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First published online 16 February 2005
doi: 10.1242/dev.01682

Development 132, 1327-1336 (2005)
Published by The Company of Biologists 2005
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A splice variant of the Wilms' tumour suppressor Wt1 is required for normal development of the olfactory system

Nicole Wagner1,*, Kay-Dietrich Wagner1,*, Annette Hammes2, Karin M. Kirschner3, Valerie P. Vidal1, Andreas Schedl1 and Holger Scholz3,{dagger}

1 INSERM U636, Centre de Biochimie, Faculté des Sciences, Nice, France
2 Max-Delbrück-Centrum für Molekulare Medizin, Berlin, Germany
3 Johannes-Müller-Institut für Physiologie, Charité-Universitätsmedizin Berlin, Germany



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  		Fig. 1. Immunostaining of Wt1, neural cell adhesion molecule (NCAM) and glial fibrillary acidic protein (GFAP) in the olfactory epithelium of a wild-type mouse embryo at E18.5. Wt1 was detected in the nuclei of a significant proportion of cells at the base of the olfactory epithelium (a). Double-immunostaining of NCAM (green fluorescence) and Wt1 (red fluorescence) revealed an overlapping pattern of both proteins (Ac). By contrast, Wt1 and glial fibrillary acidic protein (GFAP) were not co-localized in cells of the olfactory epithelium (B). The results shown are representative for the more than 20 tissue sections from five different embryos. Scale bars: 50 µm.

 


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  		Fig. 2. Haematoxylin-Eosin (HE) staining of horizontal tissue sections through the forebrain of E12.5 wild-type (Wt1+/+) embryos and embryos with complete disruption of Wt1 (Wt1-/-). (A-D) The olfactory epithelium is thinner in the Wt1-deficient than in the the Wt1+/+ embryo. (E,F) The vomeronasal organs (arrows) were normally developed in the Wt1-/- embryos. The tissue sections are representative for the three different animals of each group analysed. Scale bars: 100 µm.

 


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  		Fig. 3. HE-staining of horizontal tissue sections through the olfactory epithelia and olfactory bulbs of mouse embryos at E12.5 and E18.5. The sections were cut from wild-type embryos (Wt1+/+) and from age-matched mice with specific lack either of the Wt1(-KTS) or the Wt1(+KTS) splice variant. Strikingly, the Wt1(+KTS)-deficient embryos (I-K) exhibited thinner olfactory epithelia than wild-type animals (A-C) and embryos with lack of the Wt1(-KTS) protein (E-G). (D,H,L) The olfactory bulb in the Wt1(+KTS)-deficient (L) embryo is reduced in size and hypocellular compared with the wild-type embryos (D). Olfactory bulb morphology appeared normal in embryos with inactivation of Wt1(-KTS) (H). Representative data for five embryos from each group are shown. Scale bars: 100 µm.

 


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  		Fig. 4. Immunostaining of Wt1 (A), TUNEL-labelling of apoptotic cells (B) and Ki-67 staining of proliferating cells (C) in the olfactory epithelium of a wild-type embryo at E18.5 and in age-matched embryos with splice-specific Wt1 defects. The results shown are representative of the more than 20 tissue sections that were obtained from five animals in each group. (B) More TUNEL-positive (apoptotic) cells were present in the olfactory epithelia of embryos with lack of the +KTS isoform than in wild-type and Wt1(-KTS)-deficient mice. (C) The number of Ki-67 positive cells was slightly reduced in mouse embryos with lack of Wt1(+KTS) compared with normal and Wt1(-KTS)-deficient embryos. Scale bars: 100 µm.

 


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  		Fig. 5. Immunostaining of proneural transcription factors in the olfactory epithelia of normal embryos at E18.5 (A-C), and of mice with inactivation either of the Wt1(-KTS) (D-F) or the Wt1(+KTS) (G-I) splice variant. Expression of the mammalian homologue of achaete-scute complex, Mash1 (Ascl1), was reduced in the olfactory epithelia of Wt1(+KTS)-deficient mice (G). The basic helix-loop-helix transcription factor neurogenin1, which is activated by Mash1 (Cau et al., 1997Go), was detected in fewer olfactory epithelial cells in embryos with lack of the Wt1(+KTS) product (H) compared with wild-type (B) and Wt1(-KTS)-deficient (E) mice. By comparison, only subtle differences in the expression of Pou4f1 were detectable between the different groups (E,F,I). Scale bars: 50 µm.

 


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  		Fig. 6. Mash1 mRNA (A) and protein (B) in human embryonic kidney (HEK) 293 cells, which had been stably transfected either with empty expression vector (HEK293) or with the Wt1(-KTS) and Wt1(+KTS) splice variants, respectively. (A) Mash1, GAPDH and Wt1 transcripts were detected by reverse transcription PCR. Data shown are representative for the three independent clones that were analysed. Stable expression of Wt1(+KTS), but not of the -KTS variant, induced Mash1 mRNA in HEK293 cells. (B) Stimulation of Mash1 by the Wt1(+KTS) product was confirmed by immunoblotting with a polyclonal anti-Mash1 antibody. (C) A partially overlapping pattern of Wt1 (green) and Mash1 (red) was revealed by double-immunostaining in cells of the developing olfactory epithelium (E18.5). Scale bars: 100 µm in C, parts a, b, c; 10 µm in C, part d.

 


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  		Fig. 7. Representative HE-staining of the developing eyes of mouse embryos at E12.5 (A) and E18.5 (B). Strikingly, the Wt1(-KTS)-deficient embryos exhibited thinner retinas with fewer cells (A, parts c, d) than age-matched normal mice (A, parts a, b). For comparison, the ocular phenotype was less severe in embryos with inactivated Wt1(+KTS) (A, parts e, f). (B) Abnormal morphology of the retina, in particular the reduced cell density in the future ganglion cell layer, was clearly evident in Wt1(-KTS)-deficient embryos at E18.5 (part d; compare with b and f). Scale bars: 10 µm in B, parts b, d, f; 50 µm in A, parts b, d, f; 50 µm in B, parts a, c, e; 100 µm in A, parts a, c, e.

 


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  		Fig. 8. Immunofluorescent labelling of Wt1 and Pou4f2 in the developing retinas of mouse embryos at E18.5. Loss of retinal ganglion cells in mice with lack of Wt1(-KTS) (C,D) is reflected in the reduction of Wt1- and Pou4f2-positive cells compared with normal (Wt1+/+; A,B) and Wt1(+KTS)-deficient (E,F) embryos. Notably, the class IV POU domain factor, Pou4f2 (formerly Brn3b), was recently identified as a transcriptional target of Wt1(-KTS) (Wagner et al., 2002aGo; Wagner et al., 2003Go). Scale bars: 50 µm.

 

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© The Company of Biologists Ltd 2005