Journal: Acta oto-laryngologica

Article Title: Expression of bone morphogenetic protein 2, 4, 5, and 7 correlates with histological activity of otosclerotic foci.

doi: 10.3109/00016489.2011.653669

Figure Lengend Snippet: Figure 1. Immunofluorescent demonstration of BMP expression in active otosclerosis. (A1) Active focus of otosclerosis with hypercel- lular osteoid substance and several pseudo-vascular spaces (white arrow) (H&E, right ear). (A2) Phase-contrast (PC) microscopic view of the previous section. The cement lines show an irregular concentric (white arrow) and woven (black arrow) pattern. Immu- nofluorescent micrographs with different magnification views for BMP2 (B1, B2), BMP4 (C1, C2), BMP5 (D1, D2), and BMP7 (E1, E2) display intense granular cytoplasmic immunoreac- tions in the lesion-forming cells.

Article Snippet: Blocked sections were incubated with 1:20 monoclonal mouse antihuman BMP2 primary antibody solution (20 C, 24 h, continuous shaking) (RD-MAB2260, R&D Systems, Minneapolis, MN, USA), with 1:20 polyclonal goat anti-human BMP2/4 primary antibody solution (20 C, 24 h, continuous shaking) (RD-AF355, R&D Systems), with 1:20 polyclonal goat antihuman BMP5 primary antibody solution (20 C, 24 h, continuous shaking) (RD-AF615, R&D Systems), and finally with 1:20 monoclonal mouse antihuman BMP7 primary antibody solution (20 C, 24 h, continuous shaking) (RD-MAB3542, R&D Systems).

Techniques: Expressing

Journal: Acta oto-laryngologica

Article Title: Expression of bone morphogenetic protein 2, 4, 5, and 7 correlates with histological activity of otosclerotic foci.

doi: 10.3109/00016489.2011.653669

Figure Lengend Snippet: Figure 2. Immunofluorescent demonstration of BMP expression in inactive otosclerosis. (A1) Inactive otosclerotic focus at the posterior pole of the stapes footplate (H&E, right ear). The lesion is characterized by hypocellularity and empty cellular halos. (A2) Phase-contrast (PC) microscopic view of the previous section. Cement lines show lamellar pattern (white arrow). The remnants of osteoblasts and osteoclasts (empty halo cells) show negative immunofluorescent staining for BMP2 (B), BMP4 (C), BMP5 (D), and BMP7 (E).

Article Snippet: Blocked sections were incubated with 1:20 monoclonal mouse antihuman BMP2 primary antibody solution (20 C, 24 h, continuous shaking) (RD-MAB2260, R&D Systems, Minneapolis, MN, USA), with 1:20 polyclonal goat anti-human BMP2/4 primary antibody solution (20 C, 24 h, continuous shaking) (RD-AF355, R&D Systems), with 1:20 polyclonal goat antihuman BMP5 primary antibody solution (20 C, 24 h, continuous shaking) (RD-AF615, R&D Systems), and finally with 1:20 monoclonal mouse antihuman BMP7 primary antibody solution (20 C, 24 h, continuous shaking) (RD-MAB3542, R&D Systems).

Techniques: Expressing, Staining

Journal: Acta oto-laryngologica

Article Title: Expression of bone morphogenetic protein 2, 4, 5, and 7 correlates with histological activity of otosclerotic foci.

doi: 10.3109/00016489.2011.653669

Figure Lengend Snippet: Figure 3. Immunofluorescent demonstration of BMP expression in non-otosclerotic stapes fixation. (A) Non-otosclerotic, annular calcifi- cation of the stapes footplate with hypocellular and angled anterior pole (H&E, right ear). The osteocytes show negative immunoreactions for BMP2 (B), BMP4 (C), BMP5 (D), and BMP7 (E).

Article Snippet: Blocked sections were incubated with 1:20 monoclonal mouse antihuman BMP2 primary antibody solution (20 C, 24 h, continuous shaking) (RD-MAB2260, R&D Systems, Minneapolis, MN, USA), with 1:20 polyclonal goat anti-human BMP2/4 primary antibody solution (20 C, 24 h, continuous shaking) (RD-AF355, R&D Systems), with 1:20 polyclonal goat antihuman BMP5 primary antibody solution (20 C, 24 h, continuous shaking) (RD-AF615, R&D Systems), and finally with 1:20 monoclonal mouse antihuman BMP7 primary antibody solution (20 C, 24 h, continuous shaking) (RD-MAB3542, R&D Systems).

Techniques: Expressing

Journal: Acta oto-laryngologica

Article Title: Expression of bone morphogenetic protein 2, 4, 5, and 7 correlates with histological activity of otosclerotic foci.

doi: 10.3109/00016489.2011.653669

Figure Lengend Snippet: Figure 4. Immunofluorescent demonstration of BMP expression in bone-specific controls. (A1) Body of the incus that represents a normal structure of cement lines and blood vessels (H&E, left ear). The osteocytes represent negative immunoreactions for BMP2 (A2), BMP4 (A3), BMP5 (A4), and BMP7 (A5). (B1) Cortical bone fragment harvested from the posterior wall of the external auditory canal (H&E, left ear). The osteocytes do not display detectable expression of BMP2 (B2), BMP4 (B3), BMP5 (B4), or BMP7 (B5).

Article Snippet: Blocked sections were incubated with 1:20 monoclonal mouse antihuman BMP2 primary antibody solution (20 C, 24 h, continuous shaking) (RD-MAB2260, R&D Systems, Minneapolis, MN, USA), with 1:20 polyclonal goat anti-human BMP2/4 primary antibody solution (20 C, 24 h, continuous shaking) (RD-AF355, R&D Systems), with 1:20 polyclonal goat antihuman BMP5 primary antibody solution (20 C, 24 h, continuous shaking) (RD-AF615, R&D Systems), and finally with 1:20 monoclonal mouse antihuman BMP7 primary antibody solution (20 C, 24 h, continuous shaking) (RD-MAB3542, R&D Systems).

Techniques: Expressing

Journal: bioRxiv

Article Title: A newly-recognized population of residual neural crest cells in the adult leptomeninges is re-activated for vascular repair

doi: 10.1101/2022.12.30.522316

Figure Lengend Snippet: A. Single nuclei were extracted from the cortices of mice subjected to dMCAO 3 (n=3), 7 (n=4), 14 (n=3), and 28 (n=4) days before compared to control nuclei isolated from sham-operated age-matched mice (3 days post-operation, n=3). B. Unsupervised clustering of 107,020 nuclei, ranging from 16,411 to 29,570 nuclei across the above-mentioned time points using the R package, identified 14 clusters. PC: progenitor cells. C. Signature score analysis (z-score), using a Wnt1 -associated early neural crest gene signature, revealed a subpopulation in Cluster 11 as scoring highest ( arrowhead ). D. Cranial neural crest regulatory genes such as Tfap2b, Foxd1, Sned1 and Eya2 were found in Cluster 11 . E. Upregulated “differential gene expression (DEG)” profiling in Cluster 11 in UMAP and FLE plot. F. “Gene Ontology (GO)” enrichment analysis identified, within Custer 11 , subclusters SC2 , SC3 , and SC5 as being involved in the regulation of neural crest migration. G. Subclusters SC2 , SC3 , and SC5 were more finely resolved into Subsets 0-to-6 as follows: SC2 (Subsets 1, 2, 4, and 6), SC3 (Subsets 0 and 3), SC5 (Subset 5 alone). H. Upregulated genes in each Cluster. I. Immunohistochemical analysis showed that Klf5+ cells (Subsets 1 and 5) and Bmp7+ cells (Subsets 0 and 3) co-localized with Foxd3 (a transcriptional repressor involved in early NC commitment in the epiblast). Klf5 is a transcription factor critical for vascular survival and function. BMP7, a member of the TGF-β superfamily, plays a key role in bone homeostasis (transformation of mesenchymal cells into bone and cartilage) and is a well-accepted neural crest marker. PDGFRβ plays a critical role in the cranial neural crest contribution to craniofacial development, while Col3a1 is involved in the signaling necessary for neural crest cell migration. Ebf2 is a transcription factor that is expressed in E8.5 mesenchyme which is composed of neural crest and paraxial mesoderm. Cped1 is classified as a neural crest-associated gene though its precise function remains unknown. J. CD271+ cells were found in cortical layers I-to-IV ipsilateral to the dMCAO 3 days post-infarction. A subset of Bmp7+ cells (subsets 0 and 3) co-localized with CD271. K. Klf5+ cells were abundantly co-stained with CD271 in cortical layer II/III. L-N. The subsets of SC2 were found in the ipsilateral cortical layers II/III and IV, as confirmed by immunohistochemistry, as follows: Subset 2: Ebf2+/Cped1+ cells; Subset 4: PDGFRβ+/Col3a1+ cells; Subset 6: PDGFRβ-/Col3a1+ cells. O. CD271+ cells were isolated from leptomeninges-enriched tissue 3 days following transient focal cerebral ischemia for 60 min. P. Isolated cells were expanded in DMEM/F12 media plus 1% N2, 2% B27, FGF2 (100 ng/ml), and IGF1 (100 ng/ml). Q. Immunocytochemistry confirmed that isolated cells expressed CD271 and Foxd3. R. Representative genes in the Subsets of SC2, SC3, and SC5 were highly expressed in vascular cells ( http://betsholtzlab.org/VascularSingleCells/database.html ). S. Not unexpectedly, GSEA analysis also showed constituents of the meninges that were not neural crest-derived: Subset 4 was enriched for genes associated with bone marrow stroma and pericytes; Subsets 0 and 3 contained genes associated with neural tube-derived radial glia-like cells. T. Venn diagram of genes expressed in pericyte cluster C13 and in Subsets 2, 4, and 6. U. Cluster tree. Cluster 11 was divided into 9 distinct “subclusters”. Neural crest cell genes were enriched in Subclusters SC2, SC3, and SC5. These Subclusters were further divided into 7 distinct Subsets based on upregulated DEGs. Abbreviations: PC - Pericytes; SMC - Smooth muscle cells; MG - Microglia; FB - Vascular fibroblast-like cells; OL - Oligodendrocytes; EC - Endothelial cells; AC - Astrocytes; v - venous; capil - capillary; a - arterial; aa - arteriolar; 1,2,3-subtypes.

Article Snippet: anti-CD271 antibodies (1:100, MA5-13311, Thermo Fisher Scientific, 1:100, 839701, BioLegend), anti-Foxd3 antibody (1:200, LS-C356037-100, LSBio), anti-β-actin antibody (1:10,000, A5441, Millipore-Sigma), anti-PDGFR-β antibody (1:100, AF1042, R&D Systems), anti-smooth muscle actin antibody (1:500, ab5694, Abcam), anti-TFAP2A antibody (1:100, sc-12726, Santacruz), anti-Sox10 antibody (1:200, 14-5923-82, Thermo Fisher Scientific), anti-CD57 (HNK) antibody (1:200, ab199156, Abcam), anti-Klf5 antibody (1:100, TA811868, Origene), anti-Bmp7 antibody (1:100, AF354, R&D Systems), anti-Col3a1 antibody (1:100, NB600-594, Novus Biologicals), anti-Cped1 antibody (1:100, PA5-52903, Thermo Fisher Scientific), anti-CD31 antibody (1:50, 565629, BD Biosciences), anti-Collagen (Type-IV) antibody (1:200, 1340-01, Sourthern Biotech), anti-LYVE-1 antibody (1:500, NB100-725B, Novus Biologicals), anti-Vimentin antibody (1:200, ab92547, Abcam), anti-CD90 antibody (1:200, sc-9163, Santacruz), anti-CD200 antibody (1:500, LS-C34538, LSBio), anti-pAkt antibody (1:50, 9271S, Cell Signaling), anti-pGSK3β antibody (1:50, sc-373800, Santacruz), anti-Claudin-5 antibody (1:100, ab131259, Abcam), anti-ZO1 antibody (1:100, 33-9100, Thermo Fisher Scientific).

Techniques: Control, Isolation, Gene Expression, Migration, Immunohistochemical staining, Transformation Assay, Marker, Staining, Immunohistochemistry, Immunocytochemistry, Derivative Assay