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PTEN is essential for cell migration but not for fate determination and tumourigenesis in the cerebellum

Silvia Marino1,{dagger}, Paul Krimpenfort2, Carly Leung1, Hetty A. G. M. van der Korput3, Jan Trapman3, Isabelle Camenisch1, Anton Berns2 and Sebastian Brandner4,*,{dagger}

1 Institute of Pathology, University Hospital, 8091 Zurich, Switzerland
2 Division of Molecular Genetics and Centre of Biomedical Genetics, The Netherlands Cancer Institute, CX 1066 Amsterdam, The Netherlands
3 Department of Pathology, Erasmus University, 3000 DR Rotterdam, The Netherlands
4 Institute of Neuropathology, University Hospital, 8091 Zurich, Switzerland
* Present address: Institute of Neurology, Queen Square, London WC1N 3BG, UK



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  		Fig. 1. Generation of mice deficient for PTEN in a tissue specific fashion: (A) organisation of the PTEN wild-type locus; (B) targeting construct used for electroporation into ES cells; (C) targeted locus containing the neomycin resistance gene flanked by LoxP sites; (D) ES cells lacking the neo cassette were generated by transient cre expression, resulting in a PtenLoxP allele; (E) tissue-specific Cre-mediated recombination results in loss of exon 5. (F) Southern blot analysis of targeted clones after homologous recombination (BglII digest, probe 1; exon 4) shows a 6.6 kb wild-type band and a 5 kb band of the recombined allele in lane 1. (G) Probing with probe 2 (exon 5) of NcoI/EcoRI-digested genomic DNA resulted in a 6 kb wild-type and a 2 kb recombined band in lanes 1+2. (H) Southern blot analysis (probe 2, NcoI/EcoRI-digested genomic DNA) after transient transfection with a Cre-expressing plasmid shows a 6 kb wild-type band and a 2 kb floxed exon 5 band. (lane 3). (I) Loss of the neo cassette was confirmed after re-probing the blot with a neo probe (lane 3).

 


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  		Fig. 2. Development, proliferation and differentiation in the cerebellum of En2cre; PtenLoxP/LoxP mice. (A,B) enlarged cerebellar anlage and increased cell size (inset) of neural precursors at embryonic day 15.5 in En2cre; PtenLoxP/LoxP (B) versus controls (A). (C,D) Immunostaining for BrdU revealed reduced proliferation in mutant cerebella at embryonic day 15.5 (D), compared with littermate controls (C). (E,F) Phosphorylated AKT is highly expressed in the EGL and IGL of the developing wild type cerebellum and in all regions of the mutant cerebellum. (G,I) Widening of the Math1-expressing outer EGL (G, arrowheads) and of the p27-expressing inner EGL (I, arrowheads). Littermate controls are shown in H and J; blue arrowheads in J indicate neurons strongly positive for p27. Scale bar: 400 µm in A,B (60 µm in inset); 50 µm in C,D; 660 µm in E,F; 100 µm in G,H; 200 µm in I,J.

 


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  		Fig. 3. Analysis of En2cre; PtenLoxP/LoxP mice. (A,B) Significant enlargement of the cerebellar vermis of conditional mutant mice (195 days old) (B) when compared with age-matched controls (A). Arrowheads indicate the lateral extension of the vermis (A,B). (D,H) Sagittal sections revealed a significant increase in size of the cerebellum and loss of foliation. (C-F) Calbindin-positive Purkinje cells in mutant cerebellar areas were randomly distributed, enlarged and occasionally vacuolated (D,F) when compared with control littermates (C,E). Likewise, NeuN-positive granule cells were positioned parallel to the outer margin of the mutated cerebellum (H) and occasionally retained within the presumed molecular layer (J). Control brains are shown in G,I. Schematic illustration of the progressive loss of Purkinje cells over time (K-O): outline of the mid-sagittal cerebellum, each red dot represents one vital Purkinje cell on a representative midsagittal cerebellar section. Green dots represent vacuolated Purkinje cells in the same section. The orientation of the sections is indicated in K (a, anterior; p, posterior; d, dorsal; v, ventral). The age of the respective animals is indicated in each section. A digitised image and AnalySIS software were used to outline the section and to label vital or vacuolated Purkinje cells on a calbindin-stained slide. Scale bar: 2 mm in C,D,G,H; 100 µm in E,F,I,J; 1.8 mm in K-O.

 


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  		Fig. 4. Characterisation of pathological features in cerebella En2cre; PtenLoxP/LoxP mice. (A) Increased cerebellar size expressed as midsagittal area (mm2) in mutant (red bars) versus controls (blue bars). Evaluation of three sections each. Vertical bars indicate s.d. Significance of the size difference: 9 days, P<0.03; 15 days, P<0.02; 51 days, P<0.004; 184 days, P<0.01. (B) Graphic representation of chronic Purkinje cell loss in En2cre; PtenLoxP/LoxP mice expressed as cells per mm2 in midsagittal sections stained for calbindin. Error bars indicate s.d. (C) Abnormal accumulation of neurofilaments in Purkinje cells En2cre; PtenLoxP/LoxP mice visualised by Bielschowsky silver impregnation. (D) Occasionally, bi-nucleated neurones were encountered (synaptophysin immunostaining). (E,F) Dysplastic astrocytes, revealed by GFAP immunostaining (E) were seen in aged En2cre; PtenLoxP/LoxP mice and showed heavy accumulation of phospho-AKT (F). (G,H) Oligodendrocytes (visualised by proteolipid protein in situ hybridisation) were mainly confined to the white matter tracts and only occasionally present within the granular or molecular layer in controls (G), while En2cre; PtenLoxP/LoxP mutant mice (H) showed a random distribution of these cells, which occasionally reached the cerebellar surface. Frequently, oligodendrocytes formed pairs or clusters (inset in H; I). (I,J) In situ hybridisation of metabotropic glutamate receptor 2 (mGluR2) reveals Golgi cells (cerebellum of control mouse, I) and showed abnormal positioning but no depletion throughout lifetime in En2cre; PtenLoxP/LoxP mutant cerebella (J). Scale bar: 50 µm in C,E; 30 µm in D; 110 µm in F-H; 80 µm in I,J.

 


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  		Fig. 5. Immunoblotting of protein extracts from cerebella of L7cre; PtenLoxP/LoxP, L7cre; PtenLoxP/+ En2cre; PtenLoxP/LoxP and wild-type mice. Increased levels of phospho-Akt are only detected when Pten is deleted on both alleles in Purkinje cells (L7cre; PtenLoxP/LoxP mice) or in all cells of the vermis (En2cre; PtenLoxP/LoxP), while wild-type and L7cre; PtenLoxP/+ cerebella show no difference.

 


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  		Fig. 6. Cell density, apoptosis and proliferation in the developing cerebellum of En2cre; PtenLoxP/LoxP mice and controls at E15.5 and postnatally (P1). Although cellular density is not greatly increased at E15.5 (A), the absolute number of cells (C) in the midsagittal cerebellum is almost twice as much in mutant brains, because the total area is increased in mutants (B). Apoptosis is reduced in E15.5 mutant cerebella, compared with controls (D,E), but there is also reduced proliferation in mutant cerebella at days E15.5, P1 and P9 (F) in mutant versus control cerebella.

 


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  		Fig. 7. Analysis of L7cre; PtenLoxP/LoxP mice. (A) Transgenic construct used for microinjection. Whole-mount ß-gal staining of Purkinje cells in L7cre; ROSA26R mouse brains (B,C) and after paraffin embedding and counterstaining with nuclear Fast Red (D). Calbindin immunostaining of wild-type (E), 7-week-old L7cre; PtenLoxP/LoxP mice (F) and 16-week-old L7cre; PtenLoxP/LoxP mice (G), showing increase of cell size with thickening of dendritic processes, vacuolisation, degeneration and progressive loss of Purkinje cells. Immunostaining for phosphorylated AKT (H-J) reveals baseline levels in Purkinje cells, Bergmann glia cells and Golgi cells in controls (H), while degenerating Purkinje cells of 7-week-old mice (I) and of 16-week-old mice (J) show heavy accumulation of p-AKT. Scale bar: 100 µm in D-J.

 

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© The Company of Biologists Ltd 2002