Department of Zoology, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
Present address: Department of Biological Science, Graduate School of Science, Osaka University, Toyonaka 560-0043, Japan
The first two authors contributed equally to this work
*Author for correspondence (e-mail: satoh{at}ascidian.zool.kyoto-u.ac.jp)
Accepted May 1, 2001
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Ascidians, Cell-cell interactions, FGF signals, FGF receptor, Notochord, Mesenchyme, Nervous system, Halocynthia roretzi
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
During embryogenesis of ascidians that produce tadpole larvae, a notochord consisting of exactly 40 cells develops within a muscular tail (Satoh, 1994). Developmental processes required for notochord formation have been investigated in detail. First, the entire cell lineage responsible for generating the 40 notochord cells has been completely documented (Conklin, 1905; Nishida, 1987); the anterior 32 cells are derived from a pair of right and left A4.1 (anterior and vegetal) cells of the bilaterally symmetrical 8-cell embryo, while the posterior 8 cells are descendants of the pair of B4.1 (posterior and vegetal) cells. At the 32-cell stage, A-line endodermal cells (A6.1 and A6.3) produce a signal(s) that induces the neighboring presumptive notochord cells (A6.2 and A6.4) to be specified along the notochordal pathway (Nakatani and Nishida, 1994). At the 64-cell stage, the developmental fates of A7.3 and A7.7 are restricted to give rise to notochord cells only. Simultaneously, the Brachyury gene is activated (Yasuo and Satoh, 1993; Corbo et al., 1997), which controls downstream genes that are required for the formation of notochord (Yasuo and Satoh, 1998; Takahashi et al., 1999a; Hotta et al., 2000). The developmental fate of the B-line notochord cell B8.6 is restricted at the 110-cell stage, and then the cell expresses the Brachyury gene (Yasuo and Satoh, 1993).
One of the most important steps involved in the notochord formation in ascidian embryos is how the endoderm and the presumptive notochord cells communicate with each other so that the latter becomes specified to give rise to differentiated notochord cells. It has been shown that bFGF, but not activin A, mimics the induction event at the 32-cell stage (Nakatani et al., 1996). A6.2 and A6.4 cells isolated at the early phase of the 32-cell stage were immersed in seawater containing human recombinant bFGF, which resulted in the activation of Brachyury and subsequent differentiation of notochord cells as assessed using cell-specific antibody and changes in cell morphology. In vertebrates, it has been shown that the signal cascade triggered by bFGF binding includes the Ras pathway (Satoh et al., 1992; Pawson, 1995). Extending this previous study, Nakatani and Nishida (Nakatani and Nishida, 1997) showed that the injection of a dominant-negative form of Ras, to cause the functional inhibition of endogenous Ras, inhibited the A-line notochord cell differentiation. The mechanism for the B-line notochord cell differentiation, however, remains to be elucidated.
In addition, a recent study by Kim et al. (Kim et al., 2000) demonstrated that the differentiation of mesenchyme cells in H. roretzi embryos also requires inducing signals from endodermal cells at the 32-cell stage and that bFGF but not activin A can induce the differentiation of mesenchyme cells. Furthermore, they showed that the differentiation pathway responsible for the development of mesenchyme or notochord cells depends on whether the precursor cells contain the posterior-vegetal cytoplasm (PVC) of the eggs. PVC is present in mesenchyme precursor cells whereas notochord precursor cells do not contain PVC.
Although these experiments strongly suggest an involvement of FGF-like signals in the differentiation of notochord and mesenchyme cells in Halocynthia embryos, the reagents used in these studies were from human, not ascidian, sources. The present study was undertaken to overcome this problem and obtain direct evidence of whether or not FGF signals are involved in ascidian cellular interactions. Here, we report the isolation of a cDNA clone for a gene encoding FGF receptor (HrFGFR) from Halocynthia embryos, and an examination of the function of HrFGFR by overexpression of dominant-negative form of this receptor.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Isolation and characterization of cDNA clones for an ascidian FGF receptor gene
Polymerase chain reaction (PCR) was used to isolate fragments of HrFGFR cDNAs. Degenerate primers were designed to cover the tyrosine kinase domain, which shows the highest conservation among FGFR family members; forward primer, 5'GGNGARGGNTG-YTTYGGNCA3' and reverse primer, 5'GCYTCNGGNGCCATCCAYTT3'.
The targeted template was the reverse transcription product of an H. roretzi 110-cell stage embryo (Imai et al., 2000). Several of the cDNA fragments that were obtained were sequenced after subcloning them into pBluescript SK+ (Stratagene). One of them was found to be 592 bp, which encoded a polypeptide corresponding to part of the tyrosine kinase domain. This fragment was used to screen a 3.5x105 pfu plate containing an H. roretzi gastrula cDNA library (Shimauchi et al., 1997). Nearly 100 positive clones were obtained. Nucleotide sequences for 7 of these clones showed that they encoded an identical protein. The longest clones (about 3.2 kb) was completely sequenced. Nucleotide sequences were determined for both strands with a Big-Dye Terminator Cycle Sequencing Ready Reaction kit and ABI PRISM 377 DNA sequencer (Perkin Elmer).
Genomic Southern blotting
High-molecular mass genomic DNA was extracted from the gonad of a single specimen by the standard procedure (Sambrook et al., 1989). After digestion with EcoRI, HindIII or PstI, the DNA fragments were subjected to 0.7% agarose gel electrophoresis and transferred to a Hybond-N+ nylon membranes (Amersham). The membranes were hybridized with DNA probes at 42°C for 17 hours and then washed under high-stringency conditions (twice in 2xSSC, 0.1% SDS at 50°C). The DNA probes were excised by BamHI/BglII (nucleotide position 1314-2076, the C-terminal domain probe) or EcoRI/HindIII (nucleotide position 710-884, the Ig3 domain probe) of HrFGFR gene and labeled by [32P]dCTP using a random primed labeling kit (Roche).
RNA extraction and northern blotting
Total RNA was extracted from eggs or embryos using the acid guanidinium thiocyanate-phenol-chloroform method (Chomczynski and Sacchi, 1987). Poly(A)+ RNA was purified using Oligotex beads (Roche Japan). For northern blotting, poly(A)+ RNA was fractionated by agarose gel electrophoresis, and transferred to Hybond-N(+) membranes (Amersham). Blots were hybridized with a 32P-random-labeled DNA probe in 6x SSPE, 0.5% SDS, 5x Denhardts solution, 100 µg/ml salmon sperm DNA, and 50% formamide at 42°C. The filters were washed twice in 2x SSC/0.1% SDS at 60°C and then exposed to X-ray film (Fuji film).
Deletion constructs and microinjection of eggs
A deletion mutation of HrFGFR without the intracellular tyrosine kinase domain (dnHrFGFR) was made. FGFR that completely lacks the intracellular tyrosine kinase domain effectively abolishes the wild-type receptor function (Amaya et al., 1991). The dnHrFGFR was made by inserting the NotI-SspI fragment into the modified pBluescript-RN3 vector (Lemaire et al., 1995). By digesting at an SspI site of 12301235 bp (58 amino acids downstream from the transmembrane domain), the tyrosine kinase domain was completely deleted (Fig. 1B). As an experimental control, we injected mRNA which encodes full length HrFGFR. Injection of mRNAs into fertilized eggs was performed as described previously (Miya et al., 1997a).
|
Differentiation of muscle cells was examined using a standard histochemical reaction to detect acetylcholinesterase (AchE; Karnovsky and Roots, 1964). Embryos were fixed with 5% formaldehyde in seawater for 10 minutes at room temperature. The specimens were washed in PBT twice, and then the buffer was replaced with AChE staining buffer (65 mM sodium acetate, 3 mM copper sulfate, 0.5 mM potassium ferricyanide, 5 mM sodium citrate, pH 5.5) containing 0.2 mg/ml acetylthiocholine iodide. The reaction was performed at room temperature for 3 hours.
Antibody staining
To monitor the notochord cell differentiation of H. roretzi, we used a monoclonal antibody 5F1D5 which recognizes a notochord-specific antigen, Not-1 (Nishikata and Satoh, 1990). Indirect immunochemical staining was carried out using TSATM-DIRECT (NENTM, Life Science Products, Inc. Boston, USA) according to the instructions supplied with the kit, and observed using confocal microscopy.
Whole-mount in situ hybridization
In situ hybridization with whole-mount specimens was carried out using digoxigenin-labeled RNA probes of HrFGFR. Specimens were fixed in 4% paraformaldehyde in Mops buffer (pH 7.8), 0.2 M NaCl, 0.4 M MgCl2. After a thorough wash with PBT (phosphate-buffered saline (PBS) containing 0.1% Tween 20), the fixed specimens were treated with 2 µg/ml proteinase K (Merck) in PBT for 30 minutes at 37°C, and then they were post-fixed with 4% paraformaldehyde in PBS for 1 hour at room temperature. After a 1-hour period of prehybridization at 42°C, the specimens were allowed to hybridize with the digoxigenin-labeled antisense or sense probe for at least 16 hours at 42°C. After hybridization, the specimens were washed and treated with RNase A, then washed again extensively with PBT. The samples were then incubated for 1 hour with 1:2000 alkaline-phosphatase-conjugated anti-DIG (Roche) and the colour developed, as indicated in the protocol from Roche. The probes for HrFGFR were synthesized according to the instructions supplied with the kit (DIG RNA Labeling Kit; Roche). The gene expression patterns reported here were examined with probes produced from the coding region of the gene.
Probes used to examine HrFGFR function in experimental embryos were cDNAs for H. roretzi Brachyury gene (As-T or HrBra; Yasuo and Satoh, 1993), an epidermis-specific gene (HrEpiC; Ueki and Satoh., 1995; Ishida et al., 1996), neural tissue-specific gene (HrTBB2; Miya et al., 1997b), and cytoplasmic actin gene (HrCM1; Araki et al., 1996) which is predominantly expressed in mesenchyme cells. RNA probes were prepared with a DIG RNA Labeling Kit (Roche). Control embryos that were hybridized with sense probes did not show signal above background.
Semi-quantitative RT-PCR analysis
Semi-quantitative RT-PCR was carried out as described previously (Imai et al., 2000). Twenty embryos at the early gastrula stage were used for cDNA synthesis. PCRs were performed by utilizing primers with the following nucleotide sequence:
HrBra; 5'-CATGCAGCGTCATGCGTTTACGT-3' and
5'-GTGGTACTCTCTCCAGAACTCGA-3';
HrEF-1; 5'-CTACACGCCAGTTTTGGACTG-3' and
5'-GAAAGTGCCCAGGAAGAAATT-3'.
The reaction was performed for 30 cycles (HrBra) or 27 cycles (HrEF-1), each consisting of 94°C for 30 seconds, 55°C for 30 seconds and 72°C for 1 minute. The reactions were resolved by 5% PAGE and subjected to autoradiography.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The amino acid sequence of the tyrosine kinase domain of HrFGFR, shown in Fig. 1A, was compared with other FGFR domains and subjected to molecular phylogenetic analysis using neighbor-joining method. The mouse VEGFR2 and PDGFR were used as the outgroup. As shown in Fig. 2, HrFGFR was included within a group of FGFR family members. The tree suggested a routeness of the ascidian FGFR, as well as the amphioxus FGFR (Suga et al., 1999), before the divergence of the ancestral gene into four distinct groups present in vertebrates, FGFR1, FGFR2, FGFR3 and FGFR4.
|
When examined with the common probe, only one band was detected in the lanes of EcoRI, HindIII and PstI digestion (Fig. 3), while the specific probe resulted in one band in the lanes of EcoRI and PstI, and two bands in the lane of HindIII digestion (Fig. 3). Our HrFGFR cDNA had a restriction site for HindIII in the Ig3 domain, and a common band between the two probes was seen in the lane of EcoRI and HindIII. In addition, washing the membranes under low-stringency conditions resulted in a similar result. Therefore, it is likely that HrFGFR is present as a single copy per haploid genome of H. roretzi, and two different mRNAs are produced by alternative splicing. Nucleotide sequences of the 3' UTR are identical in the HrFGFR cDNAs characterized by Kamei et al. (Kamei et al., 2000) and that of the present study, supporting the idea that the two different mRNAs are produced by alternative splicing.
|
|
|
Effects of injection of synthetic mRNA for the dominant negative form of HrFGFR
To deduce a possible role of HrFGFR, we constructed a mutated form of HrFGFR (dnHrFGFR) in which the entire tyrosine kinase domain, starting from amino acid position of 369, was deleted from HrFGFR (Fig. 1B). This mutant is thought to act as a dominant negative form to abolish wild-type receptor function (Amaya et al., 1991). We also injected HrFGFR mRNA. Eggs injected with 1.0 µg/µl or less of synthetic mRNA cleaved normally and developed into tailbud embryos with normal morphology, and the effect of dnHrFGFR was not evident. Therefore, we examined the effect of dnHrFGFR by injecting mRNA at a concentration of 4.5 µg/µl. The following describes the effects of dnHrFGFR mRNA injections on the various tissues of the embryo.
Endoderm
Endodermal cells of the ascidian embryo differentiate autonomously and are dependent on the utilization of prelocalized maternal factors (Satoh, 1994). The gene encoding AP is specifically expressed in endodermal cells (Kumano and Nishida, 1998; Imai et al., 2000), and therefore the histochemical detection of AP is a standard method that is used to assess endoderm differentiation. As shown in Fig. 6A, AP expression was not affected by the injection of HrFGFR mRNA (Fig. 6A') in that 27 of 29 injected embryos (93%) showed distinct AP activity (Table 1). Although the injection of dnHrFGFR mRNA resulted in tail shortening of the experimental embryos, AP expression in the trunk region of these embryos did not appear to be affected (Fig. 6A'') in that 18 of 19 injected embryos (95%) showed distinct AP activity (Table 1).
|
|
Epidermis
Epidermal cell differentiation was examined by in situ hybridization using the epidermis-specific gene HrEpiC. Expression of HrEpiC begins at the 64-cell stage, and the expression is first seen in the posterior region of the embryo and later in the anterior region too (Ueki and Satoh., 1995; Ishida et al., 1996). The expression of this gene did not appear to be affected by the injection of dnHrFGFR mRNA (Fig. 6C'') as 23 of 29 injected embryos (79%) showed distinct HrEpiC expression (Table 1). However, injection of HrFGFR mRNA sometimes resulted in the slight downregulation of HrEpiC (Fig. 6C'), as about 90% of the embryos showed weak in situ signals (Table 1).
Notochord
Brachyury (As-T or HrBra) is expressed exclusively in the notochord cells of the ascidian embryo, and this gene plays a key role in notochord differentiation (Yasuo and Satoh, 1993; Yasuo and Satoh 1998). Injection of control HrFGFR mRNA did not affect HrBra expression when it was examined at the 110-cell stage (Fig. 6D'; Table 1) and at the initial tailbud stage (Fig. 6E'; Table 1). In contrast, the injection of dnHrFGFR mRNA caused the distinct downregulation of HrBra expression. In the embryo shown in Fig. 6D'', the A-line primordial notochord cells of the 110-cell embryo failed to express HrBra. As summarized in Table 1, 16 of 26 dnHrFGFR mRNA-injected embryos (62%) failed to express HrBra, while 8 of the 26 experimental embryos showed only weak signals in fewer notochord cells, usually in the B-line notochord cells, as compared with normal embryos. When examined at the initial tailbud stage, only one-third of the embryos showed the HrBra expression in few notochord cells (Fig. 6E''; Table 1). Suppression of the HrBra expression by injection of dnHrFGFR mRNA was further confirmed by quantitative RT-PCR analysis. As shown in Fig. 7, the band intensity of HrBra mRNA in dnHrFGFR-mRNA-injected gastrulae was conspicuously reduced when compared with that in normal gastrulae. Negative control (RT-) without reverse transcriptase gave no bands.
|
Mesenchyme
About 900 mesenchyme cells are formed in the posterior trunk region of the ascidian larva. A recent study by Kim et al. (Kim et al., 2000) showed that mesenchyme cells are specified by at least two factors: vegetal cytoplasm inherited from the egg, and signals emanating from embryonic endodermal cells at the 32-cell stage. They also showed that the formation of mesenchyme cells is induced by treatment with bFGF. HrCA1 encodes a cytoplasmic actin, however, its zygotic expression is not ubiquitous but is restricted to four types of cells (Araki et al., 1996). Strong in situ hybridization signals are detected in mesenchyme cells using this probe (Fig. 8A). Distinct signals are also evident in about 10 neuronal cells that are situated in a characteristic pattern in the anterior-dorsal trunk region (Fig. 8A). In addition, weak signals are evident in notochord cells and reduced signals are also detected in muscle cells (Fig. 8A).
|
The number of notochord cells capable of undergoing intercalation and showing weak HrCA1 expression also decreased (Fig. 8B-D; red dotted line). In the embryo injected with dnHrFGFR mRNA shown in Fig. 8E, notochord cells are absent, and, instead, there was an increase in HrCA1 expression in muscle cells. These results, together with the HrBra and Not1 experiments, suggest that notochord differentiation was disrupted by dnHrFGFR mRNA injection.
Nervous system
The effects of microinjection of dnHrFGFR mRNA on development of the nervous system was examined by in situ hybridization using the HrTBB2 probe, which encodes for ß-tubulin and is expressed in cells of the nervous system (Miya et al., 1997b). HrTBB2 expression is seen in the anterior-most cells, in some cells within the dorsal brain region, in pairs of several cells located in the posterior trunk region, and in pairs of cells situated in the tail region (Fig. 9A).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The degree of the disturbance of HrFGFR function caused by the microinjection of dnHrFGFR mRNA was not always absolute, and the results were not always the same in different batches of eggs (Table 1). This is mainly due to degradation of injected mRNA. In addition, as reported by Kamei et al. (Kamei et al., 2000) and shown by the present study, it is likely that the HrFGFR gene may be transcribed into two different types of mRNA by alternative splicing. If the HrFGFR that was characterized by Kamei et al. (Kamei et al., 2000) functions as a receptor of bFGF signals, it is possible that the injection of dnHrFGFR mRNA does not disrupt the FGF signaling cascade completely. This may be another reason for that the downregulation of HrBra expression by microinjection of dnHrFGFR mRNA was not always absolute.
Relationship between endoderm differentiation and notochord specification in the Halocynthia embryos
The requirement of endodermal cells for specification of notochord cells has been shown in two different ascidian species H. roretzi and Ciona intestinalis (as well as C. savignyi). In H. roretzi, it was shown that cell contact between presumptive notochord cells and endodermal cells during the early phase of the 32-cell stage is necessary and sufficient for the subsequent differentiation of notochord cells (Nakatani and Nishida, 1994). However, one intriguing result that these authors showed is that contact of two presumptive notochord cells during this stage will also result in the subsequent differentiation of notochord cells (Nakatani and Nishida, 1994). That is, in their experiments, endodermal cells are not always required for notochord differentiation. Therefore, this inductive process cannot always be explained by a scenario in which endodermal cells release FGF that functions as the ligand which binds to receptors on the presumptive notochord cells, even if exogenous bFGF can mimic normal cellular interactions (Nakatani et al., 1996). However, there is evidence that FGF regulates Brachyury in the process of mesoderm formation of vertebrate embryos (Issacs et al., 1994; Schulte-Merker and Smith, 1995; Griffin et al., 1995).
In Ciona embryos, endodermal cells are first specified by maternally derived cytoplasmic determinants, and ß-catenin is involved in the endodermal cell specification of Ciona embryos (Yoshida et al., 1998; Imai et al., 2000). During early cleavages of Ciona embryos, ß-catenin was shown to accumulate in the nuclei of vegetal blastomeres, suggesting that it plays a role in the specification of endoderm. Mis- and/or overexpression of ß-catenin induced the development of ectopic endodermal cells from presumptive notochord cells and epidermal cells. Downregulation of ß-catenin induced by the overexpression of cadherin resulted in the suppression of endodermal cell differentiation. This suppression was accompanied by the differentiation of extra epidermal cells. Both the overexpression of ß-catenin in presumptive notochord cells and the downregulation of ß-catenin in presumptive endodermal cells led to the suppression of Brachyury gene expression, resulting in the failure of notochord specification, indicating that specification of notochord cells does not take place in the absence of endoderm differentiation.
Brachyury (both HrBra and Ci-Bra) is a key regulator gene for notochord differentiation in ascidian embryos. This gene begins to be expressed at the 64-cell stage, immediately after interaction with endodermal cells at the 32-cell stage. The minimal promoter required for notochord-specific expression of this gene has been characterized both for Ci-Bra (Corbo et al., 1997) and HrBra (Takahashi et al., 1999b). Within about 500 bp upstream of the transcription start site of Ci-Bra, there are three distinct regions which regulate the notochord-specific expression of the gene: first, there is a distal region responsible for repression of expression in non-notochord mesoderm (mesenchyme and muscle) cells; second, there is an intermediate region for the activation of expression in notochord cells; and third, there is a proximal region for the activation of non-notochord mesoderm cells (Corbo et al., 1997). The notochord-specific activation domain of the Ci-Bra promoter contains the Suppressor of Hairless [Su(H)] binding site, and thereforeit has been suggested that the Notch signal cascade is involved in the interaction with endodermal cells (Corbo et al., 1998). Furthermore, the non-notochord mesoderm suppression domain of the promoter contains Snail binding sites. Fujiwara et al. (Fujiwara et al., 1998) showed that the snail gene of C. intestinalis (Ci-sna) is expressed in mesenchyme and muscle to prevent Ci-Bra expression in inappropriate lineages. Together with a scenario first proposed by Kim et al. (2000) in which endodermal cells emanate signals to induce both notochord and mesenchyme cells, the above mentioned results may explain how the Brachyury gene is expressed solely in notochord cells.
However, interaction between endoderm and notochord cells and the initiation of Brachyury gene expression in ascidian embryos seems to be much more complex than we presently can appreciate. For example, the snail gene of H. roretzi (HrSna) shows a slightly different pattern of expression when compared with Ci-sna (Wada and Saiga, 1997). In addition to its expression in muscle and mesenchyme lineages, HrSna is also expressed in an overlapping pattern with HrBra at the 64-cell and 110-cell stages, suggesting that the mechanism in which HrSna expression defines the boundary between the notochord and non-notochord mesoderm is not applicable to Halocynthia embryos despite the fact that the minimal promoter of HrBra does not contain HrSna binding sites (Takahashi et al., 1999b). Analysis of the minimal promoter of HrBra that is required for its notochord-specific expression revealed the importance of two critical sequences and gel-shift assays suggest that specific types of nuclear proteins bind to these sequences (Takahashi et al., 1999b). Therefore, it looks promising that we will be able to characterize these DNA-binding proteins that regulate the expression of HrBra.
In conclusion, the present study provides evidence for the involvement of FGF signals in the differentiation of notochord and mesenchyme cells in Halocynthia embryos. However, the results also raise many unresolved questions that need to be answered by future studies so that our understanding of the cellular and molecular mechanisms responsible for notochord differentiation of ascidian embryos can be applied to the fundamental question how the notochord arose during evolution of chordates.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Amaya, E., Musci, T. J. and Kirschner, M. W. (1991). Expression of a dominant negative mutant of the FGF receptor disrupts mesoderm formation in Xenopus embryos. Cell 66, 257-270.[Medline]
Altschul, S. F, Madden, T. L., Schaffer, A. A, Zhang, Z., Miller, W. and Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389-3402.
Araki, I., Tagawa, K., Kusakabe, T. and Satoh, N. (1996). Predominant expression of a cytoskeletal actin gene in mesenchyme cells during embryogenesis of the ascidian Halocynthia roretzi. Dev. Growth Differ. 38, 401-411.
Chomczynski, P. and Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analyt. Biochem. 162, 156-159.[Medline]
Conklin, E. G. (1905). The organization and cell lineage of the ascidian egg. J. Acad. Nat. Sci. (Philadelphia) 13, 1-119.
Corbo, J. C., Levine, M. and Zeller, R. M. (1997). Characterization of a notochord-specific enhancer from the Brachyury promoter region of the ascidian, Ciona intestinalis. Development 124, 589-602.
Corbo, J. C., Fujiwara, S., Levine, M. and Di Gregorio, A (1998). Suppressor of hairless activates Brachyury expression in Ciona embryos. Dev. Biol. 203, 358-368.[Medline]
Dierick, H. and Bejsovec, A. (1999). Cellular mechanisms of Wingless/Wnt signal transduction. Curr. Top. Dev. Biol. 43, 153-190.[Medline]
Fujiwara, S., Corbo, J. and Levine, M. (1998). The snail expression establishes a muscle/notochord boundary in the Ciona embryo. Development 125, 2511-2520.
Griffin, K., Patient, R. and Holder, N. (1995). Analysis of FGF function in normal and no tail zebrafish embryos reveals separate mechanisms for formation of the trunk and the tail. Development 121, 2983-2994.
Hotta, K., Takahashi, H., Asakura, T., Saitoh, B., Takatori, N., Satou, Y. and Satoh N. (2000). Characterization of Brachyury-downstream notochord genes in the Ciona intestinalis embryo. Dev. Biol. 224, 69-80.[Medline]
Imai, K., Takada, N. Satoh, N. and Satou, Y. (2000). ß-catenin mediates the specification of endoderm cells in ascidian embryos. Development 127, 3009-3020.
Ishida, K., Ueki, T. and Satoh, N. (1996). Spatio-temporal expression patterns of eight epidermis-specific genes in the ascidian embryo. Zool. Sci. 13, 699-709.
Isaacs, H. V., Pownall, M. E. and Slack, J. M. (1994). eFGF regulates Xbra expression during Xenopus gastrulation. EMBO J. 13, 4469-4481.[Abstract]
Kamei, S., Yajima, I., Yamamoto, H., Kobayashi, A., Makabe, K. W., Yamazaki, H., Hayashi, S-I. and Kunisada T. (2000). Characterization of a novel member of the FGF family, HrFGFR, in Halocynthia roretzi. Biochem. Biophy. Res. Comm. 275, 503-508.[Medline]
Kim, G. J., Yamada, A. and Nishida, H. (2000). An FGF signal from endoderm and localized factors in the posterior-vegetal egg cytoplasm pattern the mesodermal tissues in the ascidian embryo. Development 127, 2853-2862.
Karnovsky, M. J. and Roots, L. (1964). A direct-coloring thiocholine method for cholinesterase. J. Histochem. Cytochem. 12, 219-221.
Kumano, G. and Nishida, H. (1998). Maternal and zygotic expression of the endoderm-specific alkaline phosphatase gene in embryos of the ascidian, Halocynthia roretzi. Dev. Biol. 198, 245-252.[Medline]
Lemaire, P., Garrett, N. and Gurdon, J. B. (1995). Expression cloning of Siamois, a Xenopus homeobox gene expressed in dorsal-vegetal cells of blastulae and able to induce a complete secondary axis. Cell 81, 85-94.[Medline]
Lee, M. S., LeMaistre, A., Kantarjian, H. M., Talpaz, M., Freireich, E. J., Trujillo, J. M. and Stass, S. A. (1989). Detection of two alternative bcr/abl mRNA junctions and minimal residual disease in Philadelphia chromosome positive chronic myelgenous leukemia by polymerase chain reaction. Blood 73, 2165-2170.[Abstract]
Miya, T., Morita, K., Suzuki, A., Ueno, N. and Satoh N. (1997a). Functional analysis of an ascidian homologue of vertebrate Bmp-2/Bmp-4 suggests its role in the inhibition of neural fate specification. Development 124, 5149-5159.
Miya, T. and Satoh, N. (1997b). Isolation and characterization of cDNA clones for ß-tubulin genes as a molecular marker for neural cell differentiation in the ascidian embryo. Int. J. Dev. Biol. 41, 551-557.[Medline]
Moon, R. T., Brown, J. D. and Torres, M. (1997). WNTs modulate cell fate and behavior during vertebrate development. Trends Genet. 13, 157-162.[Medline]
Nakatani, Y. and Nishida, H. (1994). Induction of notochord during ascidian embryogenesis. Dev. Biol. 166, 289-299.[Medline]
Nakatani, Y., Yasuo, H., Satoh, N. and Nishida, H. (1996). Basic fibroblast growth factor induces notochord formation and the expression of As-T, a Brachyury homolog, during ascidian embryogenesis. Development 122, 2023-2031.
Nakatani, Y. and Nishida, H. (1997). Ras is an essential component for notochord formation during ascidian embryogenesis. Mech. Dev. 68, 6881-6889.
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.[Medline]
Nishikata, T. and Satoh, N. (1990). Specification of notochord cells in the ascidian embryo analysed with a specific monoclonal antibody. Cell Diff. Dev. 30, 43-53.[Medline]
Nugent, M. A. and Iozzo, R. V. (2000). Fibroblast growth factor-2. Int. J. Biochem. Cell Biol. 32, 115-120.[Medline]
Ornitz, D. M. (2000). FGFs, heparan sulfate and FGFRs: complex interactions essential for development. BioEssays 22, 108-112.[Medline]
Pawson, T. (1995). Protein modules and signaling networks. Nature 373, 573-580.[Medline]
Piek, E., Heldin, C. H. and Dijke, P. T. (1999). Specificity, diversity, and regulation in TGF-ß superfamily signaling. FASEB J. 13, 2105-2124.
Riese II, D. J. and Stern, D. F. (1998). Specificity within the EGF family/ErbB receptor family signaling network. BioEssays 20, 41-48.[Medline]
Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, 2nd edition. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory.
Satoh, N. (1994). Developmental Biology of Ascidians. New York: Cambridge University Press.
Satoh, T., Nakafuku, M. and Kaziro, Y. (1992). Function of Ras as a molecular switch in signal transduction. J. Biol. Chem. 267, 24149-24152.
Shen M. M. and Schier, A. F. (2000). The EGF-FGF gene family in vertebrate development. Trends Genet. 16, 303-309.[Medline]
Schulte-Merker, S. and Smith, J. C. (1995). Mesoderm formation in response to Brachyury requires FGF signalling. Curr. Biol. 5, 62-67.[Medline]
Shimauchi, Y., Yasuo, H. and Satoh, N. (1997). Autonomy of ascidian fork head/HNF-3 gene expression. Mech. Dev. 69, 143-154.[Medline]
Slack, J. (1994). Role of fibroblast growth factors as inducing agents in early embryonic development. Mol. Reprod. Dev. 39, 118-125.[Medline]
Suga, T., Hoshiyama, D., Kuraku, S., Katoh, K., Kubokawa, K. and Miyata, T. (1999). Protein ttyrosine kinase cDNAs from amphioxus, hagfish and lamprey: Isolation duplications around the divergence of cyclostomes and gnathostomes. J. Mol. Evol. 49, 601-608.[Medline]
Takahashi, H., Hotta, K., Erives, A., Di Gregorio, A., Zeller, R. W., Levine, M. and Satoh, N. (1999a). Brachyury downstream notochord differentiation in the ascidian embryo. Genes Dev. 13, 1519-1523.
Takahashi, H., Mitani, Y., Satoh G. and Satoh, N. (1999b). Evolutionary alternations of the minimal promoter for notochord-specific Brachyury expression in ascidian embryos. Development 126, 3725-3734.
Ueki, T. and Satoh, N. (1995). Sequence motifs shared by the 5' flanking regions of two epidermis-specific genes in the ascidian embryo. Dev. Growth Differ. 33, 579-604.
Wada, S. and Saiga, H. (1999). Cloning and embryonic expression of Hrsna, a snail family gene of the ascidian Halocynthia roretzi: implication in the origins of mechanisms for mesoderm specification and body axis formation in chordates. Dev. Growth Differ. 41, 9-18.[Medline]
Whittaker, J. R. and Meedel, T. H. (1989). Two histospecific enzyme expression in the same cleavage-arrested one-celled ascidian embryos. J. Exp. Zool. 250, 168-175.[Medline]
Yamaguchi, T. P., Harpal, K., Henkemeyer, M. and Rossant, J. (1994). fgfr-1 is required for embryonic growth and mesodermal patterning during mouse gastrulation. Genes Dev. 8, 3032-3044.[Abstract]
Yamaguchi, T. P. and Rossant, J. (1995). Fibroblast growth factors in mammalian development. Curr. Opin. Genet. Dev. 5, 485-491.[Medline]
Yasuo, H. and Satoh, N. (1993). Function of vertebrate T gene. Nature 364, 582-583.[Medline]
Yasuo, H. and Satoh, N. (1998). Conservation of the developmental role of Brachyury in notochord formation in a urochordate, the ascidian Halocynthia roretzi. Dev. Biol. 200, 158-170.[Medline]
Yoshida, S., Marikawa, Y. and Satoh, N. (1998). Regulation of the trunk-tail patterning in the ascidian embryo: a possible interaction of cascades between lithium/beta-catenin and localized maternal factor pem. Dev. Biol. 202, 264-279.[Medline]