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First published online 17 December 2003
doi: 10.1242/dev.00922

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DAF-5 is a Ski oncoprotein homolog that functions in a neuronal TGFß pathway to regulate C. elegans dauer development

¹ Department of Biochemistry and Molecular Biology, Rutgers University, Piscataway, NJ 08854, USA
² Cancer Institute of New Jersey, New Brunswick, NJ 08853, USA

View larger version (74K): [in a new window]   Fig. 1. DAF-5 encodes a C. elegans homolog of the Sno and Ski oncoproteins. (A) Identification of daf-5-coding region. The genetic map near daf-5 and rescued transgenic lines isolated are shown. (B) DAF-5 gene structure. At the C terminus of DAF-5, a stop codon and a deletion mutant are connected by a broken line to indicate that these two mutations were identified in a single allele. A mutation hotspot is shown; an additional mutant has an in-frame deletion of the 15 amino acids shown. Colors of amino acids in hotspot represent sequence conservation as in Fig. 1C,D. See Table S2 at http://dev.biologists.org/supplemental for complete details on mutant alleles. (C,D) Alignment of SDS and Dach boxes. Consensus at each position was defined as any set of identical and similar amino acids that were found in more than one subgroup (subgroups are indicated by spaces between the rows of sequence), and in more than three proteins in total. Bold magenta residues are identical in the primary consensus (the consensus with the most matches) and plain, magenta are similar. Green residues are the secondary consensus. The % symbols below the SDS box show conserved zinc chelating residues, and the asterisks indicate amino acids that contact Smad4 in the crystal structure of a Ski/Smad4 complex. The sequence for Dog Nem. is from an EST, and has two stop codons in frame (indicated by X). These may result from sequencing errors, or the cDNA may have come from a pseudogene. Accession Numbers and abbreviations: Dros., D. melanogaster (Iceskate: NP_651946; Snowski: NP_609166; Dachshund: NP_723972); Mosq., mosquito (A. gambiae; Iceskate-XP_317739; Snowski-XP_317545); Soy. Nem., Soybean Cyst nematode (Heterodera glycines; CA939358); Root nem., Root Knot nematode (Meloidogyne chitwoodi; CB931358); Pot. Nem., Potato Cyst nematode (Globodera rostochiensis; AY389814); Lymph Nem, Brugia malayi (AY389813); Dog Nem, dog hookworm (Ancylostoma caninum; AW735310); C. briggsae DAF-5 (found in Wormbase as CBG20832); C. elegans (DAF-5-NM_064540, NP_496941; Dachshund-NP_497266); Human (Dachshund-NP_542937; cSki-NP_003027; SnoN-NP_005405; Icy-XP_292349; Skate-XP_064560); sea squirt (Ciona intestinalis; Ski-BK001616; Dachshund-AABS01000073).

View larger version (24K): [in a new window]   Fig. 2. Binding of DAF-3 to DAF-5 in yeast two-hybrid assays. Interactions were scored by transcriptional activation of a His reporter and a ß-gal reporter. The strength of activation was assigned a score in each assay, and the symbols reflect the sum of the scores for all assays (see Materials and methods).

View larger version (85K): [in a new window]   Fig. 3. daf-5 is expressed in the nervous system and preferentially localized to nuclei. Expression of functional daf-5::GFP. (A) Functional daf-5::GFP is predominantly expressed in head ganglia. Several neurons in the head ganglia are indicated by arrowheads. (B) DAF-5 is localized to the nucleus. This panel is an enlargement of the area boxed in A. A triangle shaped neuron was glowing with DAF-5::GFP mostly found in its oval nucleus.

View larger version (104K): [in a new window]   Fig. 4. daf-5 controls cell cycle arrest in the seam cells of dauer larvae by regulating expression of a cyclin kinase inhibitor. (A,B) rnr-1::GFP expression in N2 and daf-5; daf-1 double mutants. A corresponding Nomarski image can be found in Fig. S2 at http://dev.biologists.org/supplemental. White arrowheads show seam cells that express rnr-1::GFP in N2 and in daf-5; daf-1, respectively, at early L3 stage. (C,D) cki-1::GFP expression in daf-1 and daf-5; daf-1 mutants. A corresponding Nomarski image can be found in Fig. S2 at http://dev.biologists.org/supplemental. (C) cki-1 is expressed along the seam cells in all daf-1 dauer larvae with an average of 16 green cells. White arrowheads (E) show seam cell nuclei. (D) cki-1 is expressed in a subset of daf-5; daf-1 mutants in early L3. Two daf-5; daf-1 animals are shown. The top animal is representative of the 37% of animals of this genotype that had no GFP in the seam at all. The bottom animal is representative of the 34% that expressed GFP faintly in many seam cells (between five and ten). White arrows point to the seam cells that do not express cki-1::GFP in the nuclei. Scale bars: 1 µm.

View larger version (29K): [in a new window]   Fig. 5. Model for Sno/Ski and DAF-5 function. (A) Antagonism by Sno and Ski in vertebrates. (B) Function of DAF-5 in C. elegans. See Discussion for explanation of models.