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Physiol. Genomics 19: 50-60, 2004. First published June 29, 2004; doi:10.1152/physiolgenomics.00079.2004
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Received 1 April 2004; accepted in final form 24 June 2004.
Physiological Genomics 19:50-60 (2004)
1094-8341/04 $5.00 © 2004 American Physiological Society

Mild impairment of motor nerve repair in mice lacking PTP-BL tyrosine phosphatase activity

Derick G. Wansink1, Wilma Peters1, Iris Schaafsma1, Roger P. M. Sutmuller2, Frank Oerlemans1, Gosse J. Adema2, Bé Wieringa1, Catharina E. E. M. van der Zee1 and Wiljan Hendriks1

1 Department of Cell Biology, Nijmegen Center for Molecular Life Sciences, University Medical Center Nijmegen, 6525 GA Nijmegen, The Netherlands
2 Department of Tumor Immunology, Nijmegen Center for Molecular Life Sciences, University Medical Center Nijmegen, 6525 GA Nijmegen, The Netherlands


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 GRANTS
 REFERENCES
 
Mouse PTP-BL is a large, nontransmembrane protein tyrosine phosphatase of unclear physiological function that consists of a KIND domain, a FERM domain, five PDZ domains, and a COOH-terminal catalytic PTP domain. PTP-BL and its human ortholog PTP-BAS have been proposed to play a role in the regulation of microfilament dynamics, cytokinesis, apoptosis, and neurite outgrowth. To investigate the biological function of PTP-BL enzyme activity, we have generated mice that lack the PTP-BL PTP moiety. These PTP-BL{Delta}P/{Delta}P mice are viable and fertile and do not present overt morphological alterations. Although PTP-BL is expressed in most hematopoietic cell lineages, no alterations of thymocyte development in PTP-BL{Delta}P/{Delta}P mice could be detected. Sciatic nerve lesioning revealed that sensory nerve recovery is unaltered in these mice. In contrast, a very mild but significant impairment of motor nerve repair was observed. Our findings exclude an essential role for PTP-BL as a phosphotyrosine phosphatase and rather are in line with a role as scaffolding or anchoring molecule.

FERM domain; PDZ domain; gene targeting; signal transduction; sciatic nerve


   INTRODUCTION
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 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 GRANTS
 REFERENCES
 
MOST SIGNALING EVENTS INVOLVING reversible protein tyrosine phosphorylation emanate at the cell cortex, and it should therefore come as no surprise that many protein tyrosine phosphatases (PTPs) are of the transmembrane receptor type (2). In addition to those, there is a subset of nonreceptor type PTPs, consisting of PTPH1, MEG1, PTPD1, PTPD2, and PTP-BAS in human, which is directed to the cell cortical area by virtue of a so-called FERM domain. The FERM domain is an acronym of "band 4.1, ezrin, radixin, and moesin" (9). These four proteins are capable of binding, in a regulated fashion, to transmembrane proteins and phospholipids with their NH2-terminal FERM domain and to microfilaments through their COOH-terminal actin binding domain, thereby providing a link between the cell membrane and the cytoskeleton (7). Some FERM domain-containing PTPs have in addition one (in PTPH1 and MEG1) or five (for PTP-BAS and its mouse ortholog PTP-BL) PDZ domains, small globular protein modules named after the first proteins in which they were identified: PSD95, DlgA, and ZO-1. PDZ domains can be found in a wide variety of proteins and are known to mediate protein-protein interactions by specifically recognizing COOH-terminal sequences of their targets, but some may also bind to internal peptide stretches (51). In this way, PDZ-containing proteins help to orchestrate the composition of protein complexes and thus contribute to the necessary hardware for signaling and adhesive processes (39). Recently, at the very NH2 terminus of PTP-BAS, a kinase noncatalytic C lobe domain (KIND) was identified (10), which may even further expand the anchoring and scaffolding potential of this PTP. Thus FERM and PDZ domain-containing PTPs all have the potency to be highly relevant components of cell cortical "signalosomes."

Experimental evidence for the above hypothesis is sparse, however. PTPH1 has been shown to interact with three different proteins: 14-3-3ß, vasolin-containing protein/p97/CDC48, and tumor necrosis factor {alpha}-convertase (TACE) (55–57). As a consequence, it is believed to play a role in TACE-dependent ectodomain release (57) and cell cycle progression (56). Expression studies in T cells also implicate PTPH1 in the reduction of antigen receptor signaling (25). Transfection experiments with MEG1 have pointed to inhibitory effects on cell proliferation and colony formation (24), and interaction studies revealed a potential role for MEG1 as a regulator and as a downstream signal transducer of glutamate receptors (28). For PTP-BAS/PTP-BL, potentially interacting proteins have been identified that point to a role in actin dynamics (12, 14, 20, 22, 35, 41, 50), and overexpression studies suggest regulatory roles in processes as diverse as NF{kappa}B (29, 36) and ephrin (37) signaling, cytokinesis (27), and Fas-mediated apoptosis (31, 42). Also, a gene trap insertion strategy resulted in mice that express a ß-galactosidase-tagged NH2-terminal fragment of PTP-BL, and the observed staining pattern in peripheral nerves and spinal ganglia led to the suggestion that this large intracellular PTP might play a role in neurite outgrowth (44).

The enzymatic activity of PTP-BL’s PTP domain, as demonstrated in vitro using its potential partner protein RIL ("reversion-induced LIM" protein) as substrate (12), needs to be tightly regulated since ectopic expression in COS-1 cells of the PTP domain alone, i.e., without its proper targeting signals, induces morphological abnormalities (15). As a means to study the relevance and biological function of PTP-BL-mediated dephosphorylation of phosphotyrosines at the cell cortex, we generated mice deficient in PTP-BL phosphatase activity through gene targeting in mouse embryonic stem (ES) cells. Resulting mice, which express a COOH-terminally truncated protein (PTP-BL{Delta}P) and lack the wild-type (WT) enzyme, are viable and fertile and, after backcrossing to a C57BL/6 background, do not present evident growth defects. Furthermore, no gross anatomical alterations have been observed, and lymphoid development was unimpaired. Sciatic nerve lesional studies, however, point to a mild delay in motor neuron outgrowth as a result of the targeted mutation. This finding is in line with a role for PTP-BL in neurite outgrowth.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 GRANTS
 REFERENCES
 
PTP-BL targeting vector construction and mouse production.
Mouse 129 genomic clones were identified in a cosmid library (kindly provided by M. Hofker) according to standard procedures. A 4.3-kbp EcoRI-BamHI fragment, containing exon 41 of the PTP-BL gene (Ptpn13, LocusID 19249), was subcloned in front of a neomycin phosphotransferase expression cassette (neo) (48). Downstream of the resistance cassette, a 3.4-kbp EcoRI fragment was introduced that harbors the last exon (exon 47) of the PTP-BL gene. Prior to electroporation, the targeting construct was linearized using a unique SalI site. Targeting in E14 ES cells followed by subsequent injection into C57BL/6 blastocysts and breeding with chimeric males was done as described (49).

Mice were kept at the Central Animal Facility of the University of Nijmegen in a standard room with a day/night rhythm of 06:00/18:00 h at a temperature of 21°C and a humidity of 50–60%. Mice were housed in Macrolon cages and fed ad libitum. Experiments were performed on F3 mice with a 129/Ola x C57BL/6 hybrid genetic background and on F9 animals that resulted from seven successive backcrosses onto C57BL/6 and subsequent intercross of resulting heterozygotes. All procedures involving animals were approved by the Animal Care Committee of the University Medical Center St. Radboud (Nijmegen, The Netherlands) and conformed to the Dutch Council for Animal Care and the NIH guidelines.

Genotyping cells and mice.
ES cell genomic DNA was extracted and analyzed by Southern blot analysis as described (43). Screening was performed using a 300-bp HindIII genomic fragment as a 3' probe on BamHI digested ES cell DNA, a 600-bp HindIII fragment as a 5' probe on EcoRV digests, and a 1.1-kbp XhoI fragment from the neo cassette to rule out additional integrations of the vector. DNA obtained from toe clips or tail biopsies was screened by Southern blot analysis or by PCR using primers BL-sense 5'-TGCACCTACAGGCAGCTGTGAG-3', BL-antisense 5'-CAGTAGGTGCTTGAGAAATTTGG-3', and Neo-3'-forward 5'-CTATCGCCTTCTTGACGAGTT-3'.

RNA analysis.
Total RNA from kidney, stomach, brain, and liver was isolated using the LiCl/urea-phenol-chloroform extraction method (3). For Northern blot analysis, 20 µg RNA of each sample was fractionated on a 1% (wt/vol) agarose/2.2 M formaldehyde gel and transferred to Hybond-N membrane (Amersham). Blots were probed with mouse PTP-BL cDNA fragments (26) or a 1.1-kbp XhoI fragment from the neo cassette, then exposed to film for autoradiography. RT-PCR analysis of truncated PTP-BL transcripts was done according to standard protocols using oligo(dT) and F-Bas 5'-CAAGAAGCTGAAGTTATCCAGT-3' as primers.

Antibodies.
To obtain antibodies directed against an NH2-terminal part of PTP-BL (amino acids 50–443), the corresponding cDNA fragment was cloned in-frame in the pGEX-2T vector (Amersham Biosciences UK Limited, Buckinghamshire, UK) to produce a recombinant GST-BL-N fusion protein in Escherichia coli. The recombinant protein was purified using glutathione-Sepharose CL4B beads (Amersham) and analyzed on 10% polyacrylamide gels. Polyclonal {alpha}BL-N antiserum was generated by immunizing rabbits with GST-BL-N fusion protein according to established protocols. The rabbit polyclonal {alpha}BL-PTP antiserum (recognizing the phosphatase domain) has been described previously (12).

Western blot analysis.
Protein lysates of several tissues of WT, PTP-BL+/{Delta}P, PTP-BL{Delta}P/{Delta}P mice were prepared by homogenization in lysis buffer [20 mM HEPES, pH 7.5, 2 mM EGTA, 10% glycerol, 1 mM PMSF, 10 mM NaF, 1 mM orthovanadate, and 1 protease inhibitor cocktail tablet (Roche Diagnostics, Mannheim, Germany) per 50 ml] followed by adding detergents to a final concentration of 0.5% (wt/vol) NP-40 and 0.5% (wt/vol) SDS. Whole cell homogenates were incubated on ice for 30 min, after which an equal volume of Laemmli sample buffer was added, followed by incubation for 15 min at 90°C. Protein concentration of the resulting samples was determined according to a modified Lowry procedure (38). Twenty micrograms of protein of each tissue was loaded per lane on a 6% polyacrylamide gel and after electrophoresis transferred to nitrocellulose membrane by Western blotting. Blots were processed and incubated with {alpha}BL-N or {alpha}BL-PTP followed by chemiluminescence detection using standard procedures.

Lymphocyte preparations and flow cytometry.
Single-cell suspensions were prepared by gently pressing lymphoid organs from 6- to 8-wk-old WT and PTP-BL{Delta}P/{Delta}P mice between frosted glass slides. Total lymphocytes were purified by filtration through Nitex nylon mesh (pore size, 53 µm). Cells (106) were stained with anti-CD3-PE, anti-CD4-APC, anti-CD8-FITC, anti-CD11c-APC, anti-CD25-FITC, (BD Biosciences), anti-GR-1, anti-F4/80 (Caltag Laboratories, Burlingame, CA), and anti-B220 (6B2 hybridoma supernatant) antibodies by standard procedures. Flow cytometry was performed on a BD Biosciences FACSCalibur. Propidium iodide-negative cells were analyzed using CELLQuest software.

Immunohistochemistry.
Cryosections of mouse C57BL/6 tissues snap-frozen in liquid nitrogen were cut (7 µm) and mounted on SuperFrost/Plus slides (Menzel-Glazer, Braunschweig, Germany). After drying, sections were fixed in 1% formaldehyde in 0.1 M phosphate buffer (PB) for 10 min. Sections were processed and incubated with pre-immune serum, {alpha}BL-N, or {alpha}BL-PTP according to standard procedures and embedded in Mowiol.

Histological analysis.
Various tissues from adult male and female mice were dissected, fixed in buffered formaldehyde, dehydrated, and embedded in paraffin. Sections of 6-µm thickness were stained with hematoxylin-eosin according to standard histological procedures. Brains were analyzed using parasagittal and coronal sections stained with cresyl violet.

Unilateral sciatic nerve crush lesion and functional recovery.
The sciatic nerve of the right hind paw in WT and PTP-BL{Delta}P/{Delta}P mice was subjected to a crush lesion as described previously (16, 46, 47). In short, mice were anesthetized, and the sciatic nerve was carefully exposed. With forceps, the nerve was crushed for 60 s at the sciatic notch point immediately distal from where it emerges from under the gluteus maximus muscle. The skin was sutured, and mice were returned to their home cage. Return of sensory function was determined by applying a range of small electric currents (0.1, 0.3, and 0.5 mA) to the foot sole of the mice. Absence of the foot-withdrawal reflex upon stimulation at 0.5 mA was interpreted as no recovery, whereas mice responding with the foot-withdrawal reflex at 0.1 mA current, for three consecutive days, were considered to be recovered. The withdrawal reflex was measured at postlesion day 3 and daily from day 10 onward until full recovery.

The recovery of motor function following sciatic nerve crush lesion was monitored through analysis of the individual mouse free-walking pattern. The walking test method was originally described for rats (19), and calculations were modified by De Koning and Gispen (17; see also Refs. 33 and 52). The progress of motor function recovery in the sciatic nerve was calculated using eight footprint parameters (distances in mm) and a correction factor (applicable to mice as well) according to the following formula:

where SFI is sciatic functional index (%); NTOF is normal to opposite foot; ETOF is experimental to opposite foot; NPL is normal print length; EPL is experimental print length; NTS is normal toe spreading; ETS is experimental toe spreading; NIT is normal inner toe spreading; and EIT is experimental inner toe spreading. This formula provides an SFI value of about –100% directly after the crush lesion (postlesion day 3; ETS and EIT are both set at 2 mm) and an SFI between –10% and +10% for nonlesioned control mice and when full recovery of motor function is obtained.

In the test procedure each mouse was allowed to acclimate to the experimental environment by once letting the animal walk through an inclining (10°) alley (40 x 3.5 cm) that leads into a dark box. Then, a strip of photographic paper (Kodak, Polymax II RC semi-matt) was placed on the bottom of the alley, and, after dipping the animal’s hind feet in photographic paper developer fluid (Kodak, Polymax RT), the animal was again placed at the beginning of the alley to let it walk into the dark box. Subsequently, after allowing the photographic strip to dry, mouse footprint parameters (indicated above) were measured. In this way, motor function recovery was determined at day 3 and then every second day starting from day 8 postlesion.

Neurofilament-200 immunostaining and quantitative analysis.
Following transcardial perfusion of WT and PTP-BL{Delta}P/{Delta}P mice, sciatic nerve dissection, and cryostat sectioning, a neurofilament (NF)-200 immunostaining of 8-µm-thick peripheral nerve sections was performed as described (47), using rabbit anti-NF-200 (1:1,000; Sigma Chemical, St. Louis, MO). For the quantitative analysis, digital images of the sciatic nerve sections (on coded slides) were collected using a Dialux 20 microscope (Leitz) connected to a video camera attached to a PC image-analysis system. The number of NF-200-positive nerve sprouts was counted in three nerve sections (8 µm thick, 72 µm apart) at 1, 3, and 5 mm distal to the crush site at 4 and 7 days post crush lesion. Newly outgrowing axonal sprouts, as revealed by NF-200 antibody/3-amino-9-ethylcarbazole (AEC) immunostaining, appeared under the microscope as relatively large, medium-sized, or small red dots. Counting was performed using an ocular grid with 10 squares, which covered and represented therefore a total sciatic nerve area of 6,250 µm2. The total axon number per 6,250 µm2 was calculated from counts in three to six sections per distance, per mouse, and per genotype group.

Statistics.
Data obtained from WT and PTP-BL{Delta}P/{Delta}P mice are presented as means ± SE. All data measurements were obtained blind as to genotype and subsequently analyzed using the appropriate statistical tools (one-way ANOVA, ANOVA repeated measures, Student’s t-test, paired t-test; SPSS 10.0 statistics software). Statistical significance was set at P < 0.05.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
GRANTS
 REFERENCES
 
Targeted deletion of PTP-BL phosphatase domain gene segment.
To study the function of PTP-BL as a tyrosine-specific phosphatase in the cell cortical region, we used gene targeting by homologous recombination in ES cells to produce PTP-BL{Delta}P/{Delta}P mice that would lack the PTP-BL tyrosine phosphatase moiety. Using a replacement-type targeting vector, exons 42–46 of the mPTP-BL gene, Ptpn13, encoding the phosphatase domain of PTP-BL, were replaced by the neo selection cassette (Fig. 1A). Southern blot analysis using a 3' diagnostic probe (Fig. 1B), and a 5' diagnostic probe (Fig. 1C) was used to confirm proper recombination. Furthermore, ES lines were tested for the presence of only one copy of the neo cassette (data not shown). Successfully targeted ES clones were used for blastocyst injection followed by breeding with chimeric males. F1 offspring carrying the PTP-BL{Delta}P mutation were mated with C57BL/6 mice to rapidly expand the population. Presence of the PTP-BL{Delta}P allele was confirmed in offspring from F2 heterozygote crosses by Southern blotting (Fig. 1D) and PCR analysis of genomic DNA (Fig. 1E). The PTP-BL{Delta}P allele was identified in 96 heterozygous (PTP-BL+/{Delta}P, 49%) and 49 homozygous (PTP-BL{Delta}P/{Delta}P, 25%) mice out of a total of 195 animals tested. Thus the mutant allele segregated according to Mendelian law. Subsequent breeding steps demonstrated unaltered fertility and litter sizes for heterozygous and homozygous mutant animals compared with WT littermates, indicating that removal of the PTP-BL PTP domain did not impair essential steps in development and reproduction.



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  		Fig. 1. Disruption of the PTP-BL gene by homologous recombination. A: the PTP-BL protein structure showing the various domains (KIND, FERM, PDZ I–V, and PTP) is depicted on top. A schematic diagram of the relevant part of the mouse Ptpn13 locus and the targeting strategy is shown at the bottom. Exons are indicated as small black boxes. In the targeting construct, the fragments homologous to PTP-BL genomic segments are termed 5'- and 3'-arm, respectively, and the white box termed "neo" symbolizes the neomycin phosphotransferase selection cassette. Small black bars below the wild-type (WT) allele, designated 5'p and 3'p, respectively, indicate genomic segments used as 5' and 3' diagnostic probes. Arrows indicate location of primers used for genotyping by PCR. B, BamHI; E, EcoRI; Ev, EcoRV. B: Southern blot analysis of BamHI-digested genomic DNA from WT (+/+) and correctly targeted embryonic stem (ES) cells (+/{Delta}P) using the 3' diagnostic probe, which detects the mutant 4.5-kbp BamHI fragment and the WT 10.6-kbp BamHI fragment. Size markers (kbp) are indicated on the left. C: Southern blot analysis of EcoRV-digested genomic DNA from WT and correctly targeted ES cells hybridized with the 5' diagnostic probe. Signals obtained correspond to a WT >30-kbp EcoRV fragment and an ~18-kbp mutant EcoRV fragment. D: Southern blot analysis of BamHI-digested genomic DNA from WT, heterozygous (+/{Delta}P), and homozygous ({Delta}P/{Delta}P) PTP-BL mice using the 3' diagnostic probe. E: PCR analysis on genomic DNA from +/+, +/{Delta}P, and {Delta}P/{Delta}P mice demonstrates only WT alleles (wt) in +/+ mice and targeted alleles (tg) in {Delta}P/{Delta}P mice, whereas +/{Delta}P animals contain one allele of each type. Fragment sizes are ~500 bp for the WT PCR and ~400 bp for the targeted allele.

 
The PTP-BL{Delta}P allele produces truncated mRNAs.
PTP-BL is expressed in many epithelial cell types, in brain, and in peripheral nerves and spinal ganglia (26, 44, 50). To investigate consequences of PTP-BL gene targeting at the RNA level, total RNA was isolated from several mouse tissues and analyzed by Northern blot analysis (Fig. 2). PTP-BL is not expressed in liver, which was therefore taken as a negative control. Use of the full-length PTP-BL cDNA as a probe on WT RNA revealed several different transcript isoforms that are 8–9 kb in size and most likely result from alternative splicing (4, 8, 32, 42). In PTP-BL{Delta}P/{Delta}P RNA samples, shorter versions of these transcripts, around 7–8 kb in size, result from transcription of the PTP-BL{Delta}P allele. RNA isolates from heterozygous tissues demonstrate that the WT and the PTP-BL{Delta}P mRNAs are present in comparable amounts (Fig. 2A). As expected, using a mouse PTP-BL cDNA probe consisting of the PTP-encoding part, we could detect no mRNAs in PTP-BL{Delta}P/{Delta}P mice (Fig. 2B).



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  		Fig. 2. Northern blot analysis of total RNA isolated from stomach, kidney, brain, and liver of WT (+/+), heterozygous (+/{Delta}P), and homozygous PTP-BL{Delta}P/{Delta}P ({Delta}P/{Delta}P) mice. A: a mouse PTP-BL cDNA fragment encompassing the entire coding region detected two different PTP-BL transcripts in WT mice. In PTP-BL{Delta}P/{Delta}P animals, corresponding truncated mRNAs were observed. Note equal levels of WT and truncated transcripts in the heterozygous samples. PTP-BL is not expressed in liver. Ethidium bromide staining allowed comparison of RNA loading as judged by 28S rRNA levels. Size markers (kb) are indicated on the left. B: a mouse PTP-BL PTP domain-specific probe remained without signal in PTP-BL{Delta}P/{Delta}P samples and yielded reduced signals in heterozygous samples compared with WT, confirming proper targeting. Ethidium bromide staining allowed comparison of RNA loading as judged by 28S rRNA levels. Size markers (kb) are indicated on the left. C: schematic representation of PTP-BL and PTP-BL{Delta}P protein structures, based on results obtained by RT-PCR. The PTP-BL{Delta}P protein, encoded by transcripts generated from the PTP-BL{Delta}P allele, lacks the catalytic PTP domain and contains an artificial COOH terminus (RVSMSSAIKSSSMS; represented by an asterisk) following amino acid 2100, due to altered splicing and a concomitant change in exon 47 reading frame. For protein domain nomenclature see Fig. 1A.

 
To investigate the nature of the transcripts resulting from the PTP-BL{Delta}P allele in more detail, we performed RT-PCR experiments on PTP-BL{Delta}P/{Delta}P-derived RNA and cloned and sequenced the obtained fragment. This revealed that PTP-BL{Delta}P mRNAs lack the sequence parts corresponding to exons 41–46 of the PTP-BL gene. Evidently, genomic deletion of Ptpn13 exons 42–46 resulted in PTP-BL{Delta}P primary transcripts in which the exon 40 splice donor site is used in conjunction with the splice acceptor site of the very last Ptpn13 exon, exon 47. As a result, PTP-BL{Delta}P mRNA variants are 961 bases shorter than their WT counterparts and encode PTP-BL mutant forms that, from amino acid residue 2100 onward, lack the COOH terminus containing the catalytic PTP protein part. Instead, such PTP-BL{Delta}P proteins would have a 14 amino acids long, rather serine-rich tail (-RVSMSSAIKSSSMS) due to a change in the reading frame used in exon 47-derived sequences (depicted in Fig. 2C). Database searches demonstrated that this artificially created COOH-terminal sequence is not present in any other protein reported in human or mouse.

Truncated PTP-BL protein is overrepresented in PTP-BL+/{Delta}P mice.
To determine whether PTP-BL{Delta}P transcripts indeed give rise to truncated PTP-BL protein forms, we performed Western blot analyses of protein lysates from several mouse tissues using polyvalent antisera directed against two different parts of PTP-BL (i.e., the NH2-terminal segment and the catalytic PTP segment, respectively). In WT animals two major PTP-BL protein isoforms around 270 kDa in size were detected by the two sera in brain, kidney, lung, and testis lysates, but not in liver, as expected (Fig. 3A). In tissue lysates from homozygous PTP-BL{Delta}P/{Delta}P animals no specific immunoreactivity toward the {alpha}BL-PTP antiserum was observed (Fig. 3B), but indeed two truncated forms of PTP-BL were detected using the {alpha}BL-N antiserum (Fig. 3A).



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  		Fig. 3. Truncated, PTP-BL{Delta}P protein is more stable than WT PTP-BL. A: equal protein amounts from lysates of WT (+/+), heterozygous (+/{Delta}P), and homozygous PTP-BL{Delta}P/{Delta}P ({Delta}P/{Delta}P) mouse tissues were separated by SDS-PAGE, blotted onto PVDF membranes, and immunostained with {alpha}BL-N antiserum. Two WT PTP-BL isoforms, probably different splice isoforms (arrowheads), can be detected in brain, kidney, lung, and testis samples of WT and heterozygous mice. PTP-BL is not expressed in liver. In PTP-BL{Delta}P/{Delta}P lysates stable truncated, PTP-BL{Delta}P products are clearly detectable. Signal intensity of the various bands in heterozygous material revealed that PTP-BL{Delta}P is two to four times more abundant than WT protein. B: the {alpha}BL-PTP antiserum did not yield a specific signal in PTP-BL{Delta}P/{Delta}P lysates, indicating that the corresponding epitope was absent. Asterisks indicate nonspecific signals, unrelated to PTP-BL, observed in brain, lung, and testis. When using {alpha}BL-N antiserum on brain, lung, and testis samples, the mobility of one aspecific band unfortunately coincides with that of the larger PTP-BL{Delta}P isoform. A size marker is indicated on the left (kDa).

 
Remarkably, in the PTP-BL+/{Delta}P samples the amount of PTP-BL{Delta}P proteins exceeds that of WT isoforms. Signal quantitation by densitometry revealed a two- to fourfold overrepresentation of PTP-BL{Delta}P forms compared with WT proteins. Also in PTP-BL{Delta}P/{Delta}P mice the relatively high level of PTP-BL{Delta}P proteins is evident compared with the full-length PTP-BL expression level in WT animals (Fig. 3A). This suggests that the truncated, phosphatase-dead protein is more stable, which may reflect the action of possible regulatory mechanisms responding to the loss of PTP-BL catalytic activity.

To examine the phosphorylation status of PTP-BL/PTP-BL{Delta}P and the PTP-BL substrate RIL in PTP-BL{Delta}P/{Delta}P and WT mice, lysates from brain, lung, kidney, and testis were used in immunoprecipitation experiments applying {alpha}BL-N, {alpha}BL-PTP, and {alpha}RIL antisera. Immunoprecipitates were tested on immunoblots using different phosphotyrosine-specific antibodies, but no change in tyrosine phosphorylation levels was observed (data not shown). We also investigated in these tissue lysates whether proteins that colocalize with ({alpha}-actinin, ezrin) or can bind to PTP-BL (PARG, RIL) have altered expression levels in PTP-BL{Delta}P/{Delta}P mice, but again no effect of PTP-BL phosphatase deficiency was apparent (data not shown).

WT PTP-BL protein is localized at the apical side of polarized epithelial cells in stomach and lung tissue (12). Deletion of the PTP-BL PTP domain did not alter this subcellular localization (Fig. 4), which is in line with previous observations pointing to the FERM domain in PTP-BL as being responsible for cortical targeting (6, 15).



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  		Fig. 4. Deletion of the PTP domain in PTP-BL does not alter subcellular localization. Lung cryosections of WT (A–C) and PTP-BL{Delta}P/{Delta}P (D–F) mice were stained with pre-immune serum (A and D), {alpha}BL-N (B and E), or {alpha}BL-PTP (C and F) for immunofluorescence microscopy. WT and PTP-BL{Delta}P protein were localized predominantly in bronchial epithelium, at the apical side of the cells. Note that {alpha}BL-PTP staining is nearly absent in the PTP-BL{Delta}P/{Delta}P sample (F). Bar = 50 µm.

 
Histological analysis of PTP-BL{Delta}P/{Delta}P mice.
We performed an extensive histological survey to look for possible consequences of the absence of enzymatically active PTP-BL at the tissue and cellular level. A wide variety of tissues, including brain, kidney, liver, lung, stomach, intestinal tract, testis, spleen, and thymus, were isolated, sectioned, and stained with hematoxylin-eosin. In addition, serial parasagittal and coronal sections of brain tissue were stained with cresyl violet and analyzed for abnormalities. No obvious differences between adult PTP-BL{Delta}P/{Delta}P and WT mice were discerned (data not shown).

Increased body weight in the initial cohort of PTP-BL{Delta}P/{Delta}P male mice.
Mice lacking the PTP-BL PTP domain behaved normally and appeared healthy. Over time, however, we noted that male, but not female, PTP-BL{Delta}P/{Delta}P mice, which resulted from intercrosses of heterozygous F2 animals, developed a mild obese phenotype (Fig. 5, A and C). First signs could be observed around 4 mo of age, and at the age of 15 mo the average body weight of PTP-BL{Delta}P/{Delta}P males (~42 g) differed significantly (P < 0.002) from that of WT male littermates (~33 g). PTP-BL+/{Delta}P males showed intermediate values but were also significantly heavier than WT (~37 g, P < 0.03; Fig. 5A). Macroscopic and histologic examination revealed that the increase was due to an increase of white adipose tissue and a concomitant general enlargement of other tissues, especially liver.



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  		Fig. 5. Gradual increase in body weight in F3 male PTP-BL{Delta}P/{Delta}P mice. Body weight of WT (open squares), heterozygous (open triangles), and homozygous PTP-BL{Delta}P/{Delta}P (solid circles) mice was determined at regular intervals. Average values for each genotype at each time point are plotted, and bars indicate standard errors of the mean. These data were used for curve-fitting analysis using nonlinear regression (+/+, dotted curve; +/{Delta}P, dashed curve; {Delta}P/{Delta}P, solid curve). Two different cohorts of mice were used, as follows. 1) F3 generation males (A) and females (C) resulting from intercrosses of F2 PTP-BL+/{Delta}P animals (on average a 129:C57BL/6 genetic background ratio of 1:3); n = 22, 40, and 28 for +/+, +/{Delta}P, and {Delta}P/{Delta}P male mice, respectively; n = 22, 25, and 13 for +/+, +/{Delta}P, and {Delta}P/{Delta}P female mice, respectively. 2) F9 generation males (B) and females (D) obtained after seven successive backcrosses onto C57BL/6; n = 28, 34, and 18 for +/+, +/{Delta}P, and {Delta}P/{Delta}P male mice, respectively; n = 26, 32, and 19 for +/+, +/{Delta}P, and {Delta}P/{Delta}P female mice, respectively. Body weights of F3 male mice are significantly different between {Delta}P/{Delta}P and +/+ animals (P < 0.002, Wilcoxon), and between +/{Delta}P and +/+ mice (P < 0.03). No significant difference was found when +/{Delta}P male mice were compared with {Delta}P/{Delta}P males (P = 0.56). In F3 female mice also no significant difference in body weight was observed between the genotype groups. Clearly, the almost completely overlapping curves for the F9 genotype cohorts (B and D) demonstrate equal body weights within both sex groups.

 
Subsequently, PTP-BL+/{Delta}P mice which had been backcrossed to the C57BL/6 genetic background over seven successive generations became available, and body weight measurements were repeated on littermates resulting from intercrosses between these heterozygous F9 animals. This time no differences could be detected, not even when comparing 2-yr-old animals (Fig. 5, B and D). Furthermore, introduction of a high-fat diet resulted in a comparable rate of body weight increase for the different genotype groups (data not shown). This leads us to suggest that the mild obese phenotype that was initially observed, and which segregated with the PTP-BL{Delta}P mutant allele during initial crosses, is due to a strain 129/Ola modifier locus that is either 1) present somewhere in the genome and is acting in concert with PTP-BL or 2) is in relative close proximity to Ptpn13 (mouse chromosome 5E/F; Ref. 45) and is exerting its effect independent of PTP-BL. In the latter scenario, a crossover event during the subsequent backcrosses is required to explain the segregation of this obese phenotype and the PTP-BL{Delta}P mutation.

To investigate whether changes in triglyceride or cholesterol levels in PTP-BL{Delta}P/{Delta}P males may be reminiscent of the initial observation regarding body weight, blood samples obtained from fasted and fed males of the three different genotypes were analyzed. No differences in the above parameters or in their lipoprotein profiles were detected (data not shown). Also, no alterations in fasted and fed insulin levels or leptin concentrations were detected in blood samples from PTP-BL{Delta}P/{Delta}P mice compared with WT animals (data not shown).

Normal lymphoid development in PTP-BL{Delta}P/{Delta}P mice.
The human ortholog of PTP-BL, PTP-BAS, was originally identified in basophils (32) and subsequently shown to be widely expressed in lymphoid cells (21). Furthermore, a direct and functional interaction between PTP-BAS and the human Fas death receptor has been reported in cell models (42), although this could not be confirmed in mouse (13). Therefore, to trace a possible distortion on lymphoid developmental processes, we analyzed the lymphocyte composition of thymus, spleen, and lymph nodes using flow cytometry (Table 1). Mature T cells are CD3 positive and can be classified according to the expression of CD4 and CD8 cell surface markers. Most immature T cells do not express CD4 and CD8 and thus will be double negatives. Maturation initially results in CD4/CD8 double-positive cells, representing the majority of thymocytes, which then gives rise through the mechanisms of positive and negative selection to mature single-positive cells by losing either CD4 or CD8 markers. CD3 is poorly expressed by immature thymocytes but highly expressed by mature T cells. Single-positive T cells then exit the thymus and seed peripheral lymphoid organs such as lymph nodes and the spleen. We checked for the relative contribution of the different thymocyte subsets in the various lymphoid organs (Table 1). In addition, the B lymphocyte population was monitored using a CD45 isoform-specific antibody, B220. The Gr-1 antibody allowed detection of cells from the myeloid lineage, like monocytes and granulocytes. The F4/80 antibody was used to analyze macrophages. Antigen-presenting cells (APCs) were monitored using anti-CD11c. Finally, regulatory T cells were monitored by the expression of CD4 and CD25 (40). All PTP-BL{Delta}P/{Delta}P lymphoid organs examined had mature T cells in proportions that were identical to that of WT specimen. Likewise, the subpopulations of thymocytes, the CD4+CD25+ Treg cells, the percentages of B cells, and the amounts of Gr-1-, F4/80-, or CD11c-positive cells were unchanged. Finally, the sizes of lymphoid organs were comparable between PTP-BL{Delta}P/{Delta}P and WT animals, and normal numbers of cells could be isolated. In conclusion, no obvious differences in lymphoid tissue composition were detected between samples from PTP-BL{Delta}P/{Delta}P animals and WT controls.


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  		Table 1. Lymphocyte populations in PTP-BL{Delta}P/{Delta}P mice

 
Delayed motor, but not sensory, function recovery following sciatic nerve crush in PTP-BL{Delta}P/{Delta}P mice.
The expression of PTP-BL in the developing peripheral nervous system (44) prompted us to investigate the localization of PTP-BL in the adult animal and the role of PTP-BL in nerve regeneration. Immunostaining of transversal sections of WT spinal cord and dorsal root ganglia with rabbit antiserum {alpha}BL-PTP, which recognizes the phosphatase domain, demonstrated that PTP-BL is expressed in small and large dorsal root ganglia sensory neurons (Fig. 6A) and in most spinal cord motor neurons (Fig. 6B). Similar sections from PTP-BL{Delta}P/{Delta}P mice served as specificity controls (not shown). The presence of PTP-BL in dorsal root ganglia and spinal cord motor neurons is in agreement with earlier findings (44).



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  		Fig. 6. Immunostaining of sensory and motor neurons and the sciatic nerve. PTP-BL immunostaining is visible in small (arrowheads) and large (arrows) dorsal root ganglia sensory neurons (A) and in most motor neurons in the ventral horn of the spinal cord (arrows) (B). Photomicrographs of nonlesioned sciatic nerves of WT (C) and PTP-BL{Delta}P/{Delta}P (D) mice show similar immunostaining for neurofilament (NF)-200. The individual axons are seen as red dots of variable size. At 4 days following sciatic crush lesion, newly outgrowing axons are visible as small red dots in WT (E) and PTP-BL{Delta}P/{Delta}P mice (F), here shown in nerve sections located at 5 mm distal to the crush site. Quantitative analysis of the number of newly formed axons was performed at 4 days (G) and 7 days (H) postlesion for WT (open bars) and PTP-BL{Delta}P/{Delta}P (solid bars) mice. At 4 days postlesion and 5 mm distance, a decrease in the average axon number was observed in PTP-BL{Delta}P/{Delta}P nerves, compared with the WT (*P < 0.05). Note also that the number of newly formed axons at 5 mm is outnumbering that at 3 mm distal to the crush site in 4-day lesioned WT animals, which can be attributed to branching (**P < 0.02). This axonal branching was not observed in PTP-BL{Delta}P/{Delta}P mice. Bar in A (for A and B), 50 µm; bar in C (for C–F), 50 µm.

 
To evaluate the effect of the PTP-BL{Delta}P mutation on peripheral nerve regeneration, a unilateral crush lesion of the sciatic nerve was applied. Gradual recovery of the sensory function of the sciatic nerve was assessed daily by applying a small current stimulus to the foot sole and scoring the subsequent occurrence or absence of the foot-withdrawal reflex in WT (n = 9) and PTP-BL{Delta}P/{Delta}P (n = 10) mice (Fig. 7A; showing data regarding 0.1 mA). In WT mice, the sensory function response of the nonlesioned sciatic nerve in the contralateral paw showed a fast foot-withdrawal reflex upon stimulation with a 0.1-mA current stimulus. This response was equally fast in the nonlesioned contralateral paw of PTP-BL{Delta}P/{Delta}P mice. First signs of sensory recovery in the lesioned hind paw of WT animals appeared as early as postlesion day 14, and all animals in the WT group were recovered by postlesion day 19 (average recovery, 17.2 ± 0.5 days; Fig. 7A). The first positive sensory recovery in the PTP-BL{Delta}P/{Delta}P group was scored at postlesion day 15, and all animals were finally recovered by day 24. Average recovery time for the PTP-BL{Delta}P/{Delta}P mice was 17.9 ± 0.9 days, which was not significantly different from that of the WT group.



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  		Fig. 7. Recovery of function following unilateral sciatic nerve crush lesion. A: the gradual recovery of sensory function was determined by applying a small current stimulus (0.1 mA) and scoring the foot-withdrawal reflex. Both WT (open squares) and PTP-BL{Delta}P/{Delta}P (solid circles) mice showed a similar return of sensory function, with 90–100% recovered mice at 20 days postlesion. B: the gradual recovery of motor function was measured using the "sciatic functional index" based on comparison of footprint parameters of the regenerating hind paw with the contralateral paw. The motor function recovery in PTP-BL{Delta}P/{Delta}P mice (solid circles) was significantly slower [F(1,40)=4.59, P < 0.038], and fewer animals had reached complete recovery by the fourth week postlesion, when compared with WT mice (open squares).

 
In a larger group of animals, which also included the above-mentioned mice, the gradual recovery of motor function following the sciatic nerve crush lesion was assessed by monitoring the animal’s gait in a walking alley, measuring various footprint parameters and using these to calculate the sciatic function index (SFI, see Fig. 7B). Before the lesion, WT (n = 19) and PTP-BL{Delta}P/{Delta}P (n = 23) mice showed identical gait, indicating normal motor function with regard to walking and the use of leg, paw, and toe muscles. At 3 days following sciatic nerve crush, an SFI value between –80 and –100% established the completeness of the crush lesion for all animals. The first signs of motor function recovery in WT mice became apparent at postlesion days 8 and 10 (with SFI, –76 ± 4.1% and –75 ± 6.4%, respectively; Fig. 7B). Subsequently, the motor function continued to improve over time, showing an SFI of –40 ± 5.2% at 2 wk postlesion. Full recovery was reached at postlesion day 23 (SFI, –8 ± 2.4%; Fig. 7B), which was in agreement with previous findings (33, 47).

In PTP-BL{Delta}P/{Delta}P mice the index was –77 ± 3.4% and –76 ± 2.1% at postlesion days 8 and 10, respectively (Fig. 7B). At 2 wk postlesion the average SFI was still –55 ± 4.3%. At days 23–25 in the fourth week, the SFI value remained at –16 ± 4%, indicating that not all PTP-BL{Delta}P/{Delta}P animals had reached complete recovery (Fig. 7B). By week 5, all animals were recovered (not shown). When statistically evaluated over the entire period from postlesion days 8–25, the motor function recovery in PTP-BL{Delta}P/{Delta}P mice was significantly slower than in WT animals [ANOVA repeated measures, F(1,40) = 4.59, P < 0.038].

Diminished branching of regenerating axons in PTP-BL{Delta}P/{Delta}P mice.
Subsequently, histological examination of the sciatic nerves was performed to determine the effect of the PTP-BL{Delta}P mutation on the number of peripheral nerve axons before and after crush lesion. First, NF-200 immunostaining of nonlesioned WT (Fig. 6C) and PTP-BL{Delta}P/{Delta}P (Fig. 6D) sciatic nerves showed a similar staining pattern, with NF-200-positive axons appearing as small, medium-sized, or large red dots (Fig. 6, C and D). The average number of NF-200-positive axons per 6,250 µm2 sciatic nerve area in WT (154 ± 12, n = 8) and PTP-BL{Delta}P/{Delta}P (168 ± 13, n = 9) mice was not significantly different, indicating that PTP-BL deficiency did not affect peripheral nerve development. Second, during the regeneration of the sciatic nerve, at 4 days following the crush lesion and at 5 mm distal to the crush site, WT (Fig. 6E) and PTP-BL{Delta}P/{Delta}P (Fig. 6F) mice demonstrated NF-200-positive staining of newly formed axonal sprouts. Quantitative analysis involved counting of the number of NF-200-positive axonal sprouts at 4 days (Fig. 6G; n = 4 per genotype group) and 7 days (Fig. 6H; n = 4 per genotype group) post crush lesion, at 1, 3, and 5 mm distal to the crush site, and in three to six nerve sections per distance for each mouse. At 4 days postlesion, the average axon numbers per 6,250-µm2 area at 1 and 3 mm distance were not significantly different between WT and PTP-BL{Delta}P/{Delta}P mice (Fig. 6G). However, at 5 mm distal to the crush site, the 4-day lesioned PTP-BL{Delta}P/{Delta}P mice demonstrated a lower average axon number (Fig. 6G, P < 0.05). Notably, in the 4-day lesioned WT animals the axon count at 5 mm outnumbered that at 3 mm (P < 0.02), which was not observed in PTP-BL{Delta}P/{Delta}P mice (Fig. 6G). The observation that more sprouts are, temporarily, present more distal to the crush site has been reported for rats and mice, with individual regenerating axons forming multiple regenerating fibers, of which many will be aborted during axon maturation (18, 30, 34, 47). Apparently, this temporary increase distal to the crush site is not present in PTP-BL{Delta}P/{Delta}P mice, suggesting diminished axonal branching in mice lacking PTP-BL phosphatase activity. At 7 days postlesion, the average axon numbers per 6,250-µm2 area at all three distances were not significantly different between WT and PTP-BL{Delta}P/{Delta}P mice (Fig. 6H). Also, the axonal branching effect with a higher number of axonal sprouts at 5 mm compared with 3 mm distance is no longer present in WT animals (Fig. 6H) but probably located further distal.

In summary, while sensory function recovery was normal, a small but significant delay in motor function recovery of the peripheral sciatic nerve was observed between postlesion days 8 to 25 in PTP-BL{Delta}P/{Delta}P mice, compared with WT mice. Furthermore, at 4 days postlesion at 5 mm distal to the crush site a lower average axon count was measured in combination with diminished axon branching in regenerating PTP-BL{Delta}P/{Delta}P sciatic nerves. These histological findings therefore corroborate the mild impairment of functional motor nerve repair in PTP-BL{Delta}P/{Delta}P mice.

Conclusions.
The mutation introduced in the Ptpn13 gene results in the production of truncated, protein tyrosine phosphatase activity-deficient protein, PTP-BL{Delta}P, which is more stable than full-length PTP-BL. The resulting overrepresentation of anchoring and scaffolding modules, e.g., the KIND, FERM, and PDZ domains, in the cell cortical area may in itself have bearing on the composition and activity of protein complexes involved in subcellular signaling and cell adhesion. Furthermore, the sheer reduction in tyrosine-specific phosphatase capacity in the cell cortical area could well impair signaling pathways during growth and development. Nevertheless, homozygous PTP-BL phosphatase-deficient mice are normal in their overall morphology, behavior, fertility, and life span. Also, lymphoid development patterns in the various genotype groups were indistinguishable. Lack of abnormalities in standard gross pathology has also been reported for homozygous PTP-BLgt mice (44) that carry a gene trap insertion vector in Ptpn13 intron 22, but upon careful examination these mice were found to contain normal levels of WT PTP-BL in addition to the PTP-BL/ß-galactosidase fusion protein (D. G. Wansink, W. Hendriks, T. Thomas, and P. Gruss; unpublished data). The insertion of a gene trap construct into a locus does not always cause a reduction in the levels of WT mRNA produced at this locus; for example, normal WT mRNA levels have been reported in mice homozygous for a gene trap insertion into the MAP4 locus (53).

Initially, we noted the development of an obesity syndrome milder than the tubby obesity condition (11) for male but not female PTP-BL{Delta}P/{Delta}P mice. Sexual dimorphism is not uncommon in obesity phenotypes (11), and the growth curve of heterozygous PTP-BL+/{Delta}P males even pointed to a dominant inheritance (Fig. 5A). However, a 129/Ola-derived modifier locus obviously confounded these initial body weight analyses, because subsequent backcrosses to the C57BL/6 genetic background completely eliminated the abnormalities in size and weight of male mutant mice.

Upon performing peripheral nerve lesion studies, however, we observed a small but significant impairment of sciatic motor nerve function recovery in PTP-BL{Delta}P/{Delta}P animals. The underlying mechanism remains as yet elusive, although subtle alterations in guidance cues via ephrin/EphB signaling and/or survival cues via p75 NTR may contribute (29, 37). Presumably, under normal conditions in PTP-BL{Delta}P/{Delta}P mice, other tyrosine-specific phosphatases can effectively compensate for the dephosphorylation task of PTP-BL, or, alternatively, the PTP-BL{Delta}P protein has no detrimental effects. Following sciatic nerve injury, however, cellular processes apparently are impaired in motor neurons compared with sensory cells, which may reflect differential expression of certain PTPs in these cell types as has been reported for the cell adhesion molecule-like PTP LAR (54). Still, being the largest mammalian intracellular PTP, PTP-BL contains protein stretches, such as the KIND domain, that are not present in any other family member. Furthermore, it has four more PDZ domains than comparable mammalian PTPs (i.e., PTPH1 and MEG1).

It is tempting to speculate that the main physiological contribution of PTP-BL to the functioning of cells and tissues is actually reflected by its potency to orchestrate the composition and dynamics of large protein machines through its many protein interaction modules rather than by its enzymatic activity. One could even envision that PTP-BL directly recruits other PTPs into signaling complexes, which then may overcome the PTP-BL{Delta}P enzyme mutation. Inter-PTP interactions have been noted for other family members (e.g., Refs. 5 and 23), and RPTPß, for example, possesses a COOH-terminal PDZ binding domain (1) that may well represent a target for PTP-BL PDZ domains. Characterization of mutant mice completely devoid of any of the noncatalytic PTP-BL protein parts will therefore be required to determine the physiological impact of its various scaffolding and anchoring modules.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by grant KUN 95-900 from the Dutch Cancer Society KWF (to W. Hendriks).


   ACKNOWLEDGMENTS
 
We thank Marten Hofker (Maastricht, The Netherlands) for providing the mouse 129 genomic phage library, Tim Thomas, Anne K. Voss, and Peter Gruss (Göttingen, Germany) for providing the PTP-BLgt transgenic mouse strain, Hans van der Boom and Louis Havekes (Leiden, The Netherlands) for cholesterol, triglyceride, and lipoprotein analyses, Jan Schepens for insulin and leptin measurements, Jack Fransen and Huib Croes for help with histological analyses, and our colleagues at the Central Animal Facility for help and advice.


   FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: W. Hendriks, 163 Cell Biology, Nijmegen Center for Molecular Life Sciences, Univ. Medical Center Nijmegen, Geert Grooteplein 28, 6525 GA Nijmegen, The Netherlands (E-mail: w.hendriks{at}ncmls.kun.nl).


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 GRANTS
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