Zach is developing methods for using the zebrafish as a model to analyze the function of mammalian alpha crystallin promoters, the regions of the gene that determines when and where its protein is produced.  Understanding promoter function can provide clues to how alpha crystallins are used in healthy cells, and why their levels are often increased or reduced during disease.

Why test mammalian genes in a zebrafish?  The ability to produce large numbers of transparent zebrafish embryos in a relatively small facility makes this model species less expensive and faster to use than mice.  Zach’s work will help to determine whether this approach is feasible.

Phillip has been using zebrafish to study the toxicity of two common pesticides, permethrin and atrozine.  These chemicals commonly wash off of agricultural crops into neighboring aquatic habitats.  His is the first study to examine the mixture toxicity of these two chemicals – the way they interact with each other to produce toxic effects that are not merely the addition of each chemicals individual toxicity.  And it is the first study to examine their toxicity using zebrafish embryos as a relatively new model for aquatic toxicity testing.

In addition to his honors thesis toxicology research, Phillip took part in a summer research internship at the University of North Carolina at Chapel Hill, and last summer began a project in my lab to characterize developmental changes in zebrafish lens protein expression.  He is currently applying to graduate programs.

Jackie Skiba, an undergraduate research student in our lab, has been taking the new cryostat out for its shakedown run this summer:

Jackie Skiba preparing thin sections of zebrafish larvae

Stained section through a 3-day old zebrafish eye

This was also the second year of ARVO’s meeting blog, and my second year of contributing.  This turned out to be a good way to share information from meeting veterans, and learn some new faces and names.

Two new studies in the journal Nature have leveraged the unique powers of the zebrafish as a model vertebrate to provide answers to this question.  George Streisinger of the University of Oregon first developed this cute little pet store fish as a tool to study vertebrate development and gene function in the 1970s.  It has since become a prominent player in many areas of biomedical research, and is my model of choice for studying lens development, evolution and cataract.  Its use of external fertilization and a see-through egg makes it ideal for visualizing the early stages of development.  And with basic molecular techniques you can make specific cell types light up with green fluorescent protein (GFP).  This basic approach has now been used to provide further evidence that the initial source of blood stem cells is the lining of the aorta, the largest blood vessel leaving the heart.

Previous studies in mice suggested that hematopoietic stem cells (HSCs: which will become all types of blood cells) arise from the endothelial cells lining the ventral surface of the aorta.  David Travers’ group at UCSD labelled aortic endothelial cells with GFP and used confocal microscopy to show them moving from the endothelium into the bloodstream (Movie 1).  But unlike a proposed mechanism for mammals, these zebrafish HSCs do not enter the arterial bloodstream, but instead move into a neighboring vein.  While this detail differs between zebrafish and mammals, Travers’ work shows that similar molecular signaling coordinates the production of the HSCs in both taxa.  And in a very cool experiment, they used flow cytometry to isolate these new putative HSCs from zebrafish embryos and confirmed that they indeed became blood stem cells.

Movie 1. Live imaging of green HSCs leaving the aortic endothelium.

In the second Nature paper, Kissa and Herbomel from the Pasteur Institute in Paris used confocal microscopy to detail how new HSCs can be removed from the lining of the aorta without damaging the integrity of this tube.  They document that the differentiating HSCs fold over like a burrito, bringing together the neighboring endothelial cells and joining them together before leaving the tube (Figure 1).  This study also confirms that zebrafish HSCs enter the bloodstream through the neighboring vein, not the aorta, and that the process shares similar signaling to mammals.  When the authors used synthetic RNA molecules called morpholinos to stop the expression of a known mammalian signaling molecule called Runx1, the movement of HSCs from the aortic lining was highly reduced.

Figure 1. Detachment of HSCs (labeled in green) from the endothelial lining of the zebrafish dorsal aorta. The arrowhead in panel F shows folding in the HSC pulling together two neighboring endothelial cells before it leaves the aorta.

So what do these papers add to our understanding of HSC generation?  While the source of these cells was already thought to be the endothelial lining of the aorta, these new studies provide the first live visualization and physical description of this process.  And while the physical details of the process differ between zebrafish and mammals, the molecular signaling seems to be the same, suggesting that the zebrafish can be a valuable model for further detailing the generation of HSCs and their development into blood stem cells.  These studies are just one new example of the zebrafish’s growing influence in biomedical studies.

Bertrand, J., Chi, N., Santoso, B., Teng, S., Stainier, D., & Traver, D. (2010). Haematopoietic stem cells derive directly from aortic endothelium during development Nature, 464 (7285), 108-111 DOI: 10.1038/nature08738

Kissa, K., & Herbomel, P. (2010). Blood stem cells emerge from aortic endothelium by a novel type of cell transition Nature, 464 (7285), 112-115 DOI: 10.1038/nature08761

Your average anatomy and physiology  textbook shows that all of the different cell types in our blood, such as the red blood cells that carry oxygen and the white blood cells that contribute to our immune system, develop from one stem cell type called a hemocytoblast (see figure below).  And because of the importance of understanding the function of blood stem cells to treating many diseases, such as leukemia, this area has attracted lots of research.

A textbook description of blood cell formation.

The hemocytoblast is called a multipotent stem cell because it maintains the ability to differentiate into the different types of blood cells.  This flexible stem cell “commits” to a different developmental pathway by expressing receptor proteins on its surface for different signaling molecules, that will in turn tell it what to become.  The paradigm has been that there is only one type of hemocytoblast, that only becomes committed when a receptor protein is placed on the cell surface.  But studies have hinted at the presence of more than one type of hemocytoblast, and a research team based at the Baylor School of Medicine has now identified two of them.

The researchers were able to identify and purify two mouse bone marrow stem cell types based on a difference in their interaction with a common cellular dye.  When these purified stem cell types were transplanted into mice, the researchers found that each type preferred to make either red blood cells or immune cells.  This preference was maintained in the stem cell population as each individual hemocytoblast type produced copies of itself, suggesting that the bias was programmed into the cell.

It is not known how these two stem cell types differ, or what mechanism leads to the bias in blood cell production.  The researchers found that each stem cell type responded differently to a common signaling molecule used in cellular differentiation, suggesting a possible mechanism.  It is possible that there may still be a homogenous population of hemocytoblast precursor cells that predates the differentiation into these two newly found subtypes.  But clearly, knowing about the presence of blood stem cells with different behaviors will be important for scientists attempting to harness these cells to treat human disease.  I hope to read more about it in the next edition of our textbook.

Challen, G., Boles, N., Chambers, S., & Goodell, M. (2010). Distinct Hematopoietic Stem Cell Subtypes Are Differentially Regulated by TGF-beta1 Cell Stem Cell, 6 (3), 265-278 DOI: 10.1016/j.stem.2010.02.002

Of course you do not need a lens to see.  The nautilus does just fine without one.  But the benefits of a lens are strong enough that they have evolved across the animal phyla, and even occur in at least one protist.  The function of the lens is to provide a dense structure that will refract light on its way to the retina.  A common solution for making a lens is to express proteins at very high concentrations, especially in aquatic eyes that do not get the benefit of refraction from the cornea.  But interestingly, different organisms use different proteins to do the job.  And as Darwin would have predicted, these proteins are not created de novo, but are borrowed from other parts of the body.  While debates rage about whether the developmental mechanisms to make an eye evolved once or multiple times, the lens clearly has multiple origins.  And each time, organisms have drawn from their biochemical toolbox when producing proteins at high concentration in the lens.  These densely packed lens proteins have diverse evolutionary sources, but they are all referred to as lens crystallins.

Where did lens crystallins come from?  Some are basic housekeeping enzymes, others are protective proteins produced by cells when they are stressed and others have more mysterious origins.  But many of the proteins recruited to build lenses maintain their original function in other parts of the body, and are encoded by the same gene.  That means that one DNA blueprint can simultaneously make a metabolic enzyme like lactate dehydrogenase throughout the body, but when this gene is used in the lens of a duck, its protein product becomes a structural material for bending light.  This concept of gene-sharing, where one gene contains the code for making protein with multiple functions, was first proposed to explain the evolution of lens crystallins.

Were these lens crystallins recruited because of their original enzymatic functions, or were they simply convenient building blocks for the dense packing required to make a lens?  For many crystallins the answer is – both.  My lab does research on an abundant lens crystallin family found in the vertebrate lens, the alpha crystallins.  All vertebrates contain at least two closely related alpha crystallins that resulted from a gene duplication event near the beginning of vertebrate evolution.  When the gene sequences for the alpha crystallins were resolved in the late 1980′s it was clear that they were small heat shock proteins.  This family of proteins is produced by cells that are under stress – perhaps because they are too hot, or are encountering dangerously high oxygen levels.  

So why was a stress-induced protective protein being used to make up to 30% of a vertebrate lens?  There are two reasons.  First, it turns out that alpha crystallins make great building blocks.  You can pack them in at very high concentrations and still maintain the necessary protein fluidity needed in the lens.  But second, the same protective function that they serve in other parts of the body comes in very handy in the lens.  The central cells of the lens destroy their own nuclei and other cellular machinery to prevent the scattering of light as it passes through the retina.  No nucleus means no new protein, so our lens cells must make do with the same proteins for their entire life.  Old proteins get shabby, fall apart, and then start sticking to each other.  This sticky mess interferes with the passage of light, and voila – you now have a cataract.  But alpha crystallins use their stress protective function to prevent this aggregation of old, decrepit proteins, preserving lens transparency until they are used up, generally starting at around age 50 for humans.  

At the macroevolutionary scale a wide array of proteins have been co-opted as lens building blocks because of their structural and enzymatic properties.  My lab is currently investigating the microevolutionary story.  How do small changes in alpha-crystallins alter their ability to function at different temperatures, for example?  Darwin perhaps sensed that the eye would yield excellent examples of his two great ideas: descent with modification and natural selection.

First a little background on how we are killing our oceans.  The same CO2 that is accumulating in the atmosphere from the combustion of fossil fuels and other sources, leading to global warming, is diffusing into the oceans and changing their pH.  When CO2 reacts chemically with H2O,  H+ ions are released making water more acidic. This declining pH is already adversely affecting marine organisms, which are often adapted to a narrow pH range.  Calcium carbonate, however, can react with CO2 and limit the drop in pH.  The production of calcium carbonate by microscopic organisms like coccolithophores is thought to be the major player in this regulation of ocean pH.

So where do the fish come in?  Research on the toadfish, Opsanus beta, showed that this fish produces little calcium carbonate rocks in its digestive tract.  Subsequent physiological research showed that the production of these “gut rocks” was involved in the absorption of water in the gut.  Marine fish are less salty than the surrounding ocean.  Water, therefore, diffuses out of the fish into their environment leaving them very thirsty. But when they drink they fill their guts with salty water, which would pull fluids from their bodies leaving them even thirstier. It is for a similar reason that you should not drink ocean water when stranded in a life raft (that’s when you drink your own urine instead). But the fish apparently have a trick.  They accrete some of the salts in their urine as carbonate precipitates, lowering the salinity of the water in their gut and facilitating its absorption. And then the fish defecate the rocks.

Radiographic images of a live European flounder accumulating carbonate precipitates in its gut. The fish on top was living in freshwater and lacks "gut rocks". The same fish is shown below after only three hours in seawater. Note the opacities in the gut resulting from the accretion of carbonates (white arrows).

But would this calcium carbonate release affect the Ocean’s pH balance considering the relatively low biomass of fishes compared to plankters like the coccolithophores?  In their paper Wilson et al. also calculate the total biomass of fishes in the Ocean and the amount of calcium carbonate they produce.  While these types of calculations require a good number of assumptions, the authors claim that their conservative estimate is that fishes produce 3-15% of the Ocean’s calcium carbonate.

So next time PETA tries to convince you not to eat fish because they are cute, tell them a better reason is that fish poop could help save the marine ecosystem.

R. W. Wilson, F. J. Millero, J. R. Taylor, P. J. Walsh, V. Christensen, S. Jennings, M. Grosell (2009). Contribution of Fish to the Marine Inorganic Carbon Cycle Science, 323 (5912), 359-362 DOI: 10.1126/science.1157972