Thank you for joining us today for another edition of Condo Insider that's put on by White Council of Community Associations. And today we're going to do part two with Sage Water. I'm kind of really excited to listen to hear what they have to say and to learn on the presentation because kind of interesting about replacing water pipes while the building is still being occupied and used is really kind of unique that they're able to have their little niche here in the holy market and not across the mainland that's their niche. And that's the only thing that they do. So without any further I'm going to turn it over to Jennifer Gara to start us off. Aloha. So today we're here to do actually a shortened version of an educational piece that we do for a lot of different buildings here in Hawaii. It's called How Do You Know It's Time to Repipe. And what we'll be talking about today is different things to help you identify and track different piping system issues. And a little bit about us if you want to change the screen, Chris. I am Jennifer Gara. I am the regional account executive for the Hawaii Division. You can see a little bit of information here about me. With me today is the vice president of operations, Miguel Rentus. Aloha. How's everybody doing? My name is Miguel Rentus. I've been here in Hawaii a little over 13 years now. Open up the office back in 2009, former U.S. Army Sergeant and licensed plumber here in the state of Hawaii. As Jennifer and Redding both said, we have a unique niche. What we do is multifamily occupied and repiped. That sounds a little intimidating when you hear it the first time and also kind of hard to believe when someone tells you you don't have to move out.  A lot of people are like, that's not possible. I can't live through a renovation, let alone a repipe. How's that possible? So we're going to talk about that a little bit and I'll let Jennifer go into it. But we've got a couple of slides that we'd like to show and explain when you know your building is ready for a repipe prior to actually hiring a company like ours. All right. Can we go to the page? We revolutionized page three. So this is just a little bit about what both Raylene and Miguel were talking about. We are a turnkey pipe replacement company. It is very small niche. The three things that we promise our clients is that it's one contract, one point of accountability for all of the trades. All the trades are under sage water. We do end to end service from pipe to paint. We even have cleaners on our careers. And the third thing that we promise our clients is that every night when they come home, they will have a working commode, a working sink and a place to bathe, even if they're in the middle of a project with us. And next, let's look at page four. Here's a go for it, Miguel. So here's just a couple of projects that we've done so that way you can understand that it is possible. And we're not talking about just one or two that we've done here in Hawaii. We've done well over 35 of them. We couldn't list them all here. We do have a website, sagewaterhawaii.com. If you go to that website, you'll see a list of all the jobs that we've done. One to know that it's time for your building to get the pipes changed out. That's what's going to be key for you as a building manager and for you as a board as well. So if we can go to the next slide, we'll touch on some of those. So the first 10 years of regular building, you're not really too worried about the plumbing. First 10 years, you're more focused on construction defects that you have any identifying what's really going on with your building and familiarizing yourself with your building, not just as a building manager, but as a board. So we like to break it down into three phases. So the first 10 years is phase one, you're familiarizing yourself and making yourself aware and keeping track of any and all things that are going on. Phase two is going to be the 20 or 30-year mark.  You start thinking of reserves and increasing those reserves because it's just around the corner. 40 years comes pretty quick after 20. Unfortunately, I can tell you from experience.  So once you're in that 20-year mark, it's time for you to start really focusing and nailing down what's going on with your building. What's causing the leaks? Are they just from user error? A homeowner left the faucet running? The shower was going on? Or they didn't do any maintenance to their toilet. They left the toilet just run over and over and over again. So that's you want to make sure you can identify what it is that's causing all of your leaks and your potential problems. And part of that is Jennifer's got a great leak log system that will save you what it's come up with that you can get from us and helps you identify and write down and keep track of all of your leaks and what the causes were so you can start preparing and realizing what you need to put money in the reserves for. So there's a couple of systems that most buildings have and in this next slide we'll talk more about that. So your typical building has four major systems that supply water.  You've got your hydronic piping, which is some buildings have what they call central air and they cool those systems. And so you have chiller lines that run throughout the building that have a tendency of leaking as well. Then you got your DWB, your drain waste event that takes all the wastewater out and all the bathwater out of your building that has a potential of leaking.  So those are your four major water supply component systems for your building and any one of those four systems can leak at any time. What we would like you to do as a board, as a building manager is to make it easier for yourself and to know what needs to be changed out is to keep track of these items with a leak log or just making sure you identify them accurately when you let your insurance company know should you have to file a claim or anything of that nature. Your typical life expectancy for most of these systems is on the next slide. So domestic water can last you anywhere from 30 to 50 years. When I say again domestic water I'm talking about the supply of water. So this feeds all your faucets, your washer dryer. It feeds your bathtub as well. So that's usually starting copper in some buildings as pecs. The average life expectancy of this though is still 30 to 50 years. Then you've got your drain waste and vent. Average life expectancy per drain waste and vent here on the island is proven to be about 40 to 50 years. At that point you need to start considering an alternative to change it out and getting it fixed and repaired immediately. Your next system is hydronic heating and cooling. That lasts typically anywhere from 30 to 50 years. And then lastly is your fire suppression system for most buildings. And that would typically last for the last 30 to 50 years. Jen, you can jump on the next slide. Okay. So the next slide, a couple other indicators that you will want to be aware of, signs that you've waited too long. I think we've all heard the horror stories. You have a pipe failure in a building, it takes out seven, 10, 11 units, right? It's a pretty good sign. Another one, your insurance deductible. And we see this all the time, people with deductibles not per event, but per unit of $25,000, $35,000, even $40,000 per unit. Frequent shutdowns to perform repairs if you have first onsite or one of the other remediation companies in front of your building on a regular basis. That's the sign. And when your board meetings seem to revolve around leaks, more leaks, how to pay for them, another great sign. And you will also in this day and age, most of us have social media. And you will see owners and renters venting on social media about your building. And all of these things will affect equity, which is an important thing to realize. So some of the risks of waiting too long are, and we have seen this, boards getting sued, being held legally responsible, even though board members individually usually aren't. We have seen suits against buildings because they've failed to take action for a number of years. And the other part is, is the longer your wait, the more expensive it's going to be. This is also a great picture here. If you look, it's a vent pipe. So we're talking about the drain waste and vent. This is a snap vent pipe in a high rise building. And it's also an indicator that most people don't know about. If you're having a lot of funny, like kind of disgusting smells in your building, it often could be due to a cracked vent pipe. They don't actually carry fluids in them, but the odors will come through those and people will smell those in their walls. Often that is an indicator of issues with the drain waste and vent. Can we go to the next slide? And so here's just a little bit about the drain waste and vent. We didn't really have time in this session to go over everything, but commonly is the big picture on the right was a 10 inch drain pipe. We actually took this picture in one of the buildings that we did recently. This muck in the pipe and see how thick it is. It's literally, you know, being pulled out in sheets. This is preventing the pipe from doing what it needs to do, blow these items out of your building. And this is, you know, the result of lack of maintenance over the years. This is a 10 inch pipe in there. It's a lot of stuff in there to impact something that large. We do occasionally see copper. It is much less common. We do occasionally see it in drain waste and vent systems here in the islands. And then the galvanized steel. We do see quite a bit of that actually kind of in a hybrid. See it in the vent systems. And you can see the picture in the middle here, the cracked vent. That's what it looks like when you get large cracks in your vent. And that's where those funny smells in your building will come from. So just to elaborate a little bit more on these materials. So unfortunately, the biggest question a lot of buildings have is, well, if you're going to take out the cast iron, what are you going to use back there? And it's, everyone wants to use PVC. They figure plastics will last forever. It's not biodegradable typically, and it doesn't go well in the landfills and everything else. So if it doesn't work there, then it should work great with my waste pipe, right? Because it's going to just last forever and ever and ever. Unfortunately, that's not an accurate statement. And it's actually not capable to be used in every single situation. PVC doesn't have a fire rating. So in a lot of high rise buildings, you have to take into account the fire rating of the system that you're going to install. On top of that, if you have a building that has what's called plenums, where they're, again, just big holes in the middle of the building, the lack of a fire rating doesn't allow you by code to install the PVC in there. As Jen said, so the cast iron is usually the go to for these systems. And one of the things that most companies now that do what we do, they install cleanouts so that way you can actually maintain. So a lot of the deteriorating plumbing that's going on is because of lack of maintenance. So when you're in a building and you're like, well, when was the last time we actually snaked our lines? Probably never. And some of the old slides in the pictures, you can see where like the 10 inch line that they tried to snake that one. There's actually a snake caught in that line that the plumber lost it in the line, just decided to cut it out and leave it in the line that we found when we were changing out the plumbing. But maintenance is really difficult to do when you don't have cleanouts because there's not a lot of access points to get to the plumbing. So what do we do when we do a pipe replacement? We install cleanouts so you can maintain your building and extend the life of your building. So that way, hopefully that 40 to 50 year mark that we set for cast iron has actually been extended to hopefully 60 to 70 or possibly 80. The manufacturers of cast iron say that their pipe is made to last for 100 years. Well, we would all love to see the pipe last 100 years. What they don't take into account is the lack of maintenance and everything else to help deteriorate. So part of our talks with the boards and with the building managers when we're setting up these programs is making sure that we install these cleanouts for them. Of course, they need to choose it. We recommend it. And then if they choose to install them, some people decide not to. I don't know why I can't answer that question. If you want to ask me, I won't have an answer for you. What I can tell you is that it does become humbug for some of these buildings. And the reason why is because a cleanout is not aesthetically pleasing. It's usually either a disc that's on the wall or it's an access panel.  I don't want to see an access panel or a disc on my wall. Who cares about the plumbing? That's somebody else's problem, not mine. So that's where you run into a lot of times with the buildings and stuff like that. But you know, the main thing that people need to understand when you're dealing with these issues is really identifying which system it is that is failing. Is it really the DWB that's failing? Is it the supply that's failing? Is it the hydronic lines that might be failing? Some people don't realize that a hydronic line where they have AC lines, it could actually be leaking just from its sweating so much because the installation has failed on it. And not that it actually has a pinhole leak or anything. Just the condensation from an alone will actually look like it's a running faucet. So identifying these issues allows you to make sure that you are preparing for it properly and you're putting the right money in the reserves and making sure you're also picking the correct company to replace the correct system when you put it out to bid, et cetera. And Chris, if you could put up our last slide. This is some information if you'd like to contact us. This is a shortened version of different educational presentations that we do offer. We do have one about leak logs. We do have one about condo timelines, helping you through different parts of critical infrastructure work here. With that, really, Neil, questions? Well, yeah, you talked about drain cleanouts. So how often should boards really do their cleanouts twice a year, once a year? So that's a great question. And the problem is it's not an easy answer and the reason why I say that is because you know as I do a lot of these homes are third and fourth homes for some people. So with that being said, what we recommend to buildings and build property managers and building managers is how many people, what's your occupancy? Is it 20% occupancy, 30% occupancy, 60% occupancy? Take that into account that your first step would be what is the age of your plumbing. If your plumbing, if your building is two years old, then you probably don't need to drain clean it. I would say maybe camera it to see what the condition is, how much use it actually has. If your building is 30 years old and you can't find any record of it ever being clean, then I would try to get it clean immediately. The problem is is that sometimes when you wait that long, cleaning can actually cause more problems than anything because now you start breaking down all that gunk and all that gunk will actually probably take some of the plumbing with it and then you start ending up with holes. But what we tell people after we install a new system is you should routinely clean your system out and inspect your system once a year, clean your system out at least every two years. That's what we recommend. And not snaking, the misconception is I'm just going to run a snake down there. Snaking is not always the answer. Snaking is what most people do once you already have a problem to break all the gunk up and get it down the drain. It's called hydro jetting. So what you want to do is basically take a high pressurized hose and wash and clean the inside of the pipes out so that way it pushes everything down without causing any damage to the pipes itself. So as the building ages and of course as they start seeing some blockages coming about, that's also the big red flag that, especially blockages, that's a big red flag that needs immediate attention and the time to get someone proper plumber that has a camera, especially since it may just make sure not to snake it, but to really dig down into the camera to see what other issues are going on with those pipes. 100%. It's important to investigate as much as possible to see what's really going on. And it's funny because you're saying that a blockage is a red flag, it's red alert. We've actually installed brand new plumbing and had blockages almost immediately because people are like, well, there's brand new pipes when they start throwing diapers down there. I'm like, it's a brand new pipe. It's not a brand new incinerator. That's not how it looks. But yes, you're 100% correct. As soon as you have blockages, you should hire the proper company to come out. And most of the good companies on the island that do snaking also do camera in so they can actually give you that video report and let you know what your plumbing looks like on the inside. And we can help with some of that as well. I think the board should also start being a little proactive with, you know, maybe attach a little snippet like I had one that what you don't throw down the toilet, you know, even though they say they're flushable, I said, no, not necessary. They're not always, you know. So I usually make that my role is like wipes. No, they don't go down the toilet, you know, you know, I have a list that I kind of post in my rentals.  Or do you have a cesspool or do you have a septic or anything like that? Because those are other things that people don't realize. So maybe your plumbing is just great and just fine.   Well, they don't realize that that stuff is plumber's nightmare. We actually hate that stuff because it actually deteriorates the plumbing. It shortens the life of pipe to include cast iron dramatically. And it's not the actual liquid itself. It's the gases that it produces that actually turn into bacteria that start eating and corroding the vent side of the pipe and going down into the sanitary pipe. So, you know, that's a great thing. And we tell buildings that as well, look, you should post on your website or in your bulletin board. Please do not flush.  I can put anything down there. Pickin' bones, rice. I got it. No worries. That's not the case. It's not, it's not again, it's not an incinerator. It's not going to take everything out of the building. It'll actually cause more problems. So that that's, that's, you know, the first step is to educate yourself on what you're building is actually having issues with. Is it just the waste? Some, some buildings in computer supply, you know, they're getting a lot of leaks on the supply and, and maybe it's because the anchor stops starting to leak or the supply lines are starting to leak or they haven't been changed out in forever. So, you know, people are hiring a plumber to come out and he tweaks it one way and it's never been moved that way. And so now all of a sudden it's cracked and he doesn't notice that he leaves and that next thing you know, it starts leaking. So this leak lock that we have that we've created that we hand out to buildings is a great tool so you can document and form yourself and form the board and anyone that has questions because miscommunication is just as bad as anything else. Right.  Or it was the kids that were up on the pool deck the entire time that kept on, you know, strong water over the side that actually that's where all the leaks were coming from. It wasn't even the plumbing itself. So we've got some stories for you. Well, have you guys ever, well, just as a reminder to all the condom boards that under I think 514, I think it's 138 where it's a high-risk component where there are some condos that actually once a year will go in and do a high-risk component. They will check all the angle valves, the supply lines. I know a lot of them now are recommending that on the hot water heater there's a shutoff, you know, and they check the p-traps all that kind of stuff and they have attached with it a priceless from some of the plumbers of what it would cost to do the repair. But that's always been a really useful thing. I remember one time I was like, I looked, I touched underneath the p-track and I went, oh my god, there's a hole. It took my finger to make the hole. That's how good it was, you know, but it's just stuff that you don't even realize, you know. Yeah, you bring up great points and high-risk components, you know, a lot of insurance companies now or they will actually they incentivize you as a building if you do your high-risk component inspections that they will lower some of your cost and premium and so on depending on how frequently you do it. But yeah, it's one of the things you should always check and to add to that list of high-risk components, why not schedule maintenance for the plumbing as well, you know, at minimum running camera just to see where you're at and if you even need to do it and if you're good now, well then check it again in six months. The cost of doing the inspections is minimal compared to cost the damages will happen later on if you're not doing it, you know. Vented maintenance is always cute. Just like you don't wait till your vehicle runs out of oil and overheats before you get the oil changed. No, you do it every 3,000 miles. Well, same thing with the building. That's a 3,000 miles, I'm just kidding. It got a little further now. My truck says 7,500 miles.  So I tell my go, at least you're not ruining the bottom part of your cabinet and then, you know, push a brand new cabinet and it gets more because all that water damage, you know, and I could just ruin something brand new and so I always try to put a bucket underneath a small bucket to catch the water in case at least you have some protection there. Yeah, you know, it's funny, my grandmother used to buy, you know, those big sheet pads that they use for baking and she would get those and she would keep it underneath there because it's only about an inch thick so you can still use it and it would protect the bottom but it would catch whatever's in there and then she would just line it with paper towels, keep all her stuff in there and if the paper towels got wet she knew something was going on and of course, me home, I need you to come fix it now. But you know, whether best makes a lighter underneath the cabinet, kitchen bath cabinet. Yeah, I mean, there's so many tricks to the trade, you two have got a bunch of tricks to the trade. So again, knowledge is key and knowledge is power, right? So the more you can do to prevent it and prepare for it the better it is. And one of the things that we do is help educate people as much as possible so that way you're not spending unnecessary dollars or something you don't need. Okay, great. So we're nearing our time. So I really want to thank Miguel and Jen. And it's so funny because Jen realized that we have a connection from the past through this whole process, but which is great to connect. So I want to thank you both. This is really, this is great, this is really educational. I'm going to be sending out a email blast. And Jen, if you could remember, I don't know if you sent me that, that week thing that you guys talked about the chart, the leak load. Yeah, the leak log. You want me to send you one so you can send it? Yeah, I can attach it with my if you don't mind, I can attach it with our no blast. Okay, that'd be awesome. Yeah. And then so we hope that this is going to be a very good educational experience for a lot of boards. I know some of them are teetering between this concrete placement, either they line it, you know, and I've heard some conversation about the lining. I'm not sure if I'm totally sold on it yet, but you know, but at least our white community of condos have some known alternatives now that and it's proven alternatives that has worked and has, along the way, you know, we've got down like 35 projects and we probably saved them millions and millions and millions of dollars. And one added feature that I want to remind everybody is there, I want to say almost full service because when they come in and start breaking down walls and they also have the person on their staff to replace that wall, right? Yeah, do the cleanup of the mess. So they're not like, oh, we're waiting for the other guy to come and finish this part of the job. It's not like that. It's all very controlled. So you're going to get a complete start and finish, you know, and not like, oh, we gotta wait for the other guy to come out. Why don't we put the tile guy to come back? No. So I really think that that's been added over that night top notch feature to those services. So thank you again. Have a great weekend and look forward to talking to you guys again soon. Thank you, Rayleigh. Thank you so much. Thank you, guys.


What's up everybody, Jayce here for JaspiersCaseBricks.com. We just sold out a random number of lock that features Tons of different teams here guys for Pikachu number six of a one box break of NTFL football This is a 10 team R&B that features all of these here from San Francisco to Pittsburgh the Jets Commanders, Troy Lyons, Tennessee Titans, Cincinnati Bangos, Dallas Cowboys, Jacksonville Jaguars, Houston, Texans Again, you get a piece of them via serial number guys So we go by the first number on the left side of the serial number dash remember any and all redemptions for any of These teams go to automatically the spot zero any live one-to-ones go to spot one and any non-numbering cards around the rest of the group and Nick texted me about an hour ago guys. I know it was really late But about an hour ago around 10 saying hey, I'll give away two free spots if we can sell it out tonight Which we did guys so congrats We're gonna give away two free spots of the customers that bought in get a chance Thanks to these customers here from Douglas down to Evan last by Mojo that got in now You'll still get your promo entries, which is great But on top of that we're gonna give pretty much almost, you know 350 and value backs Hopefully you get a free extra spot for buying in guys There'll be two winners at least Maybe I mean there's a couple customers. I have two spots, but all right full 12 right here. Here we go one two three four five six seven eight nine 10 12 and Boom 12 6 and a 6 12 12 Joel and Lauren Congratulations a free extra spot for both of you guys Just for buying in There you go Now 12 times on the list of names and the numbers now one two three four five six seven eight nine ten eleven twelve Joel the top done at the bottom Remember guys these teams there are just for the sorting team to know which teams are part of it You get a piece of all of them and then 12 times here zero one two three four five six seven one two three four five six seven eight nine ten eleven twelve 12 times two down to three All right, so we got 12 12 12 12 12 Joel you have spot two Douglas with nine Steven P with one and five Lauren you have four and eight Joel with any and all redemptions with zero And then Lauren you have seven Michael with six and Evan last one module with three So I'm just gonna take this off here really quick so I can help him says this And like I said, this is all the teams that are in There's a customer names and like I said guys coming up next is to break itself. Good luck everybody You


Hi and welcome to the session. Let's work out the following question. The question says solve the following differential equation That is dy by dx minus y by x is equal to 2x squared So let us start with the solution to this question. Now we are given dy by dx minus y by x is equal to 2x squared. It is a linear differential equation of the form dy by dx plus p by is equal to q where p is equal to minus 1 by x and q is equal to 2x squared Integrating factor will be e raised to power integral p dx that is e raised to power integral minus 1 upon x dx That is equal to e raised to power minus log x that is equal to e raised to power log x inverse That is equal to x inverse and that is equal to 1 upon x Now for such a linear differential equation The solution is given by y into integrating factor that is 1 upon x is equal to integral of q that is 2x squared into integrating factor that is 1 by x dx Plus a constant say c This implies that y by x is equal to this becomes 2x and 2x on integration gives x squared plus c Multiplying both the sides by x we get y is equal to x cubed plus c into x So this is our answer to this question. I hope that you understood this solution and enjoyed the session. Have a good day


A black man nervous about how cop acts, then he says to follow him.  The driver noticed the officer acting a bit odd as he talked to him about his window tint being too dark, looking around inside his car from where he was standing. Then, the officer stopped cold and told the driver to follow him down the road. Before Levante Dell was pulled over in Westland, Michigan, it may have been just a regular Monday afternoon for him. It was Officer Joshua Scallioni who noticed that Dell's car had a darker than allowed tint to its windows. When he had the lights. I did what pretty much everybody does when they get pulled over, Dell said. My heart dropped, went to my stomach. When Scallioni walked up to Dell's car, he noticed something even more troubling than the tinted windows as he started talking to him about the ticket, Dell's three-year-old daughter Lauren was not buckled into a car seat.  Consequently, Dell was asked to exit his vehicle, which really made the financially struggling dad fear the worst. As Officer Joshua Scallioni started to question Levante Dell, the policeman listened thoughtfully instead of issuing a ticket. In fact, he even saw some things in common between the two of them. As he continued to listen, the officer's heart went out to the driver and the reason behind his lack of a car seat. As Dell teared up a little, he explained that his little girl had grown out of her car seat and that he couldn't afford a new one. I took him out of the car, separated him from his family and asked him what's going on, Scallioni said. He's like, bills are building up, overtaking my income and I can't afford a car seat right now. The officer immediately saw himself in the driver's eyes, having suffered the same financial despair at one point in his own life. I related to the fact that I've been in that situation before, Scallioni explained. Rather than writing a ticket for any of the offenses, the cop asked the father to follow him. The two men drove to Walmart where they walked the aisles of the store together. You would have thought we were best friends, Dell recalled about the surprise shopping trip. No awkward silence. I learned about him. He learned about me, he added. With his own money, Scallioni bought Dell a car seat for his daughter. The compassionate cop even helped to pick out a pretty pink seat, which is Lauren's absolute favorite color. I realized this was the perfect opportunity to help him, the officer said. Dell seems like a blue-collar hardworking guy who was doing the best he could for his family. Levante Dell couldn't believe the generosity being shown to him, and he thanked his new friend in blue, praising what this kindhearted, white cop did for a black man. Don't judge a book by its cover. You'd be surprised what comes of it. He said, urging others to not be so quick to judge anyone based on race, occupation, or financial situation. Officer Scallioni didn't see skin color. He saw a citizen in need as well as himself in a sad circumstance that he could personally relate to. Rather than keeping those thoughts to himself and issuing a ticket, which would have put Dell further in debt, he chose to make someone else's day a little better. I wasn't looking for it, but it does feel good to help someone out who needs it, Scallioni said. The city of Westland Police Department was contacted about the good deed, but they had no idea who had stepped in to help Dell. In fact, Officer Joshua Scallioni hadn't informed anyone about the incident. It is clear that his sole purpose was to assist the driver, not to receive recognition. The humble officer played down his role, claiming that he was just doing his job. Indeed, it seems that acknowledgement and appreciation were not the motivations for Scallioni's kind gift. Rather than seeking praise for what he had done for Levante Dell, the officer simply requested that anybody affected by the story should just pay it forward, through their own act of charity. Whether Officer Scallioni wanted the recognition or not, he certainly earned it. I feel like he should get the recognition. Everyone should know what he did, Dell concluded, and we couldn't agree more.


OK, so good morning. So my name is Marianne Felix, and I come from the Ecole Normale Supérieure in Paris. And I work, as you will see, on the elegant evolution. And so I think my part of the course is to tell you about evolution of developmental systems in particular. And so I'll have three lectures. And today, we'll take a view at a relatively large time scale to look at the evolution of an intercellular signaling network. This is work which is already a few years old in my lab. And then the two next lectures, I go to a shorter evolutionary time scale and go to more genetic approaches. Oh no, there is a bad connection in my computer. It starts already. So we can make the announcement now. Yes. Sorry. Sorry. So I'll keep pushing the computer now. This afternoon from 5 p.m., we have another presentation. OK? So it's already close to 4 o'clock. And that's the lecture coming in the weekends. So also, I want to introduce you to Petra. Petra is helping us for this weekend as the secretary of the school. So if you have a need or a request for a complete lecture, so please just read the report to her. She is in the office number three, just before the board. And I think that's it for the moment. Thank you. OK, so as I don't know, so please interrupt me. Whenever you like. As I don't know how much you know about evolutionary biology. For today, the only thing I would like you to consider is the time scales. And so you can work at very small evolutionary time scales where you can do within a species. So here you can cross individuals and do genetics. And this we will see on the other days. You can look at very closely related species. Or you can look at, for example, different families or phyla of animals or plants. And in this case, what you have that you can use is a phylogenetic framework. So phylogeny is the tree of relationship of organisms. So here you see a tree of all animals. But it could be the whole tree of life. Or it could be a subpart of this tree. For example, just the nematoda, the nematodes here, to which C. elegans belong. And when you do what's called evolution of development, evo-devo, what you do is you look at the state of the system in some of this taxa. And then you try to reconstitute what was the history of change in the trade. So this is an inference type of science. That's not, I mean, you can never prove because we can't have access to the ancestor how it was. But you can find scenarios which are most likely. And what I will show you is that a relatively small evolutionary scale is within a small part of the nematoda. So very briefly in evolution of most of, so you have genetic variations. And the part of development here is to see how when you have genetic variation, it translates into phenotypic variation in the output of the developmental system. So here we're going to talk about variations in the phenotype, the final phenotype, the morphology of the animal or whatever. We will talk about a self-faith pattern in development. And this goes through modification of development. So today we won't talk about genetics, but more our modifications of development translate or don't translate into a phenotypic variation. And I'm saying this because I think to this audience is clear, but a lot of evolutionary biologists don't consider development as being important. Whereas a lot of features of developmental biology, of developmental systems are important to understand evolution. And I think for people who are more interested in cell or developmental biology, this is probably pretty obvious. So the two main points I want to make today, if you just take home messages of my talk, is that when you look at a developmental process and you, for example, make a quantitative model of this process, you can consider whether the evolution you're seeing in a group of species corresponds to evolution in parameter space of your model. It may be the case, or it may not be the case. For example, you may need to add variables and parameters. If you have an intercellular signaling network, for example, or a transcriptional network, you may need to add transcription factors and loops and things like this. Or you may just need to vary parameters. So it's to consider whether the explanation of just varying parameters is sufficient in the group you're looking at. The second point I'll make is that, because it is the case in the developmental system we are studying, C. elegans-volva-development, or nematode-volva-development, is that you can have evolution of development here without any change in the final product. So this is sometimes called a cryptic evolution because it's cryptic, it doesn't show at the level of the final output of development, but you do have evolution in the developmental system. So the outline of the talk is here. I start with a brief history of quantitative approaches in the evolution of morphology, and then introduce you to C. elegans-volva-development, which I'm going to tell you all along this week. Then see how, considering it's a conserved, self-made output, how we can unravel cryptic differences among species, how we could use a model to see whether the quantitative changes in model parameters were sufficient to explain the evolution. Then we'll see evolution in further, further larger evolutionary scales, and then I'll give a brief overview of other examples. Then see elegans-volva-development. So first, regarding the history, if you consider, so if you consider the fact that you have evolution of morphology and that you can quantify the final product now, the morphology, not the development, so these first approaches were not considering development really in the, and developmental parameters in the way they were seeing the evolution of morphology. So this is derived from a book by Darcy Thompson on growth and form, which appeared at the beginning of the 20th century and then was edited again in 1942, where he tries to explain the evolution of shape. So here you see between apes and human skull, for example, or different fish species, just by deforming Cartesian coordinates. So here there is no development at stake here. Slightly similarly, but on a system where actually, so if you take the snail shell, it actually develops from continuously during development. And so David Raub, who is a paleontologist, tried to classify snail shell shapes that you could find in fossils by two parameters, the width, the ratio, sorry, the ratio of size of the different coils and T, yeah, is the width of the coil. And so if you vary the parameters here, you can have very wide shells or very tiny pointed shells and you have snails like this. So here again, evolution is described as a parameter change in the final shape, even though in this system it may correspond to a developmental feature. And one point that he found if he was varying these parameters continuously, he could not find in the paleontological record every type of shape. And so in conclusion, that you have a non-random distribution of shape. And so this notion of a space of possible morphology was taken again by another paleontologist, Stephen J. Gould, who is a famous evolutionary biologist who wrote a lot of books and whom you may know. And so he gave the name of morpho space to this space of possible shapes that you get by getting here these three parameters or the translation distance between the axis and expansion. And again, so the space was not completely filled in real paleontological record. Another concept that Stephen J. Gould introduced again is this time a developmental concept which is not quantitative, it's just words, but he proposed that developmental changes in evolution could result from two mechanisms and these are not exclusive. One he called allometry, relative growth between different body parts. And the other one is heterochrony, where it's the timing of development of one part relative to the other that changes. And so example of this, for example, this neotenic salamander, which developed to a reproductive adult in a shape that corresponds to the larval stage of other species. So now what I would like to introduce is a more modern version where you really look at the developmental, you do a quantitative model of your developmental process and what you're really exploring is the parameter space not only of the final shape but of the developmental system. So here I just idealize in two dimensions two different developmental parameters one and two. And so when the genotype changes by mutation and recombination or if the environment changes you may be in different points in developmental parameter space. And this may give you different points in a final phenotype space or it may not give you a different, you may have two points here in developmental parameter space that gives rise to a single phenotype here. The mapping from here to here obviously can be funneled into one phenotype here. And this is the case of the system we're looking at but it's not necessarily the case, okay? So it's this main idea that evolution can be represented as a parameter variation in molecular and cellular models of development. You have questions at this point, is it clear? Yeah, so this is a good point. So no, these type of models are at a smaller evolutionary scale and can, I mean, it depends what novelty is and I really don't want to enter in this. It's really another field of research. So we're not trying to count, I mean novelty is, you can have novelty in a quantitative developmental system in some way, it depends how you define it, I think. Other questions? Okay, so we move to C. elegans. So C. elegans is a small nematode worm which is about one millimeter in length which is totally transparent. So you can see by normal ski optics all the nuclei of every cell. And it develops in three days. So you have three days from egg to egg at the next generation. And so someone called John Tholston followed development under the microscope continuously and found out that the cell lineage, so the pattern of divisions of the cells and what they become ultimately is practically invariant from individual to individual. So this is a great tool to study development. At the end, you have 959 somatic cells, so cells that form the body. In the germline, you don't have invariant divisions. So we're considering only the soma here. And so what we will look at is just a few, a little part here, which is formation of the vulva which is the organ for egg laying of the worm. Another thing I should tell you is that C. elegans is a cell thing amaphrodite. So this is actually kind of a female that also makes sperm at the beginning of her life, of an adult hood, and then makes all sides fertilizers internally and then lays the eggs. So the fact that it's a cell thing makes it, it's great for genetics, but it also makes that populations in the lab are totally isogenic if you want them to be. That is, given adults, if it's totally homozygous at all loci in the genome, will give rise to progeny which are all identical, except for new mutation. So you get populations of individuals which are genetically identical. You also have males if you want to do crosses. So what we are going to focus here is this part of development which occurs, so up there you have the development in the embryo, then the embryo hatches and you have four larval stages that are separated by moles. And here we're looking during the third and fourth larval stage. So these cells here which are the base here, there are six of them, and these are the six cells I'm going to represent from now on. Yes, sorry? This arrow here? No, no, this, oh, sorry. This is the pattern of division of a cell. So you take the zygote, the egg on top, and then you have a first division like this. And so these are trees of genealogy of the cells in development, okay? So at the end you get 959, but you exactly know how they were born through divisions, okay? And you also know what this cell is becoming, whether it's a neuron, a muscle, and so on. And if it's a neuron, you know which neuron it is, okay? No, we'll see examples tomorrow. This is not the point of today, but yeah. And for sure, different species of nematodes have different numbers slightly. So I mean, this is not totally fixed and yeah, that's why I put quasi-inviant. I'm interested in evolution, so in variation, so if it was always the same, it would be pretty boring. So if you consider these six cells, so they're actually situated in the ventral epidermis of the worm, so here you see a little larva with the head on the left, the tail on the right, the ventral side down and the dorsal on the top. So these six cells, so there are actually 12 of them, which are called P1P to P12P, so they're numbered from anterior to posterior. And there are six in the middle, which are called three to eight, which have the potential to make volval tissue, okay? So these are really epidermal cells in the ventral epidermis. Here you can see maybe the nuclei which are in the epidermis. And there above it is the gonad forming, the formation of the gonad, and especially we'll see one cell here, which is called the anchor cell. So what these six cells do is that they form a cell fate pattern. And basically the developmental event we're looking at is formation of this pattern of three fates. So one of the variation we'll talk about tomorrow is for P3P, but for today, we don't talk about the most anterior. So for the five other cells, we have three different fates, which are color coded here, which are also called primary, secondary and tertiary. So the primary and tertiary fates are those that actually form the vulva, and the tertiary cells is kind of a backup cell if something goes wrong with the other. So if you remove, for example, the three middle one, five, six and seven with a laser, the other ones are going to take their place and actually become primary and secondary and form a vulva. Okay. And the primary and secondary fates, basically the primary fates forms a few cells which are at the center of the vulva. I think I have the cell lineage later. So they will go on dividing each with its characteristic cell division pattern. Again, we have these trees of division. The yellow cells just divide once, whereas these ones divide us three times. And then they form the vulva. We hear the anchor cell just on top of it. So there has been a lot of work since the 80s using mostly laser ablation and genetics to understand how this cell fate pattern was obtained. And I'll go in more quantitative details in today and tomorrow or the third day. Basically, this anchor cell of the gonad, if you remove it, if you remove the anchor cell with a laser, that's why I'm using a laser here, all the cells adopt a tertiary fate. So they become yellow. So the yellow fate is the default fate. And the anchor cell is sending an inductive signal which is an EGF-like molecule which is received here in P6P with a cascade of signal transduction which is called the RASP-Mapcainase cascade. So this EGF can diffuse. It's not anchored to the membrane and it can touch the red cells. But the red cells also receive a second signal through which is, so the blue signal here activates. I think I have probably this on the next one. So the blue signal activates transcription of a molecule called delta, which I represented by a D here, which activates a receptor in the next cell, a receptor called notch. So that's one very conserved signaling pathways among animals. Like EGF-RAS, we have here delta notch. An activation of the notch pathway in the next cell activates the secondary fate. So it's transcription of different effectors and also inhibits the blue pathway, the RAS pathway. In addition, activation of the blue pathway represses notch in this cell. So you have both, you have two cross inhibitory interactions between the blue and the red pathway which locks the fate of each cell. And then you have positive feedback loops as well. If you have really an excess of EGF, you're going to see the lateral cells being induced. If you don't have enough, as we will see today, you get ultrashary or you get some kind of intermediate situation. And so after these cells have divided, yes, they form the vulva, but this is not the part we're looking at. We're really looking here at the formation of this special cell fate pattern along the anterior posterior axis. Get clear? Good, so I'll come back to these pictures. So where basically what we will see is that the fate pattern here doesn't change in the species of nematodes. The first ones I at least I'll tell you about, but we'll have evolution here in the development. So the fate pattern is conserved for these five cells before P2P8P in the whole family of nematodes to which synrubditis elegans belongs. So the question becomes, how does this pattern, which is conserved itself, but does it form in the same way in all these species? And these species are really far apart in terms of molecular divergence. For example, C elegans and the closest species known to C elegans are as distant as mouse to human in terms of molecular distance. So the advantage we have compared to mouse to human comparison is that here we have exactly the same cells. So we can have what's called homology in evolutionary biology. We can actually find a cell that we can code P4P without confusion in all these different nematodes and study the same cell in different species. So this is very powerful to have this cellular level of comparison. And so now that we, so we have these species which all have this final cell fate pattern that we can follow using the division and the morphogenesis of the vulva, how do we reveal a difference in the way it's made? So to do this, there are several methods, but so the method, so one thing you can do is to try to look to quantify the signaling pathways in the different species. Technically, this was difficult to do. So what we decided to do was to actually perturb the system to reveal differences. And so to do this, we use laser ablation. So this is a very easy thing to do. You mount your animals under the microscope. This is a Novosky optics, pretty trivial. So here you're seeing the anchor cell and P5P, P6P, and P7P nuclei. And all what you have to do is to have a laser that aligns in your microscope and so you shoot the cell that you want and then for example here we kill the anchor cell and we can see the effect. So this was historically the experiment that was done by Jelit Kimball in the 1980s to demonstrate that in C elegans, these three cells then adopt a non-volval fate, a tertiary yellow fate. If you do it early. So here now what I'm going to represent here is, so the final cell fate pattern is that one. If you remove the anchor cell very early in most of the species, you get this non-induced pattern. So in all species so far, we'll see other species later, but in the senior update is genus, all species required the gonad and the anchor cell to form the vulva. The question is if you do different types of ablation, do you get some kind of intermediate state in the middle? So it's not really an intermediate state because it's the final state after the system in the middle of the induction. And you can imagine different types of cell fate pattern that you obtain. So for example, that P6P is induced sufficiently but doesn't induce its neighbors because it's not induced enough. Or it's not induced enough, so it becomes secondary red here, which is equivalent as the signal at a distance here. And actually to my surprise, the most common patterns we got are these ones with three tertiary fate, or this one which I did not expect at the beginning, but you can completely understand, you will see later, where P6P is not induced but P5P and P7P are normally induced to the secondary fate. So these are results that you can characterize basically with what happened to P6P, yeah. So what I'm going to represent later is the proportion of the three fates in yellow, red, or blue, so primary, secondary, tertiary. That's adopted when you do an ablation in a given strain of a species. So for example, we have these species called synopditis japonica. If you do ablation at successive time, you get either this or this, but never anything else. So in this case, it goes directly from one to the other. We have another species, synopditis bryxae, where we have very clearly in the middle this pattern with two, two, two, okay. Which we confirmed in many different ways with GFP reporters and so on. And then we got this surprising pattern in species called synopditis remanai, where P6P in the middle did not form a volatile tissue and a volatile invagination, but the lateral one did adopt a secondary fate. So in my coding system, it would be like this. So in this circumstance, what we asked then was to see whether these arrows were real. So if P6P was sufficiently induced here to induce its neighbor to become secondary. And so to do this, what we did was to ablate at the same time the anchor cell plus P6P to see whether its neighbors were affected. And yes, this is the case. If you do that, then they all become tertiary. So this shows that you actually have P6P that despite the fact that it's not induced itself, is able to induce its neighbors. So now I'm going to represent, as I told you, by taking just the fate of P6P. And because we have three different fates, this is represented on a triangle. Where for the different species or different strains, different genotypes within a species, you can represent, if you do this ablations over time, taking the middle window, what's the proportion of fate adopted by P6P on this triangle. And so you see some of them, which are basal ones in the phylogeny, which adopt mostly a secondary fate. Then you've got some like synoptic breneri or japonica which are directly to the primary fate. And then remanai, where you mostly have in the middle this secondary tertiary, secondary fate. And then you have a reversal here in this part of the phylogeny to a secondary, secondary, secondary fate. So this is a kind of description of the variation that we can reveal by perturbing the system. So yes, exactly, yes. We make a perturbation, right? Yeah. Yes. Oh, sorry. So the worms develop in three days. All this happens in about 10 hours because it's a short window of level development. And so we have led this during the third level stage and we look within six hours. It's actually a very satisfying experiment to do because you have your answers immediately. Sure? No, they're not. I mean, no, sorry. Yes, they are. It depends what the question is. They can produce progeny because they're self-fertilizing amaphodites. And so what happens if they don't have a proper vulva, so to get egg laying, you need a primary fate at least. So if they don't have the primary fate here, they will, I will show you pictures another day, but they don't have a vulva, but the amaphodite self-fertilizes, the eggs are fertilized inside her.  And so yes, she can produce progeny, yes. But she cannot lay. So the question if you did not hear is, if when you laser, if I understand, when if you laser ablate a cell, are you sure that something is not leaking out of the cell? Probably, right? So my answer is no, I cannot be sure. The only thing is that if there is an effect, it must be that I remove that this part of the signal. That's the most I can say, right? And practically the cell, you can see that they undergo some kind of drastic morphological change, and then they are usually eaten by the next cell. But for sure, you may have things leaking out. Not really apoptotic, I think. It's more degeneration. I mean, a mutant of apoptosis would not result in no death of this cell. Other questions? Yes, sorry? No, no, no, no, sorry. No, no, they don't give the same output. So what gives the same output is that if you don't perturb the system, in all cases, P6P will become primary, okay? Here what I'm showing is in cases where I ablated the anchor cell, the final product here will change. It's not a natural, it's not a normal output. I'm perturbing the system. So I'm getting to cases, for example, where, so this representation may be misleading. I have to make clear. This is ablation time, and these arrows is not time of development. Is if I ablate over time at different times, I will get as final product this, or I will get as final product this, or I will get as final product this, okay? Yeah, sorry? No, no, no, this becomes irreversible because then the cells divide and it's irreversible. Yes? I mean, this is a quantitative, you may have 80% of the animals doing this, right? So you have variation among animals, subjects to which I will return tomorrow. But basically to look at this variation, you need, I mean, laser ablation is not a good tool because there is variation in laser ablation. So for this I need genetics, basically. So that's why I'm not talking about the variation today. It's mostly, we're talking about the proportion. So that's why I have a proportion. Any other questions? Fine. So the next question we asked is knowing the network and here I have simplified this entire cellular signaling network, I showed you before. Can we guess what is going on in the different species? And so I will show you the guess and then I will show you a model that actually vindicated this guess. So if in C. elegans we have this situation here in blue and now it's coded by species. In Briggs, you can imagine that the rest signaling is able at when it's at lower level to induce the secondary phase in P6P more easily and that you have maybe less inactivation of the rest pathway laterally. And this weird pattern in synoditis remani with a tertiary in the middle, you can rationalize by the fact that it's very difficult in these species to get a primary fate here but you have transcription of deltas very easily at low activity of the, of anchor cell signaling. And so you get activation of the secondary fate here in the absence of fate here induction of P6P. Yes. No, so again, all these species, the final fate is two, one, two and yes, they reproduce normally. Now, if you ablate the anchor cell, you will get these different fates and no, this doesn't develop normally if you don't have a primary fate.  So one thing we first did was to see whether on this space of P6P fate, we could move the species around just by mutation or transgenesis and so we used, so the reference strain of C elegans which is the reference species is here and we could basically move it around in this space by changing the level of activation of the RAS pathway here with this mutation, for example, or increasing it here. We could also increase, take Briggs' C Briggs' here, increase induction by the anchor cell and make it go along this axis. So this axis looked like the level of sensitivity of the RAS pathway and then to go laterally, what we could do is to make a mild gain of function in a much pathway. So you increase the activation of the lateral induction and you actually reconstitutes from C elegans the remani output. So again, these are just looking at the final fate after anchor cell ablations. So the next thing we wanted to do was to try to make a developmental model of the system and for this we collaborated with Kerry Kim and Ed Monroe at Friday Harbor and this was the work of Eric Aorios who was shared between the two labs and Josla Milos in my lab and this is a pretty old by now work about 10 years ago. So we started from this vulva signaling network architecture and we decided to include all the feedback loops and cross-inhibitor interactions but to simplify, for example, the RAS pathway is just represented by one step with an affinity component and a cooperativity one. So the fact that it's actually a cascade of kinases makes it much more sensitive and with a cooperative activation and so this is represented in the model. So our model looks like this, which is not very telling but you have in blue again the RAS pathway here, the EGF activating a map kinase to phosphorylation and then you have cross-talks between the pathways, so in green here where you have a degradation of notch inside this cell and activation of two forms of deltas. So in C. elegans you have transmembrane forms of deltas called here like two and a diffusable one, one that can be secreted without a transmembrane domain and then this will activate notch in the next cell but also in the same cell and this will inhibit here the notch pathway and we consider that here two, the outputs are here, the primary fate corresponds to this node and the secondary fate to that one. And so the level of activity of these two nodes which are called here EGLE 17 and EEP 1 just after the name of actual reporters that we use, GFP reporters, you have here depending on the level of activation of these two reporters we define that the cell at the end as a primary or a secondary fate or if both are low, a tashary fate and then you have undecided fate in the middle. So we started with a model with several cells and with a lot of parameters actually 40 parameters and what they did was to pick at random in a large range for example of two logs, each of the parameters and to see whether after running the differential equations you were getting the final fate pattern and we asked for the pattern to be stable over time in the model and so it's an approach where you basically you have a huge, huge parameter space and you look within this huge parameter space of 40 parameters whether you can pick sets of parameter at random and find a solution that actually gives the final target pattern. And just at random, even though this parameter space was huge, we found 8% of the solution of the random parameter sets that got the target pattern which is always, so 670,000 that we stored. So now we are going to later to work on this but first what they did was to see what, so what you can do with this is see whether there is a bias in the parameter that you introduced by asking for this target pattern and there isn't a lot, it's basically compared to the range we decided at the beginning we have some biases but this is really dependent on the range at the beginning and mostly it's working through the transparent range delta and with low EGF diffusion. There was a question in the back, no? Sorry. Okay, so now the main point of the model was to take these target patterns and see whether these solutions were doing it in different ways. And so now I'll come back to discussions in the silicon-volva development field on different ways to do this self-made pattern that I briefly alluded to so far but did not enter into and also using our experiments of laser ablation whether we can mimic the evolution, I mean, represent the evolution amongst another species. And so here we keep the topology of the network constant, I mean not completely because some of the parameters can go to zero so it's equivalent to removing but we don't add something. And keeping constant again the output since we only choose these solutions that give rise to the same output. So if you think about these systems there are actually two ways where the cell fate can be patterned in C elegance. One is very simple, it's the anchor cell induces the primary fate, the blue fate that activates the secondary fate. And this was suggested by experiments of some colleagues whereby they constructed animals which were genetic mosaics. So that's when you take an animal and you can make some cells mutants and not others. I will not expand on the techniques to do this. It's different with different animals, flies and worms and different things. But here what they saw is that the secondary cells, so here I represented a circle for those with the receptor of the blue signal of DGF and the square for the mutant cell. And by looking at many animals what they found was that you really needed the receptor to become a primary fated cell but you didn't need it to become the secondary cell. But the secondary cell had to be next to a primary cell. So this gave weight to these mechanisms where it's basically the EGF signal in blue is received by the primary cell which is able to activate its neighbors through the notch signaling. Another series of experiments were done about the same time in another lab where what they did was to isolate one of the cells and when I say isolate, it's not really isolated, it's killing all the other ones with a laser. So you're in a case where you don't have neighbors to induce you. And then using a transgene, they heat shocked. So you can have a transgene which is under the control of a heat shock promoter so that's inducible by heat, by raising temperature of the animals. And what they saw is that if they were not inducing the expression of the transgene, the cell became tertiary, the default fate. If they were inducing a lot it became primary as expected but in the middle at intermediate doses you could get an isolated secondary fate. And so what they suggested is that you have a kind of morphogen system here where at high doses you get a primary fate and at low doses a secondary fate. And this actually, you do require the notch pathway so this requires basically that this cell activates auto-prime signaling of delta, so the secretion of this diffusible form of delta which has the property to be able to bind to the same cells receptor. So what we did in the model is to see whether we could find solutions corresponding to both and more interestingly what were the properties of these solutions. So we played in the model with these 600,000 solutions we then removed either the lateral signal so you could only have auto-crime activation to the cell or doing the mosaic for EGF receptor. So basically reconstituting in the computer the two experiments of the previous slides and then categorized among the 667,000 solution which one were still working in which conditions. And so we defined four categories depending on whether the sequential induction was required or the morphogenetic induction was required and whether they were sufficient or not. So you have basically cases where each is sufficient so you have redundancy, you have cases where both are required, they really need to act jointly to get the pattern, if you remove one of them it doesn't work anymore but if you have the two together it works and then you have solutions where a single one is required and sufficient. So now first of all you have to look at the numbers. So by far the most common ones are those relying on sequential induction and then you have very few where the morphogen induction compared to 600,000 is required or sufficient. And if you look at the parameter distributions among these solutions you can do statistics. And what you see, so this is one example is the rate of activation here of the expression of the lateral signal by the RAS pathway here. And you're seeing that depending on the type of solution so the red and the green rely on the sequential was here the morphogenetic works by itself and so here it's in the morphogenetic case they tend to be very weak here and on the other hand no sorry this is the diffusible one so it tends to be very strong here so very low coefficients. And so you have a bias in parameter space towards a strong activation of the diffusible ligand here. So conceptually this is a 40 dimensional space but if you realize what we did so we started with nine million random parameter sets found a 660,000 that worked that produced the pattern and within this ones we have tiny much smaller than represented parts of space which rely on the different mechanisms here. So we can also look for these different mechanisms our robust they are two parameter variation and so here are color coded the four classes of requirements exclusively sequential morphogenetic both required and fully redundant and what you're seeing here is the fraction of parameter sets out in these sets which tolerate a tenfold variation in each parameter which is plotted here and so we don't need to enter in detail so basically the morphogenetic the blue or the both required in green have a much lower tolerance to parameter variation so they are much less robust especially to variation in parameters that concern EGF receptor synthesis, half live and this type of parameters which is not surprising because to get this morphogen model going you really need sharp exact concentration so that you don't get excess activation on the sides and not enough in the middle. Whereas the lateral signaling just requires a threshold of activation of the primary fate and then the second threshold of activation of the secondary fate so you have much less precise tuning for one mechanism than the other. The second thing we did was to look whether within again this 670,000 solutions we could find parameter sets that correspond to the behavior we observed in the Synapse IT species so this is a slide just summarizing this different pattern so not only we did this anchor cell ablation we also did EGF overexpression so if you overexpress EGF a lot you get all primary, not very informative but what's informative is what we get in the middle here at low dose of overexpression and so whether we get for example adjacent primary cells here or alternation of patterns and so we categorize solutions here first using the anchor cell ablation and what you're seeing here is the percentage of solution we could accumulate for corresponding to the behavior of each species and again we could then look at the distribution of the parameters to see what was biased in which species and this corresponds basically to our intuition whereby for example if you compare so in C-brainerai where you have a direct induction of the primary you have a strong induction here of the primary by the anchor cell and a strong induction of the lateral signal because you don't get just the primary by itself it's basically something we hardly ever found whereas in C-brainerai this arrow here is very difficult to activate represented by a dotted line whereas this remains very strong so at low dose here we get activation of this lateral pathway and no activation of the primary phase and finally in C-brainerai where we find two patterns what happens is that we have a strong activation of this auto-crine loop here of auto-activation of the secondary phase by the diffusible delta no so the question is whether this can be extrapolated to other cells, no not at all this is a very it's an isolated developmental system that I'm talking about the other cell there are many other type of developmental mechanisms yes so no the question I think you're asking is whether there is some kind of pleiotropy of so if the fact that this arrow is stronger for example in these species and these species I can relate to something else in the one frankly I don't see ways to do this at this evolutionary scale but this has to do with the notion of pleiotropy so the fact that if you do a genetic change it's going to reverberate in many different tissues and so one way that this does not happen is if there is a c-stregulatory element of transcription here that's specific for this cell type and not another so you can perfectly have something which is self tissue specific but the only way I see to do this is to go at much smaller evolutionary scale where you can actually probe this directly here we cannot we so far it's much too far that we can pinpoint to the genetic change that are doing this okay which is actually the good reasons to go at a smaller evolutionary scale which is what we went on doing okay so based on this actually we made some predictions one of them I mean a posteriori you can rationalize it very easily but we did not expect at the beginning is to see how easy it is to get an isolated secondary in the different species and so to do this what we do is we isolate the cell by laser ablation of all the others and we look and so we isolate the PHP the most posterior one and we ask what's the proportion of the animals where it adopts the different faiths so blue, primary and red, secondary and purple being an intermediate between the two often you have one of the daughters adopting one faith and not the other and basically in she elegance we can get an isolated cell to adopt a secondary faith but in Ramanai we cannot which looks odd because we saw that in Ramanai you have this I should have written this before maybe in Ramanai you have this so at first sight you have isolated secondary but they're not really isolated because this cell is actually signaling to them but when we actually isolate just one cell it cannot become a secondary and the same for Cibranari where you have a very strong so in Cibranari you go directly to this to the final faith pattern because you have a very strong induction here laterally we also use cell faith reporters so this is a GFP reporter for the notch pathway for the lateral pathway and you see that in C. elegance it's utterly activated in the two red cells on the side but in C. Briggs it's actually activated at a much more even level in three different cells so this fits also well with what we expected this quantification so in conclusions from this what we showed is that the quantitative variation in the same network encompasses the two mechanisms of self faith patterning in the row of six cells that different labs had been fighting about sequential induction and morphogen induction but it shows that in terms of robustness to parameter variation the first one is much stronger yet it is a fact that DGF diffuses and that's therefore morphogen induction may also participate in a kind of redundant fashion and that we also saw that we can account for the behavior of the different synorability species by just quantitative variation in network parameters so this is a sufficiency argument it doesn't show that it is the case but it's actually possible now we go back to a more evolutionary aspect to try to map this evolution on the phylogeny and actually moving away in the genus and further out of the genus do you have any question? Why not? So what you're seeing here again is only the cell is not the final self faith is always this one what I'm representing here is always this faith of cells after you remove the anchor cell at intermediate time points so what I showed you before is remind for example, brain array, Japanica, elegans is more or less like this and then Briggs and his sister species here with three red cells we went on and went out at species that branch more basally in the genus and they all like this and you will see actually outside of the genus as well so this is where you can use the phylogenetic relationship and try to map the changes that have occurred and so the inference here is that you have a direct induction here of the primary faith so you get right away a strong activation of the primary faith and lateral signaling here and you probably have a reversal in this branch that's the most likely scenario so if you go further out you will see so we did the anchor cell ablation in many other species and genus in different genus and you see that overall you always get this intermediate self faith pattern with three secondary cells I will see later with four whereby so this is equivalent to saying you have a first wave of induction of these three cells and then a second wave which is induction of the central faith so you can describe almost the mechanism as two different waves there is however an exception and this is work from a half summer when he was in Paul Standback's lab it says these species I'm going to tell you where surprisingly if you remove the anchor cell even very early in development you have absolutely nothing different happening so you get a normal self faith pattern again this self faith pattern in these species so here you have a completely different mechanisms whereby the anchor cell is not required so here you're no longer in the in the quantitative changes in parameters that I showed you was sufficient to explain evolution here you are in totally different situation and so what happens in these species is that the vulva doesn't form in the middle of the body below the anchor cell as in the other species but it forms very posteriorly so here that's the rectum of the animal and this occurs because the vulva precursor cells so P4 to P8P these five cells migrate posteriorly and yes at the end the anchor cell will connect with them but at the time where the self faith is pattern and the vulva forms it's actually very far from the anchor cell and so in these species if you remove the gonad very early even before the anchor cell is born you get a perfect normal self faith pattern so here you have to find and to be frank we still don't know exactly how this happens but from experiment from Ralph at the time what he showed was that these cells from the start are not equivalent to each other so they don't have the same potential to become primary secondary and tertiary and the most likely thing is that you have like as in any animals a patterning along the entire posterior axis especially through hoax chains through wind pathway signaling which makes these different cells different and you probably have lateral signaling between them in lateral inhibition for example you have a single primary one so here you have a very different way of making the same thing so the output of this is that there are many basically ways to go to Rome or to Trieste maybe but at the end you get the same self faith pattern but the ways to get there the developmental routes to get there can be very different so we saw the sequential mechanism and the graded mechanisms that's maybe co-occurring in C. elegans we see that in many species we seem to have more like a two step induction where you have first induction of the secondary faiths and then the second induction which induces the primary central faith and then things like which also occur probably at low level in all species but are particularly required in mesoabditis is the fact that you have some kind of self-organizations through reciprocal lateral inhibition ensuring you have a single cell that becomes primary and a pre-pattern so the fact that these cells are not equivalent at the very beginning so the conclusion of this wider evolutionary studies is that the redundancy of the different developmental mechanisms may favor rapid evolution of these mechanisms and I think it can also favor new things and so one example and I don't know here what's new and what's old because we have only two points what I showed you so far was this these are all the new methods the three of old new methods and so far we've stayed at the top here now we're going to consider the next suborder or whatever this next group in green here and there is a huge difference now in self faith pattern between this one in brown and this one is green is that in the group in brown we had this self faith pattern where P6P is primary, five and seven are secondary what we so far saw now in this other group what happens is that the central faiths the central cells ultimately in the vulva are now shared between one daughter of P6P and one of P7P and now you have it's a symmetric still self faith pattern so now you have four cells induced all together okay so here and you may have four and nine being able to replace them but not in all species actually so in this case you cannot rely on the self faith pattern patterning mechanisms where the anchor cell induces one cell that induces its neighbors and lateral inhibition whereby this one also prevents the next one to become primary because you have two adjacent cells daughter cells here which become primary so here again you have a different mechanisms to see elegance but you can imagine that this I showed you that you have so what happens in this species here if we remove the gonad we also have no induction you also have this intermediate state with all secondary and we only have later the primary faith so basically you have a one stage which where the gonad induces four cells instead of three and this we don't know exactly this may be related to positioning or to previous sensitivity of the cells most likely to receiving the signal and then we have a second signal and now the anchor cell instead of going in between the two daughters of P6Ps goes here so the fact that we have this two step mechanism that we saw is dominant basically many species in the phylogeny I showed you before have this induction it allows this displacement of the pattern in other species in another book again I don't know what's the direction of change between the four cells and the three cells induction and centering between the daughters of P6P and in between here the two daughters but it allows changes in mechanisms and in self-feed pattern in this case so to scaritize basically here many developmental routes lead to the same final pattern you have the same pattern X here and you may have changes in the rate of weights and requirements and sufficiency of different developmental routes here in these two arrows and ultimately this may allow for a change in the pattern for example if for example the red route of development doesn't allow you to reach another pattern and the green one so pattern Y and the pattern Z so the possibility of further evolution depends on the developmental routes here and I showed you this two step induction may allow for the situation in the family of Panagralamus where you have four cells and two cells actually sharing the primary fate Any questions before I get to the presumably short collection of final outcome Right, on the route, yes Mm-hmm Is it possible or is that it's going to depend on how the processes will evolve and change over time under the constant pressure? So in population, is there anything that's possible if there isn't anything there in the pathway? Yes, so So thank you that again we will come to is that can we find variation at a shorter evolutionary time scale, right? And so I'll come back to this, the third lecture I think and so one of the quick questions behind this is yes so in evolution, why are these things changing? Is that purely by drift? So because you have mutation and the processes are going to change and as long as it forms the final pattern that's under stabilizing selection, it will work or do you have something driving evolution of development? So I'll come back to this in the third lecture basically I don't have the answer but in one case I have the answer, yes that is the second, it's not neutral it's actually under selection in another tissue it's coming back to the pleiotropy problem that if you have changes in one tissue it reflects in the other questions on the vulva system. Can I give you an answer? Yes So there is a question that you have and it's indicated for another lecture. Yeah, here we'll watch the last minute so if you look at Prandler's face and you see that there's a certain strategy in more compatible with the larger fraction of Prandler's face where his male could be much more robust and so then I was wondering is it possible or common for originality a non-robust pathway or a non-robust strategy for doing something and if they do that then are there more sensitive to evolutionary change? So you're considering the aim of it could be really what the analogy is so can you hint at this one more time to have things that are... Yes, so my answer to this would be that deepened robustness to which perturbation I think so you cannot be infinitely robust to all perturbations and so yes, there are perturbations where the system will be more sensitive and evolution can drive in this direction. Now a lot of us are studying developmental systems which are extremely invariant and not sensitive to the environment but this is not just because we chose to do this because we like to have a good output but some other of my colleagues are very interested in plastic systems where the environment actually changes the output so the environment is acting on developmental processes and actually changing the output and in this case the situation is very different so in some way looking at what you may be looking at in early Josephine embryos or what we are looking at here is a special type of non-environmentally sensitive developmental systems there are also other systems which are extremely sensitive to the environment just the size of the organism for example the growth or really switches type of thing having a wing or not a wing in aphids for example and so here we are in very different situation where we expect to be in for some parameters in parts of the system which are sensitive to perturbations but for some parameters especially I don't think robustness to me you have I mean it's to which parameter you're talking about this specify is not infinitely robust there is always a compromise yeah so what basically I'm assuming this here so the question is probably what are the experimental proofs that they are I think in this group a synod that is species so this has been mostly studied in synod that is Briggs which is as far to see elegance as the other ones are and we are pretty I mean the AGF and the not signaling pathways are there and doing grossly the same thing yes so we think it's the case if we go outside of the synod that is genus that we are also looking at this at the moment we chose one other genus and did actually mutant screens for the vulva in the same way that was done for the elegance and also my colleague have summer yet another genus um what we find is that the species we looked at which is a genus not very far from synod like this we clearly have also a GF not we don't know yet in christian because the claim of how summer's lab is that the GF is not acting I'm not completely sure of of this but yeah and not we don't know either so it's it's very well possible that at a longer evolutionary scale we are talking about very different signaling pathways okay that you really have to know about pathways what does functionality mean sorry so it's based on the fact so the fact that they are part of the vulva or not it's a question of tissue definition I think it's very clear yes it's it's whether they imagine it yes so this is clear whether they become primary and secondary is really whether they form the center or they look different now there are some species where it's much more complicated to decide because each of the daughter and granddaughters there's something different so to assign a fate to the mother sale is slightly different but these are really weird exceptions so I'll continue by giving a very just small vignettes of other examples which I think are still not many where people try to take developmental systems and look at variation in evolution of these systems in the light of a parameter variation and I think so the first example I have is a very short evolutionary scale because it's a variety of chicken and this was he studied in the lab of Dennis Headon in a demo I think and so you have some chicken which don't have any feathers on the neck so they're called naked neck I think and basically what what they showed is that the system behaved as if so you have patterning of the feathers and the position of the feather is the lateral inhibition type of system well so depending on so you get this precursor of the feather it's very much like I don't know whether you heard in Drosophila of the bristle patterning it's a similar in some way and what's special in this is that it's only on the neck that the feathers disappear so there seem to be a local inhibition here and basically I think I summarized this here what they showed is that so we have summarized here the genotype in orange the developmental parameter in green and the final phenotype that's in this case changes in blue and so here the genotype is the naked neck compared to a reference the leghorn variety and the difference here is the genotype at BMP 12 so BMP is one of the another type of signaling pathway GGF beta signaling pathway and so if you look at the expression level of this signaling pathway here it's high in this genotype and low in that one and this corresponds in terms of the final phenotype in a high density here and a low here so here you have an inverse correlation and this is explained the tissue specificity is explained by the fact that locally you have another pathway yet retinoic acid and so this variation which is cryptic in another part of the body becomes non-cryptic here in the neck because in parameter space of a model so this is a kind of reaction diffusion model they did you actually go here in a region of parameter space where you completely remove first you have much less feathers and at the end you completely remove them another example that was also based on kind of reaction diffusion patterns is colors of fish and this is work from the lab of Shigeru Komdo so these are trout different types of species of trout which can have either spots which are clear white spots or they can have dark spots or they can have a kind of labyrinthine patterns and this type of patterns you can get so you can make a model again of reaction diffusion so the exact developmental process behind it is probably not a classical just reaction diffusion model because it takes I mean you have cellular components involved not just molecules but you can make a very simple model and see that if you change parameters you go from so this area of parameter space where you have white spots this one where you have black spots on the white background and the middle one where you have this labyrinthine patterns and what I find very nice in this system is that when you cross trout which have black spots and those which have white spots you get genetically in the middle by crossing them so you get here this labyrinthine patterns in the middle so I think this genetic cross is really telling you that by doing the cross you basically take a little from the two parents which gets in the middle of the parameter space here so if you take one here and one here when you cross them you are in the middle which genetically means something but I'll leave it for now and it also gives way to this model where the black spots and the white spots are obtained through this type of mechanism where you have so that the black spot and white spots are not each determined but that it's a generic pattern mechanism pattern formation mechanism and I think I have a third example which is the work of Isaac Salazar-Shudad and Yuka Yanval who look at the shape of teeth so here you can look both at the shape of teeth within one species so our different teeth may be explained by different parameters of shape formation because as you know we have different shapes of teeth along the draw but you can also look at evolution and they looked in different species especially in some marine mammals sorry the name escapes me and again they made a model of interaction between epithelium and meson chimes through a few signaling pathways and by changing parameters you can mimic the shape of different populations of so foca which is a marine mammal depending on the parameters okay and I will stop here I think if you have other questions