Can you change a chicken into a frog, a fish or a chameleon?

Gastrulation is one of the most important phases in early embryonic development. Before gastrulation, vertebrate embryos are simple two-dimensional sheets of cells. By the end of gastrulation, an embryo will have begun to differentiate distinct cell types, set up the basic axes of the body and internalize some of the precursors for organs in a three-dimensional structure. Amniotes, like chickens and humans, will have developed a primitive streak, the precursor to the brain and skin, while fish and amphibians will have developed a spherical-shaped blastopore.

Gastrulation is a feat of self-organization, requiring the ballet-like coordinated movements of hundreds to tens of thousands of cells.

But, despite its importance in development, scientists only partially understand the underlying mechanisms that coordinate this large-scale movement of cells.

Now a team of researchers from the Harvard, the University of California San Diego and the University of Dundee in the U.K., have developed a theoretical framework that can reproduce and predict the patterns associated with gastrulation in a chicken embryo.

Building on their experimental findings published earlier this year in Science Advances, and using a combination of theory and experiment, the researchers demonstrated that small changes in cell parameters and behavior can have a dramatic impact on the resulting gastrulation patterns.

The new research was also published in Science Advances.

"Linking the developmental processes underlying morphogenesis in an organism to the variations of these same processes across evolution is an old question in biology," said L. Mahadevan, the Lola England de Valpine Professor of Applied Mathematics at the Harvard John A. Paulson School of Engineering and Applied Sciences (SEAS), and Professor of Organismic and Evolutionary Biology, and of Physics in the Faculty of Arts and Sciences (FAS) and senior author of the paper.

"Moving away from the gene-centric molecular focus on this question, we asked if there are emergent biophysical principles that operate on the multi-cellular level and can help explain the self-organized gastrulation patterns from both a developmental and an evolutionary perspective."

Working closely with experimentalists, the researchers developed a theoretical and computational model that could recreate the movement of the epithelial layer of cells in chick embryos during gastrulation.

The team then identified two parameters -- one related to the initial distribution of cells in an embryo and the other related to cell behavior -- to tweak during gastrulation.

"When we changed these two parameters in the model, which was only informed by the mechanisms of chick development, it remarkably showed gastrulation patterns naturally seen in other species," said Mattia Serra, first author of the paper and former Schmidt Science fellow in the Mahadevan group.

Serra is an Assistant Professor in Physics at UC San Diego.

Consistent with the computational results, experiments show that perturbing the same parameters in vivo in a chick embryo caused the chick to form a disk-like blastopore, seen in frogs, a ring-like circular primitive streak, like those seen in fish, or an elongated elliptical canal-like streak seen in chameleon.

"Our work suggests that the general biophysical principles underlying active self-organized flows and forces during embryogenesis have the power to explain developmental processes and their evolutionary variations across different species of vertebrates," said Mahadevan.

"We were surprised to see how simple mechanochemical rules could predict highly distinct coherent flows of thousands of cells measured in a living chick embryo," said Serra.

This research not only sheds light on the principles for self- organization in early development but could also help researchers understand the evolutionary history of developmental processes and suggest ways to control the development of synthetic organoids.

This research looked at the early stages of development, when the embryo is just transforming from two-dimensions into three.

Next, Mahadevan wants to understand what happens when the embryo starts to fold and buckle to create the more complex shapes of organs and eventually whole organisms.

"Using our knowledge of developmental processes at the molecular and cellular level, we eventually hope to provide an integrated framework for how cells form into tissues and tissues into organs, towards a better understanding of morphogenesis," said Mahadevan.

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Glassfrogs achieve transparency by packing red blood cells into mirror-coated liver

New research shows that glassfrogs -- known for their highly transparent undersides and muscles -- perform their "disappearing acts" by stowing away nearly all of their red blood cells into their uniquely reflective livers. The study, led by scientists at the American Museum of Natural History and Duke University, is being published Friday in the journal Science. The work could lead to new avenues of research tied to blood clots, which the frogs somehow avoid while packing and unpacking about 90 percent of their red blood cells into their livers on a daily basis.

"There are more than 150 species of known glassfrogs in the world, and yet we're really just starting to learn about some of the really incredible ways they interact with their environment," said co-lead author Jesse Delia, a Gerstner postdoctoral fellow in the Museum's Department of Herpetology.

Glassfrogs, which live in the American tropics, are nocturnal amphibians that spend their days sleeping upside down on translucent leaves that match the color of their backs -- a common camouflage tactic. Their tummies, however, show something surprising: translucent skin and muscle that allows their bones and organs to be visible, giving the glassfrog its common name. Recent research has proposed that this adaptation masks the frogs' outlines on their leafy perches, making them harder for predators to spot.

Transparency is a common form of camouflage among animals that live in water, but it's rare on land. In vertebrates, attaining transparency is difficult because their circulatory system is full of red blood cells that interact with light. Studies have shown that ice fish and larval eels achieve transparency by not producing hemoglobin and red blood cells. But glassfrogs use an alternative strategy, according to the findings of the new study.

"Glassfrogs overcome this challenge by essentially hiding red blood cells from view," said Carlos Taboada, the study's co-lead author from Duke University. "They almost pause their respiratory system during the day, even at high temperatures."

At Duke, the researchers used a technique called photoacoustic imaging, which uses light to induce sound-wave propagation from red blood cells. This allows researchers to map the location of the cells within sleeping frogs without restraint, contrast agents, sacrifice, or surgical manipulation -- particularly important to this study because glassfrog transparency is disrupted by activity, stress, anesthesia, and death.

The researchers focused on one particular species of glassfrog, Hyalinobatrachium fleischmanni. They found that resting glassfrogs increase transparency two- to threefold by removing nearly 90 percent of their red blood cells from circulation and packing them within their liver, which contains reflective guanine crystals. Whenever the frogs need to become active again, they bring the red blood cells back into the blood, which gives the frogs the ability to move around -- at which point, light absorption from these cells breaks transparency.

In most vertebrates, aggregating red blood cells can lead to potentially dangerous blood clots in veins and arteries. But glassfrogs don't experience clotting, which raises a set of significant questions for biological and medical researchers.

"This is the first of a series of studies documenting the physiology of vertebrate transparency, and it will hopefully stimulate biomedical work to translate these frogs' extreme physiology into novel targets for human health and medicine," Delia said.

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Scientists regrow frog's lost leg

For millions of patients who have lost limbs for reasons ranging from diabetes to trauma, the possibility of regaining function through natural regeneration remains out of reach. Regrowth of legs and arms remains the province of salamanders and superheroes.

But in a study published in the journal Science Advances, scientists at Tufts University and Harvard University's Wyss Institute have brought us a step closer to the goal of regenerative medicine.

On adult frogs, which are naturally unable to regenerate limbs, the researchers were able to trigger regrowth of a lost leg using a five-drug cocktail applied in a silicone wearable bioreactor dome that seals in the elixir over the stump for just 24 hours. That brief treatment sets in motion an 18-month period of regrowth that restores a functional leg.

Many creatures have the capability of full regeneration of at least some limbs, including salamanders, starfish, crabs, and lizards. Flatworms can even be cut up into pieces, with each piece reconstructing an entire organism. Humans are capable of closing wounds with new tissue growth, and our livers have a remarkable, almost flatworm-like capability of regenerating to full size after a 50% loss.

But loss of a large and structurally complex limb -- an arm or leg -- cannot be restored by any natural process of regeneration in humans or mammals. In fact, we tend to cover major injuries with an amorphous mass of scar tissue, protecting it from further blood loss and infection and preventing further growth.

Kickstarting Regeneration

The Tufts researchers triggered the regenerative process in African clawed frogs by enclosing the wound in a silicone cap, which they call a BioDome, containing a silk protein gel loaded with the five-drug cocktail.

Each drug fulfilled a different purpose, including tamping down inflammation, inhibiting the production of collagen which would lead to scarring, and encouraging the new growth of nerve fibers, blood vessels, and muscle. The combination and the bioreactor provided a local environment and signals that tipped the scales away from the natural tendency to close off the stump, and toward the regenerative process.

The researchers observed dramatic growth of tissue in many of the treated frogs, re-creating an almost fully functional leg. The new limbs had bone structure extended with features similar to a natural limb's bone structure, a richer complement of internal tissues (including neurons), and several "toes" grew from the end of the limb, although without the support of underlying bone.

The regrown limb moved and responded to stimuli such as a touch from a stiff fiber, and the frogs were able to make use of it for swimming through water, moving much like a normal frog would.

"It's exciting to see that the drugs we selected were helping to create an almost complete limb," said Nirosha Murugan, research affiliate at the Allen Discovery Center at Tufts and first author of the paper. "The fact that it required only a brief exposure to the drugs to set in motion a months-long regeneration process suggests that frogs and perhaps other animals may have dormant regenerative capabilities that can be triggered into action."

The researchers explored the mechanisms by which the brief intervention could lead to long-term growth. Within the first few days after treatment, they detected the activation of known molecular pathways that are normally used in a developing embryo to help the body take shape.

Activation of these pathways could allow the burden of growth and organization of tissue to be handled by the limb itself, similar to how it occurs in an embryo, rather than require ongoing therapeutic intervention over the many months it takes to grow the limb.

How the BioDome Works

Animals naturally capable of regeneration live mostly in an aquatic environment. The first stage of growth after loss of a limb is the formation of a mass of stem cells at the end of the stump called a blastema, which is used to gradually reconstruct the lost body part. The wound is rapidly covered by skin cells within the first 24 hours after the injury, protecting the reconstructing tissue underneath.

"Mammals and other regenerating animals will usually have their injuries exposed to air or making contact with the ground, and they can take days to weeks to close up with scar tissue," said David Kaplan, Stern Family Professor of Engineering at Tufts and co-author of the study. "Using the BioDome cap in the first 24 hours helps mimic an amniotic-like environment which, along with the right drugs, allows the rebuilding process to proceed without the interference of scar tissue."

Next Steps in Frogs and Mammals

Previous work by the Tufts team showed a significant degree of limb growth triggered by a single drug, progesterone, with the BioDome. However, the resulting limb grew as a spike and was far from the more normally shaped, functional limb achieved in the current study.

The five-drug cocktail represents a significant milestone toward the restoration of fully functional frog limbs and suggests further exploration of drug and growth factor combinations could lead to regrown limbs that are even more functionally complete, with normal digits, webbing, and more detailed skeletal and muscular features.

"We'll be testing how this treatment could apply to mammals next," said corresponding author Michael Levin, Vannevar Bush Professor of Biology in the School of Arts & Sciences, director of the Allen Discovery Center at Tufts, and associate faculty member of the Wyss Institute.

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