Recording the score of lung development requires a symphony of teamwork

by Nick Miller 

lung

This digital rendering of a developing lung is part of the terabytes of data being assembled as part of the LungMAP project. Teams at Cincinnati Children’s needed to develop custom software to process the sheer volume of information.

Learning how a lung is made, note by molecular note, may one day lead to life-saving new abilities to repair or regenerate lung tissue—but getting there requires nothing less than a symphony of scientific cooperation.

A full orchestra of research centers, scientists and physicians are playing important roles in the multi-million-dollar LungMAP project, sponsored by the National Heart, Lung, and Blood Institute. Jeffrey Whitsett, MD, Co-Director of the Perinatal Institute at Cincinnati Children’s, serves as the head conductor.

LungMAP involves dozens of scientists and physicians at 10 research institutions who have divided the task into sections—such as different regions or developmental stages of the lung. Each is helping master the different parts of a molecular and cellular score that eventually will serve as a complete atlas of the developing lung. (Dr. Whitsett explains the LungMAP project in this video.)

Their instruments: various forms of advanced scientific technology. Single-cell genomics. Transcriptomics. Proteomics. Metabolomics. And so on. Rapid, recent advances in these technologies, plus the power of collaborative teamwork, make a project like LungMAP possible. 

“We feel strongly that understanding the cellular and molecular basis of normal tissue development will inform us about the processes involved not only in abnormal lung structure but also in repair,” says Whitsett. “This will help let us know if we can intervene, when we need to intervene, and what would be the processes that are good molecular targets for intervening.”

Roughly two years in, LungMAP has already made significant progress. However, many more mysteries have yet to be unraveled before researchers can produce a definitive blueprint of how lungs develop in people. 

“We are beginning to understand the cellular and molecular circuitry that form the tissue and tissue types that lead to lung development, but there is still a great mystery surrounding the later developmental stages when the alveolar septa are formed,” explains Whitsett.

Alveolar septa are segmented compartments of air sacs that facilitate the transfer of oxygen and carbon dioxide between the lungs and bloodstream. If these do not develop normally, trouble ensues. 

There is a stage for infants that Whitsett describes as the “perinatal transition to air breathing.” This is an especially critical time for preterm babies—who he has spent much of his career caring for at Cincinnati Children’s.

“Prematurity, infection and injury can forever change the structure of the lung,” he says, “and contribute to a lifetime of chronic health problems.”

Given that lung diseases are not confined to babies, discoveries revealed by LungMAP could one day help a wide range of people young and old. 

At first, a cacophony

Reaching the crescendo—where doctors can regenerate or repair damaged lung tissue—remains a distant goal. Currently, the orchestra is warming up in a convoluted din of work to understand how the lung’s 40 different cell types communicate across all stages of lung development. 

Take for example Steve Potter, PhD, a developmental biologist and co-leader of Cincinnati Children’s arm of LungMAP. 

potter, whitsett

Steve Potter, PhD, (left) and Jeffrey Whitsett, MD, are working with experts in bioinformatics, confocal imaging and other fields to make dramatic progress at developing a molecular-level map of lung development. Their tools include a 3D model of a mouse lung that is so data-rich it took days to print.

Potter is a champion of single-cell gene expression analysis. His work produces a quantitative readout of how each of roughly 23,000 genes functions within each and every cell. 

“You learn a tremendous amount about what is going on,” Potter explains. “Who is talking to who, who is making what, what their function is, how the whole process is working, how it’s moving forward and how it’s making a lung.”

Going a step further, scientists can use known disease-specific gene mutations to trigger human lung disease in mouse models. They then use single-cell analysis to quantify detailed expression profiles for every gene in every cell of the diseased lungs—giving them an unprecedented view of these ailments.

Finding music in the noise

Potter describes the collective accumulation of single-cell information as a “very powerful dataset” that’s also a very challenging dataset.  The sheer volume of data involved reaches into the terabytes.  A single terabyte adds up to 1 trillion bytes, or 1,000 gigabytes. 

This is where the bioinformatics experts join the concert, according to Potter. 

Yan Xu, PhD, Divisions of Neonatology and Pulmonary Biology, and Bruce Aronow, PhD, Division of Biomedical Informatics, are significant players in LungMAP.  Xu worked with , PhD, Pulmonary Biology, to create a computer program called SINCERA.

The program quickly processes and analyzes vast amounts of single-cell genomic data. It also helps identify distinct cell types, reads specific gene signatures and helps define the driving forces that cause specific cell types to form.

Freely available to scientists, SINCERA is one of two internet-based analytical tools created here for the LungMAP project. Xu worked with Yina Du, a senior bioinformatics analyst, to also develop LungGENS. LungGENS allows researchers worldwide to analyze and visualize single-gene expression profiles. It also is linked to a diverse set of other analytical programs used to interpret data, many developed by Aronow and his team, says Whitsett. 

However, this symphony goes well beyond single-cell and computer-based bioinformatics analysis. 

Many parts to play

Other investigators in Developmental Biology and Pulmonary Biology are conducting immuno-staining of high resolution confocal microscope images to identify proteins in individual lung cells and performing other experiments to validate single-cell data. 

confocal-mouse-lung

This confocal microscope image shows a developing mouse lung. Images like these are revealing new understandings of the molecular processes at work in lung formation.

Researchers working with the Confocal Imaging Core at Cincinnati Children’s (led by Matt Kofron, PhD) are producing vivid color images that highlight the details of cells and microscopic structures within developing mouse lungs. 

The teams also used a 3D printer to produce a plastic model that shows in exquisite detail a mouse’s lungs, including a surrounding lacework of vasculature. The data involved are so rich that printing the model took days.

Joe Kitzmiller, a senior research assistant in Neonatology and Pulmonary Biology, leads a project called LungImage, a web-based developmental library of mouse and human lung images available through LungGENS. 

And to promote education, collaboration and idea generation, all data are shared openly on the project website at LungMAP.net.

Other institutions working on LungMAP include Children’s Hospital Los Angeles, Pacific Northwest National Laboratory, the University of Texas at Austin, the University of Alabama at Birmingham, Yale University, the University of California, San Diego, Carnegie Mellon University and Duke University.

The next movement begins

So far, investigators have formed an increasingly robust picture of the cellular and molecular crosstalk that goes into making a mouse lung. Now researchers are beginning to work on the next step—applying what they have learned about the mouse lung to the human lung.

Obtaining human lung tissue samples requires a great deal of effort and care, as well as informed consent from families who have lost children. This critical task falls to the LungMAP Human Tissue Core at the University of Rochester Medical Center in New York.

Working with organizations such as the United Network for Organ Sharing, the tissue core is procuring, processing and distributing normal late fetal, neonatal and early childhood human lung tissue for the LungMAP project.

Whitsett says continued progress would not be possible without the help of many volunteers who make these tissue donations possible.

 “The entire LungMAP team,” he says, “is so very grateful for the donation of these precious materials, which will be used to understand the renewable functions of the human lung on which every breath depends.”