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Induction of Medulloblastomas in Mice by Sonic Hedgehog, Independent of Gli1^{1 ,,2}

Skirball Institute of Biomolecular Medicine [H. L. W., R. B., M. S. H., E. A., W. S. L., D. S., A. L. J., D. H. T.], Howard Hughes Medical Institute [A. L. J.], and Departments of Neurosurgery [H. L. W., R. B., J. R., E. A., W. S. L., D. Z.], Pathology [D. Z., D. H. T.], Cell Biology [A. L. J.], Physiology/Neuroscience [A. L. J.], and Radiology [D. H. T.], New York University School of Medicine, New York, New York 10016

	ABSTRACT

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

 
The Sonic hedgehog (Shh) signaling pathway plays a critical role in normal cerebellar development and has been implicated in medulloblastomas, common malignant childhood tumors of the cerebellum. To test whether Shh mis-expression is sufficient for medulloblastoma formation, we used ultrasound biomicroscopy-guided in utero injection of a Shh-expressing retrovirus into the cerebellum of 13.5-day mouse embryos to show that direct activation of the Shh pathway can lead to tumor formation. Significantly, medulloblastomas were observed in 76% of the mice infected with Shh-expressing retrovirus. Furthermore, contrary to recent suggestions that the Shh transcriptional target Gli1 plays a critical role in Shh-induced tumorigenesis, we found that medulloblastomas form in Gli1 null mutant mice. We have developed an efficient mouse model of medulloblastoma and shown that Gli1 is not required for tumorigenesis when Shh signaling is activated upstream in the pathway.

	Introduction

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

 
Shh⁴ is expressed from birth onwards in the Purkinje cells of the cerebellum, and Shh target genes are expressed in the adjacent granule neurons (1, 2, 3) . Shh has been shown to be sufficient to increase granule cell proliferation in vitro, and antibodies to Shh injected into the developing chick cerebellum reduce granule cell proliferation (1, 2, 3) . Furthermore, one copy of patched (PTC), which encodes a cell surface receptor and negative regulator of the pathway, is mutated in a subset of hereditary and sporadic medulloblastomas, a common malignant childhood tumor of the cerebellum thought to arise from granule cell precursors (4 , 5) . Similarly, mice heterozygous for a null mutation in Ptc develop medulloblastomas at a low frequency (10–15%; Refs. 6, 7, 8, 9 ). These facts have led to the suggestion that inappropriate maintenance of Shh signaling in the proliferative EGL cells leads to medulloblastoma formation during cerebellar development. Other results, however, suggest that the situation is more complicated. For example, during differentiation and migration of the granule neurons from the EGL to the IGL, the cells move toward the cerebellar source of Shh, the Purkinje cells. Thus, granule neurons differentiate despite continual exposure to Shh. In addition, medulloblastomas in Ptc heterozygotes do not exhibit loss-of-heteroxygosity, which indicates that other genetic pathways may contribute to tumor formation (7 , 8) . Indeed, loss of the p53 tumor suppressor gene results in early medulloblastoma formation in all Ptc heterozygous mice (10) . However, p53 mutations are not associated with human medulloblastoma, raising the question of the relevancy of this result to the human disease (11) . To test directly whether activation of the Shh pathway can lead to tumor formation, we developed a new mouse medulloblastoma model by injecting a Shh-expressing retrovirus into the embryonic mouse cerebellum using in utero UBM image guidance (12, 13, 14) .

	Materials and Methods

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Animals.
All of the animals in this study were maintained according to protocols approved by the Institutional Animal Care and Use Committee at New York University School of Medicine. Timed-pregnant Swiss Webster (Taconic, Germantown, NY) and Gli1 mutant mice (15 , 16) were used for injections. Noon of the day that a vaginal plug was detected after overnight mating was denoted E0.5. Animal care, preparation for surgery, and the use of the UBM scanner have been described in detail previously (12 , 13) .

In Utero UBM-guided Retroviral Injections.
The pseudotyped CLEG (control) and CLES (Shh-expressing) retroviral vectors used in these studies have been described previously in detail (14) . Viral stocks were prepared as described previously, and each embryo was injected with 0.4 µl of CLES viral stock, prepared to a titer of 1 x 10⁸ colony-forming units/ml. The UBM-injection technique used to inject the retroviral vector into the embryonic mouse central nervous system has been described in detail previously (12, 13, 14) .

Histological Analysis.
Mice were killed by sodium pentobarbital overdose and transcardially perfused with 4°C, 4% paraformaldehyde (PFA) in 0.1 M PBS, after which the brains were dissected and postfixed for 2 h at 4°C. The brains were then cryopreserved in 30% sucrose in PBS at 4°C, embedded in HistoPrep (Fisher Scientific, Pittsburgh, PA), and frozen on dry ice. Frozen sections were cut in a cryostat at 16–40-µm thickness. All of the subsequent analyses were carried out on near-adjacent sections. Histopathological analyses were performed on cresyl violet- or H&E-stained sections, using morphological and pathological criteria known for human tumors, by a neuropathologist (D. Z.) who was blinded as to the experimental design.

Histochemical detection of PLAP and ß-gal was performed as described previously (14 , 16) . Immunostaining protocols for Calbindin (1:4000; Sigma), GFAP (1:1000; Dako), NeuN (1:2000; Chemicon) and phospho-H3 (1:200; Santa Cruz Biotechnology) were performed as described previously (17 , 18) . All of the antibody staining was revealed with a vectastain ABC kit (Vector Laboratories) and diaminobenzidine (DAB) (Sigma). Section RNA in situ hybridization analysis was performed as described previously (19) , using probes for Shh (20) , Gli1and Gli2 (21) , and Math1 (22) . All of the in situ sections were counterstained with nuclear fast red to facilitate identification of cerebellar anatomy.

	Results and Discussion

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

 
To test whether mis-expression of Shh alone in the cerebellum is sufficient to result in medulloblastomas, we injected a Shh-expressing retrovirus directly into the mouse embryonic cerebellar anlage using in utero UBM image-guidance (Fig. 1, a and b ; Refs. 12, 13, 14 ). We previously described the pseudotyped CLES retroviral vector used in these studies, which coexpresses both full-length human SHH and a downstream reporter gene, human PLAP from an internal ribosome entry site (14) . The embryonic stage chosen for injection of the CLES retrovirus, embryonic day (E) 13.5, was the earliest stage we could accurately inject the virus specifically into the cerebellar anlage using UBM-guidance (12) . Moreover, previous injections of Shh-expressing cells and retroviruses at earlier embryonic stages resulted in transformation of the dorsal neural tube into ventral structures, which could preclude cerebellar formation (13 , 14) .

View larger version (113K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Shh-expressing retrovirus was targeted to the embryonic mouse cerebellum using UBM-guided injection. In a, the microinjection needle tip (arrow) was visualized in the E13.5 cerebellar anlage using real-time UBM. In b, a schematic of a similar section shows the mouse E13.5 mid-hindbrain region, showing the cerebellum (CB), choroid plexus (CP), and fourth ventricle (4v) [on the basis of Plate 30a of Kaufman’s Atlas of Mouse Development (28) ]. In c, PLAP-staining at E17.5 demonstrated the localization of Shh-expressing retrovirus in the cerebellum (CB), and the resulting expanded EGL on the infected side (insets from cresyl violet, CV-stained sections). Other labels: MB, midbrain; MD, medulla.

To determine the postnatal effects of Shh mis-expression from an early stage of cerebellar development, we analyzed PLAP-positive mice histologically at postnatal day (P) 14 (n = 14), P21 (n = 3) and at postnatal week 13 (n = 2). An additional group of P21 mice (n = 8) were injected at E13.5 with a control retroviral vector, CLEG, identical to CLES but without SHH. By P14, EGL cell proliferation normally has ceased and differentiation and migration to the IGL is complete.

In contrast to uninfected or CLEG-infected controls, the cerebella of P14-P21 Shh-expressing mice had regions in which the outer layer of cells resembled an EGL. These regions contained primarily, if not exclusively, Shh-expressing cells, as assessed by PLAP staining (Fig. 2, a and b) . We observed that this persistent EGL was consistently expanded in regions of heavy infection, to a thickness of 5–25 cells, at both P14 (n = 14 of 14) and P21 (n = 3 of 3; Fig. 2, c and d ). Most significantly, immunostaining for phospho-H3 revealed dividing cells only in regions of persistent EGL and not in adjacent regions of the P14–21 cerebellum, including the IGL (Fig. 2d) . The persistent EGL encroached on the subarachnoid space between adjacent folds, particularly at the depths of the cerebellar sulci. In addition, in many Shh-expressing regions, numerous finger-like extensions of cells projected between the EGL and IGL, suggesting that migration of the granule neurons was significantly still occurring (Fig. 2c) . The IGL appeared thinner in the regions adjacent to the thickened EGL and finger-like extensions. Purkinje neurons, as seen morphologically and by Calbindin staining, were PLAP negative and remained a well-defined, distinct layer above the IGL (data not shown). These morphological features show that expression of Shh in EGL cells leads to the maintenance of a proliferative state and the inhibition of, or a delay in, differentiation. Surprisingly, occasional Shh-expressing cells appeared to incorporate into the IGL in an apparently normal manner, indicating that Shh mis-expression alone might not be sufficient to maintain a persistent EGL. One possibility is that additional factors in the cerebellum normally act to render granule neurons nonresponsive to Shh during the differentiation process.

View larger version (76K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Direct injection of Shh-expressing retrovirus into the E13.5 mouse cerebellum led to abnormal persistence of the EGL and medulloblastomas. Cresyl-violet (CV)- and PLAP-stained sagittal sections of P21 Shh-expressing cerebella (a, b), showing the persistent EGL and tumors in regions of heavy infection. In many Shh-expressing regions, numerous finger-like extensions of cells projected between EGL and IGL (c, arrowheads), suggesting that migration of the granule neurons was significantly still occurring. Immunostaining for phospho-H3 (pH3) revealed dividing cells only in regions of persistent EGL and not in adjacent cerebellar regions, including the IGL (d, lower panel, pH3-staining in near adjacent section to d, upper panel). Numerous tumors were observed in cerebella infected with Shh-retrovirus, demonstrating all of the classic histological features of medulloblastoma, including marked hypercellularity, nuclear pleiomorphism, and frequent mitotic figures (e). Many dividing cells were seen in these tumors as revealed with immunostaining for phospho-H3 (f, right panel, pH3-staining in near adjacent section to f, left panel). Tumors analyzed at 13 weeks were large and exerted significant mass effect on the surrounding cerebellum; reduced immunostaining for NeuN demonstrated the tumors to be poorly differentiated when compared with normal adjacent IGL (g). RNA in situ analysis revealed patterns of Shh expression that were similar to those with PLAP staining (h, left panel, Shh in situ in near adjacent section to PLAP-stained section in right panel). Normal expression of Shh and Gli1 were complementary, with Shh expressed in Purkinje cells (PC) and Gli1 expressed in cells adjacent to the Purkinje cells (i, left panel, Shh in situ in near adjacent section to ß-gal-stained section in right panel). Tumors expressed Gli1 (j, P21 tumor), Gli2 [k, 13-week tumor (the expression of Gli2 is normal in IGL)], and Math1 (l, 13- week tumor in near adjacent section to k).

To determine whether these early tumors would progress further with age, we analyzed the cerebella of postnatal-week-13 mice, injected with CLES at E13.5. As at the earlier postnatal stages, we observed tumors near regions of heavy Shh-expressing retrovirus infection in the two PLAP-positive cerebella (Fig. 2, g and h) . These tumors exerted mass effect on the surrounding cerebellum and had the typical histological features described above, characteristic of human medulloblastoma. In addition, we observed decreased immunostaining for NeuN, a marker for many differentiated neurons, in the tumors compared with normal adjacent IGL, indicating the lesions were poorly differentiated (Fig. 2g) . We also observed a diffuse filamentous pattern of immunoreactivity for GFAP throughout the tumors (data not shown), similar to that seen in medulloblastomas in Ptc^+/- mice (7) . Finally, as in the early-stage tumors, we detected many dividing cells in these later tumors using phospho-H3 immunohistochemistry (data not shown). These results show that overexpression of Shh is sufficient for both tumor initiation and progression in the mouse cerebellum.

The Shh transcriptional target gene Gli1 is expressed in medulloblastomas and other tumors associated with activation of the Shh pathway and has been suggested to play a critical role in Shh-induced tumor formation (5 , 6 , 24) . Because Gli1 has been shown to be a transcriptional activator, even in the absence of Shh, initial activation of Gli1 transcription may be critical to tumor progression (24, 25, 26) . Once activated, Gli1 may then become Shh independent through autoregulation. Consistent with this, a recent study in frog embryos showed that continued central nervous system overproliferation, caused by transient expression of human GLI1, was dependent on endogenous Gli1 (24) . On the other hand, because Gli1 null mutant mice appear normal and Gli1 can replace Gli2 function during development, it is possible that Gli2 is sufficient for tumor formation in human patients and in our model system, in which Shh signaling is constitutively activated upstream in the pathway (15 , 27) .

Shh, Gli1, and Gli2 expression levels in normal and tumorigenic mouse cerebella were analyzed with RNA in situ hybridization. In addition, because one of the Gli1 mutant alleles was a lacZ knock-in (Gli1^lz), we also used histochemical ß-gal staining to detect Gli1 expression in Gli1^lz heterozygous mutant mice (16) . The pattern of Shh RNA expression closely matched the pattern of PLAP staining, as expected because PLAP is coexpressed with Shh in the CLES retrovirus (14) . In particular, Shh was expressed in tumors and regions of persistent EGL (Fig. 2h) , and was also detected in Purkinje cells in uninfected regions of normal cerebellum (Fig. 2i) . Normal expression of Gli1 was restricted to cells immediately adjacent to the Shh-expressing Purkinje cells (Fig. 2i) , and Gli1 was also expressed in tumors and regions of persistent EGL, in response to elevated levels of Shh (Fig. 2j) . Gli2 was expressed more broadly throughout the IGL in normal cerebellum and was also expressed in tumors and some regions of persistent EGL (Fig. 2k) . Finally, RNAs in situ for Math1, a marker of granule neuron precursors, revealed high levels of expression in tumors (Fig. 2l) , providing additional evidence that EGL cells are the target cells in these medulloblastomas.

To test the requirement for Gli1 in tumor formation, we injected the Shh-expressing retrovirus into the cerebella of E13.5 Gli1 heterozygous and homozygous null mutant embryos (15 , 16) . A similar rationale was used recently to demonstrate the requirement of Igf2 for the formation of medulloblastomas in Ptc^+/- mice (9) . Of significance, we found that infection of Gli1^-/- mice with the Shh-expressing retrovirus at E13.5 leads to both persistent EGL and tumor formation at P21 (n = 6 of 6; Fig. 3, a and b ). The tumors and regions of persistent EGL were indistinguishable from those seen in P21 wild-type and Gli1^+/- mice expressing Shh, based on cellular morphology (Fig. 3c) and phospho-H3 immunostaining (Fig. 3d) . Finally, Math1 (Fig. 3e) and Gli2 (Fig. 3e) were expressed in tumors and regions of persistent EGL in Gli1^-/- mice, as in wild-type mice.

View larger version (63K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Gli1-/- mice infected with Shh-expressing retrovirus at E13.5 also had persistent EGL and medulloblastomas. PLAP- (a, reflecting exogenous Shh expression) and near adjacent H&E- (b) stained sagittal P21 sections of a Gli1-/- cerebellum show a tumor forming in a region of heavy Shh-infection. The histopathological features of Gli1-/- cerebellar tumors were indistinguishable from those observed in wild-type Shh-infected mice [c, cresyl violet (CV)-stained cells in P21 Gli1-/- tumor], and phospho-H3 (pH3) immunostaining revealed dividing cells only in regions of persistent EGL and tumor formation, as in wild-type cerebella infected with Shh-retrovirus (d, pH3-stained cells in a region of persistent EGL). Math1 was expressed in Gli1-/- tumors, and persistent EGL (e), and Gli2 was expressed in normal IGL, tumors, and persistent EGL (f, right panel, Gli2 in situ in near adjacent section to left panel).

As a first study of components of the Shh pathway, we tested the role of Gli1 in tumor formation. Of significance, we found that Gli1, in fact, is not required for medulloblastoma formation. Thus, our studies show that, when the Shh pathway is activated upstream in the pathway, Gli1 is not required for tumorigenesis. To date, the activating mutations in the Shh pathway found in medulloblastomas have been at the level of the receptors, which should activate the pathway in a similar way to mis-expression of Shh. Such stimulation of the pathway likely leads to activation of Gli2 protein, which then induces target genes, including Gli1 (16) . Consistent with this, we found Gli1 to be expressed in tumors. In frog studies in which Gli1 was used to produce neural cell overproliferation (24) , it is likely that Gli2 cannot compensate for Gli1, because the Shh pathway is not activated upstream, and tumors form only in cells in which the endogenous Gli1 gene has become both induced and autoregulated. In conclusion, our studies show that Shh can efficiently induce medulloblastomas, and this induction is not dependent on Gli1, demonstrating that Gli1 would not be an effective drug target.

	ACKNOWLEDGMENTS

 
We thank Nicholas Gaiano and Gord Fishell (Skirball Institute, NYU School of Medicine, New York, NY) for providing the CLES and CLEG retroviruses; Chloe Duan, Roger Lichtenbaum, and Olivier Jin for technical assistance; and Brian Bai for helpful comments on the manuscript.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by National Institutes of Health Grants NS38461 (to D. H. T.) and HD035768 (to A. L. J.), and by National Science Foundation Grant IBN9728287 (to D. H. T.). A. L. J. is an Investigator of the Howard Hughes Medical Institute.

² Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org).

³ To whom requests for reprints should be addressed, at Skirball Institute of Biomolecular Medicine, New York University School of Medicine, 540 First Avenue, New York, NY 10016. Phone: (212) 263-7262; E-mail: turnbull{at}saturn.med.nyu.edu Phone: (212) 263-7290; E-mail: joyner{at}saturn.med.nyu.edu

⁴ The abbreviations used are: Shh, sonic hedgehog; EGL, external granule layer; IGL, internal granule layer; UBM, ultrasound biomicroscopy; PLAP, placental alkaline phosphatase; ß-gal, ß-galactosidase; GFAP, glial fibrillary acidic protein; phospho-H3, phospho-histone-3.

⁵ H. L. Weiner, A. L. Joyner, and D. H. Turnbull, unpublished observations.

Received 6/ 4/02. Accepted 9/27/02.

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