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Sonic Hedgehog promotes proliferation of Notch-dependent monociliated choroid plexus tumour cells

View Article: PubMed Central - PubMed

ABSTRACT

Aberrant Notch signaling has been linked to many cancers including choroid plexus (CP) tumours, a group of rare and predominantly pediatric brain neoplasms. We developed animal models of CP tumours by inducing sustained expression of Notch1 that recapitulate properties of human CP tumours with aberrant NOTCH signaling. Whole transcriptome and functional analyses showed that tumour cell proliferation is associated with Sonic Hedgehog (Shh) in the tumour microenvironment. Unlike CP epithelial cells, which have multiple primary cilia, tumour cells possess a solitary primary cilium as a result of Notch-mediated suppression of multiciliate diffferentiation. A Shh-driven signaling cascade in the primary cilium occurs in tumour cells but not in epithelial cells. Lineage studies show that CP tumours arise from mono-ciliated progenitors in the roof plate characterized by elevated Notch signaling. Abnormal SHH signaling and distinct ciliogenesis are detected in human CP tumours, suggesting SHH pathway and cilia differentiation as potential therapeutic avenues.

No MeSH data available.


Related in: MedlinePlus

Constitutive Notch 1 signaling leads to CP tumour. (a) Schematic illustration of the strategy for Notch 1 signaling activation in vivo. (b) Brain hemispheres and CPs from postnatal day 7 (P7) mice are shown. Notice that the enlarged CP from Math1-Cre;Rosa26-NICD1;Rosa26-EYFP (Mcre;NICD1;EYFP) animal contains many EYFP+ cells (arrows), while EYFP+ cells (arrowheads) are sparsely found in the CP from Math1-Cre;Rosa26-EYFP (Mcre;EYFP) animals. Scale bar: 1 mm. (c) Analysis of gene expression in CP of Math1-Cre;Rosa26-EYFP mice. EYFP expression (green) labels cells derived from Atoh1+ progenitors. The expression of Lmx1a (red), Otx2 (red), cytokeratins (red), and Aqp1 (red) labels CP epithelial cells. Scale bar: 25μm. (d) Quantitation of the percentage of EYFP+ cells or NICD1+/GFP+ cells in Otx2+ hindbrain CP epithelium of Mcre;EYFP mice or Math1-Cre;Rosa26-NICD1 (Mcre;NICD1) mice, respectively, at different time points (n=3 tumours from 3 Mcre;NICD1 animals/time point; CPs from Mcre;EYFP mice: n=4 (P0, P14), n=6 (P7, P21), n=5 (P90), data from a single experiment are shown, raw data are available in Supplementary Table 9; mean ± SEM, two-way ANOVA, **, P<0.01; ***, P<0.001; ns, non-significant). (e) H&E staining of CPs from Mcre;NICD1 and wild type (WT) mice at P0 and P14. Notice that CP epithelium exhibits the “hobnail” configuration (arrowhead), while the enlarged CP in Mcre;NICD1 mice appear “flattened” on ventricular surfaces (arrow). Vesicular sac with accumulated CSF is shown (asterisk). Scale bars: white, 500μm; black, 25μm. (f) H&E staining of human CP papilloma (CPP) and normal CP. Scale bar: 25μm. (g) Quantitative RT-PCR (Q-RT-PCR) analysis of Hes1 and Hes5 expression in CP tumours (black circles) and wild type CPs (white circles) at P0, P7, P14, and P21 (data from technical replicates of each specimen set in a single experiment are shown; experiment was not repeated. Raw data can be found in Supplementary Table 9). (h) The expression of Ki-67 is shown in CP tumours from Mcre;NICD1 mice, Lmx1a-Cre;Rosa26-NICD1 (Lcre;NICD1) mice, and normal CPs from wild type mice. Dotted lines mark the boundary of lateral ventricles. The expression of Ki-67 in human CPP and normal CP is shown. Scale bar: 50μm.
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Figure 1: Constitutive Notch 1 signaling leads to CP tumour. (a) Schematic illustration of the strategy for Notch 1 signaling activation in vivo. (b) Brain hemispheres and CPs from postnatal day 7 (P7) mice are shown. Notice that the enlarged CP from Math1-Cre;Rosa26-NICD1;Rosa26-EYFP (Mcre;NICD1;EYFP) animal contains many EYFP+ cells (arrows), while EYFP+ cells (arrowheads) are sparsely found in the CP from Math1-Cre;Rosa26-EYFP (Mcre;EYFP) animals. Scale bar: 1 mm. (c) Analysis of gene expression in CP of Math1-Cre;Rosa26-EYFP mice. EYFP expression (green) labels cells derived from Atoh1+ progenitors. The expression of Lmx1a (red), Otx2 (red), cytokeratins (red), and Aqp1 (red) labels CP epithelial cells. Scale bar: 25μm. (d) Quantitation of the percentage of EYFP+ cells or NICD1+/GFP+ cells in Otx2+ hindbrain CP epithelium of Mcre;EYFP mice or Math1-Cre;Rosa26-NICD1 (Mcre;NICD1) mice, respectively, at different time points (n=3 tumours from 3 Mcre;NICD1 animals/time point; CPs from Mcre;EYFP mice: n=4 (P0, P14), n=6 (P7, P21), n=5 (P90), data from a single experiment are shown, raw data are available in Supplementary Table 9; mean ± SEM, two-way ANOVA, **, P<0.01; ***, P<0.001; ns, non-significant). (e) H&E staining of CPs from Mcre;NICD1 and wild type (WT) mice at P0 and P14. Notice that CP epithelium exhibits the “hobnail” configuration (arrowhead), while the enlarged CP in Mcre;NICD1 mice appear “flattened” on ventricular surfaces (arrow). Vesicular sac with accumulated CSF is shown (asterisk). Scale bars: white, 500μm; black, 25μm. (f) H&E staining of human CP papilloma (CPP) and normal CP. Scale bar: 25μm. (g) Quantitative RT-PCR (Q-RT-PCR) analysis of Hes1 and Hes5 expression in CP tumours (black circles) and wild type CPs (white circles) at P0, P7, P14, and P21 (data from technical replicates of each specimen set in a single experiment are shown; experiment was not repeated. Raw data can be found in Supplementary Table 9). (h) The expression of Ki-67 is shown in CP tumours from Mcre;NICD1 mice, Lmx1a-Cre;Rosa26-NICD1 (Lcre;NICD1) mice, and normal CPs from wild type mice. Dotted lines mark the boundary of lateral ventricles. The expression of Ki-67 in human CPP and normal CP is shown. Scale bar: 50μm.

Mentions: A molecularly-defined boundary exists between the rhombic lip consisting of neural progenitors expressing the transcription factor Atonal Homolog 1 (Atoh1, also known as Math1), and the roof plate, characterized by the expression of Wnt1, Gdf7, and the transcription factor Lmx1a18–21. Some Lmx1a+ cells are present in the rhombic lip and contribute to the cerebellum20, 21. To determine whether rhombic lip progenitors contribute to the roof plate/CP lineage, we utilized Math1-Cre to drive Cre expression in Atoh1+ progenitors22 (Fig. 1a). When crossed with Rosa26-EYFP Cre reporter strain23, the resulting Math1-Cre;Rosa26-EYFP mice have cells expressing enhanced yellow fluorescent protein (EYFP) in the CP in addition to cerebellum (Fig. 1b). Though these EYFP+ cells comprise < 0.5% of hindbrain CP epithelium, they express CP markers Lmx1a, orthodenticle homeobox 2 (Otx2), cytokeratins, and Aquaporin 1 (Aqp1) (Fig. 1c, 1d, Supplementary Fig. 1a), indicating some Atoh1+ progenitors contribute to hindbrain roof plate/CP lineage.


Sonic Hedgehog promotes proliferation of Notch-dependent monociliated choroid plexus tumour cells
Constitutive Notch 1 signaling leads to CP tumour. (a) Schematic illustration of the strategy for Notch 1 signaling activation in vivo. (b) Brain hemispheres and CPs from postnatal day 7 (P7) mice are shown. Notice that the enlarged CP from Math1-Cre;Rosa26-NICD1;Rosa26-EYFP (Mcre;NICD1;EYFP) animal contains many EYFP+ cells (arrows), while EYFP+ cells (arrowheads) are sparsely found in the CP from Math1-Cre;Rosa26-EYFP (Mcre;EYFP) animals. Scale bar: 1 mm. (c) Analysis of gene expression in CP of Math1-Cre;Rosa26-EYFP mice. EYFP expression (green) labels cells derived from Atoh1+ progenitors. The expression of Lmx1a (red), Otx2 (red), cytokeratins (red), and Aqp1 (red) labels CP epithelial cells. Scale bar: 25μm. (d) Quantitation of the percentage of EYFP+ cells or NICD1+/GFP+ cells in Otx2+ hindbrain CP epithelium of Mcre;EYFP mice or Math1-Cre;Rosa26-NICD1 (Mcre;NICD1) mice, respectively, at different time points (n=3 tumours from 3 Mcre;NICD1 animals/time point; CPs from Mcre;EYFP mice: n=4 (P0, P14), n=6 (P7, P21), n=5 (P90), data from a single experiment are shown, raw data are available in Supplementary Table 9; mean ± SEM, two-way ANOVA, **, P<0.01; ***, P<0.001; ns, non-significant). (e) H&E staining of CPs from Mcre;NICD1 and wild type (WT) mice at P0 and P14. Notice that CP epithelium exhibits the “hobnail” configuration (arrowhead), while the enlarged CP in Mcre;NICD1 mice appear “flattened” on ventricular surfaces (arrow). Vesicular sac with accumulated CSF is shown (asterisk). Scale bars: white, 500μm; black, 25μm. (f) H&E staining of human CP papilloma (CPP) and normal CP. Scale bar: 25μm. (g) Quantitative RT-PCR (Q-RT-PCR) analysis of Hes1 and Hes5 expression in CP tumours (black circles) and wild type CPs (white circles) at P0, P7, P14, and P21 (data from technical replicates of each specimen set in a single experiment are shown; experiment was not repeated. Raw data can be found in Supplementary Table 9). (h) The expression of Ki-67 is shown in CP tumours from Mcre;NICD1 mice, Lmx1a-Cre;Rosa26-NICD1 (Lcre;NICD1) mice, and normal CPs from wild type mice. Dotted lines mark the boundary of lateral ventricles. The expression of Ki-67 in human CPP and normal CP is shown. Scale bar: 50μm.
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Figure 1: Constitutive Notch 1 signaling leads to CP tumour. (a) Schematic illustration of the strategy for Notch 1 signaling activation in vivo. (b) Brain hemispheres and CPs from postnatal day 7 (P7) mice are shown. Notice that the enlarged CP from Math1-Cre;Rosa26-NICD1;Rosa26-EYFP (Mcre;NICD1;EYFP) animal contains many EYFP+ cells (arrows), while EYFP+ cells (arrowheads) are sparsely found in the CP from Math1-Cre;Rosa26-EYFP (Mcre;EYFP) animals. Scale bar: 1 mm. (c) Analysis of gene expression in CP of Math1-Cre;Rosa26-EYFP mice. EYFP expression (green) labels cells derived from Atoh1+ progenitors. The expression of Lmx1a (red), Otx2 (red), cytokeratins (red), and Aqp1 (red) labels CP epithelial cells. Scale bar: 25μm. (d) Quantitation of the percentage of EYFP+ cells or NICD1+/GFP+ cells in Otx2+ hindbrain CP epithelium of Mcre;EYFP mice or Math1-Cre;Rosa26-NICD1 (Mcre;NICD1) mice, respectively, at different time points (n=3 tumours from 3 Mcre;NICD1 animals/time point; CPs from Mcre;EYFP mice: n=4 (P0, P14), n=6 (P7, P21), n=5 (P90), data from a single experiment are shown, raw data are available in Supplementary Table 9; mean ± SEM, two-way ANOVA, **, P<0.01; ***, P<0.001; ns, non-significant). (e) H&E staining of CPs from Mcre;NICD1 and wild type (WT) mice at P0 and P14. Notice that CP epithelium exhibits the “hobnail” configuration (arrowhead), while the enlarged CP in Mcre;NICD1 mice appear “flattened” on ventricular surfaces (arrow). Vesicular sac with accumulated CSF is shown (asterisk). Scale bars: white, 500μm; black, 25μm. (f) H&E staining of human CP papilloma (CPP) and normal CP. Scale bar: 25μm. (g) Quantitative RT-PCR (Q-RT-PCR) analysis of Hes1 and Hes5 expression in CP tumours (black circles) and wild type CPs (white circles) at P0, P7, P14, and P21 (data from technical replicates of each specimen set in a single experiment are shown; experiment was not repeated. Raw data can be found in Supplementary Table 9). (h) The expression of Ki-67 is shown in CP tumours from Mcre;NICD1 mice, Lmx1a-Cre;Rosa26-NICD1 (Lcre;NICD1) mice, and normal CPs from wild type mice. Dotted lines mark the boundary of lateral ventricles. The expression of Ki-67 in human CPP and normal CP is shown. Scale bar: 50μm.
Mentions: A molecularly-defined boundary exists between the rhombic lip consisting of neural progenitors expressing the transcription factor Atonal Homolog 1 (Atoh1, also known as Math1), and the roof plate, characterized by the expression of Wnt1, Gdf7, and the transcription factor Lmx1a18–21. Some Lmx1a+ cells are present in the rhombic lip and contribute to the cerebellum20, 21. To determine whether rhombic lip progenitors contribute to the roof plate/CP lineage, we utilized Math1-Cre to drive Cre expression in Atoh1+ progenitors22 (Fig. 1a). When crossed with Rosa26-EYFP Cre reporter strain23, the resulting Math1-Cre;Rosa26-EYFP mice have cells expressing enhanced yellow fluorescent protein (EYFP) in the CP in addition to cerebellum (Fig. 1b). Though these EYFP+ cells comprise < 0.5% of hindbrain CP epithelium, they express CP markers Lmx1a, orthodenticle homeobox 2 (Otx2), cytokeratins, and Aquaporin 1 (Aqp1) (Fig. 1c, 1d, Supplementary Fig. 1a), indicating some Atoh1+ progenitors contribute to hindbrain roof plate/CP lineage.

View Article: PubMed Central - PubMed

ABSTRACT

Aberrant Notch signaling has been linked to many cancers including choroid plexus (CP) tumours, a group of rare and predominantly pediatric brain neoplasms. We developed animal models of CP tumours by inducing sustained expression of Notch1 that recapitulate properties of human CP tumours with aberrant NOTCH signaling. Whole transcriptome and functional analyses showed that tumour cell proliferation is associated with Sonic Hedgehog (Shh) in the tumour microenvironment. Unlike CP epithelial cells, which have multiple primary cilia, tumour cells possess a solitary primary cilium as a result of Notch-mediated suppression of multiciliate diffferentiation. A Shh-driven signaling cascade in the primary cilium occurs in tumour cells but not in epithelial cells. Lineage studies show that CP tumours arise from mono-ciliated progenitors in the roof plate characterized by elevated Notch signaling. Abnormal SHH signaling and distinct ciliogenesis are detected in human CP tumours, suggesting SHH pathway and cilia differentiation as potential therapeutic avenues.

No MeSH data available.


Related in: MedlinePlus