Mouth to mouth

What can a frog mouth tell us about human birth defects and evolution?

As early embryos, humans bear a striking resemblance to frogs. Both species comprise three basic cell types, arranged in the same general pattern. And that isn’t surprising, considering we evolved from a common ancestor.

But where does the likeness end? The jury is still out on which developmental processes we share.

“Our wild and crazy idea is that animals as different as sea urchins and humans use the same biological mechanisms to organize their heads,” says Whitehead Member Hazel Sive.

Whitehead postdoctoral researcher Amanda Dickinson mapped how a hole pokes through in a frog to form the primary mouth, or the first opening in the organism. First, a membrane between two layers of cells dissolves where the hole is destined to be. The cells begin to mix, and some of them die as part of a thinning process. Finally, a hole forms, connecting the gut to the outside world. Photos: Amanda Dickinson

Her lab is beginning to test this idea in the frog, Xenopus, which is easy to study, and whose mouth is very similar to the human mouth.

Once researchers understand this animal, they will look at other organisms, including more primitive animals whose heads comprise just a hollow cylinder of cells, to compare processes.

Slice a frog embryo approximately 12 hours after it is fertilized and you’ll see three layers—endoderm, mesoderm and ectoderm—which eventually give rise to all of the animal’s tissues.

But the extreme front end (anterior) of the embryo lacks mesoderm, (the middle layer), just comprising the ectoderm and endoderm. Human embryos exhibit the same pattern.

In the most primitive animals, there is no mesoderm anywhere in the animal. Sive thinks that the lack of mesoderm in the extreme front of higher animals is a relic of ancient evolutionary processes, and a persistence of a process that occurred in the most primitive animals.

Sive hopes her research will yield clues about evolution. “This idea isn’t in any developmental biology textbooks yet, but the position of the cells at the front of all animal embryos is remarkably similar,” she says.

The similarity of mouth formation in frogs and humans also will shed light on the mechanisms behind human craniofacial birth defects.

Such defects, which range from cleft palates to underdeveloped jaws, account for three-fourths of all structural birth defects, according to the National Institute of Dental and Craniofacial Research. Cleft palates alone afflict roughly 8,000 newborns in the United States each year.

If frogs and humans use the same genetic circuitry to control formation of the mouth and head, then scientists might be able to apply findings from one species to the other. That’s one reason Sive’s lab is studying mouth development in the frog.

How to make a mouth

Postdoctoral researcher Amanda Dickinson managed to map the steps required to make the primary mouth, the first opening to form in the embryo, and published her results last July in the journal Developmental Biology.

The primary mouth connects the gut to the outside and allows feeding. At a later stage, the secondary mouth—which includes the jaws, teeth and tongue—grows around this hole, allowing organisms to chew.

“We think the primary mouth might play a major role in positioning other parts of the face, which is one of the reasons it’s critical to understand how this initial opening forms,” says Sive.

Although other labs had studied particular aspects of primary mouth development in a variety of model organisms, Dickinson was the first to tie this work together in a single critter. Further, she is the first to approach this process using molecular tools, with the ability to identify the genes involved.

Dickinson traced the movement of individual cells as a frog embryo’s gut tube poked through to the outside world (see the image series to the right). Initially, the endoderm cells at the end of the gut tube are encased by ectoderm. The endoderm and ectoderm are kept separate by a basement membrane between them. This basement membrane begins to disappear where the hole is destined to form, an oddity in biological terms.

Amanda Dickinson, shown here with Whitehead Member Hazel Sive, was inspired to probe the connections between species after playing with animals in Nova Scotia tide pools as a child. Photo: Kim Furnald

“Normally, the endoderm and ectoderm never mix,” says Sive. “As the basement membrane breaks down, these layers have to overcome their hatred of one another.”

The sworn enemies grow flexible as the materials between them disappear. In fact, the endoderm and ectoderm lose so much of their stiffness that they cave toward the center of the embryo, forming a dimple on its surface.

“At around the same time, the two layers begin to mix, which is truly remarkable,” says Dickinson. “It’s as if you shuffled two decks of playing cards.”

As endoderm and ectoderm cells mix, some of them begin to die. The mixed mass grows thinner and thinner until it’s just a single layer of cells. Finally, this layer, which is stretched thin like the membrane of a balloon, pops open in the middle, producing the primary mouth. Dickinson and postdoc Colin DeBakker are now working to pinpoint the networks of genes that control each step of this process.

“The animal would have real problems if holes started appearing all over the body, so we’re interested in the genetic circuitry that coordinates formation of just the right size hole in just the right place,” says Sive.

Cementing evolution

New genetic analyses tie into other work in the Sive lab on the extreme anterior region of embryos.

Postdoc Shuhong Li concentrates on the genes and proteins that control a much simpler organ—the cement gland, which sits just below the primary mouth. Sive likens this organ to an underwater Post-it note, which secrets mucus to help frog embryos stick to solid surfaces so they won’t be washed away. The Sive lab has pioneered use of the cement gland as a “marker” for the extreme anterior of the embryo.

Humans lack cement glands, but many aquatic vertebrates depend on them for survival. A cement gland consists of just one layer of cells, so it is easy to study. Li and others have identified many of the proteins that turn on the genes that make the cement gland. Li is building a detailed genetic circuit diagram that describes how this organ is positioned at the extreme anterior.

“My job is to isolate the factors that control which genes are expressed in the region, and more and more evidence shows that some of these regulatory mechanisms play a role in multiple organs and multiple species, perhaps even humans,” he says.

Thus Li’s analysis might help Dickinson and DeBakker unravel the factors that control primary mouth formation in frogs and shed light on the interactions between genes and proteins in humans. And the Sive lab’s studies highlight how basic research on seemingly esoteric organs or systems can yield information that aids human biomedical research.