, so I think we have some nice interesting new data, so I thought this is a good time to jump in and on a short notice and talk about it. Tell you a little bit about it. My lab is, I'm an associate professor in the department and I'm a researcher. My lab is located on the third floor here in the South Tower and you're very welcome to come anytime and talk to me if you have questions or yeah. And so my laboratory is interested in the role of signaling molecules during eye development. And we are specifically interested in how extracellular signals during early eye development mediate tissue-tissue interaction to ensure proper eye development. And we have a major project in the lab right now in which we specifically test the role of extracellular factors in development of the pigment epithelium, which is schematically shown here. So you know very well how important the retinal pigmented epithelium is. I will abbreviate it as RPE in the future throughout the talk. So our goal is to understand the cellular and molecular mechanism that regulate different stages of RPE development. And since there's not much, since we know quite a bit about the functions of the RPE in the adult eye, we, so we know quite a bit of adult function of the RPE or function in the adult eye, we don't know so much about development of the RPE itself. And that's the focus of my lab. So let me tell you a little bit about RPE development in general, what we know so far. So during early eye development, the first visible sign of eye development in the developing embryo is the evagination of the neuroepithelium of the ventral diencephalon. This is during early forebrain development and the eye is one of the first visible evaginations of the brain. So in the human embryos, you can see the eye about four weeks of gestation. And the evagination of the ventral diencephalon leads to the formation of the so-called optic vesicle. And at this stage, the optic vesicle is by potential. So you can see that the neuroepithelium is very homogeneous and it's one layer of cells that have this columnar epithelium that is typical for the neuroepithelium at this stage. And the whole domain of the optic vesicle can develop either into the retina or the pigmented epithelium. Subsequently, and I've depicted this in this funny pattern here in the green and brown kind of stripes. So green shows later on where the RPE will develop and the sand color will show is showing where the retina is developing. So subsequently what happens is that the distal part of the optic vesicle contacts the overlying surface actodon which is shown here in red. And this interaction is very important for subsequent evagination of this part. So this leads to the formation of the so-called optic cup and the inner layer of the optic cup or the former distal part of the optic vesicle invaginates. And you can see here at this later stage a very nice optic cup forming. And the overlying epidermis or so-called lens actoderm also invaginates, forms the vesicle and then subsequently pinches off the surface actoderm. So interestingly, both retina and RPE, so like I said, the RPE is developing here in this green domain, have a common embryonic origin. And interestingly, the RPE develops into a non-neuronal tissue subsequently. So what we know at this early stage is that there are two, at least two genes that are expressed throughout the optic vesicle that are very important during RPE development. So one of them is OTX2, which is a transcription factor. It's called orthodentical homeobox gene 2. It was originally identified in drosophila. And the other one is MITF, mycothoma is the associated transcription factor. And these genes have been shown to trans-activation pigmentation genes, so they upregulate every, the whole pigmentation machinery in the developing eye. In humans, mutations in OTX2 can cause ocular malformations that range from bilateral anothomias to retinal defects. So there's a whole diversity of malformations detectable. And in mouse, loss of function of MITF or OTX2 can result in mycothomia and or trans-differentiation. And I will come to that back later, what trans-differentiation means. So initially, these two genes are expressed throughout the whole optic vesicle. And one of the questions that we have is how is expression of MITF, for example, upregulated in the optic vesicle? Because during early embryonic development, you have overlapping gene expression. And there's actually not clear how these genes are, like MITF specifically upregulated. The expression is specifically upregulated just in this region. And a good mechanism by which this could happen is that adjacent tissues to the optic vesicle upregulate secretive signal and upregulate expression of specific genes like MITF here. And the tissue, the tissue that is a really good, could be a really good producer of the signal is the extraocular mesenchym. I'm pointing here with the arrow, red arrow to this tissue. This is just a loose accumulation of cells that migrate out from the dorsal neural tube during embryonic development. And then they migrate into the facial areas and also downwards ventrally to populate the new epithelium. And in the brain, for example, this tissue is very important for forming the meninges. And in the eye region, these cells, the extraocular mesenchym, which is derived from neurocrest and from mesodermis, important for formation of the sclera, the coreate, extraocular muscles, the corneal stroma, for example, so they migrate, surround the eye, and then later differentiate and migrate further into specific locations. And it's actually not very well understood how this is regulated to. So we think that this extraocular mesenchym, very early during eye development, plays a role in induction of RPE formation, or inducing the RPE fate in the optic vesicle. And why I think this is shown in this picture, which shows a very early section of a mouse embryo through the forebrain region. And here on this side, you can see the evaginating optic vesicle, which is shown in bluish tissue here. And it's surrounded by the mesenchym, which is labeled here in green. And it's labeled specifically for a marker at this time, AP2 alpha. And you can see that the whole optic vesicle is surrounded by mesenchymal cells. And this is really early during eye development in the mouse. So this is just when it starts. The mouse mice embryos are born after 19 days. So this is half of the embryonic time. And we can see expression of retina specific genes about a half day, three quarters of a day later. So this is way earlier before retina development actually starts. So we looked at expression at this early time point of RPE specific genes, in this case MITF. And what we found is that indeed MITF is very early expressed. And this has been not reported before. So you could just, you can extrapolate a little bit and hypothesize that at this stage, the RPE fate may be actually the default fate during eye development. And then that retina promoting signals come later and then use retina development. So to further test the role of the extractural mesenkind, we established an ex plant culture system in mouse and also in chick. I will come to that in a few minutes. So this shows a whole mouse embryo at E9.5. And you can see, so after half of the embryonic time, and you can see here the, the evaginating optic vesicles. This is the forebrain region here. And what we did is we dissected out the optic vesicles and cultured them in the presence or absence of these extracular tissues. We can enzymatically treat these tissues and then remove some, some components like the overlying surface ectoderm here. And in this case, we, which is shown here. And in this case, we also removed the extracular mesenkind shown here in green. And when we culture these optic vesicles for a couple of days and then look at the expression of eye specific genes, in this case, MITF again, we can see that in the presence of mesenkind, this RPE marker is strongly expressed. But when we remove the mesenkind, in most of the cases, MITF is not expressed. So the extracular mesenkind is really important for upregulation or at least expression of this RPE specific gene. And this here shows normal in vivo expression of MITF in the mouse embryo. So what we wanted to know also, what we are wanting to know right now is what kind of signal does the extracular mesenkind secrete to upregulate MITF expression. And we know a little bit, we have an idea from CHIC experiments where these experiments are a little bit easier. And we've started actually out doing this in CHIC and then we moved into mouse. So what we found here again is MITF expression in the optic vesicle in CHIC. And there's a little difference in CHIC which is interesting from the evolutionary standpoint maybe. In CHIC, MITF is expressed only in the future RPE domain. It's never expressed in the whole optic vesicle. So when we similarly conduct ex plant cultures with mesenkind, we can see robust MITF expression here in the ex plants with mesenkind. But when the mesenkind is removed, most of the ex plants don't express MITF. And we can rescue this defect by the addition of certain factors. And in this case, it's a TGF beta family member called active in. And this effect is very specific because others TGF beta family members like certain BMPs that were good candidate molecules don't have this effect. So this is a very strong and robust effect. And so this shows that the tissue is responsive to addition of this factor. And we have now more new data showing that it's actually required. So what we did to test this is we took this condition ex plants with mesenkind and added a molecule that inhibits specifically activation of this pathway. And this was indeed the case. So we could really, this kind of shows more biological and dodginess relevance for this pathway in RPE formation. And we also use this molecule to code beads shown here in blue with this code beads. And these beads, you have to soak these beads in this protein. It takes it up and then you can implant it into the tissue or close to your tissue of interest. And then slowly these proteins get released from the bead and they diffuse into the surrounding tissues and exert their effects. And this is very common in embryological experiments, especially in chick for example. So in control embryos in which the bead was coated with a control molecule, MITF expression is very robust and continues in the developing chick eye. But when this TGF beta or active inhibitor was introduced, you can see here that this eye shows a local down regulation of MITF expression. So this shows that really in vivo, endogenously, the TGF beta pathway plays a role in at least promoting MITF expression. And we think that this is really the first step during eye development and inducing RPE formation. So I want to go to the next developmental step. I've told you about our induction, developmental biology, you talk about induction of tissue and organ. So the next step would be how is the RPE domain or how is the RPE fate in the developing eye maintained? And I want to come back here to the concept of transdifferentiation that I mentioned before. So even though MITF, this RPE specific gene is induced and other genes like OTX2 are also necessary at this early time point, we know and pigmentation can even start. But under certain conditions, manipulations or defects, the RPE can lose its pigment specific character. So it can lose pigmentation, down regulate pigment specific genes and actually adopt a different fate. So it can develop into a second neural retina. And so this suggests, strongly suggests that there must be additional mechanism to maintain the RPE fate in the optic cup. And we are interested in these signals too, because this is important for congenital defects and also for stem cell biology. So one really good candidate pathway is the TGF beta, the wind beta ketinin pathway. So wind are secreted glycoproteins that are actually activating at least three different pathways. And one of the best characterized pathways is the wind beta ketinin pathway or so-called canonical pathway. So the winds bind to so-called frizzled receptors and LRP co-receptors. And this leads to the sequestation of a destruction complex, which is shown here. This destruction complex contains axon, APC, GSQ3, beta. So this destruction complex is pulled away from the central key player in this pathway beta ketinin. So if winds don't bind to the receptor, beta ketinin is continuously produced and degraded by this, which is mediated by this destruction complex. And when winds bind to the receptor, beta ketinin is stabilized in the cytoplasm and can translocate into the nucleus, bind to TCF left transcription factors and activate transcription of downstream target genes. So what is really nice, several groups have generated so-called reporter mice. So you can use basically a DNA sequence that activates these target genes here and connect it to a so-called reporter. So you can use fluorescence reporters, or in this case, for example, Lexi, which encodes for beta-galactosidase. And then you can do a nice color staining. And every cell that turns blue has activation of the canonical pathway. And what we found is that early during development, so this is the optic vesicle stage here in mouse, a cross-section at the optic vesicle stage. And you can see activation of the reporter in the dorsal optic vesicle, but not at earlier stages. And the RPE domain would be also a little bit more here in this direction. So we start seeing it here, and this is when MITF is already expressed. So it's not important, this pathway is not important for up-regulating MITF, but we think it's important for maintenance of MITF and OTX2 expression, or maintenance of the RPE fate. At later stages here, this is a part of the eye. Here's the lens. Here's the retina. And there's strong reporter expression in the developing RPE. This is a few days before birth. So in order to test this, the role of the wind beta-catenin pathway during RPE development, a former graduate student in my lab, Peter Vestenskow, specifically disrupted beta-catenin in the developing eye, starting at this stage. And so this is shown in this picture here. You can see a mouse optic cup little two days later at 11.5, and the red color shows an expression of a retina-specific gene, in this case V6-2. And the green color again shows expression of an MITF, in this case, of an RPE marker. And in the mouse mutants that have this pathway inactivated, you can see that the green color in the dozzle RPE is disappearing, and actually the red color is coming up. So that means that RPE-specific gene expression, and we've shown this for other RPE-specific genes too, RPE-specific gene expression disappears, and retina-specific gene expression is upregulated. And yeah, we've tested several different markers. So this is a typical example for trans-differentiation. And what is happening also is what I forgot to mention before, besides down-regulation of pigment-specific genes, also the mutant RPE starts to proliferate quite a bit. It actually really makes a second retina. It gets as thick as the second retina. So all these later differentiation markers in the retina get robustly upregulated. It's amazing how this tissue can develop into a second retina. So we were wondering whether MITF could be directly regulated by this pathway. This has never been shown that these RPE markers, like MITF or maybe even OTX2, can directly activate it by a signaling pathway. And so what Peter did is he looked on the molecular or DNA level. So in this case, the upper picture shows a gel in which he loaded samples that were treated in the following way. So he took RPE from a very early stage, grinded it up, and isolated the DNA in a way so that all the associated proteins are still bound to the DNA. So you have to be very quick. One researcher who does this very often says that the proteins actually can fall off if you're not quick enough and doing this essay quick enough. So you have to make sure that you want to see what is bound to the DNA. And so you have to sample the DNA with the associated proteins. And then the DNA is chopped up in little pieces so that it's manageable. And then you do chromatin, then you do immunoprecipitation. So you want to test whether, for example, TCFLEF, which are the transcription factors that bind to the RPE-specific genes or the promoter of these genes, if they're really bound. So you take an antibody against these transcription factors and pull out those DNA pieces together with the proteins where these transcription factors are bound. So you get an enrichment of these specific regions. And then the good thing is that we know usually what the sequence, we can estimate where these transcription factors are bound. We know the sequence and we can PCR and enrich these DNA regions. And this is shown here. So this was actually precipitated not with TCFLEF antibodies, but with beta-ketinan antibodies, which is even better because it shows that beta-ketinan binds to TCFLEF and TCFLEF binds to the MITFD promoter in this case here. So we have, we, Peter identified three different regions in the MITFD promoter that have TCFLEF, beta-ketinan bound to them. So that means in vivo, this is actually, the pathway can really activate MITF transcription of this gene or can at least bind to it. And then you have to do another test to see whether this interaction is actually an activating action. So you do a Luciferase assay where this piece of the MITFD promoter is again connected to a reporter. And then you transfect cells and like HEC293 cells and you co-transfect your modulators of interest. So in this case, beta-ketinan for example. And you can see that this reporter is strongly up-regulated when beta-ketinan is also introduced in the cells. And this is not happening when you add, in addition, a repressor of this pathway. And this is one very good control. What Peter also did is he mutated in this promoter region the binding sites and then looked what happens in this reporter assay. And you can also see that this doesn't lead to a significant up-regulation. So these are assays that we use to show a direct molecular interaction between a signaling pathway and downstream factors that are important for ocular development, in this case, RPE. So this is a little summary for this first part. So I told you about what we think about RPE induction, that this active in light molecule is produced in the extracellular mesenchyme. Right now we are testing in mouse whether the intracellular machinery of this pathway is required for RPE development. So we're doing tissue-specific inactivation of these transcription factors that mediate activation of this pathway. And this is not so easy right now. We think that maybe this early activation of this TGF beta pathway, or active in pathway, is important maybe for, could be important for up-regulating wind ligands in the RPE. And those, in turn, ensure or make sure that the RPE fate in the developing eye is stabilized by activation of the wind beta-catenin pathway. So I've shown that, hopefully, convincingly, that beta-catenin, TCF-LEF, directly transactivate the MITF, and we've shown this also for OTX-2 genes. So what we also think and what we're testing right now at later stages, we can see that the wind reporter is still active in the RPE, and at one point it gets down-regulated. So we want to test at later stages whether this pathway is required for RPE differentiation to up-regulate, for example, all these functional genes that are important in the adult eye, so RPE, like phagocytosis and epithelial transport, for example. So one question that we also have, and that leads into the second part, is where do the winds come from? Are the winds expressed that ensure maintenance of the RPE? Are they expressed in the mesenchymide? That is also possible. They can express, like I mentioned before, in the RPE or in the retina also. So they can be secreted by the neighboring tissues, and then by diffusion they activate this pathway in the RPE. And we started a new project in the lab in which we can affect all the wind production and different tissues specifically. So this shows actually what is happening to wind ligands in the producing cells. So far I've told you what is happening in the effector cell or the cells that receive wind signaling, and this is what is necessary for winds to be secreted. And so winds are modified, so they are produced in the ER or they are transported through the endoplasmatic reticulum and this purple structure here shows the different transmembrane proteins of this transmembrane domains of this protein, which is called porcupine. This is an enzyme that is located in the membrane of the endoplasmatic reticulum and it's an as a transferase that attaches lipid moieties to the wind molecule. And this happens in the endoplasmatic reticulum. This lipid modification or palmatulation is really, really important for secretion of winds. People have thought it's also important for activation of the pathway in the receiving cell, but that doesn't seem to be the case. So it seems to be really important for subsequent secretion. So there's more happening in the Golgi apparatus with other components of this pathway. So and this prerequisite, which is crucial is this lipid modification of winds in the endoplasmatic reticulum. So the other thing is that this enzyme porcupine seems to be very specific for wind signaling and it's assumed that all winds are affected, not only the ones that activate the canonical pathway, but all other pathways too. So this was very interesting for us. It's kind of, it's a more global approach, but it could show us, first of all, where are winds from which tissue they are important and second what other pathways might be important during the other wind pathways during eye development. So this enzyme is broadly expressed in the early embryo, specifically in the eye shown here. And it's, you can see that this is also suggests that specific disruption is necessary, otherwise you, it has been shown that this, if you disrupt this gene, it leads to early disruption of mouse development. So in humans, interestingly, mutations of this gene cause an X-land dominant syndrome. It's called focal dermal hypoplasia, or Galtz syndrome. And since this is X-linked, most of the female patients that have been found are heterozygous and they are usually sporadic mutations, but they're also some inherited cases, but usually they're caused by fathers that received, obtained this mutation in a mosaic manner and then they, yeah, give it to their daughters, basically. And this syndrome is highly pleiotropic and has variable congenital abnormalities and mostly these patients have patchy hypoblastic skin or dermal atrophy, for example. They have digital, ocular and dental malformations and specifically what I've mentioned here is they have fused or missing digits, split hand foot, cleft lip and palate and specifically concerning the eye, those iris and corioretinal coloboma observed in anothomia and microthomia. So this disease is rare, but also maybe underestimated because a lot of the patients might die in utero. So for us, it was very interesting to look at the rollof porcupine during eye development a little bit more closely and I think for time reasons I will not go through this, but I want to mention that again, we did this tissue-specific inactivation of porcupine and we started doing a disruption of this gene specifically in the retina domain and the effect of this experiment is shown here. So what we observed is that tissue-specific or loss of porcupine can lead to coloboma, pigment abnormalities in the RPE and also eyelid closure defects. So the left side here shows control embryos. This is a mouse embryo at E13.5, a closeup of the eye and this shows a mouse embryo at E15.5 and you can see here it looks a little bit fuzzy so that means that the eyelid is covering the eye. In porcupine mutants what can happen is that there's less pigment so in comparison to the control eye and in this case here we have a coloboma so the optic fissure doesn't close properly and what we also often observe is that the eyelid doesn't close properly during development and we thought that we have a tissue-specific deletion but unfortunately and this is quite often this case and when you use transgenic mouse lines you have to be careful and do the control experiments so we thought that we have a retina-specific deletion but it's actually not the case so we have a more global disruption of this gene because the transgenic mouse line has expression where there shouldn't be expression basically. So we basically, what is interesting is that we have a situation that is more similar to the human patients where they have a global or a mosaic heterozygous mutation of the porcupine allele and we came to this because we noticed that there were several embryonic defects that were not plausible, for example these digit defects with the synodactyly or we had tail defects open body walls, open skin, yeah. And also a cleft palate, obviously, which could be maybe explained by an early porcupine expression that somehow affects close-by tissues like the facial primordia here in this case. So we think that we have a more global deletion of this gene and we started looking at these embryos and a little bit more in detail on a molecular level and we found, for example, one obvious thing that we would look would be activation of the canonical pathway and this green labeling here shows expression of another target gene, one of the target genes of the canonical pathways, left one which is also mediator actually of the pathway and in controls at E13.5, this is the eye here, here's the lens, you can see strong expression in the meson chyme and also here in the overlying surface ectoderm and a little bit in the peripheral eye and in the mutants, we see that this surface expression or epidermal expression of left one is decreased and there's an up regulation that we can't really explain right now here in the Dorsal RPE. At later stages, at E15.5, again, here's the peripheral expression in controls. We have some expression here in the cornea, corneal epithelium, for example, and then strong expression in the eyelid meson chyme and in the mutants, you can see that the eyelid is open here, it's not connected and some of these mutants have down regulation of this effector in the cornea and meson chyme, so it would be interesting later to look at the cornea, for example, if the cornea develops properly. In terms of, I told you that our other work has shown that the canonical pathway is important for RPE differentiation and so we were interested in how does the retina and RPE in these mutants develop and what we found that in the mutants, some of the mutants show this, a dorsal down regulation of, or not only dorsal, but also in this case, in other cases here in the back of the eye of MITF, for example, another RPE specific marker shows in this case a more strong down regulation in the developing RPE and this whole tissue here is the mutant RPE, so the whole tissue is starting to develop into the retina and OTX-2 is also expressed in the retina at this stage then, early or not. And then this retina specific marker, VSX-2 shown here in green, starts to become upregulated in the RPE, so in the mutant RPE. So this shows a typical case again of trans-differentiation which is consistent with our previous results, but I told you, we think that this is a global deletion, so we still don't know which tissue provides the wind for the pathway activation in the RPE, so this is something that we are doing now, we are looking, we are trying to make sure that we have mutants that only have disruption of porcupine in one or the other tissue and what we actually find is that when we disrupt it in the retina, so these are, I call them true, tissue-specific inactivations, when we do a single disruption of porcupine in the retina only or in the RPE only or only in the mesenchym, we don't see trans-differentiation. So this suggests that at least two tissues in combination provide winds that are important for RPE development and we are doing now combinatorial tissue-specific deditions which are a little bit more difficult to get. So the other little project that kind of came up with this is the coloboma, which I think is very interesting, it's quite common in comparison to other congenital diseases, a mycothermia for example, I mean between three and 11% of blind children worldwide, depending on where you look, I think in third world countries it's quite high and several genes have been identified that can cause coloboma, but these are the minority of cases, so a lot of cases are also caused by environmental effects, so alcohol for example, or even vitamin A deficiency. So and I want to briefly go into how the optic fissure should close during development, so this shows here an optic vesicle that is developing in an optic cup and the ventral part of the optic vesicle and the optic stalk which develops into the optic nerve, needs to invaginate for this optic cup formation and this shows in the scanning electron micrograph, this is the ventral part and you can see how this invaginates and if you would take a section through here, you can see here, this is the dozzle part of the optic stalk and this is the ventral and the hyalurid artery grows here through this opening here, so these two ends, this lip here and this lip, they have to grow around and close and for this to happen, the tissue has to fuse, so this is a little later stage, so you can see here, this is the optic stalk region, this is the retina or this is a little bit more in the retina or optic cup stage, so here both lips are closely opposed so they have to attach each other and then they start to make these cytoplasmic ridges until they fuse here and here you have a basement membrane that is on top of these tissues so the basement membrane has to disintegrate and if this is not happening for whatever reason, you get a coloboma or the optic fissure doesn't close, so this is, here you can see that it's actually kind of a zipping up mechanism, here it's still open and then here it's nicely fused already, there's no basement membrane in between and this is a mouse model where coloboma is observed so you can see the lips here, they actually attach each other similar to here but it's a little bit disorganized and then later, the fissure doesn't close so you can see clear borders here and the basement membrane is not disintegrated and in this case here you can label the basement membrane with this marker laminin so this is a control and this shows basically the same cross-section and here in this region here the retina and the RPE where they come together have closed very nicely and laminin is only detectable on the outside of the optic cup but here you can see that the ends where they come together they have not fused properly and the basement membrane persists and this is the case in a mouse model that and that's also a common human mutation for coloboma this is when Pax II, the gene Pax II is disrupted so in this case you get a persistence of laminin expression but you can also get a persistence of laminin expression when there's too much Pax II so either way modulation of Pax II expression can lead to coloboma and this is what we saw here in the porcupine mutants so we actually this shows expression of Pax II in the controls and at E15.5 it should be only in the optic stalk and then in the mutants we can see an expansion of Pax II expression and in this case we see also a persistence of laminin expression in the optic cup in comparison to control so this little line here shows that the fusion has not happened properly so we would like to look in more detail actually what is happening here why does this first of all does Pax II modified or altered Pax II expression cause this failure of disintegration of the basement membrane so what is is there maybe an enzyme that needs to be regulated by Pax II that mediates degradation of the basement membrane that is somehow affected so this is one possibility could be also that salivation that the attachment is not proper and there are some salivation mutants that show coloboma for example so also the cells they have to be able to move around and make this this movement or bending for example and there are some couple of cytoskeleton mutants that exhibit coloboma so but we don't know what is actually the sequence of events that is necessary on the molecular level and I think yeah this would be very interesting to look at so yeah basically I've summarized this and these are the people in my lab so Elizabeth Bank had actually she's a technician in the lab and she did a lot of the porcupine work very nice she's a great helper in this and then Kayla George is a undergraduate in the lab who's helping her and Mary Collisanto originally identified this defect in the porcupine mutants as a rotation student in my lab so I want to also thank my collaborators so the porcupine project actually is happening in collaboration with Charlie Murtau here at the human genetics department at the university and yeah this is by support thank you very much for your attention do you have any questions or alright that's yeah that's a good question that's how you go back I mean you yeah you keep going back so the thing is that when you start out when you start out with an embryo you have the last two lab basically I mean that's a more advanced stage and the germ-like layers develop and all these tissues signal to each other so you have very early on you have signaling centers that signal like an organizer it's in the younger embryos it's called organizer in lower vertebrates organizer and so this is really early developmental biology so these organizers secrete molecules that tell the surrounding cells become this tissue and then it's the organizer still can maintain its activity for a while but then the evolving tissues tell other adjacent tissues become this so everybody is secreting signals and the wind the signals are very important the TGF beta are very important so there are only a few I mean not only a few but there are few of these signaling or growth factor families that have different functions at different stages of development in different places and they're used over and over again and often it's also a combination of different signals and the tissues change they they change their responsibility they change their intrinsic molecular how does signature so that it's there's a constant changing of all the tissues and how they interact with each other so the meson kind they migrate out they get signals from the underlying neuropathy and and and then they start producing signals by themselves and they they signal to the lens ectoderm for example also you can't make the lens develops in a specific region above the eye and why do you not get a lens somewhere else so the method it turns out that the meson kind actually suppresses lens development in the epidermis that outside of the eye so if you disrupt certain like wind signaling for example if you overexpress it in the epidermis in other regions you can induce more lens more little lenses in surrounding the eye so there's all these different interactions that are suppressive or promoting and make sure that things are in the right place and between sometimes these mechanisms are conserved that's why it's interesting to look in other animals and the molecules just the the specific molecules change a little bit but certain things you can you can test better and not necessarily in mouse mouse is not always the best system but yeah other vertebrates are good to test this so this is a region in the early embryo that kind of starts a major has a major impact on formation of the mesoderm one germ layer of the embryo so the mesoderm forms is important for meson kind for bone formation for example to the somites derived from the mesoderm so yeah that's that's formation of one layer and the organizer is very important for that and once you have the mesoderm the mesoderm actually crawls in the frog embryo you can see that even in time lapse the cells crawl underneath the ectoderm the epidermis and tell the overlying cells to become nervous system and that's the the organizer basically is important for saying telling cells hey become mesoderm and then they know what to do where to move and yeah to secrete factors that induce the nervous system basically and that's that's early yeah it goes on and on yeah you can always that's the interesting part about developmental biology yeah to look at all these interactions and to go back where does what come from I mean yeah yeah yeah I mean we are interested in the role of extracellular signals in the whole process of eye development so right now we focus on RKE development because that's very not very well understood we also look at retina development at the same time of course because if we see something and and we are interested in retina development too and we are interested in anterior eye formation because a lot of these manipulations affect the anterior like the eyelid formation lens development, ciliary body, iris, differentiation so we look at this too and we have another project that I didn't talk about today where actually the mezzanine could play a major role in the defect that we see so and yeah and the coloboma is a new project that we started in the lab and I noticed