|
|
|
|||||||
|
|||||||||
(Received for publication, January 31, 1996; and in revised form, March
26, 1996) From the
Drosophila patched is a segment polarity gene required
for the correct patterning of larval segments and imaginal discs during
fly development and has a close functional relationship with hedgehog. We have isolated a complete human PATCHED cDNA sequence, which encodes a putative protein of 1296 amino
acids, and displays 39% identity and 60% similarity to the Drosophila PATCHED protein. Hydropathy analysis suggests that
human PATCHED is an integral membrane protein with a pattern of
hydrophobic and hydrophilic stretches nearly identical to that of Drosophila patched. In the developing mouse embryo, patched is initially detected within the ventral neural tube
and later in the somites and limb buds. Expression in the limb buds is
restricted to the posterior ectoderm surrounding the zone of polarizing
activity. The results show that patched is expressed in target
tissues of sonic hedgehog, a murine homolog of Drosophila
hedgehog suggesting that patched/hedgehog interactions have been conserved during evolution. Human PATCHED maps to human chromosome 9q22.3, the candidate region
for the nevoid basal cell carcinoma syndrome. Patched expression is
compatible with the congenital defects observed in the nevoid basal
cell carcinoma syndrome.
The concept of diffusible morphogens has attracted widespread
attention in developmental biology, since it provides a useful model to
explain the patterning of tissues along a particular axis in an
organism. According to this paradigm, position-specific cell fates are
acquired due to the response of individual cells to different
concentrations of a long range signal, which is secreted by a distinct
inducing tissue (reviewed in (1) ). Drosophila patched (ptc) (
The sequence of human PTC consists of an open reading frame
of 4242 nucleotides flanked by 87 and 2238 nucleotides on the 5`- and
3`-untranslated regions, respectively (Fig. 1). The open reading
frame of human PTC cDNA encodes for a putative protein of 1414
amino acids. The first AUG codon is located 357 bases into the reading
frame and has a moderate match for the translational start consensus
sequence in vertebrates (GAGGCTAUGT in PTC versus GCCGCCAUGG(14) ). Assuming that this codon encodes for the
first amino acid of the protein, human PTC consists of 1296 amino acids
with a relative molecular weight (M
Figure 1:
Sequence of the human PTC cDNA. The sequence of PTC is shown including the open
reading frame and flanking 5` and 3` sequences. The open reading frame
(ORF) is at +1 to +3870. The first ATG in the ORF is in bold type. The poly(A) signal and mRNA destabilizing signals
are underlined.
Hydropathy analysis (15) of the entire open reading frame of
human PTC predicts the presence of eight main hydrophobic stretches (Fig. 2). Distribution of the hydrophobic blocks is remarkably
well conserved between species indicating that human PTC, like its Drosophila counterpart, is an integral membrane protein.
Figure 2:
Hydropathy plot of PTC proteins. The
hydropathy of the predicted ORF of human PTC of 1414 amino acids was
analyzed by the modified method of Kyte and
Doolittle(2, 15) . A, human PTC; B, Drosophila Ptc.
Figure 3:
Expression of PTC in selected
adult human tissues. Northern blots (2 µg of
poly(A)
Figure 4:
Expression of Ptc during murine
embryogenesis. A, expression is first detected at
Figure 5:
Details of Ptc expression during
murine embryogenesis. A, section taken through the neural tube
of a 9.5 dpc embryo. Ptc expression is detected in the ventral
neural tube (nt) and the surrounding lateral mesenchyme. Note
the absence of Ptc expression in the notochord (arrow). B, section through the tail at 11.0 dpc.
Expression of Ptc is detected in the ventral neural tube (nt) and within epithelial cells along the medial edge of each
somite (s). C, in the limb-bud (lb),
expression of Ptc is restricted to posterior
ectoderm.
The
expression pattern of Ptc points to a close relationship
between Ptc and the hedgehog family of morphogens.
This relationship was originally established in Drosophila(6) . In vertebrates, the best characterized hedgehog homolog, sonic hedgehog, has been implicated
in the induction of the floorplate and motor neurons within the ventral
neural tube (16, 17) as well as in the differentiation
of sclerotome within the somites(18) . In the limb bud, sonic hedgehog expression in the mesenchymal ``zone of
polarizing activity''' triggers antero-posterior patterning
of the limb(10) . Our data show that vertebrate PTC is
expressed in all major target tissues of sonic hedgehog, such
as the ventral neural tube, somites, and tissues surrounding the zone
of polarizing activity of the limb bud. The striking spatial
complementarity and temporal coincidence of the sonic hedgehog and Ptc expression patterns suggest that both genes might
be members of a common signaling pathway. After the completion of this
work, Goodrich et al.(19) published the sequence of a
mouse Ptc gene with an expression pattern essentially
identical to that described here. The localization of PTC in the region containing the nevoid basal cell carcinoma syndrome
(NBCCS) gene is intriguing. NBCCS is an autosomal dominant disorder,
which predisposes affected individuals to basal cell carcinomas of the
skin, medulloblastomas, and various other tumors(20) . Recent
genetic studies have placed the gene for the nevoid basal cell
carcinoma syndrome to chromosome 9q22.3, between the markers Fanconi
anemia complementation group A (21) and D9S287(22) . Several lines of evidence suggest that PTC is a candidate gene for the nevoid basal cell carcinoma
syndrome. Ptc expression is compatible with the congenital
defects commonly found in NBCCS patients. Frequent symptoms in newborns
and infants are developmental anomalies of the spine and
ribs(20) . These malformations could be due to a PTC
deficiency, expression of which coincides spatially and temporally with
the development of the neural tube and of the somites. In addition, Ptc expression in the surface ectoderm surrounding the zone of
polarizing activity is consistent with limb abnormalities often
observed in the patients with NBCCS(20) . PTC expression in all adult tissues points to a pleiotropic role of PTC in adult signal transduction pathways. Defects in these
signaling pathways could account for the symptoms that develop
postnatally(23, 24) .
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank(TM)/EMBL Data Bank with accession number(s)
U43148[GenBank].
Volume 271,
Number 21,
Issue of May 24, 1996 pp. 12125-12128
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)
is a segment polarity gene required
for the correct patterning of larval segments and imaginal discs during
fly development(2, 3) . Based on genetic studies, patched is a component of the signaling pathway of the
morphogen hedgehog(4, 5, 6) . Since
Patched is a putative membrane-spanning protein, and is expressed in hedgehog-responsive cells, it has been proposed to be the hedgehog receptor(6) . In vertebrates, several hedgehog homologs have been i
dentified. The best characterized
of them, sonic hedgehog, has been implicated in the
dorsal-ventral patterning of neural tube(7, 8) , in
the differentiation of somites (9) and in the establishing of
the anterior-posterior axis of the limb bud(10) . The
biochemical basis of hedgehog signaling in vertebrates remains
poorly understood and has been hampered largely by the lack of a proven
receptor for the molecule.
Cosmid Isolation
Cosmids used in this study were
isolated from a human chromosome 9-specific genomic cosmid library
(LL09NC01``P'', Biomedical Sciences Division, Lawrence
Livermore National Laboratory, Livermore, CA) by screening with the YAC
clone ICI-2ef8 (United Kingdom Human Genome Mapping Project Resource
Centre). This clone contains the microsatellite marker D9S287,
which has been localized to chromosome 9q22.3(11) . The
isolation of YAC DNA and hybridization was performed as described
elsewhere(12) . The localization of the cosmids was confirmed
by hybridization to YAC ICI-2ef8 resolved by means of pulsed-field gel
electrophoresis (data not shown). The 96-well plate format of the
cosmid clones that contain PTC is 42H11, 96F9, 218A8, 226G7.Library Screening
Human cDNA clones were isolated
from a fetal brain cDNA library in the 
ZAPII phage vector
(Stratagene), using standard procedures. The probes were labeled with
[
P]dCTP by random priming (Rediprime, Amersham
Corp.). Positive clones were rescued using the 704 helper
phage/pBluescript excision system (Rapid Excision Kit, Stratagene) and
sequenced. Mouse genomic clones were isolated from a 129SV FixII
library (Stratagene). Phage DNA was cut with EcoRI and
hybridized with PTC-specific probes. Mouse cDNA clones were
isolated from an 11.5 dpc mouse embryo (Swiss male) library constructed
in gt10. Hybridization was performed at 55 °C. Positive
clones were subcloned into pBluescript II SK (Stratagene) digested with NotI.Sequencing
Templates for sequencing were prepared
from overnight cultures of rescued cDNA clones and/or EcoRI
cosmid fragments subcloned in pBluescript KS(+) using a plasmid
purification kit (Qiagen). Sequencing was performed with the Taq Dyedeoxy Terminator Cycle Sequencing kit (Applied Biosystems)
according to the manufacturer's instructions. Sequencing
reactions were resolved on an Applied Biosystems Inc. model 373A
automated sequencer. Sequence analysis was performed using the GCG
software. BLAST searches were performed with the NCBI network service.Northern Hybridization
Expression of human PTC mRNA was examined by Northern hybridization of human tissue blots
(Clontech) using cDNA probes labeled with
[P]dCTP. Hybridization solution contained 5
SSPE, 10 Denhardt's solution, 100 mg/ml denatured
sheared herring sperm DNA, 50% formamide, and 2% SDS. Washes were
performed at 60 °C with 2 SSC and 0.1% SDS.Chromosomal Localization
The chromosomal
localization of human PTC was identified by PCR analysis of
DNA panels obtained from human-hamster hybrid cells. The panel
consisted of both whole chromosome 9 hybrids and deletion hybrids of
9q22.3. The primers used were PTC1 (5`-TTG CAT AAC CAG CGA GTCT 3`) and
PTC2 (5`-CAA ATG TAC GAG CAC TTC AAGG). Murine Ptc was mapped
by means of interspecific backcross mapping. The panels were provided
by the Jackson Laboratory (Bar Harbor, ME) and are the BSB panel from a
cross (C57BL/6J Mus spretus) C57BL/6J and a
similar BSS panel made up of DNA from the reciprocal backcross
(C57BL/6JEi SPRET/Ei) SPRET/Ei(25) . Mapping
was performed by means of SSCP (single strand conformation
polymorphism) analysis with the primers W18F3 (5`-CTG TCA AGG TGA ATG
GAC 3`) and W18R3 (5`-GGG GTT ATT CTG TAA AAGG 3`). PCR reactions were
performed in the presence of [P]dCTP. The
samples were resolved on a 6% acrylamide gel (2.6% cross-linking) at 4
°C at 70 watts within 1.5 h. Genetic linkage was performed by
segregation analysis.In Situ Hybridization
Whole mount in situ hybridization on mouse embryos and subsequent sectioning was
performed as described previously(13) . The mouse Ptc probe was a 706-base pair NotI/PstI cDNA
fragment, from the 5`-end of the gene, subcloned in pBluescriptII SK.
The probe was linearized with SacII, the overhang blunted by
incubation with 5 units/mg Klenow at 22 °C for 15 min, and
antisense RNA synthesized by transcribing with T7 RNA polymerase.
Cloning of a Human PTC Homolog
Cosmids used in
this study were isolated from a human chromosome 9-specific genomic
cosmid library using the YAC clone ICI-2ef8. This clone contains the
microsatellite marker D9S287, which has been localized to chromosome
9q22.3(11) . Sequencing of a 1.8-kb EcoRI fragment of
cosmid 42H11 yielded an open reading frame with significant homology to
three consecutive stretches of the Drosophila Ptc protein.
Using the 1.8-kb EcoRI fragment as a probe we have isolated
the complete human and partial mouse PTC cDNA sequences. 
) of 145
10
. It shows 39% identity and 60% similarity to its Drosophila counterpart. The 3`-un
translated region contains a
canonical polyadenylation signal (AATAAA) as well as mRNA destabilizing
ATTTA motifs. These are localized 1030 nucleotides and 167, 372, and
1144 nucleotides after the termination codon, respectively.
Chromosomal Localization of PTC
Chromosomal
localization of human PTC on 9q22.3 was confirmed by PCR
analysis of chromosome 9 hybrids, and deletion hybrids of 9q22.3,
human-hamster hybrid DNA panels. The primers used (PTC1 and PTC2) were
derived from a sequence of a 1.8-kb EcoRI fragment of cosmid
42H11. Primer PTC1 is derived from an exon sequence and PTC2 from an
intron sequence. All DNA hybridization and cDNA sequencing data suggest
that human PTC is a single copy gene (data not shown). Murine Ptc maps to a short region of chromosome 13, close to the
murine Facc locus (no recombination out of 188 meioses). This
region contains the mouse mutations flexed tail (f)
and purkinje cell degeneration (pcd), and it is
syntenic with human 9q22-q31. Both f and pcd involve
abnormal development of cells of the bone or brain and could be allelic
to Ptc.Expression of PTC
Northern blot analysis revealed
five distinct PTC transcripts in all human tissues examined.
Expression of these transcripts appears to be differentially regulated (Fig. 3). During mouse embryogenesis, expression of Ptc is first detected at E 8.0 dpc in ventral neuroepithelial tissue
in two separate domains along the midline (Fig. 4A).
Expression persists in ventral neural cells through to 9.5 dpc (Fig. 4, B-D), and transcripts are also detected
in lateral mesenchyme surrounding the neural tube (Fig. 5A). Ptc transcription is detected in
the somites soon after the time of their appearance (Fig. 4C) and follows a rostro-caudal gradient of
expression (Fig. 4, C, G, and H).
Somite expression is restricted to epithelial cells within the medial
aspects of each somite (Fig. 5B). Expression of Ptc is also detected in the posterior ectoderm of each limb bud from
10.0 dpc to 12.5 dpc (Fig. 4, E-H, and
5C). This region corresponds to surface ectoderm that covers
the zone of polarizing activity. Other sites of Ptc expression
during this period include the inner surfaces of the branchial arches
which flank the oropharyngeal region, cells surrounding the placodes of
the vibrissae and the genital eminence (data not shown). No staining
was observed with a Ptc sense probe (data not shown).
/lane) were hybridized with P-labeled 1.5-kb cDNA probe corresponding to the
nucleotides 691-2228 of human PTC ORF. K, kidney; Li, liver; Lu, lung; B, brain; H,
heart; P, placenta; M, skeletal muscle; Pa,
pancreas.
8 dpc
in neuroepithelium on either side of the neural groove. B,
8.25 dpc embryo showing expression in the ventral neural tube and
lateral mesenchyme. C, 8.5 dpc embryo (after turning) showing
expression as in B, but also with expression in the somites. D, neural expression of Ptc continues at 9.25 dpc. E, by 9.5 dpc, expression is detected in the posterior limb
bud. F-H, expression continues in the posterior limb
through to 12.5 dpc in both the forelimb (fl) and hindlimb (hl).
)
We thank Gary Smythers of the Frederick Biomedical
Supercomputing Center for assistance in the sequence analysis, Stan
Cevario for primer synthesis, Andy Greenfield for the mouse embryo cDNA
library, and Toshiya Yamada, Patrick Tam, and Leszek Wojnowski for
helpful discussions regarding the data. The chromosome-specific gene
library LL09NC01 was constructed at the Biomedical Sciences Division,
Lawrence Livermore National Laboratory Gene Library Project sponsored
by the United States Department of Energy.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  T. Uziel, F. V. Karginov, S. Xie, J. S. Parker, Y.-D. Wang, A. Gajjar, L. He, D. Ellison, R. J. Gilbertson, G. Hannon, et al. The miR-17~92 cluster collaborates with the Sonic Hedgehog pathway in medulloblastoma PNAS, February 24, 2009; 106(8): 2812 - 2817. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C.-H. Yeang, F. McCormick, and A. Levine Combinatorial patterns of somatic gene mutations in cancer FASEB J, August 1, 2008; 22(8): 2605 - 2622. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Hambardzumyan, O. J. Becher, M. K. Rosenblum, P. P. Pandolfi, K. Manova-Todorova, and E. C. Holland PI3K pathway regulates survival of cancer stem cells residing in the perivascular niche following radiation in medulloblastoma in vivo Genes & Dev., February 15, 2008; 22(4): 436 - 448. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. M. Canudas, V. Di Giorgi-Gerevini, L. Iacovelli, G. Nano, M. D'Onofrio, A. Arcella, F. Giangaspero, C. Busceti, L. Ricci-Vitiani, G. Battaglia, et al. PHCCC, a Specific Enhancer of Type 4 Metabotropic Glutamate Receptors, Reduces Proliferation and Promotes Differentiation of Cerebellar Granule Cell Neuroprecursors J. Neurosci., November 17, 2004; 24(46): 10343 - 10352. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Adolphe, M. Narang, T. Ellis, C. Wicking, P. Kaur, and B. Wainwright An in vivo comparative study of sonic, desert and Indian hedgehog reveals that hedgehog pathway activity regulates epidermal stem cell homeostasis Development, October 15, 2004; 131(20): 5009 - 5019. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Brellier, C. Marionnet, O. Chevallier-Lagente, R. Toftgard, A. Mauviel, A. Sarasin, and T. Magnaldo Ultraviolet Irradiation Represses PATCHED Gene Transcription in Human Epidermal Keratinocytes through an Activator Protein-1-Dependent Process Cancer Res., April 15, 2004; 64(8): 2699 - 2704. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Doglioni, S. Piccinin, S. Demontis, M. G. Cangi, L. Pecciarini, C. Chiarelli, M. Armellin, T. Vukosavljevic, M. Boiocchi, and R. Maestro Alterations of {beta}-Catenin Pathway in Non-Melanoma Skin Tumors: Loss of {alpha}-ABC Nuclear Reactivity Correlates with the Presence of {beta}-Catenin Gene Mutation Am. J. Pathol., December 1, 2003; 163(6): 2277 - 2287. [Abstract] [Full Text]
|
|
|
|
|
|
C Hartmann and C. Tabin Dual roles of Wnt signaling during chondrogenesis in the chicken limb Development, January 7, 2000; 127(14): 3141 - 3159. [Abstract] [PDF]
|
|
|
|
 |
|
 R.L. Johnson and M.P. Scott Control of Cell Growth and Fate by patched Genes Cold Spring Harb Symp Quant Biol, January 1, 1997; 62(0): 205 - 215. [Abstract] [PDF]
|
|
|
|
 |
|
 H. Hahn, L. Wojnowski, K. Specht, R. Kappler, J. Calzada-Wack, D. Potter, A. Zimmer, U. Muller, E. Samson, L. Quintanilla-Martinez, et al. Patched Target Igf2 Is Indispensable for the Formation of Medulloblastoma and Rhabdomyosarcoma J. Biol. Chem., September 8, 2000; 275(37): 28341 - 28344. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. P. Davies and Y. A. Ioannou Topological Analysis of Niemann-Pick C1 Protein Reveals That the Membrane Orientation of the Putative Sterol-sensing Domain Is Identical to Those of 3-Hydroxy-3-methylglutaryl-CoA Reductase and Sterol Regulatory Element Binding Protein Cleavage-activating Protein J. Biol. Chem., August 4, 2000; 275(32): 24367 - 24374. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |