Our lab studies the earliest stages of embryogenesis to understand how single-celled eggs develop into complex multicellular embryos. We focus on the choice between soma and germline, one of the first developmental decisions faced by embryos. Our goal is to identify and characterize the molecular mechanisms that polarize embryos and distinguish between somatic and germline cells. We use the nematode Caenorhabditis elegans (C. elegans) as a model system and have recently developed scalable methods for precision genome engineering in this animal.
Cell Polarity – Patterning by Local Reactions and Long-Range Diffusion
In this research we seek to understand how newly fertilized zygotes become polarized along the anterior/posterior axis and how they use this information to segregate cell fate determinants to different ends of the growing embryo. We have found that symmetry breaking involves a direct interaction between the microtubules of the sperm-donated centrosome and the polarity regulator PAR-2. Microtubules protect PAR-2 from phosphorylation, allowing PAR-2 to localize to the membrane nearest the sperm centrosome. PAR-2 at the membrane triggers the sorting of other PAR proteins, including PAR-1 kinase, into distinct membrane domains. Asymmetric PAR-1, in turn, regulates the distribution of the RNA-binding protein MEX-5 by modulating its rate of diffusion in the cytoplasm. These studies have revealed how local biochemical interactions can create asymmetries that spread over tens of microns within minutes and without the need for a polarized cytoskeleton.
RNA Granules and Germ Cell Fate – A Role for Intrinsically Disordered Proteins
Among the factors that are asymmetrically segregated in the zygote are the P granules. P granules are RNA granules that form within the germ plasm, the asymmetrically segregated cytoplasm that specifies the germline. We have identified two novel, serine-rich, intrinsically disordered proteins (MEG-3 and MEG-4) that are required to assemble P granules in germ plasm. In collaboration with Eric Betzig's lab (HHMI, Janelia Research Campus), we found that MEG-3 localizes to a dynamic domain that surrounds and penetrates each granule. These studies have led to two surprising findings: (1) P granules are nonhomogeneous structures that assemble around a perigranular scaffold regulated by phosphorylation, and (2) the essential activity of the germ plasm resides in the MEG proteins, not the P granules. We are investigating how the MEGs specify germ cell fate.
Genome Engineering – Precise and Scalable Editing of the C. elegans Genome
The remarkable efficiency of the RNA-guided Cas9 endonuclease has led to an explosion of new methods for genome engineering. One of the challenges has been to harness the power of homology-dependent repair to introduce designer mutations near Cas9 sites. We found that, under the right conditions, homology-dependent repair is surprisingly efficient in the C. elegans germline: homology arms as short as 35 bases are sufficient to introduce base- and gene-sized edits into any locus. The efficiency of homology-dependent repair is so high that markers are not required. On the basis of these findings, we have developed scalable protocols to mutate, tag, or delete any gene in the C. elegans genome.
This work is also supported by the National Institutes of Health.
As of December 22, 2014