Fig 3. ShhN and MycnT50A,S54A cooperatively induce bioluminescent medulloblastomas in vivo in BarTeL mice.
(A) BarTeL mice were injected with a mixture of chicken DF-1 cells producing RCAS-ShhN and RCAS-MycnT50A,S54A viruses and imaged for bioluminescence starting at 14 days post-injection. Shown are images at two week intervals of four mice that developed bioluminescent tumors (left) compared to three mice without tumor (right). (B) Time course of bioluminescent tumor formation in transgenic mice. A cohort of 11 transgenic mice, 10 of which were infected as in (A), were imaged weekly for 12 weeks. At each time point, net ΔLog10 flux values plotted for each mouse were determined by subtracting the mean of the log values of flux of non-tumor-bearing transgenic mice from the log of the flux of each mouse. A graph of unprocessed measurements before conversion to log values is provided separately (S2 Fig) to show the loss of bioluminescence by cGNPs during postnatal cerebellar development and the concomitant growth of tumor bioluminescence in those mice that developed medulloblastomas. (C) Medulloblastoma in whole BarTeL brain and immunohistochemistry for SHHN and MYCN. Gross appearance of a medulloblastoma in a formalin-fixed brain (second panel) compared to a normal brain (first panel) from a littermate. Staining with an anti-SHH antibody showed that only a minority of medulloblastoma cells produced SHHN protein (fourth panel). Purkinje cells in control normal (NL) adult cerebellum also stain positively with anti-SHH antibody (third panel). An anti-c-MYC antibody identified the MYC-tagged MYCNT50A,S54A protein in most cells of the same tumor (fifth panel). The magnified panels above the SHH and MYCN panels show the hypercellularity of these tumors. Bar, 10 μm. (D) Histology and immunohistochemistry of a representative medulloblastoma induced by ShhN and MycnT50A,S54A viruses. Shown are an H&E-stained tumor section (left panel) and tumor sections stained with antibodies to PGP9.5 (ubiquitin carboxyl-terminal esterase L1), Synaptophysin, Ki67 and GFAP. Bar, 10 μm.