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Embryonic expression of Tbx1, a DiGeorge syndrome candidate gene, in the lamprey Lampetrafluviatilis
Gene Expression Patterns 2 (2002) 99–103 www.elsevier.com/locate/modgep
Embryonic expression of Tbx1, a DiGeorge syndrome candidate gene, in the lamprey Lampetra fluviatilis Tatjana Sauka-Spengler a, Chantal Le Mentec a, Mario Lepage b, Sylvie Mazan a,* a
Equipe De´veloppement et Evolution des Verte´bre´s, UPRES-A 8080, Universite´ Paris- Sud, 91405 Orsay Cedex, France b AGEDRA/CEMAGREF, Cestas Gazinet, France Received 8 May 2002; received in revised form 9 August 2002; accepted 12 August 2002
Abstract We report the embryonic expression in the lamprey Lampetra fluviatilis of Tbx1, the main candidate gene involved in DiGeorge/velocardio-facial syndrome (DGS/VCFS). From the end of neurulation to stage 26, Tbx1 becomes progressively expressed in all developing pharyngeal arches, as they form. Transcripts are mainly restricted to the mesodermal core and to the posterior pharyngeal endoderm, excluding ingressing neural crest cells. They are also present in the otic vesicle, in a ventral and posterior location. From a later stage (stage 27) onwards, additional expression domains in the head mesenchyme, later contributing to labial muscle precursors, and in the cloacal region, become visible. The comparison of these data with those reported in the chick and the mouse indicates a high conservation of Tbx1 expression in the pharyngeal arches among vertebrates. q 2002 Elsevier Science B.V. All rights reserved. Keywords: Lamprey; Pharyngeal arches; Mandibular arch; Mesodermal core; Labial muscle precursor; Otic vesicle; Tbx1
1. Results and discussion The T-box is a DNA-binding structural domain, which characterizes a broad family of transcription factors, found in all triploblastic metazoans. T-box-containing genes are involved in a variety of developmental processes, such as the formation of germ layers during gastrulation, or in vertebrates, the morphogenesis of the heart and the genetic control of limb-type identity (reviewed in Papaioannou and Silver, 1998; Smith, 1999). In humans, mutations in some of these genes have also been shown to cause congenital limb, face or heart malformations. For instance, strong evidence has recently accumulated, suggesting that Tbx1 haploinsufficiency may be responsible for the major aortic arch defects, which characterize DiGeorge/velo-cardiofacial syndrome (DGS/VCFS) (Jerome and Papaioannou, 2001; Lindsay et al., 2001; Merscher et al., 2001). Using degenerate primers encoding the highly conserved TEMIV and NPFAKG protein motifs (positions 19–23 and 181–186 in the T-domain), we have amplified a 501 bp fragment from stage 19 Lampetra fluviatilis cDNA. As expected, this fragment unambiguously encodes a T-box sequence (Fig. 1A), showing the highest level of similarity with chordate Tbx1-related sequences and their Drosophila * Corresponding author. Tel.: 133-1-6915-4999; fax: 133-1-6915-6828. E-mail address: [email protected] (S. Mazan).
ortholog (88 and 70% of identities between the lamprey deduced amino acid sequence and the human Tbx1 and Drosophila Ombr-1 sequences, respectively). Phylogenetic analyses including the newly isolated LfTbx1 gene, two Tbx1-related sequences retrieved from Fugu rubripes genome sequence (termed FrTbx1a and FrTbx1b) and representatives of the major T-box families recently identified (Ruvinsky et al., 2000), confirm the assignment of the lamprey and pufferfish sequences to the Tbx1 family. Whatever the parameters and the reconstruction algorithm used (neighbor-joining, maximum parsimony, maximum likelihood), all the groups previously described are recovered. In each case, the lamprey sequence, as well as the two new Fugu sequences, cluster with all other identified chordate Tbx1 sequences and the Drosophila Ombr-1 sequence in a strongly supported monophyletic group (Fig. 1B). LfTbx1 expression was studied by whole-mount hybridization of L. fluviatilis embryos, starting from stage 21 (end of neurulation) to stage 30 (the earliest stage of ammocoete larvae). At stage 21, characterized by the presence of the first two pharyngeal pouches, a faint hybridization signal is visible in the premandibular, mandibular and hyoid segments (Fig. 2A). At stage 23, when the third pharyngeal pouch forms, this signal persists and also becomes visible at the level of the first post-otic segments (Fig. 2B). Sections show that both at pre-otic and post-otic levels,
1567-133X/02/$ - see front matter q 2002 Elsevier Science B.V. All rights reserved. PII: S 0925-477 3(02)00301-5
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Fig. 1. Sequence analysis of the lamprey Tbx1 amino acid sequence. (A) Alignment of the lamprey Tbx1 amino acid sequence with orthologous sequences over positions [19;186] of the T-box. The Tbx1-related sequences shown were retrieved from GenBank (human, NM080646; mouse, AF326960; amphioxus, AF262562; ascidian, AB073907; Drosophila Ombr-1, NM078530), or from the Fugu Genome Consortium database (http://fugu.hgmp.mrc.ac.uk/blast; FrTbx1a, scaffold no. 2988, pos. 4189–7126 and FrTbx1b, scaffold no. 4541, pos. 9386–10 374) and aligned with the LfTbx1 partial protein sequence obtained in L. fluviatilis. Dashes indicate residue identity with LfTbx1 sequence, stars indicate insertion/deletion events and dots correspond to missing sequence data. White characters on a black background correspond to residues, which appear selectively conserved among Tbx1-related proteins. Black arrows stand for protein motifs, which were used to design degenerate amplification primers, arrowheads designating the primer orientation. Numberings refer to the position in the alignment. Hs, Homo sapiens; Mm, Mus musculus; Lf, Lampetra fluviatilis; Fr, Fugu rubripes; Bf, Branchiostoma floridae; Ci, Ciona intestinalis; Dm, Drosophila melanogaster. (B) Phylogenetic relationships between Tbx1-related genes and other classes of the T-box family. A subset of sequences belonging to representatives of the six T-box classes identified in addition to the Tbx1 class and comprising in each case one actinopterygian, one sarcopterygian and when available Amphioxus or Drosophila sequences, were included in the alignment shown in (A) (data not shown; Ruvinsky et al., 2000). Like in Ruvinsky et al. (2000), divergent members of the Tbx6/16 class were not included in these analyses. Phylogenetic trees were constructed from this alignment using neighbor joining (NJ), maximum parsimony (MP) and maximum-likelihood (ML) algorithms. The tree shown is the minimal NJ tree. Only bootstrap values supporting the major groupings are shown, as squared numbers (first, second and third lines with NJ, ML and MP values, respectively). The tree is arbitrarily rooted with CeTbx9. Gene nomenclature has been homogenized with the following abbreviations: Hs, Homo sapiens; Mm, Mus musculus; Lf, Lampetra fluviatilis; Fr, Fugu rubripes; Bf, Branchiostoma floridae; Ci, Ciona intestinalis; Dm, Drosophila melanogaster; Ol, Oryzias latipes; Dr, Danio rerio; Ce, Caenorhabditis elegans.
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the transcripts are restricted to the lateral mesoderm, excluding axial and paraxial regions (data not shown). During following stages (24–26), the hybridization signal intensifies and successively appears in all forming pharyngeal arches (Fig. 2C–H). At these stages, the neural crestderived ectomesenchyme and the mesodermal core form two distinct cell populations, which do not mix and can be readily distinguished on frontal sections of the pharyngeal arches (Damas, 1944; Fig. 2J). These two cell populations have also been unambiguously identified using the AP-2 transcription factor, a genetic marker of neural crest cells in osteichthyans: in stage 24-26 lamprey embryos, a prominent AP-2 expression is present in the mesenchyme cells surrounding the mesodermal core, thus confirming their neural crest identity (Meulemans and
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Bronner-Fraser, 2002). In contrast, Tbx1 transcripts appear mainly located to the mesodermal core and the posterior endodermal wall of the forming pharyngeal arches, but excluded from the neural crest-derived ectomesenchyme, which intercalates between the mesodermal core and the pharyngeal endoderm or ectoderm (Fig. 2I,J,L,M). The developing otic vesicle is an additional LfTbx1 expression site, with transcripts restricted to its posterior half, in its ventrolateral parts (Fig. 2C–G,K). Starting from stage 27, the cell populations, which will later contribute to muscle fibers and skeletal elements in the hyoid and branchial arches, become morphologically individualized, while the mesoderm and neural crest derived cells intermingle (Damas, 1944). At this stage, the labeling in these arches regresses to their posterior parts, both in the mesenchyme,
Fig. 2. Expression pattern of LfTbx1 in stage 21–26 lamprey embryos. (A) Lateral view, stage 21. A continuous mesodermal expression domain can be detected at the level of the premandibular, mandibular and hyoid segments (sections not shown). (B) Lateral view, stage 23. The signal in the mandibular and hyoid segments, which are now individualized, persists. Transcripts also become detectable in the post-otic lateral mesoderm (sections not shown). (C,D), (E,F), (G) Lateral views, stages 24, 25 and 26, respectively. (D) and (F) are higher magnifications in the head region of the photographs shown in (C) and (E), respectively. (H) Higher magnification of the embryo shown in (G), ventral view. (I,J), (L,M) Frontal and transverse sections of stage 25 embryos, respectively. (J) is a higher magnification of the section shown in (I) at the level of the first branchial arch. As in hyoid and more posterior pharyngeal arches, the mesodermal core does not mix with the surrounding neural crest cells. (K) Frontal section of a stage 24 embryo. The planes of sections are indicated by thin lines. Starting from stage 24, the signal becomes prominent in the forming pharyngeal arches. Expression is mainly localized to the mesodermal core of the arches, but also detectable in the lateral-anterior endoderm of each branchial pouch. Transcripts are also present in the latero-posterior parts of the otic vesicle. b1–5, 1st–5th branchial arch; da, dorsal aorta; en, endoderm; end, endostyle; hy, hyoid arch; m, mouth; ma, mandibular arch; mc, mesodermal core; mm, mandibular mesoderm; nc, neural crest cells; nt, notochord; ov, otic vesicle; ph, pharynx; va, ventral aorta. Scale bars: 0.5 mm.
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Fig. 3. LfTbx1 expression pattern in stage 27–30 lamprey embryos. (A) Lateral view and (B) ventral view in the head region, stage 27, showing the signals in the posterior part of each pharyngeal arch and in the head region. (C) Lateral view in the cloacal region of a stage 30 ammocoete larvae. A prominent signal is present in the cloacal region. (D–H) Sections of stage 27 embryos. (D) Frontal section in the region of branchial arches. (E) Frontal section in the oral region. (F,G,H) Transverse sections at the levels of the mouth, the mandibular arch and the third pharyngeal pouch. Transcripts become restricted to the posterior part of the pharyngeal arches, both in the mesenchyme and the endoderm (D,H). In the head, the muscle precursors of the lower and upper lips, and to a lesser extent the velum, are labeled (E–G). Thin lines in (A) show the planes of the sections. In frontal sections anterior is to the left. In transverse sections dorsal is to the top. b1–6, 1st–6th branchial arch; da, dorsal aorta; hy, hyoid arch; llp, lower lip; lmp, labial muscle precursors; m, mouth; ma, mandibular arch; mm, mandibular mesoderm; ph, pharynx; pp3, 3rd pharyngeal pouch; vel, velum. Scale bars: 0.5 mm.
where the signal becomes faint, and in the endoderm, where it remains prominent (Fig. 3A,B,D,G,H). The mandibular arch also undergoes important transformations, which lead to the formation of the musculature of the entire mouth region. In this region, LfTbx1 transcripts are found in the head mesenchyme, both in the upper and lower lips of the ammocoete, which later contribute to labial muscles (Fig. 3E–G). A faint expression is also visible in the future velar muscles (Fig. 3E). Thus far, Tbx1 embryonic expression has only been studied in two amniotes, the chick and the mouse. Like their lamprey ortholog, these genes show prominent expression domains in the head mesenchyme, the otic vesicle, the mesodermal core and the pharyngeal endoderm, but not the neural crest derived mesenchyme of the developing pharyngeal arches. These data have led to the suggestion that the defects in neural crest migration or differentiation observed in DGS/VCFS may be related to a secondary non cell-autonomous effect of the absence of Tbx1 in the neighboring pharyngeal mesoderm and endoderm (Garg et al., 2001; Funke et al., 2001). The strong similarities displayed by Tbx1 expression pattern in the pharyngeal arches of L. fluviatilis, a lower vertebrate, with those reported in amniotes, raise new questions about the conservation of the gene functions among vertebrates.
2. Experimental procedures 2.1. Embryos Adult male and female lampreys were collected in the streams of the Gironde river, France, during the breeding season. The eggs were laid and fertilized in the laboratory and kept in fresh oxygenated water at 15 8C, until desired stages were obtained. Embryos were staged according to tables established for another closely related species Lampetra reissneri (Tahara, 1988). 2.2. Cloning of the Tbx1 fragment in the L. fluviatilis The lamprey partial Tbx1 fragment reported here was amplified by a degenerate RT–PCR approach, using as 5 0 primer 5 0 -ACNGARATGATHGT and as 3 0 primer 5 0 CCYTTNGCRAANGGRTT, which correspond respectively to the TEMIV (position 1–6 in Fig. 1A) and NPFAKG (position 162–167) protein motifs. Amplification was performed in the standard Taq DNA polymerase buffer, in the following cycling conditions: 95 8C, 1 min; 55 8C, 1 min; 72 8C, 1 min, 40 cycles. The amplified fragment was subcloned in SmaI-digested pTZ19R and recombinants were sequenced on both strands using a cycle-sequencing protocol. The sequence shown is a consensus of at least three independent clones. The LfTbx1 partial coding
T. Sauka-Spengler et al. / Gene Expression Patterns 2 (2002) 99–103
sequence has been submitted to GenBank under accession number AF508192. 2.3. Molecular phylogenetic analysis and database research The T-box protein sequences used in the analysis were retrieved from the GenBank database. Similarities to Tbx1 protein sequences were searched for in the Fugu Genome Consortium database (http://fugu.hgmp.mrc.ac.uk/blast), using the tblastn algorithm, with human Tbx1 protein sequence as input. Sequences were aligned manually using the ED program of the MUST package (Philippe, 1993). Neighbor-joining (NJ) (Saitou and Nei, 1987), maximumparsimony (MP) and maximum-likelihood (ML) phylogenetic analyses were performed using the MUST version 2000 package (http://bos.snv.jussieu.fr/must2000.html), PAUP, version 3.1.1 (Swofford, 1993), and PROTML, version 2.3 (Adachi and Hasegawa, 1996), respectively. The quick-add OTUs search with the JTT-f model of amino acid substitutions retaining the 2000 top-ranking trees was used in the former. Bootstrap proportions were calculated by analysis of 1000 replicates for NJ and MP reconstructions, and by the RELL method (Kishino et al., 1990) upon the 2000 top-ranking ML trees. 2.4. Whole-mount in situ hybridization Whole-mount in situ hybridizations of L. fluviatilis embryos (stages 21–30) were performed using digoxigenin 11-UTP labeled antisense RNA probes, with an additional bleaching step, as described in Sauka-Spengler et al. (2001). The LfTbx1 probe was generated from a SacI-linearized pTZ19R recombinant, containing the isolated fragment. 2.5. Histological sections following whole-mount hybridization Embryos were post-fixed in 4% paraformaldehyde/PBS (4 8C, overnight), rinsed in PBS, cryo-protected with 15% sucrose/PBS, embedded in 15% sucrose, 20% gelatin/PBS (37 8C, overnight) and 20% gelatin/PBS (37 8C overnight), frozen in liquid nitrogen and mounted in OCT compound (Miles, Elkhart, IN). Ten-micrometer cryosections were collected on Super Frost Plus slides (Fisher Scientific, Pittsburgh, PA), counterstained using Nuclear Fast Red (Vector Laboratories, Burlingame, CA), mounted in Eukitt (O. Kindler, Freiburg, Germany) and photographed. Acknowledgements We are grateful to Daniel Meulemans and Marianne Bronner-Fraser for communicating their result on AP-2 expression in lamprey prior to publication. We thank M. Pradels for excellent technical assistance. This work was
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supported by funds from the Centre National de la Recherche Scientifique and the Universite´ Paris-Sud, and by a Ministe`re de la Recherche et de la Technologie doctoral fellowship to T.S.-S.
References Adachi, J., Hasegawa, M., 1996. MOLPHY version 23: programs for molecular phylogenetics I PROTML: maximum likelihood inference of protein phylogeny. Comput. Sci. Monogr. 28, 1–150. Damas, H., 1944. Recherches sur le de´ veloppement de Lampetra fluviatilis L. Contribution a` l’e´ tude de la ce´ phalogene`se des verte´ bre´ s. Arch. Biol. 55, 5–284. Funke, B., Epstein, J.A., Kochilas, L.K., Lu, M.M., Pandita, R.K., Liao, J., Bauerndistel, R., Schuler, T., Schorle, H., Brown, M.C., Adams, J., Morrow, B.E., 2001. Mice overexpressing genes from the 22q11 region deleted in velo-cardio-facial syndrome/DiGeorge syndrome have middle and inner ear defects. Hum. Mol. Genet. 10, 2549–2556. Garg, V., Yamagishi, C., Hu, T., Kathiriya, I.S., Yamagishi, H., Srivastava, D., 2001. Tbx1, a DiGeorge Syndrome Candidate Gene, is regulated by sonic hedgehog during pharyngeal arch development. Dev. Biol. 235, 62–73. Jerome, L.A., Papaioannou, V.E., 2001. DiGeorge syndrome phenotype in mice mutant for the T-box gene, Tbx1. Nat. Genet. 27, 286–291. Kishino, H., Miyata, T., Hasegawa, M., 1990. Maximum likelihood inference of protein phylogeny and the origin of chloroplasts. J. Mol. Evol. 30, 151–160. Lindsay, E.A., Vitelli, F., Su, H., Morishima, M., Huynh, T., Pramparo, T., Jurecic, V., Ogunrinu, G., Sutherland, H.F., Scambler, P.J., Bradley, A., Baldini, A., 2001. Tbx1 haploinsufficieny in the DiGeorge syndrome region causes aortic arch defects in mice. Nature 410, 97–101. Merscher, S., Funke, B., Epstein, J.A., Heyer, J., Puech, A., Lu, M.M., Xavier, R.J., Demay, M.B., Russell, R.G., Factor, S., Tokooya, K., Jore, B.S., Lopez, M., Pandita, R.K., Lia, M., Carrion, D., Xu, H., Schorle, H., Kobler, J.B., Scambler, P., Wynshaw-Boris, A., Skoultchi, A.I., Morrow, B.E., Kucherlapati, R., 2001. TBX1 is responsible for cardiovascular defects in velo-cardio-facial/DiGeorge syndrome. Cell 104, 619–629. Meulemans, D., Bronner-Fraser, M., 2002. Amphioxus and lamprey AP-2 genes: implications for neural crest evolution and migration patterns. Development in press. Papaioannou, V.E., Silver, L.M., 1998. The T-box gene family. Bioessays 20, 9–19. Philippe, H., 1993. MUST, a computer package of Management Utilities for Sequences and Trees. Nucleic Acids Res. 21, 5264–5272. Ruvinsky, I., Silver, L.M., Gibson-Brown, J.J., 2000. Phylogenetic analysis of T-Box genes demonstrates the importance of amphioxus for understanding evolution of the vertebrate genome. Genetics 156, 1249–1257. Saitou, N., Nei, M., 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425. Sauka-Spengler, T., Baratte, B., Shi, D.L., Mazan, S., 2001. Structure and expression of an Otx5-related gene in the dogfish Scyliorhinus canicula: evidence for a conserved role of Otx5 and Crx genes in the specification of photoreceptors. Dev. Genes Evol. 211, 533–544. Smith, J., 1999. T-box genes: What they do and how they do it. Trends Genet. 15, 154–158. Swofford, D.L., 1993. PAUP Version 3.1, Illinois Natural History Survey, Champaign, IL. Tahara, Y., 1988. Normal stages of development in the lamprey Lampetra reissneri (Dybowski). Zool. Sci. 5, 109–111.
Embryonic expression of Tbx1, a DiGeorge syndrome candidate gene, in the lamprey Lampetra fluviatilis Tatjana Sauka-Spengler a, Chantal Le Mentec a, Mario Lepage b, Sylvie Mazan a,* a
Equipe De´veloppement et Evolution des Verte´bre´s, UPRES-A 8080, Universite´ Paris- Sud, 91405 Orsay Cedex, France b AGEDRA/CEMAGREF, Cestas Gazinet, France Received 8 May 2002; received in revised form 9 August 2002; accepted 12 August 2002
Abstract We report the embryonic expression in the lamprey Lampetra fluviatilis of Tbx1, the main candidate gene involved in DiGeorge/velocardio-facial syndrome (DGS/VCFS). From the end of neurulation to stage 26, Tbx1 becomes progressively expressed in all developing pharyngeal arches, as they form. Transcripts are mainly restricted to the mesodermal core and to the posterior pharyngeal endoderm, excluding ingressing neural crest cells. They are also present in the otic vesicle, in a ventral and posterior location. From a later stage (stage 27) onwards, additional expression domains in the head mesenchyme, later contributing to labial muscle precursors, and in the cloacal region, become visible. The comparison of these data with those reported in the chick and the mouse indicates a high conservation of Tbx1 expression in the pharyngeal arches among vertebrates. q 2002 Elsevier Science B.V. All rights reserved. Keywords: Lamprey; Pharyngeal arches; Mandibular arch; Mesodermal core; Labial muscle precursor; Otic vesicle; Tbx1
1. Results and discussion The T-box is a DNA-binding structural domain, which characterizes a broad family of transcription factors, found in all triploblastic metazoans. T-box-containing genes are involved in a variety of developmental processes, such as the formation of germ layers during gastrulation, or in vertebrates, the morphogenesis of the heart and the genetic control of limb-type identity (reviewed in Papaioannou and Silver, 1998; Smith, 1999). In humans, mutations in some of these genes have also been shown to cause congenital limb, face or heart malformations. For instance, strong evidence has recently accumulated, suggesting that Tbx1 haploinsufficiency may be responsible for the major aortic arch defects, which characterize DiGeorge/velo-cardiofacial syndrome (DGS/VCFS) (Jerome and Papaioannou, 2001; Lindsay et al., 2001; Merscher et al., 2001). Using degenerate primers encoding the highly conserved TEMIV and NPFAKG protein motifs (positions 19–23 and 181–186 in the T-domain), we have amplified a 501 bp fragment from stage 19 Lampetra fluviatilis cDNA. As expected, this fragment unambiguously encodes a T-box sequence (Fig. 1A), showing the highest level of similarity with chordate Tbx1-related sequences and their Drosophila * Corresponding author. Tel.: 133-1-6915-4999; fax: 133-1-6915-6828. E-mail address: [email protected] (S. Mazan).
ortholog (88 and 70% of identities between the lamprey deduced amino acid sequence and the human Tbx1 and Drosophila Ombr-1 sequences, respectively). Phylogenetic analyses including the newly isolated LfTbx1 gene, two Tbx1-related sequences retrieved from Fugu rubripes genome sequence (termed FrTbx1a and FrTbx1b) and representatives of the major T-box families recently identified (Ruvinsky et al., 2000), confirm the assignment of the lamprey and pufferfish sequences to the Tbx1 family. Whatever the parameters and the reconstruction algorithm used (neighbor-joining, maximum parsimony, maximum likelihood), all the groups previously described are recovered. In each case, the lamprey sequence, as well as the two new Fugu sequences, cluster with all other identified chordate Tbx1 sequences and the Drosophila Ombr-1 sequence in a strongly supported monophyletic group (Fig. 1B). LfTbx1 expression was studied by whole-mount hybridization of L. fluviatilis embryos, starting from stage 21 (end of neurulation) to stage 30 (the earliest stage of ammocoete larvae). At stage 21, characterized by the presence of the first two pharyngeal pouches, a faint hybridization signal is visible in the premandibular, mandibular and hyoid segments (Fig. 2A). At stage 23, when the third pharyngeal pouch forms, this signal persists and also becomes visible at the level of the first post-otic segments (Fig. 2B). Sections show that both at pre-otic and post-otic levels,
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Fig. 1. Sequence analysis of the lamprey Tbx1 amino acid sequence. (A) Alignment of the lamprey Tbx1 amino acid sequence with orthologous sequences over positions [19;186] of the T-box. The Tbx1-related sequences shown were retrieved from GenBank (human, NM080646; mouse, AF326960; amphioxus, AF262562; ascidian, AB073907; Drosophila Ombr-1, NM078530), or from the Fugu Genome Consortium database (http://fugu.hgmp.mrc.ac.uk/blast; FrTbx1a, scaffold no. 2988, pos. 4189–7126 and FrTbx1b, scaffold no. 4541, pos. 9386–10 374) and aligned with the LfTbx1 partial protein sequence obtained in L. fluviatilis. Dashes indicate residue identity with LfTbx1 sequence, stars indicate insertion/deletion events and dots correspond to missing sequence data. White characters on a black background correspond to residues, which appear selectively conserved among Tbx1-related proteins. Black arrows stand for protein motifs, which were used to design degenerate amplification primers, arrowheads designating the primer orientation. Numberings refer to the position in the alignment. Hs, Homo sapiens; Mm, Mus musculus; Lf, Lampetra fluviatilis; Fr, Fugu rubripes; Bf, Branchiostoma floridae; Ci, Ciona intestinalis; Dm, Drosophila melanogaster. (B) Phylogenetic relationships between Tbx1-related genes and other classes of the T-box family. A subset of sequences belonging to representatives of the six T-box classes identified in addition to the Tbx1 class and comprising in each case one actinopterygian, one sarcopterygian and when available Amphioxus or Drosophila sequences, were included in the alignment shown in (A) (data not shown; Ruvinsky et al., 2000). Like in Ruvinsky et al. (2000), divergent members of the Tbx6/16 class were not included in these analyses. Phylogenetic trees were constructed from this alignment using neighbor joining (NJ), maximum parsimony (MP) and maximum-likelihood (ML) algorithms. The tree shown is the minimal NJ tree. Only bootstrap values supporting the major groupings are shown, as squared numbers (first, second and third lines with NJ, ML and MP values, respectively). The tree is arbitrarily rooted with CeTbx9. Gene nomenclature has been homogenized with the following abbreviations: Hs, Homo sapiens; Mm, Mus musculus; Lf, Lampetra fluviatilis; Fr, Fugu rubripes; Bf, Branchiostoma floridae; Ci, Ciona intestinalis; Dm, Drosophila melanogaster; Ol, Oryzias latipes; Dr, Danio rerio; Ce, Caenorhabditis elegans.
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the transcripts are restricted to the lateral mesoderm, excluding axial and paraxial regions (data not shown). During following stages (24–26), the hybridization signal intensifies and successively appears in all forming pharyngeal arches (Fig. 2C–H). At these stages, the neural crestderived ectomesenchyme and the mesodermal core form two distinct cell populations, which do not mix and can be readily distinguished on frontal sections of the pharyngeal arches (Damas, 1944; Fig. 2J). These two cell populations have also been unambiguously identified using the AP-2 transcription factor, a genetic marker of neural crest cells in osteichthyans: in stage 24-26 lamprey embryos, a prominent AP-2 expression is present in the mesenchyme cells surrounding the mesodermal core, thus confirming their neural crest identity (Meulemans and
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Bronner-Fraser, 2002). In contrast, Tbx1 transcripts appear mainly located to the mesodermal core and the posterior endodermal wall of the forming pharyngeal arches, but excluded from the neural crest-derived ectomesenchyme, which intercalates between the mesodermal core and the pharyngeal endoderm or ectoderm (Fig. 2I,J,L,M). The developing otic vesicle is an additional LfTbx1 expression site, with transcripts restricted to its posterior half, in its ventrolateral parts (Fig. 2C–G,K). Starting from stage 27, the cell populations, which will later contribute to muscle fibers and skeletal elements in the hyoid and branchial arches, become morphologically individualized, while the mesoderm and neural crest derived cells intermingle (Damas, 1944). At this stage, the labeling in these arches regresses to their posterior parts, both in the mesenchyme,
Fig. 2. Expression pattern of LfTbx1 in stage 21–26 lamprey embryos. (A) Lateral view, stage 21. A continuous mesodermal expression domain can be detected at the level of the premandibular, mandibular and hyoid segments (sections not shown). (B) Lateral view, stage 23. The signal in the mandibular and hyoid segments, which are now individualized, persists. Transcripts also become detectable in the post-otic lateral mesoderm (sections not shown). (C,D), (E,F), (G) Lateral views, stages 24, 25 and 26, respectively. (D) and (F) are higher magnifications in the head region of the photographs shown in (C) and (E), respectively. (H) Higher magnification of the embryo shown in (G), ventral view. (I,J), (L,M) Frontal and transverse sections of stage 25 embryos, respectively. (J) is a higher magnification of the section shown in (I) at the level of the first branchial arch. As in hyoid and more posterior pharyngeal arches, the mesodermal core does not mix with the surrounding neural crest cells. (K) Frontal section of a stage 24 embryo. The planes of sections are indicated by thin lines. Starting from stage 24, the signal becomes prominent in the forming pharyngeal arches. Expression is mainly localized to the mesodermal core of the arches, but also detectable in the lateral-anterior endoderm of each branchial pouch. Transcripts are also present in the latero-posterior parts of the otic vesicle. b1–5, 1st–5th branchial arch; da, dorsal aorta; en, endoderm; end, endostyle; hy, hyoid arch; m, mouth; ma, mandibular arch; mc, mesodermal core; mm, mandibular mesoderm; nc, neural crest cells; nt, notochord; ov, otic vesicle; ph, pharynx; va, ventral aorta. Scale bars: 0.5 mm.
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Fig. 3. LfTbx1 expression pattern in stage 27–30 lamprey embryos. (A) Lateral view and (B) ventral view in the head region, stage 27, showing the signals in the posterior part of each pharyngeal arch and in the head region. (C) Lateral view in the cloacal region of a stage 30 ammocoete larvae. A prominent signal is present in the cloacal region. (D–H) Sections of stage 27 embryos. (D) Frontal section in the region of branchial arches. (E) Frontal section in the oral region. (F,G,H) Transverse sections at the levels of the mouth, the mandibular arch and the third pharyngeal pouch. Transcripts become restricted to the posterior part of the pharyngeal arches, both in the mesenchyme and the endoderm (D,H). In the head, the muscle precursors of the lower and upper lips, and to a lesser extent the velum, are labeled (E–G). Thin lines in (A) show the planes of the sections. In frontal sections anterior is to the left. In transverse sections dorsal is to the top. b1–6, 1st–6th branchial arch; da, dorsal aorta; hy, hyoid arch; llp, lower lip; lmp, labial muscle precursors; m, mouth; ma, mandibular arch; mm, mandibular mesoderm; ph, pharynx; pp3, 3rd pharyngeal pouch; vel, velum. Scale bars: 0.5 mm.
where the signal becomes faint, and in the endoderm, where it remains prominent (Fig. 3A,B,D,G,H). The mandibular arch also undergoes important transformations, which lead to the formation of the musculature of the entire mouth region. In this region, LfTbx1 transcripts are found in the head mesenchyme, both in the upper and lower lips of the ammocoete, which later contribute to labial muscles (Fig. 3E–G). A faint expression is also visible in the future velar muscles (Fig. 3E). Thus far, Tbx1 embryonic expression has only been studied in two amniotes, the chick and the mouse. Like their lamprey ortholog, these genes show prominent expression domains in the head mesenchyme, the otic vesicle, the mesodermal core and the pharyngeal endoderm, but not the neural crest derived mesenchyme of the developing pharyngeal arches. These data have led to the suggestion that the defects in neural crest migration or differentiation observed in DGS/VCFS may be related to a secondary non cell-autonomous effect of the absence of Tbx1 in the neighboring pharyngeal mesoderm and endoderm (Garg et al., 2001; Funke et al., 2001). The strong similarities displayed by Tbx1 expression pattern in the pharyngeal arches of L. fluviatilis, a lower vertebrate, with those reported in amniotes, raise new questions about the conservation of the gene functions among vertebrates.
2. Experimental procedures 2.1. Embryos Adult male and female lampreys were collected in the streams of the Gironde river, France, during the breeding season. The eggs were laid and fertilized in the laboratory and kept in fresh oxygenated water at 15 8C, until desired stages were obtained. Embryos were staged according to tables established for another closely related species Lampetra reissneri (Tahara, 1988). 2.2. Cloning of the Tbx1 fragment in the L. fluviatilis The lamprey partial Tbx1 fragment reported here was amplified by a degenerate RT–PCR approach, using as 5 0 primer 5 0 -ACNGARATGATHGT and as 3 0 primer 5 0 CCYTTNGCRAANGGRTT, which correspond respectively to the TEMIV (position 1–6 in Fig. 1A) and NPFAKG (position 162–167) protein motifs. Amplification was performed in the standard Taq DNA polymerase buffer, in the following cycling conditions: 95 8C, 1 min; 55 8C, 1 min; 72 8C, 1 min, 40 cycles. The amplified fragment was subcloned in SmaI-digested pTZ19R and recombinants were sequenced on both strands using a cycle-sequencing protocol. The sequence shown is a consensus of at least three independent clones. The LfTbx1 partial coding
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sequence has been submitted to GenBank under accession number AF508192. 2.3. Molecular phylogenetic analysis and database research The T-box protein sequences used in the analysis were retrieved from the GenBank database. Similarities to Tbx1 protein sequences were searched for in the Fugu Genome Consortium database (http://fugu.hgmp.mrc.ac.uk/blast), using the tblastn algorithm, with human Tbx1 protein sequence as input. Sequences were aligned manually using the ED program of the MUST package (Philippe, 1993). Neighbor-joining (NJ) (Saitou and Nei, 1987), maximumparsimony (MP) and maximum-likelihood (ML) phylogenetic analyses were performed using the MUST version 2000 package (http://bos.snv.jussieu.fr/must2000.html), PAUP, version 3.1.1 (Swofford, 1993), and PROTML, version 2.3 (Adachi and Hasegawa, 1996), respectively. The quick-add OTUs search with the JTT-f model of amino acid substitutions retaining the 2000 top-ranking trees was used in the former. Bootstrap proportions were calculated by analysis of 1000 replicates for NJ and MP reconstructions, and by the RELL method (Kishino et al., 1990) upon the 2000 top-ranking ML trees. 2.4. Whole-mount in situ hybridization Whole-mount in situ hybridizations of L. fluviatilis embryos (stages 21–30) were performed using digoxigenin 11-UTP labeled antisense RNA probes, with an additional bleaching step, as described in Sauka-Spengler et al. (2001). The LfTbx1 probe was generated from a SacI-linearized pTZ19R recombinant, containing the isolated fragment. 2.5. Histological sections following whole-mount hybridization Embryos were post-fixed in 4% paraformaldehyde/PBS (4 8C, overnight), rinsed in PBS, cryo-protected with 15% sucrose/PBS, embedded in 15% sucrose, 20% gelatin/PBS (37 8C, overnight) and 20% gelatin/PBS (37 8C overnight), frozen in liquid nitrogen and mounted in OCT compound (Miles, Elkhart, IN). Ten-micrometer cryosections were collected on Super Frost Plus slides (Fisher Scientific, Pittsburgh, PA), counterstained using Nuclear Fast Red (Vector Laboratories, Burlingame, CA), mounted in Eukitt (O. Kindler, Freiburg, Germany) and photographed. Acknowledgements We are grateful to Daniel Meulemans and Marianne Bronner-Fraser for communicating their result on AP-2 expression in lamprey prior to publication. We thank M. Pradels for excellent technical assistance. This work was
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supported by funds from the Centre National de la Recherche Scientifique and the Universite´ Paris-Sud, and by a Ministe`re de la Recherche et de la Technologie doctoral fellowship to T.S.-S.
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