High-throughput in vivo analysis of gene expression in Caenorhabditis elegans - PubMed (original) (raw)

doi: 10.1371/journal.pbio.0050237.

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

Affiliations

High-throughput in vivo analysis of gene expression in Caenorhabditis elegans

Rebecca Hunt-Newbury et al. PLoS Biol. 2007 Sep.

Abstract

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).

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Conflict of interest statement

Competing interests. The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Analysis Pipeline for GFP Expressing Strains

Strains with nonubiquitous embryonic expression prior to 2-fold stage were stabilized for 4-D analysis, and all postembryonic strains were briefly assessed for their expression pattern complexity and then assigned to either the confocal microscope or the stereomicroscope for detailed observations. Once the expression analysis was complete, the data were sent to the public domain, and the strains were sent to the CGC (Caenorhabditis Genetics Center).

Figure 2

Figure 2. Embryonic Cell Lineages of C45G9.13 and ZK637.11

(A) and (E) display the embryonic cell lineages of C45G9.13 and ZK637.11, respectively. (B) and (C) are Nomarski differential contrast microscopy images of C45G9.13 with a GFP overlap. The view is from the ventral side with anterior to the left of the image. The highlighted cells are a, ABprappppa; b, M5; and c, Mspapaapa. (F) and (G) show Nomarski images of ZK637.11 with ventral to the bottom and anterior to the left. The cells marked in (G) are a, ABprppa; b, ABprpap; c, MSppa; d, MSpaa; e, ABalaap; f, ABalpaa; g, ABalapp; h, ABalppa; I, ABplaap; and j, ABplapp. (D) and (H) are 3-D stick and ball models of the nuclei of the expressing cells of C45G9.13 and ZK637.11, respectively. In both panels, anterior is to the left and ventral is on the bottom. In (D), the orange ball represents M5: note that the embryo has been rotated 90° to the right compared to the above Nomarski image. In (H), the orange ball corresponds to ABalapp.

Figure 3

Figure 3. The Browse Page

The Browse page initially lists all genes (B) with the relevant information as text or links, but is searchable (A) by developmental stage or a specific tissue. The links lead to (C) the Wormbase Gene Summary, (D) a comprehensive summary page specific to that strain, and (E) the Wormbase location map.

Figure 4

Figure 4. The Gene Search Page and Search Results

The Search page (A) and (B) allows the user to search the dataset with a combination of gene names and data types. The user can enter (A) specific gene names or use an asterisk (*) to represent all genes and then choose (B) the list of data types representing specific information about the gene(s) of interest. The logic for the Field column is OR, and the results (C) will display columns for the data types. The Refine Search column (B) operates under AND logic for the selected field, and OR logic between fields, e.g., selecting “stable” transgene and “yes” Images will output all genes that are either stable or have an associated image. Thus the genes displayed are limited only to those that evaluate to True for a selected field.

Figure 5

Figure 5. A Categorization by Tissue, Summarizing the Influence of the 389 5′ DNA Sequences that Drive Expression in a Single Tissue or Cell Type

Figure 6

Figure 6. A Portrait of C. elegans General Tissue Expression Patterns, Driven by 5′ DNA::GFP Constructs

(A) Pharynx—gene C32F10.8. (B) Hypodermis (long arrow) and seam cell (short arrow)—gene F25H2.1 (inset: C29E4.8—embryonic hypodermis). (C) Body wall muscle (arrow points to muscle arm)—gene F27D9.5 (inset: W01A11.1—muscle innervation). (D) Gut—gene Y102A11A.2. (E) Stomatointestinal muscle (long arrow) and anal depressor muscle (short arrow)—gene D1081.2. (F) Vulval muscle (long arrow) and ventral nerve cord (short arrow)—gene Y32H12A.5. (G) Neural (nerve ring: short arrow, ventral nerve cord: long thin arrow, and dorsal nerve cord: thick arrow)—gene C13F10.4. (H) Excretory cell—gene F32F2.1 (inset: F48E8.3) (intersects with WormBase annotation).

Figure 7

Figure 7. A Portrait of C. elegans Neural Tissue Expression Patterns, Driven by 5′ DNA::GFP Constructs

(A) Neural network—gene B0041.7a. (B) Labial sensilla (arrow)—gene C17E4.5. (C) Amphid neuron—gene T26E3.9. (D) Ring interneuron—gene H13N06.6 (intersects with WormBase annotation). (E) Amphid socket cells (arrow)—gene Y39D8C.1. (F) Pre-anal (short arrow) and lumbar (long arrow) ganglion—gene C47A10.6 (intersects with WormBase annotation). (G) Phasmid neurons—gene W01A11.2. (H) PVT interneuron—gene M03F4.3 (intersects with WormBase annotation).

Figure 8

Figure 8. The Primordial Germ Layers' Main Sphere of Influence: Ectoderm (Neurons and Hypodermis), Mesoderm (Muscle), and Endoderm (Intestine)

From the data, we see a fairly even distribution of expression between the germ layers: 71% of the 5′ DNA sequences express in ectoderm, 63% in endoderm, 55% in mesoderm, and about half from each germ layer contributes to the intersection of the three germ layers. Within the ectoderm (see inset), we see that there is a preponderance of neural-specific expression (61%) relative to hypodermal-specific expression (14%). There are very few hypodermal 5′ DNA sequences that do not also express in either the nerve, muscle, or intestine.

Figure 9

Figure 9. A Portrait of C. elegans Reproductive Tissue Expression Patterns, Driven by 5′ DNA::GFP Constructs

(A) Vulval muscles (lateral view: long arrow, ventral view: short arrow)—gene F54A5.3a (inset F33H2.3). (B) Uterine muscles (long arrows) and body wall muscle (short arrow)—gene F10G12.5. (C) Vulva—gene Y47G6A.7. (D) VulA cell—gene F02D8.2. Inset shows VulA cel relative to other vulva cells. (E) Gonad sheath cells (arrow) and gut—gene C30F12.1. (F) Gonadal sheath cell pair 5 (arrow) and gut—gene T60A3A.9. Inset shows proximal gonadal sheath cells. (G) Spermatheca (arrow)—gene Y43B11AR.4 (inset F40F11.2). (H) Dorsal uterine cell (arrow)—gene C30F12.1. (I) Distal tip cells—gene C44B12.2. Inset shows distal tip cells at end of gonad. (J) Developing uterus—gene Y47G6A.7 (arrow indicates location of developing vulva).

Figure 10

Figure 10. Serial Deletions Series for the Promoter Regions Used To Determine the Minimal Sequence Required to Drive GFP Expression in Muscle

(A) C34E10.6 promoter length 700–118 bp drives expression in muscle and neural tissue. (B) F15G9.4a promoter length 2,869–326 bp drives expression in body wall muscle; at 132 bp, all expression was lost. (C) T27A1.4 promoter length 702–157 bp drives expression only in muscle; at 53 bp, all expression was lost. (D) T04A8.4 promoter length 2,903–995 bp drives expression in pharyngeal, vulval, and body wall muscles; promoter length 805–143 bp fails to drive expression in pharyngeal muscle; at 55 bp, all muscle had lost expression.

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