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Patterning the marginal zone of early ascidian embryos: localized maternal mRNA and inductive interactions Hiroki Nishida Summary Early animal embryos are patterned by localized egg cytoplasmic factors and cell interactions. In invertebrate chordate ascidians, larval tail muscle originates from the posterior marginal zone of the early embryo. It has recently been demonstrated that maternal macho-1 mRNA encoding transcription factor acts as a localized muscle determinant. Other mesodermal tissues such as notochord and mesenchyme are also derived from the vegetal marginal zone. In contrast, formation of these tissues requires induction from endoderm precursors at the 32-cell stage. FGF–Ras–MAPK signaling is involved in the induction of both tissues. The responsiveness for induction to notochord or mesenchyme depends on the inheritance of localized egg cytoplasmic factors. Previous studies also point to critical roles of directed signaling in polarization of induced cells and in subsequent asymmetric divisions resulting in the formation of two daughter cells with distinct fates. One cell adopts an induced fate, while the other assumes a default fate. A simple model of mesoderm patterning in ascidian embryos is proposed in comparison with that of vertebrates. BioEssays 24:613–624, 2002. ß 2002 Wiley Periodicals, Inc. Introduction Recent research using ascidian embryos has yielded insight into the mechanisms that mediate fate specification during early embryogenesis. Ascidians are simple chordates (Urochordata, Ascidiacea) and their embryogenesis shows the
Department of Biological Sciences, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama 226-8501, Japan. E-mail:
[email protected] Funding agencies: JSPS, HFSP, The Ministry of Education, Sports and Culture of Japan. DOI 10.1002/bies.10099 Published online in Wiley InterScience (www.interscience.wiley.com).
Abbreviations: macho-1, maboya no cho omoshiroi idenshi-1 in Japanese; EST, expression sequence tag; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/ extracellular signal-regulated kinase kinase: FGF, fibroblast growth factor; PVC, posterior-vegetal cytoplasm
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following characteristics (Fig. 1).(1–6) (1) Eggs develop into tadpole larvae that have the basic body plan of chordates, characterized by a dorsal neural tube and tail containing a notochord flanked by bilateral muscle tissue.(7) (2) Their organization is much simpler than that of a vertebrate. They consist of a small number of cells and cell types, and cell lineages are invariant among individuals. (3) Fate restriction of blastomeres to a single cell type is almost completed as early as the 110-cell stage.(8) (4) Precursor blastomeres of several cell types show cell-autonomous development which led to the term ‘mosaic’ development more than a century ago.(9) Thus, ascidian embryogenesis is characterized by simplicity, a feature that may enable the mechanisms of cell fate specification for the entire embryo and for every cell type at both the cellular and molecular level to be understood. Recent molecular analysis together with significant accumulation of knowledge revealed by classic experimental embryology have advanced our understanding of how developmental fates of blastomeres are specified in early ascidian embryos. Large-scale EST analysis accompanied by descriptions of the expression patterns of each gene has been carried out,(10,11) and, currently, genome sequencing projects are underway. Thus the ascidian is being recognized as a model organism in developmental biology. This article reviews the mechanisms of fate specification of mesodermal tissues in the marginal zone, emphasizing the involvement of localized maternal mRNA and inductive cell interactions. Mesodermal tissues originate from the marginal zone of the vegetal hemisphere As in frog embryos, ectoderm, mesoderm and endoderm territories are present in this order along the animal–vegetal axis of the fate map (see Fig. 4). Most animal blastomeres give rise to epidermis whose fate is specified by an unknown localized ooplasmic factor.(12) The anteriormost region of the animal hemisphere develops into brain through formation of the neural tube, and inductive interactions are required for brain formation.(13) Endoderm is derived from the vegetal pole region, whose fate is also specified by an unknown localized ooplasmic factor.(14) A recent study has indicated that bcatenin plays an important role in endoderm fate specification in ascidians.(15) b-catenin signaling pathway plays crucial
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Figure 1. Embryogenesis of the ascidian, Halocynthia roretzi. A: Adult animal. The size is approximately 15 cm. (courtesy of Dr. T. Numakunai) B: Fertilized egg. The diameter is 280 mm. C: 64-cell-stage embryo just before start of gastrulation. Vegetal view; anterior is up. D: Neurula. Anterior is to the right. Neural tube is closing from the posterior side. E: Initial tailbud. Each constituent cell is still visible. F: Tadpole larva just before hatching, 35 h after fertilization. It consists of approximately 3000 cells.
roles in maternal mechanisms that specify the dorsal–ventral axis in amphibian and fish,(16–18) and animal–vegetal axis in sea urchin.(19–21) In this regard, ascidian embryos show similarity to echinoderm embryos. The marginal zone of the vegetal hemisphere, which surrounds the central endodermal area, is mesodermal territory. The major mesodermal tissues are muscle, notochord and mesenchyme. Mesenchyme cells are preserved in the larvae, and give rise to tunic cells after metamorphosis.(22) The minor mesodermal tissues consist of trunk lateral cells and trunk ventral cells. They are precursor cells of the body wall muscle, heart and blood cells of the metamorphosed juvenile.(22) Localization of muscle determinants in the egg cytoplasm Maternal information stored in particular regions of the egg cytoplasm plays an important role in the determination of developmental fates during early animal development. The partitioning of colored egg cytoplasm into specific lineage blastomeres,(9) the autonomous differentiation of isolated and dissociated blastomeres,(23,24) and the results of transplantation of ooplasm from specific regions(3) have revealed the presence and localization of maternal determinants in the ascidian. These localized maternal determinants, which
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specify epidermis, muscle and endoderm fates, have provided an understanding of the mosaic manner of development. A great deal of interest has been concentrated on mechanisms underlying the formation of muscle cells in the larval tail, since Conklin reported in 1905 that yellow-colored myoplasm in the eggs of some species is preferentially segregated into muscle-lineage blastomeres.(9) Ooplasmic transplantation experiments have indicated that muscle determinants are present as a gradient in unfertilized eggs with the highest activity at the vegetal pole (Fig. 2A). Just after fertilization, these determinants are concentrated at the vegetal pole, and then move to the future posterior pole during ooplasmic segregation. Thus, they settle at sites that correspond to the appropriate region in the future fate map before cleavage starts. Muscle determinants are partitioned into muscle progenitor blastomeres during subsequent cleavages.(25,26) The distribution of cytoplasm that promotes muscle formation coincides with that of Conklin’s myoplasm, which is a cytoplasmic domain enriched in mitochondria, endoplasmic reticulum , cytoskeleton, and pigment granules.(27,28) macho-1 maternal mRNA as a muscle determinant A recent molecular study has identified a strong candidate for the localized maternal determinants of muscle formation in the
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Figure 2. Distribution of cytoplasmic determinants and macho-1 maternal mRNA in eggs. A: Distribution of muscle determinant during ooplasmic segregation revealed by cytoplasmic transplantation experiments. Animal pole is up and vegetal pole is down. Anterior is to the left and posterior is to the right. B: Distribution of maternal macho-1 mRNA shown by in situ hybridization at stages corresponding to A. Ani, animal pole. Veg, vegetal pole. A, anterior. P, posterior. (From Nishida H, and Sawada K. Nature 2001;409:724–729, with permission of Macmillan Magazines Ltd.)(29)
ascidian, Halocynthia roretzi.(29) In that study, a macho-1 cDNA clone was isolated by subtraction hybridization screening between the animal and vegetal hemispheres of the 8-cell embryo, and the distribution of maternal macho-1 mRNA in eggs (Fig. 2B) was found to correspond closely to the distribution of muscle determinants (Fig. 2A). Maternal macho-1 mRNA was then depleted by injection of antisense phosphorothioate oligodeoxynucleotides. The macho-1-depleted eggs showed normal ooplasmic segregation and cleavages. The eggs underwent gastrulation, and embryogenesis appeared normal up to the neurula stage. However, in tailbud embryos, tail formation was severely affected. At hatching, the trunk region appeared normal but the tail was shortened (Fig. 3A,B). The formation of most tissues in macho-1-depleted larvae, epidermis, sensory pigment cells, notochord and endoderm, was normal. However, the tail muscle cells were greatly reduced as shown by monitoring the expression of the muscle markers myosin, acetylcholinesterase, and actin (Fig. 3C,D,H,I). Although muscle was reduced, some muscle cells were always present at the tip of the tail. There are two types of muscle cell in the larval tail: primary and secondary muscle cells. Formation of the primary muscle
shows cell autonomy, and its fate is specified by localized muscle determinants. In contrast, the secondary muscle cells located at the tip of the tail are specified through cell interactions during gastrulation.(30,31) Experiments involving isolation of the primary muscle precursor blastomeres in macho-1depleted embryos indicated that only primary muscle cells were lost (Fig. 3E,F). Injection of synthetic macho-1 mRNA into macho-1 deficient embryos restored muscle formation. In a further experiment, injection of synthetic macho-1 mRNA caused ectopic muscle formation in non-musclelineage cells (Fig. 3G,J). These results indicate that macho-1 is both required and sufficient for specification of muscle fate. However, these criteria are not enough to confirm conclusively that macho-1 is the localized muscle determinant. For example, in the frog egg, b-catenin is required and sufficient for promoting the development of dorsal structures. But when the dorsal cytoplasm of b-catenin-depleted eggs is transferred to the ventral side of the intact egg, a secondary dorsal axis is still induced.(32) This observation indicates that b-catenin functions as a component of the machinery transducing the dorsal determinant, but is not the dorsal determinant itself. A similar experiment was carried out to test macho-1. The
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Figure 3. Depletion and overexpression of maternal macho-1 mRNA. A: Larvae injected with control oligo. In lower photo, tail muscle cells are stained with myosin antibody. B–D: Larvae injected with antisense oligo of macho-1 mRNA. B: Morphology. Tail is shrunken. C: Tail muscle cells that express myosin are reduced. D: Tail muscle cells detected by acetylcholinesterase histochemistry. E: Precursor blastomere of primary muscle cells (B4.1 cells) was isolated from control embryo and cultured as a partial embryo. Myosin is expressed. F: Injection of antisense oligo results in complete loss of myosin expression. G: After injection of synthetic macho-1 mRNA into eggs, the embryos develop into aberrant larvae and excess muscle forms within whole embryos. H: Muscle actin gene expression in primary muscle lineage cells of the 110-cell embryo. I: Expression of muscle actin is reduced in macho-1-deficient embryos. J: Injection of synthetic mRNA causes ectopic expression of actin genes. K: After injection of FLAG-tagged macho-1 mRNA, the protein is present in nuclei at the 110-cell stage. From Nishida H, and Sawada K. Nature 2001;409:724–729, with permission from Macmillan Magazines Ltd.(29)
posterior-vegetal cytoplasm of the fertilized egg has the ability to promote muscle formation when transferred into epidermis blastomeres. By contrast, the cytoplasm of macho-1-depleted eggs did not promote ectopic muscle formation in epidermis blastomeres. Thus maternal macho-1 mRNA satisfies the key criteria for the localized muscle-forming factor in ascidian eggs, whose existence was first proposed by Conklin one century ago. The macho-1 gene shows no zygotic expression. The macho-1 protein has five CCHH-type zinc-finger repeats in the central part that have similarity with Zic, GLI, and odd-paired proteins. All of these proteins are transcription factors.(33–35) As macho-1 protein synthesized from FLAG-tagged mRNAs accumulates in the nuclei during the cleavage stage (Fig. 3K), macho-1 is most likely a transcription factor. macho-1 may directly control the expression of muscle structural genes such as the actin and myosin genes during the initial process of muscle formation because these are
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activated as early as the 32-cell stage.(36) macho-1 may also promote the expression of regulatory genes such as ascidian Tbx6(37,38) and myogenic factor.(39,40) In ascidians, the expression of these regulatory factors occurs a little later, after the initiation of expression of the muscle structural genes. Therefore, it is suggested that these regulatory factors cooperate together to maintain muscle differentiation processes utilizing T-box-protein-binding sites and E-boxes in controlling elements of the muscle structural genes,(41) after the initial process has been triggered by macho-1. Cell interactions in ascidian embryos Cell interactions, especially inductive interactions, play crucial roles in animal embryogenesis.(42,43) During the last decade, there have been tremendous advances in understanding the various signaling molecules and pathways that mediate cell interactions during development. Previous experiments involving the isolation, dissociation and recombination of
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blastomeres have shown that ascidian embryos also utilize complex, yet conserved, cell–cell communications to specify early embryonic cell fates that are similar to those in other organisms.(3) Developmental fates are specified by cell interactions in mesodermal tissues such as notochord, mesenchyme, secondary muscle and trunk lateral cells, in ectodermal tissues such as the central and peripheral nervous systems, and also in posterior endoderm. Thus, it is now realized that the development of ascidian embryos is not entirely mosaic. The importance of cell signaling in ascidian embryos is supported by the results of treatment with an inhibitor of MEK/ MAPKK, which suppresses the formation of all of the tissues listed above.(44) By contrast, the treatment does not affect the formation of tissues whose fates are specified by maternal determinants. MEK/MAPKK is a protein kinase involved in various kinds of signal transduction by activating a mitogenactivated protein kinase (MAPK, also known as extracellular signal-regulated kinase, ERK).(45,46) Similarly, treatment with an inhibitor of fibroblast growth factor receptor (FGFR) results in loss of most of the above tissues, except for trunk lateral cells and posterior endoderm. Therefore, an FGF and MEKMAPK signaling cascade is widely involved in embryonic inductions in ascidians. Notochord induction Mesodermal tissues are derived from the marginal zone of the vegetal hemisphere. Fig. 4A–C shows a fate map of the vegetal hemisphere at the blastula stage (32- to 64-cell stage). Cell lineages that give rise to notochord and mesenchyme are shown in Fig. 4D,E. The notochord is one of the most intensively analyzed tissues in ascidian embryos because it is one of the hallmark morphological characteristics of any chordate. In ascidians, 40 notochord cells are located in the larval tail. 32 of them are called primary notochord cells and the 8 cells situated in the caudal tip region are designated secondary notochord cells. The primary notochord cells originate from four notochord precursor blastomeres (colored pink) in the anterior marginal zone of the vegetal hemisphere of the 64-cell embryo. The most-remarkable feature of studies of inductive cell interactions in ascidian embryos is that induction can be analyzed at the single cell level. Isolation and recombination of presumptive notochord blastomeres have revealed that inductive interactions mediate the determination of notochord fate.(47,48) As summarized in Table 1, this induction occurs at the 32-cell stage and notochord precursors acquire developmental autonomy at the 64-cell stage. Inducers of the primary notochord are the endoderm blastomeres (colored yellow in Fig. 4). Notochord blastomeres of 32-cell embryos can also induce notochord fates in neighboring notochord blastomeres. Only presumptive notochord blastomeres are competent and can respond to the specific kinds of endodermal signals that induce them to differentiate into notochord cells. Even in presumptive notochord blasto-
meres, competence is lost at the 44-cell stage (just after the cleavage of notochord blastomeres in the 32-cell embryo). Fibroblast growth factor (FGF), unlike activin, is a signaling molecule that mediates notochord induction.(49) Overexpression of the dominant negative form of the FGF receptor, or treatment of embryos with a specific inhibitor of FGF receptor, indicate that the inductive signal is received by the FGF receptor.(44,50) FGF signaling is known in many animals to be transduced within the cell by Ras–Raf–MEK–MAPK signaling cascade. During ascidian notochord induction, Ras and MEK are also required, and eventually MAPK is phosphorylated and activated.(44,51) Consequently, transcription of a Brachyury homolog (HrBra, formerly As-T) becomes activated in notochord cells at the 64-cell stage by an as-yet-unknown mechanism.(49,52,53) In vertebrates, Bra is known to be a transcription factor involved in mesoderm formation.(54) In ascidians, Bra is expressed exclusively in notochord precursors. It plays a central role as a transcription factor in the notochord formation process because injection of HrBra mRNA into eggs promotes ectopic notochord cell formation,(55) and, in this case, isolated blastomeres are able to differentiate autonomously into notochord without induction. Recent study has shown that another transcription factor, HNF-3, acts synergistically with HrBra during notochord differentiation, similar to what has been observed in frog embryos, although HNF-3 is expressed in most blastomeres of the vegetal hemisphere before notochord induction starts.(56) To identify the genes downstream from Brachyury, subtractive hybridization screening was carried out using Brachyury-overexpressing Ciona embryos. A total of 19 genes were found to be notochord-specific and another 20 were expressed predominantly in the notochord.(57–59) Expression of these genes starts at various stages ranging from gastrula to tailbud. At least one of them, a tropomyosin-like (Ci-trop) gene, is reported to be a direct target of Brachyury.(60) This gene has indispensable Ci-Bra-binding sites in its controlling cis-element. Along with fossil and other information, these new molecular findings will hopefully facilitate our continued quest to better understand how developmental processes evolved to generate the basic chordate body plan that is characterized by formation of the notochord. Mesenchyme induction Two bilateral mesenchyme cell clusters are located between the ventromedial endoderm and ventrolateral epidermis in the trunk region of the larva (Fig. 4C). Mesenchyme cells exclusively originate from four precursor blastomeres (colored green) in the posterior-lateral marginal zone of the vegetal hemisphere of the 64-cell embryo (Fig. 4B). Isolation and recombination of presumptive mesenchyme blastomeres was carried out.(61) Striking similarities were found between the notochord and mesenchyme inductive mechanisms, as summarized in Table 1. The similarity is noticeable just by looking at the cell
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Figure 4. Diagrams showing fates of cells in the vegetal hemisphere of ascidian embryos. A–C: The name of each blastomere is indicated. Endoderm (En)-lineage cells are colored yellow. Mesenchyme (Mes)-lineage cells are shown in green and muscle (Mus)lineage cells in red. Notochord (Not)- and nerve cord (NC)-lineage cells are colored pink and purple, respectively. A: 32-cell embryo. Vegetal view. Anterior is up. B: 64-cell embryo. Blastomeres connected with a bar are sister blastomeres. C: Tailbud embryos. Lateral views. Upper and lower diagrams illustrate midsagittal and parasaggital sections, respectively. D,E: Lineage trees in the vegetal hemisphere. As development is bilaterally symmetrical, one side of the embryo is shown. D: Lineage tree relevant to the primary notochord lineage and starting from the anterior-vegetal (A4.1) blastomere of the 8-cell embryo. E: Lineage tree starting from the posterior-vegetal (B4.1) blastomere, from which mesenchyme and primary muscle cells originate. TVC, trunk ventral cells. (From Kim GJ, Yamada A, Nishida H. Development 2000;127:2853–2862, with permission of The Company of Biologists Limited.(62))
lineage trees that generate notochord and mesenchyme (Fig. 4D,E). Inductive interactions mediate the determination of mesenchyme fate. This induction occurs at the 32-cell stage and mesenchyme precursors acquire developmental autonomy at the 64-cell stage. The inducer cells are endoderm blastomeres. Only the presumptive mesenchyme blastomeres are competent and can respond to the endodermal signal by differentiating into mesenchyme cells. Furthermore, FGF, but not activin, is an important signaling molecule in this process.(62) The signal is received by an FGF receptor(44,50) and Ras and MEK appear to be required for intracellular signaling.(44) Thus, the same signaling cascade down to MAPK is utilized in both notochord and mesenchyme inductions.
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Downstream transcription factors involved in this signaling pathway, such as Bra in notochord induction, have not yet been identified in mesenchyme induction. However, with regard to gene expression, it is intriguing that the muscle actin gene (HrMA4) is immediately downregulated after induction. The expression of HrMA4 is precociously initiated at the 32-cell stage in the muscle/mesenchyme (B6.2) blastomeres (Fig. 4A) before fate restriction.(36) The B6.2 blastomere (mesenchyme/muscle precursor) divides into the B7.3 (mesenchyme precursor) and B7.4 (muscle precursor) blastomeres of the 64-cell embryo (Fig. 4B,E). The expression continues only in the muscle blastomere, and is downregulated in the mesenchyme blastomere. The myosin heavy
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Table 1. Common features shared by notochord and mesenchyme inductions Cellular level 1. Their origins in the cell lineage tree show similar topology. 2. Inductive interactions are required. 3. The inductions start at the 32-cell stage before fate restriction. 4. The precursors acquire developmental autonomy at the 64-cell stage. 5. Competence to the inductive signal is lost at least at the 64-cell stage. 6. Endoderm blastomeres are the inducer. 7. Only the precursors have competence. 8. Only one daughter cell of the induced blastomeres assumes a notochord or mesenchyme fate. Molecular level 1. FGF is a potent inducer, but activin is not. 2. FGF receptor is required for induction. 3. Ras and MEK transduce the signal intracellularly.
chain gene is expressed in the same way. When the B6.2 cells are isolated or whole embryos are treated with MEK inhibitor to inhibit induction, actin expression continues in both daughter cells, and eventually both of them develop into muscle cells.(61) Therefore, inductive interactions cause the immediate downregulation of muscle-specific genes and suppress the muscle fate of presumptive-mesenchyme blastomeres. Suppression of the muscle fate of mesenchyme precursors by cell interactions implies that the muscle determinant, macho-1 protein, is also distributed in mesenchyme precursors. When a moderate amount of macho-1 mRNA is injected into eggs, ectopic formation of muscle occurs in non-muscle blastomeres. Under these conditions, mesenchyme and notochord precursors rarely transfate into muscle even if epidermis and endoderm precursors develop into muscle (K. Sawada and H. Nishida, unpublished data), although injection of a high dose of the macho-1 mRNA can confer a muscle fate on most embryonic cells, including mesenchyme and notochord blastomeres. This observation suggests that an endodermal signal may repress muscle fate by suppressing or modifying macho-1 function, and that emission of the signal from the vegetal blastomeres is executed independently of the endoderm fate of vegetal pole blastomeres. Responsiveness of signal-receiving blastomeres In the anterior marginal zone of the vegetal hemisphere, primary notochord is induced in the area flanked by endoderm and nerve cord blastomeres (Fig. 4). The ‘nerve cord’ designates the posterior neural tube located in the trunk and tail region of the larva with the ‘brain’ vesicle anteriorly derived from the animal hemisphere. In the posterior-lateral marginal zone, mesenchyme is induced in the area flanked by endoderm and muscle blastomeres. As mentioned before, there are striking similarities at the cellular and molecular levels
between notochord and mesenchyme inductions (Table 1). This implies that a similar mechanism symmetrically functions in both the anterior and posterior marginal zones. What, then, are the important mechanistic differences in the specification processes that underlie notochord and mesenchyme formation? In normal embryos, the notochord is induced by anterior (A-line) endoderm blastomeres, while mesenchyme is induced by posterior (B-line) endoderm. First, we examined whether the type of tissue induced depends on the inducing anterior and posterior endoderm or on the responding blastomeres by carrying out blastomere recombinations at the 32-cell stage.(62) When an anterior endoderm blastomere was recombined with a posterior mesenchyme precursor, mesenchyme was formed, and when a posterior endoderm blastomere was recombined with an anterior notochord precursor, notochord was formed. These results supported the latter possibility, i.e. that the type of tissue induced depends on the responding blastomeres. There is no difference between the inducing abilities of the anterior and posterior endoderm. The results of FGF treatment also support this idea.(62) Presumptive notochord blastomeres respond to this molecule by forming notochord; mesenchyme is never formed. Similarly, mesenchyme precursors are induced by FGF to form mesenchyme, and not to form notochord. Thus, a single signaling molecule promotes two types of response: the formation of the notochord and that of the mesenchyme. Therefore, presumptive mesenchyme and notochord blastomeres differ in their responsiveness. What, then, brings about this difference in responsiveness? Logically the difference must lie within the responding cells themselves. One possibility is that egg cytoplasmic factors differentially partitioned into each blastomere, determine the difference. To examine this possibility, removal and transplantation of egg cytoplasm was carried out by microsurgery. Egg fragments containing posterior-vegetal cytoplasm (PVC) were removed or transplanted to the anterior region of another intact egg after completion of ooplasmic segregation. PVC is the region corresponding to Conklin’s myoplasm at completion of ooplasmic segregation, and macho-1 mRNA is localized there (Fig. 2B, right panel). The experimental results are shown schematically in Fig. 5. Removal of the PVC resulted in anteriorization of the embryo. The blastomeres positioned where mesenchyme blastomeres are normally located were converted to notochord, so that central endoderm blastomeres were encircled by notochord blastomeres.(62,63) Thus, removal of the PVC causes ectopic formation of notochord and loss of mesenchyme in the posterior region (Fig. 5B). By contrast, removal of the anterior cytoplasm had no effect on embryogenesis, including notochord formation. Transplantation of the PVC to the anterior region suppressed notochord formation and promoted ectopic formation of mesenchyme in the anterior blastomeres that is never
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Figure 5. The posterior-vegetal cytoplasm (PVC) factor modifies the responsiveness of the signal-receiving blastomeres. Blastomeres are represented schematically by rectangles. For precise arrangement of blastomeres, see Fig. 4A,B. A: Normal embryos. In the anterior region, notochord (Not) is induced, while in the posterior region, mesenchyme (Mes) is induced next to endoderm (En). NC, nerve cord; Mus, Muscle. B: PVC-removed embryo. In the posterior region, notochord is induced in place of mesenchyme. C: PVCtransplanted embryo. In the anterior region, mesenchyme is induced instead of notochord.
observed in normal embryos (Fig. 5C).(62,63) By contrast, development was normal when the anterior cytoplasm was transplanted to the posterior region. In conclusion, the factors that are localized in the PVC seem to be involved in generating differences in cell responsiveness. In the presence of PVC factors, blastomeres respond to the endoderm signal by forming mesenchyme and, in their absence, blastomeres respond by developing into notochord. The results of removal and transplantation of the anterior cytoplasm suggest that no important factors are localized in the anterior region. The molecular identity of the PVC factor is still unknown. One obvious candidate is macho-1, because PVC is the region where macho-1 mRNAs is localized and macho-1 protein would also be present in mesenchyme blastomeres, as discussed above. Alternatively, the function of the PVC factor may be attributable to some of the type I postplasmic RNAs that were found in the maternal cDNA project and show a localization pattern similar to that of macho-1.(10,64) Downstream to the PVC factor, zygotic genes may work to suppress the notochord fate. Snail is a possible candidate because it is expressed in muscle and mesenchyme precursors at the 32-cell stage. Furthermore, Snail is a zinc-finger protein known to be a transcription repressor. Misexpression of Snail in notochord-lineage cells suppresses at least the expression of reporter genes driven by the Brachyury minimal promoter.(65) Therefore, even if MAPK is activated by the endodermal signal in mesenchyme blastomeres, Snail might suppress expression of the Brachyury gene. The global picture of mesoderm patterning in the marginal zone is now known. In contrast to the
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situation in vertebrates,(66) ascidian mesodermal patterning does not seem to involve a graded signal. Directed signaling and asymmetric cell divisions Another conspicuous feature of notochord and mesenchyme inductions is the asymmetric cell divisions of their progenitor cells. Recent evidence suggests that these inductions occur at the 32-cell stage. Experiments involving recombination of isolated blastomeres at various stages, and experiments determining the periods of sensitivity to FGF treatment and sensitivity to both the FGF receptor inhibitor and the MEK inhibitor, all support the idea that the inductive interactions for notochord and mesenchyme formation are initiated at the 32-cell stage. It is important to recall that, in the ascidian cell lineage tree, the fates of responding blastomeres are not yet restricted to formation of a single kind of tissue at the 32-cell stage (Figs. 4, 6). Endoderm precursors lie in the center. In the anterior region, notochord/nerve cord precursor blastomeres of the 32-cell-stage embryo divide into notochord (pink) and nerve cord (purple) precursors of the 64-cell-stage embryo. Similarly in the posterior region, mesenchyme/muscle precursor blastomeres of the 32-cell-stage embryo divide into mesenchyme (green) and muscle precursors (red) of the 64-cell-stage embryo. Therefore, the separation of each cell fate occurs at the 64-cell stage after induction has taken place, and only one daughter blastomere assumes the induced fate. Induced notochord and mesenchyme blastomeres always face the inducing endoderm at the 64-cell stage. Thus,
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Figure 6. A directed signal and asymmetric division model of the tissue specification mechanism in the vegetal hemisphere of the ascidian embryo. The model is applicable to both the anterior and posterior margins of the vegetal hemisphere. A: Vegetal view of the 32-cell embryo showing endodermal FGF signal (arrows) and presence of the PVC factor (red oblique lines) in the posterior blastomeres. B: Vegetal view of the 64-cell embryo after completion of inductions and asymmetric divisions in both the anterior and posterior regions. C: (1) Schematic drawing representing embryo at the 32-cell stage. Endoderm precursors (En) emanate an inductive FGF signal (arrows) to neighboring anterior and posterior blastomeres and polarize them. The PVC causes different response in the posterior marginal cells. (2) Asymmetric divisions occur at the 64-cell stage. In the anterior region, one daughter cell that faces the inducer and does not have the PVC assumes a notochord fate (Not). In the posterior region, one daughter cell that faces the inducer and contains the PVC adopts a mesenchyme fate (Mes). (3) Without an inductive signal, both daughter blastomeres in the anterior region assume the default nerve cord fate (NC), and those in the posterior region assume the default muscle fate (Mus). (4) When isolated blastomeres receive the FGF signal over their entire surface, both daughter cells develop into notochord or mesenchyme, depending on the absence or presence of PVC. From Minokawa T, Yagi K, Makabe KW, Nishida H. Development 2001;128:2007–2017, with permission of The Company of Biologists Limited.(67))
notochord and mesenchyme induction occurs such that only one of the daughters of the induced blastomere in the 32-cell embryo adopts a notochord or mesenchyme fate. As described previously, mesenchyme blastomeres develop into muscle when they do not receive an inductive signal. Therefore, muscle is a default fate of muscle/mesenchyme precursors. It has been reported that nerve cord is a default
fate of notochord/nerve cord precursors (Fig. 6C).(67) When notochord and nerve cord precursors are isolated at the 64-cell stage after completion of induction, and further cell division is inhibited by cytochalasin B, the notochord blastomere eventually expresses a notochord differentiation marker, Not-1 antigen, while the nerve cord blastomere expresses the neural plate marker genes HrETR-1 and HrTBB2 that encode RNA-
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binding protein(68) and b-tubulin,(69) respectively. By contrast, when notochord/nerve cord precursors are isolated at the 32-cell stage and allowed to divide once, then further cleavages are arrested, both blastomeres express neural plate markers, and not notochord marker. Treatment with inhibitors of FGFR and MEK also causes both daughter cells to assume the nerve cord fate. The autonomy of nerve cord fate specification was also confirmed by isolation of precursor blastomeres at various stages and by dissociation of embryonic cells. This was a surprise because the nerve cord of ascidian larvae is formed by neural tube closure, as in vertebrates. These findings, using HrETR-1 and HrTBB2 as molecular markers, do not tell us whether the morphogenetic processes of neural tube formation in ascidian larvae require cell interactions. However, they do indicate that the initial step of nerve cord specification during cleavage is autonomous. Treatment with FGF gives opposite results. FGF treatment of mesenchyme/muscle precursors at the 32-cell stage causes both of the daughter blastomeres to develop into mesenchyme.(62) Similar treatment of notochord/nerve cord precursors confers a notochord fate on both daughter cells.(67) Therefore, in the asymmetric divisions that occur in the anterior region, nerve cord appears to be a default cell fate and notochord is an induced fate; in the posterior region, muscle is the default fate and mesenchyme is the induced fate. Figure 6 summarizes our model. In normal embryos, the precursor cells receive an endoderm signal from the vegetal pole, and only one of the daughter cells facing the endoderm assumes an induced cell fate. Directed signals that emanate from endoderm blastomeres may polarize the responding blastomeres at the 32-cell stage and promote asymmetric divisions that operate in both the anterior and posterior regions. Presumably, FGF signaling causes localized changes in the mother cell. Then one of the daughter cells that faces the endoderm is fated to notochord or mesenchyme depending on the presence or absence of the PVC factors. This directed signaling and asymmetric division model is supported by the fact that treatment of isolated blastomeres with FGF in seawater causes both daughters to assume a mesenchyme or a notochord fate, because isolated mother blastomeres receive the signal over the entire cell surface. Similar examples can be found in the C. elegans embryo. At the 4-cell stage, the EMS blastomere receives inductive signals from the posterior P2 blastomere. Then it divides asymmetrically into the anterior MS cell (muscle, neuron, somatic gonad precursor; default fate) and posterior E cell (gut precursor; induced fate).(70–72) In this case, the signaling molecule is Wnt.(73) Moreover, it has been suggested that Wnt signaling may be globally involved in binary fate specifications that are accompanied by asymmetric cell divisions along the entire anterior-posterior axis in later embryogenesis.(74–76) Thus, directed-signal-mediated asymmetric divisions appear to be widely utilized as a mechanism to generate cell fate
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diversity in early embryos as well as later development in various kinds of animal. Future directions We can now begin to understand how developmental fates are determined at the cellular and molecular levels in most cell types in ascidian embryos. While our molecular knowledge is still incomplete, we have now characterized many of the likely key molecules involved in autonomous fate specification (localized RNAs) and cell interactions (signaling molecules). Chasing the localized maternal factor has revealed macho1 as a muscle determinant whose presence had been predicted a century ago. Similar to Drosophila and Xenopus embryos,(77–79) the localized determinant in ascidian eggs is maternal mRNA that encodes a transcription factor. So far, it seems common that localized egg mRNA plays critical roles in the initial steps of early embryogenesis. However, any conclusion should be made with care, because the isolation of many important localized RNAs may be technically simpler than finding localized proteins. Ascidian would provide a good system to analyze the mechanisms how specific mRNAs are localized and relocated in eggs and embryonic cells.(80) In order to understand the muscle-forming cascade, it will be important to analyze epistatic relationships between musclespecific genes such as Tbx6(37,38) and the myogenic factor(39,40) genes that are known to be expressed zygotically in ascidians. A search for macho-1 homologs in other organisms should also be made. We have learned much from analyses of notochord and mesenchyme inductions, and a simple model of binary specification of cell fates operating in the marginal zone of the vegetal hemisphere in ascidian embryos has been proposed. The first step depends on the presence or absence of PVC factors, and the second step is regulated by the presence or absence of inductive interactions. From a comparative viewpoint, it is remarkable that ascidians utilize cellular and molecular mechanisms to pattern mesodermal tissues that are significantly different from those in vertebrates. Ascidians seem to have adopted a ‘‘digital’’ approach, rather than the ‘‘analog’’ one in vertebrates where there is graded activity of BMP signaling.(66) This difference is probably related to the fact that ascidian embryos consist of relatively few cells, and that restriction of developmental fates occurs at a very early stage. Another reason may be egg sizes. There may not be enough distance between cells to generate graded activity of signaling. Nevertheless, it is amazing that both phylogenetically related animals can show a similar fate map and generate a similar basic body plan using substantially different mechanisms. One explanation is that the pressure of natural selection to conserve a body plan is high, but may not be so severe as to restrict the way in which the ideal body plan is attained. In induced asymmetric divisions, extracellular signaling molecules and intrinsic factors are thought to combine in a
Review articles
progenitor cell before it divides, conferring particular fates on the two progeny cells. Intrinsic factors play roles in defining the ‘responsiveness’ or ‘competence’ of the progenitor cells. In future studies of ascidian embryogenesis, identification of the PVC factor and analysis of the intracellular events involved in asymmetric divisions will be important. We observed that each blastomere shows a distinct response to the same signal, although the signaling cascade is markedly conserved among each blastomere in ascidians, as well as in other organisms that have been studied. Therefore, it will be important to understand the mechanisms that determine the way in which different blastomeres respond differently. Clarification of maternal factors will no doubt contribute to this understanding, as has been the case in notochord and mesenchyme inductions. The problem is how cells integrate the intrinsic activity of the PVC factor with the information from extrinsic cues that are delivered into the cell by the signal-transduction machinery. Directed-signal-mediated asymmetric divisions play crucial roles in the generation of cell diversity in ascidian embryos. It is still unclear how mother cells are polarized before asymmetric divisions, and cytological investigation of intracellular events will be required to answer this question. Understanding of the spatial details of activation of the MAPK pathways in signal transduction will provide clues for the study of asymmetric divisions in ascidian embryos. Acknowledgment I am grateful to the members of our HFSP group for helpful discussions.
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