Journal: eLife

Article Title: Aberrant FGF signaling promotes granule neuron precursor expansion in SHH subgroup infantile medulloblastoma

doi: 10.7554/eLife.100767

Figure Lengend Snippet: ( A ) Levels of FGF5 expression in human medulloblastoma (MB) tumors of all ages from GEO expression dataset #GSE85217 . ( B, C ) Statistical analysis of FGF5 expression levels associated with MB tumor subtypes from patients across all ages ( B ) and 0–3 years old MB patients ( C ). **p<0.01, ****p<0.0001. ( D, E ) The graph represents FGF5 expression levels in human MB SHH tumors of all ages from GEO expression dataset #GSE85217 ( D ) and corresponding plots ( E ) showing statistically higher FGF5 expression in tumors from infants with MB SHH compared to tumors from children or adults with MB SHH . ****p<0.0001. Figure 1—source data 1. Raw data for counts.

Article Snippet: RNAScope probes for Mm- Fgf5 were designed commercially by the manufacturer (Cat# 417091, Advanced Cell Diagnostics, Inc).

Techniques: Expressing

Journal: eLife

Article Title: Aberrant FGF signaling promotes granule neuron precursor expansion in SHH subgroup infantile medulloblastoma

doi: 10.7554/eLife.100767

Figure Lengend Snippet: ( A ) Pax6 (red) and DAPI (blue) immunofluorescence staining of the P0 Sufu-cKO and control cerebelli. Arrow points to severely expanded EGL region B in the P0 Sufu-cKO cerebellum. EGL regions are designated in DAPI-labeled sections as A (light blue), B (magenta), and C (yellow). Each region encompasses specific fissures: the preculminate (pc) and primary (pr) fissures for region A, the secondary (sec) fissure for region B, and the posterolateral (pl) fissure for region C. Scale bars: Scale bars = 250 μm. ( B–D ) Quantification and comparison of the cerebellar perimeter ( B ), total area occupied by densely packed Pax6+ cells ( C ), and size of specific EGL regions ( D ) between P0 Sufu-cKO and control cerebelli. ( E ) Fluorescent in situ hybridization using RNAScope probes against Fgf5 mRNA (red) shows the expansion of Fgf5 expression in the P0 Sufu-cKO cerebellum compared to controls. Sections are counterstained with DAPI to distinguish structures. Boxed areas are magnified in (F). Scale bars = 500 μm. ( F ) Fgf5 is ectopically expressed in cells within the EGL of the P0 Sufu-cKO cerebellum. Boxed areas within the EGL show DAPI-labeled cells expressing visibly high levels of Fgf5 , identified as punctate labeling (arrowheads), in the EGL of P0 Sufu-cKO cerebellum compared to controls. Scale bars = 50 μm. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Figure 2—source data 1. Raw data for counts.

Article Snippet: RNAScope probes for Mm- Fgf5 were designed commercially by the manufacturer (Cat# 417091, Advanced Cell Diagnostics, Inc).

Techniques: Immunofluorescence, Staining, Control, Labeling, Comparison, In Situ Hybridization, RNAscope, Expressing

Journal: eLife

Article Title: Aberrant FGF signaling promotes granule neuron precursor expansion in SHH subgroup infantile medulloblastoma

doi: 10.7554/eLife.100767

Figure Lengend Snippet: ( A ) Schematic diagram showing the activation of FGF signaling activity upon binding of FGF5 to extracellular domains of FGFR via the MAPK signal transduction pathway. Created with BioRender . ( B ) Double-immunofluorescence staining with Ki-67 (green) and phospho-Erk1/2 (pErk1/2; red), a marker of activated MAPK signaling in the P0 Sufu-cKO and control cerebelli. Boxed regions show pErk1/2+and Ki-67+ cells (arrowheads) in the control and Sufu-cKO EGL. Scale bars = 50 μm. ( C, D ) Quantification of pErk1/2+ cells ( C ) and double-labeled pErk1/2+and Ki-67+ cells ( D ) in the P0 Sufu-cKO and control EGL region B. **p<0.01. ( E ) Experimental design of rescue studies performed by intraventricular administration of FGFR1-3 pharmacological inhibitor, AZD4547, or vehicle controls. ( F ) Nissl staining of the P7 control and Sufu-cKO treated with either AZD4547 or vehicle, 2 days after treatment. Scale bars = 500 μm. ( G ) NeuN and Ki-67 double immunofluorescence staining of the P7 control and Sufu-cKO treated with AZD4547. Boxed regions show localization and organization of NeuN+ and Ki-67+ cells in distinct cerebellar layers. Arrows point to areas of the EGL and IGL where NeuN+ cells are beginning to be expressed. Figure 3—source data 1. Raw data for counts.

Article Snippet: RNAScope probes for Mm- Fgf5 were designed commercially by the manufacturer (Cat# 417091, Advanced Cell Diagnostics, Inc).

Techniques: Activation Assay, Activity Assay, Binding Assay, Transduction, Double Immunofluorescence Staining, Marker, Control, Labeling, Staining

Journal: eLife

Article Title: Aberrant FGF signaling promotes granule neuron precursor expansion in SHH subgroup infantile medulloblastoma

doi: 10.7554/eLife.100767

Figure Lengend Snippet: ( A ) Double-immunofluorescence staining with Pax6 (red) and γH2AX (green), a marker for double-strand DNA breaks in specific external granule layer (EGL) regions of the P0 Sufu-cKO and control cerebella. ( B ) Quantification of γH2AX+ cells in each cerebellar region of P0 control and Sufu-cKO mice. **p<0.01. ( C ) Western blot analysis of Trp53 protein levels in P0 control and Sufu-cKO cerebellar protein lysates. *p<0.05. ( F ) H&E staining of P60 control, Sufu-cKO, Sufu;Trp53-dKO cerebella. Scale bars = 500 μm. ( G, H ) Double-immunofluorescence staining against Pax6 (red) and Ki-67 (green) in the P0 control, Trp53-cKO, and Sufu;Trp53-dKO cerebellum ( G ). Boxed regions in (G) are magnified in (H), demonstrating the expansion of the EGL in the P0 Sufu;Trp53-dKO cerebellum compared to littermate controls. Scale bars = 200 μm ( A ) and 50 μm ( B ). ( I ) Fluorescent in situ hybridization using RNAScope probes against Fgf5 mRNA (red) and DAPI labeling in the P0 Sufu;Trp53-dKO and control cerebellum. Boxed areas are enlarged to show ectopic localization of Fgf5 + cells in the EGL of the Sufu-Trp53-dKO cerebellum, unlike in controls. Scale bars = 200 μm and 50 μm (boxed area). ( J ) Double-immunofluorescence staining with Ki-67 (green) and phospho-Erk1/2 (pErk1/2; red) in the P0 Sufu;Trp53-dKO and control cerebelli. Boxed regions show cells double-labeled with pErk1/2+and Ki-67+ cells in the control and Sufu;Trp53-dKO EGL region B. Scale bars = 25 μm. Figure 4—source data 1. Raw data for counts.

Article Snippet: RNAScope probes for Mm- Fgf5 were designed commercially by the manufacturer (Cat# 417091, Advanced Cell Diagnostics, Inc).

Techniques: Double Immunofluorescence Staining, Marker, Control, Western Blot, Staining, In Situ Hybridization, RNAscope, Labeling

Journal: eLife

Article Title: Aberrant FGF signaling promotes granule neuron precursor expansion in SHH subgroup infantile medulloblastoma

doi: 10.7554/eLife.100767

Figure Lengend Snippet: The schematic diagram models how Sufu loss of function (LOF) facilitates the expansion of granule neuron precursors (GNPs) (yellow cells) in the external granule layer (EGL) at the early stages of cerebellar development. We hypothesize that Fgf5 -expressing cells (blue cells, yet to be identified) send FGF5 signals to GNP to proliferate and that ectopic expression of Fgf5 in the absence of SUFU is responsible for the uncontrolled expansion of EGL localized GNPs. Created with BioRender ..

Article Snippet: RNAScope probes for Mm- Fgf5 were designed commercially by the manufacturer (Cat# 417091, Advanced Cell Diagnostics, Inc).

Techniques: Expressing