Receptor protein tyrosine phosphatase sigma inhibits axonal regeneration and the rate of axon extension

Molecular and Cellular Neuroscience 23 (2003) 681– 692

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Receptor protein tyrosine phosphatase sigma inhibits axonal regeneration and the rate of axon extension K.M. Thompson,a N. Uetani,b C. Manitt,a M. Elchebly,c M.L. Tremblay,b and T.E. Kennedya,* a

Centre for Neuronal Survival, Montreal Neurological Institute, McGill University, 3801 University Street, Montreal, Quebec, Canada H3A 2B4 b McGill Cancer Centre and Department of Biochemistry, McGill University, 3655 Drummond St., Montreal, Quebec, Canada H3G 1Y6 c Hoˆpital Sainte-Justine, Centre de Recherche-Endocrinologie, Montreal, Quebec, Canada H3T 1C5 Received 23 May 2002; revised 4 March 2003; accepted 20 March 2003

Abstract Transgenic mice lacking receptor protein tyrosine phophatase-␴ (RPTP␴), a type IIa receptor protein tyrosine phosphatase, exhibit severe neural developmental deficits. Continued expression of RPTP␴ in the adult suggests that it plays a functional role in the mature nervous system. To determine if RPTP␴ might influence axonal regeneration, the time course of regeneration following facial nerve crush in wild-type and RPTP␴ (⫺/⫺) mice was compared. Mice lacking RPTP␴ exhibited an accelerated rate of functional recovery. Immunocytochemical examination of wild-type neurons in cell culture showed RPTP␴ protein in the growth cone. To determine if RPTP␴ affects the ability of a neuron to extend an axon, the rate of axon growth in neuronal cultures derived from wild-type and RPTP␴ (⫺/⫺) embryonic mice was compared. RPTP␴ did not affect the rate of axon initiation, but the rate of axon extension is enhanced in neurons obtained from RPTP␴ (⫺/⫺) mice. These findings indicate that RPTP␴ slows axon growth via a mechanism intrinsic to the neuron and identify a role for RPTP␴ regulating axonal regeneration by motoneurons. © 2003 Elsevier Science (USA). All rights reserved.

Introduction The neuronal growth cone is a highly motile structure that explores its environment using a host of cell surface receptors. The ability of the growth cone to respond to multiple guidance cues and direct axon extension is essential for the establishment of appropriate synaptic connections in the developing nervous system. Following axotomy, similar mechanisms regulate axonal growth cones as they attempt to regenerate and reestablish connections with their targets. Tyrosine phosphorylation plays an essential role in growth cone function (reviewed by Desai et al., 1997b). Cellular phosphotyrosine levels are regulated by the opposing activities of two gene families, the protein tyrosine kinases and the protein tyrosine phosphatases (reviewed by Hunter, 1989; Stoker, 2001). The receptor protein tyrosine phosphatases (RPTPs) are * Corresponding author. Fax: ⫹1-514-398-1319. E-mail address: [email protected] (T.E. Kennedy).

a large family of proteins implicated in axon growth and guidance (reviewed by Bixby, 2000; Stoker, 2001). Genetic analyses in Drosophila melanogaster provided the first evidence that type IIa RPTPs influence axon outgrowth and guidance during development. Mutation of Drosophila type IIa RPTPs Dlar and DPTP69D caused errors in axon extension by motoneurons (Desai et al., 1996, 1997a; Krueger et al., 1996), photoreceptors (Garrity et al., 1999; Newsome et al., 2000), and commissural neurons (Sun et al., 2000a). RPTP␴ is a type IIa RPTP that is highly expressed by neurons in the developing and adult mammalian nervous system (Yan et al., 1993; Wang et al., 1995; Schaapveld et al., 1998). Two additional type IIa RPTP family members have been identified in mammals: LAR and RPTP␦ (reviewed by Stoker, 2001). Transgenic mice lacking RPTP␴ exhibit growth retardation; a high mortality rate; delayed peripheral nerve development; and altered development of the olfactory bulb, pituitary gland, and hypothalamus (Elchebly et al., 1999; Wallace et al., 1999). The CNS of mice expressing a mutant RPTP␦ that lacks the catalytic

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domain appears histologically normal but these mice exhibit impaired learning and enhanced long-term potentiation (Uetani et al., 2000). The role of RPTPs in axon guidance during development has led to interest in the role these proteins may play regulating axon growth following injury (Haworth et al., 1998; Xie et al., 2001; McLean et al., 2002). Although LAR (⫺/⫺) mice exhibit a relatively mild neural phenotype (Yeo et al., 1997; Van Lieshout et al., 2001), the absence of LAR delays axonal regeneration in vivo following injury to the sciatic nerve, a mixed sensory and motor nerve (Xie et al., 2001). In addition, McLean et al. (2002) have reported that axonal regeneration in the sciatic nerve is enhanced in the absence of RPTP␴. Furthermore, they provide evidence that appropriate axon navigation during regeneration requires RPTP␴ expression by Schwann cells in the nerve. However, this study did not determine if RPTP␴ expressed by the neuron influences axon growth. Here, we report that following facial motoneuron axotomy in vivo, RPTP␴ (⫺/⫺) mice recover significantly faster than wild-type mice. To investigate the possibility that RPTP␴ might directly affect the ability of a neuron to extend an axon, we first raised an antibody and examined the subcellular distribution of RPTP␴. We show that RPTP␴ protein is present in neuronal growth cones. Furthermore, we report enhanced axonal extension in lowdensity cultures of embryonic cortical neurons derived from RPTP␴ (⫺/⫺) mice compared to wild-type littermates. These results indicate that RPTP␴ influences the neuronal response to injury and slows the rate of axon outgrowth.

Results Accelerated regeneration in RPTP␴ (⫺/⫺) mice The cell bodies of facial motoneurons are located in the brainstem and project an axon out of the CNS through the ipsilateral peripheral facial nerve to the musculature of the face. Facial nerve crush was used to investigate a possible role for RPTP␴ regulating axonal regeneration by motoneurons in vivo. Axotomy of facial motoneurons causes ipsilateral paralysis of the vibrissae in mice. As the injured axons regenerate and reinnervate the facial musculature, whisker movement returns gradually. To examine the functional role of RPTP␴ during nerve regeneration, we compared the time course of functional recovery in RPTP␴ (⫺/⫺) mice and wild-type age-matched control animals. Functional recovery was assessed by monitoring the recovery of whisker movement using a well-established behavioral score: 0 corresponds to no whisker movement, 1 to slight whisker movement, 2 to strong but asymmetrical whisker movement, and 3 to strong symmetrical whisker movement, indicating complete recovery of function. Functional recovery occurred approximately 1 day earlier in RPTP␴ (⫺/⫺) mice (Fig. 1). In these mice, limited whisker movement was first detected ⬃8 days after injury

Fig. 1. Accelerated functional recovery in RPTP␴ (⫺/⫺) mice following facial nerve crush. Recovery of function occurs approximately 1 day earlier in RPTP␴ (⫺/⫺) mice as compared to age-matched controls. (Vertical axis) Behavioral score: 0 ⫽ lack of whisker movement, 1 ⫽ slight whisker movement, 2 ⫽ strong asymmetrical whisker movement, 3 ⫽ strong, symmetrical movement. For each time point, one score was given per animal. The mean of the scores ⫾ SEM is plotted. Horizontal axis: days following crush injury. (䡩) RPTP␴ (⫺/⫺); n ⫽ 8; (●) RPTP␴ (⫹/⫹) wild type mice; n ⫽ 7.

and full functional recovery was achieved in all animals by 10 days following injury. In wild-type animals, whisker movement was first observed on the 9th day following injury and complete recovery of function was achieved in all animals 11 days postinjury. The time required to achieve each level of behavioral recovery was significantly earlier in RPTP␴ (⫺/⫺) mice than in wild-type mice (score of 1, P ⬍ 0.001; score of 2, P ⬍ 0.01; score of 3, P ⬍ 0.001, t test). The length of the facial nerve, as measured from the point at which it exits the stylomastoid foramen to the end of the snout, was not significantly different between these two groups (not shown). The time course of functional recovery observed in wild-type animals was consistent with previous reports (Chen and Bisby, 1993; Ferri et al., 1998). Our results demonstrate that the loss of RPTP␴ results in enhanced recovery of function following facial nerve crush. Monoclonal antibody 17G7.2 binds RPTP␴ RPTP␴ is a type I transmembrane protein that is proteolytically processed and presented at the cell surface as noncovalently linked extracellular and intracellular subunits (Aicher et al., 1997). The distribution of RPTP␴ protein was examined using a monoclonal antibody (17G7.2) raised against the intracellular subunit of RPTP␴. The type IIa RPTP family consists of three closely related members in mammals, RPTP␴, RPTP␦, and LAR. The following analyses were carried out to characterize the binding specificity of monoclonal antibody 17G7.2. Western blot analysis of homogenates of newborn (data not shown) and adult mouse brain identified an ⬃80-kDa band consistent with the molecular weight of the intracellular P-subunit of RPTP␴ (Fig. 2A, lane 1; Aicher et al., 1997). Consistent with 17G7.2 binding RPTP␴, the ⬃80-kDa immunoreactive band was absent from brain homogenates derived from RPTP␴ (⫺/⫺) mice (Fig. 2A, lane 2) but detected in brain homogenates of

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Fig. 2. Monoclonal antibody 17G7.2 detects RPTP␴. (A) Western blot analyses using monoclonal antibody 17G7.2 raised against the intracellular domain of RPTP␴ (left panel) reveals an ⬃80-kDa immunoreactive band in wild-type adult mouse brain homogenate (⫹/⫹), consistent with the reported molecular weight of the intracellular subunit of RPTP␴. This band was not detected in RPTP␴ (⫺/⫺) adult whole-brain homogenate (lane 2), but was detected in whole-brain homogenate of newborn RPTP␦ (⫺/⫺) mice (lane 3). The right panel shows a Western blot of the same samples incubated with donkey antimouse secondary antibody alone (1:7500). The lower molecular weight bands are due to the secondary antibody, likely binding mouse immunoglobulins derived from blood in whole-brain homogenates (6% PAGE). (B) Western blot analysis of GST fusion proteins, GST-RPTP␴ ICD and GST-RPTP␦ ICD. The upper panel shows 17G7.2 immunoreactivity against GST-RPTP␴ ICD but not GST-RPTP␦ ICD. The lower panel shows the same blot incubated with anti-GST (7.5% PAGE). (C) The absence of an ⬃84-kDa 17G7.2 immunoreactive band (arrowhead) in homogenates of liver from wild-type or RPTP␴ (⫺/⫺) adult mice, a tissue expressing high levels of LAR, indicates that 17G7.2 does not recognize the LAR ICD (7.5% PAGE). The ⬃75-kDa immunoreactive species corresponds to the band detected by secondary antibody alone illustrated in A. The lower panel shows the same blot stained with India Ink to illustrate the protein content in each lane. Protein size standards correspond to 200, 116, 97.4, and 66.2 kDa (Bio-Rad). (C) 116, 97.4, and 66.2 kDa.

RPTP␦ (⫺/⫺) mice (Fig. 2A, lane 3). The lower molecular weight bands detected in brain homogenates in Fig. 2A are due to the donkey antimouse HRP secondary antibody alone (Fig. 2A, right panel) and are unrelated to 17G7.2 and RPTP␴. A likely possibility is that these bands correspond to endogenous mouse IgGs present in blood in the wholebrain homogenates. The intracellular domains (ICDs) of RPTP␴ and RPTP␦ share ⬃83% amino acid identity. The binding specificity of 17G7.2 was characterized further using Western blot analysis of recombinant GST fusion proteins encoding the carboxyterminal 642 amino acids of RPTP␴ (GST-RPTP␴ ICD) or the carboxyterminal 750 amino acids of RPTP␦ (GST-RPTP␦ ICD). Although the two ICDs are very similar, monoclonal antibody 17G7.2 binds GST-RPTP␴ ICD (Fig. 2B, lane 1) but not GSTRPTP␦ ICD (Fig. 2B, lane 2). Positive immunoreactivity using an antibody against GST demonstrated the presence of both GST-fusion proteins (Fig. 2B). To determine if 17G7.2 might detect LAR, protein homogenates were prepared from adult mouse liver, a tissue reported to express LAR but not RPTP␴ (Longo et al., 1993; Yan et al., 1993). The intracellular subunit of LAR has a molecular weight of ⬃84 kDa (Aicher et al., 1997). The absence of an immunoreactive band at this molecular weight indicates that the antibody does not recognize the LAR ICD (Fig. 2C). The faint immunoreactivity at ⬃75 kDa corresponds to the band detected by the secondary antibody alone illustrated in Fig. 2A, and the lightly immunoreactive bands, ⬃100 kDa and

⬃170 kDa, detected in liver homogenate are too small to correspond to full-length LAR, ⬃205 kDa (Aicher et al., 1997). Importantly, these bands were not detected in wholebrain homogenate (Fig. 2A), a tissue that also expresses LAR (Longo et al., 1993). These findings support the conclusion that 17G7.2 binds RPTP␴ and not RPTP␦ or LAR. Facial motoneurons express RPTP␴ following axotomy Expression of RPTP␴ mRNA was characterized in the facial nucleus before and after axotomy of the facial motoneurons. In situ hybridization analysis detected RPTP␴ mRNA in a population of large cells, characteristic of motoneurons, in the facial nucleus. Figure 3B illustrates the expression of RPTP␴ mRNA in the facial nucleus 3 days after facial nerve crush, whereas Fig. 3A shows expression in the contralateral nucleus that projects axons through the uncrushed nerve. RPTP␴ mRNA expression was detected simultaneously in the two nuclei using the same tissue section. In situ hybridization did not detect RPTP␴ mRNA in CNS cells morphologically characteristic of glia in the facial nucleus either before or after injury (Fig. 3A). Three days (Fig. 3B) and 5 days (not shown) after nerve crush, similar levels of RPTP␴ mRNA continued to be detected in facial motoneurons using in situ hybridization. We then assayed the level of RPTP␴ protein using micropunch dissection of the facial nuclei. Five-hundred-micron-thick coronal sections of fresh-frozen adult mouse brainstem were

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Fig. 3. Expression of RPTP␴ by facial motoneurons before and after axotomy. (A and B) RPTP␴ mRNA expression detected by in situ hybridization in the two facial motor nuclei of a single brainstem section. (A) RPTP␴ expression in the nucleus projecting to the uninjured facial nerve; (B) expression in the nucleus projecting to the nerve that had been crushed 3 days prior. Photomicrographs were taken using the same light intensity and exposure time (phase contrast, 10X objective). RPTP␴ mRNA is expressed in facial nuclei before and after injury. A similar pattern of mRNA expression was observed following in situ hybridization 5 days after crush (not shown). (C) Western blot analysis for RPTP␴ in tissue homogenates prepared from facial nuclei, uninjured and isolated 5 days after facial nerve crush (7.5% PAGE). Increased GFAP immunoreactivity is shown on the same blot as a positive control for injury. India ink staining of total protein indicates that similar amounts of total protein were loaded in each lane. Western blot analysis 3 days after crush also revealed similar levels of RPTP␴ protein (not shown). Protein size standards correspond to 116, 97.4, 66.2, 45, and 31 kDa (Bio-Rad).

cut on a cryostat and facial nuclei isolated using a 0.50-mm tissue micropunch. Western blot analysis of protein homogenates of the isolated facial nuclei revealed similar amounts of RPTP␴ protein in nuclei projecting to normal or crushed nerves (5 days postcrush, Fig. 3C, and 3 days postcrush not shown). Consistent with a previous report, examination of GFAP immunoreactivity revealed a large increase in the facial nucleus following facial motoneuron axotomy (Coers et al., 2002). Distribution of RPTP␴ protein in neuronal growth cones The neuronal growth cone contains proteins important for axon growth and guidance that include cell adhesion molecules, receptors for guidance cues, and intracellular signaling molecules. LAR (Zhang et al., 1998), HmLAR2 (Gershon et al., 1998), and a homolog of RPTP␴ in chicken, cPTP␴ (Rashid-Doubell et al., 2002), also known as CRYP␣ (Stoker et al., 1995), are present in neuronal growth cones during embryogenesis. We examined the distribution of RPTP␴ protein in embryonic day 15 (E15) mouse cortical neurons, cells that express high levels of RPTP␴ (Wang et al., 1995), following 2 days in culture. Cultures were stained with the 17G7.2 antibody (Fig. 4C) and for F-actin using rhodamine-conjugated phalloidin (Fig. 4B). RPTP␴ immunoreactivity was detected along neurites and in growth cones (Fig. 4A and B). A smooth distribution of RPTP␴ immunoreactivity was detected throughout the growth cone. In addition, bright punctate staining was present. Because monoclonal antibody 17G7.2 binds an epitope in the intracellular domain of RPTP␴, cells were permeabilized prior to incubation with the antibody. Thus, the immunoreactivity

does not distinguish between intracellular and cell surface pools of RPTP␴ protein. The elaboration of a neuronal growth cone requires the organized polymerization of actin, and axon guidance cues regulate actin-based membrane extension (reviewed by Meyer and Feldman, 2002). We therefore examined the spatial relationship between the distribution of F-actin and RPTP␴ in neuronal growth cones (Fig. 4A and C). Interestingly, close inspection of RPTP␴ and F-actin revealed intermixed, but often nonoverlapping distributions within the growth cone (Fig. 4D). RPTP␴ (⫺/⫺) cortical neurons exhibit enhanced axon outgrowth To test the hypothesis that RPTP␴ regulates axon growth, we measured axon length in low-density neuronal cultures derived from the cortex of E15 RPTP␴ (⫺/⫺) or wild-type littermates (Fig. 5). After 36 h in vitro, neurons cultured from RPTP␴ (⫺/⫺) mice extended significantly longer axons than neurons cultured from wild-type littermates. When cultured on a substrate of poly-D-lysine (PL), we observed a ⬃29% increase in axon length in RPTP␴ (⫺/⫺) neurons as compared to wild type (Fig. 5C, Table 1, P ⬍0.001, Mann–Whitney Rank Sum Test). When cultured on a substrate of poly-D-lysine plus laminin-1 (PLL), the mean axon length of RPTP␴ (⫺/⫺) neurons was ⬃16% longer than axons of wild type (Fig. 5C, Table 1, P ⬍0.001; Mann–Whitney Rank Sum Test). Axon extension by wildtype neurons on PLL was ⬃70% greater than that on PL (Table 1; P ⬍ 0.001, Mann–Whitney Rank Sum Test). RPTP␴ might produce this effect on axon length by increasing the rates of either axon extension or axon initi-

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Fig. 4. Distribution of RPTP␴ and F-actin in growth cones of embryonic cortical neurons. (A) The growth cone of a neuron isolated from E15 CD-1 mouse cortex and grown in dissociated cell culture for 48 h on a poly-D-lysine substrate. RPTP␴ immunoreactivity was detected using 17G7.2, a monoclonal antibody against the intracellular domain of RPTP␴ and an Alexa 488 coupled secondary antibody (green; A and B). F-actin was detected using rhodamine-coupled phalloidin (red; A and C, 100X objective, scale bars ⫽ 10 ␮m). (D) An enlargement of the central domain of the growth cone shown in A, illustrating the intermixed, but largely non overlapping distribution of RPTP␴ and F-actin (scale bar ⫽ 2 ␮m).

ation. To address the possibility that RPTP␴ might influence the rate of axon initiation, the number of cells having tau-immunoreactive neurites of a given length after a brief period in culture were counted. Cells isolated from E15 mouse cortex were culturd for 16 h on glass coverslips coated with PL. Following immunostaining for tau, each coverslip was divided into four quadrants and the number of cells with tau-positive neurites counted in each quadrant. Tau-immunopositive cells attached to the substrate but lacking a neurite were included in the quantification. There was

no significant difference in the percentage of tau-positive cells derived from RPTP␴ (⫹/⫹), (⫹/⫺), or (⫺/⫺) mice that extended a short neurite, at least 20 ␮m long (Fig. 6). However, significantly more neurons from RPTP␴ (⫺/⫺) mice extended at least one tau-immunopositive neurite that was greater than 30 ␮m long compared to the RPTP␴ (⫹/⫹) mice. Together with the findings indicating a significant difference in axon length (Fig. 5), these results indicate that RPTP␴ does not affect the rate of axon initiation in these culture conditions, but that it slows axon extension.

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Fig. 5. Enhanced axon outgrowth in RPTP␴ (⫺/⫺) embryonic cortical cultures. (A) Illustration of the morphology of cortical neurons isolated from E15 RPTP␴ (⫺/⫺) and wild-type littermates dissociated and plated on poly-D-lysine (PL) or poly-D-lysine plus laminin-1 (PLL) coated glass coverslips. After 36 h, cultures were fixed and stained for the microtubule associated protein tau. Cell nuclei were stained with Hoescht dye. (B) Western blot analysis of total protein homogenates derived from these cultures and probed with the 17G7.2 monoclonal antibody against RPTP␴. Anti-RPTP␴ reveals an ⬃80-kDa immunoreactive band in the homogenate of cortical neurons cultured from a wild-type embryo. This band is not detected in cortical neurons cultured from a RPTP␴ (⫺/⫺) embryo. Protein size standards correspond to 116, 97.4, 66.2, 45, and 31 kDa (Bio-Rad, 7.5% PAGE). (C) Cortical neuron axon length grown

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Table 1 Neurite extension of RPTP␴ (⫺/⫺) E15 cortical neurons is enhanced on poly-D-lysine and poly-D-lysine/laminin Mouse

Genotype

Substrate

Neurite length

n

16–46 16–41 16–45 16–46 16–41 16–45 17–4 17–2 17–8 17–4 17–2 17–8

WT KO KO WT KO KO WT KO KO WT KO KO

PL PL PL PLL PLL PLL PL PL PL PLL PLL PLL

64.4 ⫾ 1.7 87.6 ⫾ 3.4* 86.7 ⫾ 3.5* 108.8 ⫾ 3.5** 146.9 ⫾ 5.1* 123.4 ⫾ 3.4* 61.1 ⫾ 2.8 73.9 ⫾ 4.3* 70.9 ⫾ 3.1* 106.4 ⫾ 4.9** 116.8 ⫾ 3.9* 117.6 ⫾ 3.4*

184 196 163 157 139 223 121 80 141 106 152 176

Note. Neurite lengths (mean ⫾ SEM) of E15 cortical neurons after 36 h in culture on substrates of poly-D-lysine (20 ␮g/ml, PL) or poly-D-lysine plus laminin-1 (20 ␮g/ml, PLL). n is the number of cells measured. An asterisk denotes a statistically significant difference of P ⬍ 0.001 between cells cultured from wild-type (WT) and RPTP␴ (⫺/⫺) (KO) mice. Two asterisks denote a statistically significant difference of P ⬍ 0.001 between cells cultured from WT mice on PL and PLL.

Discussion RPTP␴ is essential for normal neural development, but its functional role in the adult nervous system is not known (Elchebly et al., 1999; Wallace et al., 1999). Here we investigated the possibility that RPTP␴ might regulate axon growth. We report that RPTP␴ delays functional regeneration of facial motoneurons following axotomy, that RPTP␴ protein is present in neuronal growth cones, and that axon extension is enhanced in cultures of dissociated embryonic cortical neurons lacking RPTP␴. These findings indicate that RPTP␴ slows the rate of axon growth and identify a role for RPTP␴ in regulating axon regeneration by motoneurons following injury. Role of type IIa RPTPs in axon extension and axon guidance Evidence that type IIa RPTPs influence axon extension was first obtained from genetic analysis in D. melanogaster. During embryonic development, both DPTP69D and DLAR protein are distributed along axons (Tian et al., 1991; Desai et al., 1996) and their absence causes defects in the axonal projections of motoneurons (Krueger et al., 1996; Desai et al., 1996) and photoreceptor synaptic targeting (Garrity et al., 1999; Newsome et al., 2000). DPTP69D is also required

Fig. 6. Absence of RPTP␴ does not influence axon initiation. Analysis of neurite length in cultures of E15 mouse cortical neurons indicated that RPTP␴ does not influence the rate of axon initiation. No significant difference was detected in the percentage of neurons derived from RPTP␴ (⫹/⫹), (⫹/⫺), or (⫺/⫺) mice that extended a short neurite, at least 20 ␮m long. More neurons from RPTP␴ (⫺/⫺) mice extended at least one tau immunopositive neurite that was greater than 30 ␮m long than neurons derived from the RPTP␴ (⫹/⫹) mice (P ⬍ 0.05, t test). Cells were cultured on glass coverslips coated with PL. [RPTP␴ (⫹/⫹) 265 cells counted, mean number of cells with neurites (mncn) ⫽ 52.75 ⫾ 2.53 SEM, mncn ⬎30 ␮m ⫽ 26.75 ⫾ 2.29 SEM, n ⫽ 4; RPTP␴ (⫺/⫹) 1215 cells counted, mncn ⬎20 ␮m ⫽ 51.5 ⫾ 2.07 SEM, mncn ⬎30 ␮m ⫽ 30.3 ⫾ 1.55, n ⫽ 20; RPTP␴ (⫺/⫺) 858 cells counted, mncn ⬎20 ␮m ⫽ 54.25 ⫾ 1.91, mncn ⬎30 ␮m ⫽ 33.25 ⫾ 1.65 SEM, n ⫽ 12].

for the repulsion of growth cones from the CNS midline (Sun et al., 2000). HmLAR2, a DLAR homolog in leech, has been shown to be required for appropriate axon guidance and extension (Gershon et al., 1998; Baker and Macagno 2000; Baker et al., 2000). Our findings indicate that RPTP␴ inhibits axon extension. Manipulation of cPTP␴ (Rashid-Doubell et al., 2002), also known as CRYP␣, a homolog of RPTP␴ in chicken (Stoker, 1994) and Xenopus laevis (Johnson and Holt, 2000), influences axon extension and targeting by retinal ganglion cells during embryogenesis. Our demonstration that RPTP␴ protein is present in the growth cones of mammalian neurons supports the conclusion that CRYP␣/cPTP␴ and RPTP␴ share a conserved function, in addition to sequence homology. Recently, Rashid-Doubell et al. (2002) demonstrated that expression of a secreted cPTP␴/CRYP␣ ectodomain in the chick embryo, with the aim of blocking the interaction between endogenous cPTP␴/CRYP␣ and its ligand, results in stalled axon outgrowth and targeting errors in the optic tectum. Similarly disrupting the interaction between cPTP␴/CRYP␣ and a ligand associated with the glial endfeet of retinal basement membrane reduces retinal ganglion cell axon growth by ⬃60% in vitro, leading to the

on PL and PLL was measured. For each independent experiment, the length of the axons of RPTP␴ (⫺/⫺) neurons was expressed as percentage of the wild-type values. The mean values derived from two independent experiments are presented in the histogram. The mean axonal length of wild-type neurons cultured on PLL was ⬃70% greater than that of neurons cultured on PL alone (see Table 1; P ⬍ 0.001, Mann–Whitney Rank Sum Test). When cultured on PL, the mean axon length of RPTP␴ (⫺/⫺) neurons was 29% longer than wild-type axons (columns 1 and 2). When cultured on a substrate of PLL, the axons of RPTP␴ (⫺/⫺) neurons were 16% longer than wild type (columns 2 and 3). A significant difference in axon length between RPTP␴ (⫺/⫺) and wild type was found on both substrates (P ⬍ 0.001, PL; P ⬍ 0.001, PLL; Mann–Whitney Rank Sum Test).

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hypothesis that cPTP␴/CRYP␣–ligand interactions promote axon growth (Ledig et al., 1999). Expression of a putative dominant negative CRYP␣ construct in Xenopus RGCs increased axon outgrowth by ⬃60% under similar culture conditions (Johnson et al., 2001). These findings have led to a model proposing that RPTP␴ activity slows axon outgrowth and that the interaction of RPTP␴ with its ligand suppresses phosphatase activity, thus promoting axon extension (Johnson et al., 2001; Rashid-Doubell et al., 2002). Due to the lack of identified RPTP␴ substrates and the current limited understanding of RPTP␴ ligands, it remains difficult to test certain aspects of this model directly. However, the findings we report here are consistent with previous results and support the model proposed by Johnson et al. (2001). For example, the model predicts that RPTP␴ will be active in neurons cultured in the absence of ligand, such as on a substrate of poly-D-lysine. In these conditions, neurons derived from RPTP␴ (⫺/⫺) animals extended significantly longer axons than wild-type neurons, consistent with RPTP␴ activity inhibiting the rate of axon extension.

(Yan et al., 1993; Wang et al., 1995; Schaapveld et al., 1998; Meathrel et al., 2002). Importantly, it remains to be determined how RPTP␴ influences the capacity of neurons to regenerate an axon in the nonpermissive environment of the mammalian CNS. The mammalian type IIa RPTP family member LAR regulates peripheral nerve regeneration, but unlike RPTP␴, axonal regeneration in the sciatic nerve is delayed in LAR (⫺/⫺) mice (Xie et al., 2001). Immediately following sciatic nerve injury, a decrease in LAR mRNA expression was detected in DRG sensory neurons (Haworth et al., 1998). Two weeks following sciatic nerve injury, LAR protein expression by these neurons increased (Xie et al., 2001). Although members of the type IIa RPTP family share substantial sequence similarity, their expression is differently regulated in neurons following injury, and for each of the family members this likely has different functional consequences for regenerating axons.

Role of type IIa RPTPs during axon regeneration

How RPTP␴ influences growth cone motility is not known. Genetic analyses of DLAR function in D. melanogaster suggest that type IIa RPTPs regulate cytoskeletal organization through proteins that include the guanine-nucleotide exchange factor Trio (Debant et al., 1996; Awasaki et al., 2000; Bateman et al., 2000; Liebl et al., 2000), the tyrosine kinase Abl (Wills et al., 1999a, 1999b), and the Abl substrate Enabled (Wills et al., 1999a). Downstream effectors of type IIa RPTP signaling in mammals are not known; however, the interaction of type IIa RPTP ICDs with several intracellular proteins suggests roles as downstream effectors. For example, Trio binds to LAR (Debant et al., 1996), but this has not been shown for either RPTP␴ or RPTP␦, nor has Trio been demonstrated to act as a downstream effector for any type IIa RPTP family member in mammals. Liprins are a family of coiled-coil domain proteins that interact with the intracellular domains of RPTP␴, RPTP␦, and LAR (Pulido et al., 1995; Serra Pages et al., 1998; 1995). LAR and liprin-␣ colocalize at the trailing edge of focal adhesions, but the function of this interaction is not known (Serra Pages et al., 1995). Tyrosine phosphorylation regulates both focal adhesion assembly at the leading edge of a migrating cell and disassembly at the trailing edge (reviewed by Sastry and Burridge, 2000), and protein tyrosine phosphatases play key roles regulating cell motility (reviewed by AngersLoustau et al., 1999). The specific function of type IIa RPTPs in neuronal growth cones remains to be identified, but a likely possibility is that they contribute to regulating the cytoskeletal remodeling that underlies growth cone motility. Consistent with a role in regulating growth cone motility, RPTP␴ is present in axonal growth cones and functions to slow axon extension. We demonstrate that functional recovery of facial motoneurons is accelerated in RPTP␴ (⫺/⫺) mice following axotomy, suggesting that RPTP␴ slows the

Following injury, multiple factors influence the ability of an axon to regenerate in the adult nervous system (reviewed by Schwab, 2002). Here, we demonstrate that RPTP␴ expression delays functional recovery by facial motoneurons following peripheral nerve injury. These findings are consistent with the recent report of enhanced axon regeneration following sciatic nerve injury in RPTP␴ (⫺/⫺) mice (McLean et al., 2002). Allograft experiments by these authors suggest that RPTP␴ expressed by Schwann cells contributes to guiding regenerating axons in the sciatic nerve. These studies did not determine if RPTP␴ expressed by neurons influences the rate of axon extension. Although we do not rule out an effect of RPTP␴ expression by Schwann cells on the rate of axon extension during regeneration, our analysis of axon extension in vitro indicates that neuronal RPTP␴ expression slows the rate of axon growth. This effect of RPTP␴ on the rate of axon extension likely contributes to the enhanced functional recovery following axotomy observed in RPTP␴ (⫺/⫺) mice. Evidence has been provided that expression of RPTP␴ mRNA increases in DRG sensory neurons regenerating an axon following sciatic nerve injury (Haworth et al., 1998; McLean et al., 2002). The model of RPTP␴ function described above predicts that this expression would slow the rate of axon regeneration. Our analysis of RPTP␴ expression by facial motoneurons found that RPTP␴ continues to be expressed during axon regeneration. The absence of dramatic changes in expression suggests that different neuronal cell types regulate RPTP␴ expression differently following injury, as there does not appear to be an increase in expression as was reported for dorsal root ganglion sensory neurons (Haworth et al., 1998; McLean et al., 2002). RPTP␴ is widely expressed by neurons in the adult CNS

Type IIa RPTP signaling

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rate of axon extension during regeneration. These findings identify RPTP␴ as a potential target for the development of inhibitors that aim to promote nerve regeneration following injury.

Experimental methods Animals Transgenic mice lacking RPTP␴ (⫺/⫺) were generated and bred in a Balb/C background as described (Elchebly et al., 1999). E15 CD-1 mouse embryos were obtained from Charles River Canada (QC). All procedures were performed in accordance with the Canadian Council on Animal Care guidelines for the use of animals in research. Facial nerve crush Three-month-old wild-type and RPTP␴ (⫺/⫺) knockout mice were anesthetized using ketamine and xylazine (1:1; 0.04 ml/20 g mouse; Centre de Me´ decine Ve´ te´ rinaire, StHyacinthe, Quebec) and the facial nerve on the left side of the animal was exposed through a small incision dorsal and caudal to the external ear. The nerve trunk was released from the surrounding connective tissue by gentle dissection and crushed using fine forceps, 3X for 30 s distal to the point where it exits the stylomastoid foramen. Following 24 h of postoperative recovery, the completeness of lesions were assessed prior to behavioral scoring. Whisker movement was monitored twice daily following surgery to observe the progression of regeneration and was compared between the injured and contralateral uninjured side. A well-established behavioral scoring system was used to assess whisker movement (Ferri et al., 1998): 0 is no whisker movement, 1 is slight whisker movement, 2 is strong but asymmetrical whisker movement, and 3 is strong symmetrical whisker movement, corresponding to complete functional recovery. The observer was unaware of the genotype of the mice being scored. In situ hybridization Sense and antisense cRNA probes corresponding to 478 bases of mouse RPTP␴ (nucleotides 3461–3939) were used. Transcription was carried out using T7 (New England Biolabs, MA) or T3 polymerases (Promega, WI) and digoxigenin (DIG) RNA labeling mix (Roche, QC). In situ hybridization was carried out on the facial nuclei of adult mice. Animals were anesthetized as described above and perfused transcardially with 50 ml of PBS (pH 7.5) and heparin (1 unit/ml) at 4°C. CNS tissue was then rapidly dissected and frozen by immersion in 2-methyl butane (Fisher) chilled in liquid nitrogen. Six-micrometer cryostat sections were mounted onto slides (Superfrost Plus, Fisher), briefly dried, and fixed by immersion in 4% paraformaldehyde and 15%

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picric acid (pH 8.5) in phosphate-buffered saline (PBS) for 1 h at room temperature. Following fixation, sections were rinsed in DEPC-treated 2X SSC and equilibrated for 5 min in DEPC-treated 10 mM triethanolamine (Fisher). Tissue sections were acetylated by incubation with 0.25% acetic anhydride (Sigma) in DEPC-treated 10 mM triethanolamine for 10 min at room temperature. In situ hybridization was carried out as described (Braissant and Wahli, 1998) using DIG-labeled probes. Sections were transferred to prehybridization solution (50% formamide, 5X SSC, 5X Denhardt’s, 1% SDS, and 40 ␮g/ml single-stranded salmon sperm DNA) for 30 min at room temperature. Hybridization was carried out overnight at 58.5°C in 100 ␮l of solution containing 200 ng of probe in 50% formamide, 5X SSC, and 40 ␮g/ml single-stranded salmon sperm DNA. Sections were rinsed in 2X SSC at room temperature followed by a stringent wash in 2X SSC for 1 h at 65°C. Hybridization was detected using an anti-DIG peroxidase-coupled antibody (Roche, QC), amplified using the TSA- Indirect (ISH) Tyramide Signal Amplification kit (NEN, MA), and visualized with peroxidase/DAB detection (Vector Laboratories). Facial nucleus micropunch protein analysis Western blot analysis was carried out on protein extracted from the facial nuclei of adult mice. Facial nerve crush was performed as described above. At 3 and 5 days following injury, animals were anesthetized and transcardially perfused as described. CNS tissue was rapidly dissected and frozen by immersion in 2-methylbutane chilled in liquid nitrogen. Five-hundred-micron-thick coronal brainstem sections were cut on a cryostat at the level of the facial nucleus, as determined by cresyl violet staining. Samples of control and injured facial nuclei were collected using a 0.50-mm tissue micropunch (Fine Science Tools, BC) and stored at ⫺80°C before homogenization. Tissue punches were processed for Western blot analysis as described below. Culture of dissociated embryonic cortical neurons Neocortices from RPTP␴ (⫹/⫹), (⫹/⫺), and (⫺/⫺) E15 littermates were dissected in Hanks’ balanced salt solution (HBSS; Invitrogen, ON) and diced into small pieces using a razor blade. Tail samples were collected for genotyping by polymerase chain reaction (PCR). Tissue was incubated in 0.25% trypsin (Invitrogen) in S-MEM (Invitrogen) at 37°C for 25 min and then washed in Neurobasal medium (Invitrogen) supplemented with 10% heat inactivated fetal bovine serum (iFBS, Biomedia, QC), 2 mM glutamine, 100 units/ml penicillin, and 100 ␮g/ml streptomycin. Cells were dissociated by trituration and plated at a density of 25,000 cells per glass coverslip (Carolina Biological Supply, NC). Coverslips were precoated by incubation with 20 ␮g/ml PDL (PDL; Sigma, MO) in sterile water overnight at 4°C, washed with sterile water, and allowed to dry. They were then coated with 20 ␮g/ml laminin-1 (Invitrogen) in HBSS

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(Invitrogen) at 37°C overnight. Coverslips were then washed with water and incubated with medium prior to cell plating. Cells were allowed to adhere for 2 h at 37°C, after which the medium was replaced with serum-free Neurobasal medium supplemented with 1% B27 (Invitrogen), 0.5% N2 (Invitrogen), 0.4 mM glutamine, 100 units/ml penicillin, and 100 ␮g/ml streptomycin. Cultures were maintained at 37°C for 36 h. Antibodies, immunocytochemistry, and Western blot analysis RPTP␴ immunoreactivity was detected using a monoclonal antibody (17G7.2) raised against a purified recombinant protein corresponding to the intracellular domain of mouse RPTP␴. To examine the subcellular distribution of RPTP␴, cortical cultures were washed with PBS and fixed with 4% PFA, 4% sucrose (Fisher, QC) at room temperature for 10 min. Cells were washed several times in PBS and blocked in 2% bovine serum albumin (BSA) (Fisher) and 0.2% Tween 20 (Fisher) in PBS for 1 h prior to incubation with primary antibody (17G7.2) at a dilution of 1:10 overnight at 4°C. Coverslips were then washed in blocking solution and immunofluorescence visualized using an Alexa 488 coupled secondary antibody (Calbiochem, CA) in blocking solution. For analysis of axon extension in vitro, axons were visualized using an antibody against the microtubule associated protein tau (1:500, Chemicon, CA). Cultured cells were fixed for 20 min with 4% PFA in PBS (pH 7.5) with 4% sucrose and then permeablized with PBS, 0.25% Triton-X 100 (Fisher) for 4 min prior to incubation in blocking solution (3% heat-inactivated normal goat serum, 0.125% Triton X-100 in PBS). Immunoreactivity was visualized using a Cy3-coupled secondary antibody (Jackson ImmunoResearch, PA). F-actin was visualized using 0.5 ␮g/ml rhodamine-conjugated phalloidin (Molecular Probes, OR) and cell nuclei labeled with Hoescht dye. Epifluorescent and phase-contrast images were captured using a Carl Zeiss Axiovert microscope and a MagnaFire CCD camera (Optronics, CA). Axon length was measured from phasecontrast images using Northern Eclipse software (Empix Imaging, ON). Only isolated cells from randomly selected fields were analyzed. Statistical analysis was performed using SigmaStat (SPSS Inc., IL). For Western blot analysis, tissue samples were homogenized in lysis buffer [10 mM Tris–HCl (pH 7.8) 1% NP-40, 150 mM NaCl, 1 mM EDTA, 2 mM sodium orthovanadate, 10 mM NaF, and protease inhibitors: 2 ␮g/ml leupeptin, 1 ␮g/ml pepstatin, and 2 ␮g/ml aprotinin]. Samples were centrifuged at 15,000g for 5 min at 4°C and the supernatant was collected. Protein content of homogenates was quantified using the BCA protein assay kit (Pierce, IL) and proteins were separated using either 6 or 7.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane (Amersham Pharmacia Biotech, NJ). Membranes were stained

with Ponceau S to visualize total protein and then washed and incubated in 5% milk, 2% BSA, 0.1% Tween 20 in 10 mM Tris–HCl (pH 7.5) (TBST ⫹ 2% BSA) for 1 h at room temperature. Membranes were briefly washed and incubated with primary antibody overnight in TBST plus 2% BSA at 4°C. A rabbit polyclonal antibody against GST was provided by Dr. Peter McPherson (McGill University) and used at a 1:400 dilution. A mouse monoclonal antibody against the astrocyte marker glial fibrillary acidic protein (GFAP) was used at a dilution of 1:500 (Sigma) as a positive control for injury. Immunoreactivity was visualized using a peroxidase-conjugated donkey antimouse secondary antibody (1:7500; Jackson ImmunoResearch, PA) and the Chemiluminescence Reagent Plus protein detection kit (NEN Life Science Products, MA). Following immunoblotting, membranes were stained with India ink (0.1%) in PBS with 0.4% Tween 20. Protein size standards correspond to 116, 97.4, 66.2, 45, and 31 kDa (BioRad). Preparation of RPTP␴ and RPTP␦ GST fusion proteins The intracellular domains of RPTP␴ and RPTP␦ were expressed in Escherichia coli as glutathione S–transferase (GST) fusion proteins. A cDNA fragment encoding the carboxyterminal 642 amino acids of mouse RPTP␴, corresponding to amino acids 1264 –1907 (NCBI accession number X82288; Wagner et al., 1994), was amplified using PCR and subcloned into the pGEX-2T expression contruct. Fusion protein expression was induced with 0.1 M isopropyl␤-D-thiogalactoside (IPTG) at room temperature for 1 h. Bacteria were collected, suspended in solubilizing buffer (PBS with 1% Triton-X 100, EDTA, PMSF, pepstatin, leupeptin, and aprotinin), and lysed by sonication. Bacterial debris were removed by centrifugation and the lysate was washed with PBS with 0.1% Triton-X 100, EDTA, PMSF, pepstatin, leupeptin, and aprotinin. The lysate was incubated with glutathione–Sepharose 4B (Pharmacia) beads for 30 min at 4°C. A GST-␦ (Delta-cyl) cDNA encoding the carboxyterminal 750 amino acids of mouse RPTP␦ (amino acids 542–1291, NCBI accession number BAA03003) was provided by Dr. Kazuya Mizuno (Tokyo Metropolitan Institute for Neuroscience, Japan).

Acknowledgments We thank Adriana Di Polo, Cecilia Flores, and Andrew Jarjour for comments on the manuscript, and Melanie Chagnon and Stephanie Thibault for technical assistance. This work was supported by funding from CIHR to T.E.K. and M.L.T. K.M.T. was supported by a Jean Timmins Studentship, M.L.T. is a Chercheur-Boursier from the FRSQ, and T.E.K. is a scholar of the CIHR.

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References Aicher, B., Lerch, M.M., Muller, T., Schilling, J., Ullrich, A., 1997. Cellular redistribution of protein tyrosine phosphatases LAR and PTP␴ by inducible proteolytic processing. J. Cell Biol. 138, 681– 696. Angers-Loustau, A., Cote, J.F., Tremblay, M.L., 1999. Roles of protein tyrosine phosphatases in cell migration and adhesion. Biochem. Cell Biol. 77, 493–505. Awasaki, T., Saitoh, M., Sone, M., Suzuki, E., Sakai, R., Ito, K., Hama, C., 2000. The Drosophila Trio plays an essential role in the patterning of axons by regulating their directional extension. Neuron 26, 119 –131. Baker, M.W., Macagno, E.R., 2000. RNAi of the receptor tyrosine phosphatase HmLAR2 in a single cell of an intact leech embryo leads to growth-cone collapse. Curr. Biol. 10, 1071–1074. Baker, M.W., Rauth, S.J., Macagno, E.R., 2000. Possible role of the receptor protein tyrosine phosphatase HmLAR2 in interbranch repulsion in a leech embryonic cell. J. Neurobiol. 45, 47– 60. Bateman, J., Shu, H., Van Vactor, D., 2000. The guanine nucleotide exchange factor Trio mediates axonal development in the Drosophila embryo. Neuron 26, 93–106. Bixby, J.L., 2000. Receptor protein phosphatases and axon growth and guidance. NeuroReport 11, R5–R10. Braissant, O., Wahli, W., 1998. A simplified in situ hybridization protocol using non-radioactively labeled probes to detect abundant and rare mRNAs on tissue sections. Biochemica 1, 10 –16. Chen, S., Bisby, M.A., 1993. Impaired motor axon regeneration in the C57BL/Ola mouse. J. Comp. Neurol. 333, 449 – 454. Coers, S., Tanzer, L., Jones, K.J., 2002. Testosterone treatment attenuates the effects of facial nerve transection on glial fibrillary acidic protein (GFAP) levels in the hamster facial motor nucleus. Metab. Brain Dis. 17, 55– 63. Debant, A., Serra-Pages, C., Seipel, K., O’Brien, S., Tang, M., Park, S., Streuli, M., 1996. The multidomain protein Trio binds the LAR transmembrane tyrosine phosphatase, contains a protein kinase domain, and has separate rac-specific and rho-specific guanine nucleotide exchange factor domains. Proc. Natl. Acad. Sci. USA 93, 5466 –5471. Desai, C.J., Gindhart, J.G., Goldstein, L.S.B., Zinn, K., 1996. Receptor tyrosine phosphatases are required for motor axon guidance in the Drosophila embryo. Cell 84, 599 – 609. Desai, C.J., Krueger, N.X., Saito, H., Zinn, K., 1997a. Competition and cooperation among receptor tyrosine phosphatases control motoneuron growth cone guidance in Drosophila. Development 124, 1941–1952. Desai, C.J., Sun, Q., Zinn, K., 1997b. Tyrosine phosphorylation and axon guidance: of mice and flies. Curr. Opin. Neurobiol. 7, 70 –74. Elchebly, M., Wagner, J., Kennedy, T.E., Lancot, C., Michaliszyn, E., Itie, A., Drouin, J., Tremblay, M.L., 1999. Neuroendocrine dysplasia in mice lacking protein tyrosine phosphatase ␴. Nat. Genet. 21, 330 –333. Ferri, C.G., Moore, F.A., Bisby, M.A., 1998. Effects of facial nerve injury on mouse motoneurons lacking the p75 low-affinity neurotrophin receptor. J. Neurobiol. 34, 1–9. Garrity, P.A., Lee, C.H., Salecker, I., Robertson, H.C., Desai, C.J., Zinn, K., Zipursky, S.L., 1999. Retinal axon target selection in Drosophila is regulated by receptor protein tyrosine phosphatase. Neuron 22, 707– 717. Gershon, T.R., Baker, M.W., Nitach, M., Macagno, E.R., 1998. The leech receptor protein tyrosine phosphatase HmLAR2 is concentrated in growth cones and is involved in process outgrowth. Development 125, 1183–1190. Haworth, K., Shu, K.K., Stokes, A., Morris, R., 1998. The expression of receptor tyrosine phosphatases is responsive to sciatic nerve crush. Mol. Cell. Neurosci. 12, 93–104. Hunter, T., 1989. Protein-tyrosine phosphatases: the other side of the coin. Cell 58, 1013–1016. Johnson, K.G., Holt, C.E., 2000. Expression of CRYP-alpha, LAR, PTPdelta and PTP-rho in the developing Xenopus visual system. Mech. Devel. 92, 291–294.

691

Johnson, K.G., McKinnell., I.W., Stoker, A.W., Holt, C.E., 2001. Receptor protein tyrosine phosphatases regulate retinal ganglion cell axon outgrowth in the developing Xenopus visual system. J. Neurobiol. 49, 99 –117. Krueger, N.X., Van Vector, D., Wan, H.I., Gelbart, W.M., Goodman, C.S., Saito, H., 1996. The transmembrane tyrosine phosphatase DLAR controls motor axon guidance in Drosophila. Cell 84, 611– 622. Ledig, M.M., Haj, F., Bixby, J.L., Stoker, A.W., Mueller, B.K., 1999. The receptor tyrosine phosphatase CRYP␣ promotes intraretinal axon growth. J. Cell Biol. 147, 375–388. Liebl, E.C., Forsthoefel, D.J., Franco, L.S., Sample, S.H., Hess, J.E., Cowger, J.A., Chandler, M.P., Jackson, A.M., Seeger, M.A., 2000. Dosage-sensitive, reciprocal genetic interactions between the Abl tyrosine kinase and the putative GEF trio reveal trio’s role in axon pathfinding. Neuron 25, 107–118. Longo, F.M., Martignetti, J.A., Le Beau, J.M., Zhang, J.S., Barnes, J.P., Brosius, J., 1993. Leukocyte common antigen-related receptor-linked tyrosine phosphatase: regulation of mRNA expression. J Biol. Chem. 268, 26503–26511. McLean, J., Batt, J., Doering, L.C., Rotin, D., Bain, J.R., 2002. Enhanced rate of nerve regeneration and directional errors after sciatic nerve injury in receptor protein tyrosine phosphatase ␴ knockout-mice. J. Neurosci. 22, 5481–5491. Meathrel, K., Adamek, T., Batt, J., Rotin, D., Doering, L.C., 2002. Protein tyrosine phosphatase ␴-deficient mice show aberrant cytoarchitecture and structural abnormalities in the central nervous system. J. Neurosci. Res. 70, 24 –35. Meyer, G., Feldman, E.L., 2002. Signaling mechanisms that regulate actinbased motility processes in the nervous system. J. Neurochem. 83, 490 –503. Newsome, T.P., Asling, B., Dickson, B.J., 2000. Analysis of Drosphila photoreceptor axon guidance in eye-specific mosaics. Development 127, 851– 860. O’Grady, P., Thai, T.C., Saito, H., 1998. The laminin-nidogen complex is a ligand for a specific splice isoform of the transmembrane protein tyrosine phosphatase LAR. J. Cell Biol. 141, 1675–1684. Pulido, R., Serra-Pages, C., Tang, M., Streuli, M., 1995. The LAR/PTP delta/PTP sigma subfamily of transmembrane protein-tyrosine-phosphatases: multiple human LAR, PTP delta, and PTP sigma isoforms are expressed in a tissue-specific manner and associate with the LARinteracting protein LIP. 1. Proc. Natl. Acad. Sci. USA 92, 11686 – 11690. Rashid-Doubell, F., McKinnell, I., Aricescu, A.R., Sajnani, G., Stoker, A., 2002. Chick PTPsigma regulates the targeting of retinal axons within the optic tectum. J. Neurosci. 22, 5024 –5033. Sastry, S.K., Burridge, K., 2000. Focal adhesions: a nexus for intracellular signaling and cytoskeletal dynamics. Exp. Cell Res. 261, 25–36. Schaapveld, R.Q.J., Schepens, J.T.G., Bachner, D., Attema, J., Wieringa, B., Jap, P.H.K., Hendriks, W.J.A.J., 1998. Developmental expression of the cell-adhesion molecule-like protein tyrosine phosphatases LAR, RPTP␦, and RPTP␴ in the mouse. Mech. Dev. 77, 59 – 62. Schwab, M.E., 2002. Repairing the injured spinal cord. Science 295, 1029 –1031. Serra-Pages, C., Kedersha, N.L., Fazikas, L., Medley, Q., Debant, A., Streuli, M., 1995. The LAR transmembrane protein tyrosine phosphatase and a coiled-coil LAR-interacting protein co-localize at focal adhesions. EMBO J. 14, 2827–2838. Serra-Pages, C., Medley, Q.G., Tang, M., Hart, A., Streuli, M., 1998. Liprins, a family of LAR transmembrane protein-tyrosine phosphataseinteracting proteins. J. Biol. Chem. 273, 15611–15620. Stoker, A.W., 1994. Isoforms of a novel cell adhesion molecule-like protein tyrosine phosphatase are implicated in neural development. Mech. Devel. 46, 201–217. Stoker, A.W., Gehrig, B., Haj, F., Bay, B.H., 1995. Axonal localisation of the CAM-like tyrosine phosphatase CRYP alpha: a signalling molecule of embryonic growth cones. Development 121, 1833–1844.

692

K.M. Thompson et al. / Molecular and Cellular Neuroscience 23 (2003) 681– 692

Stoker, A.W., 2001. Receptor tyrosine phosphatases in axon growth and guidance. Curr. Opin. Biol. 11, 95–102. Sun, Q.L., Bahri, S., Schmid, A., Chia, W., Zinn, K., 2000. Receptor tyrosine phosphatases regulate axon guidance across the midline of the Drosphila embryo. Development 127, 801– 812. Tian, S.S., Tsouflas, P., Zinn, K., 1991. Three receptor-linked proteintyrosine phosphatases are selectively expressed on central nervous system axons in the Drosophila embryo. Cell 67, 675– 685. Uetani, N., Kato, K., Ogura, H., Mizuno, K., Kawano, K., Mikoshiba, K., Yakura, H., Asano, M., Iwakura, Y., 2000. Impaired learning with enhanced hippocampal long term potentiation in PTPdelta-deficient mice. EMBO J. 19, 2275–2785. Van Lieshout, E.M.M., Van der Heijden, I., Hendriks, W.J.A.J., Van der Zee, C.E.E.M., 2001. A decrease in size and number of basal forebrain cholinergic neurons is paralleled by diminished hippocampal cholinergic innervation in mice lacking leukocyte common antigen-related protein tyrosine phosphatase activity. Neuroscience 102, 833– 841. Wagner, J., Boerboom, D., Tremblay, M.L., 1994. Molecular cloning and tissue-specific RNA processing of a murine receptor-type protein tyrosine phosphatase. Eur. J. Biochem. 226, 773–782. Wallace, M.J., Batt, J., Fladd, C.A., Henderson, J.T., Skarnes, W., Rotin, D., 1999. Neuronal defects and posterior pituitary hypoplasia in mice lacking the receptor tyrosine phosphatase PTP␴. Nat. Genet. 21, 334 – 337. Wang, H., Yan, H., Canoll, P.D., Silvennoinen, O., Schlessinger, J., Musacchio, J.M., 1995. Expression of receptor protein tyrosine phos-

phatase-␴ (RPTP-␴) in the nervous system of the developing and adult rat. J. Neurosci. Res. 41, 297–310. Wills, Z., Bateman, J., Korey, C., Comerm, A., Van Vactor, D., 1999a. The tyrosine kinase Abl and its substrate Enabled collaborate with the receptor phosphatase Dlar to contol motor axon guidance. Neuron 22, 301–312. Wills, Z., Marr, L., Zinn, K., Goodman, C.S., Van Vactor, D., 1999b. Profilin and the Abl tyrosine kinase are required for motor axon outgrowth in the Drosophila embryo. Neuron 22, 291–299. Yan, H., Grossman, A., Wang, H., D’Eustachio, P., Mossie, K., Musacchio, J.M., Silvennoinen, O., Schlessinger, J., 1993. A novel receptor tyrosine phosphatase-␴ that is highly expressed in the nervous system. J. Biol. Chem. 268, 24880 –24886. Yeo, T.T., Yang, T., Massa, S.M., Zhang, J.S., Honkaniemi, J., Butcher, L.L., Longo, F.M., 1997. Deficient LAR expression decreases basal forebrain cholinergic neuronal size and hippocampal cholinergic innervation. J. Neurosci. Res. 47, 348 –360. Xie, Y., Yeo, T.T., Zhang, C., Yang, T., Tisi, M.A., Massa, S.M., Longo, F.M., 2001. The leukocyte common antigen-related protein tyrosine phosphatase receptor regulates regenerative neurite outgrowth in vivo. J. Neurosci. 21, 5130 –5138. Zhang, J.S., Honkaniemi, J., Yang, T., Yeo, T.T., Longo, F.M., 1998. LAR tyrosine phosphatase receptor: a developmental isoform is present in neurites and growth cones and its expression is regional- and cellspecific. Mol. Cell. Neurosci. 10, 271–286.