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. 2018 Aug 15;440(2):152-166.
doi: 10.1016/j.ydbio.2018.05.015. Epub 2018 May 21.

Bergmann glial Sonic hedgehog signaling activity is required for proper cerebellar cortical expansion and architecture

Frances Y Cheng  1 , Jonathan T Fleming  1 , Chin Chiang  2
Affiliations

Bergmann glial Sonic hedgehog signaling activity is required for proper cerebellar cortical expansion and architecture

Frances Y Cheng et al. Dev Biol. .

Abstract

Neuronal-glial relationships play a critical role in the maintenance of central nervous system architecture and neuronal specification. A deeper understanding of these relationships can elucidate cellular cross-talk capable of sustaining proper development of neural tissues. In the cerebellum, cerebellar granule neuron precursors (CGNPs) proliferate in response to Purkinje neuron-derived Sonic hedgehog (Shh) before ultimately exiting the cell cycle and migrating radially along Bergmann glial fibers. However, the function of Bergmann glia in CGNP proliferation remains not well defined. Interestingly, the Hh pathway is also activated in Bergmann glia, but the role of Shh signaling in these cells is unknown. In this study, we show that specific ablation of Shh signaling using the tamoxifen-inducible TNCYFP-CreER line to eliminate Shh pathway activator Smoothened in Bergmann glia is sufficient to cause severe cerebellar hypoplasia and a significant reduction in CGNP proliferation. TNCYFP-CreER; SmoF/- (SmoCKO) mice demonstrate an obvious reduction in cerebellar size within two days of ablation of Shh signaling. Mutant cerebella have severely reduced proliferation and increased differentiation of CGNPs due to a significant decrease in Shh activity and concomitant activation of Wnt signaling in SmoCKO CGNPs, suggesting that this pathway is involved in cross-talk with the Shh pathway in regulating CGNP proliferation. In addition, Purkinje cells are ectopically located, their dendrites stunted, and the Bergmann glial network disorganized. Collectively, these data demonstrate a previously unappreciated role for Bergmann glial Shh signaling activity in the proliferation of CGNPs and proper maintenance of cerebellar architecture.

Keywords: Bergmann glia; Cerebellum; Neuronal-glial relationships; Sonic hedgehog.

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Figures

Figure 1
Figure 1. TNC-YFP-expressing cells are Bergmann glia
(A–A”) YFP immunohistochemistry on sagittal sections demonstrates pattern of TNC expressing cells. Boxed region denotes enlarged area in (B’). TNC-YFP expression is observed in cells that extend long processes to the pial surface (A”). (B–C”) TNC-YFP expressing cells are Blbp+ (B–B”) and Sox2+ (C–C”), indicating that they express astroglial markers. Inset shows example of co-labeled cell (B”, C”). (D–D”) TNC-YFP expressing cells are ki67+, indicating that they are proliferative. Inset shows example of co-labeled cell (D”). Abbreviation: EGL, external granular layer. Scale bars are 20 µm except A”, B”, C” and D” (100 µm).
Figure 2
Figure 2. TNCYFP-CreER;SmoF/− (Smocko) mutants display a hypoplastic cerebellum
Tamoxifen was injected at P1 and P2 in WT and Smocko mutants. Mice were analyzed as early as P3 and as late as P30. (A–B) External view of Smocko mutants and WT littermates at P5 demonstrates the reduction in overall cerebellar size. (C–F’) H&E staining of mid-sagittal sections of Smocko mutants (D, D’ F, F’) and WT (C, C’, E, E’) littermates at P5 (C–D’) and P7 (E–F’). (G–H) NeuN staining at P30 demonstrates that mutants have significant cerebellar hypoplasia. (I–L’) H&E staining of mid-sagittal sections of WT (I, I’), Smocko (J, J’), Math1CreER;SmoF/− (K, K’) and L7Cre;ShhF/− (L, L’) mutants at P7. (M) Quantification of cerebellar area of WT littermates and Smocko mutants, Math1CreER;SmoF/− mutants, and L7Cre;ShhF/− mutants. Data are mean of n=3 WT and littermate pairs for each genotype. Boxed region shows enlarged region in adjacent panel. Abbreviations: EGL, external granular layer. ML, molecular layer. PCL. Purkinje cell layer. IGL, internal granular layer. Scale bar: 20 µm.
Figure 3
Figure 3. Mutant EGL is largely agranular due to severely reduced CGNP proliferation
(A) Immunohistochemistry for pH3 (brown) at P3 and P5 in WT and Smocko mutants. (B) Quantification of pH3 staining as a percentage of total EGL cells. At P3, a 19.5% decrease (n=3, p=0.0314) in pH3 was observed. At P5, a 51.1% decrease in pH3 (n=3, p=0.0166) was observed. (C) Immunohistochemistry for p27Kip1 (brown) at P3 and P5 in WT and Smocko mutants. (D) Quantification of p27Kip1 staining as a percentage of total EGL cells. At P3, an 18.9% increase (n=3, p=0.0052) in p27Kip1-positive cells was observed. At P5, a 39.2% increase (n=3, p=0.0026) in p27Kip1-positive cells was observed. Sections in A and C were counter stained with hematoxylin to highlight the nuclei (blue). (E) Immunofluorescence staining of Ki67 (green) and Tag1 (red) at P5 in WT and Smocko mutants. (F) Quantification of Tag1 staining as a percentage of total EGL cells. A 56% increase (n=3, p=0.0615) in Tag1-positive cells was observed. In contrast, a 28.3% decrease (n=3, p=0.0128) in Ki67-positive cells was observed. Abbreviations: EGL, external granular layer. Scale bar: 20 µm.
Figure 4
Figure 4. Loss of Shh signaling is profound in CGNPs
(A) X-gal staining for β-galactosidase in Gli1lacZ and TNCYFP-CreER;SmoF/−;Gli1lacZ mutants shows that X-gal is reduced in the PCL and EGL of mutants at P3 and P4. Rectangular region in P4 panel shows enlarged area in adjacent panel. (B) β-galactosidase immunohistochemistry in WT and mutants reveals a decrease in antibody staining in the mutant EGL at P4. GFP and Calbindin staining demonstrates location of BGs and PCs, respectively. Arrowheads indicate presence of β-gal+ cells in WT EGL, note that β-gal expression is absent in the mutant EGL. Arrows indicate GFP+ BGs that no longer express β-gal+, indicating a reduction in Gli1 expression in the mutant. (C) RT-PCR of Smo and Gli1 from purified TNC-YFP population shows reduced Shh signaling in Smocko mutants. (D) Western blotting of P5 WT and mutant CGNPs shows a decrease in Gli1 and Gli2 protein levels in the mutant. (E) Western blotting of P5 cerebellar lysates shows a reduction in Gli1 protein levels in the mutant. Abbreviations: EGL, external granular layer. PCL. Purkinje cell layer. CGNPs, cerebellar granular neuron precursors. CB, cerebellum. Scale bar: 100 µm and 20 µm.
Figure 5
Figure 5. Resident astroglial cells in the EGL do not contribute significantly to the granular neuron population
(A) YFP immunohistochemistry of TNCYFP-CreER at P1 on sagittal sections demonstrates β-gal+-expressing cells in the EGL. These are Blbp+ and Sox2+, indicating that they express astroglial markers. Inset shows example of co-labeled cell. (B) β-galactosidase immunohistochemistry of TNCYFP-CreER; Gli1lacZ sagittal sections at P5 demonstrates that YFP+ cells in the EGL and PCL are β-gal+. Arrows and arrowheads indicate cells that are co-labeled with YFP and β-gal in the PCL and EGL, respectively. β-gal+ cells in the PCL and EGL are Sox2+ and Blbp+. Boxed regions indicate examples of co-labeled cells. (C–F) Lineage tracing using TNCYFP-CreER; tdTomato sagittal sections at P11. (C) The majority of tdTomato+ cells are in the PCL and are Sox2+. Boxed region labeled “1” demonstrates example of Sox2+ TNC-lineage cell in IGL. Boxed region labeled “2” shows enlarged region in adjacent panel. Arrowheads indicate co-labeled cells in PCL, indicating that TNC-lineage cells are BGs. (D) The tdTomato+ cells in the EGL show GFAP+ fibers, boxed region shows enlarged region in adjacent panel. Arrowheads indicate tdTomato+ BGs associated with GFAP+ fibers. (E) Pax2 immunohistochemistry demonstrates that TNC-lineage cells found adjacent to the EGL are immature GABAergic interneurons. Arrowheads indicate co-labeled cells. (F) NeuN immunohistochemistry demonstrates that the majority of TNC-lineage cells are not tdTomato+, indicating that TNC-lineage cells do not contribute significantly to the granular neuron population. Boxed region labeled “1” shows the only example of co-labeled cell. Boxed region labeled “2” shows enlarged region in adjacent panel, and arrowheads indicate tdTomato+ cells that are not NeuN+. (G) Lineage tracing using TNCYFP-CreER; tdTomato sagittal sections at P30 supports the data at P11 and shows that TNC-lineage cells do not contribute significantly to the granular neuron population. Arrowheads indicate tdTomato+ cells that are not NeuN+ and arrows indicate the only two examples of co-labeled cells that were observed in three lobes. Abbreviations: EGL, external granular layer. IGL, internal granular layer. PCL. Purkinje cell layer. Scale bar: Scale bar: 100 µm and 20 µm.
Figure 6
Figure 6. Smocko mutants display altered BG arrangement and cytoarchitecture
(A) Sox2 labeling (brown) in P7 Smocko mutants demonstrate positioning of BG cell bodies in Smocko mutants is irregular and aberrantly disorganized (arrowheads). Calbindin labeling (purple) indicates disrupted PC soma localization (starred). Graph shows a significantly increased number of Sox2+ BG per mm of PCL and absolute numbers of BG are not different. Data are mean of n=3 WT and littermate pairs for each genotype. (B–D) Blbp, 3-PGDH, and GFAP staining (brown) at P3, P5, and P7 demonstrate an increased complexity of BG fibers and expansion of endfeet at the pial surface starting at P5 that increased in severity at P7. Sections are counter stained with hematoxylin to highlight the nuclei. (E) Laminin staining for the basement membrane did not reveal any differences Smocko mutants, indicating the basement membrane of mutant cerebella is intact. Abbreviations: EGL, external granular layer. ML, molecular layer. IGL, internal granular layer. Scale bar, 100µm and 20 µm.
Figure 7
Figure 7. Smocko mutants have disrupted alignment and dendritic arborization of PCs
(A–B”) At P3, Calbindin immunohistochemistry shows no difference in Smocko mutant (B–B”) PC morphology, layering, or dendritogenesis. (C–C”) At P7, Calbindin immunohistochemistry shows that PC soma localization is disrupted and have a severely disrupted fiber network, with stunted, thinned dendrites and poorly branched arbors. (E) The absolute number of Calbindin+ PCs were comparable between the WT and mutant. (F) There was a significant increase in the number of PCs per mm of PCL in Smocko mutants. Data are mean of n=3 WT and littermate pairs for each genotype. (G–R) Tamoxifen injection scheme in Smocko mutant at later timepoints. One dose of tamoxifen was injected at P5 and P6 in WT (G–L) and Smocko mutants (M–R). Mice were analyzed at P8. (G, H, M, N) H&E staining shows that changes in cerebellar size and EGL area were less severe when BG Shh signaling was ablated at P5 and P6 compared to ablation at P1 and P2. (I–L, O–R) PC dendrites in later timepoint-injected Smocko mutants (O–Q) lacked secondary branching structures as revealed by Calbindin immunohistochemistry (arrowheads) and no appreciable differences in BG fibers can be observed using Blbp (I–K, O–Q) and GFAP (L, R) immunohistochemistry. Abbreviations: EGL, external granular layer. IGL, internal granular layer. PCL. Purkinje cell layer. Scale bar: 100µm and 20 µm.
Figure 8
Figure 8. Smocko mutants exhibit aberrant Wnt signaling
(A–B’) β-galactosidase immunohistochemistry in P4 TNCYFP-CreER; SmoF/−; BAT-Gal mice (B, B’) shows the presence of ectopic β-gal+ cells in the EGL of mutant mice (arrows), indicating that Smocko mutant mice have enhanced Wnt signaling in CGNPs. (C–D’) In situ hybridization for Sfrp1 in Smocko mutants (D, D’) at P4 indicates a downregulation of the mRNA in the EGL. (E) Western blotting of lysates of freshly isolated CGNPs indicates a downregulation of Sfrp1 expression. Abbreviations: EGL, external granular layer. CGNPs, cerebellar granular neuron precursors. Scale bar, 200µm and 20 µm.
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References

    1. Avanesov Andrei, Honeyager Shawn M, Malicki Jarema, Blair Seth S. The Role of Glypicans in Wnt Inhibitory Factor-1 Activity and the Structural Basis of Wif1'S Effects on Wnt and Hedgehog Signaling. In: Perrimon Norbert., editor. PLoS Genet. 2. Vol. 8. 2012. Feb 23, p. e1002503. - DOI - PMC - PubMed
    1. Bai C Brian, Auerbach Wojtek, Lee Joon S, Stephen Daniel, Joyner Alexandra L. Gli2, but Not Gli1, Is Required for Initial Shh Signaling and Ectopic Activation of the Shh Pathway. Development. 2002 Oct 1;129(20):4753–4761. - PubMed
    1. Baptista CA, Hatten ME, Blazeski R, Mason CA. Cell-Cell Interactions Influence Survival and Differentiation of Purified Purkinje Cells in Vitro. Neuron. 1994 Feb;12(2):243–260. - PubMed
    1. Bartsch S, Bartsch U, Dörries U, Faissner A, Weller A, Ekblom P, Schachner M. Expression of Tenascin in the Developing and Adult Cerebellar Cortex. The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. 1992 Mar;12(3):736–749. - PMC - PubMed
    1. Belvindrah Richard, Nalbant Perihan, Ding Sheng, Wu Chuanyue, Bokoch Gary M, Müller Ulrich. Integrin-Linked Kinase Regulates Bergmann Glial Differentiation During Cerebellar Development. Molecular and Cellular Neuroscience. 2006 Oct;33(2):109–125. doi: 10.1016/j.mcn.2006.06.013. - DOI - PubMed

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