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PHYSIOLOGICAL REVIEWS Vol. 78, No. 2, April 1998 Printed in U.S.A. Ion Channels and Early Development of Neural Cells KUNITARO TAKAHASHI AND YASUSHI OKAMURA Department of Medical Physiology, Meiji College of Pharmacy, Setagaya-ku, Tokyo; and National Institute of Bioscience and Human Technology, Agency for Industry, Science, and Technology, Tukuba, Ibaragi, Japan 308 309 309 310 312 314 314 315 317 317 317 318 318 320 320 320 Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on May 4, 2017 I. Introduction: Ion Channels, Ascidian Embryos, and Neural Induction II. The Protochordate Embryo A. A prototype of vertebrate embryos B. Cell lineage of ascidian embryos C. Development of excitability in cleavage-arrested embryos III. Ion Channels and Their Changes During Oogenesis and Fertilization A. Oocyte maturation, fertilization, and cytoplasmic movement B. Ion channels in oocytes: Ca2/, Na/, and inward and outward rectifier K/ channels IV. Early Changes in Ion Channels A. Cleavage, blastula, and animal-vegetal axis B. Egg-type Na/ and Ca2/ channels C. Various K/ channels D. Cl0 and other ion channels V. Neural Commitment and Changes in Expression of Ion Channels A. Neural induction in vertebrate embryos B. Two-cell induction system consructed from early ascidian embryos C. TuNa I channels are neuron-specific markers in the ascidian embryo and are expressed after inductive differentiation D. Quantitative difference in expression of inward rectifier K/ channels as an early sign of neural induction E. Inducer candidates for Na/ spike induction VI. Development of Other Ion Channels Than Sodium Channels A. Development of K/ and Ca2/ channels B. Comparison with cases of other vertebrate species of embryos VII. Developmental Regulation of Ion Channel Expression A. Regulation by changes in gap junctional communication B. Studies on gap junctional regulation of ion channel expression in ascidian embryos C. Regulation of ion channel expression by transcription VIII. Conclusion 321 322 324 325 325 326 327 327 327 329 330 Takahashi, Kunitaro, and Yasushi Okamura. Ion Channels and Early Development of Neural Cells. Physiol. Rev. 78: 307–337, 1998.—In this review, we underscore the merits of using voltage-dependent ion channels as markers for neuronal differentiation from the early stages of uncommitted embryonic blastomeres. Furthermore, a fairly large part of the review is devoted to the descriptions of the establishment of a simple model system for neural induction derived from the cleavage-arrested eight-cell ascidian embryo by pairing a single ectodermal with a single vegetal blastomere as a competent and an inducer cell, respectively. The descriptions are focused particularly on the early developmental processes of various ion channels in neuronal and other excitable membranes observed in this extraordinarily simple system, and we compare these results with those in other significant and definable systems for neural differentiation. It is stressed that this simple system, for which most of the electronic and optical methods and various injection experiments are applicable, may be useful for future molecular physiological studies on the intracellular process of differentiation of the early embryonic cells. We have also highlighted the importance of suppressive mechanisms for cellular differentiation from the experimental results, such as epidermal commitment of the cleavage-arrested one-cell Halocynthia embryos or suppression of epidermal-specific transcription of inward rectifier channels by neural induction signals. It was suggested that reciprocal suppressive mechanisms at the transcriptional level may be one of the key processes for cellular differentiation, by which exclusivity of cell types is maintained. 0031-9333/98 $15.00 Copyright 䉷 1998 the American Physiological Society / 9j08$$ap07 P25-7 03-18-98 08:13:31 307 pra APS-Phys Rev 308 KUNITARO TAKAHASHI AND YASUSHI OKAMURA I. INTRODUCTION: ION CHANNELS, ASCIDIAN EMBRYOS, AND NEURAL INDUCTION / 9j08$$ap07 P25-7 49). Thus morphogenesis, which is a result of migration or segregation of cells committed to various extents, establishes in turn the field of cell-cell interaction provoking new commitment of embryonic cells to specified differentiation pathways or the segregation of cytoplasmic factors into various embryonic compartments. These morphogenetic cycles, setting as both result and cause of cellular differentiation, occur repeatedly until all embryonic cells have finally differentiated. This introduces difficulties to the analysis of the mechanism of cellular differentiation during embryogenesis in vivo. Therefore, in this review, we confine the discussion to results from simple differentiating systems in which autonomous and inductive processes are experimentally separable. In these cases, cellular differentiation can be analyzed independently of a clearly definable morphogenetic field. The fact that the larva of the ascidian, a protochordate, has a simplified body organization that is essentially identical to that of the vertebrates was discovered in the mid 19th century by Kowalevsky (139). Since then, the ascidian embryo has been considered to be a prototype of the vertebrate embryo (Fig. 1, D and E). On the other hand, at the beginning of this century, the cell lineage studies by Conklin (Fig. 1C) revealed a fate map on the ascidian egg and provided the classical example of a mosaic egg (30, 31). The hypothesis that cytoplasmic factors buried in the matrix of egg cytoplasm can be segregated into various presumptive regions by cleavage and commit these regions to various cell fates has been invoked to explain autonomous determination during the development of mosaic eggs (47). The existence of cytoplasmic factors in the ascidian embryo was ingeniously proven by Whittaker (318). Whittaker (318) showed that acetylcholinesterase, one of the characteristics in striated muscle fibers of the ascidian tadpole, is actually expressed in the blastomeres containing presumptive muscular regions when cleavage of the embryo is arrested early in development, at the 8- to 64-cell stages. This report has been further supported by the analysis of other characteristics performed in our experiments, such as various kinds of electrical excitability (299). Distinctive forms of excitability appear in specific cleavage-arrested blastomeres as the result of expression of Na/, K/, and Ca2/ channels in their membranes. However, even in this ascidian embryo, a classical example of mosaic development, Rose (255) and Reverberi and Minganti (246) reported that the embryo from which presumptive notochordal blastomeres were excised, leaving the presumptive neural blastomeres intact at the eightcell stage, cannot differentiate neural tissues, such as brain vesicle, otolith, and eye. They suggested that inductive effects from the presumptive notochordal regions are required to generate neural tissues in the ascidian embryo, just as in the vertebrate neurogenesis (247). On the basis of these classical observations, we first 03-18-98 08:13:31 pra APS-Phys Rev Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on May 4, 2017 Electrical excitability involving characteristic sets of voltage-dependent ion channels is an essential property of neurons working as the elements of a highly efficient biological information processor, the brain. Thus the development of electrical excitability or expression of voltage-dependent ion channels is one of the fundamental aspects of neural differentiation. In addition, because various muscular cells, many endocrine cells (236), and recently so-called nonexcitable cells, such as glial cells (5), are all known to express voltage-dependent ion channels at certain stages of cellular differentiation, general mechanisms specifying cell types may also be understood by analyzing expression processes of ion channels in differentiating cells. Membrane differentiation is one of the key mechanisms in any type of cellular differentiation by which individual cells can be distinguished from each other through the recognition by their cell surfaces at various stages of development. Because so many molecular species have recently been identified in the plasma membrane, each type of cells is likely to be specified by a characteristic combination of functionally significant molecular species expressed on their membranes. One such example is the gene family of various voltage-dependent ion channels, which are distinctly identified by both molecular biological and electrophysiological methods (96). Thus, because of their molecular and functional distinctions, ionic channels can be useful markers for cellular differentiation not only in neural tissues but in other tissues as well. Neurogenesis in vertebrate embryos is known to be initiated by the commitment of dorsal ectoderm under inductive influence of mesoderm into neural plate formation at the beginning of gastrulation (84, 130, 288). Although ectodermal cells forming the neural plate differentiate into neuroepithelial cells, the neural plate transforms into the neural tube as a result of morphogenesis. Neuroepithelial cells further differentiate into various kinds of neuronal and glial cells under the influence of morphogenic fields formed with neural and surrounding tissues (107, 120). For example, motoneurons in the vertebrate spinal cord are induced by diffusible factors from the notochord situated closely underneath the ventral side of the cord (327), and type 2 astrocytes in the rat optic tract are known to be derived from O-2A precursor cells by the inductive influence of growth factor, ciliary neuron growth factor (CNGF), released from neighboring cells (158). Cellular differentiation is generally determined by cytoplasmic factors successively transferred from the ancestral oocyte through antecedent parent cells, or induced by factors that are produced by neighboring embryonic cells or delivered by diffusion from a more distant part of the embryo. The former is known as autonomous differentiation, and the latter as inductive differentiation (48, Volume 78 April 1998 ION CHANNELS AND EARLY DEVELOPMENT OF NEURAL CELLS 309 FIG. 1. Development of ascidian embryo, Halocynthia aurantium. A: 8-cell stage. B: initial gastrula stage. C: schematic illustration of 117-cell Cynthia partia embryo. N.p., neural plate precursor cells; ch., notochordal precursor cells; m’ch, mesenchymal precursor cells; ms., muscular precursor cells. [From Conklin (30).] D: head of a hatched tadpole larva. Black spot on left is a otolith cell, and black spot on right is a photocell-related pigment cell. E: schematic illustration of nervous system of a Ciona intestinalis tadpole. AO, adhesive organ; EN, endodermal cavity; EP, epidermis; ES, endodermal strand; G, gut primordium; ME, mesodermal pocket; NC, notochord; NS, nervous system; PH, primordial pharynx; POL, preoral lobe; V, ventricle of prosencephalon; VPC, ventral melanocyte. Scale in D is 500 mm for A and B and 125 mm for D. [From Katz (126).] II. THE PROTOCHORDATE EMBRYO A. A Prototype of Vertebrate Embryos At the middle of the 19th century, Kowalevsky (139) observed the tubular nervous system and notochordal / 9j08$$ap07 P25-7 structure in the larva of the tunicate. Both the neural tube and the notochord characterize vertebrate embryonic development (68), and their existence in tunicate larvae provided the basis for Kowalevsky to reclassify the tunicate as one of the closest relatives of the vertebrate, that is, a protochordate. The formation of the neural tube in the tunicate embryo, through folding of the neural plate consisting of the dorsal ectoderm underlined with the dorsal mesoderm, is identical to that in various vertebrate embryos, including amphibians, birds, and mammals (68). According to the evolutionary tree proposed by Garstang (67), the tunicate larva has a central role in directing the ancestral sessile animal derived from primitive echinoderms into the stem for vertebrates by evolution though neoteny. This hypothesis was further extended in ‘‘Major steps in vertebrate evolution’’ by Romer (254). According to this evolutionary history, the gill, which was an external feeding organ in the primitive echinoderms, is now internalized in the adult tunicate and forms a part of oral cavity in the freely moving tunicates, such as oikopleunids and salps, and in the cephalochordates. Although there still remains discussion whether the tunicate larva occupies a central evolutionary position or is just playing around side branches apart from the main stream (86), the close similarity of body plan to those of vertebrates and the extraordinary simplicity indicated by an extremely small cell population make tunicate embryonic development to the larval stage an excellent model system for analysis of vertebrate embryonic development (245). Recently, molecular biological approaches to the major phylogenetic branches have been allowed comparison 03-18-98 08:13:31 pra APS-Phys Rev Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on May 4, 2017 identified autonomous differentiation of respective blastomeres isolated from the cleavage-arrested 1- to 16-cell ascidian embryo (225) and then examined the alteration of developmental fates by contact with the blastomere of another type (Fig. 2) to demonstrate the inductive differentiation of neural characteristics represented by electrical excitability (224). We found that the isolated anterior animal blastomere from the eight-cell cleavage-arrested embryo, which is known to include presumptive brain vesicle region, autonomously develops long-lasting Ca2/-dependent action potentials. It was then found that the action potential was derived from the expression of slowly inactivating Ca2/ channels characteristic of epidermal differentiation of the ascidian larva (99, 225). However, when it is cultured in contact with the anterior vegetal blastomere, which is known to include presumptive notochordal region, and raised as a contacted two-cell system, the same anterior animal blastomere now develops a typical Na/ spike by expression of Na/ channels and tetraethylammonium (TEA)-sensitive delayed rectifier K/ channels (Fig. 2) (223, 224). In this review, we focus particularly on the early developmental processes of various ion channels in neuronal and other excitable membranes observed in this extraordinarily simple interacting two-cell system and compare these results with those in other significant and definable systems for neural differentiation. 310 KUNITARO TAKAHASHI AND YASUSHI OKAMURA Volume 78 of 18S rRNA among animal kingdom and provided further colored granules distributed differentially in various reinformation about the close similarity of tunicates to an gions of cytoplasm in eggs of some ascidian species, such ancestral form of vertebrates (60, 150, 310). As for the as Styla partia (31). For example, orange/yellow-colored developmental and evolutional biology of ascidians, we granules that are inferred to be mitochondial aggregates hope the readers look also to the review by Satoh (262). are initially located within the presumptive muscular regions in the posterior vegetal quadrant of the egg and finally segregated into muscle cells in the tail of the tadB. Cell Lineage of Ascidian Embryos pole larva (245). With five distinct types of colored granules, neural, muscular, mesenchymal, ectodermal, and enClassical cell lineage studies on ascidian embryos by dodermal regions can be traced distinctly (31). Conklin (31) early in this century revealed that each blasModernized cell-tracing techniques with horseradish tomere is uniquely tissue specified at the 64-cell stage. peroxidase (HRP) reveal the precise cell lineage of the Deletion of a part of an embryo resulted in an imperfect Halocynthia embryo up to the 110-cell stage at which embryo lacking the tissues involved in those presumptive point all blastomeres are committed to give rise to single regions, indicating that the ascidian embryo is a typical tissue types during subsequent development (206, 208, example of mosaic eggs with a fairly discrete fate map 209). The results confirm most of the classical studies of (30, 32). Conklin (31); however, some important details concerning The cell lineage in the ascidian embryo was classi- the caudal muscular lineage and neural lineage in dorsal cally established by tracing segregation of endogenous part of the neural tube in the tail have been revised (206). / 9j08$$ap07 P25-7 03-18-98 08:13:31 pra APS-Phys Rev Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on May 4, 2017 FIG. 2. Schematic illustration of development of H. aurantium embryos at 10⬚C and experimental design of 2-cell neural induction system. CB, isolated cleavage-arrested 8-cell blastomeres are cultured in seawater containing cytochalasin B. Nomenclature of blastomeres is according to Conklin (30). Traces on right are typical inward and outward currents with voltage steps (mV) indicated by figures on reference zero-current traces at final stages of differentiation. [From Okado and Takahashi (223).] April 1998 ION CHANNELS AND EARLY DEVELOPMENT OF NEURAL CELLS / 9j08$$ap07 P25-7 the neural tube becomes completely internalized, 170 cells of 10th to 12th generation exist in the tube, and only one further division or at most two are required to complete the neural cell lineage (201). The nervous tissue in the ascidian tadpole has been recently studied in detail by serial section of epoxy-embedded materials (203). Characteristic features of a brain or a sensory vesicles, a visceral ganglia, and the neural tube in the tail have been described by optical and electron microscopic studies, and their homology to the tubular nervous system characteristic of vertebrates has been established (75, 126, 139). Putative sensory cells in the adhesion processes at the rostral extreme possibly send axons through the ventral side of the brain vesicle to the visceral ganglion. The brain or sensory vesicle is an anterior counterpart of neural tube. The dorsoanterior wall of the vesicle consists of a thin monolayer of neuroepithelial cells, and the ventral and posterior walls include a single mechanosensory cell of the otolith (52, 55, 305, 306) and several photocells associated with a single pigment cell (4, 55), respectively. They send their axons to the visceral ganglion located just posterior to the sensory vesicle. Phototransduction potentials recorded from ascidian tadpole photocells show hyperpolarizing responses to light-on stimuli, just as in the case of vertebrate photocells (71). It is of interest in terms of vertebrate evolution that the photoresponse is hyperpolarizing in contrast to the depolarizing response observed in the invertebrates, such as crustacean photocells. Morphologically, ascidian photocells are regarded as modified ciliary cells, just as in the case of rod or cone cells of the vertebrate retina (52, 55, 71). The visceral ganglion is believed to include several motoneurons that are located ventrally and send axons down to the tail along the neural tube (203). Quite recently, the lineage of motoneurons was clarified in Halocynthia larvae, and the derivation from the anterovegetal blastomeres (A-line cells) in the eight-cell embryos was established, as described later (222). These motoneurons may be responsible for the rhythmic activity of six lines of serially connected striated muscles in the tail. Neuromuscular junctions on the striated muscle fibers have been proven to be cholinergic just as in vertebrate skeletal muscle cells (221). There are a number of sensory cells in the epidermal cell layers in the tail of the larva (305). Among the cellular populations in neural tissue, twothirds of cells in the sensory vesicle and the visceral ganglion of the trunk and most cells in the neural tube of the tail are not differentiated neurons but are neuroepithelial or ependymoglial cells with cilia protruding into the lumen of the tube (38, 126). Thus the total number of mature neurons in the central nervous system (CNS) is only Ç70 in the larva of C. intestinalis (203). The same structure of the nervous system has been described in various species, 03-18-98 08:13:31 pra APS-Phys Rev Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on May 4, 2017 The muscular lineage has been also studied by fluorescent probes for the mitochondial membrane, such as rhodamine 123 or merocyanine 540, confirming the revised lineage revealed by HRP injection (333). Early ascidian cleavages are principally symmetrical across the mid-plane through the vegetal-animal axis, which is clearly indicated by the existence of two polar bodies at the animal pole (31, 261, 262). Morphological studies of cleavage in early ascidian embryos are described with optical microscopic observations by Conklin (31). On Halocynthia roretzi embryos, Satoh and co-workers (261, 263) and Nishida (205) precisely described the observations with scanning microscope until the late gastrula stages. At the eight-cell stage, four relatively smaller animal blastomeres are located slightly anteriorly against four larger vegetal blastomeres, and thus both anteroposterior and vegetal-animal axes become apparent (Fig. 2). Until the 32-cell stage, all cell divisions are synchronized, and after this stage, cell division in the vegetal hemisphere becomes relatively asynchronous (31, 205). At the 64-cell stage, the embryo becomes disklike in shape, being compressed along vegetal-animal axis. At the 110-cell stage, the vegetal half starts to hollow slightly, indicating initiation of the morphogenic movement of gastrulation. During gastrulation, the group of animal cells most anteriorly located comes up dorsally and overlays the anterior vegetal group of cells that is in the presumptive notochordal region and responsible for neural inducing activity. The mesodermal region responsible for muscular tissue and mesenchyme is enclosed inside the blastopore and becomes located posterolaterally and ventrally. Thus the locations of various morphogenic regions, such as ventral ectoderm, neural plate, dorsal mesoderm, myogenic region, and endoderm, in this ascidian gastrula, show close similarity to those of vertebrate and particularly amphibian gastrulas (31, 139). Morphogenic movement after gastrulation was reexamined by tracing cell lineage of Halocynthia embryos with injected HRP (206) or in Ciona embryos by direct observation with Nomarski optics (201, 202). The cell-cleavage pattern and cell maps of the nervous system have been described in great detail, reviewed in Satoh (262). The cell lineage of all cells in the neural plate of Ciona intestinalis was especially determined in detail (201). The neural plate has been shown to be derived from 10 cells at the 64-cell stage. Of these, four derive from vegetal (dorsal) hemisphere and the remaining six cells from a row at the anterior animal border. After the 64-cell stage, three generations advance from 7th to 10th during gastrulation, and there are 75 neural plate cells at the start of neurulation in C. intestinalis. This number is extremely small when compared with other vertebrates, such as Xenopus embryos. By the time 311 312 KUNITARO TAKAHASHI AND YASUSHI OKAMURA including both solitary and colonial ascidian larvae. The precise number of neurons in the CNS of the larva has not been reported, except for those in the CNS identified morphologically in the case described above (203). After the metamorphosis of the larva, the sensory vesicle, visceral ganglion, and neural tube all degenerate. The undifferentiated cell mass of the cerebral ganglion situated anterodorsally to the sensory vesicle becomes the hypophysis and esophageal ganglion in adult ascidians, regulating contraction of the muscular wall (11). In summary, the neural tissue of the ascidian tadpole larva is the simplest CNS in an ancestral form of vertebrate CNS and its development provides a good model for vertebrate neurogenesis. As for the ascidian neurogenesis as a prototype of vertebrates, we hope the reader will also refer to a previous review by Okamura et al. (230). Differentiation without cell cleavage was first described by Lillie (157) for Chaetopterus eggs treated with concentrations of K/ greater than 100 mM in seawater, which causes activation without ensuring division. The cilia that characterize the ectoderm of trochophore larvae of Chaetopterus pergamentaceus are observed on the surface of cleavage-arrested, one-cell embryos at the same developmental time as in intact embryos. It was suggested that segregation of cytoplasmic components, such as ectoplasm and yolk-containing endoplasm, are produced equally in cleavage-arrested embryos, and thus differentiation occurs without cellular compartmentalization (57, 58, 157). The increased intracellular concentration of Ca2/ due to sustained depolarization by K/ may be responsible for activation of Chaetopterus eggs, but is not necessary for differentiation without cleavage after activation (17). In ascidian embryos, Whittaker (318) achieved cleavage arrest with the cytokinesis inhibitor cytochalasin B and demonstrated the expression of tissue-specific enzymes in the blastomeres, including respective presumptive regions (318). His experiment is considered to provide evidence for the existence of cytoplasmic factors in tissue determination (318). We intended to utilize cleavage-arrested embryos for analysis of membrane differentiation, especially the development of excitability, which consists of sequential expression of various ion channels, since large cells without complex cellular processes are ideal preparations for analysis of ion channel currents with voltage clamp. When Halocynthia embryos are cleavage arrested at the 16-cell stage and cultured in seawater containing cytochalasin B until control embryos hatch, the blastomere membranes reveal electrical excitability, generating Na/ and Ca2/ spikes (99, 299). / 9j08$$ap07 P25-7 The Ca2/ spikes found in the presumptive muscular blastomeres in the cleavage-arrested embryo are identical to those in muscle cells previously observed in the control larva (186). Calcium spikes in muscle cells have been confirmed by whole cell recording techniques applied to dissociated muscle cells from intact young larvae of other ascidian species (275). From these studies, it seems clear that electrical excitability represented by combinations of ion channels can be used to identify types of differentiation in distinct blastomeres in cleavage-arrested ascidian embryos. The observation that Na/ spikes appeared only in the presumptive neural region in cleavage-arrested embryos led us to consider Na/ spikes as neural and Ca2/ spikes as muscular markers in the ascidian larva, respectively. However, further studies on the cleavage-arrested embryo revealed that Ca2/-dependent action potentials appeared also in ectodermal blastomeres destined to become epidermal tissues (97, 99). Voltage-clamp studies of ectodermal blastomeres reveal that the ectodermal blastomeres show slowly inactivating Ca2/ channels without concomitant delayed rectifier K/ channels, in contrast to the case of muscularly differentiated blastomeres. Ectodermal blastomeres generating Ca2/ action potentials also bind monoclonal antibodies directed to the larval epidermis (98, 223). Thus it is concluded that the long-lasting Ca2/ action potentials in these blastomeres are different from the sharp Ca2/ spikes in muscle cells of the intact tadpole larva and characteristic of larval epidermis. The long-lasting action potential in the epidermis has been reported in other ascidian larvae by Mackie and Bone (164), and action potentials propagating over the body surface are observed in amphibian epidermal cells at the hatching stage of tadpoles (253, 260). Tables 1 and 2 illustrate the correspondence of developmental fate with the differentiation of specific classes of ion channels (99). It is clear that each blastomere has a tendency to express a particular tissue type, dependent on time of cleavage arrest and location of the blastomere in the early embryo. When the same blastomere shows more than two types of differentiation, the phenotype of each blastomere seems not to be mosaic of multiple types of characteristics but to have selected one of the possible types. The exclusivity principle in differentiation has been discussed by Weiss (314). Interesting patterns of differentiation are obtained by arresting cleavage before the first division, in which situation all cytoplasmic factors, if any, exist in one cellular compartment. It is expected that all types of tissue differentiation may occur after culturing in cytochalasin B-containing seawater. However, Crowther and Whittaker (37) have reported that all tissue types appeared to be expressed in the one-cell embryo in terms of structures visualized by electron microscopy and have suggested that multiple expression is in accordance with mosaic 03-18-98 08:13:31 pra APS-Phys Rev Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on May 4, 2017 C. Development of Excitability in Cleavage-Arrested Embryos Volume 78 April 1998 TABLE 313 ION CHANNELS AND EARLY DEVELOPMENT OF NEURAL CELLS 1. Differentiation of blastomeres of cleavage-arrested embryos Embryo Blastomere Developmental Fate Differentiation of Excitability 1 cell 2 cell 4 cell AB1 AB2 A3 B3 a4-2 (A) b4-2 (A) A4-1 (V) B4-1 (V) a5-3, 4 (A) b5-3, 4 (A) A5-1, 2 (V) B5-1 (V) B5-2 (V) All kinds of cells All kinds of cells Neural plate, epidermis, notochord, endoderm Epidermis, endoderm, muscle, mesenchyme Neural plate (brain), epidermis Epidermis, neural plate Neural plate (spinal), notocord, endoderm Muscle, mesenchyme, endoderm Neural plate (brain), epidermis Epidermis, neural plate Neural plate (spinal), notochord, endoderm Muscle, mesenchyme, endoderm Muscle, mesenchyme E E E or spike with A current* E or M* N or E E or N* nE or E or spike with A current† M or nE or E N or E E or N* nE M nE 8 cell 16 cell Names of blastomeres are according to Conklin (31). A, animal hemisphere; V, vegetal hemisphere. Differentiation was observed in cleavagearrested embryo. N, neural type; E, epidermal type; M, muscular type; nE, nonexcitable type. * From Okado and Takahashi (225, 226). † From Okada et al. (227). [Modified from Hirano et al. (99).] TABLE present even in case of Ciona, although the suppressive mechanism is not always complete and leaves only a dominant tendency to either muscular or epidermal characteristics. Actually, in the cleavage-arrested, one-cell Ciona embryo, the two muscle characteristics acetylcholinesterase and tropomyosin-containing filaments tend to appear simultaneously (36). Thus separate sets of muscle characteristics may be regulated by the same regulatory mechanism, probably through a single multigene regulatory factor, such as MyoD (313), as suggested by Crowther et al. (36), whereas genes necessary for other tissues may be suppressed as groups. Thus suppression mechanisms must be an important feature of cellular differentiation. Because a type of electrical excitability that is produced by a specific combination of ion channels can be equivalent to a set of differentiation markers that must be regulated as a group of genes, cleavage-arrested blastomeres examined by electrophysi- 2. Identification of cell type Cell Type Channel Egg Na/ channel Differentiated Na/ channel Egg Ca2/ channel Differentiated Ca2/ channel Delayed K/ channel Anomalous K/ channel Ca2/-dependent K/ channel Histochemical Acetylcholinesterase Microscopic Myofilaments Tunica Egg Epidermal Type Neural Type Muscular Type Nonexcitable Type // 0 // 0 ? / 0 /0 0 0 /// 0 /// // 0 /// 0 // // // / 0 0 0 /// ///* // / 0 0 0 0 / / 0 0 0 0 /// 0 0 0 0 // 0 0 /// 0 0 0 Shift of potential dependence and kinetics distinguished differentiated Na/ and Ca2/ channels from egg Na/ and Ca2/ channels. Ion selectivity and inactivation mechanism that distinguished differentiated Ca2/ channels from egg Ca2/ channels was from Hirano and Takahashi (98). Myofilaments were observed with an electron miscrosope. * Tetraethylammonium sensitivity that distinguished neural type from muscular type was from Shidara and Okamura (271). [Modified from Hirano et al. (99).] / 9j08$$ap07 P25-7 03-18-98 08:13:31 pra APS-Phys Rev Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on May 4, 2017 development of ascidian eggs. In the case of Halocynthia one-cell embryos, epidermal characteristics are dominantly expressed in terms of ion channel expression, whereas other characteristics are barely expressed (98, 225). The binding of antiepidermal antibodies also reveals exclusivity of epidermal differentiation (177, 211, 212), and there is evidence that other types of differentiation, such as muscular characteristics, are suppressed (212, 225). In Caenorhabditis elegans embryos, it is also known that the cytochalasin B-treated, one-cell embryos express always epidermal characters, whereas descendant cells in an untreated embryo give rise to other tissue types (35). In contrast, it has been reported that a fraction of cleavage-arrested, one-cell Ciona embryos evidently express the muscular characteristics as assessed by binding of antimuscular antibodies (212). However, it is still possible that muscular and epidermal characteristics are essentially mutually exclusive; exclusivity mechanisms may be 314 KUNITARO TAKAHASHI AND YASUSHI OKAMURA ological methods provide unique opportunities to analyze intracellular mechanisms of determination by selective suppression. III. ION CHANNELS AND THEIR CHANGES DURING OOGENESIS AND FERTILIZATION A. Oocyte Maturation, Fertilization, and Cytoplasmic Movement / 9j08$$ap07 P25-7 mechanism develops during maturation of starfish and hamster oocytes (28, 64). Calcium release mechanisms become more and more sensitive to either mechanical or chemical agents. However, this may indicate that the oocyte is ready to increase its intracellular Ca2/ concentration at the time of fertilization (19, 114, 296). In terms of electrical properties, the oocyte membrane becomes less permeable during maturation by losing K/-permeable channels, such as inward rectifier K/ channels or outward rectifier K/ channels (191, 192, 195), being more sensitive to depolarization (196). In starfish oocytes, the density of Ca2/ channels is known to increase during periods of oocyte growth before meiosis but remains relatively constant during meiotic maturation, this being contrasted to the case of K/ permeability. These changes may also derive from those in intracellular Ca2/ status during maturation. However, the essential requirement of an intracellular Ca2/ increase for maturation has been questioned and may be a result of hormonal application at least in starfish eggs (34, 319). One of the significant results of the increase in intracellular Ca2/ at the fertilization is thought to be the production of changes in electrical potential of the egg membrane: the fertilization potential. However, the rapid rise of the earliest part of fertilization potential is not because of intracellular Ca2/ increase, but to Ca2/ spikes evoked by a transient depolarization at the time of sperm attachment: the sperm attachment potential (40, 46, 72). Calcium influx through open Ca2/ channels may contribute to the initial increase in intracelluar Ca2/, at least underneath plasma membrane; however, it is known that egg activation can proceed without the initial Ca2/ influx under voltage clamp (26). In almost all species of animal eggs, the existence of various types of Ca2/ channels has been confirmed. The middle and later phases of the fertilization potential may be caused partly by a Ca2/-activated nonselective cationic conductance or by IP3-sensitive cation channels. In this phase, the coincidence between changes in potential and intracellular Ca2/ is significant in both sea urchin and ascidian eggs (18, 46). Calcium influx through these nonselective cation channels may contribute to the steady-state increase in intracellular Ca2/ (73). The depolarization produced by the fertilization potential, including both the early rapid phase and the long-lasting phase, is known to function as a fast mechanism of polyspermy block in various animal species, including echinoderms, Chaetopterus, ascidians, and amphibians (109). Apart from the membrane properties, the most obvious intracellular Ca2/-dependent change in the case of ascidian eggs is cortical ooplasmic segregation (18, 286). The segregation is fairly important for cytoplasmic determinants of later cellular differentiation. After the initial, large, transient increase in intracellular Ca2/ concentration, exocytosis of cortical granules occurs in various 03-18-98 08:13:31 pra APS-Phys Rev Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on May 4, 2017 When the oocyte membrane is situated at the starting position of differentiation or null point of development, several studies demonstrate the existence of electrical excitability similar to that observed in differentiated excitable membranes (87, 196). Thus, to study cellular differentiation in terms of electrical excitability, it is necessary to first understand various changes in ion channels of egg membranes occuring at the time of maturation or fertilization. Both membrane and cytoplasm of the oocyte may undergo considerable modulation during maturation, after which the oocytes are arrested at the first or second metaphase of meiosis, depending on animal species, and ready for fertilization by sperm (68). Upon fertilization, meiosis is mostly resumed, and the male and female pronuclei are fused (68). Developmental changes in the membrane ion channels begin at the time of fertilization. Attachment of sperm induces a long-lasting depolarization called fertilization or activation potential in various animal species, such as sea urchins, ascidians, and amphibians, possibly because of membrane permeability changes introduced by the explosive rise in intracellular Ca2/ concentration (26, 72, 269). After the fertilization potential is completed, the membrane is repolarized. The characteristic Ca2/ waves and Ca2/ pulses observed correspond to various biological phenomena, such as resumption of meiosis followed by extrusion of polar bodies, ooplasmic segregation (286, 287), and mitosis followed by cell cleavage (316). Thus the increase in intracellular Ca2/ is one of the major causes of ion channel changes triggered by fertilization. The primary drive of oocyte maturation has been recently clarified in terms of metaphase promoting factors (MPF) and cytostatic factors (CSF) arresting the mitotic process at metaphase, such as cdc2/cyclin B and CSF (27, 131, 316). In addition, involvement of intracellular Ca2/ in this maturation process has been suggested in starfish eggs and some mammalian eggs (54, 100, 127). Suppression of the changes in intracellular Ca2/ with Ca2/-chelating agents can arrest meiotic maturation at specific stages from germinal vesicle breakdown (GVBD) to metaphase of the second meiotic division in pig oocytes (127). An inositol 1,4,5-trisphosphate (IP3)-induced Ca2/ release Volume 78 April 1998 ION CHANNELS AND EARLY DEVELOPMENT OF NEURAL CELLS B. Ion Channels in Oocytes: Ca2/, Na/, and Inward and Outward Rectifier K/ Channels The ion channels that contribute to potential changes in oocyte or egg membranes, as described above, have been extensively analyzed in various animal species, including ascidians, echinoderms, amphibians, and mice (87, 196). Voltage-dependent Ca2/ channels, voltage-dependent K/ channels including inward rectifier, and in some cases Cl0 channels are known to exist in most immature oocytes. Voltage-dependent Na/ channels appear to be unique to ascidian oocytes (12, 227, 228), although tetrodotoxin (TTX)-sensitve Na/ channels have been recently reported in Xenopus oocytes (16). Ascidian oocytes are shown to have a characteristic N-shaped nonlinear current-voltage (I-V) relation produced by inward K/ rectifier channels and outward K/ rectifier channels (66, 189, 190, 219). The nonlinear I-V relation due to the inward K/ rectifier is also observed in most other marine eggs, including echinoderms (89, 90, 187, 194), annelids (88), and coelenterates (92). In starfish oocytes, the changes in expression of inward rectifier K/ and A-current channels have been analyzed in relation to maturation induced by a starfish hormone, one-methyl adenine (188, 192, 195). Major changes during maturation are the reduction of outwardly rectifying A-current channels and of inward rectifier K/ channels, resulting in a high membrane resistance in the poten- / 9j08$$ap07 P25-7 tial range from 070 to 0 mV. This higher resistance is effective in producing a larger depolarization triggered by sperm attachment (192), i.e., a larger fertilization potential. Because depolarization by the fertilization potential functions as a fast polyspermy blocker (109), these changes during maturation are favorable for monospermic fertilization. Immature oocytes are easily polyspermically fertilized, since they do not generate a depolarization to the level of the fertilization potential of mature eggs (181, 183). A current-type channels are also found in various oocytes, such as ascidians (228), starfish (192), and mice (227); however, recent studies reveal that the ‘‘A current’’ in mouse eggs may be an outward current through T-type Ca2/ channels (239). In Xenopus or frog oocytes, outward currents through various K/ channels have been identified. Channels activated by transmitters and peptides exist mostly in follicular cells connected to oocytes by gap junction (176), but some of these channels have been identified as delayed rectifier K/ channels on the oocytes themselves (162, 237). The inward K/ rectifier has not been found in Xenopus, frog, or mouse oocytes. Calcium channels exist, similar to those in excitable membranes, and Ca2/ spike potentials can be evoked by membrane depolarization above a critical potential of 050 to 040 mV in most of oocyte membranes, including those of ascidians (12, 229), echinoderms (91, 153), mice (227, 239, 332), Xenopus (15), annelids (88), and coelenterates (92). Most Ca2/ channels in oocytes exhibit fast and voltage-dependent inactivation. However, slowly inactivating channels frequently exist simultaneously (14, 42, 61). The density of Ca2/ channels increases during oocyte maturation before meiotic maturation or GVBD in starfish, that is, during the phase of oocyte growth (191). In ascidian oocytes, changes in the density of Na/ or Ca2/ channels during the maturation phase have not been examined. Extracellular Ca2/ is known to be effective in increasing the frequency of intracellular Ca2/ oscillation and is necessary for meiotic resumption after egg activation in ascidian (72, 171), murine (101, 182, 273, 304), and amphibian oocytes (69). The effects may be mediated by Ca2/ release from cortical Ca2/ stores of egg endoplasmic reticulum stimulated by Ca2/ influx (182). The channels mediating the influx have been inferred to be Ca2/-induced or IP3-mediated nonselective cation channels (331), but further analysis may be required (182). The involvement of voltage-dependent Ca2/ channels has not been excluded. Sodium channels in ascidian oocytes, which induce a Na/ and Ca2/ mixed spike in the rapidly rising phase of the ascidian fertilization potential, are known to be relatively permeable to Ca2/ (229). It is highly probable that the Ca2/ or Na/ channels that are capable of transferring divalent cations are partly responsible for the Ca2/ influx that regulates the early phase of intracellular Ca2/ 03-18-98 08:13:31 pra APS-Phys Rev Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on May 4, 2017 other species of eggs, but not in the ascidian (334). The Ca2/ increase starts at the site of sperm attachment and propagates over the whole egg cortex as a Ca2/ wave, being followed by a wave of exocytosis in the sea urchin egg (308). In ascidian eggs, the exocytosis of cortical granules is rarely observed; instead, a cortical cytoplasmic movement follows Ca2/ waves triggered by the initial transient increase in intracellular Ca2/ (286). In this cytoplasmic movement, a remarkable contraction of the cortical layer produces a new distribution of cytoplasmic components in the egg. Remarkably, the mitochondria-rich layers are transferred first to the vegetal pole and then to the posterior equatorial region, referred to as phase I and phase II (119, 259, 266). In other species, more or less cytoplasmic movement may contribute to the segregation of ooplasmic factors after activation. The involvement of an increase in intracellular Ca2/ in exocytosis is suggested by analogy to the common secretory process (9, 115). Cytoplasmic movement generated by microfilament-related structures is also considered to be promoted by an actin-myosin interaction triggered by Ca2/ increase (266). In ascidian eggs, phase I is sensitive to cytochalasin B, indicating a microfilament-related process, whereas phase II is sensitive to colcemid, indicating a microtubule-dependent process (265, 266). 315 316 KUNITARO TAKAHASHI AND YASUSHI OKAMURA / 9j08$$ap07 P25-7 285). The channels responsible for these holding currents, fertilization currents, in ascidian eggs may correspond to the nonselective cation channels activated by sperm attachment (39, 40) during the early phase or by intracellular Ca2/ release (140, 298) during the later phase. Similar cation channels are also known to produce fertilization potentials in urechis, nemertean, and sea urchin eggs (46, 110, 134, 295). The density of Na/ channels, which exist uniquely in some species of ascidian oocytes (12, 65, 220, 228, 234), doubles within 1 min after egg activation in the case of Halocynthia eggs (140); the density then decreases gradually almost to the previous level within 0.5 h (140). Calcium channels with voltage-dependent inactivation exhibit decreases in density, reaching a minimum several minutes after fertilization, probably because of Ca2/-induced Ca2/ channel suppression (140). Inward K/ rectifier channels reveal an abrupt, small, transient increase and then immediately decrease almost in parallel with Ca2/ channels (140). However, inward rectifier K/ channels gradually increase their density again after 10 min (140). This increase corresponds to the increase in permeability to K/ after the completion of the fertilization potential in echinoderms and amphibians. All those changes may be explained primarily by the increase in intracelllular Ca2/ (298), but solid evidence is still lacking. In ascidian oocytes of Boltenia villosa, a decrease in Na/ channel density and slight increase in Ca2/ and inward rectifier K/ channel densities have been described after two cell stages (12); however, the continuous observation of voltage-dependent ion channels immediately after egg activation has not been reported. In hamster oocytes, repetitive and transient increases in Ca2/-induced K/ current appear during the early phase after fertilization, in precise resister with oscillations of intracellular Ca2/ concentration (184). Evidence that Ca2/ oscillations are caused by Ca2/ release induced by IP3 (IICR) was obtained using blocking antibodies directed to the IP3 receptor (185). Similar changes in Ca2/-induced K/ current have been observed in mouse oocytes, but of much smaller amplitude than those of hamster oocytes (102). A fertilization potential as a polyspermic blocker is absent in the case of mouse eggs (113). However, the hyperpolarization induced by the K/ channels may be useful for increasing the driving force of Ca2/ influx. In murine oocytes, fast inactivating Ca2/ channels are known to exist as described above (227, 239, 332). They increase in density almost twofold within 0.5 h after sperm attachment (328), which may correspond to the later phase of intracellular Ca2/ increase, and then decrease and disappear after the eight-cell stage (178). In amphibian oocytes, as described above, Ca2/-dependent Cl0 channels increase in density as a result of the 03-18-98 08:13:31 pra APS-Phys Rev Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on May 4, 2017 release and accordingly contribute to the resumption of meiosis or the initiation of mitosis (316). Increases in Ca2/ or Na/ currents may also be effective in generating the rapid rise in fertilization potential that is essential for the fast mechanism of polyspermy block in various oocytes, such as ascidians (73), sea urchins (108), Urechis (74), Xenopus (78), and nemerteans (135). In both hamster and mouse oocytes, Na/ channels have not been reported; however, Na/ current can flow through egg Ca2/ channels when the extracellular Ca2/ concentration is reduced below 1 mM (227, 239, 332). In amphibian oocytes, Ca2/-dependent Cl0 channels producing inward current exist in addition to Ca2/ and/or Na/ cationic channels (136, 175, 238); they are increased during meiotic maturation before GVBD but tend to decrease after GVBD (309). In the environment of a natural external medium of pond water, the current through Cl0 channels is inwardly directed, and the channels in association with some K/ channels are responsible for fertilization currents in amphibian oocytes (112). In lamply oocytes, Ca2/-dependent Cl0 current has been detected during fertilization, whereas in other fish eggs, such as those of Medaka, the Cl0 current has not been identified as a component of fertilization current (137, 215). At fertilization, densities of various ion channels undergo enhancement or reduction due to changes in the intracellular environment caused by activation, often due to increased intracellular Ca2/ and/or protein kinase C activation (296). As described above, the intracellular Ca2/ concentration inside fertilized eggs usually increases in two phases: an early, large, and transient increase within a few minutes after sperm attachment and a later, relatively small and slowly oscillatory change during a few tens of minutes after fertilization in the eggs of ascidians, echinoderms, amphibians, and murines (19, 171, 182, 285, 296). These changes are now considered to be due mainly to Ca2/ release from IP3 receptor- and/or ryanodine receptor-regulated Ca2/ stores (9, 10). The increase in steady intracellular free Ca2/ concentration may be due to increased Ca2/ influx through various cation channels that are either voltage dependent or Ca2/ and/or IP3 dependent (238, 331). Correspondingly, the changes in membrane ion channel densities can be considered in two phases. In ascidian eggs, both early and later changes have been analyzed in detail by voltage clamp during activation with sperm or with Ca2/ ionophores (12, 18, 40, 72, 140, 298). The holding current at 070 mV shows a large, transient, inwardly directed increase during the early phase and a continuously relatively high level of inward current during the later phase, which produces the fertilization potential under constant-current condition (140). These two phases of changes in ionic channels may correspond to the changes in intracellular Ca2/ concentration analyzed in other ascidian species, described above (18, 171, Volume 78 April 1998 ION CHANNELS AND EARLY DEVELOPMENT OF NEURAL CELLS IV. EARLY CHANGES IN ION CHANNELS in morphogenesis, as shown in the cases of zebrafish and Drosophila embryos (122). Halocynthia embryos are synchronous until the 32cell stage; after the 64-cell stage, the cells in the animal hemisphere exhibit a shorter cell cycle, as described by Nishida (205). After the cell cycle phase in the blastula becomes asynchronous, vertebrate embryos gastrulate in the first significant morphogenetic movement, which determines the location of presumptive areas for major organs in both anteroposterior and dorsoventral axis of the vertebrate body (68). The morphogenic movement is accomplished by the activity of a morphogenetic center, Spemann’s organizer, which initiates gastrulation in vertebrate embryos (68, 288). Although the animal-vegetal axis already exists in the unfertilized ascidian egg as well as in the amphibian egg, establishment of the dorsoventral axis in the ascidian embryo occurs after the first and second phases of cytoplasmic movement just after fertilization. The cytoplasmic movement shifts the myoplasm into the posterior end of the vegetal hemisphere, triggered by an increase in intracellular Ca2/ (6, 116, 286). In the ascidian embryo, it is known that the two cells in the vegetal pole at the 64-cell stage are the pioneer cells to gastrulate (261). The presence of the gastrulation center at this vegetal pole in the ascidian embryo was recently confirmed by blocking the gastrulation at the 110cell stage with ultraviolet radiation on the vegetal pole at the period of first cytoplasmic movement, just as in the amphibian embryo (117, 118). A. Cleavage, Blastula, and Animal-Vegetal Axis As a result of activation with sperm, the egg cell undergoes first mitosis after fusion of male and female pronuclei. The initiation of mitosis requires factors similar to those required for meiosis, such as the activated cdc2 and cyclin B complex, as described in section IIIA. The increased intracellular Ca2/ concentration must play an important role on the initiation of mitosis (10, 172), probably through protein phosphorylation and dephosphorylation with calmodulin kinase II or Ca2/-dependent phasphatase IIa as shown in the case of resumption of meiosis in Xenopus eggs (161). Cell cleavage after mitosis includes formation of a contractile ring as well as new plasma membrane. The former may include actin microfilament interaction triggered by local Ca2/ increase (274, 284, 316), and the latter may induce new integration or disappearance of membrane ion channels. Cell cleavage in Xenopus and zebrafish eggs occurs synchronously within an embryo until the midblastula stage without a detectable G1 period, indicating that there is no interruption of mitosis before going into the next cell cycle (121, 200). After the midblastula stage, the cell cycle becomes asynchronous; synchrony is maintained only within a domain that shares the same functional role / 9j08$$ap07 P25-7 B. Egg-Type Na/ and Ca2/ Channels In this and the following sections, the changes in membrane ion channels are described during the blastula and early gastrula stages when cell cleavage occurs very rapidly in most animal oocyte species. In mature oocytes or eggs, there are several kinds of ion channels including Na/, Ca2/, inward rectifier K/, and delayed rectifier K/ channels. Among these ion channels, Na/ and some Ca2/ channels decrease in their densities during initial cleavage stages. In Halocynthia embryos, both Na/ and fast-inactivating Ca2/ channels decrease in density during the first 10 h at 10⬚C from fertilization to the early gastrula stage. As described in section IIIB, Na/ channels show a characteristic transient enhancement during the first few minutes after fertilization (98, 140, 233, 298). Calcium channels disappear within 10–12 h after fertilization in Halocynthia embryos (140), whereas in Boltenia embryos, the total number of channels in the embryo remains constant until the eight-cell stage and disappears at the gastrula stage (12, 276). In Halocynthia and Boltenia embryos, Na/ channels are reduced in number on the whole 03-18-98 08:13:31 pra APS-Phys Rev Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on May 4, 2017 intracellular Ca2/ increase at fertilization, causing fertilization current (19, 111, 112, 136). Intracellular Ca2/ oscillations are reflected in the oscillatory changes in Cl0 currents and in fluctuations in the fertilization potential. Although the fertilization current appears to be carried by external cations in the case of many marine oocytes, which are fertilized in high ionic strength seawater, those of amphibians spawn in freshwater are carried by anions such as Cl0, since Cl0 efflux causes an inward, depolarizing current. In Medaka eggs immersed in intravitellin fluid of relatively high ionic strength, the fertilization potential is considered to be due to cation influx of Na/ and Ca2/ (215). The functional significance of changes in other ion channels than those used for fertilization potential, such as Na/ and Ca2/ channels, are not known. However, Ca2/ influx through Ca2/-permeable Na/ channels in ascidian eggs or that through Ca2/ channels in mouse eggs may contribute to the cortical cytoplasmic contraction or secretion of cortical exocytotic granules. In summary, before initiation of embryonic development, the oocytes have already several kinds of ion channels, and relative amounts among various channels are changing depending on the stages of maturation, fertilization, and postfertilization. Some of the changes may be directly related to those changes in intracellular Ca2/ concentration. 317 318 KUNITARO TAKAHASHI AND YASUSHI OKAMURA Volume 78 / 9j08$$ap07 P25-7 03-18-98 08:13:31 pra APS-Phys Rev Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on May 4, 2017 embryonic membrane to one-fifth by the 8- to 32-cell tional inhibitor a-amanitin, a RNA polymerase II inhibitor stages. Among ascidian embryos, Ciona embryos lack Na/ (235), at the 32-cell stage blocks the increase of inward channels (2, 14). K/ rectifier (refer to Fig. 4), keeping the increase to less 2/ In amphibian embryos, the existence of Ca chan- than 2–3 times that in the egg (235). This indicates that nels was suggested by voltage-dependent activation of transcription for inward rectifier K/ channels may be initiCa2/-activated Cl0 channels, and the presence of endoge- ated as early as the 32-cell stage, probably at the time of nous Ca2/ current has been recently confirmed (15); devel- midblastula transition in the ascidian embryo. A minor opmental changes have not yet been examined during fraction of the increase appears to be derived from materearly development. In the mouse egg, a transient enhance- nal RNA, because a slight increase above the oocyte level ment of Ca2/ channel densities during the first 2 h after still remains after ample injection of a-amanitin. fertilization, as described above, is followed by a decrease This early zygotic expression of ion channels seems in Ca2/ current, and almost all Ca2/ current disappears unique, because changes in other ion channels, such as until the eight-cell stage (178). Na/ and Ca2/ channels, during early development before / With the consideration that the Na channels in Halo- 40 h (see Fig. 3) are not dependent on zygotic transcripcynthia oocytes are permeable to Ca2/ (229), the intial tion in the Halocynthia embryo (98, 233, 235). Transcriptransient increase of Na/ channels in ascidian oocytes or tion for zygotic and lineage-specific expression of other Ca2/ channels in mouse oocytes may be responsible for ion channel genes, such as Na/, Ca2/, and delayed K/ enhancing Ca2/ influx immediately after fertilization as channels, in ascidian embryos is known to be initiated suggested above. Oocytes kept in saline with a relatively after the midgastrula stage (231, 235, 276). high Ca2/ concentration, such as seawater or intercellular Resting K/ conductances of other embryos appear to fluid of extraembryonic tissues, may have an ability to increase during blastula stages, such as in Xenopus, since utilize Ca2/ influx for regulation of intracellular Ca2/ con- the resting potential usually becomes more hyperpolarcentration to maintain short mitotic cycles or other func- ized and more sensitive to K/ as development advances tions during early development. The succeeding decrease from stage 6 to stage 9 in isolated and ouabain-treated may be necessary to establish a low level of intracellular Xenopus blastomeres (7). In amphibian embryos, the inCa2/ after the initial phase so as not to disturb the progres- crease in K/ conductance associated with new membrane sion of later development. formation at the time of cell cleavage is well known (156, Quite recently, significant influences on excitabilty 312, 322). development produced by changing Ca2/ transient freIn mouse embryos, voltage-independent K/ permeabilquencies have been demonstrated in Xenopus embryonic ity increases after the eight-cell stage, and the measured spinal neurons (81, 82). The importance of intracellular resting potential becomes hyperpolarized (178). However, Ca2/ dynamics has never been questioned in early devel- no other quantitative data are available for indication of the opmental processes. However, further quantitative and early development of inward K/ rectifier except in another time-sequential studies of Ca2/ influx and intracellular ascidian embryo, Boltenia (12, 275). The significance of Ca2/ release, coordinated with analysis of changes in vari- the early transcriptional dependence of the inward rectifier ous ion channel densities, as actually carried in Xenopus is discussed in section VD with special attention to neural spinal neurons (82), are still required to attain a complete differentiation in ascidian embryos. Delayed rectifier K/ channels responsible for outunderstanding of their developmental roles in a simple differentiating system, such as ascidian blastomeres. ward current above 0 mV in the early Halocynthia embryo are not yet clearly identified. There is no evidence that the density of outward rectifier channels increases or deC. Various K/ Channels creases during early development before lineage-specific expression of ion channels. In contrast to Ca2/ and Na/ channels, inward rectifier K/ channels increase their densities gradually during the 0 blastula stage until 15 h in Halocynthia eggs as well as D. Cl and Other Ion Channels in those of other ascidians (12, 98, 235). At 15 h of development or the midgastrula stages, The resting Cl0 conductances at blastula and gastrula / the density of inward rectifier K channels in cleavage- stages have been analyzed in detail in the ascidian Bolarrested ectodermal blastomeres suddenly increases up tenia egg. At the cleavage stage, Cl0 permeability is to 20–30 times that in the unfertilized egg (235). In the closely associated with cell cleavage, showing changes intact Halocynthia gastrula embryo also, a marked in- precisely coordinated with the cell cycle (13). Cyclic crease in inward rectifier K/ current has been reported changes in kinases may be responsible for activation of by Miyazaki et al. (190). The increase is found to be tran- Cl0 channels (13). scription dependent, since the injection of the transcripAt the beginning of the blastula stage of Medaka, a April 1998 ION CHANNELS AND EARLY DEVELOPMENT OF NEURAL CELLS 319 freshwater fish, the blastocoel cavity is formed for the tive for maintaining the Cl0 equilibrium potential (272). first time. A mechanism is necessary to transport ions to In amphibian embryos, it is worth examining whether the cavity without losing them to pond water outside the Cl0 channels may have similar roles to those of Medaka. embryos. Recently, Shigemoto and Okada (272) interest- Similar Cl0 conductances have not been reported in early ingly found the Cl0 channels in the blastoderm cells, mouse embryos. which are external anion dependent (272). Here, the curGadolinium-sensitive nonselective cation channels rent cannot be activated in the membrane facing the that can be stretch activated are found in the early ascidpond water, where concentrations of anions are much ian embryos (193) and amphibians (297, 329, 330), indicatlower than those of the intracavernous solution. Only ing the possibility that the stress and strain during cell after blastocoel formation are the blastoderm cells first cleavage may activate nonselective cation conductance exposed to the extracellular fluid with high ionic concen- and thereby control cell movement or cell shape by Ca2/ trations. Then, the anion channel would become effec- influx or osmolarity-dependent volume changes. / 9j08$$ap07 P25-7 03-18-98 08:13:31 pra APS-Phys Rev Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on May 4, 2017 / FIG. 3. Development of Na channels in neurally induced cleavage-arrested 8-cell blastomeres with voltage clamp and in intact tadpole larvae with in situ hybridization. A: macroscopic Na/ currents in neurally induced cleavage-arrested a4–2 blastomeres. Modes of currents decrease with prepulse depolarization at 3 stages. Type A is found in unfertilized eggs and in both induced and uninduced blastomeres at early stages until 40 h at 10⬚C (phase 1). Type C is expressed only in induced blastomeres, correponding to neuronal specific TuNa I gene (phases 2 and 3). Type B may be a precursor for neuron-specific Na/ channels and is seen only during initially rapidly increasing phase of expression (phase 2). B: peak amplitudes of Na/ channel currents in neurally induced blastomeres are plotted against developmental time at 9⬚C, on a semilogarithmic scale. Solid circles, H. aurantium embryos; open circles, Halocynthia roretzi embryos. Dashed line indicates development of Na/ channels in uninduced epidermally differentiated blastomeres. UF, Na/ currents at the stage of unfertilized eggs. C: control in situ hybridization of myosin mRNA. D and E: in situ hybridization of TuNa I mRNA in a tail bud and a young tadpole of H. aurantium. F: an enlargement of neurons stained in another young tadpole. Camera lucida images adjacent to in situ hybridization panels indicate previously mapped positions of neurons from Nicole and Meinertzhagen (203). Arrowheads indicate positions of epidermal neurons. EN, epidermal neuron; NH, neurohypophysis; No, a row of large notochordal cells; NT, position of neural tube; OC, ocellus; OT, otolith; PSV, postsensory vesicle; SV, sensory vesicle; UD, undefined neurons in head surface; VG, visceral ganglion. Bar, 100 mm. [A and B from Okamura and Shidara (223); C–F from Okamura et al. (231).] 320 KUNITARO TAKAHASHI AND YASUSHI OKAMURA V. NEURAL COMMITMENT AND CHANGES IN EXPRESSION OF ION CHANNELS A. Neural Induction in Vertebrate Embryos B. Two-Cell Induction System Constructed From Early Ascidian Embryos In the ascidian embryo, the isolated anterior animal blastomere from the eight-cell cleavage-arrested embryo, including the presumptive brain vesicle region, autonomously develops a long-lasting action potential due / 9j08$$ap07 P25-7 to the expression of slowly inactivating Ca2/ channels characteristic for epidermal differentiation of the ascidian tadpole (225). However, when cultured in contact with the anterior vegetal blastomere, including the presumptive notochordal region, and raised as the contacted two-cell system, the same anterior animal blastomere now develops typical Na/ spikes due to expression of Na/ channels and TEA-sensitive delayed rectifier K/ channels (223, 224, 232–234, 271). Sequencing of cloned cDNAs reveals that these Na/ channels have close similarity in their primary structure to mammalian brain type II Na/ channels (231). Monoclonal antibodies directed to larval epidermal cells stain the blastomere only when cultured in isolation but show no binding when cultured as the two-cell induction system (224). To ascertain that this inductive phenomenon of Na/ spikes is equivalent to neural induction in the ascidian embryo, we examined critical periods of Na/ spike induction by changing the stages at which cells were brought into contact (224). The effective contact for induction occurs from the 64-cell stage to the midgastrula stage, or from 10 to 12.5 h of development at 10⬚C, whereas the expression of Na/ spikes appears at much later stages, from 38 to 45 h of development (224). The response-competent phase and inducer-active phase were determined separately. The responding anterior animal blastomere from various stages during 15 h from the eight-cell to the midneurula stage was contacted for 5 h with the inducing anterior vegetal blastomere from a fixed stage. Inversely, the responding blastomere from a fixed stage was contacted with inducing blastomere from various stages. The results indicate that the competent period of the presumptive neural blastomere is mainly responsible for the critical period for induction, whereas the inducer activity of the presumptive notochordal blastomere lasts much longer than the competent phase (224). Because the critical period of neural induction in the amphibian embryos occurs at the early to midgastrula stage (240), it is highly probable that the induction of Na/ channels in the ascidian embryo is equivalent to the amphibian neural induction (223, 224). Aside from the vertebrate embryo, neurogenesis in the Drosophila embryo also requires a kind of cell-cell interaction, but the major strategy is different from that of the vertebrate embryo in the following two aspects (20, 22): 1) neuroblasts are generated from the ventral ectoderm instead of the dorsal ectoderm in constrast to vertebrate embryos, and 2) the default differentiation of isolated competent ectoderm in the Drosophila is neural instead of epidermal. A group of almost 10 genes has been identified as neurogenic genes, having the same phenotype of suppressing further neuroblast formation around an antecedently differentiated neuroblast within the ventral neurogenic ectoderm (22). The Notch gene, one of the 03-18-98 08:13:31 pra APS-Phys Rev Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on May 4, 2017 Since the study of neural induction by Spemann and Mangold (289), it has been known that initiation of development of the vertebrate nervous system requires tissue interaction between the ectoderm and underlying mesoderm at the early gastrula stage. Since then, the chemical substances naturally inducing neuralization have been intensively sought, and many inducing substances have been found because of the ease of neuralization in the newt ectoderm (267). None of them has been rigidly shown to be an endogenous inducer. Recently, three substances, noggin, follistatin, and basic fibroblast growth factor (bFGF), have been proven to show neuralizing capacity and given some credit as endogenous inducers (279). Noggin was originally cloned by its effect in rescuing mesoderm for ultraviolet-irradiated and ventralized Xenopus fertilized eggs (152). Follistatin is an activin-binding protein that inhibits the effects of activin. Expression of the truncated, dominant negative form of the activin type II receptor in early Xenopus embryos promotes neuralization in explants that would otherwise become epidermis (94, 95). Both noggin and follistatin exist in the dorsal mesoderm, the Spemann organizer, at the right time and have the capability to produce neuralization of ectoderm, producing especially anterior neural structures (94, 152). On the other hand, neural induction was recently reconstituted in vitro in a microculture system composed of ectoderm and mesoderm cells dissociated from stage 10 Xenopus embryos (179, 180). Although bFGF was originally famous for inducing mesodermal tissues (280, 281), the same bFGF, at much lower concentrations than needed for mesodermal induction, was proven to be a powerful direct neuralizing agent in this system (128). The time dependence of the response is different from that for mesodermal induction, and furthermore, no mesodermal structures were detected in the microcultures even when the dose of bFGF was sufficient for neural induction (128). In light of these recent findings, we have studied intercellular mechanisms of neural induction using a simplified system of cellular interactions, consisting of two blastomeres from early ascidian embryos (223). Volume 78 April 1998 ION CHANNELS AND EARLY DEVELOPMENT OF NEURAL CELLS C. TuNa I Channels Are Neuron-Specific Markers in the Ascidian Embryo and Are Expressed After Inductive Differentiation It recently became clear that an isolated single presumptive neural blastomere from Halocynthia embryos will undergo neural differentiation without cell-cell contact if the blastomere is treated by proteolytic enzymes, serine proteases, or bFGF at the same stages when the blastomere is competent for the induction by cell contact (104, 224, 226, 230). Subtilisin, a crystallized serine protease, induces Na/ spikes with nearly 100% probability in blastomeres when it is applied at a dose of 0.03% at 10⬚C for 1 h (226). The critical period of susceptibility to proteases is identical to that for induction by cell contact, and susceptibility is lost after midgastrula stage (226). It is concluded that the protease can mimic the inducer activity of the presumptive notochordal blastomere. By using induction with protease, development of Na/ channels was quantitatively analyzed in the single isolated presumptive neural blastomere until full-sized Na/ spikes appeared (230, 233). In Figure 3, the maximum Na/ current density in the protease-induced blastomere isolated from the Halocynthia embryo is plotted against developmental time. Protease treatment was carried out at the 32- to 64-cell stage, at the start of the competent period. The unfertilized mature Halocynthia egg has Na/ channels indistinguishable from those found in common excitable membranes (228). Egg Na/ channel density nearly doubles during the first few minutes after fertilization, as mentioned above (140), but starts to decrease immediately after attaining this peak. Egg Na/ channel / 9j08$$ap07 P25-7 density is minimal at 15 h of development at 10⬚C, when the control embryo attains the late gastrula stage that corresponds to the end of the competent phase for Na/ spike induction (233). Sodium channel density then increases and shows a maximum at 25 h of development, the midneurula stage. Until this stage, Na/ channel kinetics are not distinguishable from those of egg Na/ channel. Finally, the density of egg Na/ channels shows a second minimum at Ç40 h of development when the control embryo becomes a young tadpole. After this stage, the density increases steeply, and at 50–60 developmental hours, full Na/ spikes appear. This rapid increase is transcription dependent, since it is inhibited by culturing the blastomere in the seawater containing the transcription inhibitor actinomycin D. However, modulation of egg Na/ channel density up to this stage is maternally regulated and unaffected by the transcription inhibitor (233). As shown in Figure 3A, analysis of Na/ currents at stages before and after 40 h of development reveals clear kinetic differences at both macroscopic and single-channel levels (232, 234). The Na/ channels (type C) expressed at later stages are concluded to be a neural differentiation marker, showing a slow decay phase and a burst-type opening in comparison with egg Na/ channels (232, 234). In addition, a third type of Na/ channel (type B) is identified during the steeply increasing phase of Na/ channel density, which shows characteristic burst opening at the single-channel level and extremely slow inactivation without a fast inactivation phase at the macroscopic level (233). These channels are suggested to be precursors of mature channels. Functional Na/ channels in situ are composed of at least three kinds of subunits, including pore-forming asubunits (106, 174), and it is known that a-subunits expressed alone in Xenopus oocytes show abnormally slow kinetics of inactivation (141); normal fast inactivation is observed when a- and b-subunits are coexpressed (23). Thus one possibility is that the slowly inactivating precursor channels in the Halocynthia embryo may be composed of only an a-subunit of Na/ channel protein without b-subunits (165, 233), since a-subunits appear without bsubunits in the developing rat retina before the increase in complexes of a- and b-subunits (320). Thus the development of Na/ channels in the induced blastomere is considered to consist of three phases (233). In the first phase, only egg Na/ channels are present, and developmental modulation of density is not transcription dependent. This phase is actually also observed in the uninduced animal blastomere committed to epidermal differentiation (interrupted line in Fig. 3B). In the second phase, a steep increase in density is observed, and inactivation of macroscopic Na/ current becomes extremely slow because of the presence of the third type Na/ channels, possibly precursor channels. In the third phase, the 03-18-98 08:13:31 pra APS-Phys Rev Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on May 4, 2017 best known and well-analyzed neurogenic genes, is now known to code a ligand protein expressed on the surface of ectodermal cells and inferred to act through cell-cell interaction (21, 323). In the case of the vertebrates, although the mechanisms of neurogenesis have been progressively elucidated as described above, many studies still remain to be carried out especially with respect to intracellular transduction after neuralizing signals. Thus, in ascidians, in which a smaller number of molecular cascades may underlie neural induction than in higher verteblates, the simple induction system potentially serves as an explicit model to fill in a gap between early cellular interactions and expression of neural functions, such as mRNA production for various ion channels. It is further of particular interest to study the cellular physiology during neural differentiation in the simple induction system, since its large cell size allows application of precise biophysical and cell biological techniques in addition to molecular biological techniques. 321 322 KUNITARO TAKAHASHI AND YASUSHI OKAMURA / 9j08$$ap07 P25-7 meres. The excitability was considered possibly to correspond to that evoked in the motoneurons. Because A4–1 blastomeres were potent neural inducers, as described above, the autocrine mechanism of endogenous inducers was proposed to explain the expression of this excitability and TuNa I gene transcription in A-line cells of the Halocynthia larva (222), and the significance of neuronal lineage different from ectodermal origin, such as neuronal cells included in A-line cells in the ascidian tadpole, was discussed in their report (222). D. Quantitative Difference in Expression of Inward Rectifier K/ Channels as an Early Sign of the Neural Induction Although a gene that is expressed uniquely in a particular cell type is useful as a differentiation marker, to know when the bifurcation of cell fate occurs it is also important to analyze quantitative differences in the transcription of the same gene in differentially fated pathways. Both qualitative and quantitative changes in membrane ion channels have been analyzed in neurally induced blastomeres after treatment with protease or cell contact to uncover differences between induced and uninduced blastomeres at the earliest stages. Ectodermal blastomeres, such as A-line cells derived from a4–2 blastomeres of eight-cell embryos, at the stage competent for neural induction, usually show egg-type Na/ channels and inward rectifier K/ channels (235); no differences are found until Ç5 h after treatment by the inducer, that is, the late gastrula of the control embryo or 15 h of development at 10⬚C. As shown in Figure 4B, at this time the density of inward rectifier K/ channels in cleavage-arrested a4–2 blastomeres starts to increase abruptly in both induced and uninduced ones, as decribed in section IVC; however, in induced blastomeres, they soon attain a maximum value while in uninduced blastomeres they continue to increase, resulting in a nearly threefold difference in the peak inward current values elicited at 0175 mV (235). This difference is not dependent on the method of induction, by either protease or cell contact. Blastomeres treated with the same protease after the competent phase has passed show the same level of inward rectifier as uninduced blastomeres. Thus it is concluded that the difference is a result of neural determination and the earliest functional sign expressed after neural induction. Fifteen hours of development corresponds to the end of the midgastrula stage of control embryos, which is comparable to the time of initiation of transcription of the neural marker gene TuNa I (231). In the Xenopus embryo, transcription of neural-specific genes such as neural cell adhesion molecule (NCAM) or Shaker homolog K/ channels is known to appear after the midgastrula stage as well (144, 249). 03-18-98 08:13:31 pra APS-Phys Rev Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on May 4, 2017 increment in density slows down, and Na/ spikes are fully grown. Both the second and third phases are characteristic of neural differentiation and are completely inhibited by transcriptional blockers (233). Because there are at least three types of Na/ channels in the ascidian tadpole and two of them are markers of neural differentiation, Okamura et al. (231) have recently cloned Na/ channel cDNAs amplified by polymerase chain reaction (PCR) from a cDNA library of Halocynthia tadpole (231). The PCR primers were designed from the common sequences among rat brain, electric eel, and Drosophila para gene Na/ channels (231). The resulting three putative Na/ channel clones were named TuNa I, II, and III. Analysis of the full-length sequence of TuNa I revealed high homology with rat brain type II Na/ channel (213) rather than rat muscle Na/ channel and Drosophila para gene Na/ channels. Transcription of the Na/ channel in the intact embryo was found to start after the late gastrula stage, by ribonuclease protection assay and reverse transcription PCR (231). Furthermore, as shown in Figure 3, D and E, the in situ hybridization of the TuNa I clone has demonstrated that the Na/ channels are expressed only in neuronal cells in the larval neural tissue but not in neuroepithelial cells (222, 231). Injection of antisense DNA into the induced blastomere effectively suppresses Na/ channel development in phase II and III, but not the development of delayed rectifier K/ channels (231, 233, 271). Therefore, the expression of Na/ channels and Na/ spike development in the ascidian tadpole is a specific marker of neuronal differentiation. These experiments further indicate that in the ascidian embryo neural morphogenesis, such as neural plate formation, as well as neuronal differentiation itself require induction by cell contact or by an equivalent inductive stimulus at the early gastrula stage. In addition, the default differentiation after neural induction in the two-cell system seems to be neuronal differentiation and not another type of neural cells such as neuroepithelial, glial, or pigment cells. Quite recently, Okamura et al. (222) found that TuNa I gene was also transcribed in the isolated anterior vegetal blastomere, A4–1, from the eight-cell embryo (222). The A-line cells in the Halocynthia larva derived from the A4–1 blastomere were reexamined with cell lineage tracers, and they were found to include neck motoneurons innervating tail striated muscles (222). Cleavage-arrested A4–1 blastomere frequently showed distinct excitability from that described in the induced a4–2 blastomere, or a-line cells, which are derived from a4–2 blastomeres of eight-cell embryos, having Na/ channels, transient A-current-like K/ channels, and Ca2/ channels (222). The excitability was apparently expressed without cell-contact or cell-cell interaction in contrast to the case of a4–2 blasto- Volume 78 April 1998 ION CHANNELS AND EARLY DEVELOPMENT OF NEURAL CELLS 323 To learn the cause of this difference, we determined Developmental studies of inward rectifier K/ chanthe transcription period for the inward rectifier by in- nels in relation to cell-type specification have been rejecting a-amanitin at various times from 8 to 15 h of ported in the differentiation of myocytes. In the case of development or from the 32-cell to the midgastrula stage. a mesodermal stem cell line, myogenic clones express the When a-amanitin was injected at the 32-cell stage, the inward rectifier in addition to NCAM and TTX-insensitive amount of inward rectifier expressed was markedly re- Na/ channels (146, 147). Myocytes of Xenopus embryos duced, leaving a small population of channels that appear also possess inward rectifier K/ channels (292). Cardiac to be maternally derived in both uninduced and induced myocytes also express an inward rectifier that is useful blastomeres (235). for repolarization of the cardiac action potential with a As shown in Figure 4C, if the injection was made at plateau (25). Historically, the inward rectifier was first a later developmental stage, the amount increased propor- described in adult muscle fibers (125). As shown in Figure 4B, open triangles, presumptive tional to the delay time of the injection in the uninduced blastomere, indicating that the amount of expression is mesodermal or presumptive muscular blastomeres from dependent on the duration of transcription (235). In con- the eight-cell ascidian embryo do not exhibit the rectifier trast, the delay of the injection time after protease treat- at the stage when it develops dramatically in the ectoderment in the induced blastomere does not increase the mal blastomere as described above (235). Therefore, examount of current expressed, indicating that transcription pression of the rectifier in ascidian ectodermal blastohas already been stopped by the protease treatment and meres is different in nature from that in muscular precurprobably just at the time of induction, i.e., Ç10 develop- sors. mental hours, independently of injection of a-amanitin In ascidians, not only embryonic ectodermal cells but / (235). Thus transcription of inward rectfier K channels also epidermally differentiated blastomeres frequently exor proteins necessary for the assembly of these channels hibit an inward rectifier (97–99). In addition, epidermally is instantly suppressed by the inductive stimulus. differentiated cells are characterized by Ca2/ channels, / 9j08$$ap07 P25-7 03-18-98 08:13:31 pra APS-Phys Rev Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on May 4, 2017 FIG. 4. Differential expression of inward K/ rectifier channels between neurally induced and uninduced blastomeres. A: identification of inward rectifier K/ channes in an isolated, cleavage-arrested a4–2 blastomere at early neurula stage. Inward currents elicited by hyperpolarizing pulses to 095, 0115, 0135, 0155, and 0175 mV in artificial seawater with or without K/. B: comparison of development of inward rectifier K/ channels in neurally induced (protease treated; solid circles) and uninduced (no protease treatment; open circles) a4–2 blastomeres. Peak values of inward current evoked at 0175 mV are plotted against developmental time. Timing of protease treatment is indicated by a bar. C: inhibition of inward rectifier K/ channel expression in neurally induced (solid circles) and uninduced cells (open circles) after injection of a-amanitin. Relationship between levels expressed at 20– 30 h of development, and timing of aamanitin injection is illustrated. On right side of graph, mean values and standard deviations for inward rectifier K/ channel current are shown for neurally induced (solid square) and uninduced blastomeres (open square) without injection of a-amanitin. Period of induction by protease treatment is indicated by the column. [A–C from Okamura and Takahashi (235).] 324 KUNITARO TAKAHASHI AND YASUSHI OKAMURA / 9j08$$ap07 P25-7 concentration. Our simple neural induction system of Haloncynthia embryos may be appropriate for these types of experiments. E. Inducer Candidates for Na/ Spike Induction Because the development of ion channels characteristic of the ascidian presumptive neural blastomere requires neural induction as described above, it is of particular interest to identify the inductive stimulus. The neural inducer is still much in debate, although 70 years have passed since Spemann and Mangold (289) first discovered primary induction in amphibian embryos. However, a suggestion that fibroblast growth factor (FGF) is a highly promising candidate for the inducer was recently made by Kengaku and Okamoto (128, 129). The suggestion has been further confirmed by recent reports from other laboratories (151, 155). To avoid the complexity of multicell-layered induction systems that have been used extensively, they have analyzed a micro in vitromixed culture system consisting of a small number of undifferentiated ectodermal cells and mesodermal cells dissociated from the stage 10 Xenopus embryo (179, 180). With this microculture system, they were able to reconstruct neural induction in vitro and obtain a quantitative assay system for inducer candidate substances with neuronal cell specific monoclonal antibodies as differentiation markers (180). After screening the effects of several growth factors, they found that 5–25 pM bFGF was effective in inducing differentiation of both CNS neurons and neural crest cells and mimicked well the time dependence of induction by dorsal mesodermal cells (128). Furthermore, it is convincingly proven that bFGF in the former dose expresses exclusively neuronal markers but never mesodermal markers in Xenopus ectoderm cells (129). Because the dose of bFGF for neuralization is one order of magnitude less than that required for mesodermal induction (281, 282) and the sensitive period for ectoderm is clearly later than that for mesoderm induction (282), it was suggested that the effect did not involve indirect conversion of ectodermal cells into mesodermal cells that became effective as neural inducer (128, 129). In addition, it is known that both bFGFs and their receptors exist in the appropriate regions of the early Xenopus embryo (63, 197, 280). Thus it is concluded that bFGF is the highly plausible inducer candidate in this system. Noggin and follistatin are also candidiates, especially for inducing anterior neural structures, as described previously. Basic fibroblast growth factor may induce whole neural structures only in cooperation with noggin and follistatin (94, 95, 152). This point has been partly confirmed (151, 155). In the ascidian embryo, a protease can mimic the inducer blastomere as described above (226). However, 03-18-98 08:13:31 pra APS-Phys Rev Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on May 4, 2017 intercellularly communicating gap junctions, and epidermal-specific antigens (97–99, 258). In the epidermally determined or uninduced ectodermal blastomere, the rectifier usually continues to be expressed once it has developed (98, 235). Thus, in the ascidian embryo, the rectifier may be considered as one of the early expressed epidermal characteristics. Transcriptional suppression by the neural induction indicates that neural determination can be achieved both by rapid suppressive effects on the transcription of epidermal characteristics as well as the initiation of new transcription of neural characteristics such as Na/ channel genes. Cellular commitment achieved by suppressing transcription of the characteristics for other cell types is also observed in the Xenopus tadpole. The most anterior organ, the cement gland, is known to be induced from the anterior ectoderm by the mesoderm at very early stage of the gastrula, and induced cells are located over a wider area than that occupied by the mature gland; most of the initially induced cells do not contribute to the final gland, suggesting that there is redetermination of neural cells by suppression of the intially committed developmental fate (278). In the case of MyoD transcription during a short period of time after midblastula transition, transcripts are found throughout the Xenopus embryo; during the process of mesoderm induction, the expression of MyoD becomes restricted to muscle precursor cells (93, 257). Again in the ascidian blastomere, gene regulation by transcriptional suppression of inward rectifier K/ channels may be directly related to the intrinsic mechanism of neural induction. Application of the recent success in cloning inward K/ channels by Kubo and co-workers (148, 149) to the ascidian inward rectifier motivates further experiments. With regard to the functional significance of reduced density of inward rectifier K/ channels during the early phase of Haloncynthia neural differentiation, external application of Cs/ to the uninduced and epidermally determined blastomeres to suppress inward rectifier current revealed no significant changes in epidermal determination or commitment to epidermal fate (235). However, the reduced density for inward rectifier K/ channels usually makes differentiating blastomeres tend to depolarize. Thus it may contribute to Ca2/ influx in a facilitatory or inhibitory way, depending on the situation, either by reducing the threshold for voltage-dependent Ca2/ channels or by decreasing the driving force of Ca2/ through voltage-independent nonselective cation channels. Because Ca2/ influx during a critical phase of neural differentiation has been suggested to have significant influences on the processes of maturation (82, 291), as described in section VIB, it is important to elucidate the functional significance of inward rectifier during neural differentiation especially in terms of the intracellular Ca2/ Volume 78 April 1998 ION CHANNELS AND EARLY DEVELOPMENT OF NEURAL CELLS VI. DEVELOPMENT OF OTHER ION CHANNELS THAN SODIUM CHANNELS A. Development of K/ and Ca2/ Channels Delayed rectifier K/ channels are found in the neurally differentiating a-line blastomere in which Na/ spikes are induced (224, 233). The induced K/ channels are sensitive to TEA with a half-maximal dose of 1.3 mM similar to that observed for other neurons (271). / 9j08$$ap07 P25-7 In neurally differentiating Halocynthia blastomeres, the delayed K/ channels are always coregulated with neuron-specific Na/ channels, and they appear at almost the same stages (40 developmental hours at 9⬚C) as those at which neuron-specific Na/ channels start to be expressed (233, 235, 271). The coregulation can be artificially disrupted by injecting specific antisense TuNa I RNA before its expression in the neurally determined blastomere, as described above (231). Delayed rectifier K/ channels are also expressed in muscularly differentiated blastomeres of the Halocynthia embryo, but these K/ channels are different from those expressed in neurally differentiated cells with respect to TEA sensitivity and kinetics (271). Furthermore, the development of ion channels including the delayed rectifier K/ channels in the muscularly differentiated blastomeres is not dependent on cell-cell interactions but is autonomously regulated when the cleavage-arrested blastomere from the posterior vegetal quadrant of the 16-cell embryo is used (235, 271). Such autonomous development is also reported in the case of other muscular characteristics, such as histochemical detection of acetylcholinesterase and muscle-specific antibody binding (50, 173, 207, 210). Slowly inactivating or L-type Ca2/ channels are expressed in neural, muscular, and epidermal types of excitability. In comparison to the case of Na/ channels, less differences in Ca2/ channel properties were found among various differentiation types; however, further analysis is required in the ascidian embryo. In the case of neural blastomeres, expression of Ca2/ channels seems to considerably increase upon cell-cell interactions or neural induction in contrast to that of uninduced epidermal blastomeres (unpublished data). Thus neuron-specific Na/, delayed K/, and Ca2/ channels are all regulated coordinately by the neural induction. Especially Na/ and delayed rectifier K/ channels in the Halocynthia neural blastomere are cell-type specific, as described above. If different genes specific for respective cell types exist, regulation of the combined expression of a set of membrane ion channels may be easier than the case in which only a gene is utilized for various specialized cells at various times. For example, in the former case, the promotor structure at 5ⴕ-upstream of each gene may be less complex than those of the genes for the latter case. This may be one of the reasons for the existence of many genes for similarly functioning ion channels among different cell types. The Ca2/-dependent K/ channels in addition to Ca2/ channels are expressed in epidermally differentiating blastomeres, such as the cleavage-arrested one-cell embryo, as in neurally and muscularly differentiated blastomeres. The molecular identities of Ca2/ channels and Ca2/-dependent K/ channels in the epidermis of ascidian tadpoles are not yet known, but it is possible that the same genes as transcibed in other type cells, such as in 03-18-98 08:13:31 pra APS-Phys Rev Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on May 4, 2017 the most effective enzyme, subtilisin, is obtained from Bacillus subtilis, and it is unclear whether this enzyme physiologically exists in vertebrate embryos. On the other hand, existence of bFGF in early vertebrate embryos has been convincingly demonstrated (283). From these findings, bFGF or its family proteins were worth examining for their neural-inducing activities in ascidian ectodermal blastomeres. In accordance with the results in the Xenopus embryo, we have preliminarily tested the effects of bFGF on the presumptive neural blastomere and found that FGF also mimics the inducer (104, 105). Especially in respect to the developmental time dependence, the sensitive phase of the blastomere to bFGF rather than subtilisin was identical to the competent phase for induction by cell contact (104, 105). In addition, early signs of neural determination, such as the quantitative difference of expression of inward rectifier between FGF-induced and uninduced blastomeres, are found to be identical to those in the case of induction by cell contact or by protease (104). The close similarity of the processes of Na/ spike induction in the ascidian blastomere elicited by three methods (protease, FGF, and cell contact) strongly suggests that three inductive stimuli act on a common receptor, such as a receptor tyrosine kinase. Although effects of bFGF, such as induction of neuron-specific ion channels, are tacitly assumed to directly affect the ascidian ectodermal blastomeres, there is the possibility that bFGF induces mesodermal cells and subsequently neuronal cells are induced by the mesodermal cells. Moreover, it has been reported that bFGF faciliates notochord differentiation in partial embryos from ascidian notochord precursor blastomeres, just as in vertebrate embryos (198, 199). However, it is unlikely that induction is mediated by bFGF-induced notochordal cells because notochord-specific features are not developed in bFGFtreated a4–2-derived partial embryos (199). With the use of this simple neuralizing preparation of the single ascidian blastomere, it will be interesting to identify the prototypes of endogenous neural inducers common to all vertebrate embryos and to elucidate the cascade of the intracellular neuralizing signals at the single-cell level. 325 326 KUNITARO TAKAHASHI AND YASUSHI OKAMURA neurons and muscle cells, are expressed. Thus it is at least true that the absence or suppression of neuron-specific Na/ and delayed rectifier K/ channel expression is characteristic of epidermally differentiated blastomeres in the Halocynthia embryo (224, 225). B. Comparison With Cases of Other Vertebrate Species of Embryos / 9j08$$ap07 P25-7 type reveals the extension of neurites on day 3; moreover, round cells examined electrophysiologically show fast inactivating or T-type Ca2/ current and Na/ current (145). The neurons on day 3 show fast inactivating Ca2/, slowly inactivating Ca2/, Na/, and delayed rectifier K/ currents (145). Thus the dominant presence of T-type Ca2/ channels is confirmed in precursor neurons, and the density of L-type Ca2/ channels gradually increases with neuronal maturation (145). In addition, it is known that neurally differentiated TC cells express receptors for both N-methyl-D-aspartate (NMDA) and non-NMDA-type glutamate receptors and gaminobutyric acid type A receptors as well (244, 248, 307), whereas mesoderm-like cells derived from the P19 TC cell line express adenosine 3ⴕ,5ⴕ-cyclic monophosphatedependent L-type Ca2/ channels, similar to cardiac and somatic myocytes in normal embryos (133). Thus the differentiation of TC cells in terms of membrane excitability mimics fairly exactly that of neurons and muscle cells in normal embryos. As for the earliest signs in differentiation of excitable cells, the appearance of T-type or fast and voltage-dependently inactivating Ca2/ channels has been considered significant (163) after the existence of multipe types of Ca2/ channels was widely acknowledged (62, 138, 214). The presence of T type is also observed in cells at relatively immature states of differentiation, such as mesodermal stem cell lines (146, 147), myocytes from the early chick embryo or the neonatal rat (70, 124), rat atrial myocytes (324), chick embryonic dorsal root ganglion cells, and neonatal rat hippocampal neurons (24, 325). However, the dominance of T-type Ca2/ channels over L-type channels is not exclusively characteristic of the earliest stage of excitability, but there are a few cases of CNS neurons showing this increase at later stages, such as rat cerebellar Purkinje cells and cat thalamocortical cells, in which development of T-type channels correlates with specific cellular differentiation, such as dendritic extension (79) or oscillatory activities (241). In respect to the functional significance of T-type or low-threshold Ca2/ channels during development of Xenopus spinal neurons, Gu and Spitzer (80) have proposed the triggering of spontaneous Ca2/ influx through T-type or low-threshold type Ca2/ channels because of their ease of activation due to a low threshold and their dominant presence in younger neurons. Dominance of T-type Ca2/ channels at the early stages in mammals may correspond to that of egg-type Na/ channels that were shown to be permeable to Ca2/ and fast-type Ca2/ channels in ascidians. However, it should be noted that in several experimental cases Ca2/ influx through L-type channels may also be significant for regulation of ion channel development at later stages (290, 291); one example involves the application of the Ca2/ antagonist D-600 at a critcal phase of 03-18-98 08:13:31 pra APS-Phys Rev Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on May 4, 2017 Most studies of the development of ion channels in mammals have been carried out on primary cultured embryonic CNS neurons or neuronal cell lines or myocytes, that is, cells already determined to become excitable. Developmental analysis of differentiation from undetermined embryonic cells requires systems that allow in vitro culture while preserving the morphogenetic field as in the whole embryo, or by inductive differentiation without the morphogenetic field. Teratocarcinoma (TC) cell lines, cloned from malignant teratocarcinomas, have multiple potencies to differentiate into various embryonic tissues and have been proven to be useful for studying differentiation mechanisms (204). Similar multipotent cell lines, embryonal carcinoma (EC) cell lines, can be established with transformed cells from the primitive ectoderm in embryonic 5.5-day mouse embryo (68). If the cloned TC or EC cells are injected into and mixed with the inner cell mass of the normal embryo, chimeric mice are obtained with the expression of all types of cells derived from these cell lines (103). The TC cells cultured in a medium containing retinoic acid (294) or in the medium with reduced serum concentration differentiate into neural, muscular, cartilaginous, and epidermal cells, showing multipotency for differentiation (45). The development of ion channels has been confirmed in the case of neural differentiation of TC or EC cells by whole cell recording (56, 277) and by whole cell recording combined with immunochemical staining of neurofilament as a marker for neural differentiation (145). Undetermined stem cells, which have a relatively large ratio of nucleus against cytoplasm and express no neurofilament, develop only a small amount of K/ currents with an inactivating component and express no inward current (145, 277). In contrast, round thick cells, which appear transiently on the sheet of flat polygonal cells 2 days after chemical induction by retinoic acid or after low-serum treatment, exhibit immunochemical staining for neurofilament (145). Cells with neurites are easily found at 3 days after induction that are clearly identified as neurons morphologically (145). These round cells are considered to be neuronal precursors because continuous observation of cells of this Volume 78 April 1998 ION CHANNELS AND EARLY DEVELOPMENT OF NEURAL CELLS VII. DEVELOPMENTAL REGULATION OF ION CHANNEL EXPRESSION A. Regulation by Changes in Gap Junctional Communication Although cell-cell interactions required to various types of cellular differentiation are considered to be in terms of extracellular diffusion of inducer substances, gap junctional communication (GJC) is also suggested to be one of the mechanisms mediating such interactions by transferring intracellular ions and small molecules (85, 243, 311). In many animals, at early stages of embryogenesis all cells communicate via gap junctions, and as differentiation proceeds, the extent of GJC becomes progressively restricted. For example, in the mouse embryo, dye cou- / 9j08$$ap07 P25-7 pling between the inner cell mass and trophoblast cells is observed immediately after implantation but disappears at later stages (159, 160). In rat embryos and associated extraembryonic structures, multiple species of gap junction proteins are expressed differentially at later stages (252), indicating that tissue-specific communication replaces the indiscriminant communication seen at early stages. To define the developmental role of GJC more precisely, reexamination of its quantitative changes related to various stages of cellular differentiation is required. During differentiation without cleavage as described above, a neurally differentiated Halocynthia a4–2 blastomere loses gap junctions with its paired A4–1 blastomere (a-A pair), whereas an epidermally differentiated a4–2 blastomere shows enhanced GJC with its paired and epidermally differentiated b4–2 blastomere (a-b pair) (99, 223, 224, 258). Therefore, blastomere pairs obtained from an ascidian eight-cell embryo and cultured under cleavage-arrested conditions are an ideal system for quantitative examination of the physiological role of gap junctions during differentiation. For quantitative estimation of dye coupling between two blastomeres in a pair, Saitoe et al. (258) measured the time constant of the decay of normalized difference in fluorescence intensity between blastomeres after hydrophilic dye injection. B. Studies on Gap Junctional Regulation of Ion Channel Expression in Ascidian Embryos In ascidian embryos, the presence of junctional communication at early developmental stages (from 2 to 32 cell) has been reported using conventional two-microelectrode measurements (270) and by the whole cell voltage clamp (41) in both normal and cleavage-arrested embryos. In agreement with these reports, Saitoe et al. (258) detected GJC shortly after preparation of blastomere pairs from eight-cell stage embryos using the electrophysiological methods and after 7 h of development by both electrophysiological and dye-injection methods. As described above, it has long been suggested that transfer of information by molecules moving through gap junctions may contribute to cell-cell interactions during embryonic development (311). In the cleavage-arrested Halocynthia blastomere pair a-A, the a4–2 blastomere is induced into a neural cell by contact with the A4–1 blastomere between 10 and 15 developmental hours (223, 224). The increase in GJC that occurs during the same developmental stages might be related to neural induction. However, this increase was not specific for a-A pairs but was also observed in epidermally determined a-b pairs (258). Furthermore, as described above, the fact that even a single a4–2 blastomere can be induced to differentiate 03-18-98 08:13:31 pra APS-Phys Rev Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on May 4, 2017 chick muscle cell culture, that produces enhancement of Na/ channel density but downregulation of L-type Ca2/ channels (123, 264). The result concerning Na/ channels may be related to the fact that increased intracellular Ca2/ concentration or increased electrical activity in the rat muscle cells produces downregulation of both translation of Na/ channel a-subunit proteins and transcription of Na/ channel mRNA (217), suggesting the developmental feedback regulation of Na/ channels occurs at least partly at the transcriptional level. Among various K/ currents, TEA-sensitive delayed rectifier K/ channels are reported to be characteristic of early development in amphibian spinal neurons (3, 249, 251). Delayed rectifier K/ currents in cultured Xenopus spinal neurons show a threefold increase in amplitude during the period from 5 to 27 h after dissociation at the neural plate stage, which accounts for the reduction in action potential duration (216, 251). A Xenopus homolog of a Drosophila Shaker gene, XSha1, is expressed in the neural fold region at the late gastrula stage, just after neural induction, in parallel with the transcription of the neural marker NCAM (249). This clone is not expressed in other tissues, such as the muscular lineage. Thus the molecules involved in spike production in neuronal cells are uniquely transcribed just as early as in the case of ascidian Na/ channel gene as described above. Other outward rectifier types of K/ channels, such as A-current channels, are also expressed in Xenopus spinal neurons; however, these channels appear to be assembled later than the delayed rectifier K/ channels (250). There is no evidence that A-current channels are an early marker for neural induction in ascidian blastomeres as well, although they seemed to characterize the distinct neuronal group of motoneurons (222). 327 328 KUNITARO TAKAHASHI AND YASUSHI OKAMURA / 9j08$$ap07 P25-7 FIG. 5. Schematic illustration of gap junctional regulation for expression of ion channels in ascidian neural blastomeres. A: disruption of gap junction (GJ) and development of ion channels. B: function of GJ and expression of channel proteins. (Courtesy of Dr. Minoru Saitoe.) a long delay in GJC disappearance, but also a significant increase in dye transfer rate over its level before normal GJC disappearance (Fig. 5A). As mentioned above, bFGF, a tyrosine kinase activator, was an effective inducer of Na/ spike development or neural differentiation in single a4–2 blastomeres (104, 105), and it was shown that the neurally determined a4–2 blastomere but not the A4–1 blastomere was responsible for the reduction of dye transfer rate in the aA pair (258). Therefore, if protein kinases were involved in the K252a effect, the disappearance of GJC must be caused by a serine/threonine kinase sited downstream to a FGF receptor-like tyrosine kinase. There are precedents for this model in that the disruptive effects of epidermal growth factor on gap junctions of liver epithelial cells or cardiac myocytes are mediated through a serine/threonine phosphorylation situated downstream of an epidermal growth factor receptor tyrosine kinase (154) or bFGF receptor tyrosin kinase (53). When K252a was applied during a limited period be- 03-18-98 08:13:31 pra APS-Phys Rev Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on May 4, 2017 by either protease treatment (226) or application of bFGF (104) indicates that activation of specific receptors on the extracellular surface may be required for the induction. In a variety of systems, groups of cells restrict the ability to communicate with each other as they diverge along different developmental pathways (311). In the Halocynthia, during developmental stages 25–35 h, GJC disappeared in the a-A pair, in which one cell became neuronal and the other mostly nonneuronal (possibly notochordal or endodermal). Gap junctional conductance disappears after neural determination and before expression of neuronal specific ion channels (233, 258). In contrast, in the a-b pair, in which both blastomeres became epidermal, GJC further increased (258). Extensive GJC has been observed between epidermal cells in both intact and cleavage-arrested ascidian embryos (99, 189). Therefore, the increase in GJC in a-b pairs may be indicative of epidermal determination, although the expression of characteristic epidermal markers, such as Ca2/ channels in the surface membrane and the appearance of tunic substance over the cell surface, occurred after the increase (258). Furthermore, both the increase in the a-b pair and the disappearance in the a-A pair required new transcripts, and it, therefore, suggested that these changes must be differentiation dependent (258). As mentioned above, GJC is known to become restricted within groups of cells as development proceeds (85, 311). In the blastomere triplet experiments carried by Saitoe et al. (258), when an epidermally determined b4–2 blastomere was contacted with both neural inducer A4–1 and neurally determined a4–2 blastomeres, it coupled with the A4–1 but not with the a4–2 blastomere. The dye transfer rate between the b4–2 and the A4–1 blastomeres in this case was much smaller than that between epidermal blastomeres (a-b) but was similar to that in the a-A pair at 20 developmental hours, that is, before uncoupling (258). Furthermore, although coupling is disrupted between neurally determined a4–2 and A4–1 blastomeres, preliminary results demonstrated that the junctional communication between two neurally determined a4–2 blastomeres persisted. These findings suggest that, in ascidian embryos as well, GJC is more or less limited within a group of cells after determination of cell fates. Among various agents examined, only a serine/threonine kinase inhibitor (K252a) delayed the disappearance of GJC at relatively low dose (õ1.0 mM) (258). This inhibitor has been identified as a C-kinase inhibitor (326) and found to suppress the effects of nerve growth factor (NGF) on PC-12 cells by inhibiting activity of trk kinase, which is one of several families of NGF receptors and is known to promote neurite extension (218, 301). The application of K252a for 5 h before the time of GJC disappearance in control a-A pairs not only caused Volume 78 April 1998 ION CHANNELS AND EARLY DEVELOPMENT OF NEURAL CELLS C. Regulation of Ion Channel Expression by Transcription Developmental changes in various ion channels in ascidian embryos (231, 235) appear to be primarily regulated at the transcriptional level, as shown in the rat brain (268) and Xenopus embryos (83, 249). Zygotic expression of ion channels is initiated mostly after the late gastrula stage. Some of these ion channels, particularly Na/ channels in ascidian embryos and K/ channels in Xenopus embryos, appear to be the earliest markers for neuronal differentiation (83, 231, 249). Expression of each ion channel gene seems to show a unique distribution related to both regional specificity and cell type specificity and characteristic developmental time dependence, as shown between a-line neurons and A-line motoneurons in the Halocynthia larva. In the case of rat brain Na/ channels (8, 315), within the nervous system each has a characteristic distribution of expression; type II Na/ channels are expressed mainly in the brain throughout most developmental stages, whereas type I Na/ channels are expressed mainly in the caudal region of the brain and spinal cord at later stages of development (8). Intracellular distributions between / 9j08$$ap07 P25-7 soma and axons are also subtype specific (315). Furthermore, muscle Na/ channel genes, TTX sensitive skM I and TTX-insensitive skM II, are different from those expressed in the rat brain, and they also show differential developmental expression and regulation (33, 317). In the case of ascidian embryos, three types of Na/ channels have been identified electrophysiologically (233), and three putative Na/ channel genes have been isolated (231). The TuNa I gene, for which the entire coding region has been sequenced, has been identified as a neuron-specific gene in ascidian embryos. Its transcription requires initiation by the neural inducing signal of cell contact or by inducing agents, such as bFGF or proteases, during competent phase of preneural blastomere (231). The precise mechanism of the initiation has not been elucidated; however, some suggestions may be obtained from the experiments of other preparations. The PC-12 cell line, derived from a pheochromocytoma, can be induced to differentiate into neural cells with neurites and Na/ channels by addition of NGF to the medium (51, 77). The effects of NGF are known to elicit both early and late events. The early events include activation of various kinases and expression of early and immediate genes, such as c-fos (76) and are considered to be preparatory for late events. Late events include increases in numbers of both TTX-sensitive and TTX-resistant Na/ channels (256), although Na/ channels are expressed in some untreated PC-12 cell lines. In either case, Na/ channel density increases five- to sixfold after NGF treatment. Receptors for NGF in PC12 cells have been unknown until recently, although it has been established that protein phosphorylation must be involved in the effects of NGF. In 1989, a species of receptor tyrosine kinase previously reported as a human protooncogene, trk (168, 169), was identified as the high-affinity receptor for NGF (132). Thus the induction of Na/ channels in PC-12 cells must also be mediated by the trk receptor tyrosine kinase. Furthermore, another ligand for a receptor tyrosine kinase, FGF, has similar inducing activity for neural characteristics, such as neurite extension, in a PC-12 cell line (303), although the effect does not last as long as that induced by NGF. Induction of Na/ channels by FGF has also been demonstrated (242). The inducing effects of FGF on PC-12 cells are similar for Na/ channels but qualitatively different for neurite extension, which shows less intense growth. Recent studies on neural differentiation of sympathicoadrenal progenitor cells, which may be the normal equivalent of PC-12 cells, reveal that FGF is required before the phase of NGF requirement (1, 293). Because neural inducing effects of FGF on undifferentiated ectodermal cells in Xenopus and ascidian embryos have recently been found as described, it will be interesting to elucidate the similarities and differences of FGF effects on early neurogenesis and neural crest cell differentiation. 03-18-98 08:13:31 pra APS-Phys Rev Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on May 4, 2017 tween 20 and 25 developmental hours, full expression of Na/ and K/ channels in the neurally determined a4–2 blastomere did not occur as long as GJC remained, but it attained control levels in parallel with the final disappearance of GJC at Ç80 developmental hours (258). This represents almost a 35-h delay in expression (Fig. 5A). Here, after several control experiments, Saitoe et al. (258) suggested that the persistence of GJC itself exerted suppressive effects on neural differentiation in K252atreated cell pairs and that one of the functional roles of embryonic gap junctions is to time the expression of neuron-specific ion channels in preneuronal cells, although we must remember that uncoupling is not the only factor for determining the time sequence of ion channel expression (Fig. 5B). The mechanism by which GJC suppresses the expression of neural markers is unknown at the present moment. However, because the transcription of Na/ and delayed K/ channel genes is known to be initiated before 25 developmental hours (neurula stage) in these ascidian embryos by electrophysiological methods (235) and molecular biological methods (231), the delay of ion channel expression must be the result of posttranscriptional control, such as regulation of channel protein synthesis or assembly in the membrane (Fig. 5B). For further understanding of the developmental roles of gap junctions, it will be necessary to identify the intercellular signals passing through gap junctions to control this modification of ion channel expression. 329 330 KUNITARO TAKAHASHI AND YASUSHI OKAMURA Volume 78 of those channels have been found, the neuronal type and the developmental stage of a differentiating cell can be precisely identified by the combination of ion channel species they express. 2) Most of these channel species are expressed at the early stages of neuronal differentiation, and transcripts encoding some of them appear immediately after the earliest commitment of neural induction. 3) In addition to neuronal differentiation, these ion channels may be useful for markers of other types of differentiation, such as glial cells, muscle cells, and endocrine cells, because many kinds of nonneuronal cells have been shown to have a variety of ion channels in their membrane at various stages of differentiation. 4) Both qualitative and quantitative measurements of ion channels in an embryonic cell can be performed with electrophysiological methods at any stage without disturbing ongoing developmental processes. 5) Whenever identification of molecular species is necessary, ample methods of molecular biology are available to obtain clones of interesting ion channels from the same animal species used for experiments. Finally, it was particularly stressed that the simple system of combination of cleavage-arrested large blastomeres, for which most of electronic and optical methods and various injection experiments are applicable, may be useful for future molecular physiological studies on the intracellular differentiation process of the early embryonic cells. We have also highlighted the importance of suppressive mechanisms for cellular differentiation. 1) Epidermal characteristics are dominantly expressed in cleavage-arrested one-cell Halocynthia embryos, although they must contain cytoplasmic factors for many cell types in the larva. 2) Neural induction in presumptive neural Halocynthia blastomeres simultaneously suppresses epidermal-specific transcription of inward rectifier-related genes. 3) Specific expression of type II Na/ channel genes in mammalian PC-12 cells is efficiently controlled by a silencer sequence, with which neuronal Na/ channel expression in other cell types is completely suppressed. Thus reciprocal suppressive mechanisms at the transcriptional level may be key processes for cellular differentiation, by which exclusivity of cell types is maintained. The time sequential and quantitative analysis of intracellular processes of mutual transcriptional suppression is one of the intriguing problems in molecular developmental physiology. VIII. CONCLUSION REFERENCES In this review, we have underscored the merits of using voltage-dependent ion channels as markers for neuronal differentiation. The following major points of discussion have emerged. 1) Because a large number of molecular species / 9j08$$ap07 P25-7 1. ANDERSON, D. J. Molecular control of cell fate in the neural crest: the sympathoadrenal lineage. Annu. Rev. Neurosci. 16: 129–158, 1993. 2. ARNOULT, C., AND M. VILLAZ. Differential developmental fates of the two calcium currents in early embryos of the ascidian Ciona intestinalis. J. Membr. Biol. 137: 127–135, 1994. 3. BARISH, M. E. 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