Activation of Sonic hedgehog signaling in neural progenitor cells promotes glioma development in the zebrafish optic pathway

Abstract

Dysregulation of Sonic hedgehog (Shh) signaling has been implicated in glioma pathogenesis. Yet, the role of this pathway in gliomagenesis remains controversial because of the lack of relevant animal models. Using the cytokeratin 5 promoter, we ectopically expressed a constitutively active zebrafish Smoothened (Smoa1) in neural progenitor cells and analyzed tumorigenic capacity of activated Shh signaling in both transient and stable transgenic fish. Transient transgenic fish overexpressing Smoa1 developed retinal and brain tumors, suggesting smoa1 is oncogenic in the zebrafish central nervous system (CNS). We further established stable transgenic lines that simultaneously developed optic pathway glioma (OPG) and various retinal tumors. In one of these lines, up to 80% of F1 and F2 fish developed tumors within 1 year of age. Microarray analysis of tumor samples showed upregulated expression of genes involved in the cell cycle, cancer signaling and Shh downstream targets ptc1, gli1 and gli2a. Tumors also exhibited specific gene signatures characteristic of radial glia and progenitor cells as transcriptions of radial glia genes cyp19a1b, s100β, blbp, gfap and the stem/progenitor genes nestin and sox2 were significantly upregulated. Overexpression of GFAP, S100β, BLBP and Sox2 was confirmed by immunofluorescence. We also detected overexpression of Mdm2 throughout the optic pathway in fish with OPG, therefore implicating the Mdm2-Tp53 pathway in glioma pathogenesis. In conclusion, we demonstrate that activated Shh signaling initiates tumorigenesis in the zebrafish CNS and provide the first OPG model not associated with neurofibromatosis 1.

Figure 1

GFP expression patterns in the Tg(krt5:EGFP) line. (a) Skin epithelia expression in a 24 h.p.f. embryo (side view, anterior to the left). (b) Confocal imaging of GFP-positive cells in the brain of a 72 h.p.f. larva (dorsal view, anterior left). (c) Saggital cryosection of an adult fish brain showing GFP expression in the ventricular zones, the OT and the hindbrain (HB). (d) Zoomed-in view of the white frame in (c) showing GFP-positive cells in the ventricular zones resembling radial glia cells partially positive for GFAP (red). (e) GFP expression in the ONH showing little overlap with the retinal Müller glial marker GFAP (red). (f) GFP expression in the reticular astrocytes of the ON. (g) Transverse section through the brain region of a wild-type (WT) 72h.p.f. larva showing GFP expression in the skin (arrow), brain and retina (arrowheads). (h) GFP expression in the brain and the retina was absent in the mindbomb (mb) mutant background, whereas expression in the skin was not affected. All crysections were counterstained with 4',6-diamidino-2-phenylindole (DAPI) to label nuclei (blue).

Figure 2

Phenotypic characterization of a stable transgenic line. (a) Graphic representation of the DNA constructs used for transgenesis and breeding. (b) A 24 h.p.f. embryo expresses Smoa1-EGFP in the forebrain and retinal neuroblasts. A 96 h.p.f. double transgenic embryo derived from crossing of the Tg(krt5:Gal4VP16;14 × UAS:smoa1-EGFP) and the Tg(UAS-E1b:nfsB-mCherry)^c264 stable lines expressed GFP (c) and mCherry (d) in the ONs (arrows). (e) A 6-month-old wild-type and (f) a transgenic fish with a gross tumor in its left eye (arrow). (g) Wild-type fish formed a normal optic chiasm, whereas (h) transgenic fish failed to form optic chiasm and showed dramatic expansion of the ONH region (arrow). (i) Dissected brain and eyes showing an enlarged ON associating with a gross eye tumor (arrow).

Figure 3

Retinal tumors resulted from expression of Smoa1-EGFP in the stable transgenic line. (a, a′) Hematoxylin and eosin (H&E) staining of transverse section of a fish with retinal dysplasia; note the formation of rosette-like structures (arrow). (b, b′) An ocular tumor resembling medulloepithelioma with characteristic neural tube structures. (c, c′) A tumor showing features of pigmented ocular melanoma with heavy pigmentation and bland nuclei. (d, d′) An adult fish developed unilateral primitive neuroectodermal tumor (PNET). White frames indicate areas that were enlarged (not to scale). Scale bars, 400 μm for (a–d), 40 μm for (a′–d′), respectively.

Figure 4

Stable transgenic fish developed zOPGs. (a) Hematoxylin and eosin (H&E) staining of paraffin sections from a wild-type (WT) adult retina, and (b) an eye tumor showing coexistence of retinal dysplasia and ON hyperplasia. Note the high cellularity originating from the neural retina (arrowhead) and low cellularity derived from ON (arrow). (c) Enlarged view of normal ONH showing low cellularity, and transgenic fish show ON hyperplasia to neoplasia (d–f). (c′) Enlarged view of a normal optic chiasm showing low cellularity, and transgenic fish showing disorganization and increased cellularity in chiasms (d′–f′). (g) High-magnification view of a normal ON showing low cellularity, with zOPGs showing increased cellularity, nuclear atypia and vascular proliferation (h–j, arrow). Scale bars, 200 μm for (a and b), 40 μm for (c–f′) and 20 μm for (g–j), respectively.

Figure 5

Differentially expressed genes and a radial glia cell signature of the zOPGs. (a) A heatmap generated by hierarchical clustering showing differentially expressed genes for wild-type (WT) and retinal tumors, respectively. The color scale (Z-score) is denoted on the right, with red showing upregulation and green showing downregulation. A subset of upregulated genes is listed on the right. (b) Upregulation of gli1, gli2a, gfap and wnt5a was confirmed by real-time quantitative PCR (qPCR). Microarray data represent the average expression from four eye tumors versus four control eyes. qPCR data are from the same four samples performed in quadruplicate. All data were normalized to actin b1expression. Error bars show s.d. (c–g′) Series of cryosections from a single fish with typical zOPG showed overexpression of the astrocyte/radial glia markers of Pax2 (c, c′), GFAP (d, d′), S100β (e, e′), BLBP (f, f′) and the stem cell and glioma marker Sox2 (g, g′) as compared with their respective expression in the ONH of an age-matched WT fish. Fragmented lines demarcate the ONH region. Scale bars, 40 μm.

Figure 6

Stable transgenic fish exhibited asymmetric OT development and brain tumorigenesis. (a) A wild-type adult fish exhibited symmetric development of OT, scale bar, 1000 μm. (b) A transgenic fish with gross eye tumor exhibited asymmetric OT development. (c) Comparison between left (blue bar) and right (red bar) tectal lobe diameters in the wild-type fish and between larger (blue bar) and smaller (red bar) tectal lobes in the transgenic fish showed significant difference in transgenic fish OT, but not in wild-type fish OT (n=10). NS, statistically not significant; **P<0.01. Error bars show s.d. In general, the OT from transgenic fish was smaller than wild type, because the tumor-bearing fish were smaller. (d) A 12-month-old transgenic fish showing a brain tumor (arrow); (e) hematoxylin and eosin (H&E) staining of a transverse section at the boundary of the OT and hindbrain (HB) from a wild-type fish showing symmetric OT and a small hindbrain area. (f) H&E staining of the brain tumor in (d) showing tumor formation in the HB.

Figure 7

Overexpression of Mdm2 in zOPGs and hindbrain tumor. (a) Hematoxylin and eosin (H&E) staining of a transverse section from an adult fish with a less affected eye and a gross eye tumor (arrow). (b) A fragmented line demarcates the zOPG with mild cellularity and a primitive neuroectodermal tumor (PNET) with high cellularity. Immunofluorescence revealed that only the zOPG exhibited S100β (c) and Mdm2 (d) overexpression, whereas the PNET did not. (e) H&E-stained transverse section showing the optic chiasm of the same fish. Mdm2 was only expressed in the optic nerve associated with the glioma (f, arrow). (g, h) High-magnification view of the two optic tectal lobes in (a) showing Mdm2 expression in the lobe associated with zOPG (g), but not in the contralateral lobe (h). (i) H&E staining of the hindbrain tumor. The tumor overexpressed radial glia markers S100β (j), GFAP (k) and Mdm2 (l).