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doi: 10.1242/10.1242/dev.00182

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Nmyc upregulation by sonic hedgehog signaling promotes proliferation in developing cerebellar granule neuron precursors

Anna Marie Kenney1, Michael D. Cole2 and David H. Rowitch1,3,*

1 Department of Pediatric Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA 02115, USA
2 Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA
3 Division of Newborn Medicine, Children's Hospital, Boston MA 02115, USA



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  		Fig. 1. Nmyc mRNA transcripts (N-myc in figure) are upregulated in Shh-treated CGNPs. (A) Total RNA was prepared from CGNPs (85% pure cultures) treated with Shh (3 µg/ml) for 3 hours±the protein synthesis inhibitor cycloheximide (10 µg/ml), as indicated above the lanes. Northern blot analysis was carried out using cDNA probes for candidate direct targets of Shh signaling. Representative autoradiographs for Gli1, Myc (c-myc in figure), Lmyc (L-myc in figure) and Nmyc are shown. Gapdh signal (bottom) shows equivalent loading. (B) Shh-mediated induction of Nmyc occurs in cultures enriched for CGNPs (>90% pure cultures). Cerebellar homogenates were enriched for CGNPs as described (Materials and Methods), then assayed as above for protein synthesis-independent up-regulation of Nmyc mRNA by Shh. A representative northern blot is shown (top). Gapdh signal (bottom) shows equivalent loading. (C) Nmyc expression is sustained for the duration of Shh exposure (top). The hedgehog signaling pathway is activated, as indicated by Gli1 mRNA expression (middle). Shh signaling maintains a population of immature CGNPs, as indicated by Math1 (Atoh1 — Mouse Genome Informatics) expression (bottom). Representative northern analysis autoradiographs are shown. Ethidium bromide staining indicates equivalent lane loading.

 


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  		Fig. 2. Nmyc is expressed in proliferating precursors of the developing cerebellum. (A-C) In situ hybridization for Nmyc, (A) cyclin D1 (B) and Gli1 (C) mRNA transcripts was performed on adjacent parasagittal frozen sections of PN 7 mouse cerebella. (A) Nmyc and (B) cyclin D1 expression co-localize to regions of Shh pathway activity and proliferation of CGNPs. (D-F) Nmyc expression is observed during the proliferative phase of cerebellar development at PN1 and PN7, but is undetectable in the PN15 or adult (not shown) mouse cerebellum. (D,E) Low power views of PN1 and PN7 cerebella shows the distribution of Nmyc mRNA transcripts primarily in the EGL region (white arrows). Additionally, a small Nmyc-expressing population is observed within deeper layers of the cerebellum at PN1 (arrowheads, D). (F) At PN15, Nmyc transcripts are undetectable in all cerebellar layers. DIC image at right allows discrimination of EGL and molecular layers. Arrowheads (top) indicate meninges that fail to express Nmyc. (G) At high magnification, Nmyc expression at PN7 clearly segregates to the EGLa, or proliferative compartment of the EGL. The EGLb comprises largely immature CGNPs that have recently left the cell cycle. By contrast, neither Myc (H) nor Lmyc (I) are expressed in the EGL at PN7. ant, anterior; post, posterior; EGL, external granule layer; Pur, Purkinje cells; Mol, molecular layer; Mng, meninges.

 


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  		Fig. 3. Retroviral infection of heterogeneous primary cerebellar cultures is specific for proliferating CGNPs. (A,B) Cultures pretreated with Shh vehicle (Veh) alone prior to infection with GFP-carrying retroviruses showed only 5-10% infection levels (A), compared with ~70% infection levels in cells pre-treated with Shh (B). Merge of GFP-immunolabeled cells and DAPI-stained fields (blue) are shown. (A) Several infected cells are highlighted (arrowheads). (C,D) CGNPs infected with GFP retroviruses that subsequently exit the cell cycle coexpress the neuronal marker, NeuN (FITC, green) (C), but not GFAP (FITC, green), an astroglial cell marker (D), indicating selective infection of neuronal precursors. After infection, cells were treated with vehicle alone for 48 hours prior to immunostaining. (C) Yellow pseudo-color indicates cells with overlapping expression (arrows); the inset shows infected cell with typical granule neuron morphology at higher power. (D) Arrowhead indicates a GFAP-positive cell. Note the paucity of GFAP-positive cells; we observed no astroglia that co-expressed GFP under our conditions of infection. (C',D') DAPI-staining (blue) of the fields shown in C,D.

 


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  		Fig. 4. Nmyc activity in cell cycle regulation lies downstream of Smo. Nmyc functions in a cell-autonomous manner. (A) 48 hours after infection and treatment as indicated below the graph (right), CGNPs (85% pure cultures) were harvested and processed for FACS analysis of cell cycle distribution. Percentages of cells in S-phase were normalized to levels in Shh-treated, uninfected/GFP infected samples. Bars represent average normalization from six samples per treatment. Error bars indicate standard error of the mean. *P<0.01, in comparison with Shh-treated control CGNPs. A representative autoradiograph (left) shows western blot analysis of Nmyc (N-myc in figure) and cyclin D1 expression in CGNPs infected and treated with Shh/cyclopamine as indicated above the lanes. Tubulin levels demonstrate equivalent loading of the lanes. (B) Proliferation was assessed using BrdU immunostaining of CGNP cultures enriched for granule cells and treated as indicated below the graph. Numbers of BrdU immunopositive cells in Nmyc+GFP or {Delta}MB2+GFP-infected cells treated with Shh+cyclopamine or cyclopamine alone were normalized to numbers of BrdU-positive cells in GFP-infected, Shh-treated CGNP cultures. Twenty 100-cell fields were counted per treatment, using two separate litters of animals and two coverslips per treatment per litter. Error bars indicate standard error of the mean. *P<0.01, in comparison with Shh-treated control CGNPs. (C) Phenotype of proliferating cells infected with Nmyc-carrying retrovirus. BrdU-GFP, BrdU-GFAP and GFAP-GFP immunostaining (as indicated) was carried out on Shh/cyclopamine treated cultures infected with Nmyc-GFP retrovirus. (Left) BrdU incorporation was exclusively associated with GFP expression (yellow pseudocolor indicates overlap). (Middle) BrdU incorporation was not observed in any GFAP-positive cells. (Right) No GFP-positive cells co-labeled with GFAP, indicating that glial cells were not infected. Note that yellow pseudocolor resulted in this image from a glial process underlying several neuronal cells. DAPI staining (bottom panels) shows distribution of cells.

 


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  		Fig. 5. Antagonism of Nmyc (N-myc in figure) activity reduces Shh-induced proliferation. (A) Mxi inhibits proliferation in Shh-treated cultures, as measured by flow cytometry. Thirty-six hours after infection with Mxi-SR-carrying retrovirus or controls and Shh-treatment, cycle phase distribution of cells was analyzed by flow cytometry. Percentages of cells in S phase were normalized to levels in Shh-treated, uninfected/GFP infected samples. The graph depicts average normalization from n=6 samples per treatment, per timepoint. Error bars indicate standard error of the mean. Northern blot analysis confirmed strong expression of Mxi (upper panel) in CGNPs infected as indicated. Endogenous Mxi expression can also be observed. (B) The Nmyc{Delta}MB2 mutant inhibits DNA synthesis in Shh-treated cultures, as measured by BrdU immunolabling. (Top) Immunostaining to visualize BrdU incorporation was used to measure proliferation in CGNPs infected with GFP or Nmyc{Delta}MB2+GFP retroviruses. Representative fields of BrdU immunostaining and GFP immunostaining in GFP-infected and Nmyc{Delta}MB2+GFP-infected CGNPs are shown. (Bottom) Graph shows levels of BrdU incorporation in untreated, GFP-infected or Shh-treated Nmyc{Delta}MB2+GFP-infected cells, normalized to levels in Shh-treated GFP-infected CGNP cultures. Western blot analysis (right) shows expression levels of Nmyc and Nmyc{Delta}MB2 protein levels. *P<0.01, in comparison with Shh-treated control CGNPs.

 


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  		Fig. 6. Nmyc (N-myc in figure) is upregulated in proliferating neural tube precursors exposed to Shh and in medulloblastoma.

(A-D) Hybridization for Nmyc and Shh mRNA transcripts was carried out in situ on adjacent transverse sections of spinal cord of 12.5 dpc wild-type (A) and Shh transgenic (Rowitch et al., 1999Go) embryos, which overexpress Shh in the dorsal neural tube (boxed) (C).

(B,D) Nmyc is upregulated within proliferating neural precursors exposed to ectopic Shh (red box in D). Note that Nmyc is not a general Shh transcriptional target, because expression is absent in the floorplate (fp) where Shh is strongly expressed (compare A, arrow, with B).

Additionally, Nmyc can be activated by mitogens other than Shh. Nmyc expression is observed in the dorsal spinal cord and dorsal root ganglion (drg) in the absence of adjacent Shh expression (compare B, arrow, with A; compare D with C). Thus, Nmyc is upregulated by Shh only in proliferating precursor cells. (E,F) Nmyc is upregulated in medulloblastomas of adult Ptch heterozygotes. Representative results of analysis of four animals are shown. (E) The normal adult cerebellum is devoid of Nmyc expression is at left in this image. By contrast, the tumor tissue expresses Nmyc strongly. (F) High-power view of boxed region in E shows possible area of continuity of the strongly Nmyc-expressing tumor cells (arrow) with the internal granule layer (IGL) and disruption of the molecular layer (Mol). The broken dashed line indicates the boundary between normal cerebellar tissue and medulloblastoma. ant, anterior; post, posterior; IV, fourth ventricle; cp, choroid plexus.

 

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