zebrafish Iguana (Sekimizu et al. 2004; Wolff et al. 2004).
Additionally, a Drosophila protein complex that in-
cludes Cos2 is similarly required for full pathway acti-
vation (Robbins et al. 1997; Sisson et al. 1997; Wang and
Holmgren 2000; Wang et al. 2000; Lefers et al. 2001) and
inhibition (Methot and Basler 2000; Stegman et al. 2000;
Wang et al. 2000; Lefers et al. 2001). Tectonic is the first
extracytosolic factor shown to act in this dual capacity.
Although the molecular mechanism by which Tec-
tonic functions is not clear, our double mutant analyses
suggest that it modulates Hh signal transduction at a
point fairly downstream in the pathway. As Rab23 acts
in the same region of the pathway and is thought to
control vesicle transport, it will be interesting to assess
whether it regulates the trafficking of Tectonic.
Materials and methods
The mouse embryonic stem cell line KST296 carrying an insertion of the
pGT1pfs secretory trap vector in the Tectonic gene was isolated as de-
scribed in Mitchell et al. (2001). Tectonic F1 heterozygotes were back-
crossed to C57Bl/6 mice for six generations prior to intercrossing. Geno-
typing of Tectonic was performed using genomic PCR with a pair of
wild-type-specific primers (5⬘-CGCCTCTTTAGCCCTCTGTT-3⬘ and
5⬘-AGAACCTCCACGAGAGCAGA-3⬘) and a mutant-allele-specific
primer (5⬘-TCTAGGACAAGAGGGCGAGA-3⬘). Ptch, Rab23, Shh, and
Smo embryos were genotyped as described (Chiang et al. 1996; Goodrich
et al. 1997; Eggenschwiler et al. 2001; Zhang et al. 2001).
Cos7 cells were transfected using Fugene6 (Roche) with APTag5 (Gen-
Hunter) or APTag5-TectSignal, a vector in which the SEAP signal se-
quence has been replaced with that of Tectonic. Alkaline phosphatase
activity in the supernatant was chemiluminescently measured using the
Phospha-Light Assay (Applied Biosystems) and a 20/20
RT–PCR and Northern blot analyses
RT–PCR was performed using exon-spanning primers complementary to
Tectonic cDNA 3⬘ to the gene trap insertion (5⬘-AATCCGCTGTTCC
TTCCAC-3⬘ and 5⬘-TGCGTCAGTGTGTGATTCAG-3⬘), to the ␤GEO
transcript (5⬘-CTTGGGTGGAGAGGCTATTC-3⬘ and 5⬘-AGGTGAG
ATGACAGGAGATC-3⬘), and to G3PD (5⬘-GTGTTCCTACCCCCAAT
GTG-3⬘ and 5⬘-TGTGAGGGAGATGCTCAGTG-3⬘). Northern blots
were hybridized to a Tectonic cDNA probe spanning exons 2–12 and a
probe to ␤geo.
Immunohistochemistry and in situ hybridization
X-gal staining, in situ hybridization, and immunohistochemical staining
were carried out using antibodies and protocols as previously described
(Ericson et al. 1997; Briscoe et al. 1999, 2000; Takebayashi et al. 2000;
Gritli-Linde et al. 2001) with the exception of rabbit ␣-Pax6 antibody
(Covance Research Products), which was used at 1:300. The ␣-FoxA2,
␣-Nkx2.2, ␣-Islet1/2, ␣-Msx1/2, and ␣-Pax3 antibodies were obtained
from the Developmental Studies Hybridoma Bank maintained by the
University of Iowa under contract NO1-HD-7-3263 from the NICHD.
Gene analysis and accession numbers
Sequences of Tectonic family members were aligned using ClustalW and
Boxshade 3.21. Domain analysis was performed with SignalP 3.0 and
HMMTOP 2.0. Mouse Tectonic cDNA sequence, GenBank accession
number DQ278867; human Tectonic cDNA sequence, GenBank acces-
sion number DQ278868; mouse Tect2 cDNA sequence, GenBank acces-
sion number DQ278869; human Tect2 cDNA sequence, GenBank acces-
sion number DQ278870; mouse Tect3 cDNA sequence, GenBank acces-
sion number DQ278871; human Tect3 cDNA sequence, GenBank
accession number DQ278872; Drosophila dTectonic cDNA sequence,
GenBank accession number DQ278873.
We are grateful to Debbie Pangilinan for expert mouse husbandry, to
Andrew Norman for technical assistance, and to Pao-Tien Chuang and
Andrew McMahon for the anti-Shh antibody Ab80. We thank Kevin
Mitchell, Chulho Kang, and Marc Tessier-Lavigne for help generating the
KST296 ES cell line and the Tectonic mice. This work was assisted by
valuable conversations with Gail Martin, Pao-Tien Chuang, Didier
Stainier, and Tom Kornberg. This research was supported by funding
from the Chicago Community Trust and grants from the NICHD and
NIMH. J.R. is a UCSF Fellow generously supported by the Sandler Foun-
dation and a March of Dimes Basil O’Connor Award.
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