INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Protein tyrosine phosphatases (PTPs), in concert with protein tyrosine kinases (PTKs), regulate signal transduction pathways by tyrosine phosphorylation and dephosphorylation. PTPs comprise a structurally diverse family of enzymes. One group of PTPs exhibit structural features that are also common to cell surface receptors and cell adhesion molecules (CAMs), suggesting that these receptors may play a role in cell-cell communication (4, 43). These receptor-like PTPs (RPTPs) are composed of an extracellular domain, a single transmembrane domain, and a cytoplasmic portion that contains one or two tyrosine phosphatase domains. RPTP (also known as PTP) and RPTP are two members of a subfamily of RPTPs that contain a region in their extracellular domains that has sequence homology to the enzyme carbonic anhydrase (CAH) (2, 3, 24, 25). In both RPTP and RPTP, the CAH domain is followed by a fibronectin domain type III repeat and by a long unique sequence termed the spacer domain. Three different isoforms of RPTP are expressed as a result of alternative mRNA splicing: a short and a long form that differ by the presence of a stretch of 860 amino acid residues in the spacer domain and a secreted form composed of only the extracellular domain of RPTP, also known as 3F8 proteoglycan or phosphacan. Both transmembrane RPTPs and the phosphacan isoform are predominantly expressed as chondroitin sulfate proteoglycans.

Previous studies have suggested a role for RPTP in gliogenesis and neuron-glial cell interaction, neurite outgrowth, and neuronal migration, as well as in regeneration after injury (21, 26, 43).

RPTP is expressed predominantly by glial cells, astroglia, oligodendrocytes, and Schwann cells but also by neurons throughout the developing and adult nervous system (5, 41). Both transmembrane forms of RPTP are predominantly expressed in glial progenitors cells located in the ventricular and subventricular zone, where active cell proliferation occurs. Phosphacan is expressed at high levels by more mature glial cells, which suggests that the expression of RPTP is regulated during glial cell differentiation (6). Furthermore, RPTP expressed at the surface of glial cells binds to a cell recognition complex on neurons consisting of several proteins which include contactin, Caspr (also named paranodin) (34, 35), and Nr-CAM (40). On the basis of the localization of Caspr at the paranode, it was suggested that RPTP is involved in myelination and formation of the node (10).

RPTP has been shown to bind to a variety of CAMs and matrix components such as tenascin (18), Nr-CAM (40), L1, contactin (34), and pleiotrophin (28). Overlapping localization of phosphacan and most of the binding proteins is observed in the central nervous system (CNS), suggesting that these interactions could occur in vivo and may be involved in the control of cell proliferation, migration, adhesion, neurite outgrowth, and pathfinding in the brain. It was shown that chondroitin sulfate proteoglycans and CAM are often upregulated during brain damage or nerve injury (12, 30). Furthermore, it was demonstrated that RPTP is upregulated after sciatic nerve crushes, suggesting a role of RPTP in regeneration after injury (26).

The three isoforms of RPTP are expressed throughout the developing and adult nervous system. Interestingly, phosphacan binds to neurons and inhibits adhesion and neurite outgrowth (13, 17). In contrast, the extracellular portion of RPTP has been shown to induce neurite outgrowth. RPTP induces neurite outgrowth through its interaction with contactin and Nr-CAM (40). In addition, phosphacan can also stimulate neurite outgrowth of mesencephalic and hippocampal neurons (11). It was demonstrated that heterophilic interaction between RPTP and pleiotrophin mediates cell migration of cortical neurons, a process blocked by the PTP inhibitor sodium vanadate (28). Moreover, it has been shown that mesencephalic dopaminergic (DA) neurons express phosphacan. A heterophilic interaction between phosphacan on the neurons and L1 on the fibers may be involved in the control of migration of mesencephalic DA neurons (33), suggesting that RPTP may play a more general role in cell migration.

To explore the biological function of RPTP in vivo, we have generated mice deficient in the three isoforms of RPTP. These RPTP^/ mice are viable and fertile and showed no gross anatomical alterations. We have tested the importance of RPTP in myelination, neurite outgrowth, and node formation in the adult mouse. Our results demonstrate that RPTP is not required for neurite outgrowth and paranode formation, in contrast to what has been proposed based on in vitro experiments. However, ultrastructure of the CNS nerves suggests a fragility of the myelin but with no alteration of conduction velocity.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

RPTP targeting vector. A genomic clone containing one part of the CAH domain of the RPTP gene was isolated from a FIXII mouse genomic library (129SV/Ev strain; Stratagene) by hybridization with a rat cDNA fragment corresponding to the CAH domain. A targeting vector was designed to contain 4.1 kb of 5' homologous sequence, a pgk-neo cassette (42) replacing one exon in the opposite direction to RPTP gene transcription, 2.1 kb of 3' homologous sequence, and the herpes simplex virus (HSV) thymidine kinase gene (tk). Embryonic stem (ES) cells were grown on mitomycin C-treated primary embryonic fibroblasts that had been extracted from day 15 embryos at 37°C in Dulbecco's modified Eagle's medium supplemented with 15% heat-inactivated fetal bovine serum (HyClone), 0.1 mM 2 mercaptoethanol, 1 mM sodium pyruvate, and 10³ U of leukemia inhibitory factor (GIBCO) per ml. Cells (7 × 10⁶) were electroporated in 800 µl of phosphate-buffered saline (PBS) with 32 µg of NotI-linearized targeting vector DNA at 240 V and 500 mF using a gene pulser (Bio-Rad) and plated on gelatin-coated plastic dishes. After 48 h, the cells were transferred to growth medium supplemented with G418 (150 µg/ml; GIBCO) and ganciclovir (2 µg/ml; Syntex). ES cell clones were screened by PCR using the enzyme Expand (Boehringer) and oligonucleotides located in the neo gene and in RPTP gene. Positive clones were then confirmed by Southern blot analysis. To accomplish this, genomic DNA was digested with EcoRI, transferred onto nylon filters, and hybridized with a radioactively labeled PstI fragment from the original phage clone (Fig. 1). Three independent targeted ES clones were used in embryo aggregation experiments for generation of mice. Chimeric mice were crossed to Swiss Webster mice. Heterozygous and homozygous animals were identified by PCR and Southern blot analysis.

View larger version (18K): [in this window] [in a new window]   FIG. 1.   RPTP gene organization and structure of the disrupted RPTP gene. (A) Restriction map of the mouse RPTP gene. Translated exons are represented by closed boxes and numbered I to III. E, B, H, P, RV, S, and X represent cleavage sites for EcoRI, BamHI, HindIII, PstI, EcoRV, SacI, and XbaI (not all sites given), respectively. WT, wild type. (B) Restriction map of the RPTP-targeting construct p5'PGKneo3'TK, containing 4 and 2.1 kb of homologous sequences on the 5' and 3' sites of the neo insertion, respectively. pgk-neo and HSV tk cassettes are indicated by boxes. Arrows indicate transcriptional orientation of the genes. N represents cleavage site for NotI. (C) Structure of the RPTP gene after homologous recombination and localization of probes. Horizontal bars indicate the localization of 5' and 3' hybridization probes. Small arrows represent the position of the oligonucleotide used for PCR analysis.

RNA preparation and Northern blot analysis. Total RNAs from brains of RPTP^+/+, RPTP^+/, and RPTP^/ mice were isolated by the guanidine thiocyanate method (7). RNA were electrophoresed in a 1% agarose gel containing 7% formaldehyde and transferred to a nylon membrane (Schleicher & Schuell). Hybridization was performed with a radiolabeled 500-bp mouse cDNA probe coding for the CAH domain in 0.25 M sodium phosphate buffer (pH 7.4)-7% sodium dodecyl sulfate, followed by autoradiography.

Primary antibodies. Rabbit polyclonal antibody against CasprI was a gift from E. Peles and used at a dilution of 1:5,000; rabbit polyclonal antibody against ankyrin_G, a gift from S. Lux, was used at a dilution of 1:500. Mouse monoclonal antibody against the sodium channel (previously characterized by Rasband et al. [37]) was used at a dilution of 1:10,000. Rabbit anti-rat tyrosine hydroxylase (TH) polyclonal antibody (Pel-Freeze) was used at 1:1,000.

TH staining. Mice were anesthetized and transcardially perfused with ice-cold saline followed by perfusion with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer, pH 7.4 (PB). The brains were removed, cut into 3- to 4-mm blocks containing the midbrain, and postfixed in 4% PFA for 5 h. Subsequently, the brain blocks were placed in cold 30% sucrose in 0.1 M PB overnight, and 40-µm vibratome sections were cut. Using a random start, a 1:8 series of sections was collected for TH immunocytochemistry. Free-floating sections were incubated overnight at 4°C with a rabbit anti rat-TH polyclonal antibody (Pel-Freeze) (1:1,000 in PBS-3% goat serum-0.3% Triton X-100). After removal of the primary antibody and a wash with PBS, the sections were incubated for 2 h at room temperature with anti-rabbit immunoglobulin G conjugated with biotin (Amersham) (1:200 in PBS-3% goat serum-0.3% Triton X-100). The secondary antibody was removed, sections were washed, the reaction was quenched for 5 min in 0.3% H₂O₂ in PBS, and the samples were incubated with ExtraAvidin-peroxidase (1:200; Sigma) for 1 h before being incubated with 3,3'-diaminobenzidine.

Light microscopy. Mice were deeply anesthetized and perfused through the left ventricle with 3% PFA in 0.1 M PB. Brains were removed and postfixed in the same fixative overnight, washed in PB, and embedded in paraffin. Sections of 6 µm were cut and stained with cresyl violet. For Timm staining, mice were perfused through the left ventricle with sodium sulfide solution (19) followed by 4% PFA. Then 6-µm paraffin sections were mounted and stained in darkness at 24°C as described elsewhere (19). After staining for 15 or 45 min, the sections were counterstained with cresyl, dehydrated, and coverslipped.

Electron microscopy. Mice were anesthetized with pentobarbital and perfused transcardially with a fixative consisting of 3% glutaraldehyde and 2% PFA in 0.1 M cacodylate buffer (pH 7.3). Optic nerve and spinal cord segments were dissected out, rinsed, postfixed in 1 to 2% osmium tetroxide, with or without added 1.5% potassium ferricyanide, in 0.1 M cacodylate buffer, dehydrated in a graded series of alcohols, and embedded in Araldite or an Epon-Araldite mixture. Sections of 1 µm were cut with glass knives and stained with 1% toluidine blue for survey by light microscopy. Selected areas were then sectioned at ~0.1 µm, mounted on copper grids, stained with potassium permanganate followed by ethanolic uranyl acetate, and examined in a Philips 300 electron microscope. Both cross and longitudinal sections were studied. Animals studied ranged in age from 2 to 9.5 months.

Suction electrode recording. Mouse optic nerves were dissected and placed in a recording chamber that was continuously perfused and temperature regulated. The standard Locke's solution contained NaCl (154 mM), KCl (5.6 mM), CaCl₂ (2 mM), D-glucose (5.6 mM), and HEPES (10 mM, pH 7.4). For stimulation and recording of compound action potentials (CAPs), each end of the nerve was drawn into a suction electrode. Stimuli consisted of 50-µs pulses that were adjusted to 10% above the level required for a maximal response. After a stimulus, CAPs were amplified, digitized, recorded, and analyzed with a laboratory computer. Conduction velocity was calculated as the length of the nerve divided by the time to the first peak amplitude of the CAP. For some experiments, CAPs were measured, and then nerves were fixed and used for labeling experiments.

Immunocytochemistry. Optic nerves were dissected, fixed in 4% PFA (pH 7.2) for 30 min, and soaked overnight in 20% sucrose at 4°C. The nerves were then frozen in OCT mounting medium (Miller), cut into 10-µm sections on a microtome, and dried on gelatin-coated coverslips. The sections were incubated in PBTGS (45 ml of 0.1 M PB, 150 µl of Triton X-100, 5 ml of goat serum) for 1 to 2 h to permeabilize and block. All subsequent solutions used PBTGS for dilutions or washing, and all incubations were done at room temperature. Three washes of 5 min were done after each antibody incubation. Rabbit polyclonal antibodies were applied first for 15 h. Anti-rabbit Alexa 488 (1:500; Molecular Probes, Eugene, Oreg.) was then added for 1 h. For double labeling, mouse monoclonal antibodies were added for 15 h, and anti-mouse Cy3 (1:500; Accurate Chemicals, Westbury, N.Y.) was applied for 1 h. Sections were then washed serially in PBTGS, 0.1 M PB, and 0.05 M PB and allowed to air dry. The labeled sections were mounted on slides and viewed under a Nikon Microphot-SA fluorescence microscope. Images were taken by a C4742-95 cooled charge-coupled device camera (Hamamatsu) controlled by Image Pro software (Media Cybernetics).

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Generation of RPTP-deficient mice. Using a rat cDNA fragment corresponding to the CAH domain of the RPTP gene, a clone including a 3' part of this domain was isolated from a mouse 129Sv/Ev genomic library. The targeting vector for the RPTP gene comprised 4.1 kb of 5' homologous sequence, a pgk-neo cassette in opposite direction to RPTP gene transcription (42) replacing one exon, 2.1 kb of 3' homologous sequence, and HSV tk for selection against random integration (29). Homologous recombination with this targeting vector results in a loss of exon 2 and in inadequate splicing, resulting in a null mutation.

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View larger version (35K): [in this window] [in a new window]   FIG. 2.   Southern, PCR, Northern, and immunoblot analyses of wild-type and RPTP-deficient mice. (A) Southern blot analysis. DNA from wild-type, RPTP/+, and RPTP/ mice digested with EcoRI was hybridized with a 3' probe (PstI probe). The 10-kb band represents the wild-type allele, and the 4.5-kb band represents the mutant allele. (B) PCR analyses. For easy screening, we used a PCR wherein the wild-type (WT) allele is amplified as a 500-bp DNA and the mutant allele is amplified as a 300-bp product. (C) Northern blot analysis. RNA from brains of RPTP+/+, RPTP/, and RPTP/+ mice was hybridized with a 500-bp cDNA fragment encoding the mouse CAH domain. Arrows point to the three transcripts of RPTP in both wild-type and heterozygous RNAs that are absent in RPTP/ mice. Sizes are indicated in kilobases.

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Morphological analysis of the CNS of RPTP-deficient mice. At the light microscopic level, the general morphology of brains of 2-month-old RPTP^/ mice appeared normal and indistinguishable from that of wild-type littermates. Cross sections through the hippocampi of RPTP-deficient mice displayed a normal pattern of migration of pyramidal cells in the dentate gyrus (Fig. 3A and B). In addition, we found no abnormalities in the subventricular zones of RPTP^/ mice (data not shown). In the cerebella of 2-month-old RPTP-deficient mice, the molecular layer, Purkinje cell layer, and internal granular cell layer appeared normal (Fig. 3C and D). Because RPTP is also expressed in cortical neurons (41), we also compared the lamination and organization of the cortex. We chose to look at the somatosensory cortex, where the layers are most distinguishable. The number of cells and organization in the six layers were apparently similar in wild-type and RPTP-deficient mice (Fig. 3E and F), indicating that RPTP is not necessary for cortical neuron migration. To further investigate the cortex, we examined the distribution of specific neuronal markers. Phosphacan is expressed by interneurons in the cortex (20). The calcium-binding protein parvalbumin and calbindin are expressed in distinct subpopulations of neurons. We investigated the density of inhibitory interneurons by immunocytochemistry using antibodies to the Ca²⁺-binding protein parvalbumin as a marker for a subpopulation of GABAergic (gamma amino butyric acid) neurons. The density of parvalbumin-immunoreactive cells did not differ between wild-type and mutant animals in this region (data not shown). Immunostaining with anticalbindin (data not shown) revealed no difference in number, localization, or expression pattern between RPTP^/ and control cortex samples.

View larger version (152K): [in this window] [in a new window]   FIG. 3.   Analysis of the hippocampi, cortices, and cerebella of RPTP-deficient mice by light microscopy. Sections of 6 µm through hippocampi (A and B), cerebella (C and D), and cortices (E and F) of adult wild-type (A, C, and E) and RPTP-deficient (B, D, and F) mice were stained with Nissl stain. The overall histology, number, and localization of each cell type in these regions of the brain appear normal in RPTP-deficient mice. ml, molecular layer; pl, Purkinge cell layer; gl, internal granular layer; DG, dentate gyrus.

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Normal migration of mesencephalic neurons in RPTP^/ mice. Mesencephalic DA neurons, generated in the ventricular zone of the mesencephalon, migrate first ventrally from the ventricular surface along radial glial processes and then laterally along tangentially arranged nerve fibers to their destinations, the substantia nigra pars compacta, the reticular formation, and the ventral tegmental area. The expression of phosphacan by DA neurons and its interaction with L1 and Ng-CAM have implicated a role for it in the lateral migration of DA neurons (33). This study showed that the laterally migrating substantia nigra DA neurons express phosphacan. Since the ventral tegmental DA neurons seem to migrate only radially, they may not require phosphacan for proper migration. Thus, in the absence of phosphacan, we may observe all DA neurons clustered in the ventral tegmental area, with an absence of DA neurons in the substantia nigra. Migration of DA neurons was histologically examined in RPTP-deficient mice and control animals by a series of sections through the midbrain stained with an antibody directed against TH, a DA-synthesizing enzyme. As seen in Fig. 4, DA neurons migrate properly in RPTP-deficient mice.

View larger version (65K): [in this window] [in a new window]   FIG. 4.   Localization of mesencephalic DA neurons. Coronal sections of RPTP/ and RPTP/+ adult animals were stained with anti-TH antibodies, revealing mesencephalic DA neurons. Wild-type and RPTP/ DA neurons migrated properly laterally and did not localize ventrally. SN, substantia nigra.

Electron microscopy of optic nerves of RPTP-deficient mice. RPTP is expressed by cells of the oligodendrocyte lineage during development and in the adult (6). It has been suggested that RPTP is involved in formation of the node of Ranvier (10). We evaluated myelination in 2-month-old brains stained with luxol fast blue, a stain specific for myelin, and detected no difference in staining between wild-type and RPTP^/ mice (data not shown).

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View larger version (200K): [in this window] [in a new window]   FIG. 5.   Ultrastructure of the optic nerve. (A) Survey view of wild-type optic nerve cross section (magnification, ×12,600). The myelin sheaths surrounding the larger axons appears more compact than those in panel B. (B) Survey view of RPTP/ optic nerve cross section (×12,600). In the larger fibers, myelin lamellae tend to separate. Extraneous whorls (arrow) of myelin are apposed to an oligodendrocyte. (C) Large fiber from RPTP/ optic nerve (×31,500). The sheath appears somewhat raveled (black arrows) and in one region the lamellae contain cytoplasm (white arrow). Just above, two (large 1 and 2) fibers are surrounded by sheaths that form redundant folds (small numbers). (D) Small fiber from RPTP/ optic nerve (×182,000). Both inner and outer mesaxons form tight junctions. Radial component (arrows) is visible in one quadrant of the sheath.

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View larger version (151K): [in this window] [in a new window]   FIG. 6.   Analysis of the node and paranode by electron microscopy. (A) Paranodal junction from a wild-type spinal cord (magnification, ×100,000). Some terminal loops adjoin the axolemma and form junctions containing transverse bands (arrowheads); others do not reach the axolemma. The node of Ranvier (n) is at the right. The axon (ax), paranode (pn), and myelin sheath (m) are indicated. (B) Small fiber from RPTP/ optic nerve (×79,000). The node of Ranvier (right) shows undercoated plasma membrane (arrow). Myelin lamellae form a regular succession of overlapping terminal loops. (C) Detail of paranodal junction from a large fiber in RPTP/ spinal cord (×152,000). Some terminal loops end against the axolemma, forming junctions containing periodic transverse bands. Not all terminal loops reach the axolemma, however. (D) Paranodal (pn) and juxtaparanodal (in) regions of axon in a large fiber from RPTP/ spinal cord (×87,500). Cytoplasm-containing lamellae extend beyond the paranode toward the internode (arrows).

Na²⁺ channel clustering, Caspr localization, and conduction velocity of the optic nerve. Recent studies suggested that RPTP might mediate interactions between axons and glial cells (35) through interaction with the contactin-Caspr complex. Caspr is a membrane protein highly expressed in the CNS that copurified with contactin when the CAH of RPTP was used as an affinity probe. It was shown that Caspr is an essential component of the paranode (10). Thus, RPTP could play a role in formation of the paranode and node of Ranvier. We therefore analyzed optic nerve sections, labeled for voltage-gated sodium channels to mark nodes of Ranvier and labeled for CasprI to mark paranodes (Fig. 7A). Both the nodes of Ranvier and paranodes in RPTP^/ mice exhibited normal morphology and showed similar fluorescence staining with antibody markers compared with control animals (Fig. 7A). Optic nerve sections were also labeled for ankyrin_G, a protein that links sodium channels to the cytoskeleton and is also located in the nodes of Ranvier (23). No differences in morphology or fluorescence intensity were seen with this label either. No difference was apparent in the number of labeled sites between RPTP^/ and control animals with any of the antibodies used (Fig. 7A).

View larger version (44K): [in this window] [in a new window]   FIG. 7.   Localization of Na2+ channels and Caspr in the optic nerve and conduction velocity measurements. Normal nodes and paranodes are found in optic nerves of RPTP/ mice. (A) Optic nerve sections were labeled with antibodies specific for sodium channels (red), Caspr (green), and ankyrinG. No apparent differences in labeling were seen between nerves from wild-type and RPTP/ mice (scale bars = 10 µm). (B) Conduction velocity measurements of nerves from wild-type and RPTP/ mice at 25°C (P = 0.85) or 37°C (P = 0.38). Results represent mean ± standard deviation of four nerves.

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	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

The mutation introduced in the RPTP gene abolishes expression of the three isoforms of RPTP, the two transmembrane isoforms and the soluble isoform (phosphacan), since we have abolished transcription of the RPTP gene. Nevertheless, the RPTP-deficient mice described in this study are normal in their gross general behavior and with respect to fertility, body weight, and life span.

The gross anatomy of the brain and spinal cord and the morphology of the cerebellum of RPTP-deficient mice do not show any alteration at the light microscopy level compared to their littermate controls. We could not detect aberrant localization of cells that normally express RPTP in the cerebellum, hippocampus, or cerebral cortex. Therefore, RPTP appears not to be necessary for the migration of these neural cell types to their correct locations in these areas of the CNS.

RPTP and RPTP belong to the same subfamily of RPTPs. Surprised by the lack of obvious phenotype in RPTP-deficient mice, we tested whether the expression of RPTP is altered in RPTP-deficient mice to compensate for RPTP deficiency. The expression of RPTP is restricted to the nervous system, while RPTP is ubiquitously expressed. However, certain neurons, especially cortical and hippocampal neurons, express both RPTP and RPTP. Northern blot analysis of mRNA prepared from adult RPTP^/, RPTP^/+, or RPTP^+/+ brain mRNA showed no alteration of transcription of the RPTP gene, indicating that RPTP is not altered in RPTP^/ mice (data not shown). However, we cannot rule out the possibility that RPTP function in RPTP^/ mice is compensated for by another tyrosine phosphatase.

Evidence from in vitro studies suggests that RPTP family members play a key role in neuronal migration, neurite outgrowth, and cell adhesion. The secreted form of RPTP, phosphacan, is a chondroitin sulfate, highly expressed in the brain. Phosphacan inhibits nerve growth factor-induced neurite outgrowth of PC12D in culture (22) and neurite outgrowth of dorsal root ganglia explants (14). In contrast, phosphacan promotes neurite outgrowth from mesencephalic and hippocampal neurons (8). We tested whether dorsal root ganglia prepared from day 15 embryo RPTP^/ or control samples and cultured for 2 weeks showed any changes in neurite length, but we did not detect any obvious difference (data not shown). We have also shown that the cells of the dentate gyrus, where RPTP is highly expressed, are able to produce normal MFs, as revealed by Timm and calbindin staining. Finally, we found no obvious difference in neurite length in mesencephalic neurons demonstrating, that in vivo, RPTP is not necessary for neurite growth.

RPTP isoforms are found in the developing nervous system, in patterns suggesting the involvement of these enzymes in neuronal migration and axonal guidance. RPTP was implicated in the differentiation of cortical neurons (27), the migration of olfactory neurons (32), and the migration of mesencephalic DA neurons (33). In the cortex, RPTP is expressed in layers II, III, and IV (41), and phosphacan has been found also in layers II, IV, and VI (20). Nissl stain- as well as calbindin- or parvalbumin-immunoreactive cells exhibited no difference with respect to number and localization in RPTP-deficient mice compared to control mice. We have also demonstrated that mesencephalic DA neurons migrate to their final destination in RPTP-deficient mice, suggesting that RPTP does not play a major role in neuronal migration. Of course, RPTP could regulate the migration of a particular subset of neurons that were not detected. However, phosphacan is expressed in most parvalbumin-positive cells (20), neurons that were well localized in RPTP-deficient mice.

RPTP and myelination. Previous studies suggest that RPTP could be involved in the formation of the paranode. RPTP, contactin, and Caspr/paranodin form a complex (35) and localize to the paranodal axolemma in myelinated fibers of the peripheral nervous system and CNS (10, 31). It has been suggested that RPTP is expressed in oligodendrocyte paranodal loops and can interact with the Caspr-contactin complex at the surface of the axon. RPTP may therefore participate in formation of the paranode. However, we did not detect ultrastructural abnormalities in the paranodes of RPTP^/ animals.

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