The Incredible, Malleable Embryo

by Kathleen M. Wong

Richard Harland is the C.H. Li Distinguished Professor of Genetics, Genomics and Development. Photo credit: Center for Integrative Genomics

The egg is a bona-fide miracle of biology. This single spherical cell, plump with raw nutrients and protoplasm, begins development by getting organized. It divides and elongates, establishes a back and a front, crinkles itself a spinal cord and builds a skeleton. All of this makes the early embryo the rare place where order sprouts from chaos, and entropy operates in reverse.

Richard Harland, a Berkeley professor of genetics, genomics and development, is probing the signals responsible for early embryonic development. From the pathways involved in setting up a body plan, to the formation of the spinal cord, Harland is unraveling how key molecules pattern faces, limbs, skulls and bones.

Harland performs much of his research in frogs. The large eggs of these amphibians develop outside the mother, making the earliest stages of embryonic growth easy to observe and manipulate.

"Looking at the simple early embryo of the frog is one of the ways we can get more insights than trying to understand something complicated right off the bat. We can inject things and ask what happens, and we know enough about frog development to interpret how they work," Harland says.

The eyes, brain, pigment cells, muscles, and inner ear structures of tadpoles are all clearly visible, making them good subjects for Harland's research into early development.

When he first began this research some twenty years ago, he sought genes that might influence development. "We were hoping some would induce an extra axis or an extra head." In short, Harland says, "we were looking for monsters, as all embryologists do."

To screen for these genes, Harland injected frog eggs with mRNAs, the genetic transcripts which cells read to build proteins. He hit pay dirt with a gene called noggin. When injected into a developing embryo's belly, noggin produces a tadpole with two backs. It is critical for forming the dorso-ventral axis, or what are considered the spine and belly.

Harland then tried the reverse experiment, blocking noggin and other suspected body plan factors one by one. But these experiments seemed to have no effect. Only after knocking out three of the five known factors did Harland and colleagues succeed in making a monster. The altered tadpoles failed to build a brain, spinal cord, or other back structures, forming extra belly tissue instead. These findings indicate that the five factors have overlapping functions in early development.

Harland is developing a new model organism for embryology, the frog Xenopus tropicalis. X. tropicalis has two sets of chromosomes, making it a better subject for genetic experiments than its longstanding lab model cousin X. laevis, whose four chromosome sets make gene manipulations difficult. Photo credit: Richard Harland

Noggin and its ilk, Harland discovered, are antagonists that block a molecule called BMP, or bone morphogenetic protein. Early on, BMP forms belly structures. "Here is a case where the blockage of information is just as important as an active signaling pathway," Harland says. "BMP works everywhere except the back. The antagonists block BMP there to allow the formation of the nervous system, the spine and so on."

A mouse lacking the noggin gene lays down extra cartilage in its limbs, grows thicker bones and fails to develop finger joints. Photo credit: Lisa Brunet (UC Berkeley)

Harland then knocked out noggin in mice. These animals developed normally until it came time to form the skeleton and nervous system. Their limbs were about the right overall size and length, but both cartilage and bone were grossly thickened, while the fingers lacked joints. The spinal cord was mispatterned, and the mice failed to develop mature motor neurons. Harland deduced that noggin moderates BMP activity during normal skeletal and neurological development. The find could help researchers better understand skeletal problems such as joint maintenance, excess bone deposition, and diseases that transform connective tissue into bone.

"All of these signals are used again and again in development. We've found that the same genes and signals in the frog, and in the mouse, form the body plan and the skeleton," Harland says.

Another time when noggin is crucial is during skull development. While the bones of the face fuse soon after birth, the cranium retains open sutures to permit brain growth. Sutures that stay open express noggin.

Harland wondered how noggin might alter development in an area that normally fuses early-the nose and mouth. "It did what we thought," Harland says, by blocking nose bone fusion, "but I was surprised by the extent to which the skull was different."

A normal mouse (left) and a mouse that expressed extra noggin around its nose. The gene prevents the nose bone from fusing, resulting in a wider snout. Photo credit: Steve Warren, Mike Longaker (Stanford) Lisa Brunet (UC Berkeley)

Instead of the long, pointed nose of a typical mouse, the embryo developed a broad, truncated muzzle with an eerie resemblance to the stubby snout of a bat. "These are the kinds of molecules that drive the differences in skeleton forms," Harland says forming body plans ranging from the long fingers that form the wings of bats, to the elongated neck bones of giraffe, to the pointy faces of anteaters.

"Although we are passionate about understanding what happens in a frog embryo, what we learn turns out to be interesting and applicable to both medicine and human development," Harland says.

Related Web Sites

• Virtual Harland Lab
• Harland Lab X. Tropicalis Site
• "Scientists discover that three overlapping signals in embryo help get the backbone right" by Robert Sanders, Berkeley NewsCenter, 09 March 2005
• "Single gene plays key role in neural tube defects," by Robert Sanders, Berkeley NewsCenter, 15 December 2003
• "Researchers Identify Gene that Regulates Joints," by Patricia McBroom, Berkeley NewsCenter, 19 August 1998
• "A gene that creates joints in skeletons has been identified by UC Berkeley embryologist," by Patricia McBroom, Berkeley NewsCenter, 29 May 1998

Biting into Evolution

by Kathleen M. Wong

Paleontologist Leslea Hlusko spends three to six months a year prospecting for early hominid fossils in East Africa.

Bury a body, and time will take its toll. Flesh rots and even bone returns to the dust from whence it came. Teeth, however, are tough enough to withstand the processes of decay for much longer, making them among the best-preserved and most abundant mammalian fossils.

So it's no wonder that paleontologist Leslea Hlusko studies teeth as a means to observe the processes of evolution. A professor of integrative biology at UC Berkeley, Hlusko studies variation and genetic change in the dentition of modern primates. By obtaining a better understanding of how genes control tooth anatomy in living primates, she hopes to gain insights into the fossil record and human evolution.

To do this, Hlusko has amassed a collection of more than 600 dental castings from a colony of baboons at the Southwest Foundation for Biomedical Research in Texas. Her laboratory has painstakingly measured characteristics such as length and width, the presence of extra cusps, and enamel thickness for each dentition.

Because Hlusko has pedigrees for every baboon going back several generations, she can use statistical methods to identify whether or not these dental phenotypes, or expressed traits, are inherited. She can also tell whether these traits change independently or in lockstep with other dental characteristics.

Graduate student Sarah Amugongo examines a baboon skull in the UC Museum of Vertebrate Zoology. After studying the intricacies of primate teeth, many of Hlusko's undergraduate students go on to dental or medical school. Photo credit: Leslea Hlusko

This approach is known as quantitative genetics. "It's Mendel's pea plants-looking at how variation is inherited in a family," Hlusko says. "If genetic effects underlie the variation in a trait, then we know that the variation will respond to selection," Hlusko says.

Hlusko has already applied these findings to help interpret the fossil record. For years, anthropologists have used tooth enamel thickness as a means to help sort early primate fossils into either chimpanzee or hominid lineages. Their reasoning was that modern chimpanzees have a fairly thin coating of enamel on their molars, while human teeth have a somewhat thicker coating.

Hlusko's work shows that in baboons, enamel thickness changes independently from any other tooth trait measured. Independent traits can shift rapidly from generation to generation, because no other characteristics are impacted by the change.

"We argued that paleontologists might want to be careful about using enamel thickness as such an important trait in determining whether fossils are early hominids or early chimps," Hlusko says, "because our findings suggest this trait can vary from thick to thin quite quickly over evolutionary time, perhaps just one hundred thousand years or so."

Hlusko is now determining if the inheritance patterns she has found in baboons and has gone on to model genetically in mice, hold true for other primates. She and her students are now in the midst of a project measuring the teeth of other baboons and Old World monkey specimens in museum collections across the United States.

Hlusko and Dr. Jackson Njau of the National Natural History Museum in Arusha are using satellite technology to survey Tanzania for promising new fossil sites. Tanzania's Mount Hanang can be seen in the background. Photo credit: courtesy Leslea Hlusko

"I've seen evolutionary quantitative genetics hold up in other systems such as body and wing dimensions in flies, beak and body measurements in finches, and head size in New World monkeys. But nobody's yet looked at teeth or brought in fossils before," Hlusko says. If she sees the same patterns in such distantly related living species, the principles are likely to be universal enough to use to interpret primate fossils.

Hlusko is now reaching out to other colleagues who are working on the genetics and evolution of the skeleton, around the Bay Area. To provide a framework for these meetings, Hlusko has organized a consortium of biologists called the Genetics and Evolution of the Skeleton Research Initiative. Participants so far include medical researchers, evolutionary biologists, anthropologists, and paleontologists like Hlusko. The group will hold its first symposium on March 26, and feature scientists from UC Berkeley, UC San Francisco and Lawrence Livermore National Laboratory.

"By bringing everyone together we create an opportunity to share our various research methodologies and perspectives. Such a multidisciplinary approach to skeletal biology will likely help us better design our research questions and end up with more insightful answers," Hlusko says. She has high hopes that the initiative and the symposium will yield fruitful research partnerships and interdisciplinary study opportunities for students in the future.

A program schedule for the March 26 symposium is available as a PDF: Program Schedule

Related Web Sites

• The Hlusko Lab
• The genes that lie beneath: The work of Leslea Hlusko
• "Myriad perspectives on biological differences," by Wendy Edelstein, Berkeley NewsCenter, 31 March 2005
• "Ancient man 'used tooth picks,'" BBC News, 6 November 2003
• UC Museum of Paleontology profile: Leslea Hlusko

Spiky Fish Reveals Evolution's Toolkit

by Kathleen M. Wong

Craig Miller is now studying the mechanisms that allow sticklebacks to gain features when they move to fresh water. Photo credit: Craig Miller

The threespine stickleback is one adaptable little fish. Roughly the size and shape of a sardine, with a few bony spikes protruding from its back and pelvis, it has swum the oceans of the Northern Hemisphere for over 5 million years, remaining virtually unchanged for the duration.

But this homely relative of the seahorse isn't limited to merely oceans. Over the millennia, marine sticklebacks have colonized freshwater streams and lakes from California to Canada and Eurasia. With time, these aquatic populations have evolved into suspiciously similar-looking fish.

To make the transition to freshwater, sticklebacks tend to lose many of the same traits, including the bony armor plates along their flanks; the gill rakers used to strain plankton from seawater; even the namesake dorsal spines that help prevent ingestion by larger fish.

"Along with Darwin's finches, they are a classic example of adaptive radiation," says Craig Miller, a professor of genetics, genomics and development at Berkeley.

The threespine stickleback can be found throughout coastal lakes and rivers in the Northern Hemisphere, including 58 watersheds in the Bay Area alone. Photo credit: Craig Miller

Miller is studying the steps required to morph a marine stickleback into myriad freshwater editions. By practicing a combination of fish breeding, genetics, molecular and developmental biology, he is pinpointing the exact genes and gene variants, or alleles, responsible for aquatic-adapted traits. His discoveries are revealing the means, the methods and the limits of evolutionary whimsy while shedding light on human genetics, too.

"I'm interested in using genetics to understand the variation you see in nature. I want to look beyond a model organism that's raised in the lab and try to study variation in adult form in natural populations - because adult form is the ultimate phenotype upon which natural selection acts."

The ecological forces driving stickleback change are sometimes easy to infer. Many lakes and streams lack large predators, eliminating the need for bony defenses. And since some watersheds are low in the calcium needed to build skeletons, or may have carnivorous insects that can use the spines as handles, some marine traits became liabilities in freshwater.

When marine sticklebacks colonize freshwater, they evolve very different characteristics. Freshwater fish (below) tend to lose the flank armor, dorsal and pelvic spines of their marine forebears (above). Bone is stained red. Photo credit: Pamela Colosimo and David Kingsley, Stanford University

Less clear are the genetic shifts enabling these transformations. "When evolution has a problem like freshwater adaptation to solve, how many genetic solutions are there for that problem? Thousands? Or will you see the same genetic mechanism at work again and again in different populations?" Miller asks.

To find out, Miller has studied another difference between marine and freshwater sticklebacks - their coloring. Marine fish have a uniform charcoal gray layer of pigmentation beneath silver scales. Aquatic fish tend to be a more pearly gray on the back, with a light patterning of stripes along the flanks. They also lack the spots of pigment found on the gills of their marine cousins.

By studying the offspring from a cross of one marine and one freshwater fish, Miller and collaborators were able to determine that this pigmentation pattern was under strong genetic control. With the help of genetic markers, he was able to map the gene to a small region of DNA. Referring to the recently completed stickleback genome sequence, Miller found the region contained what he calls a "smoking gun" candidate gene.

Called Kit ligand, or Kitlg, the gene is required for the migration, proliferation and survival of pigment cell precursors. The gene is also present in mammals, where it is involved in the development of pigment cells as well as the formation of germ and blood cells.

Ocean sticklebacks (above) are dark-colored fish that often migrate into freshwater. Those that colonize new lakes and streams often evolve lighter gill and skin colors (below). Photo credit: Frank Chan, Craig Miller and David Kingsley

Based on this data, Miller and colleagues investigated whether Kitlg might contribute to human skin pigmentation. Previous researchers had found one version of the gene to be present in most Africans, while another version was nearly universal in both Europeans and Asians. Lighter skin, scientists believe, enabled humans to absorb more sunlight at higher latitudes and produce sufficient levels of vitamin D. Ultimately, Miller and colleagues found the gene underlies about 20 percent of the skin pigmentation differences between Africans and Northern Europeans.

"It's a nice example of parallel evolution in fish and humans. The genetic answer that this strange fish has used again and again is also the genetic answer used in other animals like humans," Miller says.

Other researchers have traced other characteristics of freshwater sticklebacks, the armor reduction and loss of the fish pelvic spines. In each of these three cases, the same genes are used to evolve the trait.

"Not just the same genes, but even the same alleles, are used repeatedly upon freshwater adaptation," Miller says. So while Nature's variety may be nearly infinite, she may travel certain routes to get there again and again.

Related Web Sites

• Craig Miller, Department of Molecular and Cell Biology
• "Same Genetic Machinery Generates Skin Color Evolution in Fish and Humans," HHMI News, 14 December 2007

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