Neuroscience

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

Odessa R Yabut author has email address
Hector Gomez
Jessica Arela
Jesse Garcia Castillo
Thomas Ngo
Samuel J Pleasure author has email address

Department of Neurology, Weill Institute for Neuroscience, University of California San Francisco, San Francisco, United States
Department of Psychiatry, Weill Institute for Neuroscience, University of California San Francisco, San Francisco, United States
Programs in Neuroscience and Developmental Biology, Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California San Francisco, San Francisco, United States

https://doi.org/10.7554/eLife.100767.2

Open access
Copyright information

Figures and data

Upregulated FGF5 expression in MB^SHH tumors from infant patients.
(A) Levels of FGF5 expression in human MB tumors of all ages from GEO expression dataset #GSE85217(Cavalli et al., 2017). (B, C) Statistical analysis of FGF5 expression levels associated with MB tumor subtypes from patients across all ages (B) and 0-3 year old MB patients (C). **p<0.01, ****p<0.0001. (D, E) 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.

Increased FGF5 expression coincides with region-specific expansion of GNPs in the P0 Sufu-cKO cerebellum.
(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 Scale bars: Scale bars = 250 mm. (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 mm. (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 mm. **p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Ectopic activation of FGF signaling in the EGL of P0 Sufu-cKO cerebellum.
(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. (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.
Created with BioRender.com/g44j117.

Evidence of pre-neoplastic lesions and high rates of cell death in Sufu-cKO granule neuron precursors.
(A) Double-immunofluorescence staining with Pax6 (red) and (γH2AX (green), a marker for double-strand DNA breaks in specific 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 p53 protein levels in P0 control and Sufu-cKO cerebellar protein lysates. *p<0.05. (F) Hematoxylin and Eosin (H&E) staining of P60 control, Sufu-cKO, Sufu;p53-dKO cerebella. Scale bars = 500 μm. (G, H) Double-immunofluorescence staining against Pax6 (red) and Ki-67 (green) in the P0 control, p53-cKO, and Sufu;p53-dKO cerebellum (G). Boxed regions in G are magnified in H, demonstrating the expansion of the EGL in the P0 Sufu;p53-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;p53-dKO and control cerebellum. Boxed areas are enlarged to show ectopic localization of Fgf5+ cells in the EGL of the Sufu-p53-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;p53-dKO and control cerebelli. Boxed regions show cells double-labeled with pErk1/2+ and Ki-67+ cells in the control and Sufu;p53-dKO EGL Region B. Scale bars = 25 μm.

Loss of Sufu function drives excess proliferation of granule neuron precursors via FGF signaling activation.
The schematic diagram models how Sufu LOF facilitates the expansion of GNPs (yellow cells) in the EGL at the early stages of cerebellar development. We hypothesize that Fgf5-expressing cells (blue cells, yet to be identified) send FGF5 signal 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.com/h05q807.

Reduced SHH signaling activity in the P0 Sufu-cKO cerebellum.
(A) Western blot analysis of cerebellar protein lysates from P0 control and Sufu-cKO mice showing significantly lower levels of total and cleaved versions of Gli transcription factors in the P0 Sufu-cKO cerebellum. *p<0.05, **p<0.01 (B) b-galactosidase activity (blue), representing the Gli1-LacZ transgene, is largely absent in areas adjacent to the EGL along the secondary (sec) fissure of the P0 Sufu-cKO cerebellum.

Evidence of pre-neoplastic lesions and high rates of cell death in Sufu-cKO granule neuron precursors.
(A) Double-immunofluorescence staining with (γH2AX (green) and cleaved-caspase 3 (CC3; red), a marker for apoptotic cells, and DAPI labeling in regions A and B. Scale bars = 50 μm (B) Quantification of the density of cells labeled with CC3 and (γH2AX within each EGL regions. *p<0.05, ***p<0.001.

Sign up for email alerts