Zebrafish's Powerful Heart Gene Could Lead To Transplant Therapy

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 forexploring 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 developmentand are genetically programmed to evolve into one of many specialized celltypes, such as skeletal muscle cells, nerve cells, blood cells, skin cells, andliver cells.

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

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

"Discovery of a gene that could convert human embryonic stem cells intomyocardial cells would be golden," said Didier Stainier, PhD, UCSF assistantprofessor of biochemistry and biophysics, the senior author of the UCSF studyand a pioneer in the study of heart development in the transparent zebrafishembryo. "Gata5 is potentially such a gene. It appears to be sufficient todrive the entire myocardial program in certain cells not normally fated tocontribute to the heart."

Gata5's capability produces dramatic imagesStainier and colleagues' discovery of gata5's power presented powerful images:Removal of gata5 from cells normally destined to become myocardial cells causedprofound defects in the formation of the heart. Expression of gata5 throughoutthe embryo caused heart cells to form and beat -- spontaneously andrhythmically -- as far away from the head region, where the heart forms, as theanimals' lower trunk.

The finding, published in the current issue of Genes and Development¹represents a different approach to cultivating specialized cells for transplanttherapy than that being pursued in other labs. Current efforts, mostlyconducted in animal models, involve attempting to derive and aggregateembryonic stem cells, exposing them to such factors as acetic acid, allowingthem to differentiate, or specialize, and then sorting through these cells toextract the cell types of interest.

"Using different regulators, scientists have been able to induce a subset ofmyocardial characteristics in various experimental models, but never thecomplete beating phenotype, so there is something special about gata5 that cantake 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 diseaseThe finding also suggests that gata5 could be a potential new diagnostic markerfor congenital heart defects, as the researchers demonstrated that gata5regulates the expression of a gene known as nkx2.5 which, when mutated inhumans, causes human congenital heart defects and disease. Human geneticistsand cardiologists studying families with heart disease may discover, saidStainier, that a mutated form of the gata5 homologue occurs in some cases ofheart disease, in which case the mutated form of the gene could serve as amarker of predisposition to the disease.

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

The Nkx2.5 gene is the equivalent of a fly gene called "tinman," which wasdiscovered 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'sman of tin, the mutated fly gene, Bodmer discovered, led to a fly that lacked aheart.

"This is a case where scientists went from a fly mutation to a fly gene to ahuman mutation that causes congenital heart defects," said Stainier. "Itvalidates the approach of using even very distant model systems, such as thefly, to study human biology. We show that in the hierarchy of genes thatcontrol 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 tinyblue-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 handfulof labs worldwide, it is increasingly surfacing at the lab bench.

The reasons for its appeal are many, but none rival the fact that thecrystal-clear zebrafish embryo offers a view of burgeoning life that no othervertebrate model can. Less than a day after fertilization, the fertilized egghas sprouted a two-chambered beating heart, ears and eyes and a tail thatflicks. By the third day, all of its major organs have fully developed andmoved into proper position, offering scientists a view of what has beenreferred to as the zebrafish's "artful etchings."

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

Moreover, unlike other vertebrate model systems (principally the frog, chickand mouse), the animal offers the opportunity to search for mutations thatdisrupt specific biological events by a process that does not require priorknowledge of the gene or genes involved. This approach is also used ininvertebrate organisms such as Drosophila (fruit flies) and Caenorhabditiselegans (a roundworm).

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

Study pursues the genes with which a zebrafish views its worldNeuroscientist biologist Herwig Baier, PhD, UCSF assistant professor ofphysiology and formerly of the Max Planck Institute in Tubingen, is working toidentify zebrafish genes that play a role in visual perception. The fish makean ideal model for these studies, as they need no training to exhibit severaleasily recognized behaviors in response to visual cues in their waterysurroundings.

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

Heart buds nudge toward one another...to form a beating heartIn one study, researchers identified a set of genes needed to prompt the twobuds of heart cells that form early in development to migrate toward oneanother to form a single beating heart. The researchers came upon their findingserendipitously, after working out the molecular pathway, or succession ofgenes, that prompt the early-stage formation of the endoderm, one of the threelayers of cells that form the developing embryo.

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

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

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

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

These findings were reported in Current Biology² and Developmental Biology³.

Cell movements display choreography of heart formationStainier and colleagues also recently analyzed the complex process by which thetwo groups of heart cells that initially bud in the mesoderm cell layeractually form the heart, which is a complex, 3-D structure. They didn'tidentify the genes involved, but they did learn about the basic eventsinvolved. Specifically, using new heart-specific genes, they were able tofollow in detail the cell movements that lead to the formation of the heart, aswell as its divisions into two chambers, the atrium (the chamber that receivesblood and passes it to the ventricles) and the ventricle (the pumpingchamber). This work will allow the researchers to formulate specifichypotheses regarding the various cell interactions and molecules involved inthese processes. The finding was reported in Developmental Biology⁴.

With potential therapeutic implications, Stainier and a colleague also recentlydiscovered that endothelial cells, which line the blood vessels, play acritical role in inducing the development, or proper differentiation, of redblood cells, the transporters of oxygen. When endothelial cells were removedfrom the developing zebrafish embryo, red blood cells did not form. Whenendothelial cells were restored, blood cells developed. (The researchers wereable to conduct this experiment because the embryos can survive without bloodfor at least seven days.). Presumably, said Stainier, the endothelial cells actby releasing an as-yet-unidentified factor.

The finding represents the first demonstration in an animal model, Stainiersaid, that endothelial cells are necessary for the proper differentiation ofred blood cells. He said he suspects the discovery, published in Developments⁵,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 intored blood cells, which could then be used to boost the blood supply."The insights zebrafish can offer into human biology and medicine areboundless," said Stainier. "We will be learning from these animals for yearsto come."

Footnotes:

1) Co-authors of the gata5 study, published in Genes & Development, were leadauthor Jeremy Reiter, and Jonathan Alexander, both MD/PhD students funded bythe 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, TheRandall Institute, King's College London, and the late Nigel Holder, PhD, ofthe Department of Anatomy and Developmental Biology, University College London.

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

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

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

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

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

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