Matthew Sandel

Joined Program: 2005 (from UW Medical Scientist Training Program)
Previous Degree: B.S. Biology, Pennsylvania State University
Waterston Lab
mjsandel (at) u.washington.edu

Research:
Identifying Transcriptional Regulatory Networks in C. elegans Using 4-D Fluoresence Microscopy and Automated Analysis of Cell Lineage.

Research in the Waterston lab focuses on the transcriptional control of cell division, cell fate, and gene expression during the development of the nematode Caenorhabditis elegans. C. elegans (the ‘worm’) is a powerful tool for analyzing animal biology because of its stereotyped embryonic development and the tremendous body of knowledge that has built up around it. The 959-cell adult develops through an invariant lineage of which every cell division has been identified and catalogued, yet it possesses the same functional tissues as more complicated animals. My interest in the worm stems directly from the Waterston lab’s long-term experimental goal: to elucidate the mechanisms of transcriptional control of the C. elegans genome in every cell throughout its embryonic development. The lab has developed a software suite that allows us to track green fluorescent protein (GFP)-labeled cell nuclei in the worm embryo from the 4-cell embryo to the first movements within the egg (1, 2). With this software and a confocal microscope, we are currently able to create and annotate a ~300 cell embryo’s lineage with only a few hours of manual refinement. By lineaging transgenic animals that also express red fluorescent protein (RFP) driven by regulatory regions found upstream of C. elegans genes, we can study the expression of these genes in a quantitative, cell-specific way and overlay this data on the annotated lineage.

I am currently pursuing three lines of research in my efforts to develop a thesis project. The first and most immediate is to develop an RT-PCR assay system that will allow the laboratory to detect and quantify gene expression in a single embryo. This will permit me to compare the expression of our promoter-driven RFP transgene to that of the endogenous gene. It will also allow for rapid screening for changes in expression in RNAi knockdown experiments. This will identify biologically meaningful regulatory relationships and facilitate the selection of targets for analysis within our lineaging system.

My second experimental approach is directed at improving the quality of our reporter gene expression for genes with highly conserved genomic architecture. Based on preliminary data in the Waterston Lab and a paper by Nelson and colleagues, it is apparent that much of the cis-regulatory sequence of key developmental genes is not found directly upstream of the transcriptional start site (3). Thus, reporter genes driven by ~6 kb or less fragments immediately upstream of relevant genes will not necessarily produce an expression pattern that perfectly reflects the gene of interest. In collaboration with Anthony Hyman’s Laboratory at the Max Planck Institute of Molecular Cell Biology and Genetics in Dresden, we are employing recombineering to insert our reporter constructs into C. elegans genomic fosmids containing 30+ kb of flanking genomic sequence. This will allow us greater confidence in the expression patterns we generate.

The third set of experiments I am undertaking is intended to identify partially redundant activity within entire gene families using RNA interference. Specifically, I want to identify the MS-derived signals for pharynx induction in the ABara and ABalp lineages. Through genetic and cell-ablation analysis, it is known that the Notch receptor GLP-1, which is expressed on the surface of ABara and ABalp, receives a signal from the MS lineage and subsequently these cells adopt a pharyngeal fate (4). However, genetic and high-throughput RNAi experiments have not identified the ligand(s) responsible for this induction. Most likely, it is due to the activity of several redundant notch ligands. Experiments by Dudley and colleagues (5) have shown that phenotypes generated by dsRNA-induced knockdown can be generated when co-injected with as many as 7 interfering RNAs. By injecting pools of Notch ligand dsRNA, we hope to identify which are sufficient to induce pharyngeal fate in the AB lineage.

Citations
1) Bao Z, Murray JI, Boyle T, Ooi SL, Sandel MJ, Waterston RH. Automated cell lineage tracing in Caenorhabditis elegans. Proc Natl Acad Sci U S A. 2006 Feb 21;103(8):2707-12.
2) Boyle TJ, Bao Z, Murray JI, Araya CL, Waterston RH. AceTree: a tool for visual analysis of Caenorhabditis elegans embryogenesis. BMC Bioinformatics. 2006 Jun 1;7:275.
3) Nelson CE, Hersh BM, Carroll SB. The regulatory content of intergenic DNA shapes genome architecture. Genome Biol. 2004;5(4):R25.
4) Priess, J. Notch signaling in the C. elegans embryo (June 25, 2005), WormBook, ed. The C. elegans Research Community, WormBook, doi/10.1895/wormbook.1.4.1, http://www.wormbook.org.
5) Dudley NR, Labbe JC, Goldstein B. Using RNA interference to identify genes required for RNA interference. Proc Natl Acad Sci U S A. 2002 Apr 2;99(7):4191-6.