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doi: 10.1242/10.1242/dev.00183

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Transgenic mice expressing F3/contactin from the TAG-1 promoter exhibit developmentally regulated changes in the differentiation of cerebellar neurons

Antonella Bizzoca1, Daniela Virgintino2, Loredana Lorusso1, Maura Buttiglione1, Lynn Yoshida5, Angela Polizzi1, Maria Tattoli1, Raffaele Cagiano1, Ferdinando Rossi3, Serguei Kozlov4,*, Andrew Furley5,{dagger} and Gianfranco Gennarini1,{dagger}

1 Dipartimento di Farmacologia e Fisiologia Umana, Università di Bari; Università di Torino, Italy
2 Dipartimento di Anatomia Umana ed Istologia, Università di Bari; Università di Torino, Italy
3 Università di Bari; Dipartimento di Neuroscienze, Università di Torino, Italy
4 Institute of Biochemistry, University of Zurich, Switzerland
5 Centre for Developmental Genetics, Department of Biomedical Science, University of Sheffield, Western Bank, Sheffield S10 2TN, UK
* Present address: Cancer and Developmental Biology Laboratory, National Cancer Institute, Frederick, MD 21702, USA



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  		Fig. 1. Expression of F3/contactin (red) and TAG-1 (green) in postnatal day 6 cerebellar cortex. The region of TAG-1 and F3/contactin overlap is in yellow. IGL, inner granular layer; EGL, external germinal layer; iEGL, inner region of the external germinal layer. Scale bar: 40 µm.

 


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  		Fig. 2. (A) Promoter/reporter constructs generated by fusing the 5' region of the human TAX1 gene to either a lacZ reporter or the F3/contactin cDNA. (B) Expression of TAG-1 protein and lacZ reporter in the cerebellar cortex of TAG/lacZ transgenic mice at postnatal day 8. (a-c) TAG-1 immunostaining (a) and X-gal staining (c) of sequential sagittal sections obtained at the level of the vermis. In b a double TAG-1/lacZ staining is shown. In a, the lobules are indicated by roman numerals. Scale bars: 200 µm. (d-f) Expression of the TAG-1 protein (d) and of the TAX-1/lacZ transgene (f) in lobules V-VI. Double TAG-1/ lacZ staining is reported in e. Scale bars: 100 µm. (g-i) TAG-1 (g) and lacZ (i) expression in the cerebellar cortex of lobules VIII-IX. h shows a double TAG-1/X-gal staining. Scale bars: 40 µm. Sections c-f-i are counterstained with Fast Red.

 


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  		Fig. 3. Expression profile of the TAX-1/F3 transgene. (A,B) Expression of the TAX-1/F3 fusion mRNA in transgenic mice cerebellum throughout development, demonstrated by RT/PCR. (A) Amplification scheme. (A) TAX1 exon 1-derived forward primer (nt 146-169) (Kozlov et al., 1995Go) was used with an F3/contactin exon VII-derived reverse primer (nt 855-834) (Gennarini et al., 1989Go). (B) A 716 nt band could be detected throughout development. An oligonucleotide pair derived from the mouse actin gene generated a 140 nt band we used as an internal control. (C) Expression profile of F3/contactin in the cerebellar cortex of wild-type (a,c) and TAG/F3 (b,d) mice at postnatal day 4. Scale bars: 40 µm.

 


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  		Fig. 4. Cerebellar phenotype of developing wild-type and TAG/F3 mice shown in sagittal sections stained with Toluidine Blue. (A) Low magnification images of P3 (a,b), P8 (c,d), P11 (e,f) and P16 (g,h) cerebella. In sections from wild-type mice the lobules are indicated by roman numerals. Scale bars: 200 µm. (B) Differences in layer morphology between wild-type (i,k,m,o) and TAG/F3 (j,l,n,p) mice, shown in lobule VIII at P3 (i,j), P8 (k,l), P11 (m,n), P16 (o,p). The different cortical layers are indicated on the right side. EGL, external germinal layer; ML, molecular layer; PCL, Purkinje cells layer; IGL; inner granular layer. Scale bars: (i-n) 40 µm; (o,p) 100 µm. (C) Morphometric analysis of TAG/F3 and wild-type mice cerebella. The overall surface areas of the cerebellar sections, the EGL, IGL and ML, from P0 to P30, were expressed as the ratio of their values in transgenic versus wild-type mice. Asterisks refer to statistical significance of the differences estimated in the absolute values of the different parameters.

 


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  		Fig. 6. Changes in cell death in developing wild-type and TAG/F3 mice cerebella. (A) TUNEL staining in lobule IX of wild-type (WT) and TAG/F3 mice cerebellum at postnatal day 3. Arrows indicate the TUNEL-positive elements, higher magnification views of which are shown in the insets. Scale bar: 100 µm. (B) Histogram showing the density of apoptotic cells in cerebellar sections from both wild type and TAG/F3 mice from P0 to P8. Asterisks refer to statistical significance of the differences observed between the two genotypes. ***P=0.001. (C) Sample of TUNEL-stained elements (arrows) in high density primary cerebellar cultures from postnatal day 3 wild-type and TAG/F3 mice (Bar=10 µm).

 


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  		Fig. 5. (A) Analysis of precursor granule cell proliferation in the developing cerebellum of wild-type and TAG/F3 mice. 5-bromo-2'-deoxyuridine (BrdU) incorporation in lobules VIII-IX of developing cerebellar cortex from wild-type (a,c,e,g,i) and TAG/F3 (b,d,f,h,j) mice at P0 (a-b), P3 (c-d), P6 (e-f), P8 (g-h) and P11 (i-j). Scale bars: 40 µm. (B) Analysis of cell proliferation in primary cultures from postnatal day 3 cerebellum. BrdU incorporation is shown in primary cerebellar cultures from wild type (a,b) or TAG/F3 (c,d) mice labelled immediately after plating (T0) or 16 hours later (T16). Scale bars: 40 µm. In C the data are shown in graphic format. (D) Epifluorescent labelling of P8 cerebellar cortex from wild-type mice with an anti-proliferating cell nuclear antigen (PCNA) monoclonal antibody (a) and a rabbit anti-TAG-1 serum (b). (c) Confocal optical section of the same field as a and b, showing PCNA (red) and TAG-1 (green) expression. (d) Confocal optical section of P8 cerebellar cortex from wild-type mice labelled with polyclonal antibodies to phosphohistones H1 and H3 (red) and monoclonal anti-TAG-1 antibodies (green). Note that phosphohistones H1 and H3 label mitotic cells most strongly, while other phases are marked by weaker phosphohistone H1 expression alone (Lu et al., 1994Go); this image was contrast-enhanced to emphasise cells in mitosis thus de-emphasising labelling in other phases of the cycle. The pial surface is marked by a dashed line. Scale bars: 20 µm.

 


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  		Fig. 7. Developmental profile of F3/contactin expression in the cerebellum of wild-type (a,c,e,g,i,k,m,o,q,s,u,w) and TAG/F3 (b,d,f,h,j,l,n,p,r,t,v,y) mice shown at postnatal days 0 (a,b; k-n), 3 (c,d; o-r), 8 (e,f; s,t), 11 (g,h; u,v) and 16 (i,j; w,y). (A) Low and (B) high power images. In B, parts k, l, o, p correspond to lobules II and III; parts m, n, q, r correspond to lobules VI and VII. Different levels of TAX-1 promoter activity were observed in these lobules (Fig. 2B). Sections were immunostained with F3/contactin antibodies and counterstained with Hematoxylin (blue). Scale bars: (a-j) 200 µm; (k-v) 40 µm; (w-y) 100 µm.

 


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  		Fig. 8. (A) Developmental changes of Purkinje neurons in wild-type (a,c,e,g,i,k,m) and TAG/F3 (b,d,f,h,j,l,n) mice as revealed by calbindin immunostaining at postnatal day (P) 0 (a-d), P3 (e-h), P8 (i,j), P11 (k,l) and P16 (m,n) in thin (5 µm) paraffin sections from cerebellar cortex. High-power images in c and d correspond to lobules II and III, and images in g-n correspond to lobule VIII. Scale bars: (a,b,e,f) 200 µm; (c,d,g-n) 40 µm. (B) Phenotype of Purkinje cells dendritic tree shown in thick (20 µm) cryostat sections from wild type (WT) and TAG/F3 mice cerebella (lobule IX) labelled with calbindin antibodies at postnatal day 6. Bar: 20 µm.

 


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  		Fig. 9. Primary aggregate (A-D) and dissociated (E-F) cultures from postnatal day 7 cerebella derived from either wild-type (A,C,E) or TAG/F3 (B,D,F) mice, stained with anti-F3/contactin (A-D) or anti-GAP43 (E-F) rabbit antibodies. Bars: (A,B) 100 µm; (C,D) 10 µm; (E,F) 40 µm. (G) A cumulative neurite length graph in which the percentage of neurons with neurites of a given length (y axis), is plotted against neurite length (in the x axis).

 

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