(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) ()
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.
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) 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.
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)/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.
Figure 4:
Expression of Ptc during murine
embryogenesis. A, expression is first detected at 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).
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].