Receptor protein tyrosine phosphatase gamma is a marker for pyramidal cells and sensory neurons in the nervous system and is not necessary for normal development

Abstract

In order to gain insight into the biological role of receptor protein tyrosine phosphatase gamma (RPTPgamma), we have generated RPTPgamma-null mice. RPTPgamma was disrupted by insertion of the beta-galactosidase gene under the control of the RPTPgamma promoter. As the RPTPgamma-null mice did not exhibit any obvious phenotype, we made use of these mice to study RPTPgamma expression and thus shed light on potential biological functions of this phosphatase. Inspection of mouse embryos shows that RPTPgamma is expressed in a variety of tissues during embryogenesis. RPTPgamma is expressed in both embryonic and adult brains. Specifically, we detected RPTPgamma expression in cortical layers II and V and in the stratum pyramidale of the hippocampus, indicating that RPTPgamma is a marker for pyramidal neurons. Mixed primary culture of glial cells showed a lack of expression of RPTPgamma in astrocytes and a low expression of RPTPgamma in oligodendrocytes and in microglia. Interestingly, RPTPgamma expression was detected in all sensory organs, including the ear, nose, tongue, eye, and vibrissa follicles, suggesting a potential role of RPTPgamma in sensory neurons. An initial behavioral analysis showed minor changes in the RPTPgamma-null mice.

FIG. 1.

Generation of RPTPγ knockout-LacZ transgenic mice. (A) Structures of the wild-type (WT) RPTPγ mouse gene and of the targeting vector used for generating heterozygous and knockout mice. IRES, internal ribosome entry site; β-Geo, β-Gal/neomycin resistance fusion protein; CAH, carbonic anhydrase domain; FNIII, fibronectin type III domain; TM, transmembrane domain. (B) Southern blot of genomic DNA of wild-type (+/+), heterozygous knockout (+/−), and homozygous knockout (−/−) mice, using the 5′ and 3′ probes indicated in panel A. (C) Reverse transcriptase PCR analysis using total RNA prepared from WT and knockout (KO) brains. PCR was carried out on RNA incubated with (WT and KO) or without (cWT and cKO) reverse transcriptase and on purified RPTPγ cDNA (cDNA). Primers were chosen to amplify all RPTPγ isoforms (arrows on scheme). (D) Immunoprecipitation and immunoblotting for RPTPγ in extracts from WT, heterozygous (HET), and KO brains. Immunoprecipitation and immunoblotting were carried out using RPTPγ antibodies directed against the C terminus of RPTPγ (D2 domain on scheme). IgG from the immunoprecipitation is indicated. In panels C and D, base pair numbers and molecular weights, respectively, are noted at the left of blots.

FIG.2.

Expression of RPTPγ in the CNS. (A) Comparison between in situ hybridization (top) by use of an extracellular probe (indicated by the black bar in the scheme on the left) and β-Gal staining (bottom) of consecutive sagittal sections from either an E14.5 heterozygous embryo (left) or a P2 heterozygous brain (right), demonstrating RPTPγ localization. The arrows in the left panels highlight examples of structures stained by both methods. CAH, carbonic anhydrase domain; FNIII, fibronectin type III domain. (B) Whole-mount β-Gal staining at embryonic stages E10.5 and E12.5. Arrows point to the isthmus. Arrowheads indicate the nose and forelimb. (C) Sagittal sections of E14.5 and E17.5 embryos. Arrows indicate the trigeminal ganglia (top) and the left inferior ganglion of the glossopharyngeal (IX) nerve. Higher magnification of the DRG is shown in the two insets. (D) Sagittal sections of adult brains. (E) Staining of P2 (coronal section) and adult (whole mount) spinal cords. Cx, cortex; D, diencephalon; hT; hypothalamus; S, striatum; T, telencephalon; Tg, trigeminal ganglia; Th, thalamus.

FIG.2.

Expression of RPTPγ in the CNS. (A) Comparison between in situ hybridization (top) by use of an extracellular probe (indicated by the black bar in the scheme on the left) and β-Gal staining (bottom) of consecutive sagittal sections from either an E14.5 heterozygous embryo (left) or a P2 heterozygous brain (right), demonstrating RPTPγ localization. The arrows in the left panels highlight examples of structures stained by both methods. CAH, carbonic anhydrase domain; FNIII, fibronectin type III domain. (B) Whole-mount β-Gal staining at embryonic stages E10.5 and E12.5. Arrows point to the isthmus. Arrowheads indicate the nose and forelimb. (C) Sagittal sections of E14.5 and E17.5 embryos. Arrows indicate the trigeminal ganglia (top) and the left inferior ganglion of the glossopharyngeal (IX) nerve. Higher magnification of the DRG is shown in the two insets. (D) Sagittal sections of adult brains. (E) Staining of P2 (coronal section) and adult (whole mount) spinal cords. Cx, cortex; D, diencephalon; hT; hypothalamus; S, striatum; T, telencephalon; Tg, trigeminal ganglia; Th, thalamus.

FIG. 3.

Laminar expression of RPTPγ in the brain. (A) Sagittal section of adult brain. Coronal sections of the hippocampus (left insets) or the piriform cortex (right insets). H, hippocampus; Th, thalamus; S, striatum; CC, corpus callosum; DG, dentate gyrus. (B) Coronal sections of the hippocampus stained for β-Gal (top) and Nissl (bottom). (C) Staining of primary cultures of glial cells. Left, purified oligodendrocyte progenitor cells; middle, purified microglia; right, purified astrocytes.

FIG. 4.

Expression of RPTPγ in pyramidal neurons. (A) Quantification of β-Gal- and BrdU-positive cells in adult mouse brain sections injected with BrdU at E13.5 or E16.5. (Bottom) β-Gal staining of a sagittal section of an adult cortex demonstrates opposing gradients of expression in layers II and V. Data represent means ± standard deviations of measurements from three animals, summing counts from 10 sections for each animal. (B) (Left) Neutral red and β-Gal labeling of pyramidal neurons from layers II and V, with higher magnifications boxed at right. (Right) β-Gal (upper left and lower left), Hoechst (upper right), and dextran-biotin (lower left) labeling of pyramidal neurons. The panel on the lower right is a higher magnification of that on the lower left, showing β-Gal staining colocalizing with dextran-biotin.

FIG. 5.

Expression of RPTPγ in sensory neurons. (Left and right) β-Gal staining in sagittal sections of sensory organs innervated by cranial nerves, including olfactory bulb, eye, tongue, and ear, as well as nasal cavity wall and vibrissae. (Center) β-Gal staining of sagittal sections of head, showing labeling in cranial nerves I, II, III, V, VII, VIII, IX, and X. All sections are from an E14.5 embryo. T, tongue; NC, nasal cavity.

FIG. 6.

Histology of the CNS in wild-type and knockout mice. (A) Timm staining of sagittal sections of adult brains from wild-type (+/+) and knockout (−/−) mice. The lower panels show enlargements of the hippocampus (left panels, sagittal) and the somatosensory cortex (right panels, coronal). (B) Hematoxylin and eosin staining of coronal sections of the spinal cords from wild-type (+/+) and knockout (−/−) mice.

FIG. 7.

Behavioral analyses of wild-type and knockout mice. (A) Sensory-motor gating with wild-type (WT, n = 8) and knockout (KO, n = 9) mice. Percent prepulse inhibition (PPI) of startle induced by a prepulse of the indicating intensities (in dB) is shown (e.g., PP70 indicates a prepulse of 70 dB). (B) Contextual and cued fear conditioning in WT and KO mice. *, P < 0.05; **, P < 0.01