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First published online 7 November 2006
doi: 10.1242/jcs.03274

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A systematic RNA interference screen reveals a cell migration gene network in C. elegans

Department of Molecular Biology, Princeton University, Princeton, NJ 08544-1014, USA

View larger version (98K): [in this window] [in a new window]   Fig. 1. Two-step screen to identify DTC migration defects. (A) Nematodes were cultured on bacterial strains from hatching to adulthood. The primary screen was performed 48 hours post hatching and young adults were scored for the presence of clear patches using a dissecting microscope. In the secondary screen, DTC migration paths were documented by Nomarski microscopy. (B) Diagram illustrating one arm of the hermaphrodite gonad. The DTC is indicated. Cuboidal cells are maturing oocytes. Ovals are fertilized embryos. (C-G) Nomarski images showing three types of defects. Treated rrf-3(pk1426) hermaphrodites were grown on E. coli HT115(DE3) carrying an empty vector (C) or RNAi targeting vectors for act-1 (D), ced-5 (E), pat-3 (F), or dyn-1 (G). Arrows indicate paths taken by DTCs. Anterior is to the left, ventral down in all images except D and F in which anterior is to the right. Bars, 20 µm.

View larger version (94K): [in this window] [in a new window]   Fig. 2. Comparison of gonad defects with depletion of hlh-2, ppn-1 and gon-1. Nomarski microscopy was used to visualize the gonads of age-matched hermaphrodites that were hatched and grown on bacteria carrying RNAi-targeting constructs for hlh-2 (A), ppn-1 (B) and gon-1 (C). Arrows indicate lengths of paths taken by DTCs. Anterior is to the left, ventral is down in all images. Bars, 20 µm.

View larger version (81K): [in this window] [in a new window]   Fig. 3. ppn-1 expression in the DTC and regulation by hlh-2. Eggs from ppn-1::GFP nematodes were hatched and grown on bacteria carrying an RNAi construct for hlh-2 or on bacteria with a control plasmid. After 48 hours, Nomarski and fluorescence images were captured using identical exposure settings (A). DTC locations are indicated with arrowheads. DTC paths are diagrammed at the left. (B) Equal-sized ovals were centered around the DTCs on the Nomarski images and fluorescence intensities within the ovals were measured for ten animals from each condition using IPLab software. The average fluorescence ± s.d. for control and hlh-2 RNAi experiments are shown. (C) An embryo expressing ppn-1::GFP shows expression in the DTC precursor cells Z1 and Z4 indicated by arrowheads in the fluorescence image. The gonad primordium is outlined in the Nomarski image.

View larger version (42K): [in this window] [in a new window]   Fig. 4. Distributions of DTC migration genes by functional class. Functional classes for C. elegans genes as described previously (Kamath et al., 2003) are listed on the right. (A) The relative percentages for each functional class of genes that showed any RNAi phenotype are indicated. (B) Percentages for each functional class of those genes resulting in DTC phenotypes are shown. The functional class `Nucleic acid binding' includes the categories RNA binding, DNA binding, NA binding and Chromatin. `ECM' (extracellular matrix) includes collagens, ppn-1 and dig-1.

View larger version (58K): [in this window] [in a new window]   Fig. 5. Development of a DTC migration gene network. Red nodes represent the genes required for DTC migration isolated in this screen. Green nodes are a subset of interacting genes derived from a genome-wide prediction of C. elegans genetic and physical interactions (Zhong and Sternberg, 2006). For brevity, only red nodes are identified. For identities of all nodes see Fig. S1 in supplementary material. Identities of red nodes: (1) Y71G12B.11, (2) pat-3, (3) unc-112, (4) vab-10, (5) gex-3, (6) ced-5, (7) ced-10, (8) erm-1, (9) ina-1, (10) sdn-1, (11) gon-1, (12) unc-73, (13) dyn-1, (14) mig-15, (15) act-1, (16) unc-32, (17) cdc-42, (18) vab-3, (19) C16C10.6, (20) uba-2, (21) let-2, (22) rab-8, (23) cbp-1, (24) F32D1.2, (25) rpl-11.1, (26) ppn-1, (27) arf-3, (28) cogc-4, (29) apc-2, (30) ruvb-1, (31) cct-1, (32) W03H9.4, (33) F26F4.11, (34) cogc-3, (35) R53.6, (36) mcm-7, (37) icd-1, (38) Y65B4BR.5, (39) Y55F3AR.3, (40) tba-1, (41) mix-1, (42) smc-4, (43) tba-2, (44) tba-4, (45) ZK430.7, (46) H06I04.3, (47) F21C3.5, (48) snr-5, (49) T13H5.4, (50) Y59A8B.6, (51) F11A3.2, (52) tbg-1, (53) tbb-2, (54) imb-5, (55) tbb-1, (56) T04A8.3, (57) npp-9, (58) W07E6.1, (59) npp-10.

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