Regulation of Wnt Signaling and Embryo Patterning by an Extracellular Sulfatase
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Supplementary data are available on Science Online at www.sciencemag.org/cgi/content/full/293/5536/1663/DC1.
cDNA reverse transcribed from RNA prepared from segmental plate mesoderm and somites I to III of stage 12 quail embryos was polymerase chain reaction (PCR)–amplified with an arbitrary primer 5′-GCTCTTTGTC-3′ and an anchored primer 5′-GGAATTCTTTTTTTTTTTTGG-3′. PCR products were resolved by electrophoresis on 6% denaturing polyacrylamide gels and a somite-specific 241–base pair PCR product (S1) was identified and used as a probe to recover a 5.8-kb full-length QSulf1 cDNA from a stage 12 quail embryo cDNA library. Gene expression in embryos was assayed with whole mount in situ hybridization with digoxygenin-labeled riboprobes (14). Stained embryos were sectioned transversely with a vibrotome in 100-μm sections.
Phosphorothiolated QSulf-1 antisense oligonucleotides (5′-CCAAGAGGTCTTCATAG-3′ 5′-GAGCACTGCCAAGAAG-3′ and 5′-CTCTTGCTGCACACGGC-3′) were modified by phosphorothiolation of the first four and last four linkages and purified by high-performance liquid chromatography. Oligonucleotide pairs 1 and 2 and 1 and 3 inhibited QSulf-1 mRNA accumulation in stage 12 embryos. Oligonucleotide pair 2 and 3 did not disrupt expression and served as a negative control along with a random oligonucleotide: 5′-CTATGGTACGATCAACG-3′. Embryos were cultured with antisense oligonucleotides as previously described (14).
10T1/2 cells were transfected with Fugene 6 (Boerhringer/Mannheim) and transferred onto uncoated glass cover slips for 24 hours. Unpermeabilized cells were incubated for 1 hour at room temperature in a 1/500 dilution of 9E10 monoclonal antibody to myc or antibody to β-tubulin (Boerhringer/Mannheim) washed fixed in 2% paraformadehyde and incubated for 1 hour with 1/500 dilution of Cy3 anti-mouse antibody (Jackson Immuno Research) in the presence of 10% fetal bovine serum. Permeablilized cells cultured on cover slips were prefixed in 2% paraformadehyde before primary and secondary antibody staining. For Western blot analysis cell extracts and medium from transfected cultures were resolved by electrophoresis on SDS gels transferred to nitrocellulose and probed with 9E10 antibody to myc. Antibody staining was visualized with chemiluminesence (SuperSignal; Pierce Chemicals).
G. K. Dhoot et al. unpublished data.
Shimizu H., et al., Cell Growth Differ. 8, 1349 (1997).
Full-length QSulf1 cDNA was cloned into the pAG-myc vector (QSulf1-myc). C2C12 myoblasts were transfected with expression plasmids with a calcium phosphate protocol. Stably transfected clones were selected with hygromycin. Individual and pooled (200) clones were assayed for QSulf1 expression by immunostaining and Western blotting and for Wnt1 signaling activity by cotransfection with TCF luciferase reporter genes and with Renilla control vector (Promega Dual Luciferase Reporter Assay System). After 6 hours transfected cells were cocultured for 24 hours with Wnt1-expressing or control rat B1a cells in the presence or absence of heparin (Sigma; porcine intestinal mucosal) or sodium chlorate (Sigma). For some studies WT9 QSulf1-expressing C2C12 cells were retransfected with QSulf1-myc or mutant QSulf1-myc (CC89 90AA) plasmids as a mixture pAG empty vector in a total of 1.5 μg of DNA per transfection.
Single-letter abbreviations for the amino acid residues are as follows: A Ala; C Cys; D Asp; E Glu; F Phe; G Gly; H His; I Ile; K Lys; L Leu; M Met; N Asn; P Pro; Q Gln; R Arg; S Ser; T Thr; V Val; W Trp; and Y Tyr.
We thank M. Lai for excellent technical assistance; G. Esko J. Raper B. Vogelstein and T. Brown for valuable reagents; and A.-G. Borycki and B. Brunk for helpful advice. We thank the NIH for a grant to C.P.E. the Royal Veterinary College for salary support to G.K.D. during sabbatical leave at the University of Pennsylvania School of Medicine and the Royal Society and Welcome Trust for ongoing research support to G.K.D.