• Tracing Our Shifting Blueprint

    Tadpoles help scientists unravel how progenitor cells do their jobs during organ development

    by Tim Bonfield

    DNA may be known as the blueprint of the human body, but during the many stages of prenatal development that blueprint is better understood as a full-blown book with pages turning every four or five hours.

    At any given moment, only parts of the instruction manual are being used. And some parts get used over and over in combinations that scientists are just beginning to understand.

    Aaron Zorn, PhD, a researcher with the Cincinnati Children’s Division of Developmental Biology, works with this ever-shifting blueprint every day as he studies the progenitor cells that form the lung, liver, pancreas and gastrointestinal tract.

    “It turns out that the liver, pancreas and lung originate from a common set of progenitors in the early embryo,” Zorn says. “But how do these cells learn where they are supposed to go? It is clear that many, many decisions are being made right from the beginning of embryogenesis.”

    When Cells Know Their Place

    Much like the real estate business, the ability of progenitor cells to form different types of tissue depends heavily upon location, location, location. For breathing to work, developing lungs must be connected to the surrounding vasculature. For digestion to work, the liver and pancreas have to interact properly with the intestines.

    “The cell must know exactly where it is in order to coordinate its development with other tissues,” Zorn says. “We know that cells talk to each other. We also know many of the genes, growth factors and transcription factors involved in that communication. What we still do not understand is, how this is all coordinated?”

    Tiny differences in location can lead to huge differences in the fate of a progenitor cell. For example, at the equivalent of 28 days human gestation, a frog embryo has developed a simple gut tube made from nearly identical progenitor cells. Then some of these cells receive signals that tell them to become intestine — but not foregut, which later gives rise to the liver, pancreas and lung. Twelve hours later, those same signals instruct a subset of the foregut cells to become lung rather than pancreas or liver.

    “That’s an amazing thing. We are still trying to understand how is it that the same signals can produce dramatically different impacts in the space of just hours or days,” Zorn says.

    Finding the patterns within this constantly shifting blueprint requires skill, data and computing power. It also requires lots of experimentation, which is what makes frogs so important to developmental biology.

    The Power Of Xenopus

    Zorn has helped Cincinnati Children’s build a colony of about 1,000 West African frogs (Xenopus tropicalis and Xenopus laevis). These frogs produce hundreds of thousands of tadpoles a year for use in a wide variety of research projects. Zorn also is co-director of Xenbase, a huge multicenter online library of genomic data about African frogs and their use as models of human development and disease. This database was cited in nearly 900 peer-reviewed papers published in 2013.

    Frogs are especially useful in developmental biology studies because females produce thousands of eggs the day after being stimulated with an injected hormone. Once fertilized in the lab, tadpoles develop quickly. “The first indications of lung development occur at about two days in frogs,” Zorn says. “In mice, it takes nine to 10 days and 28 days in humans.”

    Not only do tadpoles grow faster and in far greater numbers than mouse embryos, they also can develop in a petri dish. “It can be difficult to observe the dynamic processes of the first trimester in utero,” Zorn says. “But with frogs, you can observe all of it in the dish. This allows you to ask more kinds of questions.”

    For example, the lining of the lung comes from embryonic tissue called endoderm while the connecting vasculature comes from nearby tissue called mesenchyme, but how the development of these two tissues are coordinated was poorly understood. By performing delicate microsurgery on tadpoles, Zorn and colleagues extracted both types of tissue and found that crucial signaling to begin lung development actually comes from the adjacent mesenchyme.

    Such findings will help clinicians better understand why human development can sometimes go awry.

    “Most principles of organ development are the same in fish, frogs, mice, and ultimately humans,” Zorn says. “In frog embryos we’ve been able to work out that this communication between cells during development actually uses a rather small handful of growth factors. They are used over and over in different combinations with dramatically different roles at different times.”

    To Grow A Lung

    This information also helps inform efforts to use induced pluripotent stem cells (iPSCs) to grow organs In recent years, Zorn has worked closely with James Wells, PhD, Director of the Pluripotent Stem Cell Center at Cincinnati Children’s. Wells’ team has had significant success in growing functional intestinal and stomach tissue from stem cells. (See related story, page 20) More recently, the two have teamed up with John Shannon, PhD, and Jeffrey Whitsett, MD, in the Division of Pulmonary Biology at Cincinnati Children’s, to work on lung development.

    “Compared to other organs, we have not had good models for studying very early stages of lung formation,” Zorn says. “Our understanding of the early stages of lung development has been murky. Now, we have about half our lab working on this.”

    The research team already has applied some of the signaling information learned from studying frogs to human stem cell cultures. This has helped them grow spheroids of lung progenitor cells that include both mesenchyme and endoderm.

    “This is very exciting because it more closely mimics the natural process of lung development than previous attempts,” Zorn says.

  • Dr. Aaron Zorn.

    Dr. Aaron Zorn.

  • Xenopus model

    Xenopus model 

  • Insights With Possibilities

    These fundamental research questions about early development are not as distant as they seem from day-to-day medical practice. Doctors at Cincinnati Children’s often care for the 3 to 4 percent of babies born each year in the U.S. with major organ defects. Understanding how organs form helps reveal how and why the process can go wrong, and how it can be corrected, says Aaron Zorn, PhD.

    ‘We still have a great deal of work to do understand these gene regulatory networks,’ Zorn says. ‘But as we do, there will be many applications.’

    • Even if it takes years to figure out how to grow full-sized replacement organs, the ability to use stem cells to grow small amounts of organ tissue opens the door to vastly improved drug screening.
    • As research reveals gene mutations that disrupt development, gene testing could inform would-be parents if they are carriers.
    • Research already shows that healthy prenatal development can be disrupted by environmental conditions, such as alcohol exposure, vitamin deficiencies or high cholesterol. Identifying the timing of key organ development signals may reveal particularly sensitive times during pregnancy where a lifestyle adjustment or a preventive treatment could save a life.
    • Improved testing could lead to earlier detection of major-but-survivable defects. This in turn could help guide expectant mothers to better-equipped hospitals and the best available surgeons.
    • Identifying the pathways used during embryonic development also could reveal how organ repair and regeneration works later in life — and suggest ways to speed up or amplify the process.