zebrafish embryo

Developing zebrafish embryo

Our understanding of how animals grow from a single cell to billions of cells has benefited tremendously from the easy-to-visualize nature of zebrafish embryos. This video begins roughly two hours after a zebrafish egg has been fertilized and covers approximately 24 hours of the embryo’s life. In less than a day, the embryo will progress through dramatic changes in shape as cells move and specialize in a process called gastrulation. By 9 hours after fertilization, the rudimentary brain will start to thicken, and by 12 hours, premature eyes form. Muscular twitches begin and exaggerate from 20 hours onward before the heart even starts beating properly. Within three days from the start of its one-cell journey, the fish will reach the length of a sesame seed before swimming in search of food.

Video by Dr. Andrei Kobitski, Dr. Jens Otte and Dr. Johannes Stegmaier, Karlsruhe Institute of Technology, Germany.

Scientists find gene vital to central nervous system development

Scientists have identified a gene that helps regulate how well nerves of the central nervous system are insulated, researchers at Washington University School of Medicine in St. Louis report.

Healthy insulation is vital for the speedy propagation of nerve cell signals. The finding, in zebrafish and mice, may have implications for human diseases like multiple sclerosis, in which this insulation is lost.

The study appears Jan. 21 in Nature Communications.

Nerve cells send electrical signals along lengthy projections called axons. These signals travel much faster when the axon is wrapped in myelin, an insulating layer of fats and proteins. In the central nervous system, the cells responsible for insulating axons are called oligodendrocytes.

The research focused on a gene called Gpr56, which manufactures a protein of the same name. Previous work indicated that this gene likely was involved in central nervous system development, but its specific roles were unclear.

In the new study, the researchers found that when the protein Gpr56 is disabled, there are too few oligodendrocytes to provide insulation for all of the axons. Still, the axons looked normal. And in the relatively few axons that were insulated, the myelin also looked normal. But the researchers observed many axons that were simply bare, not wrapped in any myelin at all.

Without Gpr56, the cells responsible for applying the insulation failed to reproduce themselves sufficiently, according to the study’s senior author, Kelly R. Monk, PhD, assistant professor of developmental biology. These cells actually matured too early instead of continuing to replicate as they should have. Consequently, in adulthood, there were not enough mature cells, leaving many axons without insulation.

Monk and her team study zebrafish because they are excellent models of the vertebrate nervous system. Their embryos are transparent and mature outside the body, making them useful for observing developmental processes.

“We first saw this defect in the developing zebrafish embryo,” said first author Sarah D. Ackerman, a graduate student in Monk’s lab. “But it’s not simply a temporary defect that only results in delayed myelination. When I looked at fish that were six months old, I still saw this problem of undermyelinated axons.”

In a companion paper in the same issue of Nature Communications, senior author Xianhua Piao, MD, PhD, of Harvard University, and her co-authors, including Monk, showed similar defects in mice without Gpr56. In past work, Piao also has shown evidence that human defects in Gpr56 lead to brain malformations related to a lack of myelin.

“These are nice studies that arrived at the same conclusion independently,” said Monk, who is also with the Hope Center for Neurological Disorders at Washington University. “Our Harvard colleagues used mouse models while we used fish models. And Dr. Piao’s research in human patients suggests that similar mechanisms are at work in people.”

Monk also said that Gpr56 belongs to a large class of cell receptors that are common targets for many commercially available drugs, making the protein attractive for further research. The investigators pointed out its possible relevance in treating diseases associated with a lack of myelin, with particular interest in multiple sclerosis.

“In the case of MS, there are areas where the central nervous system has lost its myelin,” Monk said. “At least part of the problem is that the precursor myelin-producing cells are recruited to that area, but they fail to become adult cells capable of producing nerve cell insulation. Now, we have evidence that Gpr56 modulates the switch from precursor to adult cell.”

In theory, if the precursor cells can be pushed to mature into adulthood, they may become capable of producing myelin. According to Monk and Ackerman, possible future work includes using the zebrafish model system as a drug-screening tool to search for small molecules that may flip that switch.

Zebrafish embryo

Just 22 hours after fertilization, this zebrafish embryo is already taking shape. By 36 hours, all of the major organs will have started to form. The zebrafish’s rapid growth and see-through embryo make it ideal for scientists studying how organs develop.

Image courtesy of Philipp Keller, Bill Lemon, Yinan Wan and Kristin Branson, Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, Va. Part of the exhibit Life:Magnified by ASCB and NIGMS.

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Epigenome orchestrates embryonic development

The early stages of embryonic development shape our cells and tissues for life. It is during this time that our newly formed cells are transformed into heart, skin, nerve or other cell types. Scientists are finding that this process is largely controlled not by the genome, but by the epigenome, chemical markers on DNA that tell cells when to turn genes on and off.

Now, studying zebrafish embryos, researchers at Washington University School of Medicine in St. Louis have shown that the epigenome plays a significant part in guiding development in the first 24 hours after fertilization.

The research, which appears Feb. 20 in the journal Nature Communications, may deepen understanding of congenital defects and miscarriage.

The epigenome is a bit like software that makes sense of the DNA code hard-wired into each of the body’s cells. While the DNA hardware is the same in each cell, differences in the epigenome — the software — differentiate brain cells from muscle, skin, eye or heart cells.

Using zebrafish as a model of vertebrate development, the new study is the first to map changes in the epigenome of whole embryos and their roles in gene regulation during the earliest hours of development.

“Our study suggests that an underappreciated fraction of the genome is involved in gene regulation,” said senior author Ting Wang, PhD, assistant professor of genetics. “Another surprising finding is that many of the important regions of DNA we identified are pretty far away from the genes they regulate.

“The field long has been focused on identifying genes that manufacture proteins,” Wang added. “We are showing that the epigenome is just as important and is an area that is largely uncharted.”

Wang is a principal investigator on the Roadmap Epigenomics Program, a national initiative to map the human epigenome, supported by the National Institutes of Health (NIH). Researchers leading this program recently published large data sets detailing information about human epigenetics.

“The human genome, like the zebrafish genome, is epigenetically regulated,” Wang said. “But in humans, for ethical reasons, we can only look at tissues in childhood and adulthood and describe differences between cell types. With zebrafish, we can watch the developmental process as it unfolds.”

To do so, Wang and first author Hyung Joo Lee, a graduate student in Wang’s lab, studied zebrafish embryos at five intervals after fertilization, stopping at the 24-hour mark, when the embryos start to develop separate tissues.  

At each of these points in time, the investigators measured several ways the epigenome regulates gene expression, one of the most important of which was with methyl groups. Methyl groups are organic compounds that attach to the DNA in different places. If many methyl groups are concentrated in a given area, the DNA is packaged away and gene expression is shut down. If the DNA is demethylated — with few or no methyl groups — genes are unpackaged and can be expressed.

Most studies of DNA methylation have focused on areas close to genes called promoters, which function like switches, turning gene expression on or off.

“But our data show that only 5 percent of DNA methylation changes happen in conventionally defined promoters,” Wang said.

The scientists were surprised to discover the remaining 95 percent of the methylation changes happened in regions far from genes and their promoters, in parts of the genome considered noncoding because they don’t tell the cell to make a particular protein.

Most of the changes in methylation at these distant sites involved losing methyl groups, which tends to increase gene expression. This gradual loss of methyl groups increased as the stages of development progressed, presumably turning on gene expression somewhere else. Using several techniques, the researchers were able to correlate the loss of methyl groups in one location with the dialing up of gene expression elsewhere.

This allowed them to statistically predict which noncoding regions of the genome were dialing up expression of distant genes. They determined these noncoding regions functioned as developmental enhancers, and they experimentally verified 20 of them in zebrafish. The data showed that these 20 enhancers drove expression of developmental genes in specific tissues, including the eye and parts of the brain and spinal cord.

The investigators pointed out that many developmental problems, whether they result in the loss of the embryo through miscarriage or in later disorders, can’t be pinned to a particular gene.

“This study suggests that many diseases may have an epigenetic origin,” Wang said. “Even if there is nothing wrong with the protein coding genes themselves, there are lots of different regulatory changes that could mess up gene expression and lead to disease.”

Wang noted that this study supports the trend of scientists finding more and more noncoding parts of the genome that play essential roles in gene regulation.

“I’m sure there are parts of the genome for which we may never find a function,” he said. “But when we look deep, we can derive very complex regulatory relationships between noncoding regions and the distant genes they regulate.”

Embryonic Zebrafish – a model organism for research
     into the genetic causes of neurodegeneration.
Scanning Electron Micrograph

TECHNICAL DETAILS

  • First, the sample was chemically fixed in 4% (w/v) paraformaldehyde in 0.1M Sodium Cacodylate buffer.
  • Then it was dehydrated.
  • Prior to imaging, the specimen was given a 5nm gold coating.
  • It was then imaged under a Scanning Electron Microscope 5KV.
  • A high tilt angle of 65 degrees enhanced the full structure of the head region.
  • The high-resolution digital image was imported into Photoshop and artistically colored.

Courtesy of David McCarthy (via FEI)

Neurons in a zebrafish embryo

Zebrafish have proven invaluable for understanding what we know about nerves and the brain. Observing brain development and interrogating how growing neurons find their correct targets are possible thanks to the transparent, genetically malleable nature of zebrafish embryos. Recently, scientists have developed a technique called “Brainbow” that individually colors each neuron, allowing researchers to map the start and end points of neural circuits. Applying Brainbow to zebrafish will allow researchers to visualize how neurons connect with one another during development and how different diseases disrupt this process.

Image by Dr. Albert Pan, Harvard University.

Zebrafish embryo

Just 22 hours after fertilization, this zebrafish embryo is already taking shape. By 36 hours, all of the major organs will have started to form. The zebrafish’s rapid growth and see-through embryo make it ideal for scientists studying how organs develop.

Credit: Philipp Keller, Bill Lemon, Yinan Wan and Kristin Branson, Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, Va.