gfap-cre, a transgene driven by the human gfap promoter (stock #004600; )  (Jackson Laboratory)

Journal: eLife

Article Title: Aberrant FGF signaling promotes granule neuron precursor expansion in SHH subgroup infantile medulloblastoma

doi: 10.7554/eLife.100767

Figure Lengend Snippet: Western blot analysis of cerebellar protein lysates from P0 control and Sufu-cKO mice showing significantly lower levels of total and cleaved versions of Gli transcription factors in the P0 Sufu-cKO cerebellum. *p<0.05, **p<0.01. β-Galactosidase activity (blue), representing the Gli1-LacZ transgene, is largely absent in areas adjacent to the external granule layer (EGL) along the secondary (sec) fissure of the P0 Sufu-cKO cerebellum.

Article Snippet: GFAP-cre, a transgene driven by the human GFAP promoter (Stock #004600; ), and Gli1-LacZ (Stock #008211) mice were obtained from Jackson Laboratories (Bar Harbor, ME).

Techniques: Western Blot, Control, Activity Assay

Journal: bioRxiv

Article Title: ADAM10 mediates macroglial cell fate decisions in the developing brain

doi: 10.1101/2023.02.11.527059

Figure Lengend Snippet: ( A ) Images of wild-type (WT, left), hGFAP Adam10-CKO (Hom, hGFAP-Cre;Adam10 fl/fl , right), and heterozygous (Het, hGFAP-Cre;Adam10 fl/+ , middle) mice at P4, P8, and P30. ( B ) Body weight of hGFAP Adam10-CKO (CKO) and control (Ctrl) mice from P1 to P27. ( C ) Overall survival of both CKO and control mice. ( D ) A schematic of the bar-crossing apparatus for behavioral tests. ( E ) Frequency of slips from the wide bar. ( F ) Frequency of falls from the wide bar. ( G ) Frequency of attempts to cross from one wide bar to the other via the narrow bar. ( H ) Percentage of time spent in motion on the wide bar. ( I ) Percentage of time spent in inactivity on the wide bar. ( J ) A forced crossing score on the narrow bar was calculated for control and CKO mice. ( K ) The latency until falling for control and CKO mice during the rotarod test. ( L–O ) Graphical representation of selected gait parameters of control and CKO mice. Forelimb and hindlimb steps were recorded for analysis (L). Green, forelimb; red, hindlimb. The length of forelimb steps (M), the length of hindlimb steps (N), and the velocity (O) were then determined. The analyses were based on data shown in panel L. Mouse numbers: n = 11, 17, 17, 17, 17, 17, 7, and 2 mice for each time point of the control group, and n = 8, 14, 14, 14, 14, 13, 5, and 2 mice for the CKO group in B; n = 6 for both wild type and CKO in E–I; n = 14 for both CKO and control mice; n = 8 for wild type and CKO in J; n = 9 for wild type and 8 for CKO in K; and n = 5 for both wild type and CKO in M–O. Two-tailed unpaired t -test: * p < 0.05, ** p < 0.01, *** p < 0.005; ns, not significant. Error bars, s.e.m.

Article Snippet: Ai14 (stock No. 007914), hGFAP-Cre (stock No. 004600), and Adam10-flox (stock No. 009357) were purchased from Jackson Laboratory.

Techniques: Two Tailed Test

Journal: bioRxiv

Article Title: ADAM10 mediates macroglial cell fate decisions in the developing brain

doi: 10.1101/2023.02.11.527059

Figure Lengend Snippet: ( A ) Images of the brains of hGFAP Adam10-CKO (CKO) and wild-type control (WT) mice at P0 and P5. ( B ) Images of the cerebellums of CKO and WT mice at P30. ( C ) Cerebellar weight of CKO (n = 5) and control (n = 5) mice at P30. ( D , E ) Hematoxylin and eosin (HE) staining of cerebellar sections from E17.5 control (D) and CKO (E) embryos. (a, b) Higher magnification of the boxed areas in D and E, respectively. ( F , G ) HE staining of cerebellar sections of E18.5 control and CKO embryos. Pc, Pr, Se, Pl (asterisks), the four primary fissures in the cerebellum. (c, d) Higher magnification of the boxed areas in F and G, respectively. Yellow arrowheads, EGL layers. ( H , I ) HE staining of P0 cerebellar sections of control and CKO mice. Yellow arrowheads, anterodorsal lobe and central lobe on either side of the primary fissure. (e, f) Higher magnification of the boxed areas in H and I, respectively. ( J , K ) HE staining of cerebellar sections of P7 control and CKO mice. (g, h) Higher magnification of the boxed areas in J and K, respectively. Lobes around Pr, Se, and Pl were attached in CKO mice (K, h: red arrowheads). Granule cell progenitors in EGL layers are shown (g, h: yellow arrowheads). ( L , M ) HE staining of cerebellar sections of P21 control and CKO mice. (i, j) Higher magnification of the boxed areas in L and M, respectively. Disorganized lamination of the cerebellar cortex in CKO mice (j). EGL,external granule layer (red arrowhead), ML, molecular layer; IGL, internal granule layer. Granule cell progenitors remained in the EGL in CKO mice (j, red arrowhead) but not in control mice (i, red arrowhead). Granule cell progenitor also stuck in the ML in CKO mice (j, yellow arrowhead) but migrated to IGL in control mice (i, yellow arrowhead). Dashed line in i and j, the border between the ML and IGL. ( N , O ) Representative diagram of the sagittal plane of the cerebellum of P21 control (N) and CKO (O) mice. ( P , Q ) Comparison of the cell density in the ML (P) and IGL (Q) layers from P21 cerebellums of control and CKO mice. Two-tailed unpaired t -test: *** p < 0.001. Error bars, s.e.m.

Article Snippet: Ai14 (stock No. 007914), hGFAP-Cre (stock No. 004600), and Adam10-flox (stock No. 009357) were purchased from Jackson Laboratory.

Techniques: Staining, Two Tailed Test

Journal: bioRxiv

Article Title: ADAM10 mediates macroglial cell fate decisions in the developing brain

doi: 10.1101/2023.02.11.527059

Figure Lengend Snippet: ( A–T ) Images of cerebellar sections from hGFAP-Cre;Ai14 mice at P14. Red, the fluorescent signals from tdTomato (Tdt; A, E, I, M, Q). Green, staining with anti-NeuN (B, neurons), anti-calbindin (F, Purkinje cells), anti-Blbp (J, astrocytes), anti-GFAP (N, astrocytes), and anti-Olig2 (R, oligodendrocytes and OPCs) to detect the indicated cell types. Blue, nuclei stained by Hoechst 33342 (C, G, K, O, S). Merged images shown in (D, H, L, P, T). White arrowheads in (A–T), tdTomato-expressing cells in (A), Purkinje cells in (E–H), immature astrocytes in (I–L), tdTomato-expressing cells in (M–P), and cells of the oligodendrocyte lineage in (Q–T); Yellow arrowheads in (I, K, L), glial endfeet in the EGL.

Article Snippet: Ai14 (stock No. 007914), hGFAP-Cre (stock No. 004600), and Adam10-flox (stock No. 009357) were purchased from Jackson Laboratory.

Techniques: Staining, Expressing

Journal: bioRxiv

Article Title: ADAM10 mediates macroglial cell fate decisions in the developing brain

doi: 10.1101/2023.02.11.527059

Figure Lengend Snippet: ( A ) Flow diagram of bRNA-seq of P0 and P7 cerebellums from control (Ctrl) and hGFAP Adam10-CKO (CKO) mice. ( B – F ) Principal component analysis (PCA) of bRNA-seq data from P0 (B) and P7 (E) cerebellar samples of control (n = 5, P0; n = 4, P7) and CKO (n = 5, P0; n = 4, P7) mice. (B) The top two PCs explain 33.87% and 16.8% of the total variance, respectively. (E) The top two PCs explain 39.31% and 19.17% of the total variance, respectively. Each circle indicates data from bRNA-seq of individual samples (C, F). The representative genes (e.g., Slc1a3, Olig2) are shown by a larger circle of a slightly different color. Analysis of differentially expressed genes (upregulated or downregulated) in each sample of control and hGFAP Adam10-CKO mice. (C, F) The volcano plots show downregulated (green and dark blue) and upregulated (dark pink and red) genes (FDR adjusted p < 0.05 and | log 2 FC | > 1); black vertical dashed lines highlight a FC of –1.5 and 1.5, and the black horizontal dashed line represents an adjusted p -value ( P adj) of 0.05. (D, G) Clustered heatmaps for P0 mice (D) (control, n = 5; CKO, n = 5) and P7 mice (G) (control, n = 4; CKO, n = 4). The ordinate represents individual genes, and the abscissa represents individual samples. The colors of the heatmap correspond to the expression level of genes (D, G). Red indicates high expression and blue indicates low expression. DEGs are grouped by hierarchical clustering analysis, based on Euclidean distance s and the ward.D2 method. ( H, I ) Gene Ontology (GO) biological process enrichment analysis of upregulated pathways in P0 (H) and P7 (I) mice. The process names highlighted in red are related to glial generation and development: Glial cell diff 1, glial cell differentiation; Gliogenesis 1, gliogenesis; Myelination 1, myelination; Ensheath 1, ensheathment of neurons; Ensheath 2, axon ensheathment; Oligo diff 1, oligodendrocyte differentiation; Neurogenesis, negative regulation of neurogenesis; CNS develop 1, negative regulation of nervous system development; Cell develop, negative regulation of cell development; Gliogenesis 2, regulation of gliogenesis; Glial cell diff 2, regulation of glial cell differentiation; Glial develop, glial cell development; Myelination 2, central nervous system myelination; Ensheath 3, axon ensheathment in the central nervous system; Olig develop, oligodendrocyte development; Cytokine pro 1, positive regulation of cytokine production; Leukocyte 1, leukocyte - mediated immunity; Leukocyte 2, leukocyte proliferation; Lymp prolif, lymphocyte proliferation; Mononu prolif, mononuclear cell proliferation; Leukocyte 3, myeloid leukocyte activation; Tumor nf pro 1, tumor necrosis factor production; Tumor nf pro 2, tumor necrosis factor superfamily cytokine production; Tumor nf pro 3, regulation of tumor necrosis factor production; Cytokine pro 2, regulation of tumor necrosis factor superfamily cytokine production; Glial cell diff, glial cell differentiation; T cell prolif, T - cell proliferation; IL-6 pro 1, interleukin-6 production; IL-6 pro 2, regulation of inteleukin-6 production. ( J , K ) Relative abundance of astrocyte- (J) and oligodendrocyte- (K) specific genes from P0 control (blue, n = 5 mice) and CKO (red, n = 5 mice) samples. Each circle represents individual sample. Two-tailed unpaired t -test: * p < 0.05, ** p < 0.01, *** p < 0.005. Error bars, s.e.m. ( L–O ) The Gene Set terms immature astrocyte s (L), OPCs (pre-OLs, i.e., oligodendrocyte progenitor cells and pre-myelinating oligodendrocytes) (M, O), and mature astrocytes (N) were differentially regulated in cerebellar samples from P0 and P7 CKO mice when compared with the control samples in a GSEA analysis. Vertical black bars indicate the hits in the Gene Sets as represented among all genes pre-ranked by ranking metrics. The green dashed lines in the plots indicate thresholds of p adj < 0.05 and |Log 2 (FC)| > 1.

Article Snippet: Ai14 (stock No. 007914), hGFAP-Cre (stock No. 004600), and Adam10-flox (stock No. 009357) were purchased from Jackson Laboratory.

Techniques: Expressing, Cell Differentiation, Activation Assay, Two Tailed Test

Journal: bioRxiv

Article Title: ADAM10 mediates macroglial cell fate decisions in the developing brain

doi: 10.1101/2023.02.11.527059

Figure Lengend Snippet: ( A ) Schematic diagram of analysis via single-cell RNA-seq from P2 mouse cerebellums of hGFAP-Cre;Ai14 (control, Ctrl) and hGFAP-Cre;Adam10 fl/fl ;Ai14 (CKO) mice. ( B ) Visualization of major classes of cells using nonlinear dimensionality reduction technique, uniform manifold approximation and projection (UMAP). Each dot represents an individual cell; different colors represent different cell classes. Neu 1–3, neurons; PCs, progenitor cells; Ast, astrocytes; OLs, oligodendrocytes; UBC, unipolar brush cells, Excitatory neu, excitatory neurons; GABA PCs, GABAergic progenitor cells. The total number of cells analyzed was 11,155 from CKO and control mice at P2. ( C ) Distribution of the 14 identified subclusters of cells in the cerebellums of control and CKO mice. Red, cells with tdTomato expression from CKO mice. ( D ) Dot plots of marker genes for the corresponding subclusters listed in (B). ( E ) Dot plots of marker genes for glial progenitors, astrocytes, and oligodendrocytes labeled by tdTomato in the cerebellums of control and CKO mice. PCs, progenitor cells; Ast, astrocytes; OLs, oligodendrocytes; OPC, oligodendrocyte precursor cells. ( F ) UMAP of astrocytes and oligodendrocytes from P2 control and CKO cerebellums. Streamlines show averaged and projected scVelo velocities. Each cluster type is represented by a number in the stremlines. The glial cell subclusters include six astrocyte clusters (Ast1-3, Hes + ast1, Hes + ast2, and Pbk + ast), two oligodendrocyte cell clusters (OPC, OLs), and Dll + progenitor cells (Dll + PCs). ( G ) CellRank probabilities for acquiring the terminal cell fate. Cells were colored based on the probability of reaching the differentiated state. Dark red and blue show the most differentiated and undifferentiated states, respectively. This latent time indicates internal clock of a cell and approximates the real time experienced by a cell as it differentiates. ( H ) Differentiation pathway connectivity of progenitor cells and eight different glial subclusters from the partition-based graph abstraction (PAGA) shown in (F). The proportion of the pie chart represents the probability of each terminal state, and the arrow represents the direction of cell differentiation. ( I ) Triangle projection of 1,164 tdTomato-expressing cells according to fate probabilities. Each color represents a glial subcluster. Macrostates are arranged on the edge of a triangle; each cell is placed inside the triangle according to its probability of reaching any terminal state. Cells in the center have a higher multilineage potential, whereas cells close to one of the corners are committed to differentiate into that terminal cell type. The cell types consist of astrocytes (Ast1 and Hes5 + ast1) and oligodendrocytes (OLs). ( J ) Distribution pattern of cells from the cerebellum of the CKO (red) and control (gray) mice. ( K ) Proportions of cell subpopulations of astrocytes and oligodendrocytes in control and CKO cerebellums. ( L ) Cell fate probability of differentiation into oligodendrocytes (OLs) and astrocytes (Ast 1 and Hes5 + ast 1) in control and CKO mice. Two-tailed unpaired t -test: *** p < 0.001; ns, not significant. Error bars, s.e.m. ( M ) Schematic diagram showing that Adam10 mediates the generation of oligodendrocytes (OLs) and astrocytes (Ast) from glial progenitor cells (GPCs). ADAM10 promotes astrocyte generation under physiological conditions. Once Adam10 is removed (as indicated by the scissors), the path for oligodendrocyte generation from GPCs is activated.

Article Snippet: Ai14 (stock No. 007914), hGFAP-Cre (stock No. 004600), and Adam10-flox (stock No. 009357) were purchased from Jackson Laboratory.

Techniques: RNA Sequencing Assay, Expressing, Marker, Labeling, Cell Differentiation, Two Tailed Test

Journal: bioRxiv

Article Title: ADAM10 mediates macroglial cell fate decisions in the developing brain

doi: 10.1101/2023.02.11.527059

Figure Lengend Snippet: ( A–F ) Images of cerebellar sections and quantification from the cerebellum of wild-type control (Ctrl) and hGFAP Adam10-CKO (CKO) mice at P0 (A–C) and P8 (D–F). Green, cells stained with anti-Blbp; blue, nuclei stained with Hoechst 33342 (HO). Comparisons of the density of Blbp + cells in different cerebellar subregions (EGL, ML, IGL, and WM) of control (A, D) and CKO (B, E) mice at P0 (A–C) and P8 (D–F). (a’, b’, d’, e’) Higher magnification of the boxed areas in A, B, D, E, respectively. Dashed lines indicate the margins of different layers in the cerebellum, including the external granule layer (EGL), molecular layer (ML), internal granule layer (IGL), and white matter (WM). The density (×10 4 cells/mm 3 ) of the IGL at P0: control, 15.66 ± 0.51; CKO, 6.3 ± 0.57; at P8: control, 19.75 ± 1.63; CKO, 6.30 ± 0.71. The density of the WM at P0: control, 13.14 ± 1.05; CKO, 7.51 ± 1.48; at P8: control, 13.00 ± 3.00; CKO, 5.31 ± 0.70. ( G–L ) Images of cerebellar sections from wild-type control and CKO mice at P0 (G–I) and P8 (J–L). Green, cells stained with anti-Olig2; blue, nuclei stained with Hoechst 33342 (HO). Comparisons of the density of Olig2 + cells in different cerebellar subregions (EGL, ML, IGL, and WM) of control (G, J) and CKO (H, K) mice. (g’, h’, j’, k’) Higher magnification of the boxed areas in G, H, J, K, respectively. ( M–P ) Images of cerebellar sections of wild-type control and CKO mice at P0 (M, N) and P8 (O, P). Green, myelination stained with anti-MBP; blue, nuclei stained with Hoechst 33342 (HO). Dashed lines indicate the margins of different layers in the cerebellum (EGL, ML, and IGL) of control (M, O) and CKO (N, P) mice at P0 (M, N) and P8 (O, P). (m’, n’, o’, p’) Higher magnification of the boxed areas in M, N, O, P, respectively. (m’’, n’’, o’’, p’’) Representative regions of MBP staining of oligodendrocyte myelination as indicated by the boxed areas in m’, n’, o’, p’, respectively.

Article Snippet: Ai14 (stock No. 007914), hGFAP-Cre (stock No. 004600), and Adam10-flox (stock No. 009357) were purchased from Jackson Laboratory.

Techniques: Staining