Widespread contribution of Gdf7 lineage to cerebellar cell types and implications for hedgehog-driven medulloblastoma formation

Abstract

The roof plate is a specialized embryonic midline tissue of the central nervous system that functions as a signaling center regulating dorsal neural patterning. In the developing hindbrain, roof plate cells express Gdf7 and previous genetic fate mapping studies showed that these cells contribute mostly to non-neural choroid plexus epithelium. We demonstrate here that constitutive activation of the Sonic hedgehog signaling pathway in the Gdf7 lineage invariably leads to medulloblastoma. Lineage tracing analysis reveals that Gdf7-lineage cells not only are a source of choroid plexus epithelial cells, but are also present in the cerebellar rhombic lip and contribute to a subset of cerebellar granule neuron precursors, the presumed cell-of-origin for Sonic hedgehog-driven medulloblastoma. We further show that Gdf7-lineage cells also contribute to multiple neuronal and glial cell types in the cerebellum, including glutamatergic granule neurons, unipolar brush cells, Purkinje neurons, GABAergic interneurons, Bergmann glial cells, and white matter astrocytes. These findings establish hindbrain roof plate as a novel source of diverse neural cell types in the cerebellum that is also susceptible to oncogenic transformation by deregulated Sonic hedgehog signaling.

Figure 1. Shh pathway activation in Gdf7-lineage cells leads to cerebellar hyperplasia.

Gdf7^Cre/+;SmoM2 gain-of-function mutant mice exhibit cerebellar defects. (A–B′) Hematoxylin-eosin staining of wild-type and Gdf7^Cre/+;SmoM2 mutants. Prior to P10 histological sections of mutants are similar to control. (C–E′) Gdf7^Cre/+;SmoM2 mutants over 14 days old develop ectopic foci of densely packed cells within the molecular layer of their cerebella. Higher magnification view of these foci reveals no discernible layer organization and resemblance to neoplastic lesions. Arrows in (D) indicate regions of hypercellularity. Boxed regions E and E″ are magnified and shown in the right adjacent panels. Arrows in D′ indicate nuclear molding. Arrowheads in D′ indicate apoptotic nuclei. (F-F″) The ectopic foci consist of cells of the Gdf7-lineage as indicated by their expression of SmoM2-YFP. (G-G″) Ectopic foci do not express differentiated neuronal marker NeuN. Abbreviations: EGL, external granular layer. ML, molecular layer. IGL, internal granular layer. T, tumor region. N, normal cerebellum.

Figure 2. Gdf7^Cre/;SmoM2 mice develop medulloblastoma with CGNP features.

(A–B″′) In situ hybridization of wild-type and Gdf7^Cre/+;SmoM2 mutants. The aberrant tissue foci of Gdf7^Cre/+;SmoM2 cerebella display high level Shh signaling as determined by Gli1 and Ptch1 expression, and Nmyc and Math1 expression. (C–H″′) Gdf7^Cre/+;SmoM2 mice develop medulloblastoma consisting of cells of the CGNP fate. Tumors in Gdf7^Cre/+;SmoM2 mice and adult Patched1^LacZ/+;SmoM2 mice appear very similar. Abbreviations: ML, molecular layer. PCL, Purkinje cell layer. IGL, internal granular layer. T, tumor region. N, normal cerebellum.

Figure 3. A subset of Gdf7-lineage cells express neural stem cell markers.

(A–B″′) Many cells within the tumor tissue of Gdf7^Cre/+;SmoM2 mice coexpress neural stem cell marker Nestin (red) and glial marker GFAP (green). Arrows indicate co-localization. (C–E) Tumor cell lines can be invariably established from Gdf7^Cre/+;SmoM2 cerebella. (F–G″) These cells highly express multiple neural stem cell markers Nestin and Sox2 and can undergo serial passages. (H–J) Here, one representative colony is shown when cultured for 3 and 6 days. (K–N) Gdf7^Cre/+;SmoM2 cultured cells can differentiate into mature cerebellar cell types upon switching to culture media containing 10% FBS.

Figure 4. Roof plate cells give rise to a distinct population of cerebellar vermal radial glia cells.

(A) Whole mount X-gal staining for ß-galactosidase in Gdf7^Cre/+;ROSA^lacZ embryos shows that Gdf7 lineage is present in the hindbrain roof plate at E10. (B) At E14.5, in addition to hindbrain choroid plexus, Gdf7 lineage also contributes to the ventricular zone of cerebellar vermis (red arrows). (C–F) In situ hybridization at E14.5 showing Gdf7 and its transcriptional target Msx1 are expressed in the cerebellar vermis. (G–H′) Fate-mapping of the Gdf7-lineage in Gdf7^Cre/+;ROSA^eYFP cerebella indicates that Gdf7 is present at E14.5 in a distinct population of vermal radial glia cells as highlighted by coexpression of YFP and BLBP (G-G′) or Sox2 (H-H′) Arrows indicate colocalization. (I–K) In situ hybridization at P18 shows that the Math1+ Gdf7^Cre/+;SmoM2 tumor tissue does not express Gdf7 or Msx1. Abbreviations: N, normal cerebellar tissue. T, tumor tissue. CB, cerebellum. uRL, upper rhombic lip.

Figure 5. Gdf7-lineage cells in the cerebellar rhombic lip are Lmx1a+ neural progenitor cells that give rise to CGNPs.

(A–B″) Fate-mapping studies in Gdf7^Cre/+;ROSA26^eYFP;Math1^GFP/GFP at E14.5 show that Gdf7-lineage cells (as indicated by b-gal, green) are Math1-negative (as indicated by GFP) in sagittal sections of the upper rhombic lip. However these cells retain the capacity to express Math1 in the EGL. Arrows indicate delaminating Gdf7-lineage cells from the upper rhombic lip en route to the EGL. (C–F″) Fate-mapping studies in Gdf7^Cre/;ROSA26^eYFP mice at E14.5 show that delaminating Gdf7-lineage cells (green, arrows) are present in sagittal sections of the cerebellar rhombic lip and are Pax6- (C″, red), Lmx1a+ (D″, red), Sox2+ (E″, red), and BLBP- (F″, red). (G-G″) Some delaminating cells are also Tbr2+. Arrows indicate delaminating YFP-positive cells. (H–I″) Fate-mapping studies in P6 sagittal sections show co-localization of YFP and Ki67 (red), demonstrating that Gdf7-lineage cells contribute to proliferating cells in the EGL. Arrows indicate double-positive cells. Rectangle in (H) indicates enlarged area in (I). (J-J″) Tbr2-immunostaining at P8 demonstrates that Gdf7-lineage cells can also contribute to the cerebellar population of unipolar brush cells. Abbreviations: EGL, external granular layer. uRL, upper rhombic lip. RP, roof plate. Scale bar, 20 µm.

Figure 6. Gdf7-lineage cells contribute to an extensive array of mature cerebellar cell types.

(A–F″) Fate-mapping studies in Gdf7^Cre/+;ROSA26^eYFP adult mice (age 5 months) show that Gdf7-lineage cells distribute to all the mature cerebellar tissue layers and contribute to granule neurons (A–B″), Purkinje neurons (C-C″), GABAergic interneurons (D-D″), Bergmann glia (E-E″), white matter astrocytes (F-F″), as well as non-neural hindbrain choroid plexus epithelial cells (data not shown). (G) Representative image of distribution of Gdf7-lineage cells in the adult mouse cerebellum (age 5 months). The locations of Gdf7-lineage cell nuclei (dots) were mapped by tracing from digital images of sagittal sections. Thick lines indicate the cerebellar surface; thin lines indicate the boundary between cerebellar cortex and white matter. Gdf7-lineage cells were located mainly in the internal granular layer with fewer in the PCL and ML. Cells in the IGL were concentrated in lobules III–V and IX. Roman numerals indicate lobules of the vermis. (H) Quantification of YFP+ granule neurons in the IGL per lobule. Data are mean of n = 10 sagittal sections of Gdf7^Cre/+;ROSA26^eYFP age 5 months. Abbreviations: EGL, external granular layer. ML, molecular layer. IGL, internal granular layer. WM, white matter. Scale bar, 100 µm.