Journal: Arthritis Research & Therapy

Article Title: Interactions between IL-32 and tumor necrosis factor alpha contribute to the exacerbation of immune-inflammatory diseases

doi: 10.1186/ar2074

Figure Lengend Snippet: Primers used in the real-time PCR

Article Snippet: Recombinant human TNFα, IL-12, IL-18, IL-23, granulocyte-macrophage colony-stimulating factor, and IL-4 were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques:

Journal: Arthritis Research & Therapy

Article Title: Interactions between IL-32 and tumor necrosis factor alpha contribute to the exacerbation of immune-inflammatory diseases

doi: 10.1186/ar2074

Figure Lengend Snippet: Examination of tissue and cell expression of IL-32 by quantitative real-time PCR. (a) Tissue expression of IL-32. WBC, white blood cells. (b) Human peripheral blood mononuclear cells (PBMCs) expressed IL-32. PBMCs were cultured with or without concanavalin A. PBMCs were also stimulated by immobilized anti-human CD3 and anti-human CD28 antibodies. Cont, control. (c) IL-32 expression of monocytes and B cells after the depletion of CD3 + cells. (d) Peripheral CD4 + T cells were cultured with the indicated inflammatory cytokines for 24 hours. (e) Human monocyte-derived dendritic cells (MoDCs) were cultured with lipopolysaccharide (LPS) or tumor necrosis factor alpha (TNFα) for 24 hours to induce maturation. The data are representative of at least three independent studies.

Article Snippet: Recombinant human TNFα, IL-12, IL-18, IL-23, granulocyte-macrophage colony-stimulating factor, and IL-4 were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Cell Culture, Control, Derivative Assay

Journal: Arthritis Research & Therapy

Article Title: Interactions between IL-32 and tumor necrosis factor alpha contribute to the exacerbation of immune-inflammatory diseases

doi: 10.1186/ar2074

Figure Lengend Snippet: IL-32 was abundantly expressed in the synovial tissues of rheumatoid arthritis patients. (a) In situ hybridization of the synovial tissues from rheumatoid arthritis (RA) patients. IL-32β was expressed in the synovial-infiltrated lymphocytes of RA patients. HE stain, hematoxylin and eosin stain. We examined the tissue samples from four RA patients, and show representative examples. (b) IL-32 expression of the synovial fibroblasts derived from four RA patients in response to human tumor necrosis factor alpha (hTNFα).

Article Snippet: Recombinant human TNFα, IL-12, IL-18, IL-23, granulocyte-macrophage colony-stimulating factor, and IL-4 were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: In Situ Hybridization, H&E Stain, Expressing, Derivative Assay

Journal: Arthritis Research & Therapy

Article Title: Interactions between IL-32 and tumor necrosis factor alpha contribute to the exacerbation of immune-inflammatory diseases

doi: 10.1186/ar2074

Figure Lengend Snippet: Inflammatory cytokines were induced by human IL-32β in mice. (a) Raw 267.4 was cultured with the supernatant of human IL-32β (h IL-32β) or mock-transfected mammalian cells (293T) for 24 hours. Left, relative expression of mouse tumor necrosis factor alpha (mTNFα), compared with β-actin; right, secreted TNFα protein level measured by ELISA. (b) We generated hIL-32β overexpressed mice by transplantation of hIL-32β-transduced bone marrow cells. The expression of green fluorescent protein, was analyzed by flow cytometry 6–9 weeks after transplantation. (c) Expression of mTNFα, mIL-1β and mIL-6 in the cultured splenocytes of the control group (white bars; n = 3), or bone-marrow chimeric mice of the mock group (BM-Mock mice) (gray bars; n = 4), or hIL-32β (BM-hIL-32) (black bars; n = 4) with or without 1 μg/ml lipopolysaccharide (LPS). Concentrations of indicated cytokines of the cultured supernatants are shown in the right-hand figures. (d) Serum concentration of mTNFα determined in control mice ( n = 4), in BM-Mock mice ( n = 8), and in BM-hIL-32 mice ( n = 8). (e) Expression of mTNFα in splenic F4/80 + CD11c - macrophages of BM-Mock mice (gray bars; n = 4) and in BM-hIL-32 mice (black bars; n = 4). (f) Expression of mTNFα, mIL-1β, and mIL-6 in LPS-stimulated splenic F4/80 + CD11c - macrophages and CD11c + , CD3 - , and CD19 - dendritic cells in BM-Mock mice (gray bars; n = 4), and in BM-hIL-32 mice (black bars; n = 4). Data are representative of at least three independent studies. * P < 0.05, ** P < 0.01, BM-hIL-32 mice versus BM-Mock mice or control mice.

Article Snippet: Recombinant human TNFα, IL-12, IL-18, IL-23, granulocyte-macrophage colony-stimulating factor, and IL-4 were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Cell Culture, Transfection, Expressing, Enzyme-linked Immunosorbent Assay, Generated, Transplantation Assay, Flow Cytometry, Control, Concentration Assay

Journal: Nature Communications

Article Title: LSD1 co-repressor Rcor2 orchestrates neurogenesis in the developing mouse brain

doi: 10.1038/ncomms10481

Figure Lengend Snippet: ( a ) X-gal staining to detect Rcor2 expression patterns. Whole-embryo staining at E11.5 stage shows Rcor2 is mainly expressed in the CNS. Scale bar, 1 mm. ( b ) Western blot analysis of Rcor2 expression levels during brain development. The decreased expression of Rcor2 with embryonic development is noteworthy. β-Actin is used as an endogenous control. ( c ) In situ hybridization to detect endogenous Rcor2 mRNA expression patterns in cortical development at E11.5, E13.5, E15.5 and E17.5. Insets show high-magnification image of Rcor2 expression in the neocortex at E13.5. VZ, ventricular zone; SVZ, subventricular zone; CP, cortical plate. Scale bar, 100 μm. ( d ) Confocal images of immunofluorescence to detect cellular localization of Rcor2 in the neocortex at E13.5. Rcor2 localized mainly in the nucleus at interphase and metaphase, and localized between separated chromosomes in anaphase of dividing cells in VZ. Dotted lines circle the shape of nuclei. Scale bar, 5 μm. ( e ) Western blot analysis of Rcor2 expression level in Rcor2 fl/fl and Rcor2 cko brains at E13.5 and E15.5, respectively. Rcor2 expression was depleted in Rcor2 cko brains. β-Actin is used as an endogenous control. ( f ) Representative images of Rcor2 fl/fl and Rcor2 cko brain size at different stages of development. Rcor2 cko mice show severe brain growth retardation at E13.5 and E15.5. Scale bar, 1 mm. ( g ) Representative images of Rcor2 fl/fl and Rcor2 cko cortex at E15.5 by Nissl staining. Structural abnormalities of lamination with reduced cortical thickness are observed in Rcor2 cko cortex. Scale bar, 200 μm. ( h ) RT–qPCR analysis of knockdown efficiencies of the two shRNAs targeting Rcor2. Transcripts were normalized to the control group. Data are shown as mean±s.e.m., t -test, **** P <0.0001, n =3. ( i ) Confocal images of E16.5 cortical sections electroporated with shControl (red), shRcor2-a (red) and shRcor2-b (red) plasmids at E13.5. Knockdown of Rcor2 results in impaired cortical development. IZ, intermediate zone. Scale bar, 20 μm. ( j ) Quantification of the percentage of RFP + cells in different regions of the developing neocortex after electroporation as shown in i . proportion of RFP + cells in different zones ( y axis). Data are shown as mean±s.e.m., t -test, * P <0.05, ** P <0.01, *** P <0.001 and **** P <0.0001, n =3 individual experiments.

Article Snippet: Primary antibodies used were as follows: Rcor2 (1:50, catalogue number NBP1-74099, Novus Biological); Nes (1:300, catalogue number Rat-401, DSHB); Pax6 (1:100, DSHB); Sox2 (1:500, catalogue number sc-17319, Santa Cruz); BrdU (1:500, catalogue number ab6326, Abcam); Ki67 (1:300, catalogue number ab9260, Millipore); Satb2 (1:500, catalogue number ab34735, Abcam); Olig2 (1:200, catalogue number ab109186, Abcam); Shh (1:100, catalogue number sc-33943, Santa Cruz); Ptch1 (1:100, catalogue number sc-6149, Santa Cruz); Dlx2 (1:500, catalogue number ab117546, Abcam); Nkx2.1 (1:300, catalogue number ab76013, Abcam); Tbr1 (1:500, catalogue number ab31940, Abcam); Dcx (1:500, catalogue number ab18723, Abcam); Tbr2 (1:500, catalogue number ab23345, Abcam); Flag M2 (1:1,000, catalogue number F1804, Sigma); Map2 (1:500, catalogue number ab32454, Abcam); and Tuj1 (1:1,000, catalogue number mms-435p, Convance).

Techniques: Staining, Expressing, Western Blot, Control, In Situ Hybridization, Immunofluorescence, Quantitative RT-PCR, Knockdown, Electroporation

Journal: Nature Communications

Article Title: LSD1 co-repressor Rcor2 orchestrates neurogenesis in the developing mouse brain

doi: 10.1038/ncomms10481

Figure Lengend Snippet: ( a ) Immunostaining images of Sox2 at E13.5. Sox2 is dramatically reduced on Rcor2 depletion. VZ, ventricular zone; SVZ, subventricular zone. Scale bar, 20 μm. ( b ) Quantification of Sox2 + cell ratios in VZ/SVZ regions shown in a . Data are shown as mean±s.e.m., t -test, ** P <0.01, n =3 individual experiments. ( c ) Confocal images of Tbr2 expression at E13.5. Tbr2 is dramatically reduced on Rcor2 depletion. Scale bar, 20 μm. ( d ) Quantification of Tbr2 + cell ratios in VZ/SVZ regions shown in c , respectively. Data are shown as mean±s.e.m., t -test, * P <0.05, n =3 individual experiments. ( e ) Immunostaining images of Nestin, Sox2 and Tbr2 in cultured Rcor2 fl/fl and Rcor2 cko NPCs, all of which exhibit significantly reduced expression in the Rcor2 cko NPCs. Scale bar, 20 μm. ( f ) Confocal images of immunofluorescence for Ki67 and PHH3 in Rcor2 fl/fl and Rcor2 cko cortex at E13.5 and E15.5. Ki67 signals (red), but not PHH3 signals (green), are dramatically reduced in Rcor2 cko developing brains. Scale bar, 20 μm. ( g ) Quantification of Ki67 + cells in the VZ/SVZ regions of the developing neocortex as shown in f . Data are shown as mean±s.e.m., t -test, ** P <0.01, n =3 separate stainings from three independent brains. ( h ) Confocal images of BrdU (green) and Ki67 (red) staining in Rcor2 fl/fl and Rcor2 cko cortex 24 and 48 h after BrdU incorporation. Scale bar, 100 μm. ( i ) Quantification of the cell cycle exit by percentage of BrdU + and Ki67 + NPCs divided by BrdU + cells shown in h . Data are shown as mean±s.e.m., t -test, *** P <0.001 and **** P <0.0001, n =3 individual experiments. ( j , k ) Representative images ( j ) and quantification of ( k ) of Rcor2 fl/fl and Rcor2 cko neurosphere sizes. The neurospheres'radius of Rcor2 cko are much smaller than those of Rcor2 fl/fl , t -test, **** P <0.0001, n =12. Scale bar, 50 μm. ( l ) Representative time-lapse imaging of the RGC dividing process in the sections of the cerebral cortex electroporated with RFP-shControl (upper panels) and RFP-shRcor2 (lower panels). The radial glial dividing pattern is abnormal on Rcor2 knockdown, resulting in cell death. Arrows, mother RGCs. Arrowheads, two daughter cells. Scale bar, 50 μm. ( m ) Representative time-lapse images of Rcor2 fl/fl cortex sections electroporated with EGFP-Control (upper panels) and EGFP-Cre (lower panels). Loss of cells is observed with Rcor2 knockout by Cre recombinase electroporation. Scale bar, 50 μm.

Article Snippet: Primary antibodies used were as follows: Rcor2 (1:50, catalogue number NBP1-74099, Novus Biological); Nes (1:300, catalogue number Rat-401, DSHB); Pax6 (1:100, DSHB); Sox2 (1:500, catalogue number sc-17319, Santa Cruz); BrdU (1:500, catalogue number ab6326, Abcam); Ki67 (1:300, catalogue number ab9260, Millipore); Satb2 (1:500, catalogue number ab34735, Abcam); Olig2 (1:200, catalogue number ab109186, Abcam); Shh (1:100, catalogue number sc-33943, Santa Cruz); Ptch1 (1:100, catalogue number sc-6149, Santa Cruz); Dlx2 (1:500, catalogue number ab117546, Abcam); Nkx2.1 (1:300, catalogue number ab76013, Abcam); Tbr1 (1:500, catalogue number ab31940, Abcam); Dcx (1:500, catalogue number ab18723, Abcam); Tbr2 (1:500, catalogue number ab23345, Abcam); Flag M2 (1:1,000, catalogue number F1804, Sigma); Map2 (1:500, catalogue number ab32454, Abcam); and Tuj1 (1:1,000, catalogue number mms-435p, Convance).

Techniques: Immunostaining, Expressing, Cell Culture, Immunofluorescence, Staining, BrdU Incorporation Assay, Imaging, Knockdown, Control, Knock-Out, Electroporation

Journal: Nature Communications

Article Title: LSD1 co-repressor Rcor2 orchestrates neurogenesis in the developing mouse brain

doi: 10.1038/ncomms10481

Figure Lengend Snippet: ( a ) Confocal images of Satb2 and Tbr1 expressions in Rcor2 fl/fl and Rcor2 cko cortex at E15.5, which exhibit significant reduction on Rcor2 knockout. Scale bar, 50 μm. ( b ) Quantification of Satb2 + and Tbr1 + cells in Rcor2 fl/fl and Rcor2 cko cortex at E15.5 in a indicates Satb2 and Tbr1 expressions are decreased on Rcor2 depletion during development. Data are shown as mean±s.e.m., t -test, ** P <0.01, n =3 individual experiments. ( c ) Western blot to analyse Dcx, Satb2 and Tbr1 expressions in Rcor2 fl/fl and Rcor2 cko cortex at E15.5. β-Actin is used as an endogenous control. ( d ) Representative images of Map2 and Tuj1 immunostaining in cultured neurons directly dissociated from Rcor2 fl/fl and Rcor2 cko brain cortex at E15.5. Decreased expression of both markers and reduced neurofilaments can be observed in Rcor2 cko cultured neurons. Scale bar, 20 μm. ( e ) Confocal images of in-vitro cultured Rcor2 fl/fl and Rcor2 cko NPCs 5 days post spontaneous differentiation using neuronal marker Map2 and Tuj1 antibodies, both of which are significantly reduced in the differentiated Rcor2 cko NPCs. Scale bar, 20 μm. ( f , g ) RT–qPCR analysis of neuronal markers expression in both Rcor2 fl/fl and Rcor2 cko neocortex at E15.5 stage ( f ) and in-vitro -cultured Rcor2 fl/fl and Rcor2 cko NPCs 5 days post spontaneous differentiation ( g ). Transcripts were normalized to Rcor2 fl/fl group. Data are shown as mean±s.d., t -test, * P <0.05, ** P <0.01 and *** P <0.001, n =3.

Article Snippet: Primary antibodies used were as follows: Rcor2 (1:50, catalogue number NBP1-74099, Novus Biological); Nes (1:300, catalogue number Rat-401, DSHB); Pax6 (1:100, DSHB); Sox2 (1:500, catalogue number sc-17319, Santa Cruz); BrdU (1:500, catalogue number ab6326, Abcam); Ki67 (1:300, catalogue number ab9260, Millipore); Satb2 (1:500, catalogue number ab34735, Abcam); Olig2 (1:200, catalogue number ab109186, Abcam); Shh (1:100, catalogue number sc-33943, Santa Cruz); Ptch1 (1:100, catalogue number sc-6149, Santa Cruz); Dlx2 (1:500, catalogue number ab117546, Abcam); Nkx2.1 (1:300, catalogue number ab76013, Abcam); Tbr1 (1:500, catalogue number ab31940, Abcam); Dcx (1:500, catalogue number ab18723, Abcam); Tbr2 (1:500, catalogue number ab23345, Abcam); Flag M2 (1:1,000, catalogue number F1804, Sigma); Map2 (1:500, catalogue number ab32454, Abcam); and Tuj1 (1:1,000, catalogue number mms-435p, Convance).

Techniques: Knock-Out, Western Blot, Control, Immunostaining, Cell Culture, Expressing, In Vitro, Marker, Quantitative RT-PCR

Journal: Nature Communications

Article Title: LSD1 co-repressor Rcor2 orchestrates neurogenesis in the developing mouse brain

doi: 10.1038/ncomms10481

Figure Lengend Snippet: ( a ) Schematic overview of strategy to generate an Rcor2 Flag knock-in allele by CRISPR/Cas9. The sgRNA sequence site is shown as a green arrowhead. The start codon of Rcor2 is indicated and capitalized. The oligo donor contained 50 bp homologies on both sides flanking the DSB, in which 3 × Flag sequences are labelled as a red box. ( b ) Western blot analysis to validate FLAG, RCOR2 and LSD1 expressions in Rcor2 Flag knock-in neocortex using Flag-M2 antibody. β-Actin was used as an endogenous control. ( c ) Pie chart depicts distribution of Rcor2 occupancies in genome-wide scale in FLAG ChIP-seq results using Rcor2 Flag knock-in neocortex at E13.5. ( d ) WebLogos of consensus binding motifs of Rcor2 generated by Multiple EM for Motif Elicitation (MEME) motif analysis tool. ( e ) GO analysis for Rcor2-binding regions in genome-wide scale revealed by Flag ChIP-seq results using Rcor2 Flag brain. ( f ) GO analysis for LSD1 occupancy in genome-wide scale revealed by LSD1 ChIP-seq results using Rcor2 Flag brain. ( g ) Density plots analysis of H3K4me1 signal change in promoter regions (−2- to ∼0.5 kb from TSS) on Rcor2 depletion. Compared with all genes, the change of H3K4me1 signal is significantly ( P <0.0005, Kolmogorov–Smirnov test) enhanced in the promoter regions of Shh pathway-related genes on Rcor2 depletion. H3K4me1 signal change on Rcor2 depletion ( x axis); H3K4me1 signal density ( y axis). ( h ) Gene tracks of Rcor2, LSD1 and H3K4me1 enrichments by ChIP-seq analysis at core promoter regions of Dlx2 and upstream regulatory regions of Shh, which are closely related to Shh signalling. ( i ) ChIP–qPCR analysis of Rcor2 Flag and Rcor2 cko cortex at E13.5 using specific FLAG-M2 antibody. Significant enrichments of the Rcor2 at the regulatory regions of Dlx2 and Shh gene locus detected in g in the Rcor2 flag samples are worth noting. Fold enrichments of Rcor2 occupancy compared with input ( y axis). Data are shown as mean±s.d., t -test, *** P <0.001, n =3.

Article Snippet: Primary antibodies used were as follows: Rcor2 (1:50, catalogue number NBP1-74099, Novus Biological); Nes (1:300, catalogue number Rat-401, DSHB); Pax6 (1:100, DSHB); Sox2 (1:500, catalogue number sc-17319, Santa Cruz); BrdU (1:500, catalogue number ab6326, Abcam); Ki67 (1:300, catalogue number ab9260, Millipore); Satb2 (1:500, catalogue number ab34735, Abcam); Olig2 (1:200, catalogue number ab109186, Abcam); Shh (1:100, catalogue number sc-33943, Santa Cruz); Ptch1 (1:100, catalogue number sc-6149, Santa Cruz); Dlx2 (1:500, catalogue number ab117546, Abcam); Nkx2.1 (1:300, catalogue number ab76013, Abcam); Tbr1 (1:500, catalogue number ab31940, Abcam); Dcx (1:500, catalogue number ab18723, Abcam); Tbr2 (1:500, catalogue number ab23345, Abcam); Flag M2 (1:1,000, catalogue number F1804, Sigma); Map2 (1:500, catalogue number ab32454, Abcam); and Tuj1 (1:1,000, catalogue number mms-435p, Convance).

Techniques: Knock-In, CRISPR, Sequencing, Western Blot, Control, Genome Wide, ChIP-sequencing, Binding Assay, Generated, ChIP-qPCR

Journal: Nature Communications

Article Title: LSD1 co-repressor Rcor2 orchestrates neurogenesis in the developing mouse brain

doi: 10.1038/ncomms10481

Figure Lengend Snippet: ( a , b ) Scatter plot analysis of genome-wide expression profiles of Rcor2 cko versus Rcor2 fl/fl samples at E13.5 ( a ) and E15.5 ( b ). Dots above or below the dash line indicate upregulated or downregulated genes on Rcor2 depletion, respectively. Red dots or green dots highlight the significantly differentially expressed genes on Rcor2 depletion. Raw counts ( x axis); gene expression fold changes on Rcor2 depletion ( y axis). ( c ) Venn diagrams of upregulated genes (left) and downregulated genes (right) in Rcor2 cko samples compared with Rcor2 fl/fl samples. ( d ) The profiles of Rcor2 and H3k4me1 enrichments analysed in ChIP-seq results shown in in regulatory regions of genome-wide scale (red) and of the upregulated genes (purple) according to RNA-seq results. ( e ) Correlation network of overlapped upregulated genes in both E13.5 and E15.5 samples. Lines indicate the correlations between two connected genes with R >0.55. Genes were analysed by GO analysis and divided into different categories.

Article Snippet: Primary antibodies used were as follows: Rcor2 (1:50, catalogue number NBP1-74099, Novus Biological); Nes (1:300, catalogue number Rat-401, DSHB); Pax6 (1:100, DSHB); Sox2 (1:500, catalogue number sc-17319, Santa Cruz); BrdU (1:500, catalogue number ab6326, Abcam); Ki67 (1:300, catalogue number ab9260, Millipore); Satb2 (1:500, catalogue number ab34735, Abcam); Olig2 (1:200, catalogue number ab109186, Abcam); Shh (1:100, catalogue number sc-33943, Santa Cruz); Ptch1 (1:100, catalogue number sc-6149, Santa Cruz); Dlx2 (1:500, catalogue number ab117546, Abcam); Nkx2.1 (1:300, catalogue number ab76013, Abcam); Tbr1 (1:500, catalogue number ab31940, Abcam); Dcx (1:500, catalogue number ab18723, Abcam); Tbr2 (1:500, catalogue number ab23345, Abcam); Flag M2 (1:1,000, catalogue number F1804, Sigma); Map2 (1:500, catalogue number ab32454, Abcam); and Tuj1 (1:1,000, catalogue number mms-435p, Convance).

Techniques: Genome Wide, Expressing, Gene Expression, ChIP-sequencing, RNA Sequencing

Journal: Nature Communications

Article Title: LSD1 co-repressor Rcor2 orchestrates neurogenesis in the developing mouse brain

doi: 10.1038/ncomms10481

Figure Lengend Snippet: ( a ) qPCR analysis of the expression of genes related to the Shh signalling pathway in the cortex of Rcor2 fl/fl and Rcor2 cko brains during development. Significant upregulation of these genes on Rcor2 depletion is noteworthy. Transcripts were normalized to Rcor2 fl/fl group. Data are shown as mean±s.d., t -test, * P <0.05, ** P <0.01 and *** P <0.001, n =3. ( b ) Confocal images of Shh and Ptch1 expressions in Rcor2 fl/fl and Rcor2 cko cortex. Enhanced Shh and Ptch1 signals are observed in Rcor2 cko compared with Rcor2 fl/fl neocortex at E13.5 and E15.5. Insets show high-magnification images of the outlined regions. Scale bars, 50 μm. ( c ) Dlx2 expression in Rcor2 fl/fl and Rcor2 cko cortex detected by immunofluorescence analysis at E15.5. Dlx2 + cells were observed in the neocortex on Rcor2 depletion. Scale bars, 50 μm. ( d ) Confocal images of Shh, Ptch1 and Dlx2 expressions in in-vitro -cultured Rcor2 cko NPCs. Scale bar, 20 μm. ( e ) Western blot analysis of expression levels of Dlx2, Shh and Ptch1 in Rcor2 fl/fl and Rcor2 cko cortex at E15.5. β-Actin is used as an endogenous control.

Article Snippet: Primary antibodies used were as follows: Rcor2 (1:50, catalogue number NBP1-74099, Novus Biological); Nes (1:300, catalogue number Rat-401, DSHB); Pax6 (1:100, DSHB); Sox2 (1:500, catalogue number sc-17319, Santa Cruz); BrdU (1:500, catalogue number ab6326, Abcam); Ki67 (1:300, catalogue number ab9260, Millipore); Satb2 (1:500, catalogue number ab34735, Abcam); Olig2 (1:200, catalogue number ab109186, Abcam); Shh (1:100, catalogue number sc-33943, Santa Cruz); Ptch1 (1:100, catalogue number sc-6149, Santa Cruz); Dlx2 (1:500, catalogue number ab117546, Abcam); Nkx2.1 (1:300, catalogue number ab76013, Abcam); Tbr1 (1:500, catalogue number ab31940, Abcam); Dcx (1:500, catalogue number ab18723, Abcam); Tbr2 (1:500, catalogue number ab23345, Abcam); Flag M2 (1:1,000, catalogue number F1804, Sigma); Map2 (1:500, catalogue number ab32454, Abcam); and Tuj1 (1:1,000, catalogue number mms-435p, Convance).

Techniques: Expressing, Immunofluorescence, In Vitro, Cell Culture, Western Blot, Control

Journal: Nature Communications

Article Title: LSD1 co-repressor Rcor2 orchestrates neurogenesis in the developing mouse brain

doi: 10.1038/ncomms10481

Figure Lengend Snippet: ( a ) Knockdown of Rcor2 impairs cortical neurogenesis, which can be partially rescued by knockdown of Shh during cortical development. In-utero electroporation with RFP-shControl (red)/GFP-shControl (green), RFP-shRcor2 (red)/GFP-shControl (green), RFP-shControl (red)/GFP-shShh (green) or RFP-shRcor2 (red)/GFP-shShh (green) plasmids was performed at E13.5. Cerebral sections were fixed and imaged at E16.5. VZ, ventricular zone; SVZ, subventricular zone; CP, cortical plate. Scale bar, 50 μm. ( b ) Quantification of the percentage of RFP + /GFP + cells in different regions of the developing cortex after electroporation shown in a . Data are shown as mean±s.e.m., t -test, ** P <0.01 and *** P <0.001, n =3 individual experiments. ( c ) Inhibition of Shh by Cyclopamine can partially rescue neurogenesis defects caused by Rcor2 downregulation during cortical development. Rcor2 was knocked down at the lateral ventricle in the brain by in-utero electroporation with RFP-shRcor2 plasmids at E13.5. Cerebral sections were collected at E14.5 and then treated with cyclopamine to inhibit Shh activity for 48 h. Scale bar, 50 μm. ( d ) Quantification of the percentage of RFP + cells in different regions of the developing neocortex after knockdown of Rcor2 or inhibition of Shh shown in c . Data are shown as mean±s.e.m., t -test, * P <0.05, n =3 individual experiments. ( e ) Representative images depicting neurosphere size is partially rescued in the in-vitro -cultured Rcor2 cko NPCs after treatment with Cyclopamine. Scale bar, 50 μm. ( f ) Histogram depicting cell numbers of in-vitro -cultured Rcor2 fl/fl and Rcor2 cko NPCs with or without Cyclopamine treatment for 48 h. Cells (5 × 10 5 ) are seeded initially. Data are shown as mean±s.e.m., t -test, ** P <0.01, n =3. ( g ) Confocal images of Tuj1 expression in the differentiated cells from in-vitro -cultured Rcor2 fl/fl and Rcor2 cko NPCs with or without Cyclopamine treatment. Tuj1 expressions are partially restored in Cyclopamine-treated Rcor2 cko cells. Scale bar, 20 μm. ( h ) Model of Rcor2 function in neurogenesis in the developing neocortex. Rcor2 safeguards cortical neurogenesis by recruiting LSD1 complex to the regulatory regions of Dlx2 and Shh genes, to inhibit the Shh pathway activation during development. The absence of Rcor2 leads to inhibition release of these genes and thus ectopic activation of Shh signalling in the developing neocortex, resulting in cortical neurogenesis defects.

Article Snippet: Primary antibodies used were as follows: Rcor2 (1:50, catalogue number NBP1-74099, Novus Biological); Nes (1:300, catalogue number Rat-401, DSHB); Pax6 (1:100, DSHB); Sox2 (1:500, catalogue number sc-17319, Santa Cruz); BrdU (1:500, catalogue number ab6326, Abcam); Ki67 (1:300, catalogue number ab9260, Millipore); Satb2 (1:500, catalogue number ab34735, Abcam); Olig2 (1:200, catalogue number ab109186, Abcam); Shh (1:100, catalogue number sc-33943, Santa Cruz); Ptch1 (1:100, catalogue number sc-6149, Santa Cruz); Dlx2 (1:500, catalogue number ab117546, Abcam); Nkx2.1 (1:300, catalogue number ab76013, Abcam); Tbr1 (1:500, catalogue number ab31940, Abcam); Dcx (1:500, catalogue number ab18723, Abcam); Tbr2 (1:500, catalogue number ab23345, Abcam); Flag M2 (1:1,000, catalogue number F1804, Sigma); Map2 (1:500, catalogue number ab32454, Abcam); and Tuj1 (1:1,000, catalogue number mms-435p, Convance).

Techniques: Knockdown, In Utero, Electroporation, Inhibition, Activity Assay, In Vitro, Cell Culture, Expressing, Activation Assay

Journal: Developmental cell

Article Title: In vitro and in vivo development of the human airway at single cell resolution

doi: 10.1016/j.devcel.2020.01.033

Figure Lengend Snippet: A) Schematic of lung epithelial development. As the airways extend, bud tip progenitors are maintained as progenitors in the tips of branching buds, and leave cells behind that give rise to the intrapulmonary airways. Late in development, remaining bud tip progenitors differentiate into alveolar cells. No bud tip progenitors are present in the adult lung. B) Schematic of creation of epithelium-only bud tip progenitor organoids from 12 week fetal lungs. C) Feature plots for bud tip progenitor marker genes SFTPC, ID2 and HMGA1 from scRNA-seq of day 0 bud tip progenitor organoids maintained for 3 weeks in culture. Additional feature plots from this same data set are shown in Figure 4A. D) mRNA expression by QRT-PCR of basal cell marker TP63 in bud tip progenitor organoids treated for 3 days with serum-free basal medium supplemented with DMSO (control) or with signaling factors known to be important for lung development and cellular differentiation. DMSO (1:1,000 dilution), FGF7 (10 ng/mL), ‘3F’ (FGF7 10ng/mL, CHIR99021 3μM, ATRA 50 nM), Dexamethasone (25 ng/mL), IL6 (10 ng/mL), IL2 (50 U/mL), Smoothened Agonist (SAG; 500 nM), EGF (100 ng/mL), FGF10 (500 ng/mL), CHIR99021 (2 μM), IFNγ (10 ng/mL), TGFβ1 (100 ng/mL), BMP4 (100 ng/mL), DAPT (10 μM), Hydrocortisone (100 ng/mL). Gene expression is reported as arbitrary units. Treatment with TGFβ1 led to a significant increase in the expression of TP63 (one-way Analysis of Variance (ANOVA) (alpha=0.05, p<0.0001, F=14.7. Dunnett’s test of multiple comparison’s compared the mean of each group to the mean of the DMSO control group.) Estimated p values are shown on the graph. Error bars are plotted to show mean +/− the standard error of the mean. N=3 independent biological specimens. Data is from a single experiment. E) Bud tip progenitor organoids were treated with FGF7 (10 ng/mL), a permissive environment for TP63 expression compared to maintenance in bud tip progenitor medium (‘3F’), or with FGF7 (10 ng/mL) plus factors to inhibit SMAD signaling (A8301 [1 μM] and NOGGIN [100 ng/mL]) and TP63 gene expression was evaluated by QRT-PCR after 10 days in culture. A one-way Analysis of Variance was used followed by Tukey’s multiple comparison test to compare the means of each group to the mean of every other group. Estimated p values are reported on the graph. Error bars are plotted to show mean +/− the standard error of the mean. N=3 independent biological specimens. Data is from a single experiment. F) Bud tip progenitor organoids were treated for 3 days with SMAD activation or inhibition conditions and expression of TP63 was evaluated by QRT-PCR for all treatment groups. All media contained 3F components (FGF7 10ng/mL, CHIR99021 3μM, ATRA 50 nM), with individual groups containing combinations of: DMSO (1:1000 dilution), TGFβ1 (100 ng/mL), BMP4 (100 ng/mL), SB431542 (10 μM), LDN212854 (200 nM). One-way ANOVA alpha=0.05, F=21.19, p<0.0001; Tukey’s multiple comparisons of the mean of each group versus the mean in all other groups, estimated p values are reported on the graph. 3 days TGFβ1 and BMP4 is referred to as ‘dual SMAD activation’, or ‘DSA’. Data is plotted as arbitrary units. Error bars are plotted to show mean +/− the standard error of the mean. N=3 independent biological specimens. Data is from a single experiment and is representative of n=3 experiments. G) QRT-PCR for markers of canonical differentiated lung epithelial cell types showing DMSO (gray bars) and DSA treated (blue bars) organoids after 3 total days of treatment. Data is plotted as fold change over DMSO controls. Two-sided Mann-Whitney Tests were performed to compare the mean of the DMSO group to the Dual Smad Activation group (alpha=0.05). Error bars represent the mean +/− the standard error of the mean. n=3 independent biological specimens, and data is from a single experiment and is representative of n=3 experiments. H) Protein staining of DMSO treated (control) fetal bud tip progenitor organoids (top row) and 3 days of DSA treatment (bottom row) for TP63+ protein (green), KRT5 (pink) and DAPI (blue). Scale bar represents 50 μm. I) Quantification of (g). Total number of TP63+ cells were counted for 3–9 individual organoids across 3 biological replicates. n=3 independent biological specimens. A two-sided Mann Whitney test (alpha=0.05) was used to compare the means of each sample. For all graphs, p values are reported as follows: * p<0.05; ** p<0.01, *** p<0.001, **** p<0.0001.

Article Snippet: Growth factors and small molecules were used at the following concentrations: FGF10 (500 ng/mL, made in-house as previously described), A8301 (1 μM, Stem Cell Technologies cat. no. 72024), NOGGIN (100 ng/mL, R&D Systems, cat. no. 6057), Y27632 (APExBIO cat. no. A30008), LDN212854 (200 nM, R&D Systems cat. no. 6151/10), SB431542 (10 μM, Stemgent cat. no. 04–0010), TGFβ1(100 ng/mL, R&D systems cat. no. 240-B-002), BMP4 (100 ng/mL, R&D systems cat. no. 314-BP-050) Dexamethasone (25 ng/mL, Stem Cell Technologies cat. no. 72092), IL6 (10 ng/mL, R&D Systems, cat. no. 206-IL-010), IL2 (50 U/mL, R&D Systems, cat. no. 202-IL-010), Smoothened Agonist (SAG; 500 nM, R&D Systems, cat. no. 4366/1), EGF (100 ng/mL, R&D Systems cat. no. 236-EG-200), IFNγ (10 ng/mL, R&D Systems cat. no. {"type":"entrez-protein","attrs":{"text":"CAA31639","term_id":"32692","term_text":"CAA31639"}} CAA31639 ), DAPT (10 μM, R&D Systems cat. no. 2634/10), Hydrocortisone (100 ng/mL, Stem Cell Technologies cat. no. 74142).

Techniques: Marker, Expressing, Quantitative RT-PCR, Control, Cell Differentiation, Gene Expression, Comparison, Activation Assay, Inhibition, MANN-WHITNEY, Staining

Journal: Developmental cell

Article Title: In vitro and in vivo development of the human airway at single cell resolution

doi: 10.1016/j.devcel.2020.01.033

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Growth factors and small molecules were used at the following concentrations: FGF10 (500 ng/mL, made in-house as previously described), A8301 (1 μM, Stem Cell Technologies cat. no. 72024), NOGGIN (100 ng/mL, R&D Systems, cat. no. 6057), Y27632 (APExBIO cat. no. A30008), LDN212854 (200 nM, R&D Systems cat. no. 6151/10), SB431542 (10 μM, Stemgent cat. no. 04–0010), TGFβ1(100 ng/mL, R&D systems cat. no. 240-B-002), BMP4 (100 ng/mL, R&D systems cat. no. 314-BP-050) Dexamethasone (25 ng/mL, Stem Cell Technologies cat. no. 72092), IL6 (10 ng/mL, R&D Systems, cat. no. 206-IL-010), IL2 (50 U/mL, R&D Systems, cat. no. 202-IL-010), Smoothened Agonist (SAG; 500 nM, R&D Systems, cat. no. 4366/1), EGF (100 ng/mL, R&D Systems cat. no. 236-EG-200), IFNγ (10 ng/mL, R&D Systems cat. no. {"type":"entrez-protein","attrs":{"text":"CAA31639","term_id":"32692","term_text":"CAA31639"}} CAA31639 ), DAPT (10 μM, R&D Systems cat. no. 2634/10), Hydrocortisone (100 ng/mL, Stem Cell Technologies cat. no. 74142).

Techniques: Transduction, Control, Virus, Plasmid Preparation, Recombinant, RNAscope, Multiplex Assay, In Situ Hybridization, Generated, Software

Journal: Nature Communications

Article Title: Cytoglobin regulates NO-dependent cilia motility and organ laterality during development

doi: 10.1038/s41467-023-43544-0

Figure Lengend Snippet: a Schematic of Cygb2-NO-sGC signaling outlining experimental approaches to activate and inhibit the pathway. Panel generated with BioRender.com. b mRNA expression of nos2b relative to β-actin at 8–10 somites normalized to wt expression quantified by qRT-PCR. Means are ± SD ( n = 6–12, each n is the lysate of 50 embryos). c Immunostaining of acetylated tubulin (white), Cygb2 (red) and Nos2b (green) on whole mount embryos. d Whole mount fluorescence in situ hybridization labeling dand5 (pink), cygb2 (yellow) and nos2b (cyan) transcripts using RNAscope. c , d show representative images of three experiments. e , h Immunostaining of acetylated tubulin in the KV of scrambled control and nos2b or gucy1a morphants. The KV is outlined with a white dashed line. f , i Average cilia length, means are ± SD ( n = 10–13 embryos). Student’s t test, two-tailed, **** P < 0.0001, *** P < 0.001. g , j Percentage of embryos with left-sided hearts in control morphants (co-MO) versus nos2b (ATG MO) and gucy1a (ATG MO) morphants, means are ± SD ( n = 4–5, each n representing an independent experiment consisting of 50 embryos). Student’s t test, two-tailed, ** P < 0.01, * P < 0.05. k Experimental schematic demonstrating the timing of morpholino (MO) injection, KV formation and left/right patterning phenotype. Panel generated with BioRender.com. l cGMP levels were measured by ELISA comparing wt to cygb2 pt801a with and without 250 μM DETA/NO treatment. Means are ± SD ( n = 5–17 independent replicates with each sample representing 50 pooled embryos at 8–10 somites). Student’s t test, two-tailed, * P < 0.05, *** P < 0.001. Image orientation: A- anterior, P- posterior, L- left, R- right. Scale bar = 10 μm. Source data are provided as source data file.

Article Snippet: The qPCR reaction used TaqMan Universal PCR Master Mix (Applied Biosystems) and TaqMan gene expression assays (Thermo Fisher): cygb2 (Dr03090731-m1), nos2b (Dr03431350_m1), iNOS (Mm00440502_m1), eNOS (Mm00440502_m1).

Techniques: Generated, Expressing, Quantitative RT-PCR, Immunostaining, Fluorescence, In Situ Hybridization, Labeling, RNAscope, Control, Two Tailed Test, Injection, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: Cytoglobin regulates NO-dependent cilia motility and organ laterality during development

doi: 10.1038/s41467-023-43544-0

Figure Lengend Snippet: a Beads were injected into the KV between 8–10 somites and velocity was measured over time. Representative bead tracks of the KV comparing wt, cygb2 801a embryos, wt treated with 500 μM cPTIO and DMSO as control and cygb2 801a treated with 250 μM DETA/NO and NaOH/PBS as control ( n = 4 embryos per treatment). The dot indicates the end of the bead track after ten seconds of tracking. Scale bar = 20 μm. Boundaries of the KV are demarcated by a white dashed line. b Quantification of bead velocities ( n = 20–35 beads over 4 embryos). Means are ± SD. One-way Anova, *** P = 0.003; **** P < 0.0001. c Analysis of cardiac laterality defects represented as a percentage of embryos with the correct heart looping (on the left) analyzed at 2 dpf. Wt and cygb 801a treated with 250 µM DETA/NO or 500 μM cPTIO at the end of the blastula period. Means are ± SD, ( n = 4). Student’s t test, two-tailed, ns = not significant, * P < 0.05, ** P < 0.01. d Experimental schematic of the developmental time course drug treatment with 250 µM DETA/NO. Panel generated with BioRender.com. e Percentage of embryos with right, midline or left heart orientation following treatment with DETA/NO beginning at different developmental stages. The total number of embryos analyzed is shown in red above the graph. The Chi-squared test was used to determine statistical significance. f Quantification of the percentage of embryos with left hearts comparing wt to cygb2 pt801a with and without treatment of 80 µM cinaciguat. Means are ± SD, ( n = 4–6). Student’s t test, two-tailed, ns = not significant, ** P < 0.01. g Quantification of the percentage of embryos with left heart comparing wt to cygb2 pt801a injected with nos2b mRNA compared to uninjected controls. Means are ± SD, ( n = 6–7). Student’s t test, two-tailed, * P < 0.05, ns = not significant. In ( c , f , g ): each sample represent an independent experiment consisting of 50 embryos. Source data are provided as source data file.

Article Snippet: The qPCR reaction used TaqMan Universal PCR Master Mix (Applied Biosystems) and TaqMan gene expression assays (Thermo Fisher): cygb2 (Dr03090731-m1), nos2b (Dr03431350_m1), iNOS (Mm00440502_m1), eNOS (Mm00440502_m1).

Techniques: Injection, Control, Two Tailed Test, Generated

Journal: Nature Communications

Article Title: Cytoglobin regulates NO-dependent cilia motility and organ laterality during development

doi: 10.1038/s41467-023-43544-0

Figure Lengend Snippet: a Immunostaining of Cygb in mouse airway epithelium visualized by immunofluorescence. Scale bar = 10 μm. b Quantification of cilia length from wt and Cygb mutant (ko) mouse ( n = 4). Means are ± SD. Student’s t test, two-tailed, ** P < 0.01. c Chemiluminescence detection of NO production by triiodide assays using mouse trachea lysates as samples comparing wt to ko with injection volume indicated. On the right, quantification of nitrite concentration normalized by protein amount in each tissue ( n = 4). Means are ± SD. Student’s t test, two-tailed, * P < 0.05. d Reaction of 8 µM ferric CYGB in the presence of 250 µM NADPH. Increased absorbance at around 543 and 575 nm is consistent with the reduction of CYGB and formation of the CYGB Fe 2+ -O 2 complex. On the left, the reaction with CYGB and NADPH only. On the right, the same reaction in presence of 12.5 nM mouse iNOS. e Traces indicating the reduction of ferric CYGB in the presence of different reducing systems: zebrafish Nos2b, mouse iNOS or NADPH alone. f L-Citrulline assay indicating NO production by iNOS in presence or absence of CYGB ( n = 3–5). Means are ± SD. Student’s t test, two-tailed, ** P < 0.01, **** P < 0.0001. Source data are provided as source data file.

Article Snippet: The qPCR reaction used TaqMan Universal PCR Master Mix (Applied Biosystems) and TaqMan gene expression assays (Thermo Fisher): cygb2 (Dr03090731-m1), nos2b (Dr03431350_m1), iNOS (Mm00440502_m1), eNOS (Mm00440502_m1).

Techniques: Immunostaining, Immunofluorescence, Mutagenesis, Two Tailed Test, Injection, Concentration Assay

Journal:

Article Title: A novel balanced chromosomal translocation found in subjects with schizophrenia and schizotypal personality disorder: altered L-serine level associated with disruption of PSAT1 gene expression

doi: 10.1016/j.neures.2010.10.003

Figure Lengend Snippet: Chromosomal translocation and molecular profiles of the proband and her son. A) Fluorescent in situ hybridization positions the chromosome 9q21.2 breakpoint. A metaphase spread (left) and interphase nucleus (right) stained with 4',6-diamidino-2-phenylindole (DAPI) (blue). A green fluorescently labeled probe generated from RP11-45D15 BAC clone DNA is shown hybridized to the normal chromosome 9 and derived chromosomes 3 and 9 on a metaphase spread (left) stained blue with DAPI. This triple signal, also seen in an interphase nucleus (right), indicates that the DNA probe sequence spans the breakpoint. The relative proportions of signals on the two derived chromosomes suggest that the breakpoint is located towards the centromeric end of the DNA probe sequence. B) Schematic representation of the two breakpoint loci. The BAC and fosmid clones flanking the chromosome 3 breakpoint within the BC036229 transcript are shown as black rectangles. The magnified genomic context of the disruption is shown below including the location and orientation of nearby genes (nearest genes, grey block arrows: other genes, white block arrows). The chromosome 9 breakpoint does not directly disrupt a gene but is located close to PSAT1. The proximity to a segmental duplication pair (the black arrow indicates the centromeric component and the dashed line indicates the direction of the telomeric component ~1.8 Mb downstream) may indicate potential chromosome instability in this region. C) Serum level of L-serine, D-serine, and other relevant amino acids determined by HPLC. *p=0.013, Mann-Whitney U-test. Control, n=16. The open diamond indicates the proband. D) mRNA expression of the genes that are adjacent to the breakpoints. Expression level was determined by quantitative real-time RT-PCR and presented as expression relative to GAPDH mRNA. PSAT1 mRNA expression level was decreased significantly in the subjects (proband and her son) (**p=0.0053, Mann-Whitney U-test). No significant difference was observed for bobby sox homolog (Drosophila) (BBX) and centrosomal protein 78 kDa (CEP78) mRNA expression. Control: n=26 for PSAT1, n=10 for BBX and CEP78. The open diamond indicates the proband. mRNA expression of coiled-coil domain containing 54 (CCDC54), guanine nucleotide binding protein q polypeptide (GNAQ), and BC036229 was not detected in either subject or control lymphoblastoid cells. See Figure 1B for the location of each gene.

Article Snippet: Assay IDs for each target were as follows: BBX: Hs00329131_m1, CCDC54: Hs00540426_s1, CEP78: Hs00397220_m1, GNAQ Hs00387073_m1, and PSAT1: Hs00253548_m1.

Techniques: Translocation Assay, In Situ Hybridization, Staining, Labeling, Generated, Derivative Assay, Sequencing, Clone Assay, Disruption, Blocking Assay, MANN-WHITNEY, Control, Expressing, Quantitative RT-PCR, Binding Assay

Journal:

Article Title: A novel balanced chromosomal translocation found in subjects with schizophrenia and schizotypal personality disorder: altered L-serine level associated with disruption of PSAT1 gene expression

doi: 10.1016/j.neures.2010.10.003

Figure Lengend Snippet: Effects of PSAT1 knockdown by RNAi on L-serine level. A) Successful knockdown of PSAT1 by siRNA in primary rat astrocytes. Primary rat astrocytes were transfected with siRNA against PSAT1 or control siRNA. Cells were harvested on day 7 and examined for PSAT1 protein expression by Western blot analysis by using anti-PSAT1 antibody. GAPDH expression level was examined for internal control. (-); no transfection. Bar graph shows PSAT1 expression level relative to GAPDH. B) Amino acids concentration in culture supernatants of primary rat astrocytes with PSAT1 siRNA. Astrocytes transfected with either PSAT1 siRNA or control siRNA was determined at 24 h after the last medium change (day 7 post-transfection) by a high-performance liquid chromatography (HPLC) system. Error bars represent standard deviation. NT; non-treated, Con; treated with control siRNA, and Exp; treated with PSAT1 siRNA. **p=0.00175, Student t-test. Data are representative of three independent experiments.

Article Snippet: Assay IDs for each target were as follows: BBX: Hs00329131_m1, CCDC54: Hs00540426_s1, CEP78: Hs00397220_m1, GNAQ Hs00387073_m1, and PSAT1: Hs00253548_m1.

Techniques: Knockdown, Transfection, Control, Expressing, Western Blot, Concentration Assay, High Performance Liquid Chromatography, Standard Deviation

Journal: Disease Models & Mechanisms

Article Title: Tfap2a -dependent changes in mouse facial morphology result in clefting that can be ameliorated by a reduction in Fgf8 gene dosage

doi: 10.1242/dmm.017616

Figure Lengend Snippet: Alterations in gene expression in Neo/Null mice. (A,B) Volcano plots from the (A) maxillary and (B) nasal microarrays. Orange shows all values called ‘above absent’, blue all values called ‘present’, with a P -value of 0.05 (adjusted) and a fold change >1.25. Positive fold change is increased expression in Neo/Null compared with Neo/Wt. (C) Fold change of Fgf pathway members in Neo/Null E10.5 nasal prominences compared with control embryos. (D–K) In situ hybridization for Fgf8 (D–G) and Dusp6 (H–K) expression in Neo/Wt (D,F,H,J) and Neo/Null (E,G,I,K) E10.5 embryos showing frontal (D,E,H,I) and lateral (F,G,J,K) views. Arrow in G shows increased Fgf8 expression at the lateral edge of nasal pit. (L,M) Fgf8 staining visualized by OPT in E10.5 Neo/Wt (L) and Neo/Null (M) samples. (N) qRT-PCR examination of Fgf8 and Wnt9b transcript levels from E10.5 facial prominences. * P <0.05.

Article Snippet: For real-time PCR, pre-validated probes and primers were used to examine Tfap2a levels targeting exons 2–3 and 6–7 (IDT PrimeTime assay Mm.PT.56a.1580695 and 12995135, Integrated DNA Technologies, Coralville, IA) as well as Fgf8 (TaqMan gene expression assay Mm00438922_m1, Applied Biosystems, San Francisco, CA) and Wnt9b (TaqMan gene expression assay Mm00457102_m1).

Techniques: Gene Expression, Expressing, Control, In Situ Hybridization, Staining, Quantitative RT-PCR

Journal: Disease Models & Mechanisms

Article Title: Tfap2a -dependent changes in mouse facial morphology result in clefting that can be ameliorated by a reduction in Fgf8 gene dosage

doi: 10.1242/dmm.017616

Figure Lengend Snippet: Reduced Fgf8 gene dosage leads to a rescue of bilateral cleft lip. (A) Numbers of various genotypes and phenotypes observed from the Tfap2a (neo/neo) × Tfap2a (+/−) ; Fgf8 (+/−) crosses (L and R are left- and right-sided cleft, respectively). (B,C) Frontal images of the heads of Neo/Null; Fgf8 wt (B) and Neo/Null; Fgf8 het (C) P0 pups. Black arrow shows the unilateral cleft line. (D–F) Ventral view of skulls using μCT for Neo/Wt; Fgf8 wt (D), Neo/Null; Fgf8 wt (E) and Neo/Null; Fgf8 het (F) P0 pups. Red arrows show clefting at points where the premaxilla and the palatal process of the premaxilla should be fused.

Article Snippet: For real-time PCR, pre-validated probes and primers were used to examine Tfap2a levels targeting exons 2–3 and 6–7 (IDT PrimeTime assay Mm.PT.56a.1580695 and 12995135, Integrated DNA Technologies, Coralville, IA) as well as Fgf8 (TaqMan gene expression assay Mm00438922_m1, Applied Biosystems, San Francisco, CA) and Wnt9b (TaqMan gene expression assay Mm00457102_m1).

Techniques:

Journal: Disease Models & Mechanisms

Article Title: Tfap2a -dependent changes in mouse facial morphology result in clefting that can be ameliorated by a reduction in Fgf8 gene dosage

doi: 10.1242/dmm.017616

Figure Lengend Snippet: Morphometric analysis of the genetic interactions between Tfap2a and Fgf8 in facial development. (A) Morphometric analysis of E10.5 embryos from Tfap2a (neo/neo) × Tfap2a (+/−) ; Fgf8 (+/−) crosses. CVA showing between-group differences. The wireframes visualize +10 of the axis in black and 0 in gray and the warp images show −6 and +6, respectively, along the axes. (B) PCA of E10.5 Neo/Null; Fgf8 het compared with Neo/Wt; Fgf8 wt. Wireframes and warp embryo images show −0.05 and +0.05 along PC1. (C) PCA of E10.5 Neo/Null; Fgf8 het compared with Neo/Null; Fgf8 wt. Wireframes and warp embryo images show −0.06 and +0.06 along PC1.

Article Snippet: For real-time PCR, pre-validated probes and primers were used to examine Tfap2a levels targeting exons 2–3 and 6–7 (IDT PrimeTime assay Mm.PT.56a.1580695 and 12995135, Integrated DNA Technologies, Coralville, IA) as well as Fgf8 (TaqMan gene expression assay Mm00438922_m1, Applied Biosystems, San Francisco, CA) and Wnt9b (TaqMan gene expression assay Mm00457102_m1).

Techniques:

Journal: Disease Models & Mechanisms

Article Title: Tfap2a -dependent changes in mouse facial morphology result in clefting that can be ameliorated by a reduction in Fgf8 gene dosage

doi: 10.1242/dmm.017616

Figure Lengend Snippet: Analysis of variance and integration of facial morphology associated with Tfap2a and Fgf8 mutations. (A) Trace of the variance-covariance matrix to measure variation in shape at E10.5. P -value was determined based on overlap between the curves based on 1000 repetitions of the analysis. (B) Two-block PLS analysis to determine integration at E10.5 between the maxilla and the nasal prominences, the two regions with the largest differences in shape. Statistics can be found in supplementary material Table S5 . The data concerning E10.5 C57Bl/6J is presented as an additional control for background or strain effects and was obtained by re-landmarking scans generated previously .

Article Snippet: For real-time PCR, pre-validated probes and primers were used to examine Tfap2a levels targeting exons 2–3 and 6–7 (IDT PrimeTime assay Mm.PT.56a.1580695 and 12995135, Integrated DNA Technologies, Coralville, IA) as well as Fgf8 (TaqMan gene expression assay Mm00438922_m1, Applied Biosystems, San Francisco, CA) and Wnt9b (TaqMan gene expression assay Mm00457102_m1).

Techniques: Blocking Assay, Control, Generated