The Seydoux 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 1-cell embryos 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.
A Novel Class of Intrinsically-Disordered Proteins Scaffold RNA Granules in Germ Cells
Among the factors that are asymmetrically segregated in the zygote are the P granules. P granules are micron-sized, ribonucleoprotein complexes in the cytoplasm. We have identified two novel, intrinsically-disordered proteins (MEG-3 and MEG-4) that scaffold the P granules. In collaboration with Eric Betzig's lab (HHMI, Janelia Research Campus), we found that MEG-3 forms a dynamic cage-like structure around each granule. Despite the lack of a recognizable RNA-binding domain, MEG-3 binds RNA and is required (with its homolog MEG-4) to recruit specific mRNAs (such as nanos) to the P granules. These studies reveal a novel role for intrinsically-disordered proteins in RNA localization and regulation during development.
Genome Engineering – Precise and Scalable Editing of the C. elegans Genome Using Recombineering
A major goal of genetic engineering is to develop methods to modify the genome of animals at will. CRISPR/Cas9 technology has greatly accelerated progress in genome engineering but a major challenge remains: the harnessing of endogenous DNA repair pathways to introduce complex edits easily in the genome. We have found that homology-dependent repair (HDR) is a highly efficient mechanism with two important characteristics: HDR requires templates with short (35bp) homology arms and is prone to template switching. We have exploited these characteristics to develop new recombineering-based methods for genome editing that employ oligonucleotides to target PCR fragments to precise genomic sites. Recombineering streamlines genome editing and offers a new platform to study mechanisms of DNA repair in animals.
This work is also supported by the National Institutes of Health.
As of May 4, 2016