- Email: [email protected]
Developmental gene activities in ascidian embryos
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Developmental gene activities in ascidian embryos Yutaka Satou and Nori Satoh* The fertilized egg of ascidians develops quickly into a tadpoletype larva consisting of several distinct types of tissues including epidermis, central nervous system, endoderm, mesenchyme, notochord, and muscle. This architecture of the ascidian larva represents the most simplified chordate body plan. Taking advantage of simple, well-defined cell lineages, the expression of developmental genes is analyzed at single-cell level. Advances in the methodology promote the ascidian embryo as a useful system for studying transcriptional control involved in the specification of embryonic cells and pattern formation of the embryo. Addresses Department of Zoology, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan *e-mail: [email protected] Correspondence: Nori Satoh Current Opinion in Genetics & Development 1999, 9:542–547 0959-437X/99/$ — see front matter © 1999 Elsevier Science Ltd. All rights reserved. Abbreviations bFGF basic fibroblast growth factor bHLH basic helix-loop-helix bmp-2/4 bone morphogenetic protein-2/4 CAB centrosome-attracting body CNS central nervous system pem posterior end mark SH3 Src-homology 3 Su(H) Suppressor-of-Hairless
Introduction Ascidians (sea squirts) are ubiquitous marine animals that have served for more than a century as a model system for classic embryological studies — in particular, the mosaic mode of embryogenesis [1–4]. Ascidian embryos exhibit bilateral cleavage and a highly determinate mode of development. Gastrulation initiates around the 118-cell stage, followed by neurulation which is accomplished by folding of the presumptive neural cells (as occurs in vertebrate embryos). The tailbud embryo is then formed and it eventually develops into a tadpole-type larva. The ascidian tadpole consists of ~2,600 cells that form several distinct types of tissues (Table 1). The tadpole is organized into a trunk and tail: the trunk contains a dorsal central nervous system (CNS) with two sensory organs (otolith and ocellus), endoderm, mesenchyme and trunk lateral cells, whereas the tail contains a notochord flanked dorsally by the nerve cord, ventrally by an endodermal strand, and bilaterally by three rows of muscle cells. The entire surface of the animal is covered by an epidermis. The ascidian larva represents the simplest chordate body plan. The complete ascidian cell lineage has been described up to the early gastrula stage [1,2]. In addition, the lineages of epidermis, CNS, notochord and muscle are documented.
The fate restriction in ascidian embryos takes place relatively early. At the 64-cell stage, blastomeres appear that are restricted to their various individual fates: epidermis, endoderm, notochord, muscle, nerve cord, mesenchyme and trunk lateral cells. Recent studies have provided convincing evidence for the specification patterns of embryonic cells [1,2]. As shown in Table 1, epidermal, endodermal and B-line muscle cells are specified autonomously as mediated by prelocalized egg cytoplasmic factors or determinants. In particular, the posterior–vegetal cytoplasm of the fertilized egg contains muscle determinants, factors for the anteroposterior axis establishment, and those for initiation of gastrulation [2]. On the other hand, cells of the CNS, notochord and mesenchyme are specified conditionally via cell–cell interactions. In addition, advances in the methodology promote the ascidian embryo as a useful model for studying transcriptional control involved in embryonic cell specification and pattern formation. Whole-mount in situ hybridization shows that the signal for zygotic transcription first appears in the nucleus of blastomeres of a defined lineage. Therefore, the spatio-temporal pattern of developmental gene expression is determined precisely at the single-cell level [5,6]. Overexpression of genes via RNA injection has been used to deduce their function [7], and antisense methods have been used to disrupt their function [8]. Electroporation and microinjection methods permit the efficient incorporation of transgenic DNA into developing embryos [9,10]. Transgenesis has been used to characterize cis-regulatory DNA elements that mediate tissue-specific and lineage-specific patterns of gene expression [9,10]. This technique is also useful for producing mutant phenotypes via ectopic expression of regulatory genes [11••]. Here, we summarize some recent advances in the regulation of developmental gene expression responsible for the formation of the ascidian tadpole.
Maternal genes with developmental functions As mentioned above, the ascidian egg contains prelocalized maternal factors responsible for differentiation of muscle, endoderm and epidermis, and factors for anteroposterior axis establishment [1,2]. Therefore, the molecular identification of the maternal factors, the elucidation of the machinery responsible for the localization, and the exploration of the mode of action of the factors are essential for the study of transcriptional regulation involved in the specification of embryonic cells and pattern formation of the embryo. Centrifugation of unfertilized Ciona savignyi eggs yielded four types of fragments: a large nucleated red fragment, and small enucleated black, clear and brown fragments. Fusion experiments of these fragments showed that factors for epidermis development are contained in red fragments,
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Table 1 Cell types, specification and specific genes in ascidian embryos. Cell types
No. of cells
Specification
Specific structural genes
Epidermal CNS
800 ~330
Autonomous Conditional
HrEpiA~H [59] TuNa1 [60] HrTBB2 [61]
Sensory pigment cell Endodermal Mesenchymal Trunk lateral Notochord
2
Conditional
Tyrosinase [63]
~500 ~900 ~32 40
Autonomous Conditional Conditional
Alkaline phosphatase [65] HrCA1 (cytoplasmic-actin) [66] Specific genes [67] Specific genes [11••]
β-catenin [13•]
Muscle B-line A- and b-line
42 (28) (4, 10)
HrMA4 [10] HrMHC [38]
MyoD (AMD1 [42], Ciona MyoD [43]) Snail (Ci-sna [35], Hrsna [39])
Autonomous Conditional
whereas factors for development of muscle and endoderm cells are found in black fragments [12]. Differential screening of cDNA libraries of red and black fragments identified a novel maternal gene named posterior end mark (pem), the transcript of which is initially concentrated in the posterior–vegetal cytoplasm of the fertilized egg, and later in the posterior end of developing embryos [7]. Overexpression of pem by RNA microinjection resulted in the development of larvae with deficiencies of the anterior-most adhesive organ, dorsal brain and sensory pigment cells, suggesting that pem is involved in pattern formation of the embryo [7]. Further experiments involving treatment of the egg with LiCl suggested that pem function involves the signaling pathways of the wnt/β-catenin cascade [13•]. In addition, pem-2, pem-4, pem-5, and pem-6 [14], and pem-3 [15] were isolated from a cDNA library of C. savignyi maternal mRNAs subtracted with gastrula mRNAs. PEM-2 contains a nuclear localization signal and an src-homology 3 (SH3) domain. PEM-3 contains two KH-domains, and is a putative homolog of Caenorhabditis elegans MEX-3 [16]. PEM-4 has a nuclear localization signal and three putative C2H2-type zinc-finger motifs. Furthermore, analysis of the cDNA of maternally expressed genes in Halocynthia roretzi demonstrated that maternal transcripts of HrWnt-5 [17] and a gene for serine/threonine kinase [18] are localized in the posterior cortex of the early embryo. Namely, most of the localized maternal transcripts identified to date show a localization pattern similar to that of pem, although some other genes (CsEndo1~3) have maternal mRNAs localized in the ‘endoplasm’ [19]; this suggests a common machinery for the localization of a variety of maternal mRNAs in the vegetal cortex. The centrosome-attracting body (CAB) is a unique structure in the posterior cortex of the posteriormost blastomeres [20•,21]. This structure is formed in the unequally dividing posterior-most blastomere pair and is responsible for this unequal division. The CAB is one of a few candidate structures that are involved in trapping of the
Genes for transcription factor and others
Hroth [50], HrHox1 [51], HrPax-258 [52••], HrPax-37 [53], Hrlim [54], Cihox5 [49], HrBMPa [62], HrBMPb [57], Notch [64], PPax-6 [55]
Brachyury (As-T [5], Ci-Bra [9]) Su(H) [30]
many maternal messages within the posterior cortex of the early embryo. Molgula oculata and M. occulta are closely related ascidian species with different larval forms [22]. M. oculata develops a conventional, tailed tadpole larva, whereas M. occulta produces a tailless larva. As the tailed species represents the ancestral condition in ascidians, ‘tail-forming’ genes might be downregulated in the tailless species. Differential screening of cDNA libraries of gonads of both species with a subtracted probe yielded three genes — Cymric, lynx and Manx — that are maternally expressed only in early embryos of the tailed species ([23]; KW Makabe et al., unpublished data). The Cymric gene encodes a Shark family protein-tyrosine kinase and the lynx gene encodes a leucine zipper protein. The Manx gene is expressed both maternally and zygotically and encodes a novel zinc-finger protein. Zygotic Manx expression is restricted to embryonic primordia that generate the CNS and tail tissues in the tailed species, is downregulated in the tailless species, and is restored in hybrids [8]. An antisense-mediated gene disruption method showed that Manx is required for development of the tailed phenotype [8]. Interestingly, the Manx gene is part of a locus of unusual organization: it also contains the DEAD box RNA helicase gene bobcat [24•]. Alternative splicing in the Manx–bobcat gene complex appears to be responsible for producing mature bobcat and Manx mRNAs. The bobcat gene is required for the same selection of developmental features as Manx, indicating that the Manx–bobcat gene locus plays a role in the development of tailed species features.
Genetic circuitry underlying notochord formation The ascidian tadpole contains a notochord comprising just 40 cells, of which lineage have been described completely [1,2]. As the notochord is the most characteristic feature of chordates, the elucidation of the genetic circuitry underlying its formation in ascidian embryos will lead to an insight of
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possible molecular developmental mechanisms of chordate evolution [3,25]. Brachyury encodes a sequence-specific activator that contains a T-box DNA-binding domain and is critical for notochord differentiation in vertebrate embryos [26,27]. Brachyury is expressed exclusively in the notochord precursor cells of two divergent ascidians, H. roretzi (As-T) [5] and C. intestinalis (Ci-Bra) [9]. The spatial and temporal patterns of expression coincide with the clonal restriction of the notochord lineages. In H. roretzi, notochord formation is induced at the 32-cell stage by signals emanating from the adjacent endoderm [28]. Overexpression of As-T via RNA injection results in notochord formation without a requirement for the inductive event at the 32-cell stage [29•]. In addition, misexpression of As-T [29•] as well as Ci-Bra [11••] causes transformation of endoderm and neuronal lineages into notochord, indicating that the ascidian Brachyury gene is a critical determinant of the notochord. Therefore, the upstream and downstream regulatory systems of the ascidian Brachyury genes should be investigated. Upstream of Ci-Bra and As-T
The 434 bp upstream promoter region of Ci-Bra contains three different cis-elements, that is, ectopic repression element, notochord activation element and ectopic activation element [9]. The notochord activation element contains two Suppressor-of-Hairless (Su[H]) binding sites, which are responsible for upregulation of the Ci-Bra gene [30]. There are two Ci-Sna-binding sites (the product of the C. intestinalis snail gene) neighbouring Su(H)-binding sites [31•]. It was shown that Ci-Sna binds to these sites and represses the expression of Ci-Bra in muscle cells, suggesting that Ci-Sna defines the boundary between muscle and notochord [31•]. On the other hand, bFGF (basic fibroblast growth factor) mediates notochord differentiation via activation of As-T in Halocynthia embryos [32]. Examination of the minimal promoter of As-T showed a module between –290 and –250 that promotes notochordspecific expression of the reporter gene [33]. Interestingly, the 5′ flanking region of As-T contains a potential T-binding motif (–ACCTAGGT–) [34] around –160, which is responsible for autoactivation of the gene. Superficially, these results suggest striking difference between the minimal promoters of Ci-Bra and As-T with respect to their notochord-specific expression. Downstream of Ci-Bra
The Ci-fkh is a fork head gene of C. intestinalis, which is expressed in the endodermal tissues, notochord and nerve cord [35]. With the aid of Ci-fkh promoter, Ci-Bra is misexpressed in the endodermal tissues. This causes transformation of endoderm cells into notochord cells [11••]. Subtractive hybridization screens were conducted to identify potential Brachyury target genes that are induced upon Ci-Bra misexpression. Of 501 independent cDNA clones that were surveyed, 38 were specifically expressed in notochord cells. These potential genes downstream of Ci-Bra genes appear to encode a broad spectrum of divergent proteins associated with notochord formation. Discrimination
between the direct and indirect targets and systematic analyses of the regulatory machinery of the downstream genes will provide a better understanding of the genetic circuitry underlying Brachyury-mediated notochord formation.
Gene regulation involved in muscle-cell differentiation In H. roretzi, 42 unicellular and striated muscle cells are formed in the larval tail. Lineage analysis shows that 28 muscle cells of anterior and middle tail parts are derived from B4.1 blastomeres of the 8-cell stage embryo. The B-line muscle cells have an extensive potential for autonomous differentiation dependent on muscle determinants in the ‘myoplasm’. The ascidian larval muscle cells therefore provide an experimental system with which to explore an intrinsic genetic program for autonomous specification of embryonic cells, from the beginning of maternal factor appearance until the activation of specific structural genes [36]. One of the features in ascidian muscle differentiation is very early expression of muscle-specific structural genes: muscle actin genes as well as myosin heavy chain gene are first expressed at the 32-cell stage [6]. Analysis of the minimal promoter of the H. roretzi actin gene HrMA4a, revealed that two short motifs (designated the B- and Mregions) are necessary and sufficient for muscle-specific expression of the reporter gene [10,37]. The sequences of the B- and M-regions are 5′–TCGCACTTC–3′ and 5′–GTGATAACAACTG–3′, respectively. The motifs were found in the minimal promoter of a muscle actin gene (CsMA1) of C. savignyi, suggesting the conservation between two divergent ascidian species of the upstream regulatory system of the genes (Y Satou, N Satoh, unpublished data). The B-region was also found in the promoter of Halocynthia myosin heavy chain gene [38]. Ascidian homologs of snail — Ci-Sna [31•] in C. intestinalis and HrSna [39] in H. roretzi — are expressed in the B-line muscle cells. The Ci-Sna expression is activated by a 500 bp B-linespecific enhancer [40]. The enhancer contains three AC-core E-boxes, two of which are critical for expression. The E-box (CANNTG) is known to be a binding site for bHLH transcription factors including MyoD. The M-region of the muscle actin gene also contains an E-box with an AC-core. The sequences (5′–GTGgcgACAACTG–3′ and 5′–GTGcagtACAACTG–3′) around the critical E-boxes in the Ci-Sna enhancer are similar to that of the motif of the actin genes, suggesting that the same factors bind to these elements to control the expression of the genes. In vertebrates, myogenic bHLH factors such as MyoD have a critical role in muscle differentiation [41]. Ascidian homologs of myogenic bHLH factors were identified in H. roretzi [42] and C. intestinalis [43], and they are single copy genes. The genes begin to be expressed around the 64-cell stage. As the expression of muscle-specific structural genes begins at the 32-cell stage, prior to that of the myogenic factor, it is likely that another bHLH or non-bHLH factor binds to this site to activate the musclespecific structural gene expression. MEF2 is essential for muscle differentiation in vertebrate embryos [41]. An ascidian
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Figure 1 Comparison of brain organization and gene expression between mouse and ascidian, indicating hypothesized homology of brain regions. The gene expression patterns suggest that the ascidian CNS is subdivided into at least three regions: the sensory vesicle or brain, the neck between the brain and visceral ganglion, and the visceral ganglion and the caudal nerve cord, which correspond to the forebrain and midbrain, the midbrain and an anterior part of hindbrain, and the hindbrain and spinal cord of the vertebrate central nervous system, respectively (reproduced with permission from [52••]).
Ascidian
Mouse
Forebrain
Otx
Sensory vesicle
Midbrain Pax-2/5/8
Neck
Hindbrain Visceral ganglion and tail nerve cord
Hox
Spinal cord
Hroth or Otx
Overlap of Otx and Pax-2/5
HrPax-258 or Pax-2/5/8
HrPax-37
HrHox1 or Hoxb-1
Hrlim Current Opinion in Genetics & Development
homolog (As-MEF2) of MEF2 is maternally expressed and the maternal mRNA does not show any localization pattern (I Araki et al., unpublished data). The function of these factors remains to be determined. The T-box genes encode a novel family of transcriptional factors that play a crucial roles in various developmental processes, in particular, mesoderm formation of chordate embryos [44]. In H. roretzi, three T-box genes identified are As-T for Brachyury cognate, As-T2 for Tbx6 cognate [45] and maternal T-box gene As-mT [46]. Interestingly, As-T is expressed exclusively in the notochord precursor cells whereas As-T2 is expressed in muscle precursor cells and cells in the tip of the tail. Misexpression of As-T2 induces an ectopic expression of the muscle actin gene as well as of the myosin heavy chain gene [47].
Pattern of gene expression in the ascidian CNS According to elaborated lineage studies [48], the complement of the C. intestinalis larval CNS numbers some 330 cells, comprising ~215 cells on the sensory vesicle or the brain, 50 cells in the visceral or tail ganglion or the brain stem, and ~65 ciliated ependymal cells of the caudal nerve cord or spinal cord. Recent characterization of developmental genes has presented evidence that the ascidian larval CNS has features common to the vertebrate CNS. An ascidian homolog of fork head/HNF-3β is expressed in a ventral row of nerve-cord cells, reminiscent of the floor plate of vertebrate embryos [35]. A C. intestinalis Hox-related gene Cihox5 is also involved in
nerve cord regionalization [49]. The expression patterns of ascidian homologs of Otx (Hroth) [50], Hoxb-1 (HrHox1) [51], Pax-2/5/8 (HrPax-258) [52••], Pax-3/7 (HrPax-37) [53], and Hrlim [54] are summarized in Figure 1. The patterns suggest that the ascidian CNS is subdivided into at least three regions, which are comparable to vertebrate counterparts: the sensory vesicle or the brain corresponds to the forebrain and midbrain, the neck between the brain and visceral ganglion is comparable to the midbrain and an anterior part of hindbrain, and the visceral ganglion and the caudal nerve cord correspond to the hindbrain and spinal cord [52••]. An ascidian Pax-6 (PPax-6) is also expressed in cells of sensory pigment cell lineage [55]. Differentiation of the ascidian CNS requires an inductive event during gastrulation [1,2]. Overexpression of HrPax-37 induces ectopic expression of the dorsal neural marker tyrosinase gene, although it cannot completely dorsalize the CNS [56]. An ascidian homolog (HrBMPb) of bmp-2/4 (bone morphogenetic protein-2/4) does not function as a ventralizing factor in early embryos but functions as a neural inhibitor and an epidermal inducer [57]. These and other studies suggest that the mechanism involved in specification of nerve cells is also conserved between ascidians and vertebrates. The small number of constituent cells in addition to their well-defined lineage may promote the ascidian embryo as a model system to explore molecular mechanisms underlying the formation of CNS of higher vertebrates.
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Conclusions A recent estimation suggests that C. intestinalis possesses a small, compact genome (~180 Mb and 15,500 genes) [58] which is comparable in size to those of Drosophila melanogaster and C. elegans. A harmony of developmental gene expression manifests the architecture of the ascidian tadpole, which represents the most simplified chordate body plan. The small number of constituent cells as well as a welldocumented cell lineage enable the easy analysis of the expression pattern of developmental genes at the single-cell level. Together with advances in methods to deduce their function, the ascidian embryo may offer an experimental system to explore the entire genetic cascade from the egg cytoplasmic determinants to tissue-specific gene expression.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest •• of outstanding interest
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58. Simmen MW, Leitgeb S, Clark VH, Jones SJ: Gene number in an invertebrate chordate, Ciona intestinalis. Proc Natl Acad Sci USA 1998, 95:4437-4440. 59. Ueki T, Yoshida S, Marikawa Y, Satoh N: Autonomy of expression of epidermis-specific genes in the ascidian embryo. Dev Biol 1994, 164:207-218. 60. Okamaura Y, Ono F, Okazaki R, Chong JA, Mandel G: Neural expression of a sodium channel gene requires cell-specific interactions. Neuron 1994, 13:937-948.
44. Papaioannou VE, Silver LM: The T-box gene family. Bioessays 1998, 20:9-19.
61. Miya T, Satoh N: Isolation and characterization of cDNA clones for β-tubulin genes as a molecular marker for neural cell differentiation in the ascidian embryo. Int J Dev Biol 1997, 41:551-557.
45. Yasuo H, Kobayashi M, Shimauchi Y, Satoh N: The ascidian genome contains another T-domain gene that is expressed in differentiating muscle and the tip of the tail of the embryo. Dev Biol 1996, 180:773-779.
62. Miya T, Morita K, Ueno N, Satoh N: 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 1996, 57:181-190.
46. Takada N, Tagawa K, Takahashi H, Satoh N: Characterization of an ascidian maternal T-box gene, As-mT. Int J Dev Biol 1998, 42:1093-1100.
63. Sato S, Masuya H, Numakunai T, Satoh N, Ikeo K, Gojobori T, Tamura K, Ide H, Takeuchi T, Yamamoto H: Ascidian tyrosinase gene: its unique structure and expression in the developing brain. Dev Dynamics 1997, 208:363-374.
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Mitani Y, Takahashi H, Satoh N: An ascidian T-box gene As-T2 is related to the Tbx6 subfamily and is associated with embryonic muscle cell differentiation. Dev Dyn 1999, 215:62-68.
48. Nicol DR, Meinertzhagen IA: Development of the central nervous system of the larva of the ascidian, Ciona intestinalis L. II. Neural plate morphogenesis and cell lineages during neurulation. Dev Biol 1988, 130:737-766. 49. Gionti M, Ristoratore F, Di Gregorio A, Aniello F, Branno M, Di Lauro R: Cihox5, a new Ciona intestinalis Hox-related gene, is involved in regionalization of the spinal cord. Dev Genes Evol 1998, 207:515523. 50. Wada S, Katsuyama Y, Sato Y, Itoh C, Saiga H: Hroth an orthodenticle-related homeobox gene of the ascidian, Halocynthia roretzi: its expression and putative roles in the axis formation during embryogenesis. Mech Dev 1996, 60:59-71.
64. Hori S, Saitoh T, Matsumoto M, Makabe KW, Nishida H: Notch homologue from Halocynthia roretzi is preferentially expressed in the central nervous system during ascidian embryogenesis. Dev Genes Evol 1997, 207:371-380. 65. Kumano G, Nishida H: Maternal and zygotic expression of the endoderm-specific alkaline phosphatase gene in embryos of the ascidian, Halocynthia roretzi. Dev Biol 1998, 198:245-252. 66. Araki I, Tagawa K, Kusakabe T, Satoh N: Predominant expression of a cytoskeletal actin gene in mesenchyme cells during embryogenesis of the ascidian Halocynthia roretzi. Dev Growth Differentiation 1996, 38:401-411. 67.
Takahashi H, Ishida K, Makabe KW, Satoh N: Isolation of cDNA clones for genes that are expressed in the tail region of the ascidian tailbud embryo. Int J Dev Biol 1997, 41:691-698.
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Developmental gene activities in ascidian embryos Yutaka Satou and Nori Satoh* The fertilized egg of ascidians develops quickly into a tadpoletype larva consisting of several distinct types of tissues including epidermis, central nervous system, endoderm, mesenchyme, notochord, and muscle. This architecture of the ascidian larva represents the most simplified chordate body plan. Taking advantage of simple, well-defined cell lineages, the expression of developmental genes is analyzed at single-cell level. Advances in the methodology promote the ascidian embryo as a useful system for studying transcriptional control involved in the specification of embryonic cells and pattern formation of the embryo. Addresses Department of Zoology, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan *e-mail: [email protected] Correspondence: Nori Satoh Current Opinion in Genetics & Development 1999, 9:542–547 0959-437X/99/$ — see front matter © 1999 Elsevier Science Ltd. All rights reserved. Abbreviations bFGF basic fibroblast growth factor bHLH basic helix-loop-helix bmp-2/4 bone morphogenetic protein-2/4 CAB centrosome-attracting body CNS central nervous system pem posterior end mark SH3 Src-homology 3 Su(H) Suppressor-of-Hairless
Introduction Ascidians (sea squirts) are ubiquitous marine animals that have served for more than a century as a model system for classic embryological studies — in particular, the mosaic mode of embryogenesis [1–4]. Ascidian embryos exhibit bilateral cleavage and a highly determinate mode of development. Gastrulation initiates around the 118-cell stage, followed by neurulation which is accomplished by folding of the presumptive neural cells (as occurs in vertebrate embryos). The tailbud embryo is then formed and it eventually develops into a tadpole-type larva. The ascidian tadpole consists of ~2,600 cells that form several distinct types of tissues (Table 1). The tadpole is organized into a trunk and tail: the trunk contains a dorsal central nervous system (CNS) with two sensory organs (otolith and ocellus), endoderm, mesenchyme and trunk lateral cells, whereas the tail contains a notochord flanked dorsally by the nerve cord, ventrally by an endodermal strand, and bilaterally by three rows of muscle cells. The entire surface of the animal is covered by an epidermis. The ascidian larva represents the simplest chordate body plan. The complete ascidian cell lineage has been described up to the early gastrula stage [1,2]. In addition, the lineages of epidermis, CNS, notochord and muscle are documented.
The fate restriction in ascidian embryos takes place relatively early. At the 64-cell stage, blastomeres appear that are restricted to their various individual fates: epidermis, endoderm, notochord, muscle, nerve cord, mesenchyme and trunk lateral cells. Recent studies have provided convincing evidence for the specification patterns of embryonic cells [1,2]. As shown in Table 1, epidermal, endodermal and B-line muscle cells are specified autonomously as mediated by prelocalized egg cytoplasmic factors or determinants. In particular, the posterior–vegetal cytoplasm of the fertilized egg contains muscle determinants, factors for the anteroposterior axis establishment, and those for initiation of gastrulation [2]. On the other hand, cells of the CNS, notochord and mesenchyme are specified conditionally via cell–cell interactions. In addition, advances in the methodology promote the ascidian embryo as a useful model for studying transcriptional control involved in embryonic cell specification and pattern formation. Whole-mount in situ hybridization shows that the signal for zygotic transcription first appears in the nucleus of blastomeres of a defined lineage. Therefore, the spatio-temporal pattern of developmental gene expression is determined precisely at the single-cell level [5,6]. Overexpression of genes via RNA injection has been used to deduce their function [7], and antisense methods have been used to disrupt their function [8]. Electroporation and microinjection methods permit the efficient incorporation of transgenic DNA into developing embryos [9,10]. Transgenesis has been used to characterize cis-regulatory DNA elements that mediate tissue-specific and lineage-specific patterns of gene expression [9,10]. This technique is also useful for producing mutant phenotypes via ectopic expression of regulatory genes [11••]. Here, we summarize some recent advances in the regulation of developmental gene expression responsible for the formation of the ascidian tadpole.
Maternal genes with developmental functions As mentioned above, the ascidian egg contains prelocalized maternal factors responsible for differentiation of muscle, endoderm and epidermis, and factors for anteroposterior axis establishment [1,2]. Therefore, the molecular identification of the maternal factors, the elucidation of the machinery responsible for the localization, and the exploration of the mode of action of the factors are essential for the study of transcriptional regulation involved in the specification of embryonic cells and pattern formation of the embryo. Centrifugation of unfertilized Ciona savignyi eggs yielded four types of fragments: a large nucleated red fragment, and small enucleated black, clear and brown fragments. Fusion experiments of these fragments showed that factors for epidermis development are contained in red fragments,
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Table 1 Cell types, specification and specific genes in ascidian embryos. Cell types
No. of cells
Specification
Specific structural genes
Epidermal CNS
800 ~330
Autonomous Conditional
HrEpiA~H [59] TuNa1 [60] HrTBB2 [61]
Sensory pigment cell Endodermal Mesenchymal Trunk lateral Notochord
2
Conditional
Tyrosinase [63]
~500 ~900 ~32 40
Autonomous Conditional Conditional
Alkaline phosphatase [65] HrCA1 (cytoplasmic-actin) [66] Specific genes [67] Specific genes [11••]
β-catenin [13•]
Muscle B-line A- and b-line
42 (28) (4, 10)
HrMA4 [10] HrMHC [38]
MyoD (AMD1 [42], Ciona MyoD [43]) Snail (Ci-sna [35], Hrsna [39])
Autonomous Conditional
whereas factors for development of muscle and endoderm cells are found in black fragments [12]. Differential screening of cDNA libraries of red and black fragments identified a novel maternal gene named posterior end mark (pem), the transcript of which is initially concentrated in the posterior–vegetal cytoplasm of the fertilized egg, and later in the posterior end of developing embryos [7]. Overexpression of pem by RNA microinjection resulted in the development of larvae with deficiencies of the anterior-most adhesive organ, dorsal brain and sensory pigment cells, suggesting that pem is involved in pattern formation of the embryo [7]. Further experiments involving treatment of the egg with LiCl suggested that pem function involves the signaling pathways of the wnt/β-catenin cascade [13•]. In addition, pem-2, pem-4, pem-5, and pem-6 [14], and pem-3 [15] were isolated from a cDNA library of C. savignyi maternal mRNAs subtracted with gastrula mRNAs. PEM-2 contains a nuclear localization signal and an src-homology 3 (SH3) domain. PEM-3 contains two KH-domains, and is a putative homolog of Caenorhabditis elegans MEX-3 [16]. PEM-4 has a nuclear localization signal and three putative C2H2-type zinc-finger motifs. Furthermore, analysis of the cDNA of maternally expressed genes in Halocynthia roretzi demonstrated that maternal transcripts of HrWnt-5 [17] and a gene for serine/threonine kinase [18] are localized in the posterior cortex of the early embryo. Namely, most of the localized maternal transcripts identified to date show a localization pattern similar to that of pem, although some other genes (CsEndo1~3) have maternal mRNAs localized in the ‘endoplasm’ [19]; this suggests a common machinery for the localization of a variety of maternal mRNAs in the vegetal cortex. The centrosome-attracting body (CAB) is a unique structure in the posterior cortex of the posteriormost blastomeres [20•,21]. This structure is formed in the unequally dividing posterior-most blastomere pair and is responsible for this unequal division. The CAB is one of a few candidate structures that are involved in trapping of the
Genes for transcription factor and others
Hroth [50], HrHox1 [51], HrPax-258 [52••], HrPax-37 [53], Hrlim [54], Cihox5 [49], HrBMPa [62], HrBMPb [57], Notch [64], PPax-6 [55]
Brachyury (As-T [5], Ci-Bra [9]) Su(H) [30]
many maternal messages within the posterior cortex of the early embryo. Molgula oculata and M. occulta are closely related ascidian species with different larval forms [22]. M. oculata develops a conventional, tailed tadpole larva, whereas M. occulta produces a tailless larva. As the tailed species represents the ancestral condition in ascidians, ‘tail-forming’ genes might be downregulated in the tailless species. Differential screening of cDNA libraries of gonads of both species with a subtracted probe yielded three genes — Cymric, lynx and Manx — that are maternally expressed only in early embryos of the tailed species ([23]; KW Makabe et al., unpublished data). The Cymric gene encodes a Shark family protein-tyrosine kinase and the lynx gene encodes a leucine zipper protein. The Manx gene is expressed both maternally and zygotically and encodes a novel zinc-finger protein. Zygotic Manx expression is restricted to embryonic primordia that generate the CNS and tail tissues in the tailed species, is downregulated in the tailless species, and is restored in hybrids [8]. An antisense-mediated gene disruption method showed that Manx is required for development of the tailed phenotype [8]. Interestingly, the Manx gene is part of a locus of unusual organization: it also contains the DEAD box RNA helicase gene bobcat [24•]. Alternative splicing in the Manx–bobcat gene complex appears to be responsible for producing mature bobcat and Manx mRNAs. The bobcat gene is required for the same selection of developmental features as Manx, indicating that the Manx–bobcat gene locus plays a role in the development of tailed species features.
Genetic circuitry underlying notochord formation The ascidian tadpole contains a notochord comprising just 40 cells, of which lineage have been described completely [1,2]. As the notochord is the most characteristic feature of chordates, the elucidation of the genetic circuitry underlying its formation in ascidian embryos will lead to an insight of
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possible molecular developmental mechanisms of chordate evolution [3,25]. Brachyury encodes a sequence-specific activator that contains a T-box DNA-binding domain and is critical for notochord differentiation in vertebrate embryos [26,27]. Brachyury is expressed exclusively in the notochord precursor cells of two divergent ascidians, H. roretzi (As-T) [5] and C. intestinalis (Ci-Bra) [9]. The spatial and temporal patterns of expression coincide with the clonal restriction of the notochord lineages. In H. roretzi, notochord formation is induced at the 32-cell stage by signals emanating from the adjacent endoderm [28]. Overexpression of As-T via RNA injection results in notochord formation without a requirement for the inductive event at the 32-cell stage [29•]. In addition, misexpression of As-T [29•] as well as Ci-Bra [11••] causes transformation of endoderm and neuronal lineages into notochord, indicating that the ascidian Brachyury gene is a critical determinant of the notochord. Therefore, the upstream and downstream regulatory systems of the ascidian Brachyury genes should be investigated. Upstream of Ci-Bra and As-T
The 434 bp upstream promoter region of Ci-Bra contains three different cis-elements, that is, ectopic repression element, notochord activation element and ectopic activation element [9]. The notochord activation element contains two Suppressor-of-Hairless (Su[H]) binding sites, which are responsible for upregulation of the Ci-Bra gene [30]. There are two Ci-Sna-binding sites (the product of the C. intestinalis snail gene) neighbouring Su(H)-binding sites [31•]. It was shown that Ci-Sna binds to these sites and represses the expression of Ci-Bra in muscle cells, suggesting that Ci-Sna defines the boundary between muscle and notochord [31•]. On the other hand, bFGF (basic fibroblast growth factor) mediates notochord differentiation via activation of As-T in Halocynthia embryos [32]. Examination of the minimal promoter of As-T showed a module between –290 and –250 that promotes notochordspecific expression of the reporter gene [33]. Interestingly, the 5′ flanking region of As-T contains a potential T-binding motif (–ACCTAGGT–) [34] around –160, which is responsible for autoactivation of the gene. Superficially, these results suggest striking difference between the minimal promoters of Ci-Bra and As-T with respect to their notochord-specific expression. Downstream of Ci-Bra
The Ci-fkh is a fork head gene of C. intestinalis, which is expressed in the endodermal tissues, notochord and nerve cord [35]. With the aid of Ci-fkh promoter, Ci-Bra is misexpressed in the endodermal tissues. This causes transformation of endoderm cells into notochord cells [11••]. Subtractive hybridization screens were conducted to identify potential Brachyury target genes that are induced upon Ci-Bra misexpression. Of 501 independent cDNA clones that were surveyed, 38 were specifically expressed in notochord cells. These potential genes downstream of Ci-Bra genes appear to encode a broad spectrum of divergent proteins associated with notochord formation. Discrimination
between the direct and indirect targets and systematic analyses of the regulatory machinery of the downstream genes will provide a better understanding of the genetic circuitry underlying Brachyury-mediated notochord formation.
Gene regulation involved in muscle-cell differentiation In H. roretzi, 42 unicellular and striated muscle cells are formed in the larval tail. Lineage analysis shows that 28 muscle cells of anterior and middle tail parts are derived from B4.1 blastomeres of the 8-cell stage embryo. The B-line muscle cells have an extensive potential for autonomous differentiation dependent on muscle determinants in the ‘myoplasm’. The ascidian larval muscle cells therefore provide an experimental system with which to explore an intrinsic genetic program for autonomous specification of embryonic cells, from the beginning of maternal factor appearance until the activation of specific structural genes [36]. One of the features in ascidian muscle differentiation is very early expression of muscle-specific structural genes: muscle actin genes as well as myosin heavy chain gene are first expressed at the 32-cell stage [6]. Analysis of the minimal promoter of the H. roretzi actin gene HrMA4a, revealed that two short motifs (designated the B- and Mregions) are necessary and sufficient for muscle-specific expression of the reporter gene [10,37]. The sequences of the B- and M-regions are 5′–TCGCACTTC–3′ and 5′–GTGATAACAACTG–3′, respectively. The motifs were found in the minimal promoter of a muscle actin gene (CsMA1) of C. savignyi, suggesting the conservation between two divergent ascidian species of the upstream regulatory system of the genes (Y Satou, N Satoh, unpublished data). The B-region was also found in the promoter of Halocynthia myosin heavy chain gene [38]. Ascidian homologs of snail — Ci-Sna [31•] in C. intestinalis and HrSna [39] in H. roretzi — are expressed in the B-line muscle cells. The Ci-Sna expression is activated by a 500 bp B-linespecific enhancer [40]. The enhancer contains three AC-core E-boxes, two of which are critical for expression. The E-box (CANNTG) is known to be a binding site for bHLH transcription factors including MyoD. The M-region of the muscle actin gene also contains an E-box with an AC-core. The sequences (5′–GTGgcgACAACTG–3′ and 5′–GTGcagtACAACTG–3′) around the critical E-boxes in the Ci-Sna enhancer are similar to that of the motif of the actin genes, suggesting that the same factors bind to these elements to control the expression of the genes. In vertebrates, myogenic bHLH factors such as MyoD have a critical role in muscle differentiation [41]. Ascidian homologs of myogenic bHLH factors were identified in H. roretzi [42] and C. intestinalis [43], and they are single copy genes. The genes begin to be expressed around the 64-cell stage. As the expression of muscle-specific structural genes begins at the 32-cell stage, prior to that of the myogenic factor, it is likely that another bHLH or non-bHLH factor binds to this site to activate the musclespecific structural gene expression. MEF2 is essential for muscle differentiation in vertebrate embryos [41]. An ascidian
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Figure 1 Comparison of brain organization and gene expression between mouse and ascidian, indicating hypothesized homology of brain regions. The gene expression patterns suggest that the ascidian CNS is subdivided into at least three regions: the sensory vesicle or brain, the neck between the brain and visceral ganglion, and the visceral ganglion and the caudal nerve cord, which correspond to the forebrain and midbrain, the midbrain and an anterior part of hindbrain, and the hindbrain and spinal cord of the vertebrate central nervous system, respectively (reproduced with permission from [52••]).
Ascidian
Mouse
Forebrain
Otx
Sensory vesicle
Midbrain Pax-2/5/8
Neck
Hindbrain Visceral ganglion and tail nerve cord
Hox
Spinal cord
Hroth or Otx
Overlap of Otx and Pax-2/5
HrPax-258 or Pax-2/5/8
HrPax-37
HrHox1 or Hoxb-1
Hrlim Current Opinion in Genetics & Development
homolog (As-MEF2) of MEF2 is maternally expressed and the maternal mRNA does not show any localization pattern (I Araki et al., unpublished data). The function of these factors remains to be determined. The T-box genes encode a novel family of transcriptional factors that play a crucial roles in various developmental processes, in particular, mesoderm formation of chordate embryos [44]. In H. roretzi, three T-box genes identified are As-T for Brachyury cognate, As-T2 for Tbx6 cognate [45] and maternal T-box gene As-mT [46]. Interestingly, As-T is expressed exclusively in the notochord precursor cells whereas As-T2 is expressed in muscle precursor cells and cells in the tip of the tail. Misexpression of As-T2 induces an ectopic expression of the muscle actin gene as well as of the myosin heavy chain gene [47].
Pattern of gene expression in the ascidian CNS According to elaborated lineage studies [48], the complement of the C. intestinalis larval CNS numbers some 330 cells, comprising ~215 cells on the sensory vesicle or the brain, 50 cells in the visceral or tail ganglion or the brain stem, and ~65 ciliated ependymal cells of the caudal nerve cord or spinal cord. Recent characterization of developmental genes has presented evidence that the ascidian larval CNS has features common to the vertebrate CNS. An ascidian homolog of fork head/HNF-3β is expressed in a ventral row of nerve-cord cells, reminiscent of the floor plate of vertebrate embryos [35]. A C. intestinalis Hox-related gene Cihox5 is also involved in
nerve cord regionalization [49]. The expression patterns of ascidian homologs of Otx (Hroth) [50], Hoxb-1 (HrHox1) [51], Pax-2/5/8 (HrPax-258) [52••], Pax-3/7 (HrPax-37) [53], and Hrlim [54] are summarized in Figure 1. The patterns suggest that the ascidian CNS is subdivided into at least three regions, which are comparable to vertebrate counterparts: the sensory vesicle or the brain corresponds to the forebrain and midbrain, the neck between the brain and visceral ganglion is comparable to the midbrain and an anterior part of hindbrain, and the visceral ganglion and the caudal nerve cord correspond to the hindbrain and spinal cord [52••]. An ascidian Pax-6 (PPax-6) is also expressed in cells of sensory pigment cell lineage [55]. Differentiation of the ascidian CNS requires an inductive event during gastrulation [1,2]. Overexpression of HrPax-37 induces ectopic expression of the dorsal neural marker tyrosinase gene, although it cannot completely dorsalize the CNS [56]. An ascidian homolog (HrBMPb) of bmp-2/4 (bone morphogenetic protein-2/4) does not function as a ventralizing factor in early embryos but functions as a neural inhibitor and an epidermal inducer [57]. These and other studies suggest that the mechanism involved in specification of nerve cells is also conserved between ascidians and vertebrates. The small number of constituent cells in addition to their well-defined lineage may promote the ascidian embryo as a model system to explore molecular mechanisms underlying the formation of CNS of higher vertebrates.
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Conclusions A recent estimation suggests that C. intestinalis possesses a small, compact genome (~180 Mb and 15,500 genes) [58] which is comparable in size to those of Drosophila melanogaster and C. elegans. A harmony of developmental gene expression manifests the architecture of the ascidian tadpole, which represents the most simplified chordate body plan. The small number of constituent cells as well as a welldocumented cell lineage enable the easy analysis of the expression pattern of developmental genes at the single-cell level. Together with advances in methods to deduce their function, the ascidian embryo may offer an experimental system to explore the entire genetic cascade from the egg cytoplasmic determinants to tissue-specific gene expression.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
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38. Araki I, Satoh N: cis-Regulatory elements conserved in the proximal promoter region of an ascidian embryonic muscle myosin heavy-chain gene. Dev Genes Evolution 1996, 206:54-63.
55. Glardon S, Callaerts P, Halder G, Gehring WJ: Conservation of Pax6 in a lower chordate, the ascidian Phallusia mammillata. Development 1997, 124:817-825.
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63. Sato S, Masuya H, Numakunai T, Satoh N, Ikeo K, Gojobori T, Tamura K, Ide H, Takeuchi T, Yamamoto H: Ascidian tyrosinase gene: its unique structure and expression in the developing brain. Dev Dynamics 1997, 208:363-374.
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