Genetic instructions from mom set the pattern for embryonic development

Posted on by Catherine Zandonella
Micrograph of a zebrafish organ called the Kupffer's vesicle

By the Department of Molecular Biology

A new study indicates an essential role for a maternally inherited gene in embryonic development. The study found that zebrafish that failed to inherit specific genetic instructions from mom developed fatal defects earlier in development, even if the fish could make their own version of the gene. The study by researchers at Princeton University was published Nov. 15 in the journal eLife.

When female animals form egg cells inside their ovaries, they deposit messenger RNAs (mRNAs) – a sort of genetic instruction set – in the egg cell cytoplasm. After fertilization, these maternally supplied mRNAs can be translated into proteins required for the early stages of embryonic development, before the embryo is able to produce mRNAs and proteins of its own.

More than thirty years ago, researchers discovered that mRNAs encoding a protein called Vg1 are deposited in the cytoplasm of frog eggs. “vg1 is famous for being one of the first recognized maternal mRNAs,” said Rebecca Burdine, associate professor of molecular biology at Princeton. “Many papers have been written on how this RNA is localized and regulated, but it was never clear what the Vg1 protein actually does in the developing embryo.”

Two zebrafish embryos
Compared to a normal zebrafish embryo (right), an embryo lacking gdf3 (left) inherited from mom shows major defects resulting from its inability to form mesoderm and endoderm cells early in development. Credit: Pelliccia et al., 2017.

In the study, Burdine and two graduate students Jose Pelliccia and Granton Jindal used CRISPR/Cas9 gene editing to remove Vg1, known as Gdf3 in zebrafish. Embryos that couldn’t produce any Gdf3 of their own–but received a healthy portion of the gdf3 mRNA from their mothers–developed perfectly normally. But embryos that didn’t receive maternal gdf3 mRNA showed major defects early on in their development, dying just three days after fertilization.

“If gdf3 is not supplied to the egg by the mother, the fertilized egg cannot produce two of the three major types of cells required for development,” Burdine said. “The embryos lack all [cell types known as] mesoderm and endoderm and are left with skin and some neural tissue, [which derive from the third major cell type, the ectoderm].”

Vg1/Gdf3 is a member of the TGF-beta family of cell-signaling molecules. Two other members of this family, Ndr1 and Ndr2, are required to form the mesoderm and endoderm early in zebrafish development. Embryos lacking maternally supplied gdf3 look very similar to embryos lacking both of these proteins, which are analogous to the Nodal 1 and 2 proteins in mammals.

The researchers found that maternal gdf3 is required for Ndr1 and Ndr2 to signal at the levels necessary to properly induce the formation of mesoderm and endoderm cells in early zebrafish embryos. In the absence of gdf3, Ndr1 and Ndr2 signaling is dramatically reduced and embryonic development goes awry.

Nodal signaling is also required later in zebrafish development when it helps to establish differences between the left and right sides of the developing embryo. It does this, in part, by directing the formation of an organ known as Kupffer’s vesicle, whose asymmetric shape helps determine the embryo’s left and right sides. Subsequently, Nodal signaling induces the expression of a third Nodal protein, called southpaw, in a group of mesoderm cells on the left-hand side of the embryo.

To investigate whether maternally supplied gdf3 mRNA also plays a role in left-right patterning, the researchers used a series of experimental tricks to supply embryos with enough Gdf3 protein to form the mesoderm and endoderm and survive until the later stages of embryonic development.

As predicted, these embryos showed defects in left-right patterning. Their Kupffer’s vesicles were abnormally symmetric in shape, and southpaw expression was greatly reduced, suggesting that gdf3 is also required for optimal Nodal signaling during later stages of embryonic development. At this stage, however, embryonic gdf3 seems to be capable of doing the job if maternally supplied gdf3 is absent.

Nodal and Vg1 proteins are known to bind to each other in other species. “Thus, we hypothesize that Gdf3 combines with Ndr1 and Ndr2 to facilitate Nodal signaling during zebrafish development, acting as an essential factor in embryonic patterning,” said Pelliccia, a graduate student in molecular biology. Co-author Jindal earned his Ph.D. in chemical and biological engineering in 2017.

At the same time as Burdine and colleagues, two other research groups, led by Joe Yost at the University of Utah and Alex Schier at Harvard University, made similar findings on the role of gdf3 during zebrafish development. “All three groups worked together to co-submit and co-publish in eLife, allowing the students involved to all get credit for their hard work,” Burdine said. “It’s a great example of how science should be done.”

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The research was funded by the Eunice Kennedy Shriver National Institute of Child Health and Human Development (grant R01HD048584) and the National Science Foundation (graduate research fellowship DGE 1148900).

Citation: Pelliccia, J.L., G.A. Jindal, and R.D. Burdine. Gdf3 is required for robust Nodal signaling during germ layer formation and left-right patterning. eLife. 6: e28635 (2017). DOI: 10.7554/eLife.28635

Researchers’ Sudoku strategy democratizes powerful tool for genetics research (Nature Communications)

Posted on by Catherine Zandonella
Princeton University researchers Buz Barstow (left), graduate student Kemi Adesina and undergraduate researcher Isao Anzai ’17,
Princeton University researchers Buz Barstow (left), graduate student Kemi Adesina and undergraduate researcher Isao Anzai, Class of 2017, with colleagues at Harvard Universiy, have developed a strategy called “Knockout Sodoku” for figuring out gene function.

By Tien Nguyen, Department of Chemistry

Researchers at Princeton and Harvard Universities have developed a way to produce the tools for figuring out gene function faster and cheaper than current methods, according to new research in the journal Nature Communications.

The function of sizable chunks of many organisms’ genomes is a mystery, and figuring out how to fill these information gaps is one of the central questions in genetics research, said study author Buz Barstow, a Burroughs-Wellcome Fund Research Fellow in Princeton’s Department of Chemistry. “We have no idea what a large fraction of genes do,” he said.

One of the best strategies that scientists have to determine what a particular gene does is to remove it from the genome, and then evaluate what the organism can no longer do. The end result, known as a whole-genome knockout collection, provides full sets of genomic copies, or mutants, in which single genes have been deleted or “knocked out.” Researchers then test the entire knockout collection against a specific chemical reaction. If a mutant organism fails to perform the reaction that means it must be missing the particular gene responsible for that task.

It can take several years and millions of dollars to build a whole-genome knockout collection through targeted gene deletion. Because it’s so costly, whole-genome knockout collections only exist for a handful of organisms such as yeast and the bacterium Escherichia coli. Yet, these collections have proven to be incredibly useful as thousands of studies have been conducted on the yeast gene-deletion collection since its release.

The Princeton and Harvard researchers are the first to create a collection quickly and affordably, doing so in less than a month for several thousand dollars. Their strategy, called “Knockout Sudoku,” relies on a combination of randomized gene deletion and a powerful reconstruction algorithm. Though other research groups have attempted this randomized approach, none have come close to matching the speed and cost of Knockout Sudoku.

“We sort of see it as democratizing these powerful tools of genetics,” said Michael Baym, a co-author on the work and a Harvard Medical School postdoctoral researcher. “Hopefully it will allow the exploration of genetics outside of model organisms,” he said.

Their approach began with steep pizza bills and a technique called transposon mutagenesis that ‘knocks out’ genes by randomly inserting a single disruptive DNA sequence into the genome. This technique is applied to large colonies of microbes to ensure the likelihood that every single gene is disrupted. For example, the team started with a colony of about 40,000 microbes for the bacterium Shewanella oneidensis, which has approximately 3,600 genes in its genome.

Barstow recruited undergraduates and graduate students to manually transfer 40,000 mutants out of laboratory Petri dishes into separate wells using toothpicks. He offered pizza as an incentive, but after a full day of labor, they only managed to move a couple thousand mutants. “I thought to myself, ‘Wait a second, this pizza is going to ruin me,’” Barstow said.

Instead, they decided to rent a colony-picking robot. In just two days, the robot was able to transfer each mutant microbe to individual homes in 96-well plates, 417 plates in total.

But the true challenge and opportunity for innovation was in identifying and cataloging the mutants that could comprise a whole-genome knockout collection in a fast and practical way.

DNA amplification and sequencing is a straightforward way to identify each mutant, but doing it individually quickly gets very expensive and time-consuming. So the researchers’ proposed a scheme in which mutants could be combined into groups that would only require 61 amplification reactions and a single sequencing run.

But still, after sequencing each of the pools, the researchers had an incredible amount of data. They knew the identities of all the mutants, but now they had to figure exactly where each mutant came from in the grid of plates. This is where the Sudoku aspect of the method came in. The researchers built an algorithm that could deduce the location of individual mutants through its repeated appearance in various row, column, plate-row and plate-column pools.

Knockout sodoku helps find genes' functions.

But there’s a problem. Because the initial gene-disruption process is random, it’s possible that the same mutant is formed more than once, which means that playing Sudoku wouldn’t be simple. To find a solution for this issue, Barstow recalled watching the movie, “The Imitation Game,” about Alan Turing’s work on the enigma code, for inspiration.

“I felt like the problem in some ways was very similar to code breaking,” he said. There are simple codes that substitute one letter for another that can be easily solved by looking at the frequency of the letter, Barstow said. “For instance, in English the letter A is used 8.2 percent of the time. So, if you find that the letter X appears in the message about 8.2 percent of the time, you can tell this is supposed to be decoded as an A. This is a very simple example of Bayesian inference.”

With that same logic, Barstow and colleagues developed a statistical picture of what a real location assignment should look like based on a mutant that only appeared once and used that to rate the likelihood of possible locations being real.

“One of the things I really like about this technique is that it’s a prime example of designing a technique with the mathematics in mind at the outset which lets you do much more powerful things than you could do otherwise,” Baym said. “Because it was designed with the mathematics built in, it allows us to get much, much more data out of much less experiments,” he said.

Using their expedient strategy, the researchers created a collection for microbe Shewanella oneidensis. These microbes are especially good at transferring electrons and understanding their powers could prove highly valuable for developing sustainable energy sources, such as artificial photosynthesis, and for environmental remediation in the neutralization of radioactive waste.

Using the resultant collection, the team was able to recapitulate 15 years of research, Barstow said, bolstering their confidence in their method. In an early validation test, they noticed a startlingly poor accuracy rate. After finding no fault with the math, they looked at the original plates to realize that one of the researchers had grabbed the wrong sample. “The least reliable part of this is the human,” Barstow said.

The work was supported by a Career Award at the Scientific Interface from the Burroughs Wellcome Fund and Princeton University startup funds and Fred Fox Class of 1939 funds.

Read the full article here:

Baym, M.; Shaker, L.; Anzai, I. A.; Adesina, O.; Barstow, B. “Rapid construction of a whole-genome transposon insertion collection for Shewanella oneidensis by Knockout Sudoku.” Nature Comm. Available online on Nov. 10, 2016.

“Kurly” protein keeps cilia moving, oriented in the right direction (Cell Reports)

Posted on by Catherine Zandonella
Cilia with a mutant form of the Kurly protein are wild and disorganized.
Cilia with a mutant form of the Kurly protein are wild and disorganized.

By Catherine Zandonella, Office of the Dean for Research

A new study of a protein found in cilia – the hair-like projections on the cell surface – may help explain how genetic defects in cilia play a role in developmental abnormalities, kidney disease and a number of other disorders.

The researchers at Princeton University and Northwestern University found that the protein, which goes by the name C21orf59 or “Kurly,” is needed for cilia to undulate to keep fluid moving over the surface of cells. They also found that the protein is needed during development to properly orient the cilia so that they are facing the right direction to move the fluid.

“It’s extremely exciting that we’ve found a single protein that is responsible for these two functions – orientation and motility – in cilia,” said Rebecca Burdine, an associate professor of molecular biology at Princeton University. “Despite their importance in human disease, very little is known about how cilia motility and orientation are coordinated, so this protein will provide an important gateway into looking at this process.” The finding is published online and in the March 1 issue of the journal Cell Reports.

Kurly panels
Caption: Staining of cilia (hair-like projections in green and nuclei in blue) in zebrafish kidney tubules show cilia are disorganized and oriented incorrectly in fish with mutated Kurly protein (bottom panel) versus normal Kurly (top panel). Image courtesy of the Burdine lab.

The studies were conducted in zebrafish at Princeton and in African clawed frogs (Xenopus laevis) at Northwestern. In the zebrafish kidney, the researchers found that the Kurly protein enabled cilia to orient themselves in a uniform direction, and most importantly, in the proper direction to facilitate the flow of fluid along the narrow channels in the kidney. In frogs, the cilia on skin cells help move fluid along the surface of the animal during its larval stage. In both cases, knocking out the gene for Kurly caused the cilia to orient incorrectly thereby losing their ability to move in the waving fashion that helps push fluid along.

The discovery of Kurly’s role in cilia movement and orientation stemmed from work in the Burdine lab on fetal organ development, specifically an investigation of mutations that alter the left-right asymmetric orientation of the heart. Such mutations can result in an organ that is working properly but is an exact mirror image of a normal heart. During a search for genes involved in this left-right patterning, the Burdine team discovered that mutations in a gene they called kur, which codes for the Kurly protein, were linked to errors in left-right orientation in zebrafish heart.

When the kurly protein is mutated, the cilia cannot orient and move properly.
Image credit: Burdine lab

As the team investigated kur, they noted that the mutation also affected the function of cilia. It has been known for some time that cilia are important for a number of jobs, from sensing the environment to facilitating fluid flow, to ensuring that the lungs excrete inhaled contaminants. Cilia genetic defects are linked to a number of human diseases, including polycystic kidney disease, respiratory distress, hearing loss, infertility, and left-right patterning disorders such as the one Burdine studies.

Researchers in Burdine’s laboratory found that Kurly’s role in cilia movement stems from its ability to ensure proteins called dynein arms are correctly located in the cilia. When the researchers knocked out the kur gene, the dynein proteins failed to form in the proper location.

The finding that a single protein is involved in both movement and orientation is surprising, said co-first author Daniel Grimes, a postdoctoral research associate in the Burdine lab. “These are two aspects that are both required to generate fluid flow, and we’d like to know how they are linked molecularly. This work adds a new gene that aids this discovery.”

The gene for Kurly has also been detected in relation to human cilia disorders, so the work may have an impact on understanding the mechanisms of human disease, Grimes added. The researchers also found that the mutation they discovered rendered the Kurly protein sensitive to temperature, and used this trait to find that the Kurly protein may be involved in initiating movement rather than keeping the cilia moving once they’ve started.

The team also explored proteins that interact with Kurly. The Northwestern team showed that when the kur gene was inactivated using a gene-editing technique called CRISPR-Cas9, the lack of a functioning Kurly protein led to the mis-positioning of a second protein on the cell surface called Prickle2, which helps cells know which direction they face. Without proper Prickle2 positioning, the cilia pushed fluid in the wrong direction.

The study of the Kurly protein involved Grimes as well as two additional co-authors, Kimberly Jaffe and Jodi Schottenfeld-Roames, a former postdoctoral researcher and graduate student respectively, in the Burdine lab. The initial studies on the Kurly protein were conducted as part of an undergraduate research project by Tse-shuen (Jade) Ku, Class of 2007. Additional work was contributed by Nicholas Morante and José Pelliccia, graduate students in the Burdine lab.

The work at Northwestern University was performed in the laboratory of Brian Mitchell with the assistance of Michael Werner and Sun Kim.

The research was supported by a National Institutes of Health (NIH) Ruth L. Kirschstein Institutional National Research Service Award grant to K. Jaffe (#1F32HD060396-01A1), an NIH National Institute of General Medical Sciences grant to B. Mitchell (#2R01GM089970), and an NIH Eunice Kennedy Shriver National Institute of Child Health and Human Development grant to R. Burdine (#2R01HD048584).

Read the article.

Kimberly M. Jaffe, Daniel T. Grimes, Jodi Schottenfeld-Roames, Michael E. Werner, Tse-Shuen J. Ku, Sun K. Kim, Jose L. Pelliccia, Nicholas F.C. Morante, Brian J. Mitchell, Rebecca D. Burdine.c21orf59/kurly controls both cilia motility and polarization. Cell Reports (2016), http://dx.doi.org/10.1016/j.celrep.2016.01.069. In Press Corrected Proof.