bmp7 r d systems  (R&D Systems)

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

Journal: Development (Cambridge, England)

Article Title: HOXA13 regulates the expression of bone morphogenetic proteins 2 and 7 to control distal limb morphogenesis.

doi: 10.1242/dev.01327

Figure Lengend Snippet: Fig. 1. Spatiotemporal expression of Hoxa13 correlates with sites of malformation and decreased BMP expression. (A-D) Analysis of Hoxa13 expression by in situ hybridization using an exon 1-specific riboprobe. Hoxa13 expression is present in the distal interdigital mesenchyme and peridigital tissues (A, arrowheads). (C) Hoxa13 localizes to the distal joint/nail bed in E14.5 limbs (arrowheads). (B,D) In homozygous mutants, elevated levels of Hoxa13 exon 1 transcripts were detected throughout the autopod. (E,F) Alcian Blue staining of E15.5 mutant limbs reveals defects in distal digit separation (F, arrows) and chondrogenesis (arrowheads), when compared with wild-type controls. Pi, pisiform carpal element. (G-J) Bmp7 expression is reduced (arrows) in the distal interdigital tissues at E12.5 (G,H) and in the peridigital tissues at E13.5 (I,J) of homozygous Hoxa13 mutants, when compared with age-matched wild-type controls (arrowheads). (K-N) Bmp2 expression is reduced (arrows) in the interdigital tissues of E12.5 Hoxa13 mutant embryos, and in the distal joints/nail beds of E14.5 Hoxa13 mutants, when compared with wild-type controls (arrowheads). (O-R) Msx2, a target of BMP signaling, exhibits reduced interdigital expression in E12.5- E13.5 mutant limbs (arrows) compared with age-matched controls (arrowheads). (S,U) BMP7 (red, arrowhead) and HOXA13-GFP (green) co- localize (yellow cells in U and V) in the interdigital tissues of E12.5 limbs. (T,V) Age-matched homozygous mutants exhibit reduced numbers of BMP7-positive cells in the same interdigital regions (arrow), a finding consistent with the reduced Bmp7 transcripts in these same tissues (compare H and T). Scale bar: 50 µm.

Article Snippet: Antibodies to BMP2/4 (AF355) and BMP7 (AF354) were purchased from R&D Systems, and were used at 0.02 μg/ml on frozen limb sections.

Techniques: Expressing, In Situ Hybridization, Staining, Mutagenesis

Journal: Development (Cambridge, England)

Article Title: HOXA13 regulates the expression of bone morphogenetic proteins 2 and 7 to control distal limb morphogenesis.

doi: 10.1242/dev.01327

Figure Lengend Snippet: Fig. 3. HOXA13 activates transcription from the Bmp2 and Bmp7 enhancer regions, and associates with these enhancers in vivo. Co- transfection of NG108-15 cells with luciferase reporter plasmids containing forward or reverse orientations of the Bmp2 enhancer sequence (A,C), or the Bmp7 enhancer sequence (B,C), and pCMV-A13 resulted in 2.5- and 1.8-fold (Bmp2), and 1.8- and 2.0-fold (Bmp7), increases in RLA, when compared with identical transfections with the control pCMV vector. Luciferase activity was normalized for transfection efficiency using a Renilla Luciferase control plasmid in all co-transfection assays. Bars represent the standard deviation of results derived from four transfection assays. (D) Western blot analysis of protein lysates derived from Hoxa13 wild-type (+/+), heterozygous mutant (+/–) and homozygous mutant (–/–) tissues confirms that the Hoxa13 antibody recognizes proteins of the correct molecular weight for wild-type HOXA13 (43 kDa) and mutant HOXA13-GFP (64 kDa). (E) The Hoxa13 antibody immunoprecipitates HA-tagged full-length HOXA13. (F-I) Immunostaining of cultured limb mesenchyme from HOXA13-GFP mutant mice, using the Hoxa13 antibody, reveals strong nuclear co- localization (H, yellow, arrows) between the endogenous HOXA13-GFP protein (F) and the Hoxa13 antibody (G,H). Nuclei are stained with DAPI (I). (J-L) Chromatin immunoprecipitation using the Hoxa13 antibody confirms that wild-type (+/+) HOXA13 binds the Bmp2 (J) and Bmp7s1 (K) enhancer regions in the developing limb, whereas immunoprecipitates from mutant limbs (–/–) lacking the HOXA13 DNA-binding domain did not contain the Bmp2 and Bmp7s1 enhancer regions. (L) The absence of Bmp7s2 sequences in wild-type immunoprecipitates confirms HOXA13 specificity for the TAAT-containing sequences in Bmp2 and Bmp7s1. The TAAT-containing sequences present in the Bmp2, Bmp7s1 and Bmp7s2 regions are listed below panels J, K, and L. NC, negative PCR control; PC, positive PCR control.

Article Snippet: Antibodies to BMP2/4 (AF355) and BMP7 (AF354) were purchased from R&D Systems, and were used at 0.02 μg/ml on frozen limb sections.

Techniques: In Vivo, Cotransfection, Luciferase, Sequencing, Transfection, Control, Plasmid Preparation, Activity Assay, Standard Deviation, Derivative Assay, Western Blot, Mutagenesis, Molecular Weight, Immunostaining, Cell Culture, Staining, Chromatin Immunoprecipitation, Binding Assay

Journal: Development (Cambridge, England)

Article Title: HOXA13 regulates the expression of bone morphogenetic proteins 2 and 7 to control distal limb morphogenesis.

doi: 10.1242/dev.01327

Figure Lengend Snippet: Fig. 5. IPCD is delayed in Bmp7 homozygous mutant limbs. (A,B) TUNEL analysis of IPCD in E13.5 Bmp7 mutants (B) revealed a delay in IPCD between digits II and III (arrow), when compared with age-matched heterozygous controls (A, arrowhead). (C,D) Bright-field analysis of the same Bmp7 heterozygous and mutant limbs shown in A and B. (E-G) Analysis of Hoxa13 induction by exogenous BMP2 or BMP7. Wild-type limbs treated with 0.1 mg/ml BMP2 (E) or BMP7 (F) did not exhibit any increase in Hoxa13 expression when compared with PBS controls (G), indicating that in the developing autopod, BMP2 and BMP7 do not regulate Hoxa13 expression.

Article Snippet: Antibodies to BMP2/4 (AF355) and BMP7 (AF354) were purchased from R&D Systems, and were used at 0.02 μg/ml on frozen limb sections.

Techniques: Mutagenesis, TUNEL Assay, Expressing