We have been studying the molecular and cellular mechanisms underlying the development and functioning of the vertebrate central nervous system. The scope of our interests encompasses regional specification in the retina, neuronal differentiation, cellular migration, path-finding and target recognition of axons, formation and refinement of specific synapses, and also various functions of the matured brain.
I. Molecular mechanism for topographic retinotectal projection
A. Regional specification in the developing retina and topographic projection
Topographic maps are a fundamental feature of
neural networks in the nervous system. Understanding the molecular
mechanisms by which topographically ordered neuronal connections
are established during development has long been a major challenge
in developmental neurobiology. The retinotectal projection of
lower vertebrates including birds has been used as a readily accessible
model system. In this projection, the temporal (posterior) retina
is connected to the rostral (anterior) part of the contralateral
optic tectum, the nasal (anterior) retina to the caudal (posterior)
tectum, and likewise the dorsal and ventral retina to the ventral
and dorsal tectum, respectively. Thus, images received by the
retina are precisely projected onto the tectum in a reversed manner.
Since 1992, we have been devoting our efforts to searching for topographic molecules which show an asymmetrical distribution in the embryonic chick retina. In the first-round screening using a cDNA subtractive hybridization technique, we identified two winged-helix transcriptional regulators, CBF-1 and CBF-2, expressed in the nasal and temporal retina, respectively. Furthermore, our misexpression experiments using a retroviral vector showed that these two transcription factors determine the regional specificity of the retinal ganglion cells, namely, the directed axonal projections to the appropriate tectal targets along the anteroposterior axis.
To further search for topographic molecules in the embryonic retina, we next performed a large-scale screening using a new cDNA display system called Restriction Landmark cDNA Scanning (RLCS) in 1997. With the assistance of a computer image-processing software, we successfully identified 33 molecules along the nasotemporal axis and 20 molecules along the dorsoventral axis, with various asymmetrical expression patterns in the developing retina. We have elucidated the primary structures of all these cDNA clones and examined their expression patterns during development. Included were many novel together with known molecules: transcription factors (CBF-2, COUP-TFII, etc.), receptor proteins (EphA3, EphB3, etc.), secretory factors, intracellular proteins, and so on. Some of them have already been characterized as described below.
Among them, we identified a novel retinoic acid (RA)-generating enzyme, RALDH-3, which is specifically expressed in the ventral region of the retina, together with a dorsal-specific enzyme RALDH-1. In chick and mouse embryos, the expression of Raldh-3 is observed first in the surface ectoderm overlying the ventral portion of the prospective eye region earlier than that of Raldh-1 in the dorsal retina, and then the Raldh-3 expression shifts to the ventral retina. Furthermore, we found that Raldh-3 is a downstream target of Pax6 which is known to be the master gene for the development of the eye in many species. RA is essential for the eye to develop. These results suggest that RALDH-3 and RALDH-1 are the key enzymes for the dorsoventral patterning at the early stage of retinal development.
Furthermore, we identified a novel secretory protein, Ventroptin, which has BMP-4-neutralizing activity. Ventroptin is expressed in the retina with a ventral high-dorsal low gradient at the early stages (E2-E5). This expression pattern is complementary to that of BMP-4. From E6, with the disappearance of BMP-4 from the dorsal retina, expression of Ventroptin begins to show a nasal high-temporal low gradient. Ventroptin thus shows a double-gradient expression profile along the dorsoventral and anteroposterior axes at later stages. Misexpression of Ventroptin altered expression patterns of several topographic genes: BMP-4, Tbx5 and cVax along the dorsoventral axis and ephrin-A2 along the anteroposterior axis. Consistently, in these embryos, projection of the retinal ganglion cell axons to the tectum was changed along both axes.
For some time, downstream topographic genes and molecular mechanisms by which CBF-1 controls the expression of them have not been elucidated. We addressed this question by using electroporation of recombinant retrovirus DNA constructs into the developing chick optic vesicle at stage 8. Very recently, we successfully showed that misexpression of CBF-1 represses the expression of EphA3 and CBF-2, and induces that of SOHo-1, GH6, ephrin-A2, and ephrin-A5. CBF-1 controls ephcin-A5 by a DNA binding-dependent mechanism, ephrin-A2 by a DNA binding-independent mechanism, and CBF-2, SOHo-1, GH6, and EphA3 by dual mechanisms. In addition, we found BMP-2 expression begins double-gradiently in the retina from E5 in a complementary pattern to the Ventroptin expression. Ventroptin antagonizes BMP-2 as well as BMP-4. CBF-1 interferes in BMP-2 signaling and thereby induces expression of ephrin-A2. Our data suggest that CBF-1 is located at the top of the gene cascade for the regional specification along the nasotemporal (N-T) axis in the retina, and distinct BMP signals play pivotal roles in the topographic projection along both axes. The topographic retinotectal projection along the two axes thus appears to be controlled, not separately but in a highly concerted manner, by the sequential interplay between BMPs and Ventroptin.
B. Molecular mechanisms underlying the morphogenesis and behavior of the retinal axon
Among the molecules with region-specific expression
in the developing retina, some genes are supposed to be involved
in axogenesis or synaptogenesis. We recently identified a novel
subtype for CRMP3, and subsequently all for CRMP1-4 (named CRMP-As),
which are amino (N)-terminal variants of the CRMPs originally
reported (here renamed CRMP-Bs). In neurons, CRMP2B is distributed
in axons and dendrites, while CRMP2A is only in axons. We studied
functional differences between CRMP2A and CRMP2B by using two
different cellular systems.
Overexpression of CRMP2A and CRMP2B in chick embryonic fibroblasts induced orientated and disoriented patterns of microtubules, respectively. Moreover, sequential overexpression of another subtype overcame the effect of the former expression of the countersubtype. Overexpression experiments in cultured chick retinae showed that CRMP2B promoted axon branching and suppressed axon elongation of ganglion cells, while CRMP2A blocked these effects when co-overexpressed. Our findings suggest that the opposing activities of CRMP2A and CRMP2B contribute to the cellular morphogenesis including neuronal axonogenesis through remodeling of microtubule organization.
C. Layer-specific projections
After reaching their appropriate target zones
along the rostrocaudal and dorsoventral axes of the tectum, retinal
axons begin to seek their appropriate termination sites among
15 distinct laminae within the tectum, of which only three or
four receive retinal projections. The molecular and cellular basis
of such a discrete choice is poorly understood.
In 1994, we screened for monoclonal antibodies that recognize one of these retinal termination laminae. We found three clones, TB5, TB2 and TB4, which labeled laminae B, D and F, respectively. cDNA cloning and immunochemical analysis revealed that the TB4 antigen was ezrin, a cytoskeletal-membrane linker molecule belonging to the ezrin-radixin-moesin family. Ezrin was selectively expressed in a subset of retinal ganglion cells that project to lamina F. A similar subset-selective expression and resultant laminae-selective distribution of ezrin were observed in the lamina-specific central projections from the dorsal root ganglia. The staining pattern for TB4 in the dorsal root ganglia and spinal cord indicated that the expression of ezrin was restricted to cutaneous sensory neurons. Since ezrin modulates cell morphology and cell adhesion profiles by linking specific membrane proteins with the cytoskeleton, it was suggested that ezrin may be involved in the formation and/or maintenance of lamina-specific connections for neuronal subsets in the visual and somatosensory systems.
D. Genetic labeling of specific axonal pathways
We generated transgenic mouse lines in which
several sensory systems in the brain are specifically visualized
genetically. We employed GAP-lacZ as an axon-targeted reporter
protein that was constructed by fusing the membrane-anchoring
domain of the GAP-43 protein to lacZ. The reporter gene was introduced
into the genome under the control of a promoter element of Brn3b
transcription factor to establish transgenic mouse lines. The
individual lines thus generated afforded clear images of specific
axonal pathways of the visual, vomeronasal, pontocerebellar, and
auditory systems. The reporter protein labeled the entire axonal
process as well as the cell body of developing and mature neurons
on staining with X-gal. We showed that these lines facilitate
the developmental and anatomical study of these neural systems.
This strategy should be applicable to a variety of neural systems
by using various specific promoter elements.
We expect that our studies will lead to elucidation of the molecular mechanism underlying the formation of the retinotectal projection, and ultimately help to uncover the basic principles for establishing complicated but extremely precise neural networks in the nervous system.
II. Functional roles of protein tyrosine phosphatase ζ (Ptprz/PTPζ/PTPβ)
A. PTPζ in the brain
Protein tyrosine phosphorylation plays crucial
roles in various aspects of brain development and function. The
level of tyrosine phosphorylation is determined by the balance
between the activities of protein tyrosine kinases and protein
tyrosine phosphatases. Many types of receptor-like protein tyrosine
phosphatases (RPTP) have been cloned and characterized. On the
other hand, proteoglycans are a family of proteins bearing sulfated
glycosaminoglycans, which bind many extracellular matrix components
and growth factors through their core protein and glycosaminoglycan
portions. We have been interested in the functional roles of the
brain-specific proteoglycans in the development of the nervous
system because they are the major extracellular matrix components
in the tissue.
In 1994, we found that PTPζ/RPTPβ abundant in the nervous system, is expressed as a chondroitin sulfate proteoglycan in the brain. The extracellular region of PTPζ consists of a carbonic anhydrase-like domain, a fibronectin-type III-like domain, and a serine-glycine-rich region which is considered to be the chondroitin sulfate attachment region. There exist three splice variants of this molecule: the full-length transmembrane form (PTPζ-A); the short transmembrane form which has a deletion in the extracellular region compared with PTPζ-A (PTP-B); and the soluble form (PTP-S), which is also known as 6B4 proteoglycan/phosphacan. PTPζ is expressed from the early developmental stage to adulthood. This suggests that the gene plays variegated roles in the development and function of the brain.
The RPTP subfamily, R5 has another member, PTPγ which shows a high degree of structural similarity to PTPζ. To reveal its molecular diversity, we cloned rat PTPγ cDNAs and identified four splicing variants in 1997. We designated these forms PTPγ-A, -B, -C and -S. PTPγ-A was the longest species and had a similar structure to human and mouse PTPγ. PTPγ-B was devoid of the intracellular juxtamembrane 29 amino acids of PTPγ-A. PTPγ-C had a single phosphatase domain, and PTPγ-S was a secretory-type PTPγ. mRNAs of these four species were expressed in the brain, kidney, lung and heart. PTPγ is thus comparable to PTPζ with regard not only to structure but also to the presence of both secretory and transmembrane isoforms. However, no PTPγ variants are expressed as proteoglycans, in contrast to PTPζ variants.
We began by searching for ligand molecules of PTPζ using 6B4 proteoglycan-Sepharose. We found in 1996 that PTPζ binds pleiotrophin/HB-GAM and midkine, closely related heparin-binding growth factors which share many biological activities. The chondroitin sulfate portion of PTPζ is essential for the high affinity binding (Kd = ~0.25 nM) to these growth factors, and removal of chondroitin sulfate chains results in a marked decrease of binding affinity (Kd = ~13 nM). We further revealed that chondroitin sulfate interacts with Arg78 in Cluster I, one of the two heparin-binding sites in the C-terminal half domain of midkine. This is the first demonstration that chondroitin sulfate plays an important regulatory role in growth factor signaling. Next, we examined the roles of pleiotrophin/midkine-PTPζ interaction in neuronal migration using the glass fiber assay and Boyden chamber cell migration assay. Pleiotrophin and midkine on the substratum stimulated the migration of neurons in these assays. Experiments using various midkine mutants with various affinities for PTPζ indicated that the strength of binding and the neuronal migration-inducing activity are highly correlated. These results suggest that PTPζ is involved in migration as a neuronal receptor for pleiotrophin and midkine.
In order to reveal the intracellular signaling mechanism of PTPζ we performed yeast two-hybrid screening using the intracellular region of PTPζ as bait in 1998. We found that PTPζ interacts with PSD-95/SAP90 family members, SAP102, PSD-95/SAP90 and SAP97/hDlg, which are concentrated in the central synapses mediating protein-protein interactions to form large synaptic macromolecular complexes. Here, the C-terminus of PTPζ binds to PSD-95/SAP90 proteins through the second PDZ domain. This suggests that PTPζ is involved in the regulation of synaptic function. However, PSD-95/SAP90 family members are not likely to be the substrate for PTPζ because they are not tyrosine-phosphorylated.
To identify the substrate molecules of PTPζ, we then developed a yeast substrate-trapping system. This system is based on the yeast two-hybrid system with two essential modifications: conditional expression of v-src to tyrosine-phosphorylate the prey proteins and screening using a substrate-trap mutant of PTPζ as bait. Using this system, we isolated a number of candidate clones for substrate molecules or interacting molecules. Among them, we first identified GIT1/Cat-1 as a PTPζ substrate. GIT1/Cat-1 has ADP-ribosylation factor-GTPase-activating protein (ARF-GAP) activity. When B103 neuroblastoma cells were stimulated with pleiotrophin-coated beads, a transient increase in tyrosine phosphorylation of GIT1 was observed after 30 min. GIT1 is known to be tyrosine-phosphorylated by FAK and Src, although its physiological role is not clear. Overexpression of GIT1 in fibroblast and epithelial cells causes a loss of paxillin from the Rho-containing focal complex and stimulates cell motility. Cat-1 modulates Pak signaling, a serine threonine kinase which serves as a target for the small GTP-binding proteins, Cdc42 and Rac, and is implicated in a wide range of cellular events including cell adhesion and cell morphological change. Pleiotrophin, PTPζ and GIT1/Cat-1 may regulate the neuronal migration and neurite extension by controlling the Pak signaling pathway. We are continuing efforts to characterize the other candidate clones.
To further study the physiological function of PTPζ in vivo, in 1997 we generated PTPζ-deficient mice in which the PTPζ gene was replaced with the LacZ gene. First, we examined the cell types expressing PTPζ by investigating the expression of LacZ in heterozygous PTPζ-deficient mice. Throughout development from the early stage of embryogenesis, LacZ staining was essentially restricted to the nervous system. On embryonic day 12.5 (E12.5), LacZ staining was observed in the forebrain, midbrain, hindbrain and spinal cord. Examination of the cerebral cortex at higher magnification indicated that subsets of neurons including pyramidal neurons expressed LacZ. At the early postnatal stages, subsets of neurons and astrocytes in the brain including pyramidal cells in the hippocampus expressed LacZ. Both neurons and astrocytes were positive for LacZ in primary cultures of cells from the fetal cerebral cortex. From these results, we concluded that many neurons as well as astrocytes express PTPζ.
We are currently studying the phenotype of PTPζ-deficient mice biochemically, anatomically, physiologically and ethologically, and have already found abnormalities in behavior, learning and memory, etc.
B. PTPζ in the stomach
We found that protein tyrosine phosphatase receptor type Z (Ptprz, also called PTP-ζ or RPTP-β) is expressed in the stomach, where Ptprz-B is the major species. The level of expression of Ptprz-B is, however, lower by one order of magnitude than that in the brain. It is well known that Helicobacter pylori infects the stomach of more than 50% of the human population worldwide. The vacuolating cytotoxin VacA produced by H. pylori causes massive cellular vacuolation in vitro and gastric tissue damage in vivo, leading to gastric ulcers, when administered intragastrically. In 2001, we found that mice deficient in Ptprz do not show mucosal damage by VacA, although VacA is incorporated into the gastric epithelial cells to the same extent as in wild-type mice. Primary cultures of gastric epithelial cells from Ptprz+/+ and Ptprz-/- mice also showed similar incorporation of VacA, cellular vacuolation and reduction in cellular proliferation, but only Ptprz+/+ cells showed marked detachment from a reconstituted basement membrane from 24 h after treatment with VacA. VacA bound to Ptprz, and the levels of tyrosine phosphorylation of the G protein-coupled receptor kinase-interactor 1 (Git1), a Ptprz substrate, were higher after treatment with VacA, indicating that VacA behaves as a ligand for Ptprz. Furthermore, pleiotrophin (PTN), an endogenous ligand of Ptprz, also induced gastritis specifically in Ptprz+/+ mice when administered orally. Taken together, these data indicate that erroneous Ptprz signaling induces gastric ulcers.
III. Physiological roles of Nax sodium channel
channel, formerly called NaG/SCL11 (rat), Nav2.3 (mouse), and Nav2.1 (human), has been classified as a subfamily of voltage-gated
sodium channels, although the primary structure of Nax is significantly different from
other voltage-gated sodium channels, including in the regions
important for voltage sensing and inactivation. However, the functional
properties of the channel was enigmatic for some time, because
all attempts at the functional expression of Nax in heterologous systems failed. We generated Nax gene-knockout mice by inserting
the lacZ gene in-frame, and found that this channel is expressed
in neurons in the circumventricular organs (CVOs) like the subfornical
organ (SFO) and organum vasculosum laminae terminalis (OVLT) which
are known to be important regions for the control of body fluid
balance. The Nax-deficient
mice showed hyperactivity of neurons in these areas under thirst
conditions and ingested excessive salt as compared with wild-type
mice. This led us to hypothesize that Nax is involved in the sodium-level sensing mechanism in
We verified this possibility by imaging analysis of changes in the intracellular sodium-ion concentration [Na+]i when [Na+]o was raised from the normal level. When [Na+]o was increased from the control level of 145 mM to 170 mM (high sodium solution) by bath application of NaCl solution, [Na+]i of the dissociated SFO neurons derived from wild-type mice showed a marked increase from ~10 mM to ~ 30 mM: all of the [Na+]i response-positive cells were Nax-immunoreactive cells. The threshold value for the [Na+]o was 157 mM. In contrast, none of the SFO neurons derived from Nax-deficient mice showed such responses. The findings were confirmed by the whole-cell current clamp technique. The inward currents with an average amplitude of 8.4 pA were observed only from the wild-type neurons. The current amplitude was consistent with that estimated from the ion-imaging study (6.7 pA).
These results strongly suggest physiological roles for the Nax channel. It is well known that the sodium level and plasma osmolarity increase by 5-10% under thirst conditions. The sensitivity and threshold of Nax for [Na+]o is in the range of physiological change. SFO and OVLT are regions where the blood-brain barrier is deficient and directly accessible to monitor body fluid conditions. Thus, Nax is a newly identified type of sodium channel that is sensitive to an increase in the extracellular sodium concentration, and is likely to be the sodium-level sensor of body fluids in the brain.
Next, we examined the localization of Nax throughout the visceral organs at the cellular level. In visceral organs including lung, heart, intestine, bladder, kidney and tongue, a subset of Schwann cells within the peripheral nerve trunks wee highly positive for Nax. An electron microscopic study indicated that these Nax-positive cells were non-myelinating Schwann cells. In the lung, Nax-positive signals were also observed in the alveolar type II cells, which actively absorb sodium and water to aid gas exchange through the alveolar surface. It was thus suggested that the Nax sodium channel is involved in controlling the local extracellular sodium level through sodium absorption activity.