Loss of Smarc proteins impairs cerebellar development

Abstract

SMARCA4 (BRG1) and SMARCB1 (INI1) are tumor suppressor genes that are crucially involved in the formation of malignant rhabdoid tumors, such as atypical teratoid/rhabdoid tumor (AT/RT). AT/RTs typically affect infants and occur at various sites of the CNS with a particular frequency in the cerebellum. Here, granule neurons and their progenitors represent the most abundant cell type and are known to give rise to a subset of medulloblastoma, a histologically similar embryonal brain tumor. To test how Smarc proteins influence the development of granule neurons and whether this population may serve as cellular origin for AT/RTs, we specifically deleted Smarca4 and Smarcb1 in cerebellar granule cell precursors. Respective mutant mice displayed severe ataxia and motor coordination deficits, but did not develop any tumors. In fact, they suffered from a severely hypoplastic cerebellum due to a significant inhibition of granule neuron precursor proliferation. Molecularly, this was accompanied by an enhanced activity of Wnt/β-catenin signaling that, by itself, is known to cause a nearly identical phenotype. We further used an hGFAP-cre allele, which deleted Smarcb1 much earlier and in a wider neural precursor population, but we still did not detect any tumor formation in the CNS. In summary, our results emphasize cell-type-dependent roles of Smarc proteins and argue against cerebellar granule cells and other progeny of hGFAP-positive neural precursors as the cellular origin for AT/RTs.

Keywords: AT/RT; Smarc; brain; cerebellum; development; tumor.

Figure 1.

Loss of Smarcb1 in cerebellar granule cells results in a severe reduction of the cerebellar size. Cerebellar histopathology of control mice is normal (A, F) with the typical cortical layering (G, J) as visualized by H&E stains of P7 and P20 sagittal sections. Cre is strongly expressed in rostral parts (B), but less extensive in caudal lobes of control mice (C), and nuclear staining of Smarcb1 is retained in all control cells (D, E). In contrast, Math1-cre::Smarcb1^fl/fl mice display a severe phenotype in the Cre-expressing rostral parts of the cerebellum. At P7 and P20, a loss of normal architecture and blurred cortical layers are apparent (K, Q and R, U, respectively), which is caused by a loss of Smarcb1 protein expression in rostral parts and, to a lesser extent, in caudal parts of the cerebellum (L, M). Smarcb1 is particularly lost in Cre-positive cells (N–P), the overall number of NeuN-positive cells is severely decreased in Math1-cre::Smarcb1^fl/fl mice, and Purkinje cells are randomly distributed within the cerebellar cortex as compared with controls (H, I, S, T). With respect to histomorphology (V–X and AA–CC) and expression of NeuN and Calbindin (Y, Z, DD, EE), Math1-cre::Smarca4^fl/fl mice display changes that are similar, if not identical, to Math1-cre::Smarcb1^fl/fl mice. oEGL, Outer external granular layer; iEGL, inner external granular layer; ML, molecular layer; PCL, Purkinje cell layer; IGL, inner granular layer. Scale bars: A, K, 250 μm; B–E, L, M, 50 μm; F, Q, 500 μm; N–P, 10 μm; G–J, R–U, W–Z, BB, CC, DD, EE, 100 μm; V, AA,1 mm.

Figure 2.

Loss of Smarcb1 impairs proliferation and activates Wnt signaling in cerebellar granule cell precursors. Cerebellar granule cell precursors were isolated from Smarcb1^fl/fl mice on day P5, cultured, and transduced with IRES-GFP or Cre-IRES-GFP viruses. Thirty-six hours after transduction, cells were sorted for GFP-positive cells and experiments were performed. Cre-IRES-GFP-transduced cells displayed a severe reduction of Smarcb1 expression, as expected (A). Furthermore, proliferation was significantly decreased (B), and cells within the Cre-IRES-GFP condition were characterized by a massive upregulation of Wnt target genes Tcf4, Lef1, Dkk1, and Axin2. No significant changes were observed regarding the expression of Ink4a, Gli1, Gli2, Gli3 or Cyclin D1 (C).

Figure 3.

Loss of Smarcb1 in hGFAP-positive precursor cells similarly results in hypoplastic cerebella, but not in brain tumor formation. Cerebellar growth in hGFAP-cre::Smarcb1^fl/fl was dramatically decreased compared with controls (H&E stains of sagitally cut brains; A, B vs E, F) with lack of foliation and cortical lamination (C, D vs G, H). Moreover, we detected severe lamination disturbances and a thinning of the cerebral cortex (A vs E), indicating that Smarcb1 is similarly important for the development of the forebrain. Scale bars: A, E, 2 mm; B, 500 μm; F, 300 μm; C, D, G, H, 100 μm.