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The Ascidian Genome Contains Another T-Domain Gene That Is Expressed in Differentiating Muscle and the Tip of the Tail of the Embryo
DEVELOPMENTAL BIOLOGY 180, 773 –779 (1996) ARTICLE NO. 0345
RAPID COMMUNICATION The Ascidian Genome Contains Another T-Domain Gene That Is Expressed in Differentiating Muscle and the Tip of the Tail of the Embryo Hitoyoshi Yasuo,1 Mari Kobayashi, Yoshie Shimauchi, and Noriyuki Satoh2 Department of Zoology, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-01, Japan
The vertebrate Brachyury (T ) gene is transiently expressed in nascent and migrating mesoderm, in the differentiating notochord, and in the tail bud, reflecting its independent functions. In contrast, the expression of an ascidian Brachyury gene (As-T ) is restricted to differentiating notochord. The present study revealed that the genome of Halocynthia roretzi contains another T-domain gene (As-T2) which encodes a divergent T-domain protein. The transient expression of As-T2 was detected in the endoderm- and muscle-lineage blastomeres of the early embryo and the transcript was retained by involuting and differentiating muscle cells until it became undetectable by the mid-tailbud stage. In addition, As-T2 was expressed transiently in cells that form the tip of the newly forming tail. Interestingly, the combined pattern of spatial expression of As-T and As-T2 appears to correspond to that of a single vertebrate Brachyury gene. q 1996 Academic Press, Inc.
INTRODUCTION Among a dozen genes implicated in the formation of the chordamesoderm, Brachyury (T) is involved in notochord and posterior mesoderm formation (reviewed by Herrmann and Kispert, 1994; Herrmann, 1995; Smith et al., 1995). The cloning of mouse Brachyury was followed by isolation and characterization of its homologs in chick (Ch-T; Kispert et al., 1995a), Xenopus (Xbra; Smith et al., 1991), zebrafish (ZfT or no tail; Schulte-Merker et al., 1992, 1994), amphioxus (AmBra; Holland et al., 1995; Terazawa and Satoh, 1995), ascidian (As-T; Yasuo and Satoh, 1993, 1994), sea urchin (HpTa; Harada et al., 1995), and Drosophila (Trg; Kispert et al., 1994). The Brachyury protein acts as a transcriptional factor, which is achieved through DNA binding activity of the ‘‘T-domain,’’ a highly conserved region in the N-terminal half of T gene products (Kispert et al., 1995b; Conlon et al., 1996). The expression of vertebrate and cephalochordate 1 Present address: Developmental Biology Institute of Marseille, Campus de Luminy, Case 907, F-13288 Marseille Cedex 9, France. 2 To whom correspondence should be addressed. Fax: 81-75-7051113. E-mail: [email protected].
(amphioxus) Brachyury genes is conserved. The mouse Brachyury gene is expressed in all cells ingressing through the primitive streak. Soon after ingression, Brachyury expression becomes undetectable in the migrating mesoderm and endoderm cells, but it continues in the notochord and in the tail bud. Like mouse Brachyury, chick Brachyury, Xenopus Xbra, and zebrafish ntl are transiently expressed in nascent and migrating mesoderm and endoderm and continuously in the notochord and in the tail bud. The expression of the gene is closely associated with its functions (e.g., Wilson et al., 1995); vertebrate Brachyury is required in notochord differentiation and in posterior mesoderm formation. Ascidians (urochordates) are one of the three chordate groups. In the previous studies, we showed that the ascidian Halocynthia roretzi conserves the Brachyury (As-T) gene, that As-T is expressed exclusively in blastomeres of notochord lineage, and that the As-T transcripts become detectable immediately after the developmental fate of the blastomeres is restricted to give rise to the notochord (Yasuo and Satoh, 1993, 1994). The notochord-restricted expression of As-T appears to represent one of the two domains of vertebrate Brachyury expression and function. We therefore
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pointed out that the primary function of Brachyury is to specify embryonic cells to differentiate into notochord (Yasuo and Satoh, 1993). However, the notochord-restricted expression of As-T raises an intriguing question of how the difference in the Brachyury expression pattern between vertebrates and ascidians emerged. In this study we reexamined whether the ascidian genome contains T-domain genes other than As-T. We show here that the H. roretzi genome contains another T-domain gene, As-T2. Although As-T2 encodes a divergent T-domain protein, it is transiently expressed in differentiating muscle cells and in cells at the tip of the elongating tail.
fragments were determined by dideoxy chain termination using Sequenase version 2.0 (USB Corp.). The PCR-derived clones were random-labeled with [32 P]dCTP (Amersham). The cDNA libraries were screened using the probes under high stringency conditions (hybridization: 61 SSPE, 0.1% SDS, 11 Denhardt’s solution, 0.1 mg/ml salmon sperm DNA, 50% formamide at 427C; washing: 21 SSC, 0.1% SDS at 657C). Several positive clones were obtained. The longest cDNA clones were prepared for sequencing by controlled nested deletion from either the T3 or the T7 side and sequenced using the ABI Prism dye primer cycle sequencing kit (Perkin Elmer).
In Situ Hybridization
MATERIALS AND METHODS Ascidian Eggs and Embryos Naturally spawned eggs of H. roretzi were fertilized and then raised in filtered seawater at about 127C. Embryogenesis proceeded synchronously in various batches of eggs. The first cleavage occurred about 2 hr after insemination, and the embryo divided at about hourly intervals. They became gastrulae about 12 hr after fertilization and developed into early tailbud embryos about 24 hr after fertilization. Tadpole larvae hatched at about 40 hr of development.
PCR Amplification of Brachyury-Related Fragments, Isolation of cDNA Clones and Sequencing Amino acid sequences of T-domain of the T gene products are highly conserved among mouse (Herrmann et al., 1990), chick (Kispert et al., 1995a), Xenopus (Smith et al., 1991), zebrafish (Schulte-Merker et al., 1992), amphioxus (Holland et al., 1995; Terazawa and Satoh, 1995), ascidian (Yasuo and Satoh, 1993, 1994), sea urchin (Harada et al., 1995), and Drosophila (Kispert et al., 1994). We synthesized the sense-strand oligonucleotide [5*-TA(T/ C)(A/G)TNCA(T/C)CCNGA(T/C)TCNCC-3*] that corresponds to the amino acid sequence Y(I/V)HPDSP and the antisense oligonucleotide [5*-A(A/G)N(C/G)C(T/C)TTNGC(A/G)AANGG(A/G)TT3*] that corresponds to the amino acid sequence NPFAK(G/A)(L/F) using an automated DNA synthesizer (Applied Biosystems Inc.). We used these degenerate oligonucleotides as PCR primers. Target fragments were amplified from an H. roretzi gastrula cDNA library constructed with Lambda ZAPII (Stratagene) and an early tailbudembryo cDNA library constructed with uni-ZAPII (Stratagene). Amplification proceeded for 30 cycles of 947C (60 sec), 407C (60 sec), 727C (60 sec). PCR products were purified by gel electrophoresis and cloned into pBluescript II (SK/ ) (Stratagene). Sequences of the
Eggs and embryos at appropriate stages were fixed in 4% paraformaldehyde in 0.5 M NaCl, 0.1 M Mops buffer at 47C for 12 hr. In situ hybridization of whole mount specimens proceeded essentially as described (Miya et al., 1996). Usually, reacted embryos were dehydrated in a graded ethanol series and cleared with a 1:2 mixture (v/v) of benzyl alcohol and benzyl benzoate. Some embryos were embedded in paraffin and sectioned at 8 mm to confirm the localization of the hybridization signals.
RESULTS AND DISCUSSION Using degenerate oligonucleotide primers corresponding to the shared T-domain sequences, we amplified target fragments from cDNA libraries of H. roretzi gastrulae and tailbud embryos by means of PCR. Sequencing the amplified fragments after subcloning them into plasmid vectors revealed that the libraries contained at least two independent cDNA clones that encoded different types of T-domains. The deduced amino acid sequence of a group of clones was identical to that determined as As-T (Yasuo and Satoh, 1994; Fig. 1A). Sequence of the other group was considerably different from those of vertebrate Brachyury gene products (Fig. 1A). The corresponding ascidian gene was named AsT2. Over the amplified domain, As-T showed 79% amino acid sequence identity to the mouse T. In contrast, As-T2 showed only 48% identity to the mouse T. In addition, AsT2 appeared to be divergent even from Drosophila optomotor-blind (the identity was 51%). Northern blot analysis suggested that the As-T2 transcript was about 2.5 kb in length (data not shown). Screening 2 1 105 plasmid clones of the tailbud cDNA library with the PCR-derived As-T2 fragment yielded several positive clones. The entire sequence of the longest was determined.
FIG. 1. (A) Comparisons of predicted amino acid sequences of T-domain encoded by As-T and As-T2 with those of other T-domain genes. All the amino acid residues are shown for mouse T and only residues different from these are shown for other T-domains. Asterisks represent identical amino acid residues. Gaps are introduced for alignment with maximum similarity. (B) Nucleotide and predicted amino acid sequences of a cDNA clone for the As-T2 gene. The sequence of the cDNA encompasses 2465 bp including 18 adenylyl residues. The ATG at the position 142– 144 represents the putative start codon of the As-T2-encoded protein. An asterisk indicates the termination codon. The predicted As-T2 protein consists of 681 amino acids. The T-domain is underlined. The nucleotide sequence data of As-T2 reported in this paper will appear in the DDBJ, EMBL, and GenBank Nucleotide Sequence Databases with the Accession No. D83265.
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As shown in Fig. 1B, the insert consisted of 2465 bp including 18 adenylyl residues. It contained a single long, open reading frame of 2043 nucleotides that encode a polypeptide consisting of 681 amino acids. Most of the Brachyury genes encode polypeptides of about 440– 480 amino acids (Herrmann and Kispert, 1994; Holland et al., 1995; Yasuo and Satoh, 1994; Harada et al., 1995). Therefore, the As-T2 appeared to be divergent from the other Brachyury gene products on the basis not only of its amino acid sequence, but also of its length. Genomic Southern analysis also suggested that As-T and As-T2 are independent and exist as a single copy within the H. roretzi genome (data not shown). The molecular phylogenetic analysis using confidently aligned sites of 149 amino acids of the T-domain suggested that the As-T is an ortholog of the mouse Brachyury while As-T2 appeared to be divergent from any other T-domain genes (data not shown). In ascidians, every blastomere of early embryos is distinguishable and the lineage of the embryonic cells is completely known up to the early gastrula stage (Nishida, 1987). Therefore, we determined that As-T is expressed exclusively in blastomeres of notochord lineage and that the timing of As-T expression coincides with that of developmental fate restriction of the blastomeres (Yasuo and Satoh, 1993, 1994). In situ hybridization of whole-mount specimens revealed that As-T2 is expressed first in cells of endoderm and muscle lineages and later in cells located at the tip of the tail. No hybridization signals were found in differentiating notochord cells. Hybridization signals were undetectable above the background level in unfertilized and fertilized eggs, and early embryos up to the 16-cell stage (data not shown). Signals were first detected in a pair of cells in the vegetal hemisphere of the 32-cell stage embryos (Figs. 2A and 2E). They were cells of a B6.1 pair (right B6.1 and left B6.1 of the bilaterally symmetrical embryo) and their developmental fate is endoderm and endodermal strand. At the 44-cell stage, the B6.1 pair divides into B7.1 and B7.2 pairs, and signals were detected in these pairs (Figs. 2B and 2F). However, as development proceeded, the As-T2 signals in cells of the endoderm lineage became less intense (Figs. 2C, 2D, 2G, and 2H) and undetectable by the neurula stage (Fig. 2I). In contrast, hybridization signals in cells of muscle lineage became intense as embryogenesis proceeded. Some of the 32-cell stage embryos showed a very weak signal in the B6.2 pair, which gives rise to muscle, mesenchyme, and notochord cells. A weak but distinct signal was evident in
B7.4 and B6.4 pairs of muscle lineage in the 44-cell stage embryo (Figs. 2B and 2F). At the 64-cell stage, B6.4 divides into B7.7 and B7.8, the former being of mesenchyme lineage and the latter being of muscle lineage (Fig. 2C). Signals were detectable in mesenchymal B7.7 at this stage but soon became undetectable (Fig. 2G). During gastrulation, signals in cells of muscle lineage became intense. The As-T2-expressing muscle cells moved inside the embryo and increased in number (Figs. 2H – 2J and 2L– 2O). At the neurula stage, more than 10 primordial muscle cells on each side of the embryo showed intense signals (Fig. 2I). In early tailbud embryos, signals were evident in differentiating muscle cells located at the right and left sides of the notochord in the tail (Fig. 2J). However, as the tail elongated, the signals became less intense and finally undetectable by the late tailbud stage (data not shown). As shown in Fig. 2J, As-T2 transcripts were evident in a few cells at the tip of the tail of the early tailbud embryo. An examination of histological sections revealed that the hybridization signals were distributed in cells of the epidermis (Fig. 2K). The distribution of As-T2 transcripts in this region was traced back to the midgastrula stage. At this stage, hybridization signals were evident in three pairs of epidermal lineage at the posterior animal region of the embryo (Fig. 2M). These pairs may be b9.41, b9.49, and b9.53. During gastrulation, these cells moved from both sides to the midline of the embryo at the posterior tip (Figs. 2N and 2O). At the neurula stage, a few cells of the posterior tip showed distinct signals (Fig. 2I). During tail elongation, the cells at the tip of the tail retained the signals (Figs. 2J and 2K). In Xenopus, the ‘‘tail’’ is formed as a continuation of gastrulation (Gont et al., 1993). This appears to be true in the mouse (Wilson et al., 1995). It is generally agreed that the tail bud of amniotes is an undifferentiated blastema, that directly gives rise to all tissues of the tail (Griffith et al., 1992). However, development of the ascidian larval tail proceeds without a structure like the vertebrate tail bud. As-T2-expressing cells at the tip of tail are epidermal and they never differentiate into muscle or notochord (Fig. 2K; reviewed by Satoh, 1994). Thus, there appears to be no direct relationship between these cells and the vertebrate tail bud. The As-T2-expressing cells at the tip of the tail elongate along apical – basal polarity at the larval stage and form the tail fin (data not shown). This appears to be the case in amphioxus (Conklin, 1932). Therefore, the role of As-T2 in these epidermal cells might relate to a change in cell shape.
FIG. 2. Spatial localization of As-T2 transcripts in ascidian embryos. (A– D) Diagrams of blastomeres expressing As-T2 relative to the lineage of endoderm (marked by yellow) and muscle (marked by orange). Vegetal hemispheres of (A) 32-cell, (B) 44-cell, (C) 64-cell, and (D) 110-cell embryos. The nomenclature of blastomeres is based on Conklin’s description (1905) and blastomeres of only the left half of the embryos are shown because the embryo is bilaterally symmetrical. (E – J) Localization of As-T2 transcripts in (E) 32-cell, (F) 44-cell, (G) 64-cell, (H) and 110-cell embryos, (I) neurula, and (J) early tailbud embryo. (E) A 32-cell embryo viewed from the vegetal side, showing distinct hybridization signals around the nuclei of a pair of presumptive endoderm cells (B6.1). (F) At the 44-cell and (G) 64-cell stages, distinct signals were found in two pairs of endoderm cells (B7.1 and B7.2), while weak signals are seen in two or three pairs of muscle
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lineage cells (B7.4 and B6.4 at the 44-cell stage; B7.4, B7.7 and B7.8 at the 64-cell stage). (H) At the 110-cell stage, intense signals are seen in cells of muscle lineage while signals in the cells of endoderm lineage are weak at this stage. (I) In a neurula and (J) tailbud embryoviewed from the dorsal side (anterior is to the right), distinct signals are evident in muscle cells (Mu) and in cells of the tip of the tail (arrow). (K) A section of an early tailbud embryo showing that the cells with signals in the tip of tail are epidermal (arrow). (L –O) Localization of As-T2 transcripts during gastrulation; (L) an early gastrula, dorsal view, (M) a midgastrula, posterior pole view, (N) late gastrula, dorsal view, and (O) neural-plate stage embryo, dorsal view. These samples were not rendered transparent to show the localization of As-T2 transcripts in cells that give rise to caudal tip cells (arrows). Scale bar, 50 mm.
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As revealed in this study, the H. roretzi genome contains at least two Brachyury genes, As-T and As-T2. Although AsT2 encodes a divergent T-domain protein, it is transiently expressed in differentiating muscle cells and in cells at the tip of the elongating tail. Interestingly, the combined pattern of spatial expression of As-T and As-T2 appears to correspond to that of a single vertebrate Brachyury gene. However, it seems unlikely that two independent, differentially expressed genes (As-T and As-T2) joined to become one gene with the different functions of vertebrate Brachyury (e.g., Rennebeck et al., 1995). We suspect that vertebrates also contain a few T-domain genes other than Brachyury. The T-domain itself is not unique to Brachyury, but shared with optomotor-blind (omb) of Drosophila (Pflugfelder et al., 1992). In addition, recent clonings of genes containing the T-domain other than Brachyury from the mouse (Bollag et al., 1994; Bulfone et al., 1995), chicken (Gibson-Brown et al., 1996), and nematodes (Wilson et al., 1994) suggest a novel gene family of T-related genes. Further cloning might disclose the presence of Brachyury and other T-domain genes with various expression patterns and functions.
ACKNOWLEDGMENTS This research was supported by a Grant-in Aid for Specially Promoted Research (No. 07102012) from the Ministry of Education, Science, Sports and Culture of Japan to N.S. H.Y. and M.K. were supported by a predoctoral fellowship from the Japan Society for the Promotion of Science for Japanese Junior Scientists with Research Grants 3082 and 4585, respectively.
REFERENCES Bollag, R. C., Siegfried, Z., Cebra-Thomas, J. A., Garvay, N., Davison, E. M., and Silver, L. M. (1994). An ancient family of embryonically expressed mouse genes sharing a conserved protein motif with the T locus. Nature Genet. 7, 383 –389. Bulfone, A., Smiga, S. M., Shimamura, K., Peterson, A., Puelles, L., and Rubenstein, J. L. R. (1995). T-Brain-1: A homolog of Brachyury whose expression defines molecularly distinct domains within the cerebral cortex. Neuron 15, 63– 78. Conklin, E. G. (1905). Organization and cell-lineage of the ascidian egg. J. Acad. Nat. Sci. (Philadelphia) 13, 1 –119. Conklin, E. G. (1932). The embryology of amphioxus. J. Morphol. 54, 69– 151. Conlon, F. L., Sedgwick, S. G., Weston, K. M., and Smith, J. C. (1996). Inhibition of Xbra transcription activation causes defects in mesodermal patterning and reveals autoregulation of Xbra in dorsal mesoderm. Development 122, 2427 – 2435. Gibson-Brown, J. J., Agulnik, S. I., Chapman, D. L., Alexiou, M., Garvey, N., Silver, L. M., and Papaioannou, V. E. (1996). Evidence of a role for T-box genes in the evolution of limb morphogenesis and the specification of forelimb/hindlimb identity. Mech. Dev. 56, 93– 101. Gont, L. K., Steinbeisser, H., Blumberg, B., and De Robertis, E. M. (1993). Tail formation as a continuation of gastrulation: The mul-
tiple cell populations of the Xenopus tailbud derive from the late blastopore lip. Development 119, 991– 1004. Griffith, C. M., Wiley, M. J., and Sanders, E. J. (1992). The vertebrate tail bud: three germ layers from one tissue. Anat. Embryol. 185, 101–113. Harada, Y., Yasuo, H., and Satoh, N. (1995). A sea urchin homologue of the chordate Brachyury (T) gene is expressed in the secondary mesenchyme founder cells. Development 121, 2747 – 2754. Herrmann, B. G. (1995). The mouse Brachyury (T) gene. Semin. Dev. Biol. 6, 385– 394. Herrmann, B. G., Labeit, S., Poustka, A., King, T. R., and Lehrach, H. (1990). Cloning of the T gene required in mesoderm formation in the mouse. Nature 343, 617–622. Herrmann, B. G., and Kispert, A. (1994). The T genes in embryogenesis. Trends Genet. 10, 280– 286. Holland, P. W. H., Koschorz, B., Holland, L. Z., and Herrmann, B. G. (1995). Conservation of Brachyury(T) genes in amphioxus and vertebrates: Developmental and evolutionary implications. Development 121, 4283 – 4291. Kispert, A., Herrmann, B. G., Leptin, M., and Reuter, R. (1994). Homologs of the mouse Brachyury gene are involved in the specification of posterior terminal structures in Drosophila, Tribolium, and Locusta. Genes Dev. 8, 2137 –2150. Kispert, A., Ortner, H., Cooke, J., and Herrmann, B. G. (1995a). The chick Brachyury gene: Developmental expression pattern and response to axial induction by localized activin. Dev. Biol. 168, 406–415. Kispert, A., Koschorz, B., and Herrmann, B. G. (1995b). The T protein encoded by Brachyury is a tissue-specific transcription factor. EMBO J. 14, 4763 – 4772. Miya, T., Morita, K., Ueno, N., and Satoh, N. (1996). An ascidian homologue of vertebrate BMPs-5-8 is expressed in the midline of the anterior neuroectoderm and in the midline of the ventral epidermis of the embryo. Mech. Dev. 57, 181–190. Nishida, H. (1987). Cell lineage analysis in ascidian embryos by intracellular injection of a tracer enzyme. III. Up to the tissue restricted stage. Dev. Biol. 121, 526– 541. Pflugfelder, G. O., Roth, H., and Poeck, B. (1992). A homology domain shared between Drosophila optomotor-blind and mouse Brachyury is involved in DNA binding. Biochem. Biophys. Res. Commun. 186, 918–925. Rennebeck, G. M., Lader, E., Chen, Q., Bohm, R. A., Cai, Z. S., Faust, C., Magnuson, T., Pease, L. R., and Artzt, K. (1995). Is there a Brachyury the second? Analysis of a transgenic mutation involved in notochord maintenance in mice. Dev. Biol. 172, 206– 217. Satoh, N. (1994). ‘‘Developmental Biology of Ascidians,’’ Cambridge Univ. Press, New York. Schulte-Merker, S., Ho, R. K., Herrmann, B. G., and Nu¨sslein-Volhard, C. (1992). The protein product of the zebrafish homologue of the mouse T gene is expressed in nuclei of the germ ring and the notochord of the early embryo. Development 116, 1021 – 1032. Schulte-Merker, S., van Eeden, F. J. M., Halpern, M. E., Kimmel, C. B., and Nu¨sslein-Volhard, C. (1994). no tail (ntl) is the zebrafish homologue of the mouse T (Brachyury) gene. Development 120, 1009 – 1015. Smith, J. C., Price, B. M. J., Green, J. B. A., Weigel, D., and Herrmann, B. G. (1991). Expression of a Xenopus homolog of Brachyury (T) is an immediate-early response to mesoderm induction. Cell 67, 79– 87.
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Smith, J. C., Cunliffe, V., O’Reilly, M-A. J., Schulte-Merker, S., and Umbhauer, M. (1995). Xenopus Brachyury. Semin. Dev. Biol. 6, 405–410. Terazawa, K., and Satoh, N. (1995). Spatial expression of the amphioxus Brachyury (T) gene during early embryogenesis of Branchiostoma belcheri. Dev. Growth Differ. 37, 395– 401. Wilson, R., Ainscough, R., Anderson, K., et al. (1994). 2.2 Mb of contiguous nucleotide sequence from chromosome III of C. elegans. Nature 368, 32– 38. Wilson, V., Manson, L., Skarnes, W. C., and Beddington, R. S. P.
(1995). The T gene is necessary for normal mesodermal morphogenetic cell movements during gastrulation. Development 121, 877–886. Yasuo, H., and Satoh, N. (1993). Function of vertebrate T gene. Nature 364, 582–583. Yasuo, H., and Satoh, N. (1994). An ascidian homolog of the mouse Brachyury (T) gene is expressed exclusively in notochord cells at the fate restricted stage. Dev. Growth Differ. 36, 9– 18. Received for publication June 17, 1996 Accepted October 1, 1996
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RAPID COMMUNICATION The Ascidian Genome Contains Another T-Domain Gene That Is Expressed in Differentiating Muscle and the Tip of the Tail of the Embryo Hitoyoshi Yasuo,1 Mari Kobayashi, Yoshie Shimauchi, and Noriyuki Satoh2 Department of Zoology, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-01, Japan
The vertebrate Brachyury (T ) gene is transiently expressed in nascent and migrating mesoderm, in the differentiating notochord, and in the tail bud, reflecting its independent functions. In contrast, the expression of an ascidian Brachyury gene (As-T ) is restricted to differentiating notochord. The present study revealed that the genome of Halocynthia roretzi contains another T-domain gene (As-T2) which encodes a divergent T-domain protein. The transient expression of As-T2 was detected in the endoderm- and muscle-lineage blastomeres of the early embryo and the transcript was retained by involuting and differentiating muscle cells until it became undetectable by the mid-tailbud stage. In addition, As-T2 was expressed transiently in cells that form the tip of the newly forming tail. Interestingly, the combined pattern of spatial expression of As-T and As-T2 appears to correspond to that of a single vertebrate Brachyury gene. q 1996 Academic Press, Inc.
INTRODUCTION Among a dozen genes implicated in the formation of the chordamesoderm, Brachyury (T) is involved in notochord and posterior mesoderm formation (reviewed by Herrmann and Kispert, 1994; Herrmann, 1995; Smith et al., 1995). The cloning of mouse Brachyury was followed by isolation and characterization of its homologs in chick (Ch-T; Kispert et al., 1995a), Xenopus (Xbra; Smith et al., 1991), zebrafish (ZfT or no tail; Schulte-Merker et al., 1992, 1994), amphioxus (AmBra; Holland et al., 1995; Terazawa and Satoh, 1995), ascidian (As-T; Yasuo and Satoh, 1993, 1994), sea urchin (HpTa; Harada et al., 1995), and Drosophila (Trg; Kispert et al., 1994). The Brachyury protein acts as a transcriptional factor, which is achieved through DNA binding activity of the ‘‘T-domain,’’ a highly conserved region in the N-terminal half of T gene products (Kispert et al., 1995b; Conlon et al., 1996). The expression of vertebrate and cephalochordate 1 Present address: Developmental Biology Institute of Marseille, Campus de Luminy, Case 907, F-13288 Marseille Cedex 9, France. 2 To whom correspondence should be addressed. Fax: 81-75-7051113. E-mail: [email protected].
(amphioxus) Brachyury genes is conserved. The mouse Brachyury gene is expressed in all cells ingressing through the primitive streak. Soon after ingression, Brachyury expression becomes undetectable in the migrating mesoderm and endoderm cells, but it continues in the notochord and in the tail bud. Like mouse Brachyury, chick Brachyury, Xenopus Xbra, and zebrafish ntl are transiently expressed in nascent and migrating mesoderm and endoderm and continuously in the notochord and in the tail bud. The expression of the gene is closely associated with its functions (e.g., Wilson et al., 1995); vertebrate Brachyury is required in notochord differentiation and in posterior mesoderm formation. Ascidians (urochordates) are one of the three chordate groups. In the previous studies, we showed that the ascidian Halocynthia roretzi conserves the Brachyury (As-T) gene, that As-T is expressed exclusively in blastomeres of notochord lineage, and that the As-T transcripts become detectable immediately after the developmental fate of the blastomeres is restricted to give rise to the notochord (Yasuo and Satoh, 1993, 1994). The notochord-restricted expression of As-T appears to represent one of the two domains of vertebrate Brachyury expression and function. We therefore
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pointed out that the primary function of Brachyury is to specify embryonic cells to differentiate into notochord (Yasuo and Satoh, 1993). However, the notochord-restricted expression of As-T raises an intriguing question of how the difference in the Brachyury expression pattern between vertebrates and ascidians emerged. In this study we reexamined whether the ascidian genome contains T-domain genes other than As-T. We show here that the H. roretzi genome contains another T-domain gene, As-T2. Although As-T2 encodes a divergent T-domain protein, it is transiently expressed in differentiating muscle cells and in cells at the tip of the elongating tail.
fragments were determined by dideoxy chain termination using Sequenase version 2.0 (USB Corp.). The PCR-derived clones were random-labeled with [32 P]dCTP (Amersham). The cDNA libraries were screened using the probes under high stringency conditions (hybridization: 61 SSPE, 0.1% SDS, 11 Denhardt’s solution, 0.1 mg/ml salmon sperm DNA, 50% formamide at 427C; washing: 21 SSC, 0.1% SDS at 657C). Several positive clones were obtained. The longest cDNA clones were prepared for sequencing by controlled nested deletion from either the T3 or the T7 side and sequenced using the ABI Prism dye primer cycle sequencing kit (Perkin Elmer).
In Situ Hybridization
MATERIALS AND METHODS Ascidian Eggs and Embryos Naturally spawned eggs of H. roretzi were fertilized and then raised in filtered seawater at about 127C. Embryogenesis proceeded synchronously in various batches of eggs. The first cleavage occurred about 2 hr after insemination, and the embryo divided at about hourly intervals. They became gastrulae about 12 hr after fertilization and developed into early tailbud embryos about 24 hr after fertilization. Tadpole larvae hatched at about 40 hr of development.
PCR Amplification of Brachyury-Related Fragments, Isolation of cDNA Clones and Sequencing Amino acid sequences of T-domain of the T gene products are highly conserved among mouse (Herrmann et al., 1990), chick (Kispert et al., 1995a), Xenopus (Smith et al., 1991), zebrafish (Schulte-Merker et al., 1992), amphioxus (Holland et al., 1995; Terazawa and Satoh, 1995), ascidian (Yasuo and Satoh, 1993, 1994), sea urchin (Harada et al., 1995), and Drosophila (Kispert et al., 1994). We synthesized the sense-strand oligonucleotide [5*-TA(T/ C)(A/G)TNCA(T/C)CCNGA(T/C)TCNCC-3*] that corresponds to the amino acid sequence Y(I/V)HPDSP and the antisense oligonucleotide [5*-A(A/G)N(C/G)C(T/C)TTNGC(A/G)AANGG(A/G)TT3*] that corresponds to the amino acid sequence NPFAK(G/A)(L/F) using an automated DNA synthesizer (Applied Biosystems Inc.). We used these degenerate oligonucleotides as PCR primers. Target fragments were amplified from an H. roretzi gastrula cDNA library constructed with Lambda ZAPII (Stratagene) and an early tailbudembryo cDNA library constructed with uni-ZAPII (Stratagene). Amplification proceeded for 30 cycles of 947C (60 sec), 407C (60 sec), 727C (60 sec). PCR products were purified by gel electrophoresis and cloned into pBluescript II (SK/ ) (Stratagene). Sequences of the
Eggs and embryos at appropriate stages were fixed in 4% paraformaldehyde in 0.5 M NaCl, 0.1 M Mops buffer at 47C for 12 hr. In situ hybridization of whole mount specimens proceeded essentially as described (Miya et al., 1996). Usually, reacted embryos were dehydrated in a graded ethanol series and cleared with a 1:2 mixture (v/v) of benzyl alcohol and benzyl benzoate. Some embryos were embedded in paraffin and sectioned at 8 mm to confirm the localization of the hybridization signals.
RESULTS AND DISCUSSION Using degenerate oligonucleotide primers corresponding to the shared T-domain sequences, we amplified target fragments from cDNA libraries of H. roretzi gastrulae and tailbud embryos by means of PCR. Sequencing the amplified fragments after subcloning them into plasmid vectors revealed that the libraries contained at least two independent cDNA clones that encoded different types of T-domains. The deduced amino acid sequence of a group of clones was identical to that determined as As-T (Yasuo and Satoh, 1994; Fig. 1A). Sequence of the other group was considerably different from those of vertebrate Brachyury gene products (Fig. 1A). The corresponding ascidian gene was named AsT2. Over the amplified domain, As-T showed 79% amino acid sequence identity to the mouse T. In contrast, As-T2 showed only 48% identity to the mouse T. In addition, AsT2 appeared to be divergent even from Drosophila optomotor-blind (the identity was 51%). Northern blot analysis suggested that the As-T2 transcript was about 2.5 kb in length (data not shown). Screening 2 1 105 plasmid clones of the tailbud cDNA library with the PCR-derived As-T2 fragment yielded several positive clones. The entire sequence of the longest was determined.
FIG. 1. (A) Comparisons of predicted amino acid sequences of T-domain encoded by As-T and As-T2 with those of other T-domain genes. All the amino acid residues are shown for mouse T and only residues different from these are shown for other T-domains. Asterisks represent identical amino acid residues. Gaps are introduced for alignment with maximum similarity. (B) Nucleotide and predicted amino acid sequences of a cDNA clone for the As-T2 gene. The sequence of the cDNA encompasses 2465 bp including 18 adenylyl residues. The ATG at the position 142– 144 represents the putative start codon of the As-T2-encoded protein. An asterisk indicates the termination codon. The predicted As-T2 protein consists of 681 amino acids. The T-domain is underlined. The nucleotide sequence data of As-T2 reported in this paper will appear in the DDBJ, EMBL, and GenBank Nucleotide Sequence Databases with the Accession No. D83265.
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As shown in Fig. 1B, the insert consisted of 2465 bp including 18 adenylyl residues. It contained a single long, open reading frame of 2043 nucleotides that encode a polypeptide consisting of 681 amino acids. Most of the Brachyury genes encode polypeptides of about 440– 480 amino acids (Herrmann and Kispert, 1994; Holland et al., 1995; Yasuo and Satoh, 1994; Harada et al., 1995). Therefore, the As-T2 appeared to be divergent from the other Brachyury gene products on the basis not only of its amino acid sequence, but also of its length. Genomic Southern analysis also suggested that As-T and As-T2 are independent and exist as a single copy within the H. roretzi genome (data not shown). The molecular phylogenetic analysis using confidently aligned sites of 149 amino acids of the T-domain suggested that the As-T is an ortholog of the mouse Brachyury while As-T2 appeared to be divergent from any other T-domain genes (data not shown). In ascidians, every blastomere of early embryos is distinguishable and the lineage of the embryonic cells is completely known up to the early gastrula stage (Nishida, 1987). Therefore, we determined that As-T is expressed exclusively in blastomeres of notochord lineage and that the timing of As-T expression coincides with that of developmental fate restriction of the blastomeres (Yasuo and Satoh, 1993, 1994). In situ hybridization of whole-mount specimens revealed that As-T2 is expressed first in cells of endoderm and muscle lineages and later in cells located at the tip of the tail. No hybridization signals were found in differentiating notochord cells. Hybridization signals were undetectable above the background level in unfertilized and fertilized eggs, and early embryos up to the 16-cell stage (data not shown). Signals were first detected in a pair of cells in the vegetal hemisphere of the 32-cell stage embryos (Figs. 2A and 2E). They were cells of a B6.1 pair (right B6.1 and left B6.1 of the bilaterally symmetrical embryo) and their developmental fate is endoderm and endodermal strand. At the 44-cell stage, the B6.1 pair divides into B7.1 and B7.2 pairs, and signals were detected in these pairs (Figs. 2B and 2F). However, as development proceeded, the As-T2 signals in cells of the endoderm lineage became less intense (Figs. 2C, 2D, 2G, and 2H) and undetectable by the neurula stage (Fig. 2I). In contrast, hybridization signals in cells of muscle lineage became intense as embryogenesis proceeded. Some of the 32-cell stage embryos showed a very weak signal in the B6.2 pair, which gives rise to muscle, mesenchyme, and notochord cells. A weak but distinct signal was evident in
B7.4 and B6.4 pairs of muscle lineage in the 44-cell stage embryo (Figs. 2B and 2F). At the 64-cell stage, B6.4 divides into B7.7 and B7.8, the former being of mesenchyme lineage and the latter being of muscle lineage (Fig. 2C). Signals were detectable in mesenchymal B7.7 at this stage but soon became undetectable (Fig. 2G). During gastrulation, signals in cells of muscle lineage became intense. The As-T2-expressing muscle cells moved inside the embryo and increased in number (Figs. 2H – 2J and 2L– 2O). At the neurula stage, more than 10 primordial muscle cells on each side of the embryo showed intense signals (Fig. 2I). In early tailbud embryos, signals were evident in differentiating muscle cells located at the right and left sides of the notochord in the tail (Fig. 2J). However, as the tail elongated, the signals became less intense and finally undetectable by the late tailbud stage (data not shown). As shown in Fig. 2J, As-T2 transcripts were evident in a few cells at the tip of the tail of the early tailbud embryo. An examination of histological sections revealed that the hybridization signals were distributed in cells of the epidermis (Fig. 2K). The distribution of As-T2 transcripts in this region was traced back to the midgastrula stage. At this stage, hybridization signals were evident in three pairs of epidermal lineage at the posterior animal region of the embryo (Fig. 2M). These pairs may be b9.41, b9.49, and b9.53. During gastrulation, these cells moved from both sides to the midline of the embryo at the posterior tip (Figs. 2N and 2O). At the neurula stage, a few cells of the posterior tip showed distinct signals (Fig. 2I). During tail elongation, the cells at the tip of the tail retained the signals (Figs. 2J and 2K). In Xenopus, the ‘‘tail’’ is formed as a continuation of gastrulation (Gont et al., 1993). This appears to be true in the mouse (Wilson et al., 1995). It is generally agreed that the tail bud of amniotes is an undifferentiated blastema, that directly gives rise to all tissues of the tail (Griffith et al., 1992). However, development of the ascidian larval tail proceeds without a structure like the vertebrate tail bud. As-T2-expressing cells at the tip of tail are epidermal and they never differentiate into muscle or notochord (Fig. 2K; reviewed by Satoh, 1994). Thus, there appears to be no direct relationship between these cells and the vertebrate tail bud. The As-T2-expressing cells at the tip of the tail elongate along apical – basal polarity at the larval stage and form the tail fin (data not shown). This appears to be the case in amphioxus (Conklin, 1932). Therefore, the role of As-T2 in these epidermal cells might relate to a change in cell shape.
FIG. 2. Spatial localization of As-T2 transcripts in ascidian embryos. (A– D) Diagrams of blastomeres expressing As-T2 relative to the lineage of endoderm (marked by yellow) and muscle (marked by orange). Vegetal hemispheres of (A) 32-cell, (B) 44-cell, (C) 64-cell, and (D) 110-cell embryos. The nomenclature of blastomeres is based on Conklin’s description (1905) and blastomeres of only the left half of the embryos are shown because the embryo is bilaterally symmetrical. (E – J) Localization of As-T2 transcripts in (E) 32-cell, (F) 44-cell, (G) 64-cell, (H) and 110-cell embryos, (I) neurula, and (J) early tailbud embryo. (E) A 32-cell embryo viewed from the vegetal side, showing distinct hybridization signals around the nuclei of a pair of presumptive endoderm cells (B6.1). (F) At the 44-cell and (G) 64-cell stages, distinct signals were found in two pairs of endoderm cells (B7.1 and B7.2), while weak signals are seen in two or three pairs of muscle
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lineage cells (B7.4 and B6.4 at the 44-cell stage; B7.4, B7.7 and B7.8 at the 64-cell stage). (H) At the 110-cell stage, intense signals are seen in cells of muscle lineage while signals in the cells of endoderm lineage are weak at this stage. (I) In a neurula and (J) tailbud embryoviewed from the dorsal side (anterior is to the right), distinct signals are evident in muscle cells (Mu) and in cells of the tip of the tail (arrow). (K) A section of an early tailbud embryo showing that the cells with signals in the tip of tail are epidermal (arrow). (L –O) Localization of As-T2 transcripts during gastrulation; (L) an early gastrula, dorsal view, (M) a midgastrula, posterior pole view, (N) late gastrula, dorsal view, and (O) neural-plate stage embryo, dorsal view. These samples were not rendered transparent to show the localization of As-T2 transcripts in cells that give rise to caudal tip cells (arrows). Scale bar, 50 mm.
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As revealed in this study, the H. roretzi genome contains at least two Brachyury genes, As-T and As-T2. Although AsT2 encodes a divergent T-domain protein, it is transiently expressed in differentiating muscle cells and in cells at the tip of the elongating tail. Interestingly, the combined pattern of spatial expression of As-T and As-T2 appears to correspond to that of a single vertebrate Brachyury gene. However, it seems unlikely that two independent, differentially expressed genes (As-T and As-T2) joined to become one gene with the different functions of vertebrate Brachyury (e.g., Rennebeck et al., 1995). We suspect that vertebrates also contain a few T-domain genes other than Brachyury. The T-domain itself is not unique to Brachyury, but shared with optomotor-blind (omb) of Drosophila (Pflugfelder et al., 1992). In addition, recent clonings of genes containing the T-domain other than Brachyury from the mouse (Bollag et al., 1994; Bulfone et al., 1995), chicken (Gibson-Brown et al., 1996), and nematodes (Wilson et al., 1994) suggest a novel gene family of T-related genes. Further cloning might disclose the presence of Brachyury and other T-domain genes with various expression patterns and functions.
ACKNOWLEDGMENTS This research was supported by a Grant-in Aid for Specially Promoted Research (No. 07102012) from the Ministry of Education, Science, Sports and Culture of Japan to N.S. H.Y. and M.K. were supported by a predoctoral fellowship from the Japan Society for the Promotion of Science for Japanese Junior Scientists with Research Grants 3082 and 4585, respectively.
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(1995). The T gene is necessary for normal mesodermal morphogenetic cell movements during gastrulation. Development 121, 877–886. Yasuo, H., and Satoh, N. (1993). Function of vertebrate T gene. Nature 364, 582–583. Yasuo, H., and Satoh, N. (1994). An ascidian homolog of the mouse Brachyury (T) gene is expressed exclusively in notochord cells at the fate restricted stage. Dev. Growth Differ. 36, 9– 18. Received for publication June 17, 1996 Accepted October 1, 1996
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