Patterns of life

By Yaakov Zinberg

Celeste Nelson has been upending assumptions about developmental biology since she opened her lab at Princeton some 16 years ago.

As part of her research into how lungs develop before birth, she and an undergraduate mentee found they could measure the effect of a particular chemical on the patterns of lung growth in chicken embryos. They excitedly submitted their work for peer review but were met with a lukewarm response.

“One of the reviewers said that this is fine,” recalled Nelson, “but this had already been known, because folks had observed similar phenomena in mice, and mice are the same as chickens.” Because the conventional wisdom at the time said that chicken lungs form and behave like mouse and human lungs, Nelson’s findings in chickens were considered unremarkable.

Instead of accepting this status quo, Nelson became motivated to further investigate how lung development, also known as lung morphogenesis, differs across species. “It was in part because of that comment that we decided to dive deeply into these possible differences between birds, mammals, and reptiles,” said Nelson, who today is the Wilke Family Professor in Bioengineering at Princeton. She and her research team have since identified unique mechanisms that govern how lungs are built in mice, chickens, and, most recently, a lizard called the brown anole. Rather than focus on the molecules that are involved in these processes, they use physics and materials science to understand how lung tissue bends and folds into lungs that can support breathing immediately after birth.

She and her research team have found that members of the animal kingdom have efficient designs for building lungs that can change the way we approach human tissue engineering. Evolutionary diversity in the development of lungs could inspire new ways of constructing human lung tissue outside the body, which could be used to treat people suffering from chronic lung diseases.

Variations in lung development across species may inspire new ways of constructing human lung tissue outside the body, which could be used to treat people suffering from chronic lung diseases.

“Conventional tissue engineering would be to say, ‘How does the mouse do it? How does the human do it? Let’s do it that way,’” said Nelson. But nature, says Nelson, has found many ways to build lungs, and we can learn something from each of those ways. “There’s beauty in diversity,” she added, “and we can construct an engineering toolbox from the diversity of mechanisms we uncover.”

Balloon animals

No synthetic material can perfectly match the properties and function of lungs, but Nelson compares lungs to balloons: both balloons and lungs take in and expel air and are insulated from the outside environment by a thin layer of material, be it plastic or lung cells. To shape a simple balloon into a more complex structure — think balloon animals — you’d need to know the balloon’s material properties, like how elastic and compressible it is, as well as the amount and kinds of forces to apply to the material. The same factors dictate how lungs are formed.

Nelson found that a type of stiff tissue called smooth muscle, which was previously assumed to lack a role in lung development, is critical for the formation of branches in mouse lungs. Mammalian lungs contain millions of microscopic tubes called bronchioles that sprout like tree limbs from larger airways.

Using mouse cells that express fluorescent proteins, Nelson’s team created a time-lapse video that showed smooth muscle wrapping around the elongating bronchiole like a telephone cord, forcing the softer bronchiole to fork into two daughter branches that, fittingly, together resemble Mickey Mouse ears. This process occurs at the terminus of each growing branch, resulting in millions of such branching events over the course of lung development. The researchers found that this mechanism results from the elasticity of the different tissues and the physical forces within the budding lung.

Nelson’s lab’s latest model organism, the brown anole, has provided surprising insights into how simple lung development can be. This lizard is about the length of a pencil as an adult and is recognizable by its dewlap, a vibrant orange flap of scaly skin hanging from the throat. Because these anoles are highly invasive in Florida, two graduate students from the lab traveled there to capture some and road-tripped through the night back to Princeton with their new companions. (It turns out you can’t bring a dozen anoles aboard a plane or check into a hotel with them.)

“Just about all our major findings have come from being completely ignorant about how biology is supposed to work, and actually seeing the system with fresh eyes.”

—Celeste Nelson

Rather than compare anole lungs to a balloon, Nelson and her lab found that they more closely resemble a different material: a stress ball — specifically, the kind with a mesh exterior that bulges into little orbs of goo when squeezed. Anole lungs do not contain the intricate branches found in mouse lungs. Instead, the lung precursor is a lobe of thin tissue known as epithelium that develops bumps across its surface. Smooth muscle forms a hexagonal lattice through which the bumps of epithelium protrude due to the force of fluid pressure from within the lung. Not more than two days after this process begins — dubbed by Nelson’s group as “stress-ball morphogenesis” — the lungs are fully formed, ready to perform gas exchange through the bumps of epithelium.

Much more research is needed before scientists can construct lungs outside the body. Building the delicate blood vessels that keep tissue alive, for example, is an especially tricky puzzle bioengineers haven’t yet cracked. Nelson’s work of studying lung development in different species helps identify models of biological design that can potentially inspire biotechnology applications; though anole and human biology have very little in common, the unexpected (and previously unknown) speed and elegance of stress ball morphogenesis could prove useful for engineers trying to simplify the design of artificial human lungs.

Seeing the system with fresh eyes

Most academic research groups are highly specialized: biologists are typically the only ones who’d work in a biology lab, and chemists and physicists will similarly stick to their own kind. Nelson’s research group, however, is remarkably diverse, and includes biologists, physicists, mathematicians, bioengineers and herpetologists (reptile experts) who collaborate on a regular basis. For Nelson, creating an interdisciplinary environment is key to the research process.

“What I like about putting them all together in one lab is that they start to question each other’s assumptions,” Nelson said.

“Everyone comes in with a certain level of ignorance, and that ignorance is a massive superpower, because they don’t know what the dogmas are,” she added. “Just about all our major findings have come from being completely ignorant about how biology is supposed to work, and actually seeing the system with fresh eyes.”

Within the biology community, for instance, it’s traditionally been assumed that conserved traits — those shared by different species and selected by evolution on multiple occasions — are inherently important, while nature’s outliers are less worthy of study. Because many in Nelson’s group were trained outside the world of biology, they’re not beholden to its assumptions and have no issue prioritizing the study of unconserved traits, like mechanisms of lung development.

Bezia Lemma, a postdoctoral research associate working with Nelson, exemplifies the team’s versatility. With a Ph.D. in physics, Lemma wanted to apply his background to study developmental diseases in chickens. Everyone told him the two were incompatible, but he found a home in Nelson’s group, where he is now investigating the thermodynamics of chicken lung development. He credits his new colleagues with catching him up to speed on techniques not in the arsenal of your average physicist, like dissecting chicken embryos.

“People were very forgiving of my lack of knowledge about developmental biology, and were very willing to both speak to me in my language of physics, and also try to bring me into the language of developmental biology,” Lemma said of his labmates.

Lemma compared a typical lab meeting to an introductory foreign language class. “Everybody in the room is trying to learn French, but nobody in the room speaks the same language, and we all have to be okay stumbling over French together. And then there are a few French people in the room who somehow agree to walk us through it,” said Lemma, where “French” could be materials science one day and molecular biology the next.

These communication skills enable the scientists to discuss their research with each other, and Nelson hopes the team will use these skills to communicate their future research to the general public once they leave Princeton. Skills like explaining jargon, breaking down difficult concepts into digestible parts, and distilling data into a story about a single research question never become obsolete for the scientist interested in sharing their research, even if the methods of conducting research do.

“These are skills that aren’t typically taught to scientists and engineers,” said Nelson, “and they really are, I think, some of the most important training we can give. Whatever career path they choose, being able to communicate is essential.”

Branching out

It was thanks to a generous graduate student that Nelson set her sights on a career in research. As an undergraduate at the Massachusetts Institute of Technology, she had a work-study job washing glassware in a biology lab. She always had an interest in biology, but thought of it as something you learned in a classroom, not a hands-on undertaking in which a person could make discoveries — and certainly not something out of which you could build a successful career. During the fall of her sophomore year, a grad student in the lab asked if she wanted to help with an experiment, a relatively simple one to measure how cells interact with different proteins. Nelson was hooked.

“I was just so excited,” she said. “I flew home for winter break, and I was just jumping up and down, telling my mom that I was going to get to work with antibodies. There was just something intoxicating about going into the lab and setting up an experiment and then seeing a result for the first time. That drew me into science.”

Nelson said this enthusiasm is shared by Princeton scientists. “It’s the one place I’ve ever been where people are legitimately excited each and every time they talk about science and are eager to help,” said Nelson. This makes collaborations between different labs commonplace on campus, and Nelson works closely with two Princeton colleagues. One is Andrej Košmrlj, associate professor in mechanical and aerospace engineering, who leads a group that specializes in building computational models to simulate the forces behind biological processes like lung morphogenesis. By incorporating data about the material properties of lung tissue, these models show how the tissue responds to forces within the embryo.

The other member of the trio is Jared Toettcher, associate professor of molecular biology, who uses optogenetics, a technique to control gene expression using light signals, to monitor and manipulate cell behaviors. As part of the anole study, Toettcher and his team engineered optogenetic components that successfully enabled control over when and where smooth muscle contracts, which could be the first step toward controlling how engineered human lungs grow.

The lungs aren’t the only organ to grow via a branching pattern, and Nelson is investigating some of the others as well. She recently received the National Institutes of Health Director’s Pioneer Award to investigate how the timing of lung, pancreas, and kidney development is coordinated such that all three organs have fully matured by birth. She calls this her “Thanksgiving dinner” project.

“If you think about Thanksgiving dinner, you want everything done at dinner time: you want the turkey done at the same time as the stuffing at the same time as the veggies at the same time as the gravy. If something’s done too early, then it gets dry or cold. If something’s done too late, then people are complaining because they’re waiting.”

Flaws in the timing of organ development are associated with birth defects and chronic disease, yet it’s still a mystery how organs develop at just the right pace. “The embryo manages to solve the Thanksgiving dinner problem each and every time, no matter what the species is,” Nelson said.

The lungs, however, will always have Nelson’s heart. And there are many more kinds of lungs to figure out. Her lab is now beginning to study lung development in tadpoles, which are born with gills but grow lungs as they metamorphose into frogs. Nelson and her lab are a diverse team of experts working out yet another mechanism of lung development to further reveal nature’s endless diverse beauty.