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Distinct expression of two types of Xenopus Patched genes during early embryogenesis and hindlimb development
Mechanisms of Development 98 (2000) 99±104
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Gene expression pattern
Distinct expression of two types of Xenopus Patched genes during early embryogenesis and hindlimb development Takashi Takabatake a, Tadashi C. Takahashi b, Yuka Takabatake c, Kazuto Yamada c, Masanori Ogawa c, Kazuhito Takeshima a,* a
Radioisotope Research Center, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan b Biohistory Research Hall, Takatsuki, Osaka 569-1125, Japan c Graduate School of Human Informatics, Nagoya University, Chikusa, Nagoya 464-8601, Japan Received 28 April 2000; received in revised form 21 June 2000; accepted 25 July 2000
Abstract Patched (Ptc) is a putative twelve transmembrane domain protein that is both a Hedgehog (Hh) receptor and transcriptional target of Hh. In this study, we isolated Xenopus Ptc cDNAs, Ptc-1 and Ptc-2, and carried out comparative analyses on their expression patterns. The putative Ptc-2 protein has a long C-terminal extension that has similarities in both length and sequence to those of Ptc-1 proteins in mouse, chick and human. In both early embryogenesis and hindlimb development, Ptc-2 expression is restricted to cells that receive a Hh signal, a pattern similar to that of Gli-1. Ptc-1, however, shows a broader distribution, mainly non-overlapping with that of Ptc-2. Despite the difference in their expression patterns, both are induced in animal cap explants synergistically by Shh and Noggin, showing a conserved regulation in their activation mechanisms. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Xenopus laevis; Hedgehog signalling; Patched; Limb development
1. Results The signalling molecule Hedgehog (Hh) mediates various important cell-cell interactions during invertebrate and vertebrate development (reviewed in Hammerschmidt et al., 1997). Direct biochemical evidence (Marigo et al., 1996; Stone et al., 1996) suggests that transduction of Hh signals is achieved initially by the reception of Hh proteins by a multiple-pass transmembrane protein patched (Ptc), thereby suppressing the inhibitory effect of Ptc on another transmembrane protein smoothened (Smo) (reviewed by Ingham, 1998). In addition, since Ptc expression is up-regulated in response to Hh activity, Ptc is believed to limit the range of Hh action by sequestering any free Hh proteins (Chen and Struhl, 1996; Goodrich et al., 1999; Lewis et al., 1999b). Drosophila has a single Ptc gene, but there are at least two types of Ptc genes in vertebrates (Concordet et al., 1996; Takabatake et al., 1997; Lewis et al., 1999a; Carpenter et al., 1998; Smyth et al., 1999; Zaphiropoulos et al., 1999). Here we present the molecular cloning and the spatiotemporal expression characteristics of possible Xenopus * Corresponding author. Tel.:181-52-789-2572; fax: 181-52-789-2567. E-mail address: [email protected] (K. Takeshima).
orthologs, Ptc-1 and Ptc-2. The open reading frames of Xenopus Ptc-1 (Xptc-1) and Ptc-2 (Xptc-2) are predicted to encode 1258 and 1413 amino acids, respectively (Fig. 1A). The overall amino acid identity between Xptc-1 and Xptc-2 is 63%. Like all the other Ptc proteins, both Xenopus Ptc proteins are predicted to contain 12 hydrophobic membrane-spanning domains with two large extracellular loops. Both Xenopus proteins have eight cysteine residues, conserved in all the other Ptc proteins and exhibit extensive similarity (36% identical in Xptc-1 and 34% identical in Xptc-2) in a region containing ®ve predicted transmembrane domains 2±6 to a potential sterol-sensing domain in Niemann-Pick type C protein, that has been implicated in intracellular traf®cking of cholesterol (Carstea et al., 1997; Loftus et al., 1997; Johnson and Scott, 1998). The most obvious structural difference between the two Xenopus proteins is the C-terminal extension present in Ptc-2. Whereas Ptc-1, but not Ptc-2, has a C-terminal extension in mouse, chick and human, phylogenic analysis (Fig. 1B) clearly indicates close relationships of Xptc-1 to Ptc-1 proteins in mouse, chick and human. Like zebra®sh Ptc-2 (ZFptc-2), Xptc-1 lacks the C-terminal extension. On the other hand, Xptc-2 shows highest similarity (80% identical) to zebra®sh Ptc1 (ZFptc-1). Although Xptc-2 has a long C
0925-4773/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(00)00436-6
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Fig. 1. Comparison of the amino acid sequences of different Ptc proteins. (A) Sequence alignment of Xenopus Ptc proteins. Identities are enclosed in boxes, and gaps in the sequence are indicated by hyphens. Putative transmembrane domains are indicated by blue lines above the sequence. Residues shaded with pink are conserved cysteine residues that are conserved also in Ptc proteins in other species. Yellow shading indicates potential N-glycosylation sites that are conserved between Xenopus Ptc-1 and Ptc-2. Light blue shading indicates the sites that are conserved among Xenopus Ptc-1, human Ptc-1, mouse Ptc-1, chick Ptc-1 and Zebra®sh Ptc-2. Dots under the Xenopus Ptc-2 sequence represent the identical residues within C terminal cytoplasmic domain among Xenopus Ptc-2, human Ptc1, mouse Ptc-1 and chick Ptc-1. (B) Phylogenic tree. A phylogenic tree based on the amino acid sequences of the open reading frames of different Ptc proteins. The phylogenic tree was generated by Unweighted Pair-Group Method with Arithmetic mean (UPGMA) method. Neighbor±Joining (NJ) method showed essentially the same divergent pro®le (data not shown). Ptc proteins containing a long C terminal cytoplasmic domain are indicated with red characters.
terminal cytoplasmic domain similar in both length and sequence to those of Ptc-1 proteins in mouse, chick and human (Fig. 1A), the overall amino acid sequence of Xptc-2 is less similar to Ptc-1 (about 60% identical) than to Ptc-2 (about 70% identical) proteins in mouse and human.
In addition, Xptc-2 has only two putative glycosylation sites, like ZFptc-1 and Ptc-2 proteins in mouse and human, while Xptc-1 has an additional three sites that are conserved among ZFptc-2 and Ptc-1 proteins in mouse, chick and human (Fig. 1A). Judging from these character-
T. Takabatake et al. / Mechanisms of Development 98 (2000) 99±104
Fig. 2. RT-PCR analysis showing embryonic (A) and adult (B) expression of Ptc and Hh transcripts. ODC and EF1-a were ampli®ed as internal controls. The lane marked Ptc-1 (2RT) indicates all the ingredients of Ptc-1 except for reverse transcriptase. n. retina, neural retina; p. retina, retinal pigment epithelium. Nucleotide sequences used for the design of Chh primers are identical between Chh and Hh-4. Thus the PCR products in the line of Chh should be derived from both mRNAs.
istics, Xptc-1 appear to be the ortholog of ZFptc-2 and Ptc-1 in mouse, chick and human, and Xptc-2 would belong to the same or closely related class of Ptc-2 in mouse and human. These data suggest that a common ancestral form of the two types of Ptc proteins might have had a long C-terminal extension that was eliminated from some Ptc proteins after gene duplication during vertebrate evolution. The temporal expression of Xptc-1 and Xptc-2 in normal development (Fig. 2A) and adult tissue distribution (Fig. 2B) were analyzed by RT-PCR. In these experiments, we compared their expression patterns to those of Xenopus Hh genes, Shh, Bhh and Chh (Ekker et al., 1995) to examine the correlation of Ptc expression on Hh activities. Consistent with Ekker et al. (1995), Hh mRNAs were detected after mid-blastula transition (MBT) with signi®cantly weaker levels of Bhh in embryos than in adult tissues; similar signal intensities of Bhh required four additional PCR cycles in Fig. 2A compared to those in Fig. 2B. While Xptc-2 expression was detected only zygotically, the presence of a significant amount of maternal transcript was noted with Xptc-1. The maternal transcript was probably present uniformly in early embryos, as suggested by RT-PCR analysis on dissected embryonic regions (data not shown). Similarly, Xptc-1 was widely distributed in all adult tissues examined. The spatial expression pattern was also analyzed, using whole-mount in situ hybridization. At midneurula stages (stage ,14), Xptc-1 was expressed mainly throughout the neural plate outside the midline (Fig. 3A,V) whereas expression of Xptc-2 was observed as longitudinal stripes immediately adjacent to the midline (Fig. 3D), similarly to Gli-1 expression (Fig. 3J). A transverse section of a stained midneurula embryo showed that Xptc-2 was also expressed in somitogenic mesodermal cells proximal to the notochord (Fig. 3W). At tail bud stages (stage 26±30), Xptc-1 expression was detected in the dorsal aspect of the neural tube especially in the midbrain and hindbrain, sensory organs (olfactory placode, optic vesicle, otic vesicle), cranial neural crest derivatives (branchial arches, cranial ganglia), the
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excretory system (pronephros, proctodeum), around the tail bud, and very weakly in the dermatome in each somite and in the epidermis (Fig. 3B,C,S,X). In a pattern reminiscent to Gli-1, Xptc-2 was expressed in cells adjacent to the Shh (compare Fig. 3T and 3U). At stage 26, both Xptch-2 and Gli-1 were expressed relatively broadly in somites in a graded manner with the highest levels at the posterior end along the rostral-caudal axis and proximal to the notochord (Fig. 3E,K). At stage 31, the expression of both genes was more restricted to the cells closest to the notochord (Fig. 3F,L,Y). In contrast, we found that Gli-3 expression was down-regulated in somites during these stages, leaving an expression that was complementary to those of Gli-1 and Xptc-2 (Fig. 3Q,R). In the neural tube, Xptc-2 was expressed more ventrally than Xptc-1, but the expression did not extend into the ¯oor plate (Fig. 3X,Y). Expression patterns of Xptc-1 and Xptc-2 during hindlimb development also differed signi®cantly (Fig. 4). Initial hindlimb expression of Xptc-1 was detected predominantly throughout the surface ectoderm (Fig. 4 inset) in a graded manner with highest levels in the distal limb buds (,stage 52). As limb outgrowth proceeded (stage 52±55), the surface expression became weaker, leaving a strong expression in the anterior proximal region. At later stages, strong
Fig. 3. Whole-mount in situ hybridization showing the localization of Ptc-1 (A±C,S,V,X), Ptc-2 (D±F,T,W,Y), Shh (G±I,U,Z), Gli-1 (J±L), Gli-2 (M± O) and Gli-3 (P±R). (A,D,G,J,M,P) Dorsal views at stages 14. (B,E,H,K,N,Q) Lateral views at stage 26. (C,F,I,L,O,R) Lateral views at stage 31. (S±U) Lateral views of facial regions at stage 31. (V, W) Transverse sections at stage 14. (X±Z) Transverse sections through spinal cord and notochord at stage 27. Embryos in (B,C,E,F,H,I,K,L,N,O,Q±U) were cleared by benzyl alcohol-benzyl benzoate.
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tissues in the animal caps (reviewed in Sasai and De Robertis, 1997). Like the newt Ptc genes (Takabatake et al., 1997; Sakuma et al., 1999), the expression of both Xenopus Ptc genes was weakly detected in control uninjected caps and conjugates, and was up-regulated in response to Hh activity (Fig. 5). In addition, we found a synergistical effect of Shh and Noggin on Ptc induction. Unexpectedly, both Ptc genes, together with Shh and Gli-1, were weakly induced in the animal cap explants (data not shown) and conjugates misexpressing noggin alone. We con®rmed that Noggin could also upregulate the expression of newt Ptc genes weakly in newt animal cap explants (data not shown). The expression of both Ptc genes was activated most strongly in the conjugate with the highest dose of Shh (1 ng), although Xptc-1 Fig. 4. Expression of Ptc, Gli-1 and Hh genes in developing hindlimbs. All are dorsal views. An inset in Xptc-1 (stage ,52) is a transverse section of the limb bud oriented with the posterior and dorsal sides facing down and to the left, respectively.
expression of Xptc-1 was observed in all the interdigital regions. Unlike Xptc-1, interdigital expression of Xptc-2 was prominent only in the most posterior one. In earlier limb buds, Xptc-2 expression was similar to that of Gli-1. As in embryos, the expression domains of Xptc-2 and Gli-1 were closely correlated with Shh expression at the posteriordistal mesenchyme. In addition, both were expressed in the mesenchyme as three stripes along the anterior-posterior axis at stages 52±53. To examine the possibility that these stripes may re¯ect the expression of Hh genes other than Shh, we analyzed the expression of Bhh and Chh. No significant signal for Chh could be detected in limbs at least during stages 51±56, while Bhh was expressed as several clear spots during stage 51±53. Bhh expression changed from sites proximal to distal as limb outgrowth proceeded. Subsequently Bhh was expressed in mesenchyme surrounding the digital primordia (stage ,55) and as a single spot in each digital mesenchyme (stage ,56). It is possible that the three stripes of Xptc-2 expression at stages 52±53 correlate with the spot-like expressions of Bhh. The number of spots along the proximo-distal axis corresponds to the number of the main cartilaginous elements in the adult limb. It is interesting to note that Bhh is most similar to Ihh among mouse Hh proteins (Ekker et al., 1995), which plays crucial roles in the regulation of chondrogenic development in higher vertebrates (Vortkamp et al., 1996; St-Jacques et al., 1999). To address whether the transcription of Xenopus Ptc genes were activated in response to Hh activity, we examined their expressions in animal cap explants injected with changing amounts of Shh mRNA. In this experiment, we conjugated the cap expressing Shh mRNA with one expressing Noggin mRNA. Since both Ptc genes were expressed most eminently in the anterior neural tissues, we decided to inject Noggin mRNA in order to induce anterior neural
Fig. 5. Shh and Noggin synergistically stimulate the expression of Ptc genes in animal cap explants of Xenopus. Half of indicated amounts of Noggin and/or Shh RNA was injected into the animal pole bilaterally at the two-cell stage. At the late-blastula stage (7 h after fertilization), animal caps were isolated, cultured alone or conjugated. They were cultured until the sibling control embryos reached stage 26. The lane labeled `Embryo' is total RNA from stage 26 embryos used as a positive control. The lane marked `Control' contains all the ingredients of `Embryo' except for reverse transcriptase. Endogenous transcripts of Shh was ampli®ed using primers corresponding to the 5 0 untranslated sequences of Xenopus Shh, that were absent within the injected mRNA.
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responded to Hh signal only weakly compared with the response of Xptc-2. On the other hand, Pax-6 expression was clearly induced by noggin and was suppressed in the conjugate with the highest or a tenfold lower dose of Shh.
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was performed as described (Harland, 1991), but modi®ed to include the use of BM purple substrate (Boehringer), and that of limbs as described (Sone et al., 1999). 2.3. RNA expression constructs
2. Experimental procedures 2.1. Isolation of Xenopus Ptc cDNAs PCR fragments of Xenopus Ptc genes were initially isolated from cDNA synthesized using tailbud RNA as a template. Four kinds of cDNAs obtained were designated as Ptc-1, Ptc-1 0 , Ptc-2 and Ptc-2 0 , based on the similarities with mouse Ptc genes. Ptc-1 and Ptc-1 0 as well as Ptc-2 and Ptc-2 0 have strong similarities with each other (96.9 and 94.6%), suggesting that Ptc-1 0 and Ptc-2 0 are pseudovariant genes of Ptc-1 (Xptc-1) and Ptc-2 (Xptc-2), respectively. Xptc-1 (4.96 kb) and Xptc-2 (4.76 kb) cDNAs were isolated from a Xenopus neurula cDNA library (Yamada et al., 1999) using modi®ed enrichment procedures (Takabatake et al., 1996). Xptc-1 cDNA had a frameshift mutation, revealed by comparison with RT-PCR products of the corresponding region, which was caused by one base (adenosine) insertion within a 7-base stretch of adenosines (nucleotides 1073± 1079). The accession numbers for Xptc-1, Xptc-1 0 , Xptc-2 and Xptc-2 0 are AB037686, AB037687, AB037688 and AB037689, respectively. 2.2. RT-PCR and whole-mount in situ hybridization analyses The primer sequences used in RT-PCR analysis were as follows: Xptc-1forward 5 0 -GGACAAGAATCGCAGAGCTG-3 0 , reverse 5 0 -GGATGCTCAGGGAACCTTAC3 0 ; Xptc-2 forward 5 0 -TTGTTCATTGGATTGC-TGGTG3 0 , reverse 5 0 -CTCTTCCTGGTAGATATGCCA-3 0 ; Gli-1 forward 5 0 -GAGCTAGTGACCCTGCAAG-3 0 , reverse 5 0 CATCGGGACCTGCTGTTTCC-3 0 ; Pax-6 forward 5 0 CTACCACACCAGTGTCCTCA-3 0 , reverse 5 00 -TTGGCCAGTACTGAGACATG-3 0 . The primers for Shh, Bhh, Chh and EF1-a were the same as those reported by Takabatake et al. (1997) except for Fig. 5, in which the endogenous transcript of Shh was distinguished from the injected mRNA using the following primers corresponding to the 5 0 untranslated region of Shh (Stolow and Shi, 1995): forward 5 0 -TACTGTCTCGTCTCTACACC-3 0 , reverse 5 0 CATCTCGTCCGAGCGAAGC-3 0 . The primers for NCAM and a-actin were the same as described previously (Hemmati-Brivanlou and Melton, 1994). The primers for Ornithine decarboxylase (ODC) were the same as those reported by Yamada et al. (1999). The PCR cycle numbers were: Xptc-1, 25 cycles; Xptc-2, 25 cycles; Shh, 26 cycles; Bhh, 30 cycles except for Fig. 2B (26 cycles); Chh, 26 cycles; Gli-1, 30 cycles; Pax-6, 24 cycles; ODC, 18 cycles; EF1-a , 18 cycles; NCAM, 23 cycles; and a-actin, 19 cycles. Whole-mount in situ hybridization of embryos
Expression constructs of Shh/RN3P were made by replacing the Xenopus Eomesodermin cDNA in the pEob1/RN3P (a gift from Dr K. Ryan; Ryan et al., 1996) by the PCR fragment ampli®ed from Shh cDNA (a gift from Dr R.T. Moon; Ekker et al., 1995a). Shh/RN3P was cut with S® I and transcribed with T3 polymerase. Expression constructs for Xenopus Noggin and LacZ RNAs were used as described (Mizuno et al., 1997). Acknowledgements We would like to thank Drs R.T. Moon for providing plasmids of hedgehog cDNAs, K. Ryan for providing plasmids including pEob1/RN3P and E. Amaya for reading the draft and making a number of helpful suggestions. This work was supported in part by a Grant-in-aid for Scienti®c Research (C) (No. 11680720) to K.T. from the Ministry of Education, Science, Sports and Culture, Japan. References Carpenter, D., Stone, D.M., Brush, J., Ryan, A., Armanini, M., Frantz, G., Rosenthal, A., De Sauvage, F.J., 1998. Characterization of two patched receptors for the vertebrate hedgehog protein family. Proc. Natl. Acad. Sci. USA 95, 13630±13634. Carstea, E.D., Morris, J.A., Coleman, K.G., Loftus, S.K., Zhang, D., Cummings, C., Gu, J., Rosenfeld, M.A., Pavan, W.J., Krizman, D.B., Nagle, J., Polymeropoulos, M.H., Sturley, S.L., Ioannou, Y.A., Higgins, M.E., Comly, M., Cooney, A., Brown, A., Kaneski, C.R., BlanchetteMackie, E.J., Dwyer, N.K., Neufeld, E.B., Chang, T.Y., Liscum, L., Tagle, D.A., 1997. Niemann-Pick C1 disease gene: homology to mediators of cholesterol homeostasis. Science 277, 228±231. Chen, Y., Struhl, G., 1996. Dual roles for patched in sequestering and transducing hedgehog. Cell 87, 553±563. Concordet, J.-P., Lewis, K.E., Moore, J.W., Goodrich, L.V., Johnson, R.L., Scott, M.P., Ingham, P.W., 1996. Spatial regulation of a zebra®sh patched homologue re¯ects the roles of sonic hedgehog and protein kinase A in neural tube and somite patterning. Development 122, 2835±2846. Ekker, S.C., McGrew, L.L., Lai, C.-J., Lee, J.J., von Kessler, D.P., Moon, R.T., Beachy, P.A., 1995. Distinct expression and shared activities of members of the hedgehog gene family of Xenopus laevis. Development 121, 2337±2347. Goodrich, L.V., Jung, D., Higgins, K.M., Scott, M.P., 1999. Overexpression of ptc1 inhibits induction of Shh target genes and prevents normal patterning in the neural tube. Dev. Biol. 211, 323±334. Hammerschmidt, M., Brook, A., McMahon, A.P., 1997. The world according to hedgehog. Trends Genet. 13, 14±21. Harland, R.M., 1991. In situ hybridization: an improved whole-mount method for Xenopus embryos. In: Kay, B., Peng, H. (Eds.). Methods in Cell Biology, Vol. 36. Academic Press, San Diego, CA, pp. 685± 695. Hemmati-Brivanlou, A., Melton, D.A., 1994. Inhibition of activin receptor signaling promotes neuralization in Xenopus. Cell 77, 273±281.
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Ingham, P.W., 1998. Transducing Hedgehog: the story so far. EMBO J. 17, 3505±3511. Johnson, R.L., Scott, M.P., 1998. Control of cell growth and fate by patched genes. Cold Spring Harb. Symp. Quant. Biol. 62, 205±215. Lewis, K.E., Concordet, J.P., Ingham, P.W., 1999a. Characterisation of a second patched gene in the zebra®sh Danio rerio and the differential response of patched genes to hedgehog signalling. Dev. Biol. 208, 14± 29. Lewis, K.E., Currie, P.D., Roy, S., Schauerte, H., Haffter, P., Ingham, P.W., 1999b. Control of muscle cell-type speci®cation in the zebra®sh embryo by hedgehog signalling. Dev. Biol. 216, 469±480. Loftus, S.K., Morris, J.A., Carstea, E.D., Gu, J.Z., Cummings, C., Brown, A., Ellison, J., Ohno, K., Rosenfeld, M.A., Tagle, D.A., Pentchev, P.G., Pavan, W.J., 1997. Murine model of Niemann-Pick C disease: mutation in a cholesterol homeostasis gene. Science 277, 232±235. Marigo, V., Davey, R., Zuo, Y., Cunningham, J., Tabin, C., 1996. Biochemical evidence that patched is the hedgehog receptor. Nature 384, 176± 179. Mizuno, M., Takabatake, T., Takahashi, T.C., Takeshima, K., 1997. pax-6 gene expression in newt eye development. Dev. Genes Evol. 207, 167± 176. Ryan, K., Garrett, N., Mitchell, A., Gurdon, J.B., 1996. Eomesodermin, a key early gene in Xenopus mesoderm differentiation. Cell 87, 989± 1000. Sakuma, Y., Kimura, M., Takabatake, T., Takeshima, K., Fujimura, H., 1999. Expression and secretion of a biologically active mouse sonic hedgehog protein by the methylotrophic yeast Pichia pastoris. Appl. Microbiol. Biotechnol. 52, 410±414. Sasai, Y., De Robertis, E.M., 1997. Ectodermal patterning in vertebrate embryos. Dev. Biol. 182, 5±20. Smyth, I., Narang, M.A., Evans, T., Heimann, C., Nakamura, Y., ChenevixTrench, G., Pietsch, T., Wicking, C., Wainwright, B.J., 1999. Isolation and characterization of human Patched2 (PTCH2), a putative tumour
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Gene expression pattern
Distinct expression of two types of Xenopus Patched genes during early embryogenesis and hindlimb development Takashi Takabatake a, Tadashi C. Takahashi b, Yuka Takabatake c, Kazuto Yamada c, Masanori Ogawa c, Kazuhito Takeshima a,* a
Radioisotope Research Center, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan b Biohistory Research Hall, Takatsuki, Osaka 569-1125, Japan c Graduate School of Human Informatics, Nagoya University, Chikusa, Nagoya 464-8601, Japan Received 28 April 2000; received in revised form 21 June 2000; accepted 25 July 2000
Abstract Patched (Ptc) is a putative twelve transmembrane domain protein that is both a Hedgehog (Hh) receptor and transcriptional target of Hh. In this study, we isolated Xenopus Ptc cDNAs, Ptc-1 and Ptc-2, and carried out comparative analyses on their expression patterns. The putative Ptc-2 protein has a long C-terminal extension that has similarities in both length and sequence to those of Ptc-1 proteins in mouse, chick and human. In both early embryogenesis and hindlimb development, Ptc-2 expression is restricted to cells that receive a Hh signal, a pattern similar to that of Gli-1. Ptc-1, however, shows a broader distribution, mainly non-overlapping with that of Ptc-2. Despite the difference in their expression patterns, both are induced in animal cap explants synergistically by Shh and Noggin, showing a conserved regulation in their activation mechanisms. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Xenopus laevis; Hedgehog signalling; Patched; Limb development
1. Results The signalling molecule Hedgehog (Hh) mediates various important cell-cell interactions during invertebrate and vertebrate development (reviewed in Hammerschmidt et al., 1997). Direct biochemical evidence (Marigo et al., 1996; Stone et al., 1996) suggests that transduction of Hh signals is achieved initially by the reception of Hh proteins by a multiple-pass transmembrane protein patched (Ptc), thereby suppressing the inhibitory effect of Ptc on another transmembrane protein smoothened (Smo) (reviewed by Ingham, 1998). In addition, since Ptc expression is up-regulated in response to Hh activity, Ptc is believed to limit the range of Hh action by sequestering any free Hh proteins (Chen and Struhl, 1996; Goodrich et al., 1999; Lewis et al., 1999b). Drosophila has a single Ptc gene, but there are at least two types of Ptc genes in vertebrates (Concordet et al., 1996; Takabatake et al., 1997; Lewis et al., 1999a; Carpenter et al., 1998; Smyth et al., 1999; Zaphiropoulos et al., 1999). Here we present the molecular cloning and the spatiotemporal expression characteristics of possible Xenopus * Corresponding author. Tel.:181-52-789-2572; fax: 181-52-789-2567. E-mail address: [email protected] (K. Takeshima).
orthologs, Ptc-1 and Ptc-2. The open reading frames of Xenopus Ptc-1 (Xptc-1) and Ptc-2 (Xptc-2) are predicted to encode 1258 and 1413 amino acids, respectively (Fig. 1A). The overall amino acid identity between Xptc-1 and Xptc-2 is 63%. Like all the other Ptc proteins, both Xenopus Ptc proteins are predicted to contain 12 hydrophobic membrane-spanning domains with two large extracellular loops. Both Xenopus proteins have eight cysteine residues, conserved in all the other Ptc proteins and exhibit extensive similarity (36% identical in Xptc-1 and 34% identical in Xptc-2) in a region containing ®ve predicted transmembrane domains 2±6 to a potential sterol-sensing domain in Niemann-Pick type C protein, that has been implicated in intracellular traf®cking of cholesterol (Carstea et al., 1997; Loftus et al., 1997; Johnson and Scott, 1998). The most obvious structural difference between the two Xenopus proteins is the C-terminal extension present in Ptc-2. Whereas Ptc-1, but not Ptc-2, has a C-terminal extension in mouse, chick and human, phylogenic analysis (Fig. 1B) clearly indicates close relationships of Xptc-1 to Ptc-1 proteins in mouse, chick and human. Like zebra®sh Ptc-2 (ZFptc-2), Xptc-1 lacks the C-terminal extension. On the other hand, Xptc-2 shows highest similarity (80% identical) to zebra®sh Ptc1 (ZFptc-1). Although Xptc-2 has a long C
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Fig. 1. Comparison of the amino acid sequences of different Ptc proteins. (A) Sequence alignment of Xenopus Ptc proteins. Identities are enclosed in boxes, and gaps in the sequence are indicated by hyphens. Putative transmembrane domains are indicated by blue lines above the sequence. Residues shaded with pink are conserved cysteine residues that are conserved also in Ptc proteins in other species. Yellow shading indicates potential N-glycosylation sites that are conserved between Xenopus Ptc-1 and Ptc-2. Light blue shading indicates the sites that are conserved among Xenopus Ptc-1, human Ptc-1, mouse Ptc-1, chick Ptc-1 and Zebra®sh Ptc-2. Dots under the Xenopus Ptc-2 sequence represent the identical residues within C terminal cytoplasmic domain among Xenopus Ptc-2, human Ptc1, mouse Ptc-1 and chick Ptc-1. (B) Phylogenic tree. A phylogenic tree based on the amino acid sequences of the open reading frames of different Ptc proteins. The phylogenic tree was generated by Unweighted Pair-Group Method with Arithmetic mean (UPGMA) method. Neighbor±Joining (NJ) method showed essentially the same divergent pro®le (data not shown). Ptc proteins containing a long C terminal cytoplasmic domain are indicated with red characters.
terminal cytoplasmic domain similar in both length and sequence to those of Ptc-1 proteins in mouse, chick and human (Fig. 1A), the overall amino acid sequence of Xptc-2 is less similar to Ptc-1 (about 60% identical) than to Ptc-2 (about 70% identical) proteins in mouse and human.
In addition, Xptc-2 has only two putative glycosylation sites, like ZFptc-1 and Ptc-2 proteins in mouse and human, while Xptc-1 has an additional three sites that are conserved among ZFptc-2 and Ptc-1 proteins in mouse, chick and human (Fig. 1A). Judging from these character-
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Fig. 2. RT-PCR analysis showing embryonic (A) and adult (B) expression of Ptc and Hh transcripts. ODC and EF1-a were ampli®ed as internal controls. The lane marked Ptc-1 (2RT) indicates all the ingredients of Ptc-1 except for reverse transcriptase. n. retina, neural retina; p. retina, retinal pigment epithelium. Nucleotide sequences used for the design of Chh primers are identical between Chh and Hh-4. Thus the PCR products in the line of Chh should be derived from both mRNAs.
istics, Xptc-1 appear to be the ortholog of ZFptc-2 and Ptc-1 in mouse, chick and human, and Xptc-2 would belong to the same or closely related class of Ptc-2 in mouse and human. These data suggest that a common ancestral form of the two types of Ptc proteins might have had a long C-terminal extension that was eliminated from some Ptc proteins after gene duplication during vertebrate evolution. The temporal expression of Xptc-1 and Xptc-2 in normal development (Fig. 2A) and adult tissue distribution (Fig. 2B) were analyzed by RT-PCR. In these experiments, we compared their expression patterns to those of Xenopus Hh genes, Shh, Bhh and Chh (Ekker et al., 1995) to examine the correlation of Ptc expression on Hh activities. Consistent with Ekker et al. (1995), Hh mRNAs were detected after mid-blastula transition (MBT) with signi®cantly weaker levels of Bhh in embryos than in adult tissues; similar signal intensities of Bhh required four additional PCR cycles in Fig. 2A compared to those in Fig. 2B. While Xptc-2 expression was detected only zygotically, the presence of a significant amount of maternal transcript was noted with Xptc-1. The maternal transcript was probably present uniformly in early embryos, as suggested by RT-PCR analysis on dissected embryonic regions (data not shown). Similarly, Xptc-1 was widely distributed in all adult tissues examined. The spatial expression pattern was also analyzed, using whole-mount in situ hybridization. At midneurula stages (stage ,14), Xptc-1 was expressed mainly throughout the neural plate outside the midline (Fig. 3A,V) whereas expression of Xptc-2 was observed as longitudinal stripes immediately adjacent to the midline (Fig. 3D), similarly to Gli-1 expression (Fig. 3J). A transverse section of a stained midneurula embryo showed that Xptc-2 was also expressed in somitogenic mesodermal cells proximal to the notochord (Fig. 3W). At tail bud stages (stage 26±30), Xptc-1 expression was detected in the dorsal aspect of the neural tube especially in the midbrain and hindbrain, sensory organs (olfactory placode, optic vesicle, otic vesicle), cranial neural crest derivatives (branchial arches, cranial ganglia), the
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excretory system (pronephros, proctodeum), around the tail bud, and very weakly in the dermatome in each somite and in the epidermis (Fig. 3B,C,S,X). In a pattern reminiscent to Gli-1, Xptc-2 was expressed in cells adjacent to the Shh (compare Fig. 3T and 3U). At stage 26, both Xptch-2 and Gli-1 were expressed relatively broadly in somites in a graded manner with the highest levels at the posterior end along the rostral-caudal axis and proximal to the notochord (Fig. 3E,K). At stage 31, the expression of both genes was more restricted to the cells closest to the notochord (Fig. 3F,L,Y). In contrast, we found that Gli-3 expression was down-regulated in somites during these stages, leaving an expression that was complementary to those of Gli-1 and Xptc-2 (Fig. 3Q,R). In the neural tube, Xptc-2 was expressed more ventrally than Xptc-1, but the expression did not extend into the ¯oor plate (Fig. 3X,Y). Expression patterns of Xptc-1 and Xptc-2 during hindlimb development also differed signi®cantly (Fig. 4). Initial hindlimb expression of Xptc-1 was detected predominantly throughout the surface ectoderm (Fig. 4 inset) in a graded manner with highest levels in the distal limb buds (,stage 52). As limb outgrowth proceeded (stage 52±55), the surface expression became weaker, leaving a strong expression in the anterior proximal region. At later stages, strong
Fig. 3. Whole-mount in situ hybridization showing the localization of Ptc-1 (A±C,S,V,X), Ptc-2 (D±F,T,W,Y), Shh (G±I,U,Z), Gli-1 (J±L), Gli-2 (M± O) and Gli-3 (P±R). (A,D,G,J,M,P) Dorsal views at stages 14. (B,E,H,K,N,Q) Lateral views at stage 26. (C,F,I,L,O,R) Lateral views at stage 31. (S±U) Lateral views of facial regions at stage 31. (V, W) Transverse sections at stage 14. (X±Z) Transverse sections through spinal cord and notochord at stage 27. Embryos in (B,C,E,F,H,I,K,L,N,O,Q±U) were cleared by benzyl alcohol-benzyl benzoate.
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tissues in the animal caps (reviewed in Sasai and De Robertis, 1997). Like the newt Ptc genes (Takabatake et al., 1997; Sakuma et al., 1999), the expression of both Xenopus Ptc genes was weakly detected in control uninjected caps and conjugates, and was up-regulated in response to Hh activity (Fig. 5). In addition, we found a synergistical effect of Shh and Noggin on Ptc induction. Unexpectedly, both Ptc genes, together with Shh and Gli-1, were weakly induced in the animal cap explants (data not shown) and conjugates misexpressing noggin alone. We con®rmed that Noggin could also upregulate the expression of newt Ptc genes weakly in newt animal cap explants (data not shown). The expression of both Ptc genes was activated most strongly in the conjugate with the highest dose of Shh (1 ng), although Xptc-1 Fig. 4. Expression of Ptc, Gli-1 and Hh genes in developing hindlimbs. All are dorsal views. An inset in Xptc-1 (stage ,52) is a transverse section of the limb bud oriented with the posterior and dorsal sides facing down and to the left, respectively.
expression of Xptc-1 was observed in all the interdigital regions. Unlike Xptc-1, interdigital expression of Xptc-2 was prominent only in the most posterior one. In earlier limb buds, Xptc-2 expression was similar to that of Gli-1. As in embryos, the expression domains of Xptc-2 and Gli-1 were closely correlated with Shh expression at the posteriordistal mesenchyme. In addition, both were expressed in the mesenchyme as three stripes along the anterior-posterior axis at stages 52±53. To examine the possibility that these stripes may re¯ect the expression of Hh genes other than Shh, we analyzed the expression of Bhh and Chh. No significant signal for Chh could be detected in limbs at least during stages 51±56, while Bhh was expressed as several clear spots during stage 51±53. Bhh expression changed from sites proximal to distal as limb outgrowth proceeded. Subsequently Bhh was expressed in mesenchyme surrounding the digital primordia (stage ,55) and as a single spot in each digital mesenchyme (stage ,56). It is possible that the three stripes of Xptc-2 expression at stages 52±53 correlate with the spot-like expressions of Bhh. The number of spots along the proximo-distal axis corresponds to the number of the main cartilaginous elements in the adult limb. It is interesting to note that Bhh is most similar to Ihh among mouse Hh proteins (Ekker et al., 1995), which plays crucial roles in the regulation of chondrogenic development in higher vertebrates (Vortkamp et al., 1996; St-Jacques et al., 1999). To address whether the transcription of Xenopus Ptc genes were activated in response to Hh activity, we examined their expressions in animal cap explants injected with changing amounts of Shh mRNA. In this experiment, we conjugated the cap expressing Shh mRNA with one expressing Noggin mRNA. Since both Ptc genes were expressed most eminently in the anterior neural tissues, we decided to inject Noggin mRNA in order to induce anterior neural
Fig. 5. Shh and Noggin synergistically stimulate the expression of Ptc genes in animal cap explants of Xenopus. Half of indicated amounts of Noggin and/or Shh RNA was injected into the animal pole bilaterally at the two-cell stage. At the late-blastula stage (7 h after fertilization), animal caps were isolated, cultured alone or conjugated. They were cultured until the sibling control embryos reached stage 26. The lane labeled `Embryo' is total RNA from stage 26 embryos used as a positive control. The lane marked `Control' contains all the ingredients of `Embryo' except for reverse transcriptase. Endogenous transcripts of Shh was ampli®ed using primers corresponding to the 5 0 untranslated sequences of Xenopus Shh, that were absent within the injected mRNA.
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responded to Hh signal only weakly compared with the response of Xptc-2. On the other hand, Pax-6 expression was clearly induced by noggin and was suppressed in the conjugate with the highest or a tenfold lower dose of Shh.
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was performed as described (Harland, 1991), but modi®ed to include the use of BM purple substrate (Boehringer), and that of limbs as described (Sone et al., 1999). 2.3. RNA expression constructs
2. Experimental procedures 2.1. Isolation of Xenopus Ptc cDNAs PCR fragments of Xenopus Ptc genes were initially isolated from cDNA synthesized using tailbud RNA as a template. Four kinds of cDNAs obtained were designated as Ptc-1, Ptc-1 0 , Ptc-2 and Ptc-2 0 , based on the similarities with mouse Ptc genes. Ptc-1 and Ptc-1 0 as well as Ptc-2 and Ptc-2 0 have strong similarities with each other (96.9 and 94.6%), suggesting that Ptc-1 0 and Ptc-2 0 are pseudovariant genes of Ptc-1 (Xptc-1) and Ptc-2 (Xptc-2), respectively. Xptc-1 (4.96 kb) and Xptc-2 (4.76 kb) cDNAs were isolated from a Xenopus neurula cDNA library (Yamada et al., 1999) using modi®ed enrichment procedures (Takabatake et al., 1996). Xptc-1 cDNA had a frameshift mutation, revealed by comparison with RT-PCR products of the corresponding region, which was caused by one base (adenosine) insertion within a 7-base stretch of adenosines (nucleotides 1073± 1079). The accession numbers for Xptc-1, Xptc-1 0 , Xptc-2 and Xptc-2 0 are AB037686, AB037687, AB037688 and AB037689, respectively. 2.2. RT-PCR and whole-mount in situ hybridization analyses The primer sequences used in RT-PCR analysis were as follows: Xptc-1forward 5 0 -GGACAAGAATCGCAGAGCTG-3 0 , reverse 5 0 -GGATGCTCAGGGAACCTTAC3 0 ; Xptc-2 forward 5 0 -TTGTTCATTGGATTGC-TGGTG3 0 , reverse 5 0 -CTCTTCCTGGTAGATATGCCA-3 0 ; Gli-1 forward 5 0 -GAGCTAGTGACCCTGCAAG-3 0 , reverse 5 0 CATCGGGACCTGCTGTTTCC-3 0 ; Pax-6 forward 5 0 CTACCACACCAGTGTCCTCA-3 0 , reverse 5 00 -TTGGCCAGTACTGAGACATG-3 0 . The primers for Shh, Bhh, Chh and EF1-a were the same as those reported by Takabatake et al. (1997) except for Fig. 5, in which the endogenous transcript of Shh was distinguished from the injected mRNA using the following primers corresponding to the 5 0 untranslated region of Shh (Stolow and Shi, 1995): forward 5 0 -TACTGTCTCGTCTCTACACC-3 0 , reverse 5 0 CATCTCGTCCGAGCGAAGC-3 0 . The primers for NCAM and a-actin were the same as described previously (Hemmati-Brivanlou and Melton, 1994). The primers for Ornithine decarboxylase (ODC) were the same as those reported by Yamada et al. (1999). The PCR cycle numbers were: Xptc-1, 25 cycles; Xptc-2, 25 cycles; Shh, 26 cycles; Bhh, 30 cycles except for Fig. 2B (26 cycles); Chh, 26 cycles; Gli-1, 30 cycles; Pax-6, 24 cycles; ODC, 18 cycles; EF1-a , 18 cycles; NCAM, 23 cycles; and a-actin, 19 cycles. Whole-mount in situ hybridization of embryos
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