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Keywords:

  • proteomics;
  • ascidian embryo;
  • sperm axonemes;
  • mass spectrometry;
  • peptide mass fingerprinting;
  • two-dimensional gel electrophoresis;
  • Ciona intestinalis

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. IMPORTANCE OF PROTEOMIC APPROACHES IN ASCIDIAN DEVELOPMENTAL BIOLOGY
  5. TECHNICAL OVERVIEW OF PROTEOMICS
  6. ADVANCES IN SPERM PROTEOMICS FOR STUDYING THEIR MOLECULAR ARCHITECTURE AND FUNCTIONAL CHANGES DURING FERTILIZATION IN C. INTESTINALIS
  7. PROFILES OF PROTEIN EXPRESSION DURING CIONA DEVELOPMENT
  8. CIONA PROTEOMICS FOR THE COMPREHENSIVE UNDERSTANDING OF ASCIDIAN DEVELOPMENT
  9. Acknowledgements
  10. REFERENCES

Ascidians have been providing a unique experimental system for a variety of fields, including reproductive biology, developmental biology, neurobiology, immunology, and evolutional biology. Recent progress in the genome sequencing of Ciona intestinalis has led to the development of a great tool for investigating the gene functions and expressions involved in several biological events in ascidians. The disclosure of genomic information has ushered in the postgenomic era, spearheaded by extensive protein analysis. The characterization of the function, localization, and molecular interaction of cellular proteins results in a more direct description of the molecular mechanism underlying several biological processes. Proteomics in ascidians, however, has just recently appeared and is not well established yet. In this study, we give an outline of the technical processes used in proteomics and review the recent status of ascidian proteomics. Developmental Dynamics 236:1782–1789, 2007. © 2007 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. IMPORTANCE OF PROTEOMIC APPROACHES IN ASCIDIAN DEVELOPMENTAL BIOLOGY
  5. TECHNICAL OVERVIEW OF PROTEOMICS
  6. ADVANCES IN SPERM PROTEOMICS FOR STUDYING THEIR MOLECULAR ARCHITECTURE AND FUNCTIONAL CHANGES DURING FERTILIZATION IN C. INTESTINALIS
  7. PROFILES OF PROTEIN EXPRESSION DURING CIONA DEVELOPMENT
  8. CIONA PROTEOMICS FOR THE COMPREHENSIVE UNDERSTANDING OF ASCIDIAN DEVELOPMENT
  9. Acknowledgements
  10. REFERENCES

Ascidians are primitive chordates whose phylogenetic position during evolution has been drawing considerable attention. Due to their simple and unique developmental process, they have been providing elegant experimental systems for the study of the mechanism of development for more than a century (reviewed in Satoh et al.,2003). They undergo a typical mosaic development and grow into tadpole larvae that show simple cell organization. Detailed investigation of cell fate during development has enabled the elucidation of cell differentiation based on cell–cell interactions and signal transduction (Conklin,1905; Nishida,1987). In terms of the early determination of cell fate, research on ascidians has shed light on the mechanism by which the distribution of maternal substances, including the determinant of zygotic gene expression, controls the formation of the body axes, such as the anterior–posterior, the dorsal–ventral, and the left–right axes (reviewed in Sardet et al.,2005; Nishida,2005). The study of the principle of cellular organization in chordates has also taken advantage of the simple body plan of ascidian larvae. Especially the simple nerve system with a dorsal tubular nerve organization in tadpole larvae has served as a miniature system for elucidating the nerve system of vertebrates (Meinertzhagen and Okamura,2001).

Another important feature of ascidians in developmental biology is that they provide a model system for studying the function of gametes and their interaction. The activation of sperm motility and the chemotaxis of sperm to egg during fertilization are clearly observed in the ascidian Ciona intestinalis (Miller,1975; Yoshida et al.,1993; Nomura et al.,2000). This species could be an appropriate model for studying not only the common mechanism of chemotaxis but also signal transduction in the regulation of cell motility (Dey and Brokaw,1991; Inaba,2003; Satouh et al.,2005). Furthermore, sperm mitochondria move along the sperm flagella and are excluded from the sperm cell during fertilization. This unique phenomenon, called sperm reaction, contributes to the discrimination of paternal mitochondria in embryonic development (Lambert and Koch,1988). Also, ascidians show gamete self-incompatibility to prevent self-fertilization (Morgan,1923). This finding is effected by a strict interaction between the sperm surface and the oocyte vitelline coat (Fuke,1983; Marino et al.,1999).

Recent progress in Ciona genomics accelerates the search for detailed knowledge of the molecular mechanism underlying ascidian development. One of the advantages of genomic information is that it can serve as the molecular basis for proteomics. However, extensive analyses of Ciona proteins have been limited to a few studies on Ciona testis or sperm (Hozumi et al.,2004,2006; Satouh et al.,2005). Because the composition and dynamics of proteins constitute the molecular and cellular basis of ascidian development, we have recently started to carry out proteomic analysis at every stage of embryogenesis, in larvae and in the adult tissues of C. intestinalis. In this review, an outline of the technical background of proteomics is given and the recent status of ascidian proteomics is presented.

IMPORTANCE OF PROTEOMIC APPROACHES IN ASCIDIAN DEVELOPMENTAL BIOLOGY

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. IMPORTANCE OF PROTEOMIC APPROACHES IN ASCIDIAN DEVELOPMENTAL BIOLOGY
  5. TECHNICAL OVERVIEW OF PROTEOMICS
  6. ADVANCES IN SPERM PROTEOMICS FOR STUDYING THEIR MOLECULAR ARCHITECTURE AND FUNCTIONAL CHANGES DURING FERTILIZATION IN C. INTESTINALIS
  7. PROFILES OF PROTEIN EXPRESSION DURING CIONA DEVELOPMENT
  8. CIONA PROTEOMICS FOR THE COMPREHENSIVE UNDERSTANDING OF ASCIDIAN DEVELOPMENT
  9. Acknowledgements
  10. REFERENCES

First, the cosmopolitan species C. intestinalis was selected as the organism for the ascidian genome project (Dehal et al.,2002; http://genome.jgi-psf.org/ciona4/ciona4.home.html; http://ghost.zool.kyoto-u.ac.jp/indexr1.html; http://www.ensembl.org/). The genome of C. intestinalis contains the basic set of vertebrate genes with a very compact genome constitution: the size of the genome is 160 Mb, with ∼16,000 deduced genes. The assembly of the Ciona genome information was supported by an extensive analysis of the expressed sequence tags (∼700,000 ESTs) from several adult tissues and from embryos at several stages of development. Extensive information on ESTs enables us to compare the gene expression profiles of several developmental stages and adult tissues and to identify tissue-specific genes (Satou et al.,2003). The genome analysis of another Ciona species, C. savignyi, has also progressed and has become a strong resource for comparative genomics (http://www.broad.mit.edu/annotation/ciona/; http://www.ensembl.org/).

The extensive analysis of gene expression reveals the profile of the genes expressed at a given embryonic stage and the spatiotemporal regulatory mechanism of transcription during embryonic development (Imai et al.,2006). The genomic information is also widely used for gene knockout/knockdown techniques to investigate gene function (Yamada et al.,2003). However, the expression levels of genes and proteins are not always parallel in many cells (Gygi et al.,1999). Understanding the molecular mechanism underlying many of the cellular processes becomes possible only when information on the function, localization, and regulation of proteins is available.

Most proteins function as protein complexes that serve the machinery which promotes certain intracellular processes. These complexes serve as functional platforms or scaffolds in the cytoplasm or the organelles. Many proteins exert their regulatory functions through posttranslational modifications, such as phosphorylation/dephosphorylation, methylation, tyrosination, glycylation, and glycosylation (Jensen,2004). These modifications also include intramolecular and intermolecular covalent bonding, in the form of transglutamination, ubiquitination, and SUMOlylation (Seo and Lee,2004). Reversely limited proteolysis is also a prerequisite for the expression of protein function (Ehrmann and Clausen,2004). One example of the sophisticated regulation exerted by proteins is the cell-cycle–dependent regulation of the protein machinery: the cell cycle is regulated by a well-organized network of multiple protein complexes comprising cyclin-dependent kinases, kinase inhibitors, the APC/C-SCF ubiquitin–ligase system, and the ubiquitin–proteasome degradation system (Peters,2002). Thus, it is obvious that protein complexes and the dynamics effected by them through various modifications are the cellular engine driving cell proliferation and differentiation; ultimately, they directly manage the spatiotemporal differentiation of cells during development.

Ascidians have provided a unique and simple system for studying developmental biology, but some phenomena can be understood only by proteomic approach. For example, the proteins essential for sperm behavior at fertilization can be extensively analyzed by proteomic techniques (Hozumi and Inaba,2001). Furthermore, the egg undergoes cytoplasmic segregation and reorganization, resulting in the postplasmic localization of organelles and substances, including cortical mRNA (Sardet et al.,2005). The mRNAs become concentrated into a structure called the centrosome-attracting body (CAB; Hibino et al.,1998; Nishikata et al.,1999). Because the CAB appears to be involved in both the unequal cleavage and the accumulation of specific mRNAs, a proteomic approach to the identification of the proteins in this structure would be the key to the elucidation of cell differentiation in ascidian development. The ascidian embryo or larva provides several interesting experimental systems for proteomics that would potentially bring a breakthrough in our understanding of the molecular mechanism of ascidian development: fragmentation into specific egg regions (Marikawa et al.,1994); the in vitro differentiation of specific cell types, as in notochord differentiation (Nakatani and Nishida,1994); the behavior of adhesive papillae during metamorphosis (Cloney,1979); and the development of the nerve system in larvae, including the sensory organs otolith and ocellus (Nicol and Meinertzhagen,1991).

TECHNICAL OVERVIEW OF PROTEOMICS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. IMPORTANCE OF PROTEOMIC APPROACHES IN ASCIDIAN DEVELOPMENTAL BIOLOGY
  5. TECHNICAL OVERVIEW OF PROTEOMICS
  6. ADVANCES IN SPERM PROTEOMICS FOR STUDYING THEIR MOLECULAR ARCHITECTURE AND FUNCTIONAL CHANGES DURING FERTILIZATION IN C. INTESTINALIS
  7. PROFILES OF PROTEIN EXPRESSION DURING CIONA DEVELOPMENT
  8. CIONA PROTEOMICS FOR THE COMPREHENSIVE UNDERSTANDING OF ASCIDIAN DEVELOPMENT
  9. Acknowledgements
  10. REFERENCES

Proteomics is the large-scale analysis of proteins; therefore, efficient strategies for the separation and identification of proteins are indispensable. Recent progress in two-dimensional gel electrophoresis (2DE) using immobilized gel strips and mass spectrometry (MS) has made possible protein profiling at the quantitative level. The devices used for protein separation in conjunction with several types of mass spectrometry have become sophisticated, and the techniques are now so diverse as to be applicable to several proteomic strategies (for a review, see Gorg et al.,2000; Reinders et al.,2004; Steen and Mann,2004; Gingras et al.,2005).

There are two basic methods of identifying proteins by MS: one is peptide mass fingerprinting (PMF), and the other is the identification of sequence tags, or de novo sequencing, using tandem MS (MS/MS; Fig. 1). For ensuring the accuracy of peptide mass evaluation, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) is often used. The identification of proteins by PMF requires a database. Accurate research can only be performed if the amino acid sequence of the protein in question is available. Before PMF, proteins must be purified by the appropriate biochemical method, such as 2DE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), or liquid chromatography (LC; Fig. 1). One of the conventional methods for such analysis is the separation of proteins by 2DE, followed by the proteolytic digestion and MS of the products. The advantage of protein separation by 2DE is that it allows the quantitative comparison of protein spots from tissues at different differentiation stages or from embryos at different developmental stages. Recently, fluorescent dye-based differential displays for protein comparison have begun to be used to detect protein changes between two different cellular conditions (Lilley and Friedman,2004). For PMF, the protein must be purified as a single protein spot on 2DE or a single protein band on SDS-PAGE. The 2DE using isoelectric focusing followed by SDS-PAGE is most often used, but a combination of fractionation by column chromatography and SDS-PAGE can also be applied to PMF. Although 2DE is a powerful technique for separating proteins at high resolution, the range of usable proteins is limited in some cases. For example, some membrane proteins cannot be properly analyzed by 2DE due to their low solubility in the lysis buffer for isoelectric focusing. In such cases, it is necessary to purify the proteins by column chromatography under denaturing conditions or by SDS-PAGE (Wu and Yates,2003).

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Figure 1. Overview of mass-spectrometry–based proteomics. Proteins from cells or tissues are separated by two-dimensional gel electrophoresis or liquid column chromatography. Immunoprecipitation or affinity purification is also an efficient procedure for isolating functional protein complexes. The protein band in a gel or the protein peak from a column is digested by a protease, most commonly by trypsin. Protein mixtures can be digested by protease, followed by separation with tandem liquid chromatography. Proteolytic fragments are analyzed by mass spectrometry. To identify the proteins, the patterns of the mass values of the proteolytic fragments are searched in the database (peptide mass fingerprinting, PMF). Alternatively, the fragmented ions from a certain proteolytic fragment can be searched against a database to obtain amino acid sequence tags or de novo sequences (MS/MS, see text).

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Protein bands or spots are usually digested “in gel” by a protease, most often by trypsin, and analyzed by MALDI-TOF/MS. The set of the mass values of the protein fragments is then introduced in a protein database, and the deduced molecular masses of the fragments are calculated from the amino acid sequences in silico. Several software programs for protein identification by PMF are available. The most conventional ones are those equipped with Internet-based tools, such as ProteinProspector from UCSF (http://prospector.ucsf.edu/), MASCOT from Matrix Science, Ltd. (http://www.matrixscience.com/), and the program on the ExPASy server (http://tw.expasy.org/tools/). These programs generally provide powerful tools for protein identification by PMF, especially for proteins from organisms for which only the genome sequence of the protein coding regions has been completely identified. However, MS data from organisms for which even fewer genome sequences have been introduced into the database result in low identification efficiency.

An alternative method of identifying proteins takes advantage of the molecular masses of the peptide fragments further generated by postsource decay (PSD) from a proteolytic fragment. MALDI-TOF/MS identifies the PSD fragment ions generated from both the N-terminus (b-ions) and the C-terminus (y-ions). Sequence information is obtained by comparing the molecular masses of these PSD fragment ions with the help of the protein database. On the other hand, the MS/MS analysis of proteins enables de novo sequencing. The mass measurement of fragments generated from collision-induced dissociation (CID) or PSD allows the determination of partial amino acid sequences by computation. The limitations of protein identification by PMF can be overcome through separation by SDS-PAGE, followed by MS/MS. However, a more powerful and high-throughput strategy for MS/MS has been developed: the sequential combination of multidimensional LCs and MS/MS. This process is the so-called MudPIT, or shotgun proteomics (Wolters et al.,2001). Another combination is also possible: separation by SDS-PAGE, followed by LC-MS/MS. For example, proteolytic digests from crude samples or sometimes whole-cell homogenate could be separated by tandem LC (Fig. 1). A single peak is automatically analyzed by MS/MS.

Posttranslational modifications are known to be important for the appropriate functioning, regulation, and assembly of proteins. They are wide-ranging, and there are more than 200 types of modification. Technical advances in proteomics have targeted posttranslational modifications, but a high coverage rate is required to distinguish the mass of intact peptides from that of modified peptides. For example, protein phosphorylation results in an 80-Da increase in the molecular mass of a peptide fragment, due to the added phosphate group. Because the possible protein modifications can be included in the PMF search, modified peptide candidates can be easily assigned. Subsequent MS/MS further confirms the modified sites. Extensive identification of proteins with specific types of modification can be performed if the modified peptides are isolated on some affinity columns, such as the immobilized metal-ion affinity column (IMAC; Annan and Carr,1996; Ficarro et al.,2002).

Protein identification by 2DE/MS or LC-MS/MS covers an extensive number of protein species existing in the cell; however, the limitations of such proteomic analysis are also made obvious by the fact that identification efficiency depends highly on the cell contents: proteins with a more minor presence, or minor proteins, are difficult to identify by high-throughput analysis. This problem can be overcome by the process of “concentration,” which comprises a variety of biochemical procedures, such as the isolation of stable cellular substructures, immunoprecipitation, the isolation of protein complexes by column chromatography, and affinity purification using several ligands (Fig. 1). Approaches using antibodies or affinity columns can produce corroborating information on protein–ligand or protein–protein interactions, which is quite important in functional proteomics.

ADVANCES IN SPERM PROTEOMICS FOR STUDYING THEIR MOLECULAR ARCHITECTURE AND FUNCTIONAL CHANGES DURING FERTILIZATION IN C. INTESTINALIS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. IMPORTANCE OF PROTEOMIC APPROACHES IN ASCIDIAN DEVELOPMENTAL BIOLOGY
  5. TECHNICAL OVERVIEW OF PROTEOMICS
  6. ADVANCES IN SPERM PROTEOMICS FOR STUDYING THEIR MOLECULAR ARCHITECTURE AND FUNCTIONAL CHANGES DURING FERTILIZATION IN C. INTESTINALIS
  7. PROFILES OF PROTEIN EXPRESSION DURING CIONA DEVELOPMENT
  8. CIONA PROTEOMICS FOR THE COMPREHENSIVE UNDERSTANDING OF ASCIDIAN DEVELOPMENT
  9. Acknowledgements
  10. REFERENCES

Initial proteomic analysis in ascidians has been carried out using the sperm of C. intestinalis (Hozumi et al.,2004; Satouh et al.,2005). First, a local database of the Ciona testis proteins was established. By combining the testis EST information (Inaba et al.,2002) with the genome draft sequence (Dehal et al.,2002), we constructed a local database for the protein primary structure and for the mass of tryptic fragments (MSCITS), along with a Perl-based program, called PerMS, for protein identification of testicular proteins in C. intestinalis (Hozumi et al.,2004). The protein identification efficiency we have attained so far is nearly 80%, which is almost equivalent to that of other known Internet-based tools. An example of our results in protein identification is shown in Figure 2. Ciona proteins, such as creatine kinase (spot 14), have already been registered in the NCBI database and could be easily identified by PMF using online tools such as MS-Fit. Proteins, such as tubulin (spot 12), which are highly conserved through evolution, show high identity over their entire length in distantly related species and could also be identified by MS-Fit, even though the Ciona tubulin sequence was not registered in the database. The amino-acid sequence of β-tubulin showed 95% identity over its entire length between Ciona and human. This identity seems to be high enough for protein identification by MS-Fit. However, the identity of other proteins, such as aconitase (spot 4), enolase (spot 13), aldolase (spot 15), or glyceraldehyde-3-phosphate dehydrogenase (spot 18), between Ciona and human was 68%, 70%, 67%, or 78%, respectively. These identities were high enough to allow protein identification by BLASTP sequence homology, but were too low for identification by PMF using databases for other organisms. By means of MSCITS and PerMS, all of these proteins could be efficiently identified (Hozumi et al.,2004). Thus, except for highly conserved proteins, protein identification can be performed by PMF only if the sequence data are available in the database. The construction of a local database and a simple search program for Ciona testis proved to be useful, especially for the PMF identification of the proteins of organisms with limited genomic or protein information.

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Figure 2. Identification of Ciona sperm proteins by proteomics. Two-dimensional gel electrophoresis separates the proteins of Ciona sperm into ∼400 major spots (left; first isoelectric focusing with an immobilized pH gradient of 3–10, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with 10% polyacrylamide separating gel). The protein spots are cut off and subjected to tryptic digestion in gel, followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). The mass values of the fragmented proteins are searched against a local database MSCITS by the search program PerMS. Some of the sperm proteins that we have identified are summarized (right).

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The biochemical fractionation of protein complexes or subcellular structures enables us to carry out more sophisticated proteomics in relation to the function of each protein. This step may also lead to the identification of important proteins with a minor presence in the cell. For example, we have isolated one of the molecular motor units, the outer arm dynein, and a substructure with a regulatory complex for dynein, called the radial spoke, from the axonemes of Ciona sperm flagella (Satouh et al.,2005; Hozumi et al.,2006). Proteomic identification revealed not only the functions of novel proteins, such as a dynein-related coiled-coil protein, a protein with a calmodulin-binding site and a ubiquitin-binding site (CMUB), and a novel MORN repeat protein, but also unexpected functions of well-known proteins, such as the heat shock protein HSP40 and nucleoside diphosphate kinase (NDK; Fig. 3).

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Figure 3. Identification of protein components in two important axoneme substructures. The outer arm dyneins (ODA) are extracted from the Ciona sperm axonemes and separated by sucrose density gradient centrifugation or gel filtration. The components of the ODA are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by peptide mass fingerprinting (PMF; Hozumi et al.,2006). For the radial spokes (RS), the KI extract of the axonemes is separated using a Superose 6 gel filtration column to isolate the subcomplex of the RS. Similar subcomplexes can be isolated by immunoprecipitation from the KI extract using an antibody against the radial spoke protein RSP3 (Satouh et al.,2005). The components of the subcomplex are separated by SDS-PAGE and identified by PMF. A thin electron microscopy image of the axonemes is shown to the left, with the positions of both ODA and RS.

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The set of protein sequences deduced from the Ciona genome is available at the Ciona genome database (http://genome.jgi-psf.org/ciona4/ciona4.home.html; http://ghost.zool.kyoto-u.ac.jp/indexr1.html). To identify proteins in embryos at several developmental stages or adult tissues by PMF, we have started the construction of a system that identifies proteins by Mascot using the amino acid sequences in the Ciona proteome. The efficiency of protein identification by PMF is less than that obtained by the MSCITS/PerMS system (Hozumi et al.,2004) for the Ciona testis, suggesting that the database for the Ciona proteome still needs to be improved. It is necessary to complete the Ciona gene model for the PMF-based identification of Ciona proteins. To this end, it may be essential to include alternative splicing or trans-splicing information (Vandenberghe et al.,2001; Satou et al.,2006).

PROFILES OF PROTEIN EXPRESSION DURING CIONA DEVELOPMENT

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. IMPORTANCE OF PROTEOMIC APPROACHES IN ASCIDIAN DEVELOPMENTAL BIOLOGY
  5. TECHNICAL OVERVIEW OF PROTEOMICS
  6. ADVANCES IN SPERM PROTEOMICS FOR STUDYING THEIR MOLECULAR ARCHITECTURE AND FUNCTIONAL CHANGES DURING FERTILIZATION IN C. INTESTINALIS
  7. PROFILES OF PROTEIN EXPRESSION DURING CIONA DEVELOPMENT
  8. CIONA PROTEOMICS FOR THE COMPREHENSIVE UNDERSTANDING OF ASCIDIAN DEVELOPMENT
  9. Acknowledgements
  10. REFERENCES

Recent genomic analysis of Ciona intestinalis has revealed the expression profiles of the genes from fertilized eggs to larvae. Some of the genes showed a dramatic increase or decrease in expression and could be responsible for cell differentiation or morphogenesis (Kawashima et al.,2005; Imai et al.,2006). These findings, however, are not always comprehensive in terms of proteins, because the correlations between mRNA levels and protein levels are poor (Gygi et al.,1999; Awad and Gruppu,2000), possibly due to posttranscriptional regulation. Maternally inherited transcripts and proteins are stored during oogenesis. They continue to perform essential functions or interact with newly expressed genes or proteins through maternal-to-zygotic transition. It is interesting to see how each of the encoded proteins changes during the mosaic-type development of the ascidian embryo.

We carried out 2DE of the Ciona embryo and detected ∼600 main protein spots (Fig. 4). The patterns of major proteins in fertilized eggs, cleaved embryos, and embryos at later stages are not greatly different form each other. These proteins may serve as housekeeping proteins, as in mice (Greene et al.,2002), Drosophila (Gong et al.,2004), and zebrafish (Link et al.,2006). They include ribosomes, metabolic enzymes, and cytoskeletal elements. Although yolk proteins in ascidian eggs are not well characterized yet, they may include abundant yolk proteins, as in Drosophila (Gong et al.,2004) and zebrafish (Link et al.,2006). The major proteins in the ascidian embryo should be good targets for initial proteomic analysis, not only to describe the general nature of the ascidian egg or blastomere but also to use as landmarks of molecular mass and isoelectric points for the 2D comparison of proteins during development. Although there are few differences in the amount of major proteins between fertilized eggs and cleaved embryos or between cleaved embryos and tail bud embryos, some minor changes in the amount or isoelectric point of the proteins could be detected: these proteins may play a key role in the cell cycle, cell differentiation, or morphogenesis. Analysis of these proteins promises a breakthrough in the elucidation of the cell differentiation mechanism during development.

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Figure 4. Two-dimensional comparison of the proteins of unfertilized eggs, fertilized eggs, cleaved embryos, mid-tailbud embryos, and swimming larvae. The proteins from dechorionated eggs (0 min), fertilized eggs (25 min), and eight-cell stage embryos (150 min) were separated by isoelectric focusing (IEF) with an immobilized pH gradient of 3–10, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with 10% polyacrylamide separating gel. For the separation of proteins from mid-tailbud embryos and swimming larvae, IEF with an immobilized pH gradient of 3–10 and SDS-PAGE with 12% polyacrylamide separating gel were used for the first and second separation, respectively.

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CIONA PROTEOMICS FOR THE COMPREHENSIVE UNDERSTANDING OF ASCIDIAN DEVELOPMENT

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. IMPORTANCE OF PROTEOMIC APPROACHES IN ASCIDIAN DEVELOPMENTAL BIOLOGY
  5. TECHNICAL OVERVIEW OF PROTEOMICS
  6. ADVANCES IN SPERM PROTEOMICS FOR STUDYING THEIR MOLECULAR ARCHITECTURE AND FUNCTIONAL CHANGES DURING FERTILIZATION IN C. INTESTINALIS
  7. PROFILES OF PROTEIN EXPRESSION DURING CIONA DEVELOPMENT
  8. CIONA PROTEOMICS FOR THE COMPREHENSIVE UNDERSTANDING OF ASCIDIAN DEVELOPMENT
  9. Acknowledgements
  10. REFERENCES

As described above, the profiles of proteins expressed in embryos and adult tissues, along with their structural and functional information, should shed new light on the molecular mechanism of ascidian development. For the comprehensive description of protein expression during embryogenesis, proteomic analysis and a comparison of both abundant and minor proteins by 2DE would be a powerful strategy. Furthermore, ascidian embryos have been used as excellent model systems for studying maternal determinants through the dissection or dissociation of blastomeres. Ascidian embryos with defects in a certain gene or its expression could be experimentally obtained through chemical treatment (Katsuyama and Saiga,1998; Hinman and Degnan,1998; Kim and Nishida,2001), gene overexpression (e.g., Yoshida et al.,1996), and gene knockdown by a morpholino antisense oligo (e.g., Yamada et al.,2003). Mutagenesis by ENU (Nakatani et al.,1999; Moody et al.,1999) or insertion using Minos (Sasakura et al.,2003; Awazu et al.,2004; Matsuoka et al.,2004) results in the isolation of mutants lacking a certain gene, with consequent morphological or functional defects. The isolation or fractionation of structures that seem to be important in mosaic development (Marikawa et al.,1994; Hibino et al.,1998; Nishikata et al.,1999) and metamorphosis (Cloney,1979) would also prove to be interesting if combined with the proteomic approach. All of these experimental and genetic systems for ascidian research should be coupled with the latest proteomic techniques to identify the set of proteins responsible for tissue differentiation and morphogenesis.

In conclusion, the information from proteomic analysis is becoming indispensable to the study of the development mechanism of Ciona intestinalis at the cell biology level. This information promises to bring about progress in the understanding of the function of each protein in Ciona development, and further integration with the genome or cDNA database should establish the molecular basis for a comprehensive description of several interesting and important events in ascidian biology.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. IMPORTANCE OF PROTEOMIC APPROACHES IN ASCIDIAN DEVELOPMENTAL BIOLOGY
  5. TECHNICAL OVERVIEW OF PROTEOMICS
  6. ADVANCES IN SPERM PROTEOMICS FOR STUDYING THEIR MOLECULAR ARCHITECTURE AND FUNCTIONAL CHANGES DURING FERTILIZATION IN C. INTESTINALIS
  7. PROFILES OF PROTEIN EXPRESSION DURING CIONA DEVELOPMENT
  8. CIONA PROTEOMICS FOR THE COMPREHENSIVE UNDERSTANDING OF ASCIDIAN DEVELOPMENT
  9. Acknowledgements
  10. REFERENCES

We thank Dr. Toshifusa Toda for his contribution to the commencement of the Ciona proteome project. We also thank Yuhkoh Satouh, Yuji Ushimaru, Maiko Kaizu, Aru Konno, and Potturi Padma for their cooperation in the proteomic research. The proteomic work on the Ciona testis was supported in part by Grants-in-Aid to K.I. from MEXT, Japan. K.I. was also funded by a grant for Ciona protein database from JST-BIRD.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. IMPORTANCE OF PROTEOMIC APPROACHES IN ASCIDIAN DEVELOPMENTAL BIOLOGY
  5. TECHNICAL OVERVIEW OF PROTEOMICS
  6. ADVANCES IN SPERM PROTEOMICS FOR STUDYING THEIR MOLECULAR ARCHITECTURE AND FUNCTIONAL CHANGES DURING FERTILIZATION IN C. INTESTINALIS
  7. PROFILES OF PROTEIN EXPRESSION DURING CIONA DEVELOPMENT
  8. CIONA PROTEOMICS FOR THE COMPREHENSIVE UNDERSTANDING OF ASCIDIAN DEVELOPMENT
  9. Acknowledgements
  10. REFERENCES