 The next presenter is Andrea Blitzer. She's a postdoc research fellow. She's going to be presenting primary cilia dynamics during patterning and repair of the corneal endothelia. So I'm going to go from the most clinical relevant talk to a very basic research talk here. So I'm Andrea Blitzer. I'm a recent graduate from Loyola in Chicago. And now I'm doing a postdoc year here with David Kudai. So as part of that postdoc project, we're looking at steroid-induced ocular hypertension. I'm not going to talk about that today because that data is really preliminary, though. I'd be happy to talk about it with anybody who's interested. But instead, I'm going to discuss some research that I did at Mount Sinai School of Medicine in New York over the course of about a year or so, looking at the corneal endothelium and how primary cilia are needed during patterning and repair of that tissue. I have no disclosure. So my PI there, Carlo Iomini, he was not very tech-savvy, but he used to make these really great watercolors to describe what he was thinking. So these are some of them. But this is just an overview of what the talk's going to be. So there's two time points during endothelium that I'm going to talk about, the first is the development of it. So when we're all born, our corneal endothelial cells are all different shapes, not very uniform. And as we progress to an adult tissue, they become these really nice hexagonal honeycomb structure. And the second is in repair. So these cells do not divide. So if they're lost through fused dystrophy or FACO or any reason why you would lose a corneal endothelial cell, the only way that that tissue can repair is by the existing cells migrating to fill the gaps. So I'm going to talk about that a little bit as well and how primary cilia may play a role in that. So as an introduction to the cilia, if you were at the translational research day on Friday, Dr. Bear gave a much better introduction to cilia than I ever could. But the basic points here are that it's a cytoskeletal structure that grows from the basal body. There's two types, motile and primary. What we'll be talking about is primary. Another example of a primary cilia is the outer segment of photoreceptors, which is a modified primary cilia. This is what it looks like on electron microscopy. And I just want to draw your attention to these dark areas. These are what are called IFT particles or interflagellar transport particles. And these IFT particles show proteins up and down the cilium. But in addition to that, they're required to grow the cilium in the first place. So later I kind of look at some mutations in these IFT particles. And ciliopathies affect lots and lots of systems in the human body. The genes that we were looking at are kind of most related to Bartlett-Biedel syndrome. And these patients can show problems in the cornea, but RP is much more prominent in them. So I don't really need to introduce the cornea so much to you guys, but what I'll be talking about is just the endothelium. So just this innermost single cell layer that faces the aqueous humor. And it's responsible for pumping water out of the cornea in order to maintain clarity. So once you lose a critical number of these cells, the cornea will start to become cloudy and eventually require surgical intervention. So this is what an adult, human, cornea endothelium looks like. So this is electron microscopy and this is the kind of honeycomb, hexagonal structure that I'm talking about. And in 2004, it was shown that these cells occasionally have acilia, not all the time, but every now and then we see acilium on one of the cells. And that was really exciting to my PI at the time because he was a cilia guy and there'd never been cilia seen on endothelial cells. Unfortunately he was misguided because the endothelium of the cornea is not a true endothelium, it's actually an epithelial cell. And cilia and epithelial cells are quite common, but regardless of this, that's kind of why we got started in this. So when we look at the development of the cornea endothelium, right now we're basically sitting in the anterior chamber looking at it. We look at two different areas, the periphery and the center. So even though these are all the same cell type, there is a polarity to this structure. I just wanna kinda introduce you to the system that we used and the staining that I'll be talking about for the next few slides. So this is just a normal adult mouse. And what I wanna highlight here is what we have, how we do it. So we take out the cornea, we dissect out the cornea, we can stain it with antibodies, cut it for a flat mount, put it on a slide and take these really beautiful images under the microscope. So in red here, this is just kind of to show the outline of the cell. This is a cell marker called ZO1. In green is acetylated tubulin. So that is usually around the nucleus, but if there's acilium in the cell, like actually I kinda see one on this bottom cell here, it would be at the acilium as well. These red dots in the center of the cells, those are the basal bodies from where cilia grow. And blue is just the nucleus. So let's look at the development of a mouse cornea endothelium. So this is a very young mouse at two days. On the left, we have kind of the center, if we're looking at the center of the cornea and the right would be the periphery. And I think the most important thing is the note here or if we look at the red outline of the cells, these are not those nice hexagons that I was showing you before. These are very irregular cells. Also, especially at the periphery, you see these really nice long cilia. That's the acetylated tubulin staining I was talking about. If we go a little later in development, now we're at 12 days. If you look at the red outline, I think they're starting to become more normal looking. And certainly at the periphery, the cilia are getting shorter. At the center, however, the cilia are now getting longer. And even later, so now we're at 35 days, this is pretty much the normal adult form. Cells are pretty hexagonal. There's no cilia or if you do see cilia, they're very, very short. And this can be quantified. So this is just looking over the course of development how the length of the cilia changes. And if I highlight just this, this is the periphery of the cornea. We see that it starts really long and eventually you lose these cilia as you progress to adulthood. The same pattern is seen at the center of the cornea, though the longest cilia kind of happened a little bit later. So this suggests to us that this is a polar system that develops kind of from the periphery to the center over time. And these are just my first conclusions, which I've already kind of talked about, that you have these irregular polygons becoming this honeycomb shape that cells are initially cilia and then they lose those cilia. And that the loss of this cilia is both time and location dependent. So this brings us to a question, are these cilia required? We know they're there, but are they actually necessary for the development of this system? And unfortunately I can't really go into all the methods we use to answer that question, although the answer is yes they are. So we were able to get some mu and mice that have mutations in these IFT particles that I talked about earlier that are necessary to grow the cilia. And we were also able to make interference RNA that we could inject into the anterior chamber of eyes. So even after they'd been developed and they were maybe seven days old, we could inject this RNA into the anterior chamber and knock out any of the cilia that were already there and see what happened. And this is very busy and there don't need to really look too much at it. But I think the important things to see is on the top here we have a control eye and these two are mutated eyes. Oops, excuse me. And if you just look at the nuclei, you see that the distribution is definitely off. There are areas where there are no cells at all. There are areas where cells kind of clump together. So absolutely cilia are required for this normal development. So in the beginning of my talk, I mentioned we talk about two things. One is the development. But the second is how it responds to an injury. How these cells in adulthood will fill in any gaps that it needs to. So to do that, we found a way to wound just the endothelium or hopefully just the endothelium. So we would put just a very small needle into the anterior chamber and with that we could scratch the endothelium. And where you see a W here, that represents the wound that we've made. So on the left here, this is a cornea that we've wounded. The right is that same cornea but just far away from the wound to show what it looks like normally. And what we found is very interesting. So in the wound, the bottom image here is just a blow up of the top. All the cells, or at least most of the cells nearest the wound, re-grew acilia. And you can see that that's not true typically. So we looked at this a little bit more in depth. And what we see here is if you look at different hours after you do the wound. So in both cases, these are two different eyes with the wound on the right side of the image. So in both cases, at 18 hours or 30 hours after the wound, they have cilia. But if you look a little more closely, at 18 hours, these cilia seem to be kind of in the center of the cell. At 30 hours they've migrated. These cilias typically in most cells have moved towards the wound. So that kind of leads me to our little model, which is that during development, these cells are pretty much oscillated. And those cilia are required in order to make the proper organization of an adult cornea endothelium. And when they do, that cilium regresses. But those cells maintain the ability to form a cilium again. And if there's an injury to that endothelium, they will re-grow acilia, which will then orient towards the side of the wound and instruct that cell where it needs to migrate in order to fix the cornea endothelium and maintain integrity of that structure. So that's basically it. I wish I knew what it was that makes the cilia orient that way. I suspect it's some type of inflammatory cytokine or some gradient that I don't know what it is yet, but that's the basis of it. So these are my final conclusions. That defects in ciliary proteins, which we could see through a mutant mice, or we could look at with RNAi, definitely affect the overall organization of the endothelium. So these cilia are absolutely required. We also saw that cornea endothelial cells regain the ability to grow out primary cilium and they do if they need to during tissue repair and that the basal body and thus the cilia orient towards the direction in which these cells need to migrate in order to maintain the system. So with that, these are my acknowledgments. So this is actually the Bonneville salt flats, which look a lot like a cornea endothelium to me. Not my picture. And Parliomini is who I worked with. He's my PI I'm outside and these are other members of the lab and we collaborated at SUNY Downstate. If anybody has any questions.