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doi: 10.1242/10.1242/dev.00540

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SDF-9, a protein tyrosine phosphatase-like molecule, regulates the L3/dauer developmental decision through hormonal signaling in C. elegans

Kiyotaka Ohkura^?, Norio Suzuki^*, Takeshi Ishihara and Isao Katsura

^? Structural Biology Center, National Institute of Genetics and School of Genetics, Faculty of Life Sciences, Graduate University for Advanced Studies, Mishima, Shizuoka 411-8540, Japan
^* Present address: Laboratory for Cell Migration, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe 605-0074, Japan

View larger version (92K): [in a new window]   Fig. 1. Nomarski micrographs of the pharynx and the lateral cuticle of mutant dauer larvae. (A) sdf-9(ut163) dauer-like larva. (B) daf-9(e1406) dauer-like larva. (C) daf-7(e1372) dauer larva. Left and right panels show the pharynx and the alae, respectively. In the left panels the arrows indicate the terminal bulb and the arrowheads the isthmus. The pharynxes of sdf-9 and daf-9 mutants are similar to the L3 pharynx and thicker than the daf-7 dauer pharynx, which looks like the pharynx of the wild-type dauer larva. In addition to pharyngeal morphology, the pharynx of normal dauer larvae (e.g. wild type, daf-7, and daf-2) never pumps, whereas those of sdf-9 and daf-9 dauer larvae pump. The arrowheads in the right panels indicate longitudinal ridges called alae. They are present at the L1, dauer, and adult stages but absent at the L3 stage. Scale bar: 10 µm.

View larger version (18K): [in a new window]   Fig. 2. Molecular cloning of sdf-9. (A) Genetic and physical map of the sdf-9 region. The arrows indicate genes predicted by the C. elegans genome project. Genomic DNA fragments containing the predicted gene Y44A6D.4 rescued the Daf-c phenotype of unc-31(e169); sdf-9(ut187) double mutants. (B) Structure of sdf-9 gene and reporter constructs. Boxes represent exons. pKO2 and pKOG7 rescued the unc-31(e169); sdf-9(ut187) Daf-c phenotype.

View larger version (28K): [in a new window]   Fig. 3. cDNA and deduced amino acid sequence of sdf-9. The SL1 trans-splice leader sequence at the 5' end is boxed. The PTP domain is underlined and the active site sequence is doubly underlined. The DNA bases and the amino acid residues that are changed in the sdf-9 mutants are marked with asterisks and arrowheads, respectively.

View larger version (71K): [in a new window]   Fig. 4. Alignment of the PTP domains of SDF-9 and known PTPs. Conserved residues and homologous residues (gray) are boxed. The active site of PTP is underlined. The cysteine residue (Cys 223) that is essential for phosphatase activity is replaced by serine in SDF-9. HsRPTPA-D1: human receptor PTP alpha proximal domain, HsRPTPA-D2: human receptor PTP alpha distal domain, HsPTP1B: human PTP1B.

View larger version (63K): [in a new window]   Fig. 5. Expression pattern of sdf-9. (A) Expression of sdf-9p::GFP2 (pKOG9) in the head region of an L3 larva. The left-hand cell, which is in focus in this micrograph, is strongly fluorescent, while the right-hand cell, which is also strongly fluorescent, is out of focus. The arrows indicate short dendrite-like structures. (B) Expression of sdf-9p::GFP2 expression in the head region of an L1 larva. The right-hand cell that express the GFP fusion gene is indicated. The arrow shows the dendrite-like process, which extends to the tip of the head. (C) Expression of SDF-9::GFP (pKOG7) in two head cells of a daf-9(e1406) dauer larva. One of the two dendrite-like processes extends posteriorly. Both cells have processes, but those of the left-hand cell are out of focus. The images in the right white boxes in (A) and (B) are magnified versions of those in the left white boxes, respectively, and the bottom image is a magnified version of that in the white box in (C). Scale bar: 10 µm.

View larger version (52K): [in a new window]   Fig. 6. Identification of the sdf-9-expressing cells. (A) Expression of sdf-9p::RFP (left) and DAF-9::GFP (middle) in an adult animal. Only the left-hand cell is in focus. A merged image (right) shows that RFP (nuclear) and GFP (perinuclear) are expressed in the same cell. Scale bar: 10 µm. (B) Cell lineage of the candidates of sdf-9-expressing cells (not boxed) and the neurons that express the GFP markers (boxed). The candidates are selected by their positions in the head and by the absence of che-2 expression (Fig. 7). Mosaic experiments indicated that sdf-9-expressing cells are XXXL and XXXR, which are indicated by asterisks. a: anterior, p: posterior, d: dorsal, v: ventral, l: left, r: right. (C) Cell lineages of the cells expressing sdf-9::RFP and the GFP marker cells as deduced from the mosaic experiments. Only the topology but not the left-right relationship in these lineages is relevant. The sublineages of five cells in the most left lineage and three cells in the most right lineage could not be determined from the data shown in Table 4.

View larger version (34K): [in a new window]   Fig. 7. Candidates of the sdf-9-expressing cells considered in the mosaic experiment [modified from figure 2 by White et al. (White et al.,1986)]. The figures show the cell bodies on the ventral side near the anterior bulb (metacorpus) of the pharynx. The top and the bottom diagrams show the left-hand and the right-hand views, respectively. Hatched cells are reported to express che-2::GFP (Fujiwara et al., 1999), and hence are excluded from the list of candidates.