Zebrafish's powerful heart gene could lead to transplant therapy

December 08, 1999

Researchers have discovered a gene in zebrafish so powerful it can be used to redirect the fate of cells in the developing embryo to become beating heart cells, suggesting that a similar gene in humans could be used to generate heart cells in culture for transplant in ailing people.

The finding, the first discovery of a so-called "master" gene for myocardial, or heart muscle, cells in an animal model, puts researchers on track for exploring the capability of homologous genes in mice and humans.

The gene, known as gata5, acts in embryonic cells, which are primordial, unspecialized cells that form in the earliest stage of embryonic development and are genetically programmed to evolve into one of many specialized cell types, such as skeletal muscle cells, nerve cells, blood cells, skin cells, and liver cells.

Normally, the gene acts only in those embryonic cells destined to become myocardial cells. The research shows, however, that gata5 can reprogram otherwise predestined embryonic cell types to become beating heart cells.

If zebrafish gata5, or its human equivalent, could prompt a particular type of embryonic cell, known as a human embryonic stem cell, to become a beating heart cell, researchers could theoretically use this technique to cultivate and harvest such genetically modified cells in the petri dish and then transplant them into people with failing hearts.

"Discovery of a gene that could convert human embryonic stem cells into myocardial cells would be golden," said Didier Stainier, PhD, UCSF assistant professor of biochemistry and biophysics, the senior author of the UCSF study and a pioneer in the study of heart development in the transparent zebrafish embryo. "Gata5 is potentially such a gene. It appears to be sufficient to drive the entire myocardial program in certain cells not normally fated to contribute to the heart."

Gata5's capability produces dramatic images

Stainier and colleagues' discovery of gata5's power presented powerful images: Removal of gata5 from cells normally destined to become myocardial cells caused profound defects in the formation of the heart. Expression of gata5 throughout the embryo caused heart cells to form and beat -- spontaneously and rhythmically -- as far away from the head region, where the heart forms, as the animals' lower trunk.

The finding, published in the current issue of Genes and Development, represents a different approach to cultivating specialized cells for transplant therapy than that being pursued in other labs. Current efforts, mostly conducted in animal models, involve attempting to derive and aggregate embryonic stem cells, exposing them to such factors as acetic acid, allowing them to differentiate, or specialize, and then sorting through these cells to extract the cell types of interest.

"Using different regulators, scientists have been able to induce a subset of myocardial characteristics in various experimental models, but never the complete beating phenotype, so there is something special about gata5 that can take a cell that's not supposed to become a heart cell to actually become one," said Stainier. "We're very excited about this finding."

Gata5 a potential diagnostic marker for congenital heart disease

The finding also suggests that gata5 could be a potential new diagnostic marker for congenital heart defects, as the researchers demonstrated that gata5 regulates the expression of a gene known as nkx2.5 which, when mutated in humans, causes human congenital heart defects and disease. Human geneticists and cardiologists studying families with heart disease may discover, said Stainier, that a mutated form of the gata5 homologue occurs in some cases of heart disease, in which case the mutated form of the gene could serve as a marker of predisposition to the disease.

The researchers identified the human gata5 gene and mapped it to chromosome 20 as a first step towards identifying human mutations.

The Nkx2.5 gene is the equivalent of a fly gene called "tinman," which was discovered in 1993, by Ralph Bodmer, PhD, in the laboratory of Yuh Nung Jan, PhD, UCSF professor of physiology and biochemistry,. Like the Wizard of Oz's man of tin, the mutated fly gene, Bodmer discovered, led to a fly that lacked a heart.

"This is a case where scientists went from a fly mutation to a fly gene to a human mutation that causes congenital heart defects," said Stainier. "It validates the approach of using even very distant model systems, such as the fly, to study human biology. We show that in the hierarchy of genes that control myocardial differentiation, gata5 acts early in the pathway."

Diaphanous zebrafish embryo reveals its 'artful etchings'

The significance of the UCSF discoveries dramatizes the importance of the tiny blue-and-silver striped denizen of India's Ganges River - and many an aquarium -- as a model for biomedical studies. Until recently studied in only a handful of labs worldwide, it is increasingly surfacing at the lab bench.

The reasons for its appeal are many, but none rival the fact that the crystal-clear zebrafish embryo offers a view of burgeoning life that no other vertebrate model can. Less than a day after fertilization, the fertilized egg has sprouted a two-chambered beating heart, ears and eyes and a tail that flicks. By the third day, all of its major organs have fully developed and moved into proper position, offering scientists a view of what has been referred to as the zebrafish's "artful etchings." (See images, below.)

Less dramatic but of great import, zebrafish, like mice and humans, are vertebrate - having a backbone and a tubular nervous system divided into the brain and spinal cord - and are therefore more likely to be genetically similar to humans than non-vertebrate models such as yeast, roundworms and fruit flies (even though the latter have provided valuable information about mechanisms in vertebrate).

Moreover, unlike other vertebrate model systems (principally the frog, chick and mouse), the animal offers the opportunity to search for mutations that disrupt specific biological events by a process that does not require prior knowledge of the gene or genes involved. This approach is also used in invertebrate organisms such as Drosophila (fruit flies) and Caenorhabditis elegans (a roundworm).

So desirable is the zebrafish as a scientific model that the National Institutes of Health recently launched the NIH Zebrafish Initiative Website, offering funding for studies of cancer, cardiovascular, blood and pulmonary diseases, eye development and disease, gene function, circadian rhythms, aging, longevity, immune system development and function, addiction, hearing, balance, smell and taste. http://www.nih.gov/science/models/zebrafish/

Study pursues the genes with which a zebrafish views its world Neuroscientist biologist Herwig Baier, PhD, UCSF assistant professor of physiology and formerly of the Max Planck Institute in Tubingen, is working to identify zebrafish genes that play a role in visual perception. The fish make an ideal model for these studies, as they need no training to exhibit several easily recognized behaviors in response to visual cues in their watery surroundings.

Stainier and colleagues, meanwhile, recently reported other findings in the zebrafish embryo that reveal basic mechanisms of development and could lead to an understanding of some birth defects.

Heart buds nudge toward one another...to form a beating heart

In one study, researchers identified a set of genes needed to prompt the two buds of heart cells that form early in development to migrate toward one another to form a single beating heart. The researchers came upon their finding serendipitously, after working out the molecular pathway, or succession of genes, that prompt the early-stage formation of the endoderm, one of the three layers of cells that form the developing embryo.

"Discovering a molecular pathway of endoderm formation was unchartered territory in itself," said Stainier. "This is the first time a group of scientists has been able to bring a set of genes together and show how they fit together to control the formation in this layer of the developing embryo."

But then Stainier and colleagues showed that the endoderm was also somehow responsible for the migration of the heart cell buds in the layer of cells above it, known as the mesoderm. And that, said Stainier, was unexpected.

Normally, two heart buds emerge on either side of the mesoderm, and ultimately merge to form a single heart. But the researchers discovered, much to their surprise, that when the underlying endoderm layer was not in place, the heart buds did not migrate toward one another; instead, they formed two hearts, a condition known as cardia bifida.

Researchers have known that the embryo's three layers form simultaneously, in a rhythm dependent on cell-signaling cross talk. But the degree of interaction was not clear. "We thought we were studying hearts but we really were studying the role of the endoderm," said Stainier. "That's one of the exciting things about genetics - you don't know where it will take you."

These findings were reported in Current Biology and Developmental Biology.

Cell movements display choreography of heart formation

Stainier and colleagues also recently analyzed the complex process by which the two groups of heart cells that initially bud in the mesoderm cell layer actually form the heart, which is a complex, 3-D structure. They didn't identify the genes involved, but they did learn about the basic events involved. Specifically, using new heart-specific genes, they were able to follow in detail the cell movements that lead to the formation of the heart, as well as its divisions into two chambers, the atrium (the chamber that receives blood and passes it to the ventricles) and the ventricle (the pumping chamber). This work will allow the researchers to formulate specific hypotheses regarding the various cell interactions and molecules involved in these processes. The finding was reported in Developmental Biology.

With potential therapeutic implications, Stainier and a colleague also recently discovered that endothelial cells, which line the blood vessels, play a critical role in inducing the development, or proper differentiation, of red blood cells, the transporters of oxygen. When endothelial cells were removed from the developing zebrafish embryo, red blood cells did not form. When endothelial cells were restored, blood cells developed. (The researchers were able to conduct this experiment because the embryos can survive without blood for at least seven days.). Presumably, said Stainier, the endothelial cells act by releasing an as-yet-unidentified factor.

The finding represents the first demonstration in an animal model, Stainier said, that endothelial cells are necessary for the proper differentiation of red blood cells. He said he suspects the discovery, published in Development,5 will apply to the immune system's white blood cells, as well.

Identifying the factor released by endothelial cells could lead, said Stainier, to a mechanism for prompting naive, or undifferentiated, cells to develop into red blood cells, which could then be used to boost the blood supply. "The insights zebrafish can offer into human biology and medicine are boundless," said Stainier. "We will be learning from these animals for years to come."

1) Co-authors of the gata5 study, published in Genes & Development, were lead author Jeremy Reiter, and Jonathan Alexander, both MD/PhD students funded by the NIH Medical Scientific Training Program and students in Stainier's lab, Deborah Yelon, PhD, a postdoctoral fellow in Stainier's lab, Adam Rodaway, PhD, and Roger Patient, PhD, both of Developmental Biology Research Centre, The Randall Institute, King's College London, and the late Nigel Holder, PhD, of the Department of Anatomy and Developmental Biology, University College London.

The study was funded by the American Heart Association, the National Institutes of Health, the Packard Foundation, the Life and Health Medical Insurance Fund and the Sandler Foundation.

2) The co-author of the study revealing the molecular pathway of endoderm development, published in Current Biology, (September) was Jonathan Alexander. The study was funded by the American Heart Association and the Packard Foundation

3) The co-authors of the role of the endoderm in heart formation study, published Developmental Biology, (September) were Jonathan Alexander, Michael Rothenberg, a graduate student in Jun's lab, and Gilbert L. Henry, PhD, of the Department of Molecular and Cellular Biology at Harvard University. The study was funded by the American Heart Association and the Packard Foundation.

4) Co-authors of the study of heart structure development, published in Developmental Biology (May), were Deborah Yelon and Sally Horne, a graduate student. The study was funded by NIH.

5) The co-author of the study revealing the role of endothelial cells in blood cell development, published in Development (September) was Leon Parker, PhD, at the time a UCSF postdoctoral fellow in Stainier's lab. The study was funded by the National Institutes of Health and the Packard Foundation.

University of California - San Francisco

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