Research
Overview
Our lab studies embryogenesis; the process by which a fertilized egg becomes a multicellular organism with distinctive tissues and organs. Most of our work focuses on the development of a microscopic "worm" - more accurately a soil nematode - called Caenorhabditis elegans. C. elegans has become an important model system for animal biology, and is described in almost 8000 research papers. The 2002 Nobel Prize in Medicine was awarded for work on the C. elegans cell death (apoptosis) pathway. C. elegans progresses from a fertilized egg into a newly hatched larva in only 12 hours. During this time the embryo develops a nervous system, muscles, and digestive system that, at the cellular level, show a great deal of similarity with tissues in higher organisms, including humans. Because C. elegans develops into a fertile adult in only three days, it is possible to dissect developmental processes with genetic and molecular tools that would be impossible or impractical to use on higher animals with longer generation times.
We have worked on several aspects of C. elegans development, including morphogenesis (changes in cell shape that transform the round embryo into a long, thin "worm"), gastrulation (how the early embryo distinguishes inside from outside), specification of the germ line, and specification of cell fates during early embryogenesis. Some of the current work in the lab is in the following areas.
Germ Cell Specification
During embryonic development most cells differentiate into various somatic cell types, such as muscles, neurons, etc. However, a small group of special cells are set aside that remain totipotent; these form the germ cells that eventually produce sperm or oocytes. Germ cells, and germ cell precursors in C. elegans contain cytoplasmic structures of unknown function called P granules; morphologically similar structures are associated with germ cells in many other animals. We have shown that these structures are associated with nuclear pores on germ cell nuclei in adult gonads, and contain multiple mRNAs that are synthesized maternally and used during embryonic development. We would like to learn what molecular functions the P granules serve, and why P granules are associated specifically with germ cells.
Panel A is an electron micrograph of a germ cell nucleus at the pachytene stage of meiosis. Arrows point to examples of nuclear pores. Panel B shows an immunofluorescent micrograph of a similar nucleus with DNA shown in blue, a P granule component (PGL-1) in green, and a nuclear pore component in red (mAb414). Panel C shows a low magnification image of an oocyte nucleus as P granules detach from the envelope and distribute to the cytoplasm.
P granules detach from the nuclear envelope during oogenesis, and in newly fertilized eggs are segregated into the posterior cytoplasm that is incorporated into germ cells. one-cell embryo We have shown that a novel, cytoplasmic/nuclear protein called PIE-1 is similarly localized to the posterior cytoplasm. The PIE-1 protein is critical for germline development, and in the absence of PIE-1 germ cell precursors instead adopt somatic cell fates . In wild-type embryos, PIE-1 is localized asymmetrically to the germ cell lineage beginning shortly after fertilization. The protein MEX-5 is required for the localization of PIE-1, and is itself localized asymmetrically in a reciprocal pattern. In current studies we are addressing how MEX-5 regulates PIE-1 asymmetry at the 1-cell stage, and how PIE-1 functions to regulate germ cell development.
Somatic Cell specification Though Notch Signaling and Pop-1 Asymmetry
We and others have identified maternally-expressed transcription factors that are localized asymmetrically in the early embryo, and that promote somatic cell fates. However, cell-cell interactions also play a major role in creating diversity between early embryonic cells. One of the major interaction pathways in C. elegans and other animals is called the Notch pathway. We and others have characterized several Notch-mediated interactions in the early embryo with roles that include mesodermal (muscle) induction, morphogenesis of the intestine, and head formation [reviewed at WormBook]. To understand the logic of the Notch signaling network, we are interested in identifying Notch targets. We recently showed that a family of bHLH transcription factors, called the REF-1 family, is a major effector of Notch signaling in the embryo, and are currently analyzing how expression of this family is regulated.
Example of Notch signaling in the developing C. elegans intestine. The four cells (E4) that will produce the intestine express the receptor LIN-12/Notch (shown in red) at their cell surface. The left two E4 cells contact neighboring cells that express the ligand LAG-2/Delta (shown in blue), and in response turn on the target REF-1 (green). REF-1 in turn down- regulates LIN-12/Notch in the left cells, such that LIN-12/Notch is expressed only in the right E8 cells at the next cell cycle A second interaction occurs at the third cell cycle, when cells express a new ligand, APX-1/Delta. Because the first interaction restricted expression of the receptor to the right cells, REF-1 comes on only on the right side of the developing intestine at the E16 stage.
We are also interested in the targets of REF-1-mediated repression, and have identified T-box transcription factors that are repressed by Notch signaling in one interaction, but act cooperatively with Notch signaling in a second interaction.
We are also interested in the targets of REF-1-mediated repression, and have identified T-box transcription factors that are repressed by Notch signaling in one interaction, but act cooperatively with Notch signaling in a second interaction.
The second major cell-cell interaction system leads to the asymmetric distribution of a transcription factor called POP-1, a protein related to Tcf or Lef in vertebrates. We have shown that POP-1 is distributed unequally between sister nuclei in most embryonic cell divisions, and that POP-1 acts combinatorially to create anterior/posterior differences between sister cells in mulitple cell divisions.
The top panels show an embryo stained for POP-1 (left) and with DAPI (right) to visualize nuclei. In addition, the embryo was stained with an antibody that recognizes the midbody between sister cells (small arrows in right panel). Note that in each pair of sisters (indicated by white bars on the left), POP-1 is present at higher levels in the more anterior sister. The development of POP-1 asymmetry is summarized in the lower panel for an embryonic cell called AB, with light blue indicating high levels of POP-1 and dark blue indicating low levels. Wnt signaling initiates POP-1 asymmetry when there are eight descendants of AB (the AB8 stage), but Wnt signaling is not essential for later POP-1 asymmetry.
The four panels show two cells (1 and 2) during division (time indicated in minutes at lower right). The cells express the MOM-5 protein tagged with GFP.
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