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Identification by differential display of a chicken tolloid-related metalloprotease specifically expressed in the caudal notochord
Mechanisms of Development 96 (2000) 101±105
www.elsevier.com/locate/modo
Gene expression pattern
Identi®cation by differential display of a chicken tolloid-related metalloprotease speci®cally expressed in the caudal notochord Laurence Liaubet, Nicolas Bertrand, FrancËois Medevielle, Fabienne Pituello* Centre de Biologie du DeÂveloppement, UMR-5547 CNRS-Universite P. Sabatier, 118 Route de Narbonne, 31062 Toulouse, Cedex 04, France Received 2 May 2000; received in revised form 6 June 2000; accepted 8 June 2000
Abstract While the ventralizing factor Sonic hedgehog is expressed in the entire notochord (Development 121 (1995) 2537) the latter displays distinct ventralizing activities along its rostrocaudal axis. Hence, in HH stage-10 chicken embryo, the caudal notochord exhibits ¯oor plate inducing capacities lost by rostral regions (Development 117 (1993) 205). Therefore, we hypothesize that the caudal notochord produces some cofactors which may contribute to its ventralizing properties. In order to identify such molecules we applied the differential display strategy and isolated a secreted Tolloid-related metalloprotease displaying a regionalized expression in the notochord. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Tolloid-related metalloprotease; Astacin; Notochord; Floor plate; Dorso-ventral patterning; Neural tube; Spinal cord; Somite; Chicken embryo; Differential display
1. Results and discussion 1.1. Identi®cation of genes speci®cally expressed in the caudal notochord The aim of this study was to identify secreted factors which, contrary to Sonic hedgehog (Marti et al., 1995), will be restricted to the caudal notochord correlating with its speci®c ventralizing capacities (Placzek et al., 1993). Therefore, we compared the messenger contents of caudal notochord vs. those of an embryo lacking it (Fig. 1A). In two independent experiments, a complementary DNA fragment of 391 bp was speci®cally ampli®ed in caudal notochord samples (Fig. 1A). A region of its nucleotide sequence (about 140 bp) displayed a high degree of homology (.80%) with the sequence encoding the EGF (epidermal growth factor)-like 2 domain of certain metalloprotease of the astacin family (Bond and Beynon, 1995). We then isolated the full-length cDNA corresponding to our PCR fragment. It displays an open reading frame of 3027 bp encoding a protein of 1009 amino acids. Analysis of the protein structure (Fig. 1B) reveals the presence of a signal peptide, suggesting that the protein can be secreted, a proregion followed by a protease domain displaying the signature * Corresponding author. Tel.: 133-561-55-67-42; fax: 133-561-55-6507. E-mail address: [email protected] (F. Pituello).
sequence of the metalloproteases of the astacin family (Bond and Beynon, 1995) and ®ve non-catalytic interaction domains including two EGF-like domains and three CUB (complement subcomponents C1r/C1s, embryonic sea urchin protein Uegf, BMP-1) domains. The entire protein displays more than 80% identity with human Tolloid-like 1 (Scott et al., 1999), mammalian Tolloid-like 1 (Takahara et al., 1996) and Zebra®sh Tolloid/Mini ®n (Blader et al., 1997; Connors et al., 1999) proteins (Fig. 1B). Furthermore, comparison of signature sequences of the proregion (Fig. 1C) and of the protease domain (Fig. 1D) reveals 100% identity at the amino acid level with the corresponding domains of human, mouse and zebra®sh tolloid-like1 proteins. Therefore, we have probably isolated a chicken orthologue of human and mammalian tolloid-like 1 genes. We called this gene Colloid-like 1 for chicken Tolloid-like1. Northern blot analysis reveals that a unique transcript of around 7 kb encodes the protein (data not shown). 1.2. Spatial and temporal dynamics of Colloid-like 1 expression As expected from the differential display data, in situ hybridization analysis performed on HH stage-10 embryos shows that transcripts are restricted to the caudal notochord, expanding between Hensen's node and the last individualized somite (Fig. 2). No expression can be detected at the ventral midline of the neural plate (Fig. 2C±E).
0925-4773/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(00)00382-8
102
L. Liaubet et al. / Mechanisms of Development 96 (2000) 101±105
Fig. 1. Identi®cation of a gene speci®cally transcribed in the caudal notochord. (A) Schematic representation of a HH stage-10 embryo (HH, (Hamburger and Hamilton, 1992)) showing the region of the notochord (red color) used for differential display analysis. (B) Differential display of mRNA from samples of 102 and 108 notochords (nc) vs. sample of an embryo without that notochordal region (embr-nc). The blue arrow marks a PCR product present in both samples and the red arrow the PCR product speci®cally ampli®ed from the notochord samples. (B) Schematic representation of the protein structure. The yellow box represents the signal peptide. P1±P3 corresponds to the regions used to synthesize the riboprobes. The EMBL Nucleotide Sequence Database accession number for Colloid-like 1 is AJ012462. Comparison of Colloid-like 1 with the astacin family members. The numbers represent the percent of amino acid identities for each region and for the entire protein (last number on the right). The numbers noted in sub-index corresponds to the length of the amino acid sequence in the proregion used for the comparison. Notice the high degree of identities with human tolloid-like 1 (hTLL1), mammalian tolloid-like 1 (mTLL1) and zebra®sh tolloid (zTLD). (C,D) Comparison of the sequences of Colloid-like 1 with the proregion (C) and protease (D) signature domains suggests that Colloid-like 1 is the orthologue of mTLL1 and hTLL1.
L. Liaubet et al. / Mechanisms of Development 96 (2000) 101±105
Fig. 2. Colloid-like 1 transcripts are restricted to the caudal notochord in HH stage-10 embryos. (A) Whole mount in situ hybridization showing Colloid-like 1 transcripts. Only the caudal notochord is stained. (B) Higher magni®cation of the caudal region of the embryo in (A) Note the decreasing caudorostral gradient of Colloid-like 1 expression. The transcripts become undetectable around the last formed somite. Bars represent the levels of sections on (C±E). (C±E) Cross-sections through the embryo shown in (A). Colloid-like 1 is expressed in the notochord but not at the ventral midline of the neural tube.
In HH stage-6 embryos, a barely detectable level of transcripts is observed in the notochord (data not shown). In HH stage-8 embryos, transcripts are clearly detected in the whole notochord (Fig. 3A±D), in the ¯oor of the pharynx (Fig. 3B), in individualized somites (Fig. 3A,C) and in the adjacent lateral plate mesoderm (Fig. 3C). In HH stage-9 embryos (data not shown), the staining vanishes from the somites, while transcripts become restricted to the caudal region of the notochord
103
as described above for HH stage-10 embryos. This regionalized expression in the notochord persists at least until HH stage-15 (Fig. 4G±I). From HH stage-12, a faint staining is detected in the ®rst somites, this staining becoming clearly visible in HH stage-13 embryos (Fig. 4A). Cross-sections analysis reveals that transcripts are restricted to the dermamyotome (Fig. 4B). A third domain of Colloid-like 1 expression is observed at this stage in the roof of the caudal diencephalon. This expression domain subsequently expands caudally to the mesencephalon, as clearly visualized on HH stage-15 embryos (Fig. 4D,E). Colloid-like 1 is still expressed in the dermatome (Fig. 4F). At all the stages tested, transcripts are strongly expressed in the caudal notochord. In chick embryo, the expression pattern of another Tolloid-related metalloprotease, BMP1/TLD, has recently been reported (Marti, 2000). In HH stage-10 embryos, BMP1/TLD expression is detected within the notochord, but at the somite level, and in the FP and ventrolateral adjacent cells, as its mouse orthologue (Takahara et al., 1994). Transcripts are also present in the ectoderm ¯anking the neural plate and subsequently in neural folds along the entire length of the closing neural tube. Finally, BMP1/TLD is expressed in the myotome whereas Colloid-like 1 is present in the dermatome. Therefore, it appears that BMP1/TLD and Colloid-like 1 have distinct rather than overlapping regions of expression. In conclusion, we have isolated a secreted Tolloid-related metalloprotease speci®cally expressed in the caudal notochord. Injection of Colloid-like 1 in Xenopus leads to a mild ventralization of the embryos (data not shown) suggestive of a cleavage activity of chordin as reported for its closest orthologue (Scott et al., 1999). Our goal is now to identify Colloid-like 1 function in relationship with its remarkable domain of expression in the notochord.
2. Experimental procedures 2.1. Cloning of Colloid-like 1 The differential display procedure was performed according to (Liang et al., 1993; Liang and Pardee, 1992). The primers giving the 391 bp fragment were 5 0 -CTGATCCATG-3 0 for the forward primer and 5 0 -(T)12GC-3 0 for the reverse primer. That fragment was used to screen a HH stage-17 chicken embryo cDNA library (Stein and Kessel, 1995). Rapid ampli®cation of cDNA ends (RACE)-PCR experiments were performed to obtain the entire ORF.
Fig. 3. Expression of Colloid-like 1 at HH stage-8. (A) Whole-mount in situ hybridization showing the expression of Colloid-like 1 in the entire notochord and somites. Bars represent the levels of sections in (B±D). (B±D) Cross-sections of an equivalent embryo reveal a faint level of transcription in the ¯oor of the pharynx (B), all along the notochord (B±D) but also in the somitic and adjacent lateral plate mesoderm (C).
2.2. Whole-mount in situ hybridization The whole-mount in situ procedure was performed according to (Wilkinson, 1992) using three different probes (Fig. 1B).
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L. Liaubet et al. / Mechanisms of Development 96 (2000) 101±105
Fig. 4. New domains of Colloid-like 1 expression appear at later stages. (A±C) Pattern of Colloid-like 1 expression in HH stage-13 embryo. Three domains of expression are identi®ed: the roof of the caudal diencephalon (A), the dermamyotome (A,B) and the caudal notochord (A,C). Bars represent the level of crosssections shown in (B,C). (D±I) Pattern of expression of Colloid-like 1 in HH stage-15 embryos. The staining in the roof of the caudal diencephalon has extended caudally to the mesencephalon (D,E). The dermatome of rostral somites is clearly stained (F) while the newly individualized somites do not express Colloidlike 1 (G,H). The caudal notochord still displays a high level of Colloid-like 1 transcripts (G±I).
Acknowledgements The authors are extremely grateful to Dr A.M. Duprat for strong scienti®c support. We thank F. Foulquier for technical assistance, P. Cochard, J. Smith and E. Agius for critical comments on the manuscript. This work was supported by the Centre National de la Recherche Scienti®que, the Minis-
teÁre de la Recherche et de l'Enseignement SupeÂrieur, the Fondation pour la Recherche MeÂdicale and the Association pour la Recherche sur le Cancer. References Blader, P., Rastegar, S., Fischer, N., Strahle, U., 1997. Cleavage of the
L. Liaubet et al. / Mechanisms of Development 96 (2000) 101±105 BMP-4 antagonist chordin by zebra®sh tolloid. Science 278, 1937± 1940. Bond, J.S., Beynon, R.J., 1995. The astacin family of metalloendopeptidases. Protein Sci. 4, 1247±1261. Connors, S.A., Trout, J., Ekker, M., Mullins, M.C., 1999. The role of tolloid/mini ®n in dorsoventral pattern formation of the zebra®sh embryo. Development 126, 3119±3130. Hamburger, V., Hamilton, H.L., 1992. A series of normal stages in the development of the chick embryo. Dev. Dyn. 195, 231±272. Liang, P., Pardee, A.B., 1992. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257, 967± 971. Liang, P., Averboukh, L., Pardee, A.B., 1993. Distribution and cloning of eukaryotic mRNAs by means of differential display: re®nements and optimization. Nucleic Acids Res. 21, 3269±3275. Marti, E., 2000. Expression of chick BMP-1/Tolloid during patterning of the neural tube and somites. Mech. Dev. 91, 415±419. Marti, E., Takada, R., Bumcrot, D.A., Sasaki, H., McMahon, A.P., 1995. Distribution of Sonic hedgehog peptides in the developing chick and mouse embryo. Development 121, 2537±2547. Placzek, M., Jessell, T.M., Dodd, J., 1993. Induction of ¯oor plate differ-
105
entiation by contact-dependent, homeogenetic signals. Development 117, 205±218. Scott, I.C., Blitz, I.L., Pappano, W.N., Imamura, Y., Clark, T.G., Steiglitz, B.M., Thomas, C.L., Maas, S.A., Takahara, K., Cho, K.W., Greenspan, D.S., 1999. Mammalian BMP-1/Tolloid-related metalloproteinases, including novel family member mammalian Tolloid-like 2, have differential enzymatic activities and distributions of expression relevant to patterning and skeletogenesis. Dev. Biol. 213, 283±300. Stein, S., Kessel, M., 1995. A homeobox gene involved in node, notochord and neural plate formation of chick embryos. Mech. Dev. 49, 37± 48. Takahara, K., Lyons, G.E., Greenspan, D.S., 1994. Bone morphogenetic protein-1 and a mammalian tolloid homologue (mTld) are encoded by alternatively spliced transcripts which are differentially expressed in some tissues. J. Biol. Chem. 269, 32572±32578. Takahara, K., Brevard, R., Hoffman, G.G., Suzuki, N., Greenspan, D.S., 1996. Characterization of a novel gene product (mammalian tolloidlike) with high sequence similarity to mammalian tolloid/bone morphogenetic protein-1. Genomics 34, 157±165. Wilkinson, D.G., 1992. Situ Hybridization: A Practical Approach. IRL Press, Oxford, pp. 75±82.
www.elsevier.com/locate/modo
Gene expression pattern
Identi®cation by differential display of a chicken tolloid-related metalloprotease speci®cally expressed in the caudal notochord Laurence Liaubet, Nicolas Bertrand, FrancËois Medevielle, Fabienne Pituello* Centre de Biologie du DeÂveloppement, UMR-5547 CNRS-Universite P. Sabatier, 118 Route de Narbonne, 31062 Toulouse, Cedex 04, France Received 2 May 2000; received in revised form 6 June 2000; accepted 8 June 2000
Abstract While the ventralizing factor Sonic hedgehog is expressed in the entire notochord (Development 121 (1995) 2537) the latter displays distinct ventralizing activities along its rostrocaudal axis. Hence, in HH stage-10 chicken embryo, the caudal notochord exhibits ¯oor plate inducing capacities lost by rostral regions (Development 117 (1993) 205). Therefore, we hypothesize that the caudal notochord produces some cofactors which may contribute to its ventralizing properties. In order to identify such molecules we applied the differential display strategy and isolated a secreted Tolloid-related metalloprotease displaying a regionalized expression in the notochord. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Tolloid-related metalloprotease; Astacin; Notochord; Floor plate; Dorso-ventral patterning; Neural tube; Spinal cord; Somite; Chicken embryo; Differential display
1. Results and discussion 1.1. Identi®cation of genes speci®cally expressed in the caudal notochord The aim of this study was to identify secreted factors which, contrary to Sonic hedgehog (Marti et al., 1995), will be restricted to the caudal notochord correlating with its speci®c ventralizing capacities (Placzek et al., 1993). Therefore, we compared the messenger contents of caudal notochord vs. those of an embryo lacking it (Fig. 1A). In two independent experiments, a complementary DNA fragment of 391 bp was speci®cally ampli®ed in caudal notochord samples (Fig. 1A). A region of its nucleotide sequence (about 140 bp) displayed a high degree of homology (.80%) with the sequence encoding the EGF (epidermal growth factor)-like 2 domain of certain metalloprotease of the astacin family (Bond and Beynon, 1995). We then isolated the full-length cDNA corresponding to our PCR fragment. It displays an open reading frame of 3027 bp encoding a protein of 1009 amino acids. Analysis of the protein structure (Fig. 1B) reveals the presence of a signal peptide, suggesting that the protein can be secreted, a proregion followed by a protease domain displaying the signature * Corresponding author. Tel.: 133-561-55-67-42; fax: 133-561-55-6507. E-mail address: [email protected] (F. Pituello).
sequence of the metalloproteases of the astacin family (Bond and Beynon, 1995) and ®ve non-catalytic interaction domains including two EGF-like domains and three CUB (complement subcomponents C1r/C1s, embryonic sea urchin protein Uegf, BMP-1) domains. The entire protein displays more than 80% identity with human Tolloid-like 1 (Scott et al., 1999), mammalian Tolloid-like 1 (Takahara et al., 1996) and Zebra®sh Tolloid/Mini ®n (Blader et al., 1997; Connors et al., 1999) proteins (Fig. 1B). Furthermore, comparison of signature sequences of the proregion (Fig. 1C) and of the protease domain (Fig. 1D) reveals 100% identity at the amino acid level with the corresponding domains of human, mouse and zebra®sh tolloid-like1 proteins. Therefore, we have probably isolated a chicken orthologue of human and mammalian tolloid-like 1 genes. We called this gene Colloid-like 1 for chicken Tolloid-like1. Northern blot analysis reveals that a unique transcript of around 7 kb encodes the protein (data not shown). 1.2. Spatial and temporal dynamics of Colloid-like 1 expression As expected from the differential display data, in situ hybridization analysis performed on HH stage-10 embryos shows that transcripts are restricted to the caudal notochord, expanding between Hensen's node and the last individualized somite (Fig. 2). No expression can be detected at the ventral midline of the neural plate (Fig. 2C±E).
0925-4773/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(00)00382-8
102
L. Liaubet et al. / Mechanisms of Development 96 (2000) 101±105
Fig. 1. Identi®cation of a gene speci®cally transcribed in the caudal notochord. (A) Schematic representation of a HH stage-10 embryo (HH, (Hamburger and Hamilton, 1992)) showing the region of the notochord (red color) used for differential display analysis. (B) Differential display of mRNA from samples of 102 and 108 notochords (nc) vs. sample of an embryo without that notochordal region (embr-nc). The blue arrow marks a PCR product present in both samples and the red arrow the PCR product speci®cally ampli®ed from the notochord samples. (B) Schematic representation of the protein structure. The yellow box represents the signal peptide. P1±P3 corresponds to the regions used to synthesize the riboprobes. The EMBL Nucleotide Sequence Database accession number for Colloid-like 1 is AJ012462. Comparison of Colloid-like 1 with the astacin family members. The numbers represent the percent of amino acid identities for each region and for the entire protein (last number on the right). The numbers noted in sub-index corresponds to the length of the amino acid sequence in the proregion used for the comparison. Notice the high degree of identities with human tolloid-like 1 (hTLL1), mammalian tolloid-like 1 (mTLL1) and zebra®sh tolloid (zTLD). (C,D) Comparison of the sequences of Colloid-like 1 with the proregion (C) and protease (D) signature domains suggests that Colloid-like 1 is the orthologue of mTLL1 and hTLL1.
L. Liaubet et al. / Mechanisms of Development 96 (2000) 101±105
Fig. 2. Colloid-like 1 transcripts are restricted to the caudal notochord in HH stage-10 embryos. (A) Whole mount in situ hybridization showing Colloid-like 1 transcripts. Only the caudal notochord is stained. (B) Higher magni®cation of the caudal region of the embryo in (A) Note the decreasing caudorostral gradient of Colloid-like 1 expression. The transcripts become undetectable around the last formed somite. Bars represent the levels of sections on (C±E). (C±E) Cross-sections through the embryo shown in (A). Colloid-like 1 is expressed in the notochord but not at the ventral midline of the neural tube.
In HH stage-6 embryos, a barely detectable level of transcripts is observed in the notochord (data not shown). In HH stage-8 embryos, transcripts are clearly detected in the whole notochord (Fig. 3A±D), in the ¯oor of the pharynx (Fig. 3B), in individualized somites (Fig. 3A,C) and in the adjacent lateral plate mesoderm (Fig. 3C). In HH stage-9 embryos (data not shown), the staining vanishes from the somites, while transcripts become restricted to the caudal region of the notochord
103
as described above for HH stage-10 embryos. This regionalized expression in the notochord persists at least until HH stage-15 (Fig. 4G±I). From HH stage-12, a faint staining is detected in the ®rst somites, this staining becoming clearly visible in HH stage-13 embryos (Fig. 4A). Cross-sections analysis reveals that transcripts are restricted to the dermamyotome (Fig. 4B). A third domain of Colloid-like 1 expression is observed at this stage in the roof of the caudal diencephalon. This expression domain subsequently expands caudally to the mesencephalon, as clearly visualized on HH stage-15 embryos (Fig. 4D,E). Colloid-like 1 is still expressed in the dermatome (Fig. 4F). At all the stages tested, transcripts are strongly expressed in the caudal notochord. In chick embryo, the expression pattern of another Tolloid-related metalloprotease, BMP1/TLD, has recently been reported (Marti, 2000). In HH stage-10 embryos, BMP1/TLD expression is detected within the notochord, but at the somite level, and in the FP and ventrolateral adjacent cells, as its mouse orthologue (Takahara et al., 1994). Transcripts are also present in the ectoderm ¯anking the neural plate and subsequently in neural folds along the entire length of the closing neural tube. Finally, BMP1/TLD is expressed in the myotome whereas Colloid-like 1 is present in the dermatome. Therefore, it appears that BMP1/TLD and Colloid-like 1 have distinct rather than overlapping regions of expression. In conclusion, we have isolated a secreted Tolloid-related metalloprotease speci®cally expressed in the caudal notochord. Injection of Colloid-like 1 in Xenopus leads to a mild ventralization of the embryos (data not shown) suggestive of a cleavage activity of chordin as reported for its closest orthologue (Scott et al., 1999). Our goal is now to identify Colloid-like 1 function in relationship with its remarkable domain of expression in the notochord.
2. Experimental procedures 2.1. Cloning of Colloid-like 1 The differential display procedure was performed according to (Liang et al., 1993; Liang and Pardee, 1992). The primers giving the 391 bp fragment were 5 0 -CTGATCCATG-3 0 for the forward primer and 5 0 -(T)12GC-3 0 for the reverse primer. That fragment was used to screen a HH stage-17 chicken embryo cDNA library (Stein and Kessel, 1995). Rapid ampli®cation of cDNA ends (RACE)-PCR experiments were performed to obtain the entire ORF.
Fig. 3. Expression of Colloid-like 1 at HH stage-8. (A) Whole-mount in situ hybridization showing the expression of Colloid-like 1 in the entire notochord and somites. Bars represent the levels of sections in (B±D). (B±D) Cross-sections of an equivalent embryo reveal a faint level of transcription in the ¯oor of the pharynx (B), all along the notochord (B±D) but also in the somitic and adjacent lateral plate mesoderm (C).
2.2. Whole-mount in situ hybridization The whole-mount in situ procedure was performed according to (Wilkinson, 1992) using three different probes (Fig. 1B).
104
L. Liaubet et al. / Mechanisms of Development 96 (2000) 101±105
Fig. 4. New domains of Colloid-like 1 expression appear at later stages. (A±C) Pattern of Colloid-like 1 expression in HH stage-13 embryo. Three domains of expression are identi®ed: the roof of the caudal diencephalon (A), the dermamyotome (A,B) and the caudal notochord (A,C). Bars represent the level of crosssections shown in (B,C). (D±I) Pattern of expression of Colloid-like 1 in HH stage-15 embryos. The staining in the roof of the caudal diencephalon has extended caudally to the mesencephalon (D,E). The dermatome of rostral somites is clearly stained (F) while the newly individualized somites do not express Colloidlike 1 (G,H). The caudal notochord still displays a high level of Colloid-like 1 transcripts (G±I).
Acknowledgements The authors are extremely grateful to Dr A.M. Duprat for strong scienti®c support. We thank F. Foulquier for technical assistance, P. Cochard, J. Smith and E. Agius for critical comments on the manuscript. This work was supported by the Centre National de la Recherche Scienti®que, the Minis-
teÁre de la Recherche et de l'Enseignement SupeÂrieur, the Fondation pour la Recherche MeÂdicale and the Association pour la Recherche sur le Cancer. References Blader, P., Rastegar, S., Fischer, N., Strahle, U., 1997. Cleavage of the
L. Liaubet et al. / Mechanisms of Development 96 (2000) 101±105 BMP-4 antagonist chordin by zebra®sh tolloid. Science 278, 1937± 1940. Bond, J.S., Beynon, R.J., 1995. The astacin family of metalloendopeptidases. Protein Sci. 4, 1247±1261. Connors, S.A., Trout, J., Ekker, M., Mullins, M.C., 1999. The role of tolloid/mini ®n in dorsoventral pattern formation of the zebra®sh embryo. Development 126, 3119±3130. Hamburger, V., Hamilton, H.L., 1992. A series of normal stages in the development of the chick embryo. Dev. Dyn. 195, 231±272. Liang, P., Pardee, A.B., 1992. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257, 967± 971. Liang, P., Averboukh, L., Pardee, A.B., 1993. Distribution and cloning of eukaryotic mRNAs by means of differential display: re®nements and optimization. Nucleic Acids Res. 21, 3269±3275. Marti, E., 2000. Expression of chick BMP-1/Tolloid during patterning of the neural tube and somites. Mech. Dev. 91, 415±419. Marti, E., Takada, R., Bumcrot, D.A., Sasaki, H., McMahon, A.P., 1995. Distribution of Sonic hedgehog peptides in the developing chick and mouse embryo. Development 121, 2537±2547. Placzek, M., Jessell, T.M., Dodd, J., 1993. Induction of ¯oor plate differ-
105
entiation by contact-dependent, homeogenetic signals. Development 117, 205±218. Scott, I.C., Blitz, I.L., Pappano, W.N., Imamura, Y., Clark, T.G., Steiglitz, B.M., Thomas, C.L., Maas, S.A., Takahara, K., Cho, K.W., Greenspan, D.S., 1999. Mammalian BMP-1/Tolloid-related metalloproteinases, including novel family member mammalian Tolloid-like 2, have differential enzymatic activities and distributions of expression relevant to patterning and skeletogenesis. Dev. Biol. 213, 283±300. Stein, S., Kessel, M., 1995. A homeobox gene involved in node, notochord and neural plate formation of chick embryos. Mech. Dev. 49, 37± 48. Takahara, K., Lyons, G.E., Greenspan, D.S., 1994. Bone morphogenetic protein-1 and a mammalian tolloid homologue (mTld) are encoded by alternatively spliced transcripts which are differentially expressed in some tissues. J. Biol. Chem. 269, 32572±32578. Takahara, K., Brevard, R., Hoffman, G.G., Suzuki, N., Greenspan, D.S., 1996. Characterization of a novel gene product (mammalian tolloidlike) with high sequence similarity to mammalian tolloid/bone morphogenetic protein-1. Genomics 34, 157±165. Wilkinson, D.G., 1992. Situ Hybridization: A Practical Approach. IRL Press, Oxford, pp. 75±82.