A combined approach exploring gene function based on worm-human orthology

Abstract

Background: Many aspects of the nematode Caenorhabditis elegans biology are conserved between invertebrates and vertebrates establishing this particular organism as an excellent genetic model. Because of its small size, large populations and self-fertilization of the hermaphrodite, functional predictions carried out by genetic modifications as well as RNAi screens, can be rapidly tested.

Results: In order to explore the function of a set of C. elegans genes of unknown function, as well as their potential functional roles in the human genome, we performed a phylogenetic analysis to select the most probable worm orthologs. A total of 13 C. elegans genes were subjected to down-regulation via RNAi and characterization of expression profiles using GFP strains. Previously unknown distinct expression patterns were observed for four of the analyzed genes, as well as four visible RNAi phenotypes. In addition, subcellular protein over-expression profiles of the human orthologs for seven out of the thirteen genes using human cells were also analyzed.

Conclusion: By combining a whole-organism approach using C. elegans with complementary experimental work done on human cell lines, this analysis extends currently available information on the selected set of genes.

Figure 1

Maximum parsimony and Neighbor joining trees for F41D9.1 (A, B) and C13F10.4 genes (C,D). Illustration of the criteria that has been applied in order to select the genes. Only genes able to produce trees as shown in A and B (F41D9.1) were subjected to experimental work.

Figure 2

F41D9.1::GFP expression. Widespread through the neural system. Panel A shows the general expression pattern in an L1 stage animal. Many cells in the nerve ring, the ventral nerve cord (vnc) and the tail region express GFP. Panel B presents the tail region in greater detail, scanning through the animal at three focal planes from right (Bi) to left (Biii), with the ventral side facing down. A cluster of laterally symmetrical cells is visible in panels Bi and Biii, whereas in Bii cells of the vnc are visible. Magnification is 100×. Fig C presents 3 focal planes from dorsal (Panel Ci) to ventral (Panel Ciii) through the worm head, with the posterior pharyngeal bulb to the left. The GFP images have undergone deconvolution to increase resolution. Cells of the dorsal ganglion are visible in Ci, the retrovesicular ganglion is marked in Cii, and processes leading to it are indicated in Ciii. Scale bar represents10 μm.

Figure 3

C17E4.3::GFP reporter expression. Present in the head and tail regions of larval stages. Panel Ai shows an overview of an L1 animal, with distinct cells near the anterior pharyngeal bulb as well as in the tail. Close up of the head reveals GFP expressing cells (Panel Bii), including muscle cells located in the posterior pharyngeal bulb (Panel Biv, labeled 'p'). Sheath/socket cells located at the anterior pharyngeal bulb are indicated in panels Biv, Dii and Diii. Dye-filling tests (see Materials and Methods) to stain sensory amphid (head) and phasmid (tail) neurons are shown in panels C and D. The amphids are specifically visualized in panel Civ (red fluorescence), and under an FITC filter in Ciii (yellow fluorescence, not in nucleus) and green with an EGFP filter, and are not the same as the cells expressing the C17E4.3::GFP reporter (arrow in panels Cii and Ciii). In the tail, phasmids (labeled 'ph') are clearly seen in panel Div just below the anus (cl), but are not expressing GFP, which is instead present in several hypodermal cells (arrow panel Diii). Scale bar represent 10 μm.

Figure 4

ZK795.3::GFP reporter expression. Evident in the excretory cell system (panel A) in about 20% of animals, but was always present in the excretory cell (labeled 'ex', panels A and Bi). The anal sphincter and/or depressor cell around the anus ('cl') also expressed GFP (panel Bii), as did the juvenile vulva ('v', panel Biii) and the spermathecae (panel C). Scale bar represents 10 μm.

Figure 5

C09D4.1::GFP expression. Limited to the nuclei of intestinal cells. The top figure is an overlay of the DIC and GFP images. Scale bar represents 10 μm.

Figure 6

Subcellular localization of recombinant fusion proteins in human cell lines. Human protein NP_219484 (C. elegans protein ZK795.3) was detected in transfected HeLa cells with goat anti-V5 conjugated with FITC and co-localized with human anti-fibrillarin subsequently detected by donkey anti-rabbit Cy3 (A). Nuclei were stained with DAPI. This protein displays both a nucleolar (top row) and nuclear speckle (bottom row) pattern (A). NP_848545 (C. elegans protein C17E4.3) was detected in HEK293 cells with mouse anti-V5 and goat anti-mouse Alexa 488 (B). Cells were costained with rabbit anti-Lamin and donkey anti-rabbit Cy3 illustrating a colocalization with the nuclear envelope (B). Partial ER and nuclear distributions were observed for NP_115683 by co-detection with rabbit anti-calreticulin in HeLa cells (C). AAP34400.1 was detected in HeLa cells at the plasma membrane by co-staining with Annexin II (left panel, D) and a partial colocalization with beta-actin was also detected (right panel, D). Scale bars represent 10 μm.