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Biological significance of a universally conserved transcription mediator in metazoan developmental signaling pathways

Department of Biology, Yonsei University, 134 Shinchon-dong, Seodaemun-ku, Seoul 120-749, Korea
^* Author for correspondence (e-mail: Leej{at}yonsei.ac.kr )

View larger version (18K): [in a new window]   Fig. 1. Identification of the med-6 mutation. (A) The physical and genetic maps of the med-6 region. The yeast artificial chromosome (YAC) Y57E12 contained the med-6 gene. The cosmid gap within the YAC is indicated. The genetic deficiencies, sDf20 and sDf30, delete both unc-68 and odr-2, whereas the deficiency, nDf32, does not. Lethal mutations, let-322, let-339, let-343, let-404, let425, let-438 and let-468, which were mapped in that region, are shown. The map is not drawn to an exact scale. (B) Genomic structure of med-6 and the mutation identified in let-425. The med-6 gene contains three exons separated by two introns. The med-6 gene isolated from the homozygous let-425 animals contained a point mutation that changes C to T, causing a change in the amino acid residue from Arg to a stop codon. The numbers above the genomic structure are the amino acid numbers encoded by the gene.

View larger version (75K): [in a new window]   Fig. 2. Typical phenotypes of let-425 homozygotes (A-C), Nomarski images of the animals at the L3 molt stage, when the induced vulval precursors cells have divided to form four progeny. (A) Wild type, (B) homozygous let-425 mutant animals and (C) a wild-type animal affected by med-6 RNAi. (D,E) Nomarski images of the vulval tissue at the L4 stage, when invagination of the induced vulval precursor cells occurs. (D) Wild type, (E) a homozygous let-425 mutant animal, showing an abnormal invagination caused by less than wild-type induction of VPCs. (F) Nomarski image of a homozygous let-425 mutant animal at the adult stage. Hypo-induced VPCs have undergone morphogenesis and formed a protruding vulva. Scale bar: 25 µm.

View larger version (173K): [in a new window]   Fig. 3. med-6 is involved in regulating genes in the Ras pathway: vulval phenotypes before and after med-6 RNAi in various genetic backgrounds. The left panels show the vulval tissues at the L4 stage before med-6 RNAi, and the right panels, after RNAi. (A,B) lin-3 (e1417); (C,D) lin-3 (syIs1); (E,F) let-23(sa62); and (G,H), let-60(n1700). This figure shows representative animals only. For example, (A) shows a wild-type vulva of an animal of the genotype lin-3(e1417), whose typical phenotype is 70-90% vulvaless, with only 10-30% wild-type vulval induction. For detailed data, see Table 1. The arrows indicate vulval tissue invagination. Wild-type animals always have only one invagination. Scale bar: 25 µm.

View larger version (147K): [in a new window]   Fig. 4. med-6 is involved in ray development. The locations where the V6-derived rays are supposed to be found are indicated by the lines in all figures. (A) A typical wild-type ray structure. (B) The ray phenotype caused by med-6 RNAi in the rde-1 background. Development of the V6-derived rays is affected by RNAi. (C) Defective V6-derived rays in a pal-1 (e2091) mutant animal. (D) The ray phenotype caused by med-6 RNAi in a pal-1(e2091) mutant animal. (E) The restored V6-derived rays in the pal-1; sop-1 double mutant animal. (F) The ray phenotype caused by med-6 RNAi in the pal-1; sop-1 double mutant background. The V6-derived rays are defective. Scale bar: 25 µm.

View larger version (14K): [in a new window]   Fig. 5. Experimental scheme of the rde-1 background RNAi. (A) Control experiment. Homozygous rde-1 unc-32 animals were microinjected with med-6 dsRNA, and the total number of eggs laid was counted. Individual progeny were transferred to individual plates, and phenotypes were examined after 4 days. (B) Maternal rescue RNAi. Homozygous rde-1 unc-32 animals were microinjected with med-6 dsRNA and were mated with wild-type males. The total number of eggs laid was counted. Non-Unc cross progeny were transferred to new plates individually, and checked for their sex and phenotypes. The percentages of dead embryos were determined by the differences in the numbers of eggs and viable animals.

View larger version (36K): [in a new window]   Fig. 6. An action model of the mediator-related proteins in yeast and C. elegans: the MED-6-containing mediator complex as a convergence point of transcriptional regulation. (A) In yeast, yeast-specific mediator proteins such as Gal11 and Hrs1 relay signals from transcription activators through the universally conserved mediator complex that contains MED-6 to the general transcription factors to regulate transcription. (B) In the nematode vulval development pathway, SUR-2 is a metazoan-specific mediator-related protein that interacts with outside signals (activated by EGF) and relays them through the conserved mediator complex containing MED-6 to the general transcription factors that regulate genes associated with the Ras pathway. The SynMuv proteins including the histone deacetylase complex proteins may counteract the mediator functions. (C) In the nematode ray developmental pathway, SOP-1 is a mediator-related protein that represses the Wnt-mediated transcriptional activation, which is dependent on the universally conserved mediator complex that contains MED-6.