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First published online 8 June 2005
doi: 10.1242/dev.01891

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PTEN deletion in Bergmann glia leads to premature differentiation and affects laminar organization

¹ Department of Molecular and Medical Pharmacology, University of California, Los Angeles, CA 90095, USA
² Molecular Biology Institute, University of California, Los Angeles, CA 90095, USA
³ Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, CA 90095, USA
⁴ Department of Pathobiological Sciences and Waisman Center, University of Wisconsin, Madison, WI 53705, USA
⁵ Department of Pathology and Laboratory Medicine, University of California, Los Angeles, CA 90095, USA

View larger version (64K): [in a new window]   Fig. 1. hGfap-Cre-mediated Pten deletion leads to macrocephaly and cerebella layering defects. (A) Significant increase of brain mass of hGfap-cre+/–;Ptenloxp/loxp mice at P21. (B) Western blot analysis of protein extracts from freshly isolated granule neurons and cerebellar glia at P6. Both granule neurons and glia of Pten mutant showed a significant decrease in PTEN expression, accompanied by increased P-AKT levels. (C, a-d') The sagittal sections revealed a significant increase in size of the cerebella of Pten mutants by Hematoxylin and Eosin staining (rostral is upwards and anterior is towards the left). Mutants also showed the loss of foliation and the disruption of the IGL after P6 (C, c',d'). (D, a'-d') Calbindin immunostaining showed Purkinje cells misplacement in Pten mutant mice over time. At P3, Purkinje cell layer was relatively normal in mutant mice (D, b'). At P9, many ectopic Purkinje cells were seen in Pten mutants (D, d'). Scale bar: 100 µm.

View larger version (98K): [in a new window]   Fig. 2. hGfap-cre+/–;Ptenloxp/loxp mice show severe granule neuron ectopia but normal neuronal differentiation. (A) Sagittal section of P14 mouse cerebella (rostral is upwards and anterior is towards the left). High-magnification view of Pten mutant cerebella (from framed areas in A, a,b) showed increased number of cells in the ML (A,b') compared with littermate controls (A,a'). (B) p27 immunostaining confirmed ectopic cells in Pten mutants were granule neurons. (C) The expression patterns for axon extension (Tuj1) and early granule neuron differentiation (TAG1). Control versus Pten mutant. (D) The expression of GABAA receptor 6 at different time points. Control versus Pten mutant. EGL, external granule layer; ML, molecular layer; IGL, internal granule layer. Scale bar: 50 µm.

View larger version (82K): [in a new window]   Fig. 3. hGfap-cre+/–;Ptenloxp/loxp mice show defects in granule neuron migration in vivo. (A) Granule cell migration was analyzed by BrdU pulse labeling. Mice were injected 100 mg/kg BrdU peritoneally and distribution of BrdU-labeled neurons was determined at 20 hours and 110 hours after injection. At 110 hours post-BrdU labeling, increased number of granule cells was seen in the ML of PTEN mutant mice (A, b') compared with littermate controls (A, a'). A histogram (quantitation of BrdU-labeled neurons within the ML) indicates that Pten mutant mice had a significant increase in cell number at 110 hours post-BrdU injection (P<0.001). (B) Ki67 and phospho-histone H3 immunostaining indicated that the proliferation zone in the EGL was largely maintained with no apparent ectopic proliferation within the ML. EGL, external granule layer; ML, molecular layer; PL, Purkinje cell layer; IGL, internal granule layer; WM, white matter. Scale bar: 50 µm.

View larger version (28K): [in a new window]   Fig. 4. Pten-null neurons are highly resistant to apoptosis. Cerebellar neurons (Tuj1 positive) grown for 7 days were switched to a serum-free medium containing either 30 mM KCl or 5 mM KCl for 24 hours. (A) Neuronal viability was estimated by DAPI staining of the apoptotic nuclei. Fewer apoptotic neurons could be identified in mutants when compared with littermate controls (lower panel). Mutant neurons were also significantly larger than littermate control granule neurons (upper panel). (B) Histogram (quantitation of apoptotic nuclei) indicates Pten-null neurons are highly resistant to low K+-induced cell death under the serum deprivation condition (P<0.001).

View larger version (62K): [in a new window]   Fig. 5. Pten-null granule neurons display normal migratory properties. (A) High-magnification time-lapse video microscopy revealed the migrating neurons attached to laminin-coated glass fiber. (B) Competitive migration assay showed similar migration capacity between Pten-null and control granule neurons. Representative neuron migration from reaggregate after a 24 hour incubation was shown on micrograph (top panel). Comparisons of numbers of control versus mutant neurons that migrated to each bin are shown on the histogram (bottom). A value of 1 represents an equal ratio of control and mutant neurons in a specific bin. (C) Pten-null granule neurons exhibited the typical migrating profile on organotypic cerebellar slice culture. Ptenloxp/loxp slices from P6 mice were infected with a recombinant retrovirus expressing CRE-GFP and DsRed. After 48-72 hours incubation, the infected granule neurons with nuclear GFP and cytosol DsRed expression were visualized using a confocal microscope. (D) Pten-null and wild-type granule neurons were co-cultured with cerebellar astroglia. (D, a,a') pure cerebellar glia 7 days in vitro. (b,b') Wild-type granule neurons were co-cultured with either wild-type or mutant glia for 36 hours, wild-type neurons (Tuj1) formed a close apposition with GFAP-positive mutant glial fibers during migration. (c,c') Mutant granule neurons were co-cultured with wild-type or mutant glia, and showed normal migrating profile and axon extension along with GFAP-positive astroglial fibers. Arrowheads indicate migrating neurons closely apposite with glial fibers. Scale bars: 50 µm.

View larger version (116K): [in a new window]   Fig. 6. Developmental stage-dependent Bergmann glia defects in Pten-null animals. (A,A') Sagittal sections immunolabeled with anti-BLBP antibody and revealed significant enlargement of the immature Bergmann glial fibers in Pten mutant at P3 (A'). (B,B') Bergmann glial scaffold defects in Pten mutants at P7. In control littermates, BLBP-positive Bergmann glia aligned as a single layer next to Purkinje neurons, whereas many Pten mutant Bergmann glia were localized within the deeper IGL region (arrowheads, B'). (C,C') Bergmann glia prematurely transformed into an astrocytic morphology (arrowhead); shown at P14 in the mutant (C'). Some astrocytic Bergmann fibers still retained end-feet on the pial surface. EGL, external granule layer; ML, molecular layer; IGL, internal granule layer. Scale bars: 50 µm.

View larger version (94K): [in a new window]   Fig. 7. Bergmann glia scaffold defects lead to granule neuron migration defects. (A) Sagittal sections immunostained with BLBP (a) and counterstained with DAPI (a'). In Pten mutants (P7), severe Bergmann glia layering defect (b) coincides with major granule neuron accumulation within the ML (b'). Arrow indicates normal positioned Bergmann glial cell body; arrowhead pointed to ectopic Bergmann glial cell body. (B, a-c) Bergmann glia defects after adenovirus mediated Pten deletion were associated with increased granule neurons within the ML. HDA.Cre/YFP virus was injected into Ptenloxp/loxp;Rosa26floxed-Stop-lacZ cerebella at P3 and the tissues were processed 8 days after injection. Bergmann glial scaffold defects were a direct consequence of viral CRE-mediated Pten deletion (B, a,b, area indicated by paired arrows) and coincides with granule neuron heterotopia in the ML (B, a-c; red boxed area for injected side; blue boxed area for uninjected control). Granule neurons accumulated within the ML were negative in X-gal staining (red boxed areas in B, a,c). EGL, external granule layer; ML, molecular layer; BG, Bergmann glia layer; IGL, internal granule layer. Scale bars: 50 µm.

View larger version (52K): [in a new window]   Fig. 8. Bergmann glia premature differentiation is a cell-autonomous event. HDA.Cre/YFP virus was injected into Ptenloxp/loxp;Rosa26floxed-Stop-lacZ at P7 and tissues were analyzed 7 or 14 days after injection. (A) Good correlation between virus infection (YFP, shown in green, upper panel) and CRE recombinase-mediated loxp excision (lacZ+, lower panel) in injection site. (B, a-c') Increased Bergmann fiber thickening and lateral branching (arrow) were evident one week after Pten deletion (a'). These Bergmann glia showed astrocytic morphology (arrow) 2 weeks after infection (b'). (c,c') DAPI staining performed 7 days after injection showed no apparent granule neuron migration defect within the ML; compare lacZ (+) (c') and lacZ (–) (c) regions. (C) Purkinje cell positioning was not influenced by Bergmann glia Pten deletion after P7. Normally positioned Purkinje cells (arrows) are present in lacZ (+) area when compared with the adjacent uninfected lacZ (–) area. EGL, external granule layer; ML, molecular layer; BG, Bergmann glia layer; IGL, internal granule layer. Scale bars: 50 µm.