Download Ion Channels and Early Development of Neural Cells

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. Differentiation of voltage-gated potassium current
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
In PC-12 cell lines, the signal transduction pathway
following activation of the receptor tyrosine kinase with
NGF has been partly elucidated, and the roles of protooncogenes, such as src, ras, and raf, have been confirmed
(143, 302, 321). However, each neural phenotype seems
not always to require participation of all of those protooncogenes but, rather, to be regulated in such a specific way
that the NGF receptor, Src, Ras, and Raf are activated in
a linear order by NGF, and these signaling components
define branch points in the pathway to specific gene induction events (43). In the case of Na/ channel induction
in PC-12 cell lines, two types of Na/ channels expressed,
type II and the peripheral type, are driven by different
branches of the signal transduction pathway activated by
stimulation with NGF (44).
Furthermore, one of the best studies of transcriptional regulation of changes in ion channels is that of Na/
channels in a PC-12 cell line stimulated by NGF mentioned
above.
One of the major classes of Na/ channels expressed
in NGF-treated PC-12 cells is type II, as described above
(166). The 5ⴕ-upstream region of type II Na/ channel gene
has been cloned, and about one thousand base pairs have
been sequenced (170). The functional domains of this region have been analyzed with a reporter gene, CAT. A
specific sequence has been found to work as a negative
element to prevent expression of type II Na/ channels in
nonneuronal cell lines, such as a muscle cell line in which
Na/ channels different from type II are expressed (142).
Transfection of this negative element upstream of the
CAT reporter gene in muscle L6 cell line reveals no CAT
activity; however, transfection of the reporter gene without the negative element produces substantial activity in
the same cell line (142). Further analysis reveals a key
sequence of several base pairs that is important for silencing the gene. The sequence is unique and shows no homology to known silencer sequences but is now known to be
included in the 5ⴕ-upstream region of other neuron-specific genes, such as peripherin (142). The binding protein
to this silencer region was recently found, and the molecular nature has been characterized (29, 300).
Thus negative elements may be an important regulatory component for neuronal expression of various
characteristics (167). As for further discussion about
this silencer, we hope the readers look for other reviews
(59, 167).
April 1998
4.
5.
6.
7.
8.
9.
10.
11.
12.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
and modulation of excitability in cultured amphibian spinal neurones. J. Physiol. (Lond.) 375: 229–250, 1986.
BARNES, S. N. Fine structure of the photoreceptor of the ascidian
tadpole during development. Cell Tissue Res. 155: 27–45, 1974.
BARRES, B. A., L. L. CHUN, AND D. P. COREY. Ion channels in
vertebrate glia. Annu. Rev. Neurosci. 13: 441–474, 1990.
BATES, W. R., AND W. R. JEFFERY. Polarization of ooplasmic segregation and dorsal-ventral axis determination in ascidian embryos.
Dev. Biol. 130: 98–107, 1988.
BAUD, C. Ionic basis of membrane potential in developing ectoderm of the Xenopus blastula. J. Physiol. (Lond.) 393: 525–544,
1987.
BECKH, S., M. NODA, H. LUBBERT, AND S. NUMA. Differential
regulation of three sodium channel messenger RNAs in the rat
central nervous system during development. EMBO J. 8: 3611–
3616, 1989.
BERRIDGE, M. J. Inositol trisphosphate and calcium signalling. Nature 361: 315–325, 1993.
BERRIDGE, M. J. Calcium signalling and cell proliferation. Bioessays 17: 491–500, 1995.
BERRILL, N. J. The Tunicata. With an Account of the British Species. London: The Ray Society, 1950.
BLOCK, M. L., AND W. J. MOODY. Changes in sodium, calcium and
potassium currents during early embryonic development of the
ascidian Boltenia villosa. J. Physiol. (Lond.) 393: 619–634, 1987.
BLOCK, M. L., AND W. J. MOODY. A voltage-dependent chloride
current linked to the cell cycle in ascidian embryos. Science 247:
1090–1092, 1990.
BOSMA, M. M., AND W. J. MOODY. Macroscopic and single-channel
studies of two Ca2/ channel types in oocytes of the ascidian Ciona
intestinalis. J. Membr. Biol. 114: 231–243, 1990.
BOURINET, E., F. FOURNIER, J. NARGEOT, AND P. CHARNET.
Endogenous Xenopus-oocyte Ca-channels are regulated by protein
kinases A and C. FEBS Lett. 299: 5–9, 1992.
BOURINET, E., J. NARGEOT, AND P. CHARNET. Electrophysiological characterization of a TTX-sensitive sodium current in native
Xenopus oocytes. Proc. R. Soc. Lond. B Biol. Sci. 250: 127–132,
1992.
BRACHET, J., AND S. DENIS-DONINI. The effects of diverse agents
(Ca2/ and K/ ionophores, organomercurials, dithiols, colchicine,
and cytochalasin B) on the maturation and differentiation without
cleavage in Chaetopterus eggs. Compt. Rendus Hebdomadaires
Seances Acad. Sci. D Sci. Naturel. 284: 1091–1096, 1977.
BROWNLEE, C., AND B. DALE. Temporal and spatial correlation
of fertilization current, calcium waves and cytoplasmic contraction
in eggs of Ciona intestinalis. Proc. R. Soc. Lond. B Biol. Sci. 239:
321–328, 1990.
BUSA, W. B. Involvement of calcium and inositol phosphates in
amphibian egg activation. J. Reprod. Fertil. 42, Suppl.: 155–161,
1990.
CAMPOS-ORTEGA, J. A. Cellular interactions during early neurogenesis of Drosophila melanogaster. Trends Neurosci. 11: 400–
405, 1988.
CAMPOS-ORTEGA, J. A. Mechanisms of early neurogenesis in Drosophila melanogaster. J. Neurobiol. 24: 1305–1327, 1993.
CAMPOS-ORTEGA, J. A., AND Y. N. JAN. Genetic and molecular
bases of neurogenesis in Drosophila melanogaster. Annu. Rev.
Neurosci. 14: 399–420, 1991.
CANNON, S. C., A. I. MCCLATCHEY, AND J. F. GUSELLA. Modification of the Na/ current conducted by the rat skeletal muscle alpha
subunit by coexpression with a human brain beta subunit. Pflügers
Arch. 423: 155–157, 1993.
CARBONE, E., AND H. D. LUX. A low voltage-activated calcium
conductance in embryonic chick sensory neurons. Biophys. J. 46:
413–418, 1984.
CARMELIET, E. K/ channels and control of ventricular repolarization in the heart. Fundam. Clin. Pharmacol. 7: 19–28, 1993.
CHAMBERS, E. L. Fertilization in voltage-clamped sea urchin eggs.
In: Mechanisms of Egg Activation, edited by R. Nuccitelli, G. N.
Cherr, and W. H. Clark. New York: Plenum, 1989, p. 1–18.
CHANG, F., AND P. NURSE. Finishing the cell cycle: control of
mitosis and cytokinesis in fission yeast. Trends Genet. 9: 333–335,
1993.
/ 9j08$$ap07
P25-7
331
28. CHIBA, K., R. T. KADO, AND L. A. JAFFE. Development of calcium
release mechanisms during starfish oocyte maturation. Dev. Biol.
140: 300–306, 1990.
29. CHONG, J. A., J. TAPIA-RAMIREZ, S. KIM, J. J. TOLEDO-ARAL, Y.
ZHENG, M. C. BOUTROS, Y. M. ALTSHULLER, M. A. FROHMAN,
S. D. KRANER, AND G. MANDEL. REST: a mammalian silencer protein that restricts sodium channel gene expression to neurons. Cell
80: 949–957, 1995.
30. CONKLIN, E. G. Mosaic development in ascidian eggs. J. Exp. Zool.
2: 145–223, 1905.
31. CONKLIN, E. G. The organization and cell-lineage of the ascidian
egg. J. Acad. Nat. Sci. 13: 1–119, 1905.
32. CONKLIN, E. G. The development of centrifuged eggs of ascidians.
J. Exp. Zool. 60: 3–119, 1931.
33. COOPERMAN, S. S., S. A. GRUBMAN, R. L. BARCHI, R. H. GOODMAN, AND G. MANDEL. Modulation of sodium-channel mRNA levels in rat skeletal muscle. Proc. Natl. Acad. Sci. USA 84: 8721–
8725, 1987.
34. CORK, R. J., M. F. CICIRELLI, AND K. R. ROBINSON. A rise in cytosolic calcium is not necessary for maturation of Xenopus laevis
oocytes. Dev. Biol. 121: 41–47, 1987.
35. COWAN, A. E., AND J. R. MACINTOSH. Mapping the distribution
of differentiation potential for intestine, muscle, and hypodermis
during early development of Caenorhabditis elegans. Cell 41: 923–
932, 1985.
36. CROWTHER, R. J., T. H. MEEDEL, AND J. R. WHITTAKER. Differentiation of tropomyosin-containing myofibrils in cleavage-arrested
ascidian zygotes expressing acetylcholinesterase. Development
109: 953–959, 1990.
37. CROWTHER, R. J., AND J. R. WHITTAKER. Differentiation without
cleavage: multiple cytospecific ultrastructural expressions in individual one-celled ascidian embryos. Dev. Biol. 117: 114–126, 1986.
38. CROWTHER, R. J., AND J. R. WHITTAKER. Structure of the caudal
neural tube in an ascidian larva: vestiges of its possible evolutionary
origin from a ciliated band. J. Neurobiol. 23: 280–292, 1992.
39. DALE, B. Fertilization channels in ascidian eggs are not activated
by Ca. Exp. Cell Res. 172: 474–480, 1987.
40. DALE, B., AND L. DE FELICE. Sperm-activated channels in ascidian
oocytes. Dev. Biol. 101: 235–239, 1984.
41. DALE, B., L. SANTELLA, AND E. TOSTI. Gap-junctional permeability in early and cleavage-arrested ascidian embryos. Development
112: 153–160, 1991.
42. DALE, B., R. TALEVI, AND L. J. DEFELICE. L-type Ca2/ currents
in ascidian eggs. Exp. Cell Res. 192: 302–306, 1991.
43. D’ARCANGELO, G., AND S. HALEGOUA. A branched signaling pathway for nerve growth factor is revealed by Src-, Ras-, and Rafmediated gene inductions. Mol. Cell. Biol. 13: 3146–3155, 1993.
44. D’ARCANGELO, G., K. PARADISO, D. SHEPHERD, P. BREHM, S.
HALEGOUA, AND G. MANDEL. Neuronal growth factor regulation
of two different sodium channel types through distinct signal transduction pathways. J. Cell Biol. 122: 915–921, 1993.
45. DARMON, M., J. BOTTENSTEIN, AND G. SATO. Neural differentiation following culture of embryonal carcinoma cells in a serumfree defined medium. Dev. Biol. 85: 463–473, 1981.
46. DAVID, C., J. HALLIWELL, AND M. WHITAKER. Some properties
of the membrane currents underlying the fertilization potential in
sea urchin eggs. J. Physiol. (Lond.) 402: 139–154, 1988.
47. DAVIDSON, E. H. Gene Activity in Early Development. New York:
Academic, 1986.
48. DAVIDSON, E. H. How embryos work: a comparative view of diverse modes of cell fate specification. Development 108: 365–389,
1990.
49. DAVIDSON, E. H. Spatial mechanisms of gene regulation in metazoan embryos. Development 113: 1–26, 1991.
50. DENO, T., H. NISHIDA, AND N. SATOH. Autonomous muscle cell
differentiation in partial ascidian embryos according to the newly
verified cell lineages. Dev. Biol. 104: 322–328, 1984.
51. DICHTER, M. A., A. S. TISCHLER, AND L. A. GREENE. Nerve
growth factor-induced increase in electrical excitability and acetylcholine sensitivity of a rat pheochromocytoma cell line. Nature
268: 501–504, 1977.
52. DILLY, N. Electron microscope observations of the receptors 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
13.
ION CHANNELS AND EARLY DEVELOPMENT OF NEURAL CELLS
332
53.
54.
55.
56.
57.
58.
59.
61.
62.
63.
64.
65.
66.
67.
68.
69.
70.
71.
72.
73.
74.
75.
the sensory vesicle of the ascidian tadpole. Nature 191: 786–788,
1961.
DOBLE, B. W., Y. CHEN, D. G. BOSC, D. W. LITCHFIELD, AND E.
KARDAMI. Fibroblast growth factor-2 decreases metabolic coupling and stimulates phosphorylation as well as masking of connexin43 epitopes in cardiac myocytes. Circ. Res. 79: 647–658, 1996.
DOREE, M., J. C. CAVADORE, AND A. PICARD. Facts and hypotheses of calcium regulation of MPF activity during meiotic maturation
of starfish oocytes. J. Reprod. Fertil. 42, Suppl.: 135–140, 1990.
EAKIN, R. M., AND A. KUDA. Ultrastucture of sensory receptors in
ascidian tadpoles. Z. Zellforsch. 112: 287–312, 1971.
EBIHARA, L., AND W. C. SPEERS. Ionic channels in a line of embryonal carcinoma cells induced to undergo neuronal differentiation. Biophys. J. 46: 827–830, 1984.
ECKBERG, W. R. The effects of cytoskeleton inhibitors on cytoplasmic localization in Chaetopterus pergamentaceus. Differentiation 19: 55–58, 1981.
ECKBERG, W. R., AND Y.-H. KANG. A cytological analysis of differentiation without cleavage in cytochalasin B- and colchicine-treated
embryos of Chaetopterus pergamentaceus. Differentiation 19:
154–160, 1981.
EGGEN, B. J., AND G. MANDEL. Regulation of sodium channel gene
expression by transcriptional silencing. Dev. Neurosci. 19: 25–26,
1997.
FIELD, K. G., G. J. OLSEN, D. J. LANE, S. J. GIOVANNONI, M. T.
GHISELIN, E. C. RAFF, N. R. PACE, AND R. A. RAFF. Molecular
phylogeny of the animal kingdom. Science 239: 748–753, 1988.
FOX, A. P., AND S. KRASNE. Two calcium currents in Neanthes
arenaceodentatus egg cell membranes. J. Physiol. (Lond.) 356:
491–505, 1984.
FOX, A. P., M. C. NOWYCKY, AND R. W. TSIEN. Single-channel recordings of three types of calcium channels in chick sensory neurones. J. Physiol. (Lond.) 394: 173–200, 1987.
FRIESEL, R., AND I. B. DAWID. cDNA cloning and developmental
expression of fibroblast growth factor receptors from Xenopus
laevis. Mol. Cell. Biol. 11: 2481–2488, 1991.
FUJIWARA, T., K. NAKADA, H. SHIRAKAWA, AND S. MIYAZAKI.
Development of inositol trisphosphate-induced calcium release
mechanism during maturation of hamster oocytes. Dev. Biol. 156:
69–79, 1993.
FUKUSHIMA, Y. Identification and kinetic properties of the current
through a single Na/ channel. Proc. Natl. Acad. Sci. USA 78: 1274–
1277, 1981.
FUKUSHIMA, Y. Blocking kinetics of the anomalous potassium
rectifier of tunicate egg studied by single channel recording. J.
Physiol. (Lond.) 331: 311–331, 1982.
GARSTANG, W. The morphology of the tunicata, and its bearings
on the phylogeny of the chordata. Q. J. Microsc. Sci. 72: 51–187,
1928.
GILBERT, S. F. Developmental Biology. Sunderland, MA: Sinauer,
1991.
GIRARD, S., AND D. CLAPHAM. Acceleration of intracellular calcium waves in Xenopus oocytes by calcium influx. Science 260:
229–232, 1993.
GONOI, T., AND S. HASEGAWA. Post-natal disappearance of transient calcium channels in mouse skeletal muscle: effects of denervation and culture. J. Physiol. (Lond.) 401: 617–637, 1988.
GORMAN, A. L. F., J. S. REYNOLDS, AND S. N. BARNES. Photoreceptors in primitive chordates: fine structure, hyperpolarizing receptor potentials, and evolution. Science 172: 1052–1054, 1971.
GOUDEAU, H., M. GOUDEAU, AND N. GUIBOURT. The fertilization
potential and associated membrane potential oscillations during
the resumption of meiosis in the egg of the ascidian Phallusia
mammillata. Dev. Biol. 153: 227–241, 1992.
GOUDEAU, M., AND H. GOUDEAU. In the egg of the ascidian Phallusia mammillata, removal of external Ca2/ modifies the fertilization potential, induces polyspermy, and blocks the resumption of
meiosis. Dev. Biol. 160: 165–177, 1993.
GOULD-SOMERO, M., L. A. JAFFE, AND L. Z. HOLLAND. Electrically mediated fast polyspermy block in eggs of the marine worm,
Urechis caupo. J. Cell Biol. 82: 426–440, 1979.
GRAVE, C. Amaroucium constellatum (Verrill). II. The structure
and organization of the tadpole larva. J. Morphol. 36: 71–101, 1921.
/ 9j08$$ap07
P25-7
Volume 78
76. GREENBERG, M. E., L. A. GREENE, AND E. B. ZIFF. Nerve growth
factor and epidermal growth factor induce rapid transient changes
in proto-oncogene transcription in PC12 cells. J. Biol. Chem. 260:
14101–14110, 1985.
77. GREENE, L. A., AND A. S. TISCHLER. Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which
respond to nerve growth factor. Proc. Natl. Acad. Sci. USA 73:
2424–2428, 1976.
78. GREY, R. D., M. J. BASTIANI, D. J. WEBB, AND E. R. SCHERTEL.
An electrical block is required to prevent polyspermy in eggs fertilized by natural mating of Xenopus laevis. Dev. Biol. 89: 475–484,
1982.
79. GRUOL, D. L., C. R. DEAL, AND A. J. YOOL. Developmental changes
in calcium conductances contribute to the physiological maturation
of cerebellar Purkinje neurons in culture. J. Neurosci. 12: 2838–
2848, 1992.
80. GU, X., AND N. C. SPITZER. Low-threshold Ca2/ current and its
role in spontaneous elevations of intracellular Ca2/ in developing
Xenopus neurons (published erratum appears in J. Neurosci. 14,
1994) J. Neurosci. 13: 4936–4948, 1993.
81. GU, X., AND N. C. SPITZER. Distinct aspects of neuronal differentiation encoded by frequency of spontaneous Ca2/ transients. Nature
375: 784–787, 1995.
82. GU, X., AND N. C. SPITZER. Breaking the code: regulation of neuronal differentiation by spontaneous calcium transients. Dev. Neurosci. 19: 33–41, 1997.
83. GURANTZ, D., A. B. RIBERA, AND N. C. SPITZER. Temporal regulation of Shaker- and Shab-like potassium channel gene expression
in single embryonic spinal neurons during K/ current development.
J. Neurosci. 16: 3287–3295, 1996.
84. GURDON, J. B. Embryonic induction-molecular prospects. Development 99: 285–306, 1987.
85. GUTHRIE, S. C., AND N. B. GILULA. Gap junctional communication
and development. Trends Neurosci. 12: 12–16, 1989.
86. GUTMANN, W. F. Das Tunicaten-Modell. Zool. Beitragung 21: 279–
303, 1975.
87. HAGIWARA, S., AND L. A. JAFFE. Electrical properties of egg cell
membranes. Annu. Rev. Biophys. Bioeng. 8: 385–416, 1979.
88. HAGIWARA, S., AND S. MIYASAKI. Changes in excitability of the
cell membrane during ‘‘differentiation without cleavage’’ in the egg
of the annelid, Chaetopterus pergamentaceus. J. Physiol. (Lond.)
272: 197–216, 1977.
89. HAGIWARA, S., S. MIYAZAKI, W. MOODY, AND J. PATLAK.
Blocking effects of barium and hydrogen ions on the potassium
current during anomalous rectification in the starfish egg. J. Physiol. (Lond.) 279: 167–185, 1978.
90. HAGIWARA, S., S. MIYAZAKI, AND N. P. ROSENTHAL. Potassium
current and the effect of cesium on this current during anomalous
rectification of the egg cell membrane of a starfish. J. Gen. Physiol.
67: 621–638, 1976.
91. HAGIWARA, S., S. OZAWA, AND O. SAND. Voltage clamp analysis
of two inward current mechanisms in the egg cell membrane of a
starfish. J. Gen. Physiol. 65: 617–644, 1975.
92. HAGIWARA, S., S. YOSHIDA, AND M. YOSHII. Transient and delayed potassium currents in the egg cell membrane of the coelenterate, Renilla koellikeri. J. Physiol. (Lond.) 318: 123–141, 1981.
93. HARVEY, R. P. Widespread expression of MyoD genes in Xenopus
embryos is amplified in presumptive muscle as a delayed response
to mesoderm induction. Proc. Natl. Acad. Sci. USA 88: 9198–9202,
1991.
94. HEMMATI-BRIVANLOU, A., O. G. KELLY, AND D. A. MELTON. Follistatin, an antagonist of activin, is expressed in the Spemann organizer and displays direct neuralizing activity. Cell 77: 283–295, 1994.
95. HEMMATI-BRIVANLOU, A., AND D. A. MELTON. Inhibition of activin receptor signaling promotes neuralization in Xenopus. Cell 77:
273–281, 1994.
96. HILLE, B. Ionic Channels of Excitable Membrane. Sunderland, MA:
Sinauer, 1992.
97. HIRANO, T., AND K. TAKAHASHI. Comparison of properties of
calcium channels between the differentiated 1-cell embryo and the
egg cell of ascidians. J. Physiol. (Lond.) 347: 327–344, 1984.
98. HIRANO, T., AND K. TAKAHASHI. Development of ionic channels
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
60.
KUNITARO TAKAHASHI AND YASUSHI OKAMURA
April 1998
99.
100.
101.
102.
103.
104.
105.
107.
108.
109.
110.
111.
112.
113.
114.
115.
116.
117.
118.
119.
120.
121.
122.
123.
124.
and cell-surface antigens in the cleavage-arrested one-cell embryo
of an ascidian. J. Physiol. (Lond.) 386: 113–133, 1987.
HIRANO, T., K. TAKAHASHI, AND N. YAMASHITA. Determination
of excitability types in blastomeres of the cleavage-arrested but
differentiated embryos of an ascidian. J. Physiol. (Lond.) 347: 301–
325, 1984.
HOMA, S. T., J. CARROLL, AND K. SWANN. The role of calcium in
mammalian oocyte maturation and egg activation. Hum. Reprod.
8: 1274–1281, 1993.
IGUSA, Y., AND S. MIYAZAKI. Effects of altered extracellular and
intracellular calcium concentration on hyperpolarizing responses
of the hamster egg. J. Physiol. (Lond.) 340: 611–632, 1983.
IGUSA, Y., S. MIYAZAKI, AND N. YAMASHITA. Periodic hyperpolarizing responses in hamster and mouse eggs fertilized with mouse
sperm. J. Physiol. (Lond.) 340: 633–647, 1983.
ILLMENSEE, K., AND B. MINTZ. Totipotency and normal differentiation of single teratocarcinoma cells cloned by injection into blastocysts. Proc. Natl. Acad. Sci. USA 73: 549–553, 1976.
INAZAWA, T., Y. OKAMURA, AND K. TAKAHASHI. Basic fibroblast
growth factor causes neural induction on ascidian ectodermal blastomeres in the same manner as a natural inducer. Jpn. J. Physiol.
44, Suppl.: S158, 1994.
INAZAWA, T., T. SHIGEMOTO, AND K. TAKAHASHI. Effect of bFGF
and protein tyrosine kinase inhibitors on the differentiation of ascidian
ectodermal blastomeres. Jpn. J. Physiol. 42, Suppl.: S120, 1992.
ISOM, L. L., K. S. DE JONGH, D. E. PATTON, B. F. REBER, J. OFFORD, H. CHARBONNEAU, K. WALSH, A. L. GOLDIN, AND W. A.
CATTERALL. Primary structure and functional expression of the
beta 1 subunit of the rat brain sodium channel. Science 256: 839–
842, 1992.
JACOBSON, M. Developmental Neurobiolgy. New York: Plenum,
1979.
JAFFE, L. A. Fast block to polyspermy in sea urchin eggs is electrically mediated. Nature 261: 68–71, 1976.
JAFFE, L. A., AND N. L. CROSS. Electrical regulation of sperm-egg
fusion. Annu. Rev. Physiol. 48: 191–200, 1986.
JAFFE, L. A., M. GOULD-SOMERO, AND L. HOLLAND. Ionic mechanism of the fertilization potential of the marine worm, Urechis
caupo (Echiura). J. Gen. Physiol. 73: 469–492, 1979.
JAFFE, L. A., R. T. KADO, AND L. MUNCY. Propagating potassium
and chloride conductances during activation and fertilization of the
egg of the frog, Rana pipiens. J. Physiol. (Lond.) 368: 227–242,
1985.
JAFFE, L. A., AND L. C. SCHLICHTER. Fertilization-induced ionic
conductances in eggs of the frog, Rana pipiens. J. Physiol. (Lond.)
358: 299–319, 1985.
JAFFE, L. A., A. P. SHARP, AND D. P. WOLF. Absence of an electrical polyspermy block in the mouse. Dev. Biol. 96: 317–323, 1983.
JAFFE, L. F. Sources of calcium in egg activation: a review and
hypothesis. Dev. Biol. 99: 265–276, 1983.
JAFFE, L. F. The path of calcium in cytosolic calcium oscillations:
a unifying hypothesis. Proc. Natl. Acad. Sci. USA 88: 9883–9887,
1991.
JEFFERY, W. R. Calcium ionophore polarizes ooplasmic segregation in ascidian eggs. Science 216: 545–547, 1982.
JEFFERY, W. R. Ultraviolet irradiation during ooplasmic segregation prevents gastrulation, sensory cell induction, and axis formation in the ascidian embryo. Dev. Biol. 140: 388–400, 1990.
JEFFERY, W. R. An ultraviolet-sensitive maternal mRNA encoding
a cytoskeletal protein may be involved in axis formation in the
ascidian embryo. Dev. Biol. 141: 141–148, 1990.
JEFFERY, W. R. A gastrulation center in the ascidian egg. Development Suppl. 53–63, 1992.
JESSELL, T. M., AND D. A. MELTON. Diffusible factors in vertebrate
embryonic induction. Cell 68: 257–270, 1992.
KANE, D. A., AND C. B. KIMMEL. The zebrafish midblastula transition. Development 119: 447–456, 1993.
KANE, D. A., R. M. WARGA, AND C. B. KIMMEL. Mitotic domains
in the early embryo of the zebrafish. Nature 360: 735–737, 1992.
KANO, M., R. SATOH, AND Y. NAKABAYASHI. Calcium channels
in embryonic chick skeletal muscle cells after cultivation with calcium channel blocker. Neurosci. Lett. 144: 161–164, 1992.
KANO, M., K. WAKUTA, AND R. SATOH. Two components of cal-
/ 9j08$$ap07
P25-7
125.
126.
127.
128.
129.
130.
131.
132.
133.
134.
135.
136.
137.
138.
139.
140.
141.
142.
143.
144.
145.
146.
147.
148.
03-18-98 08:13:31
333
cium channel current in embryonic chick skeletal muscle cells developing in culture. Dev. Brain Res. 47: 101–112, 1989.
KATZ, B. Les constantes electriques de la membrane du muscle.
Arch. Sci. Physiol. 3: 285–300, 1949.
KATZ, M. J. Comparative anatomy of the tunicate tadpole, Ciona
intestinalis. Biol. Bull. 164: 1–27, 1983.
KAUFMAN, M. L., AND S. T. HOMA. Defining a role for calcium in
the resumption and progression of meiosis in the pig oocyte. J.
Exp. Zool. 265: 69–76, 1993.
KENGAKU, M., AND H. OKAMOTO. Basic fibroblast growth factor
induces differentiation of neural tube and neural crest lineages of
cultured ectoderm cells from Xenopus gastrula. Development 119:
1067–1078, 1993.
KENGAKU, M., AND H. OKAMOTO. bFGF as a possible morphogen
for the anteroposterior axis of the central nervous system in Xenopus. Development 121: 3121–3130, 1995.
KINTNER, C. Molecular bases of early neural development in Xenopus embryos. Annu. Rev. Neurosci. 15: 251–284, 1992.
KIRSCHNER, M. W. The biochemical nature of the cell cycle. Important Adv. Oncol. 3–16, 1992.
KLEIN, R., S. Q. JING, V. NANDURI, E. O’ROURKE, AND M. BARBACID. The trk proto-oncogene encodes a receptor for nerve
growth factor. Cell 65: 189–197, 1991.
KLEPPISCH, T., A. M. WOBUS, C. STRUBING, AND J. HESCHELER.
Voltage-dependent L-type Ca channels and a novel type of nonselective cation channel activated by cAMP-dependent phosphorylation in mesoderm-like (MES-1) cells. Cell. Signal. 5: 727–734,
1993.
KLINE, D., L. A. JAFFE, AND R. T. KADO. A calcium-activated sodium conductance contributes to the fertilization potential in the
egg of the nemertean worm Cerebratulus lacteus. Dev. Biol. 117:
184–193, 1986.
KLINE, D., L. A. JAFFE, AND R. P. TUCKER. Fertilization potential
and polyspermy prevention in the egg of the nemertean, Cerebratulus lacteus. J. Exp. Zool. 236: 45–52, 1985.
KLINE, D., AND R. NUCCITELLI. The wave of activation current in
the Xenopus egg. Dev. Biol. 111: 471–487, 1985.
KOBAYASHI, W., Y. BABA, T. SHIMOZAWA, AND T. S. YAMAMOTO.
The fertilization potential provides a fast block to polyspermy in
lamprey eggs. Dev. Biol. 161: 552–562, 1994.
KOSTYUK, P. G. Diversity of calcium ion channels in cellular membranes. Neuroscience 28: 253–261, 1989.
KOWALEVSKY, A. Entwicklingsgeschichite der einfachen Ascidian.
Mem. Acad. Imperiale Sci. St. Petersburg 10: 1–19, 1866.
KOZUKA, M., AND K. TAKAHASHI. Changes in holding and ionchannel currents during activation of an ascidian egg under voltage
clamp. J. Physiol. (Lond.) 323: 267–286, 1982.
KRAFTE, D. S., T. P. SNUTCH, J. P. LEONARD, N. DAVIDSON, AND
H. A. LESTER. Evidence for the involvement of more than one
mRNA species in controlling the inactivation process of rat and
rabbit brain Na channels expressed in Xenopus oocytes. J. Neurosci. 8: 2859–2868, 1988.
KRANER, S. D., J. A. CHONG, H. J. TSAY, AND G. MANDEL. Silencing the type II sodium channel gene: a model for neural-specific
gene regulation. Neuron 9: 37–44, 1992.
KREMER, N. E., G. D’ARCANGELO, S. M. THOMAS, M. DEMARCO,
J. S. BRUGGE, AND S. HALEGOUA. Signal transduction by nerve
growth factor and fibroblast growth factor in PC12 cells requires
a sequence of src and ras actions. J. Cell Biol. 115: 809–819, 1991.
KRIEG, P. A., D. S. SAKAGUCHI, AND C. R. KINTNER. Primary
structure and developmental expression of a large cytoplasmic domain form of Xenopus laevis neural cell adhesion molecule
(NCAM). Nucleic Acids Res. 17: 10321–10335, 1989.
KUBO, Y. Development of ion channels and neurofilaments during
neuronal differentiation of mouse embryonal carcinoma cell lines.
J. Physiol. (Lond.) 409: 497–523, 1989.
KUBO, Y. Electrophysiological and immunohistochemical analysis
of muscle differentiation in a mouse mesodermal stem cell line. J.
Physiol. (Lond.) 442: 711–741, 1991.
KUBO, Y. Comparison of initial stages of muscle differentiation in
rat and mouse myoblastic and mouse mesodermal stem cell lines.
J. Physiol. (Lond.) 442: 743–759, 1991.
KUBO, Y., T. J. BALDWIN, Y. N. JAN, AND L. Y. JAN. Primary struc-
pra
APS-Phys Rev
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on May 4, 2017
106.
ION CHANNELS AND EARLY DEVELOPMENT OF NEURAL CELLS
334
149.
150.
151.
152.
153.
154.
155.
157.
158.
159.
160.
161.
162.
163.
164.
165.
166.
167.
168.
169.
170.
171.
ture and functional expression of a mouse inward rectifier potassium channel. Nature 362: 127–133, 1993.
KUBO, Y., E. REUVENY, P. A. SLESINGER, Y. N. JAN, AND L. Y.
JAN. Primary structure and functional expression of a rat G-protein-coupled muscarinic potassium channel. Nature 364: 802–806,
1993.
LAKE, J. A. Origin of the Metazoa. Proc. Natl. Acad. Sci. USA 87:
763–766, 1990.
LAMB, T. M., AND R. M. HARLAND. Fibroblast growth factor is
a direct neural inducer, which combined with noggin generates
anterior-posterior pattern. Development 121: 3627–3636, 1995.
LAMB, T. M., A. K. KNECHT, W. C. SMITH, S. E. STACHEL, A. N.
ECONOMIDES, N. STAHL, G. D. YANCOPOLOUS, AND R. M. HARLAND. Neural induction by the secreted polypeptide noggin. Science 262: 713–718, 1993.
LANSMAN, J. B. Calcium current and calcium-activated inward current in the oocyte of the starfish Leptasterias hexactis. J. Physiol.
(Lond.) 390: 397–413, 1987.
LAU, A. F., M. Y. KANEMITSU, W. E. KURATA, S. DANESH, AND
A. L. BONYTON. Epidermal growth factor disrupts gap-junctional
communication and induces phosphorylation of connexin43 on serine. Mol. Biol. Cell 3: 865–867, 1992.
LAUNAY, C., V. FROMENTOUX, D.-L. SHI, AND J.-C. BOUCAUT. A
truncated FGF receptor blocks neural induction by endogenous
Xenopus inducers. Development 122: 869–880, 1996.
LEE, S. C., AND R. A. STEINHARDT. Observations on intracellular
pH during cleavage of eggs of Xenopus laevis. J. Cell Biol. 91: 414–
419, 1981.
LILLIE, F. R. Differentiation without cleavage in the egg of the
annelid Chaetopterus pergamentaceus. Arch. Entwickellungs
Mechanik Organismen 14: 477–499, 1902.
LILLIEN, L. E., AND M. C. RAFF. Analysis of the cell-cell interactions that control type-2 astrocyte development in vitro. Neuron 4:
525–534, 1990.
LO, C. W., AND N. B. GILULA. Gap junctional communication in the
preimplantation mouse embryo. Cell 18: 399–409, 1979.
LO, C. W., AND N. B. GILULA. Gap junctional communication in the
postimplantation mouse embryo. Cell 18: 411–422, 1979.
LORCA, T., F. H. CRUZALEGUI, D. FESQUET, J. C. CAVADORE, J.
MERY, A. MEANS, AND M. DOREE. Calmodulin-dependent protein
kinase II mediates inactivation of MPF and CSF upon fertilization
of Xenopus eggs. Nature 366: 270–273, 1993.
LU, L., C. MONTROSE-RAFIZADEH, T. C. HWANG, AND W. B. GUGGINO. A delayed rectifier potassium current in Xenopus oocytes.
Biophys. J. 57: 1117–1123, 1990.
LUX, H. D. Studies on the development of voltage-activated calcium
channels in vertebrate neurons. Puerto Rico Health Sci. J. 7: 116–
121, 1988.
MACKIE, G. O., AND Q. BONE. Skin impulses and locomotion in
an ascidian tadpole. J. Mar. Biol. Assoc. 56: 751–768, 1976.
MANDEL, G. Sodium channel regulation in the nervous system:
how the action potential keeps in shape. Curr. Opin. Neurobiol.
3: 278–282, 1993.
MANDEL, G., S. S. COOPERMAN, R. A. MAUE, R. H. GOODMAN,
/
AND P. BREHM. Selective induction of brain type II Na channels
by nerve growth factor. Proc. Natl. Acad. Sci. USA 85: 924–928,
1988.
MANDEL, G., AND D. MCKINNON. Molecular basis of neural-specific gene expression. Annu. Rev. Neurosci. 16: 323–345, 1993.
MARTIN-ZANCA, D., G. MITRA, L. K. LONG, AND M. BARBACID.
Molecular characterization of the human trk oncogene. Cold Spring
Harbor Symp. Quant. Biol. 51: 983–992, 1986.
MARTIN-ZANCA, D., R. OSKAM, G. MITRA, T. COPELAND, AND
M. BARBACID. Molecular and biochemical characterization of the
human trk proto-oncogene. Mol. Cell. Biol. 9: 24–33, 1989.
MAUE, R. A., S. D. KRANER, R. H. GOODMAN, AND G. MANDEL.
Neuron-specific expression of the rat brain type II sodium channel
gene is directed by upstream regulatory elements. Neuron 4: 223–
231, 1990.
MCDOUGALL, A., AND C. SARDET. Function and characteristics of
repetitive calcium waves associated with meiosis. Curr. Biol. 5:
318–328, 1995.
/ 9j08$$ap07
P25-7
Volume 78
172. MEANS, A. R. Calcium, calmodulin and cell cycle regulation. FEBS
Lett. 347: 1–4, 1994.
173. MEEDEL, T. H., R. J. CROWTHER, AND J. R. WHITTAKER. Determinative properties of muscle lineages in ascidian embryos. Development 100: 245–260, 1987.
174. MESSNER, D. J., AND W. A. CATTERALL. The sodium channel from
rat brain. Separation and characterization of subunits. J. Biol.
Chem. 260: 10597–10604, 1985.
175. MILEDI, R., AND I. PARKER. Chloride current induced by injection
of calcium into Xenopus oocytes. J. Physiol. (Lond.) 357: 173–
183, 1984.
176. MILEDI, R., AND R. M. WOODWARD. Effects of defolliculation on
membrane current responses of Xenopus oocytes. J. Physiol.
(Lond.) 416: 601–621, 1989.
177. MITA-MIYAZAWA, I., T. NISHIKATA, AND N. SATOH. Cell- and tissue-specific monoclonal antibodies in eggs and embryos of the
ascidian Halocynthia roretzi. Development 99: 155–162, 1987.
178. MITANI, S. The reduction of calcium current associated with early
differentiation of the murine embryo. J. Physiol. (Lond.) 363: 71–
86, 1985.
179. MITANI, S., AND H. OKAMOTO. Embryonic development of Xenopus studied in a cell culture system with tissue-specific monoclonal
antibodies. Development 105: 53–59, 1989.
180. MITANI, S., AND H. OKAMOTO. Inductive differentiation of two
neural lineages reconstituted in a microculture system from Xenopus early gastrula cells. Development 112: 21–31, 1991.
181. MIYAZAKI, S. Fast polyspermy block and activation potential. Electrophysiological bases for their changes during oocyte maturation
of a starfish. Dev. Biol. 70: 341–354, 1979.
182. MIYAZAKI, S. Repetitive calcium transients in hamster oocytes.
Cell Calcium 12: 205–216, 1991.
183. MIYAZAKI, S., AND S. HIRAI. Fast polyspermy block and activation
potential. Correlated changes during oocyte maturation of a starfish. Dev. Biol. 70: 327–340, 1979.
184. MIYAZAKI, S., AND Y. IGUSA. Ca-mediated activation of a K current
at fertilization of golden hamster eggs. Proc. Natl. Acad. Sci. USA
79: 931–935, 1982.
185. MIYAZAKI, S., H. SHIRAKAWA, K. NAKADA, AND Y. HONDA. Essential role of the inositol 1,4,5-trisphosphate receptor/Ca2/ release
channel in Ca2/ waves and Ca2/ oscillations at fertilization of mammalian eggs. Dev. Biol. 158: 62–78, 1993.
186. MIYAZAKI, S., K. TAKAHASHI, AND K. TSUDA. Calcium and sodium
contributions to regenerative responses in the embryonic excitable
cell membrane. Science 176: 1441–1443, 1972.
187. MIYAZAKI, S. I., H. OHMORI, AND S. SASAKI. Action potential and
non-linear current-voltage relation in starfish oocytes. J. Physiol.
(Lond.) 246: 37–54, 1975.
188. MIYAZAKI, S. I., H. OHMORI, AND S. SASAKI. Potassium rectifications of the starfish oocyte membrane and their changes during
oocyte maturation. J. Physiol. (Lond.) 246: 55–78, 1975.
189. MIYAZAKI, S. I., K. TAKAHASHI, AND K. TSUDA. Electrical excitability in the egg cell membrane of the tunicate. J. Physiol. (Lond.)
238: 37–54, 1974.
190. MIYAZAKI, S. I., K. TAKAHASHI, K. TSUDA, AND M. YOSHII. Analysis of non-linearity observed in the current-voltage relation of the
tunicate embryo. J. Physiol. (Lond.) 238: 55–77, 1974.
191. MOODY, W. J. The development of calcium and potassium currents
during oogenesis in the starfish, Leptasterias hexactis. Dev. Biol.
112: 405–413, 1985.
192. MOODY, W. J., AND M. M. BOSMA. Hormone-induced loss of surface membrane during maturation of starfish oocytes: differential
effects on potassium and calcium channels. Dev. Biol. 112: 396–
404, 1985.
193. MOODY, W. J., AND M. M. BOSMA. A nonselective cation channel
activated by membrane deformation in oocytes of the ascidian Boltenia villosa. J. Membr. Biol. 107: 179–188, 1989.
194. MOODY, W. J., AND S. HAGIWARA. Block of inward rectification
by intracellular H/ in immature oocytes of the starfish Mediaster
aequalis. J. Gen. Physiol. 79: 115–130, 1982.
195. MOODY, W. J., AND J. B. LANSMAN. Developmental regulation of
Ca2/ and K/ currents during hormone-induced maturation of starfish oocytes. Proc. Natl. Acad. Sci. USA 80: 3096–3100, 1983.
196. MOODY, W. J., L. SIMONCINI, J. L. COOMBS, A. E. SPRUCE, AND
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
156.
KUNITARO TAKAHASHI AND YASUSHI OKAMURA
April 1998
197.
198.
199.
200.
201.
202.
203.
205.
206.
207.
208.
209.
210.
211.
212.
213.
214.
215.
216.
217.
218.
M. VILLAZ. Development of ion channels in early embryos. J. Neurobiol. 22: 674–684, 1991.
MUSCI, T. J., E. AMAYA, AND M. W. KIRSCHNER. Regulation of
the fibroblast growth factor receptor in early Xenopus embryos.
Proc. Natl. Acad. Sci. USA 87: 8365–8369, 1990.
NAKATANI, Y., AND H. NISHIDA. Induction of notochord during
ascidian embryogenesis. Dev. Biol. 166: 289–299, 1994.
NAKATANI, Y., H. YASUO, N. SATOH, AND H. NISHIDA. Basic
fibroblast growth factor induces notochord formation and the expression of As-T, a Brachyury homolog, during ascidian embryogenesis. Development 122: 2023–2031, 1996.
NEWPORT, J., AND M. KIRSCHNER. A major developmental transition in early Xenopus embryos. I. Characterization and timing of
cellular changes at the midblastula stage. Cell 30: 675–686, 1982.
NICOL, D., AND I. A. MEINERTZHAGEN. Development of the central nervous system of the larva of the ascidian, Ciona intestinalis
L. I. The early lineages of the neural plate. Dev. Biol. 130: 721–
736, 1988.
NICOL, D., AND I. A. MEINERTZHAGEN. Development of the central nervous system of the larva of the ascidian, Ciona intestinalis
L. II. Neural plate morphogenesis and cell lineages during neurulation. Dev. Biol. 130: 737–766, 1988.
NICOL, D., AND I. A. MEINERTZHAGEN. Cell counts and maps in
the larval central nervous system of the ascidian Ciona intestinalis
(L.). J. Comp. Neurol. 309: 415–429, 1991.
NICOLAS, J. F., P. AVNER, J. GAILLARD, J. L. GUENET, H. JAKOB,
AND F. JACOB. Cell lines derived from teratocarcinomas. Cancer
Res. 36: 4224–4231, 1976.
NISHIDA, H. Cell division pattern during gastrulation of the ascidian, Halocynthia roretzi. Dev. Growth Differ. 28: 191–201, 1986.
NISHIDA, H. Cell lineage analysis in ascidian embryos by intracellular injection of a tracer enzyme. III. Up to the tissue restricted
stage. Dev. Biol. 121: 526–541, 1987.
NISHIDA, H. Determinative mechanisms in secondary muscle lineages of ascidian embryos: development of muscle-specific features
in isolated muscle progenitor cells. Development 108: 559–568,
1990.
NISHIDA, H., AND N. SATOH. Cell lineage analysis in ascidian embryos by intracellular injection of a tracer enzyme. I. Up to the
eight-cell stage. Dev. Biol. 99: 382–394, 1983.
NISHIDA, H., AND N. SATOH. Cell lineage analysis in ascidian embryos by intracellular injection of a tracer enzyme. II. The 16- and
32-cell stages. Dev. Biol. 110: 440–454, 1985.
NISHIKATA, T., I. MITA-MIYAZAWA, T. DENO, AND N. SATOH.
Muscle cell differentiation in ascidian embryos analysed with a
tissue-specific monoclonal antibody. Development 99: 163–171,
1987.
NISHIKATA, T., I. MITA-MIYAZAWA, T. DENO, K. TAKAMURA,
AND N. SATOH. Expression of epidermis-specific antigens during
embryogenesis of the ascidian, Halocynthia roretzi. Dev. Biol. 121:
408–416, 1987.
NISHIKATA, T., I. MITA-MIYAZAWA, AND N. SATOH. Differentiation expression in blastomeres of cleavage-arrested embryos of the
ascidian Halocynthia roretzi. Dev. Growth Differ. 30: 371–381,
1988.
NODA, M., T. IKEDA, T. KAYANO, H. SUZUKI, H. TAKESHIMA, M.
KURASAKI, H. TAKAHASHI, AND S. NUMA. Existence of distinct
sodium channel messenger RNAs in rat brain. Nature 320: 188–
192, 1986.
NOWYCKY, M. C., A. P. FOX, AND R. W. TSIEN. Three types of
neuronal calcium channel with different calcium agonist sensitivity.
Nature 316: 440–443, 1985.
NUCCITELLI, R. The wave of activation current in the egg of the
medaka fish. Dev. Biol. 122: 522–534, 1987.
O’DOWD, D. K., A. B. RIBERA, AND N. C. SPITZER. Development
of voltage-dependent calcium, sodium, and potassium currents in
Xenopus spinal neurons. J. Neurosci. 8: 792–805, 1988.
OFFORD, J., AND W. A. CATTERALL. Electrical activity, cAMP, and
cytosolic calcium regulate mRNA encoding sodium channel alpha
subunits in rat muscle cells. Neuron 2: 1447–1452, 1989.
OHMICHI, M., S. J. DECKER, L. PANG, AND A. R. SALTIEL. Inhibition of the cellular actions of nerve growth factor by staurosporine
/ 9j08$$ap07
P25-7
219.
220.
221.
222.
223.
224.
225.
226.
227.
228.
229.
230.
231.
232.
233.
234.
235.
236.
237.
238.
239.
240.
241.
242.
03-18-98 08:13:31
335
and K252a results from the attenuation of the activity of the trk
tyrosine kinase. Biochemistry 31: 4034–4049, 1992.
OHMORI, H. Inactivation kinetics and steady-state current noise in
the anomalous rectifier of tunicate egg cell membranes. J. Physiol.
(Lond.) 281: 77–99, 1978.
OHMORI, H. Unitary current through sodium channel and anomalous rectifier channel estimated from transient current noise in the
tunicate egg. J. Physiol. (Lond.) 311: 289–305, 1981.
OHMORI, H., AND S. SASAKI. Development of neuromuscular transmission in a larval tunicate. J. Physiol. (Lond.) 269: 221–254, 1977.
OKADA, T., H. HIRANO, K. TAKAHASHI, AND Y. OKAMURA. Distinct neuronal lineages of the ascidian embryo revealed by expression of a sodium channel gene. Dev. Biol. 1282: 7936–7711, 1997.
OKADO, H., AND K. TAKAHASHI. A simple ‘‘neural induction’’
model with two interacting cleavage-arrested ascidian blastomeres.
Proc. Natl. Acad. Sci. USA 85: 6197–6201, 1988.
OKADO, H., AND K. TAKAHASHI. Induced neural-type differentiation in the cleavage-arrested blastomere isolated from early ascidian embryos. J. Physiol. (Lond.) 427: 603–623, 1990.
OKADO, H., AND K. TAKAHASHI. Differentiation of membrane excitability in isolated cleavage-arrested blastomeres from early ascidian embryos. J. Physiol. (Lond.) 427: 583–602, 1990.
OKADO, H., AND K. TAKAHASHI. Neural differentiation in cleavagearrested ascidian blastomeres induced by a proteolytic enzyme. J.
Physiol. (Lond.) 463: 269–290, 1993.
OKAMOTO, H., K. TAKAHASHI, AND N. YAMASHITA. Ionic currents through the membrane of the mammalian oocyte and their
comparison with those in the tunicate and sea urchin. J. Physiol.
(Lond.) 267: 465–495, 1977.
OKAMOTO, H., K. TAKAHASHI, AND M. YOSHII. Membrane currents of the tunicate egg under the voltage-clamp condition. J.
Physiol. (Lond.) 254: 607–638, 1976.
OKAMOTO, H., K. TAKAHASHI, AND M. YOSHII. Two components
of the calcium current in the egg cell membrane of the tunicate.
J. Physiol. (Lond.) 255: 527–561, 1976.
OKAMURA, Y., H. OKADO, AND K. TAKAHASHI. The ascidian embryo as a prototype of vertebrate neurogenesis. Bioessays 15: 723–
730, 1993.
OKAMURA, Y., F. ONO, R. OKAGAKI, J. A. CHONG, AND G. MANDEL. Neural expression of a sodium channel gene requires cellspecific interactions. Neuron 13: 937–948, 1994.
OKAMURA, Y., AND M. SHIDARA. Kinetic differences between Na
channels in the egg and in the neurally differentiated blastomere
in the tunicate. Proc. Natl. Acad. Sci. USA 84: 8702–8706, 1987.
OKAMURA, Y., AND M. SHIDARA. Changes in sodium channels
during neural differentiation in the isolated blastomere of the ascidian embryo. J. Physiol. (Lond.) 431: 39–74, 1990.
OKAMURA, Y., AND M. SHIDARA. Inactivation kinetics of the sodium channel in the egg and the isolated, neurally differentiated
blastomere of the ascidian. J. Physiol. (Lond.) 431: 75–102, 1990.
OKAMURA, Y., AND K. TAKAHASHI. Neural induction suppresses
early expression of the inward-rectifier K/ channel in the ascidian
blastomere. J. Physiol. (Lond.) 463: 245–268, 1993.
OZAWA, S., AND O. SAND. Electrophysiology of excitable endocrine
cells. Physiol. Rev. 66: 887–952, 1986.
PARKER, I., AND I. IVORRA. A slowly inactivating potassium current in native oocytes of Xenopus laevis. Proc. R. Soc. Lond. B
Biol. Sci. 238: 369–381, 1990.
PARKER, I., AND R. MILEDI. Inositol trisphosphate activates a voltage-dependent calcium influx in Xenopus oocytes. Proc. R. Soc.
Lond. B Biol. Sci. 231: 27–36, 1987.
PERES, A. The calcium current of mouse egg measured in physiological calcium and temperature conditions. J. Physiol. (Lond.)
391: 573–588, 1987.
PHILLIPS, C. R. Neural induction. Methods Cell Biol. 36: 329–346,
1991.
PIRCHIO, M., S. LIGHTOWLER, AND V. CRUNELLI. Postnatal development of the T calcium current in cat thalamocortical cells. Neuroscience 38: 39–45, 1990.
POLLOCK, J. D., M. KREMPIN, AND B. RUDY. Differential effects
of NGF, FGF, EGF, cAMP, and dexamethasone on neurite outgrowth and sodium channel expression in PC12 cells. J. Neurosci.
10: 2626–2637, 1990.
pra
APS-Phys Rev
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on May 4, 2017
204.
ION CHANNELS AND EARLY DEVELOPMENT OF NEURAL CELLS
336
KUNITARO TAKAHASHI AND YASUSHI OKAMURA
/ 9j08$$ap07
P25-7
268. SCHEINMAN, R. I., V. J. AULD, A. L. GOLDIN, N. DAVIDSON, R. J.
DUNN, AND W. A. CATTERALL. Developmental regulation of sodium channel expression in the rat forebrain. J. Biol. Chem. 264:
10660–10666, 1989.
269. SCHLICHTER, L. C. Ionic currents underlying the action potential
of Rana pipiens oocytes. Dev. Biol. 134: 59–71, 1989.
270. SERRAS, F., C. BAUD, M. MOREAU, P. GUERRIER, AND J. A. M.
VAN DEN BIGGELAAR. Intercellular communication in the early
embryo of the ascidian Ciona intestinalis. Development 102: 55–
63, 1988.
271. SHIDARA, M., AND Y. OKAMURA. Developmental changes in delayed rectifier K/ currents in the muscular- and neural-type blastomere of ascidian embryos. J. Physiol. (Lond.) 443: 277–305, 1991.
272. SHIGEMOTO, T., AND Y. OKADA. External-anio-dependent anionic
current in blastoderm cells of early medaka fish embryo. J. Physiol.
(Lond.) 495: 51–63, 1996.
273. SHIINA, Y., M. KANEDA, K. MATSUYAMA, K. TANAKA, M. HIROI,
2/
2/
AND K. DOI. Role of the extracellular Ca on the intracellular Ca
changes in fertilized and activated mouse oocytes. J. Reprod. Fertil.
97: 143–150, 1993.
274. SILVER, R. B. Calcium and cellular clocks orchestrate cell division.
Ann. NY Acad. Sci. 582: 207–221, 1990.
275. SIMONCINI, L., M. L. BLOCK, AND W. J. MOODY. Lineage-specific
development of calcium currents during embryogenesis. Science
242: 1572–1575, 1988.
276. SIMONCINI, L., AND W. J. MOODY. Dependence of Ca2/ and K/
current development on RNA and protein synthesis in muscle-lineage cells of the ascidian Boltenia villosa. J. Neurosci. 11: 1413–
1420, 1991.
277. SIMONNEAU, M., C. DISTASI, L. TAUC, AND C. POUJEOL. Development of ionic channels during mouse neuronal differentiation. J.
Physiol. 80: 312–320, 1985.
278. SIVE, H. L., K. HATTORI, AND H. WEINTRAUB. Progressive determination during formation of the anteroposterior axis in Xenopus
laevis. Cell 58: 171–180, 1989.
279. SLACK, J. M. Developmental biology. The inducer that never was.
Nature 369: 279–280, 1994.
280. SLACK, J. M., B. G. DARLINGTON, L. L. GILLESPIE, S. F. GODSAVE, H. V. ISAACS, AND G. D. PATERNO. Mesoderm induction
by fibroblast growth factor in early Xenopus development. Philos.
Trans. R. Soc. Lond. 327: 75–84, 1990.
281. SLACK, J. M., B. G. DARLINGTON, J. K. HEATH, AND S. F. GODSAVE. Mesoderm induction in early Xenopus embryos by heparinbinding growth factors. Nature 326: 197–200, 1987.
282. SLACK, J. M., H. V. ISAACS, AND B. G. DARLINGTON. Inductive
effects of fibroblast growth factor and lithium ion on Xenopus
blastula ectoderm. Development 103: 581–590, 1988.
283. SLACK, J. M. W., AND D. TANNAHILL. Mechanism of anteroposterior axis specification in vertebrates: lessons from the amphibians.
Development 114: 285–302, 1992.
284. SNOW, P., AND R. NUCCITELLI. Calcium buffer injections delay
cleavage in Xenopus laevis blastomeres. J. Cell Biol. 122: 387–394,
1993.
285. SPEKSNIJDER, J. E., D. W. CORSON, C. SARDET, AND L. F. JAFFE.
Free calcium pulses following fertilization in the ascidian egg. Dev.
Biol. 135: 182–190, 1989.
286. SPEKSNIJDER, J. E., C. SARDET, AND L. F. JAFFE. The activation
wave of calcium in the ascidian egg and its role in ooplasmic segregation. J. Cell Biol. 110: 1589–1598, 1990.
287. SPEKSNIJDER, J. E., C. SARDET, AND L. F. JAFFE. Periodic calcium waves cross ascidian eggs after fertilization. Dev. Biol. 142:
246–249, 1990.
288. SPEMANN, H. Embryonic Development and Induction. New York:
Hafner, 1967.
289. SPEMANN, H., AND H. MANGOLD. Uber Induktion von Embryonalanlagen durch Implantation artfremder Organsatoren. Arch. f.
mikr. Anat. u. Entw. Mech. 100: 599–638, 1924.
290. SPITZER, N. C. Spontaneous Ca2/ spikes and waves in embryonic
neurons: signaling systems for differentiation. Trends Neurosci. 17:
115–118, 1994.
291. SPITZER, N. C., X. GU, AND E. OLSON. Action potentials, calcium
transients and the control of differentiation of excitable cells. Curr.
Opin. Neurobiol. 4: 70–77, 1994.
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
243. POTTER, D. D., E. J. FURSHPAN, AND E. S. LENNOX. Connections
between cells of the developing squid as revealed by electrophysiological methods. Proc. Natl. Acad. Sci. USA 55: 328–336, 1966.
244. RAY, W. J., AND D. I. GOTTLIEB. Expression of ionotropic glutamate receptor genes by P19 embryonal carcinoma cells. Biochem.
Biophys. Res. Commun. 197: 1475–1482, 1993.
245. REVERBERI, G. Experimental Embryology of Marine and Freshwater Invertebrates. London: North-Holland, 1971, p. 507–550.
246. REVERBERI, G., AND A. MINGANTI. Fenomeni di evocatione nello
sviluppo dell’uovo di Ascidie. Risultati dell’indagine sperimentale
sull’uovo di Ascidielle aspersa e di Ascidia malaca allo stadio di
otto blastomeri. Pubbl. Stazione Zool. Napoli 20: 199–252, 1946.
247. REVERBERI, G., G. ORTOLANI, AND N. FARINIELLA-FERRUZZA.
The causal formation of the brain in the ascidian larva. Acta
Embryol. Morphol. Exp. 3: 296–336, 1960.
248. REYNOLDS, J. N., P. J. RYAN, A. PRASAD, AND G. D. PATERNO.
Neurons derived from embryonal carcinoma (P19) cells express
multiple GABAA receptor subunits and fully functional GABAA receptors. Neurosci. Lett. 165: 129–132, 1994.
249. RIBERA, A. B. A potassium channel gene is expressed at neural
induction. Neuron 5: 691–701, 1990.
250. RIBERA, A. B., AND N. C. SPITZER. Differentiation of IKA in amphibian spinal neurons. J. Neurosci. 10: 1886–1891, 1990.
251. RIBERA, A. B., AND N. C. SPITZER. Differentiation of delayed rectifier potassium current in embryonic amphibian myocytes. Dev.
Biol. 144: 119–128, 1991.
252. RISEK, B., AND N. B. GILULA. Spatiotemporal expression of three
gap junction gene products involved in fetomaternal communication during rat pregnancy. Development 113: 165–181, 1991.
253. ROBERTS, A., AND C. A. STIRLING. The properties and propagation
of a cardiac-like impulse in the skin of young tadpoles. Z. Vergl.
Physiol. 71: 295–310, 1971.
254. ROMER, A. S. Major steps in vertebrate evolution. Science 158:
1629–1637, 1967.
255. ROSE, S. M. Embryonic induction in the Ascidia.. Biol. Bull. 77:
216–232, 1939.
256. RUDY, B., B. KIRSCHENBAUM, A. RUKENSTEIN, AND L. A.
GREENE. Nerve growth factor increases the number of functional
Na channels and induces TTX-resistant Na channels in PC12 pheochromocytoma cells. J. Neurosci. 7: 1613–1625, 1987.
257. RUPP, R. A., AND H. WEINTRAUB. Ubiquitous MyoD transcription
at the midblastula transition precedes induction-dependent MyoD
expression in presumptive mesoderm of X. laevis. Cell 65: 927–
937, 1991.
258. SAITOE, M., T. INAZAWA, AND K. TAKAHASHI. Neuronal expression in cleavage-arrested ascidian blastomeres requires gap junctional uncoupling from neighbouring cells. J. Physiol. (Lond.) 491:
825–842, 1996.
259. SARDET, C., J. SPEKSNIJDER, S. INOUE, AND L. JAFFE. Fertilization and ooplasmic movements in the ascidian egg. Development
105: 237–249, 1989.
260. SATO, E., O. NAKAMURA, AND S. ITO. Ionic dependence and transmission of epidermal action potentials in a newt embryo. Dev. Biol.
97: 460–467, 1983.
261. SATOH, N. Cellular morphology and architecture during early morphogenesis of the ascidian egg: an SEM study. Biol. Bull. 155: 608–
614, 1978.
262. SATOH, N. Developmental Biology of Ascidians. Cambridge, UK:
Cambridge Univ. Press, 1994.
263. SATOH, N., AND T. DENO. Periodic appearance and disappearance
of microvilli associated with cleavage cycles in the egg of the ascidian, Halocynthia roretzi. Dev. Biol. 102: 488–492, 1984.
264. SATOH, R., Y. NAKABAYASHI, AND M. KANO. Chronic treatment
with D600 enhances development of sodium channels in cultured
chick skeletal muscle cells. Neurosci. Lett. 138: 249–252, 1992.
265. SAWADA, T., AND G. SCHATTEN. Microtubules in ascidian eggs
during meiosis, fertilization, and mitosis. Cell Motil. Cytoskeleton
9: 219–230, 1988.
266. SAWADA, T., AND G. SCHATTEN. Effects of cytoskeletal inhibitors
on ooplasmic segregation and microtubule organization during fertilization and early development in the ascidian Molgula occidentalis. Dev. Biol. 132: 331–342, 1989.
267. SAXEN, L. Neural induction. Int. J. Dev. Biol. 33: 21–48, 1989.
Volume 78
April 1998
ION CHANNELS AND EARLY DEVELOPMENT OF NEURAL CELLS
/ 9j08$$ap07
P25-7
313. WEINTRAUB, H., R. DAVIS, S. TAPSCOTT, M. THAYER, M.
KRAUSE, R. BENEZRA, T. K. BLACKWELL, D. TURNER, R. RUPP,
AND S. HOLLENBERG. The myoD gene family: nodal point during
specification of the muscle cell lineage. Science 251: 761–766, 1991.
314. WEISS, P. Principles of Development. A Text in Experimental
Embryology. New York: Holt, 1939.
315. WESTENBROEK, R. E., D. K. MERRICK, AND W. A. CATTERALL.
Differential subcellular localization of the RI and RII Na/ channel
subtypes in central neurons. Neuron 3: 695–704, 1989.
316. WHITAKER, M., AND R. PATEL. Calcium and cell cycle control.
Development 108: 525–542, 1990.
317. WHITE, M. M., L. Q. CHEN, R. KLEINFIELD, R. G. KALLEN, AND
R. L. BARCHI. SkM2, a Na/ channel cDNA clone from denervated
skeletal muscle, encodes a tetrodotoxin-insensitive Na/ channel.
Mol. Pharmacol. 39: 604–608, 1991.
318. WHITTAKER, J. R. Segregation during ascidian embryogenesis of
egg cytoplasmic information for tissue-specific enzyme development. Proc. Natl. Acad. Sci. USA 70: 2096–2100, 1973.
319. WITCHEL, H. J., AND R. A. STEINHARDT. 1-Methyladenine can consistently induce a fura-detectable transient calcium increase which
is neither necessary nor sufficient for maturation in oocytes of the
starfish Asterina miniata. Dev. Biol. 141: 393–398, 1990.
320. WOLLNER, D. A., R. SCHEINMAN, AND W. A. CATTERALL. Sodium
channel expression and assembly during development of retinal
ganglion cells. Neuron 1: 727–737, 1988.
321. WOOD, K. W., H. QI, G. D’ARCANGELO, R. C. ARMSTRONG, T. M.
ROBERTS, AND S. HALEGOUA. The cytoplasmic raf oncogene induces a neuronal phenotype in PC12 cells: a potential role for
cellular raf kinases in neuronal growth factor signal transduction.
Proc. Natl. Acad. Sci. USA 90: 5016–5020, 1993.
322. WOODWARD, D. J. Electrical signs of new membrane production
during cleavage of Rana pipiens eggs. J. Gen. Physiol. 52: 509–
531, 1968.
323. XU, T., I. REBAY, R. J. FLEMING, T. N. SCOTTGALE, AND S. ARTAVANIS-TSAKONAS. The Notch locus and the genetic circuitry involved in early Drosophila neurogenesis. Genes Dev. 4: 464–475,
1990.
324. XU, X., AND P. M. BEST. Postnatal changes in T-type calcium current density in rat atrial myocytes. J. Physiol. (Lond.) 454: 657–
672, 1992.
325. YAARI, Y., B. HAMON, AND H. D. LUX. Development of two types
of calcium channels in cultured mammalian hippocampal neurons.
Science 235: 680–682, 1987.
326. YAMADA, K., K. IWAHASHI, AND H. KASE. K252a, a new inhibitor
of protein kinase C, concomitantly inhibits 40 K protein phosphorylation and serotonin secretion in phorbol ester-stimulated platelets.
Biochem. Biophys. Res. Commun. 144: 35–40, 1987.
327. YAMADA, T., S. L. PFAFF, T. EDLUND, AND T. M. JESSELL. Control
of cell pattern in the neural tube: motor neuron induction by diffusible factors from notochord and floor plate. Cell 73: 673–686, 1993.
328. YAMASHITA, N. Enhancement of ionic currents through voltagegated channels in the mouse oocyte after fertilization. J. Physiol.
(Lond.) 329: 263–280, 1982.
329. YANG, X. C., AND F. SACHS. Block of stretch-activated ion channels
in Xenopus oocytes by gadolinium and calcium ions. Science 243:
1068–1071, 1989.
330. YANG, X. C., AND F. SACHS. Characterization of stretch-activated
ion channels in Xenopus oocytes. J. Physiol. (Lond.) 431: 103–
122, 1990.
331. YAO, Y., AND I. PARKER. Inositol trisphosphate-mediated Ca2/ influx into Xenopus oocytes triggers Ca2/ liberation from intracellular
stores. J. Physiol. (Lond.) 468: 275–295, 1993.
332. YOSHIDA, S. Permeation of divalent and monovalent cations
through the ovarian oocyte membrane of the mouse. J. Physiol.
(Lond.) 339: 631–642, 1983.
333. ZALOKAR, M., AND C. SARDET. Tracing of cell lineage in embryonic
development of Phallusia mammillata (Ascidia) by vital staining
of mitochondria. Dev. Biol. 102: 195–205, 1984.
334. ZUCKER, R. S., AND R. A. STEINHARDT. Calcium activation of the
cortical reaction in sea urchin eggs. Nature 279: 820–821, 1979.
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
292. SPRUCE, A. E., AND W. J. MOODY. Developmental sequence of expression of voltage-dependent currents in embryonic Xenopus
laevis myocytes. Dev. Biol. 154: 11–22, 1992.
293. STEMPLE, D. L., N. K. MAHANTHAPPA, AND D. J. ANDERSON. Basic FGF induces neuronal differentiation, cell division, and NGF
dependence in chromaffin cells: a sequence of events in sympathetic development. Neuron 1: 517–525, 1988.
294. STRICKLAND, S., AND V. MAHDAVI. The induction of differentiation in teratocarcinoma stem cells by retinoic acid. Cell 15: 393–
403, 1978.
295. SWANN, K., D. H. MCCULLOH, A. MCDOUGALL, E. L. CHAMBERS,
AND M. WHITAKER. Sperm-induced currents at fertilization in sea
urchin eggs injected with EGTA and neomycin. Dev. Biol. 151: 552–
563, 1992.
296. SWANN, K., AND M. J. WHITAKER. Second messengers at fertilization in sea-urchin eggs. J. Reprod. Fertil. 42, Suppl.: 141–153, 1990.
297. TAGLIETTI, V., AND M. TOSELLI. A study of stretch-activated channels in the membrane of frog oocytes: interactions with Ca2/ ions.
J. Physiol. (Lond.) 407: 311–328, 1988.
298. TAKAHASHI, K., AND M. YOSHII. Effects of internal free calcium
upon the sodium and calcium channels in the tunicate egg analysed
by the internal perfusion technique. J. Physiol. (Lond.) 279: 519–
549, 1978.
299. TAKAHASHI, K., AND M. YOSHII. Development of sodium, calcium
and potassium channels in the cleavage-arrested embryo of an ascidian. J. Physiol. (Lond.) 315: 515–529, 1981.
300. TAPIA-RAMIREZ, J., B. J. EGGEN, M. J. PERAL-RUBIO, J. J. TOLEDO-ARAL, AND G. MANDEL. A single zinc finger motif in the
silencing factor REST represses the neural-specific type II sodium
channel promoter. Proc. Natl. Acad. Sci. USA 94: 1177–1182, 1997.
301. TAPLEY, P., F. LAMBALLE, AND M. BARBACID. K252a is a selective
inhibitor of the tyrosine protein kinase activity of the trk family of
oncogenes and neurotrophin receptors. Oncogene 7: 371–381, 1992.
302. THOMAS, S. M., M. DEMARCO, G. D’ARCANGELO, S. HALEGOUA,
AND J. S. BRUGGE. Ras is essential for nerve growth factor- and
phorbol ester-induced tyrosine phosphorylation of MAP kinases.
Cell 68: 1031–1040, 1992.
303. TOGARI, A., G. DICKENS, H. KUZUYA, AND G. GUROFF. The effect
of fibroblast growth factor on PC12 cells. J. Neurosci. 5: 307–316,
1985.
304. TOMBES, R. M., C. SIMERLY, G. G. BORISY, AND G. SCHATTEN.
Meiosis, egg activation, and nuclear envelope breakdown are differentially reliant on Ca2/, whereas germinal vesicle breakdown is
Ca2/ independent in the mouse oocyte. J. Cell Biol. 117: 799–811,
1992.
305. TORRENCE, S. A., AND R. A. CLONEY. Nervous system of ascidian
larvae: caudal primary sensory neurons. Zoomorphology 99: 103–
115, 1982.
306. TORRENCE, S. A., AND R. A. CLONEY. Ascidian larval nervous system: primary sensory neurons in adhesive papillae. Zoomorphology
102: 111–123, 1983.
307. TURETSKY, D. M., J. E. HUETTNER, D. I. GOTTLIEB, M. P. GOLDBERG, AND D. W. CHOI. Glutamate receptor-mediated currents and
toxicity in embryonal carcinoma cells. J. Neurobiol. 24: 1157–1169,
1993.
308. TURNER, P. R., L. A. JAFFE, AND A. FEIN. Regulation of cortical
vesicle exocytosis in sea urchin eggs by inositol 1,4,5-trisphosphate
and GTP-binding protein. J. Cell Biol. 102: 70–76, 1986.
309. VILAIN, J. P., M. MOUMENE, AND M. MOREAU. Chloride current
modulation during meiosis in Xenopus oocytes. J. Exp. Zool. 250:
100–108, 1989.
310. WADA, H., AND N. SATOH. Details of the evolutionary history from
invertebrates to vertebrates, as deduced from the sequences of 18S
rDNA. Proc. Natl. Acad. Sci. USA 91: 1801–1804, 1994.
311. WARNER, A. The gap junction. J. Cell Sci. 89: 1–7, 1988.
312. WEBB, D. J., AND R. NUCCITELLI. A comparative study of the
membrane potential from before fertilization through early cleavage in two frogs, Rana pipiens and Xenopus laevis. Comp. Biochem. Physiol. A Physiol. 82: 35–42, 1985.
337