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

Letting go of the (genetic) apron strings (Cell)

Posted on by Catherine Zandonella

Researchers explore the shift from maternal genes to the embryo’s genes during development

By Catherine Zandonella, Office of the Dean for Research

Fruit fly embryo
Cells in an early-stage fruit fly embryo. (Image courtesy of NIGMS image gallery).

A new study from Princeton University researchers sheds light on the handing over of genetic control from mother to offspring early in development. Learning how organisms manage this transition could help researchers understand larger questions about how embryos regulate cell division and differentiation into new types of cells.

The study, published in the March 12 issue of the journal Cell, provides new insight into the mechanism for this genetic hand-off, which happens within hours of fertilization, when the newly fertilized egg is called a zygote.

“At the beginning, everything the embryo needs to survive is provided by mom, but eventually that stuff runs out, and the embryo needs to start making its own proteins and cellular machinery,” said Princeton postdoctoral researcher in the Department of Molecular Biology and first author Shelby Blythe. “We wanted to find out what controls that transition.”

Blythe conducted the study with senior author Eric Wieschaus, Princeton’s Squibb Professor in Molecular Biology, Professor of Molecular Biology and the Lewis-Sigler Institute for Integrative Genomics, a Howard Hughes Medical Institute investigator, and a Nobel laureate in physiology or medicine.

Researchers have known that in most animals, a newly fertilized egg cell divides rapidly, producing exact copies of itself using gene products supplied by the mother. After a short while, this rapid cell division pauses, and when it restarts, the embryonic DNA takes control and the cells divide much more slowly, differentiating into new cell types that are needed for the body’s organs and systems.

To find out what controls this maternal to zygotic transition, also called the midblastula transition (MBT), Blythe conducted experiments in the fruit fly Drosophila melanogaster, which has long served as a model for development in higher organisms including humans.

These experiments revealed that the slower cell division is a consequence of an upswing in DNA errors after the embryo’s genes take over. Cell division slows down because the cell’s DNA-copying machinery has to stop and wait until the damage is repaired.

Blythe found that it wasn’t the overall amount of embryonic DNA that caused this increase in errors. Instead, his experiments indicated that the high error rate was due to molecules that bind to DNA to activate the reading, or “transcription,” of the genes. These molecules stick to the DNA strands at thousands of sites and prevent the DNA copying machinery from working properly.

To discover this link between DNA errors and slower cell replication, Blythe used genetic techniques to create Drosophila embryos that were unable to repair DNA damage and typically died shortly after beginning to use their own genes. He then blocked the molecules that initiate the process of transcription of the zygotic genes, and found that the embryos survived, indicating that these molecules that bind to the DNA strands, called transcription factors, were triggering the DNA damage. He also discovered that a protein involved in responding to DNA damage, called Replication Protein A (RPA), appeared near the locations where DNA transcription was being initiated. “This provided evidence that the process of awakening the embryo’s genome is deleterious for DNA replication,” he said.

The study also demonstrates a mechanism by which the developing embryo ensures that cell division happens at a pace that is slow enough to allow the repair of damage to DNA during the switchover from maternal to zygotic gene expression. “For the first time we have a mechanistic foothold on how this process works,” Blythe said.

The work also enables researchers to explore larger questions of how embryos regulate DNA replication and transcription. “This study allows us to think about the idea that the ‘character’ of the DNA before and after the MBT has something to do with the DNA acquiring the architectural features of chromatin [the mix of DNA and proteins that make up chromosomes] that allow us to point to a spot and say ‘this is a gene’ and ‘this is not a gene’,” Blythe said. “Many of these features are indeed absent early in embryogenesis, and we suspect that the absence of these features is what allows the rapid copying of the DNA template early on. Part of what is so exciting about this is that early embryos may represent one of the only times when this chromatin architecture is missing or ‘blank’. Additionally, these early embryos allow us to study how the cell builds and installs these features that are so essential to the fundamental processes of cell biology.”

This work was supported in part by grant 5R37HD15587 from the Eunice Kennedy Shriver National Institute of Child Health and Human Development.

Read the abstract

Blythe, Shelby A. & Eric R. Wieschaus. Zygotic Genome Activation Triggers the DNA Replication Checkpoint at the Midblastula Transition. Cell. Published online on March 5, 2015. doi:10.1016/j.cell.2015.01.050. http://www.sciencedirect.com/science/article/pii/S0092867415001282