QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Lütolf, S. Articles by Taylor, V. Search for Related Content PubMed PubMed Citation Articles by Lütolf, S. Articles by Taylor, V.

Notch1 is required for neuronal and glial differentiation in the cerebellum

Simone Lütolf^1,*, Freddy Radtke^2,*, Michel Aguet², Ueli Suter¹ and Verdon Taylor^1,

¹ Institute of Cell Biology, Department of Biology, Swiss Federal Institute of Technology, CH-8093 Zurich, Switzerland
² Swiss Institute of Experimental Cancer Research, 1066 Epalinges, Switzerland
^* These authors contributed equally to this work

View larger version (23K): [in a new window]   Fig. 1. Notch1 and the transgenic constructs. Notch1 is a type-I transmembrane protein with a cleavable signal peptide followed by 36 EGF-like repeats and three Lin/Notch domains (LN). The intracellular domain contains ankyrin-like repeats, a polyglutamine stretch (Opa) and a protein stability PEST sequence. The first coding exon of the Notch1 gene was flanked by LoxP sequences (triangles) to generate the Floxed Notch1 allele (1) and the null allele for Notch1 generated after Cre-induced recombination (2). EcoRI (RI) restriction sites flanking the Floxed coding exon are shown. The Cre-recombinase was driven from the engrailed-2 promoter enhancer (En2-Cre), which restricted expression to the neuroepithelium of the midbrain-hindbrain boundary (Zinyk et al., 1998). The ROSA26-R Cre-reporter allele (R26R) was used in combination with the En2-Cre transgene and Floxed Notch1 alleles for cell fate tracing. The R26R comprises a Floxed PGK promoter driving a neomycin cassette containing four polyadenylation sequences (4XpA) and a ß-galactosidase coding region (lacZ) with polyadenylation site (bpA) introduced into the ROSA26 locus by homologous recombination (Soriano, 1999). Cre-mediated recombination deletes the PGK neo cassette and results in constitutive expression of ß-galactosidase.

View larger version (47K): [in a new window]   Fig. 2. Determination of the En2-Cre expression domain using the R26R reporter and restricted ablation of Notch1 signaling. (A) Heterozygous Floxed Notch1 (/wt) animals carrying an En2-Cre and R26R allele showed a restricted distribution of Cre-expressing cells (blue staining) and their progeny in the midbrain-hindbrain region of the developing brain at E10. The plane of the sections used in the subsequent analysis is depicted in red. (B) Homozygous Floxed Notch1 (/) littermates carrying an En2-Cre and R26R allele showed a similar distribution of cells expressing ß-galactosidase (blue) in the midbrain-hindbrain region of the brain to control embryos at E10. (C) In situ RNA hybridization on cross-sections through the neural tube, dorsal to the left and ventral to the right, of E10 mutant animals (/) showed normal expression of Notch1 mRNA in the ventral region of the hindbrain neural tube but loss of expression in the medial cerebellar primordium (arrow). (D) Control Floxed Notch1 (lox/lox) animals without an En2-Cre transgene showed normal distribution of Notch1 transcripts throughout the dorsal and ventral region of the hindbrain neural tube including the cerebellar primordium (arrow). (E) The downregulation of Notch1 expression in the medial portion of the cerebellar primordium of the mutants coincides with the region of recombination as indicated by expression of ß-galactosidase from the R26R allele on adjacent sections (F). (G) Hes5 expression was strongly reduced in the medial cerebellar primordium of mutant embryos (/) (arrow). By contrast, control embryos (lox/lox) showed homogeneous expression of Hes5 in the cerebellar primordium (H). Scale bars: in C, 100 µm for C,D,G,H; in E 100 µm for E,F.

View larger version (73K): [in a new window]   Fig. 3. Expression patterns of Notch signaling cascade components at E10. In situ RNA hybridization on cross-sections through the neural tube, dorsal to the left and ventral to the right, of E10 mutant (A,C,E,G) and control (B,D,F,H) embryos show an upregulation of the Notch ligands Dll1 (arrows, A) and Dll3 (arrows, C) in the medial aspect of the mutant cerebellar primordium. Mash1 (E,F) and Math1 (G,H) expression were also upregulated in the mutant cerebellar primordium compared to control littermates. (I,J) Whole-mount in situ RNA hybridization confirmed the ectopic expression of Math1 in the mutant compared with control embryos (arrow, I). Scale bars: in A, 100 µm for A-D; in E, 100 µm for E-H.

View larger version (85K): [in a new window]   Fig. 4. Differentiation state of cells within the cerebellar primordium. Immunofluorescent analysis of cross-sections through the neural tube at E10, dorsal to the left and ventral to the right, shows a reduction in the expression of the intermediate filament protein nestin in the medial aspect of the mutant cerebellar primordium (arrow, A) compared with control littermates (B). Examination of neuronal differentiation genes, such as those encoding ß-tubulinIII (TuJ1) (C,D), and neurofilament 160 (E,F), failed to reveal ectopic formation of differentiated neurons in the cerebellar primordium between E10 mutant and control animals. Calbindin D 28k expression could not be detected in E10 mutant animals at the protein (G) or RNA (H) levels, indicating the absence of precociously differentiated postmitotic Purkinje cells. Bars in A for A,B and in C for C-H are 100 µm.

View larger version (76K): [in a new window]   Fig. 5. Expression patterns of Notch signaling cascade components and neuronal markers calbindin D 28k and ß-tubulinIII (TuJ1) at E12.5. In situ RNA hybridization on cross-sections through the neural tube, dorsal to the left and ventral to the right, of E12.5 mutant (/) (A,C,E,G) and control embryos (lox/lox) (B,D,F,H) show that the extended expression of Dll1 (A,B) observed in E10 mutants compared with controls was no longer seen at E12.5 and the induction of Dll3 (arrow, C,D) and Mash1 (E,F) was detectable but less pronounced than at E10. In situ RNA hybridization did not reveal precocious expression of calbindin D 28k (G,H) in the mutant compared with control animals. Expression of the pan-neuronal marker ß-tubulin (TuJ1) (I,J) revealed differentiated cells in the lateral aspects of the cerebellum (arrows) but no precocious neurogenesis in the mutants. Scale bar: 100 µm.

View larger version (58K): [in a new window]   Fig. 6. Cell survival and fate analysis in the mutant cerebellum. TUNEL analysis (green) for dying cells and nestin (red) for neuroepithelial progenitor cells in the cerebellum failed to show a difference between mutant (A) and control animals (B) at E10. However, at E12.5, mutant animals (C) contained a greatly increased number of TUNEL-positive cells (green; arrow) within the medial aspect of the cerebellar primordium compared to control animals (D). These TUNEL-positive cells were in a position analogous to differentiating neuroblasts and had downregulated expression of nestin. (E,F) The lineage tracing Cre-reporter transgene R26R revealed an extensive number of ß-galactosidase-expressing cells in the midbrain-hindbrain region of control animals carrying heterozygous Floxed Notch1, En2-Cre and R26R transgenes (E). A substantial reduction in the number of ß-galactosidase-expressing cells was observed in the E12.5 mutant embryos (F; homozygous Floxed Notch1, En2-Cre and R26R), compared with control embryos. Scale bars: in A, 100 µm for A,B; in C, 50 µm for C,D.

View larger version (133K): [in a new window]   Fig. 7. Differentiation and fate of Notch1-ablated cells in the cerebellar primordium at E15. (A,B) In situ RNA hybridization for calbindin D 28k expression on sagittal sections of E15 mutant (A; homozygous Floxed Notch1 En2-Cre) and control embryos (B; heterozygous Floxed Notch1 En2-Cre) show no differences in the expression of the Purkinje cell marker. (C,D) Cell fate analysis of mutant (C) and control animals (B) at E15 using the lineage tracer Cre-reporter transgene R26R revealed an absence of ß-galactosidase expressing cells from the cerebellum (CB) of mutant compared with control embryos, where most of the cerebellar cells had undergone R26R recombination. Some ß-galactosidase-expressing cells were detected in the isthmus (IST) and posterior midbrain (pMB) region of the E15 mutant brain. However, the control embryos also showed substantially more ß-galactosidase-expressing cells in these brain regions. (E,F) TUNEL analysis (green; arrows) for dying cells and nestin (red) for neuroepithelial progenitor cells on cross-sections of the hemi-cerebellum (midline to the right and dorsal to the top) at E15 showed no significant difference between mutant (E) and control animals (F). Scale bars: in A, 100 µm for A-D; in E, 100 µm for E,F.

View larger version (70K): [in a new window]   Fig. 8. Morphological analysis of the postnatal cerebellum and determination of cell loss. Examination of the cerebellum of P4 animals by in situ RNA hybridization with a calbindin D 28k probe (A,B) showed a significant reduction in the length of the Purkinje cell layer in mutant (A) compared with control (B) animals (reduced by 50%) without obvious changes in cell density. (C-F) Morphological analysis of the cerebellum from adult homozygous Floxed Notch1 En2-Cre mutant (C,E) and control, homozygous Floxed Notch1, littermates (D,F) revealed a marked reduction in the size and foliation of the medial cerebellum (arrow, C). Histological examination of midline sagittal sections showed aberrant lobulation of the adult mutant cerebellum (E) with a loss of lobe VII compared with control (F) littermates. (G-I) In situ RNA hybridization with a calbindin D 28K probe (G,H) and quantification of lobe length (I), revealed a significant (*; P<0.05 Student’s t-test) reduction in the length of lobe 2/3 and a highly significant (**; P<0.01 Student’s t-test) reduction in the other lobes of the mutant (black bars) with the exception of lobe 10. Owing to the loss of lobe 7, lobe 6 and 7 were measured together, from the posterior end of lobe 5 to the anterior end of lobe 8. Analysis of Purkinje cell number and density (J; data not shown) in adult animals showed a highly significant (**) reduction in cell number within the anterior (A, lobes 2-5) and central (C, lobes 6-8) lobes of the mutant (black bars) as well as over the total cerebellum, but no significant difference in the posterior lobes (P, lobes 9 and 10). (K,L) Lineage tracing using the Cre-reporter transgene R26R confirmed that few Notch1-deficient, ß-galactosidase-expressing Purkinje cells were apparent in the medial cerebellum of adult mutants (homozygous Floxed Notch1 En2-Cre and R26R) (arrow, K), compared with control animals carrying heterozygous Floxed Notch1 En2-Cre and R26R transgenes (L). C, inferior colliculus; GL, granule cell layer; ML, molecular layer; PL, Purkinje cell layer. Scale bars: in A, 1 mm for A,B; in E, 1 mm for E-H; in K, 1 mm for K,L.

View larger version (106K): [in a new window]   Fig. 9. Effects of Notch1 ablation on glial cell formation. (A,B) Immunofluorescent analysis of GFAP expression in the vermis of adult mutant (A) and control (B) animals revealed no obvious differences in the glial cell population or density of Bergmann glia fibers, suggesting that although the size of the vermis is reduced, the proportion and distribution of glial cells is not substantially affected in the mutants. (C) Confocal analysis of midline sagittal sections of adult control (heterozygous Floxed Notch1, En2-Cre, R26R) cerebellum immunostained with anti-GFAP (green) and anti-ß-galactosidase (red) antibodies revealed co-expression in a population of Bergmann glial cells (arrowhead). The soma of a Purkinje cell expressing ß-galactosidase but negative for GFAP is also indicated (asterisk). (D) Quantification of GFAP ß-galactosidase double-positive cells on midline sagittal sections through the cerebellar vermis showed a highly significant (**P<0.01; Student’s t-test) reduction in recombined glial cells in the mutant (7±3%; homozygous Floxed Notch1, En2-Cre, R26R (/) compared with the control animals (66±6%; heterozygous Floxed Notch1, En2-Cre, R26R (/wt)). (E) Isolation of glial cells from the vermis region of the cerebellum of P4 control animals and immunostaining with anti-GFAP (red) and anti-ß-galactosidase (green) antibodies supported the presence of glial cells derived from Cre-recombinase-expressing progenitor cells (arrowheads). ß-Galactosidase-expressing GFAP-negative, putative neurons are indicated (arrows in E). (F) Quantification of four individual experiments to identify GFAP ß-galactosidase-expressing cells in control (/wt) and mutant (/) animals. The percentage of GFAP ß-galactosidase double-positive cells is shown, as is the total number of GFAP-positive cells in each culture (n) and the average of all four experiments (Avr.). Note that the total number of cells in the cultures from mutants was lower than that from control animals due to the 50% reduction in size of the mutant vermis at P4. GL, granule cell layer; ML, molecular layer; W, white matter. Scale bar in A, 0.5 mm for A,B.