AudioHelicase Podcast: Pulin Li on Creating Multicellular Patterns in a Petri Dish

In this episode of AudioHelicase, Whitehead Institute Member Pulin Li talks about how her lab engineers cells in Petri dishes to communicate with each other and form patterns, recreating processes seen in embryo development—and how this work could eventually inform efforts to grow tissues in the lab.

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Conor Gearin: Welcome to AudioHelicase, the podcast from Whitehead Institute where we unwind the science and the people behind some of the Institute’s most exciting discoveries. How does a developing embryo know where the arms and the legs should go? To make a fully formed body, cells have to communicate with each other to form patterns. And these patterns become the building blocks of tissues and organs.

I’m Conor Gearin, digital media specialist at Whitehead Institute. In this episode, I’m talking with Pulin Li, the newest Member of the Institute. Pulin studies how cells communicate with each other to form multicellular patterns. She focuses on molecules called morphogens. These molecules are the primary guide for developing tissues, providing the blueprint for the body. Morphogens help give direction to the embryo, guiding where the head and the limbs should develop, as well as controlling the finer details of organs with many cell types like the brain. Before coming to Whitehead Institute, Li earned her Ph.D. in Chemical Biology at Harvard University in the lab of Leonard Zon and completed a postdoctoral fellowship with Michael Elowitz at California Institute of Technology. By growing cells in Petri dishes and genetically engineering them to form patterns, Li hopes to uncover the fundamental rules for tissue formation, which could address longstanding questions about development and potentially prove useful to learning how to regenerate body parts or heal damaged tissues.

Pulin, welcome to AudioHelicase. Can you describe what your lab is focused on?

Pulin Li: My lab right now is trying to build upon this methodology I developed during my postdoc time, which is engineering cells to create tissue patterning or developmental modules. The approach we’re taking is bottom up. We take cells that naturally cannot form any patterns, and then we engineer some of the cells to become sender cells so they can secrete morphogens. And then the other cells are receiver cells. So we provide them with the capability to respond to the signal. We also engineered fluorescent reporters, meaning that when cells receive the signal they will glow, and then they will glow to different levels based on the signal they have received. So using this approach, we can co-culture the senders and receivers in a Petri dish so they can communicate with each other. And then we can really watch the communication happening in real time under the microscope.

Gearin: So you have the sender cells, which secrete morphogens, and then the receiver cells react to this morphogen based on how much of the morphogen signal they receive. It’s a bit like a cell phone tower broadcasting a signal, with cell phones receiving more or less depending on how far away they are?

Li: Right. We’re trying to understand how cells communicate with each other in the tissue context, and what properties of the signaling molecules control how far they can go. And also, how different tissue contexts might play a role in modulating the communication distance. The distance between the different cells that can talk to each other can play a very important role in maintaining tissue homeostasis or in cancer progression. Within this umbrella, we’re also interested in the evolutionary perspective, because cell-cell communication is really fundamental for building a multicellular system. Life started with a unicellular system, and then different cell types come together to form a multicellular system where different cell types have specialized functions. So, you can think about how these cell-cell communication pathways evolved.

Gearin: How did you first become interested in studying development?

Li: When I was a grad student, initially I was inspired by discovering drugs for stimulating stem cells to help leukemia patients with bone marrow transplants. So that’s a very direct biomedical application. So that’s where we started. But we were using zebrafish as a model organism to approach this question. I did some screens and found some interesting drugs that could potentially help stem cells to engraft the hosts. But then I kept asking questions about how the drugs worked. That got me really into the fundamental mechanisms of signaling pathways. I started to use the developing embryo as a model to study cell-cell communication in that context. I think that’s the first time I realized developmental biology is really interesting, one of the most beautiful biological systems. I remember the first time when I was watching embryo development from a single cell to a larva within a couple days, it was just really the most amazing thing that occurred. Within 24 hours you start to see a little heart pumping in that tiny embryo, and then you just can’t stop wondering about how all these cells learned what they are becoming, and how every single embryo can follow this genetic program precisely and always develop into this beautiful organism.

Gearin: And within development, morphogens have a really central importance, because they form gradients that instruct cells on what cell type to become. They’re a central focus for your lab. So, when did scientists first learn about morphogens?

Li: The history of morphogens started in the 1950s. The first person who coined the name morphogen was actually a mathematician, Alan Turing. He was asking a question about pattern formation just in the natural system. He proposed some very simple equations — we call them reaction diffusion equations — to explain how you can generate periodic patterns with just two diffusing species or molecules. But at that time, that was really way before modern molecular biology, so we had no idea about whether they actually exist in a biological system or if it’s just a mathematician’s fantasy about how things work. The morphogen concept was under debate for decades because some people felt like diffusion is not robust enough to create a precise tissue pattern and generate such a complex system. But then came the boom of molecular biology in the late ’70s, ’80s, ’90s, when we started to be able to identify all these different molecules and genes, people figured out the identity of morphogen molecules. Now we know very well about a limited set of morphogens. They’re being used over and over again to pattern different tissues in our body.

One very interesting aspect about morphogens is that even though we have such complex tissue structures in our bodies, and different organisms also take on very different shapes and have different tissue types, they are all patterned by a limited number of morphogen families — probably only 5 or 6 families of these molecules. Most of them are proteins, with very few exceptions that are small molecules. It’s another puzzle which is related to what I’m interested in the lab as well, which is how do you use this small set of molecules to generate complex patterns, and how cells integrate different information together to make complex decisions.

Gearin: What questions are you currently exploring related to morphogens and cell-cell communication?

Li: Right now, we’re heavily invested in what controls the communication length scale and time scale in a multicellular system. You could imagine this question involves understanding cell-cell communication aspect outside the cells. The morphogen gradient has to match the tissue size in order to properly pattern the tissue. So understanding what controls the size of the gradient, its shape, would have direct impact on understanding the proportion of different cell types within a tissue. The second question we’re really interested in understanding is how cells take this quantitative information and convert it into fate decisions. During development, when you have a continuous gradient, cells receive graded information — it’s a continuous spectrum. But in the end, cells have to convert it into a discrete decision, with two or three different outputs. So how cells convert this information is something we’re also very interested in.

A third question would be taking these communication systems to the next level, which is how cells integrate multiple signaling information to make a collective decision. Within a multicellular system, cells are exposed to very complex environment. Oftentimes, they have to compute more than one, or two, or three inputs. So, what are the genetic circuits that enable cells to make different decisions based on a particular combination of signaling inputs?

Because development is a very dynamic process, in order to understand the whole system we need to basically watch the whole developmental process in real time. It’s challenging to do in an embryo except in a few simpler organisms. We’re taking a bottom-up approach to simplify the complex system into well-controlled, well-defined components—and asking, if we put these components together, do we produce multicellular behavior? And if we perturb a parameter, what is the direct outcome of this perturbation? Importantly, we can directly image the whole process in the petri dish over multiple days and get really quantitative data out of the experiments.

Gearin: One cell signaling pathway that you’ve been able to study in great detail is called the Hedgehog pathway. Can you tell us a bit about how this pathway works as an example of a key morphogen signaling system?

Li: The Hedgehog pathway is one of the most classic morphogen gradients. It forms a gradient in many developmental systems such as central nervous system to specify different types of neurons, and also controls the number of digits in our limbs. Mutations in the Hedgehog pathway can lead to developmental defects and also cancer — so the pathway itself is very interesting medically. And finally, the Hedgehog pathway itself has a unique signal transduction logic compared to other pathways. In many other pathways, when you think about how cells communicate with each other, usually you have a ligand that binds to the receptor, and then this receptor activates the signal inside the cells. But in the Hedgehog pathway, that logic is a little bit reversed. In the absence of ligand, the receptor inhibits the signal. And then when the ligand binds to the receptor, they mutually inhibit each other. That actually de-represses the signal inside the cells and allows the cell to activate downstream signaling activity.

The pathway also has a really evolutionarily conserved negative feedback loop. Meaning that whenever signal is on, the cells always try to express more of the receptor. That would allow the cells to adjust the level of the receptor based on the ligand. 

So why is the pathway designed in such a different way, what kind of capability or function could it provide given this unique architecture? So that’s why, for all these different reasons, we started with the Hedgehog pathway – and tried to first to break it down into simplest signal transduction logic, and then try to build the negative feedback loop back into the system, and to ask the question, with or without the negative feedback loop, how does it behave in terms of forming the morphogen gradient?

Gearin: Digging into that a bit further, this double-negative logic in the Hedgehog pathway — it isn’t the simplest solution that evolution could have provided. So why do we end up with these seemingly more complex solutions to development problems?

Li: It turns out that both our mathematical modeling and our experiments showed that this complex, counterintuitive pathway architecture lets it form a robust morphogen gradient—despite variations in the level of the Hedgehog ligand itself. Intuitively, when a tissue has a little more of ligand or less ligand, then you might have a different size of gradient. But when you have this negative feedback loop, it actually allows the tissue to respond to level of the morphogen gradient, and then tune the receptor level. That would allow the tissue to precisely control the morphogen gradient itself. Then you’ll always form the morphogen gradient with a particular size and shape. That would allow the organism to develop robustly, develop the stereotypical tissue pattern that will be required for the later functionality of the tissue.

Gearin: So thinking broadly, thinking of evolution, there are benefits, sometimes from more complex solutions?

Li: Definitely. The question is how can we understand the rationale, why are pathways wired in a certain way? We are really good at figuring out how the genes are interacting with each other, but understanding why these interactions exist and how they actually have evolved, what kind of function they can provide — I feel like those are the next big questions.

Gearin: If we are able to understand the systems that evolution has developed, will that help address problems facing the field of tissue engineering? Basically, what’s stopping us from growing complete organs in the lab?

Li: It is a very complex question. You’re asking a lot for the cells to do outside of their normal developmental context. So far, the tissue engineering approach is to provide a scaffold of a certain size and shape. Then you seed the cells on top of the scaffold. Basically, you hard-program something before you have the cells. That has been useful for certain applications, but it’s generally limited to simple structures like skin. But thinking of the kidney or liver, each tissue or organ is composed of many different cell types. How could you precisely define which cell types would go to where within a complex tissue? But these kinds of engineering goals have been achieved by embryos over and over again using cell-cell communication.

So, we’re hoping to program the cells to be able to communicate with each other and then make decisions so we can pattern the cells—and let the cells pattern themselves—by giving them the genetic programs. What we’re trying to do is to take a bottom up approach, to program the cells and recapitulate these developmental processes. Even though the question we’re asking is a developmental biology question, we’re hoping the principles we can reveal or the molecular tools or engineering tools we can develop will eventually be useful for tissue engineering.

Gearin: You can learn more about Pulin Li’s research and Whitehead Institute science on our website at Stay tuned for future episodes AudioHelicase and catch up on past episodes on SoundCloud and iTunes. Thanks for listening.

Produced by Conor Gearin

Music: Pierce Murphy, “Versailles” (CC-BY 4.0)

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Pulin Li’s primary affiliation is with Whitehead Institute for Biomedical Research, where her laboratory is located and all her research is conducted. She is also an assistant professor of biology at Massachusetts Institute of Technology.

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