PPubMed11 Sep 2007Cited 388×

High-Throughput In Vivo Analysis of Gene Expression in Caenorhabditis elegans Gene Expression in <i> C. elegans </i>

Rebecca Hunt-Newbury, Ryan Viveiros, Robert Johnsen, Allan Mah, Dina Anastas, Lily Fang, Erin Halfnight, David Lee, John Lin, Adam Lorch, Sheldon McKay, H. Mark Okada, Jie Pan, Ana K Schulz, Domena Tu, Kim Wong, Z Zhao, Andrey Alexeyenko, Thomas Burglin, Eric Sonnhammer, Ralf Schnabel, Steven J Jones, Marco A Marra, David L Baillie, Donald G Moerman, John Sulston

Abstract

'Dina Anastas' 'Lily Fang' 'Erin Halfnight' 'David Lee' 'John Lin' 'Adam Lorch' 'Sheldon McKay' 'H. Mark Okada' 'Jie Pan' 'Ana K Schulz' 'Domena Tu' 'Kim Wong' 'Z Zhao' 'Andrey Alexeyenko' 'Thomas Burglin' 'Eric Sonnhammer' 'Ralf Schnabel' 'Steven J Jones' 'Marco A Marra' 'David L Baillie' 'Donald G Moerman' 'John Sulston'] Using DNA sequences 5′ to open reading frames, we have constructed green fluorescent protein (GFP) fusions and generated spatial and temporal tissue expression profiles for 1,886 specific genes in the nematode Caenorhabditis elegans. This effort encompasses about 10% of all genes identified in this organism. GFP-expressing wild-type animals were analyzed at each stage of development from embryo to adult. We have identified 5′ DNA regions regulating expression at all developmental stages and in 38 different cell and tissue types in this organism. Among the regulatory regions identified are sequences that regulate expression in all cells, in specific tissues, in combinations of tissues, and in single cells. Most of the genes we have examined in C. elegans have human orthologs. All the images and expression pattern data generated by this project are available at WormAtlas ([http://gfpweb.aecom.yu.edu/index]()) and through WormBase ([http://www.wormbase.org]()).

A figure from High-Throughput In Vivo Analysis of Gene Expression in Caenorhabditis elegans Gene Expression in <i> C. elegans </i> — fig. from the paper

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The study generated GFP reporter expression profiles for 1,886 C. elegans genes (~10% of the genome), revealing tissue‑specific and broadly active regulatory regions.

What’s new

i.Created GFP promoter::GFP fusions for 1,886 C. elegans genes and recorded their spatial‑temporal expression
ii.Built a publicly accessible database of expression patterns via WormAtlas and WormBase
iii.Identified 414 tissue‑specific regulatory regions and characterized tissue‑specific versus broad promoters
iv.Determined minimal promoter lengths required for muscle expression (143‑326 bp)

How they did it

The 5′ regulatory regions (up to 3 kb upstream of the ATG) were amplified and fused to GFP using PCR stitching, then microinjected into wild‑type C. elegans to generate transgenic lines. GFP‑expressing animals were imaged at each developmental stage with low‑power and confocal microscopes, recording expression in at least 20 replicates per stage. All data were curated, annotated, and uploaded to an interactive web site and WormBase.

What they found

Expression profiles obtained for 1,886 genes (~10% of the C. elegans genome)
80% of benchmark genes matched WormBase annotations; 10% showed partial overlap and 10% little or no agreement
414 tissue‑specific regulatory regions identified, representing 20% of the dataset
Neural‑specific promoters: 155; intestinal‑specific promoters: 136; other tissue‑specific counts also reported
Minimal muscle promoter lengths ranged from 143 bp to 326 bp
516 genes showed no detectable expression
Only 15% of strains are stable integrants, the rest are extrachromosomal arrays

Caveats

✕Analysis limited to ≤3 kb upstream sequences; downstream, intronic, or distant enhancers were not examined
✕85% of strains carried extrachromosomal concatamer arrays, causing mosaic expression
✕Only 15% of strains were stable integrants, limiting long‑term reproducibility
✕Mosaicism required analysis of many replicates, and some expression patterns may still be missed

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