Compared to a normal zebrafish embryo (right), an embryo lacking gdf3 (left) shows major defects resulting from its inability to form mesoderm and endoderm cells early in development. Pelliccia et al., 2017
When female animals form egg cells inside their ovaries, they deposit numerous messenger RNAs (mRNAs) in the egg cell cytoplasm. Once the eggs are fertilized, these maternally supplied mRNAs can be translated into many of the 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,” says Rebecca Burdine, an Associate Professor in Princeton University’s Department of Molecular Biology. “There are a wealth of papers studying how this RNA is localized and regulated, but it was never clear what the Vg1 protein actually does in the developing embryo.”
In a study just published in the journal eLife, Burdine and two of her graduate students, Jose Pelliccia and Granton Jindal, used CRISPR/Cas9 gene editing to remove Vg1 from zebrafish, where it is known as Gdf3. 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 any maternal gdf3 mRNA showed major defects early on in their development, dying just 3 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 explains. “The embryos lack all mesoderm and endoderm and are left with skin and some neural tissue, [which derive from the third major cell type, the ectoderm]. It’s a very striking phenotype.”
Vg1/Gdf3 is a member of the TGFb 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 looked very similar to embryos lacking both of these proteins, which are orthologs of a mammalian signaling protein called Nodal.
Pelliccia et al. 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 ortholog, called southpaw, in a group of mesoderm cells on the left–hand side of the embryo.
To investigate whether maternally supplied gdf3 also plays a role in left-right patterning, Pelliccia et al. used a series of experimental tricks to supply embryos with enough Gdf3 to form the mesoderm and endoderm and survive until the later stages of embryonic development.
Sure enough, 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 dimerizes with Ndr1 and Ndr2 to facilitate Nodal signaling during zebrafish development, acting as an essential factor in embryonic patterning,” explains Pelliccia.
At the same time as Burdine and colleagues, two other research groups, namely Joe Yost’s laboratory at the University of Utah and Alex Schier’s group 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,” says Burdine. “It’s a great example of how science should be done.”
Compared to a normal zebrafish embryo (right), an embryo lacking gdf3 (left) shows major defects resulting from its inability to form mesoderm and endoderm cells early in development. Pelliccia et al., 2017.