Figure 1.
GFP-tagged embryonic muscle cells purified by FACS.
MYO-3::GFP expression in the body wall muscle cells of an RW1596 embryo (A,B), and in purified embryonic muscle cells after isolation and FAC sorting (C,D) visualized by Fluorescence microscopy (A,C) and DIC microscopy (B,D). Final fluorescence intensity scatter plot of wild-type (non-GFP) cells (E) and MYO-3::GFP positive cells (F). Cells in box P3 were sorted as GFP positive.
Figure 2.
Reproducibility of SAGE biological replicate libraries.
Comparison of two Long SAGE biological replicate libraries for embryonic muscle cells in C. elegans (SW031 and SWEM1). A total of 88% of the sequence tags are similarly expressed with a correlation coefficient of 0.94. DiscoverySpace was used to generate the plot [68].
Figure 3.
Comparison of the embryonic muscle SAGE profile with other tissue-specific SAGE libraries and Affymetrix GeneChip data.
The overlap between genes found in tissue specific SAGE libraries for C. elegans muscle (SWEM1 and SW031), intestinal (SWEG1) and pan neuronal (SW028) cells are shown in (A). Only 11%–16% of the genes identified are unique for each cell type. (B) Comparison of both muscle SAGE libraries to previously published Affymetrix GeneChip data [26] on sorted embryonic muscle cells. Less than half (45%) of the 7,974 genes detected by SAGE were also detected by GeneChip technology.
Figure 4.
Schematic diagram for the two RNAi by feeding screens.
The muscle transcriptome includes 3,297 genes that overlap with the Ahringer RNAi feeding library, the SAGE dataset and the Affymetrix GeneChip dataset. (A) The protocol used for a high-throughput liquid RNAi feeding screen to identify genes essential for embryonic muscle formation and (B) the protocol used for an RNAi feeding screen on solid media for defects in myosin localization using RW1596, a MYO-3::GFP expressing strain. See Materials and Methods for details.
Figure 5.
Body wall muscle structure in wild-type and RNAi-treated animals.
(A) Myofilaments visualized by polarized light microscopy in wild-type animals treated with the empty RNAi feeding vector L4440 showing nicely organized filament structure (arrow). In animals treated with dsRNA for T27B1.2 (B), F31D5.3 (C) or T28B4.3 (D), filaments are disorganized and gaps are present in the myofilament (asterisk).
Figure 6.
Sarcopenia in aging nematodes.
Synchronized animals were observed for irregularities in filament organization with age using the GFP-tagged MYO-3 marker strain RW1596. Fluorescence images are shown for L4/young adult (A), 1-day adult (B), 2-day adult (C), and 3-day adult (D) animals. As the filament structure is nicely organized in L4/young adult animals (arrow), both the severity of myofilament disorganization (asterisk) as well as the percentage of animals affected increase during aging (E). To preclude any artifacts caused by GFP itself, we visualized disorganized filaments in 3-day adult RW1596 animals using polarized light (F, asterisk). No defects are visible in 3-day adult N2 animals (G, arrow), indicating an early onset of sarcopenia in RW1596 muscle filaments.
Figure 7.
Diagram summarizing the results of the RNAi screen for defects in myosin localization.
(A) The results of the preliminary screen were grouped into four classes according to the proportion of animals showing irregularities in myosin localization after RNAi treatment. High class (HC) for [75–100], intermediate class (IC) for [50–75], low class (LC) for [25–50], and wild-type class (WT) for [0–25] percent affected animals per scoring. Only genes resulting in IC (248 genes) and HC (42 genes) were considered as “muscle affecting” to be further analyzed in the re-screen. Feeding of 45 different RNAi constructs resulted in embryonic or adult lethality (green); no animals could be scored in the F1 generation and these genes were included in a modified re-screen (see Materials and Methods for details). (B) Summarized results of re-screening 335 genes identified as muscle affecting in the preliminary screen. Twelve genes remained in the HC category, 6 of which have been previously reported to affect C. elegans muscle structure, and 107 genes were scored in the intermediate class, including 7 known muscle-affecting genes. The remaining 216 genes did not reach the increased stringency and dropped under the 50% cutoff.
Figure 8.
RNAi induced myofilament abnormalities visualized by MYO-3::GFP.
Representative fluorescent images are shown for (A) the empty vector plasmid L4440, resulting in proper wild-type filament organization and (B) GFP RNAi resulting in strong reduction of GFP expression. The MYO-3::GFP abnormalities observed in the RNAi screen were grouped into three categories. (C) and (D) showing general disorganization of myofilaments with minor aggregations, (E) and (F) exhibit well ordered and small aggregations (arrow) along otherwise well organized filaments, and (G) and (H) large GFP clumps or deposits.