antibody human chd7 pa5–72964  (Thermo Fisher)

Journal: Human Genetics

Article Title: CHD7 variants associated with hearing loss and enlargement of the vestibular aqueduct

doi: 10.1007/s00439-023-02581-x

Figure Lengend Snippet: Seven variants were identified in CHD7 in seven families with bilateral sensorineural hearing loss and bilateral enlargement of the vestibular aqueduct (EVA)

Article Snippet: The preparations were then permeabilized for 30 min in PBS with 0.5% Triton X-100, blocked for 1 h at RT in PBS containing 1% BSA and 10% donkey serum, then incubated with the primary antibodies diluted in the same solution for 24 h at 4 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$^\circ{\rm C}$$\end{document} ∘ C . Rabbit monoclonal anti-CHD7 (D3F5, #6505S, LOT: 1, Cell signaling Technology, Danvers, MA, USA, raised against a recombinant protein corresponding to the N-terminal region of human CHD7 protein), Alexa Fluor 594-conjugated rabbit anti-CHD7 (#NBP1-77393AF594, LOT: A-030520-AF594, Novus, Littleton, CO, USA, raised against the N-terminal region of the human CHD7 protein (within residues 25–200) [Swiss-Prot Q9P2D1]), and goat anti-FOXI1 (RRID:AB_732416, ab20454, Abcam, Cambridge, MA, USA) antibodies were used as primary antibodies at 1:50, 1:200 and 1:200 dilutions, respectively.

Techniques: Variant Assay, Mutagenesis

Journal: Human Genetics

Article Title: CHD7 variants associated with hearing loss and enlargement of the vestibular aqueduct

doi: 10.1007/s00439-023-02581-x

Figure Lengend Snippet: CHD7 variants and inner ear phenotypes. A Schematic representation of the coding exons of CHD7 and reported functional domains of the CHD7 protein. The variants NM_017780.4: c.3553A > G [p.(Met1185Val)] and c.5390G > C [p.(Gly1797Ala)] were identified in our EVA cohort in families 276 and 388, and are likely causal. The other variants presented in Table not thought to be pathogenic are not shown. B Segregation of EVA, SNHL, and CHD7 variants in families 276 and 388. Variants in bold are thought to be causal. One subject in family 388, who did not enroll in the study, was reported to have had bilateral hearing loss since 10 years of age which progressed to severe levels requiring bilateral hearing aids by the age of 39 years. This functional phenotype was not evaluated by radiologic imaging but was considered to be consistent with EVA (grey symbol). Other subjects had reported hearing loss whose onset and progression in adulthood was more consistent with aging as the primary etiology (presbycusis) rather than EVA (striped symbol). The genotype is indicated for all the gDNA available. “ + ” denotes a wild-type allele. C Chromatograms showing the presence of the c.3553A > G and c.5390G > C heterozygous variants of CHD7 detected in families 276 and 388. D Conservation of human CHD7 amino acid residues Met1185 and Gly1797 among vertebrate and invertebrate ( Drosophila melanogaster , Caenorhabditis elegans ) species. Blue highlighting reflects conservation among 11 (light blue) or 13–14 (dark blue) of the 14 orthologs shown. Sequences obtained through Uniprot website were analyzed in Jalview. Alignment was performed using ClustalW. Protein sequences identifiers for CHD7 orthologues are Q9P2D1 for Homo sapiens , F6PP91 for Macaca mulatta , F1PWD8 for Canis familiaris , G3UE09 for Loxodonta africana , F7G444 for Monodelphis domestica , A2AJK6 for Mus musculus , A0A452E916 for Capra hircus , A0A674K692 for Terrapene carolina triunguis , Q06A37 for Gallus gallus , U3JST3 for Ficedula albicollis , A0A1L8FT46 for Xenopus laevis , F1QGL1 for Danio rerio , M9NEL3 for Drosophila melanogaster , O61845 for Caenorhabditis elegans

Article Snippet: The preparations were then permeabilized for 30 min in PBS with 0.5% Triton X-100, blocked for 1 h at RT in PBS containing 1% BSA and 10% donkey serum, then incubated with the primary antibodies diluted in the same solution for 24 h at 4 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$^\circ{\rm C}$$\end{document} ∘ C . Rabbit monoclonal anti-CHD7 (D3F5, #6505S, LOT: 1, Cell signaling Technology, Danvers, MA, USA, raised against a recombinant protein corresponding to the N-terminal region of human CHD7 protein), Alexa Fluor 594-conjugated rabbit anti-CHD7 (#NBP1-77393AF594, LOT: A-030520-AF594, Novus, Littleton, CO, USA, raised against the N-terminal region of the human CHD7 protein (within residues 25–200) [Swiss-Prot Q9P2D1]), and goat anti-FOXI1 (RRID:AB_732416, ab20454, Abcam, Cambridge, MA, USA) antibodies were used as primary antibodies at 1:50, 1:200 and 1:200 dilutions, respectively.

Techniques: Functional Assay, Imaging

Journal: Human Genetics

Article Title: CHD7 variants associated with hearing loss and enlargement of the vestibular aqueduct

doi: 10.1007/s00439-023-02581-x

Figure Lengend Snippet: Radiological images of temporal bones of affected individuals with p.(Met1185Val) and p.(Gly1797Val) variants of CHD7 . Montage of axial computed tomography (CT) (rows 1–4) and magnetic resonance (MR) (row 5) temporal bone imaging in a cohort subject with normal labyrinths and vestibular aqueducts (row 1), and from subjects with CHD7 variant p.(Met1185Val) (row 2) and CHD7 variant p.(Gly1797Ala) (rows 3–5). In row 1, normal anatomy is shown for reference, including the lateral semicircular canals (white arrows), which are often hypoplastic or absent in patients with CHARGE syndrome, and the normal vestibular aqueducts (white arrowheads) with a midpoint diameter less than 1 mm. For each patient (1810, 2106–2108), the lateral semicircular canals are well-formed, indistinguishable from normal (row 1, white arrow) and the vestibular aqueducts are enlarged (rows 2–5, white arrowheads) measuring 3 to 6 mm at the midpoint in each case. R right ear, L left ear

Article Snippet: The preparations were then permeabilized for 30 min in PBS with 0.5% Triton X-100, blocked for 1 h at RT in PBS containing 1% BSA and 10% donkey serum, then incubated with the primary antibodies diluted in the same solution for 24 h at 4 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$^\circ{\rm C}$$\end{document} ∘ C . Rabbit monoclonal anti-CHD7 (D3F5, #6505S, LOT: 1, Cell signaling Technology, Danvers, MA, USA, raised against a recombinant protein corresponding to the N-terminal region of human CHD7 protein), Alexa Fluor 594-conjugated rabbit anti-CHD7 (#NBP1-77393AF594, LOT: A-030520-AF594, Novus, Littleton, CO, USA, raised against the N-terminal region of the human CHD7 protein (within residues 25–200) [Swiss-Prot Q9P2D1]), and goat anti-FOXI1 (RRID:AB_732416, ab20454, Abcam, Cambridge, MA, USA) antibodies were used as primary antibodies at 1:50, 1:200 and 1:200 dilutions, respectively.

Techniques: Computed Tomography, Imaging, Variant Assay

Journal: Human Genetics

Article Title: CHD7 variants associated with hearing loss and enlargement of the vestibular aqueduct

doi: 10.1007/s00439-023-02581-x

Figure Lengend Snippet: Structural models of human CHD7 in active and inactive states. A Structural models based upon the alignment shown in Fig. S3 of CHD7 containing two chromodomains (CRD 1 and 2, purple), an ATPase motor (lobe 1 in orange and lobe 2 in green) and a SANT–SLIDE domain (pink) that binds DNA. Domains are colored as in Farnung et al. (Farnung et al. ). The coordinates of the histone complex (shown in gray cylinders) and the double strand DNA (blue) in CHD7 active form were obtained for purpose of visual display, after structural superimposition of the active template structure onto the model obtained. For the inactive model, the coordinates of the histone complex and the double strand DNA were obtained after structural superimposition of lobe 1 of the ATPase motor and SANT–SLIDE domains of the active template onto the inactive model. C-alpha atoms of residues Met1185 and Gly1797 are shown as blue spheres, while the atoms forming the nucleotides are shown as yellow (C), red (O), orange (P), blue (N) spheres. BeF3 is shown as cyan spheres (right model). Structural changes in CHD7 between the active and inactive states are indicated with arrows: the 40° rotation of lobe 2 in the ATPase motor and the 15° rotation of the chromodomains with respect to the DNA. B , C Close-up views of the structural models of inactive and active states of wild-type CHD7, and with the substitutions p.(Met1185Val) ( B ) and p.(Gly1797Ala) ( C ). The residues at positions 1185 and 1797 as well as those within 6 Å of the variant site are shown as sticks. The interacting networks involving Met1185 and Gly1797, Val1185 and Ala1797 are indicated as dashed lines. The corresponding C-alpha atom of each of these residues is shown as a blue sphere

Article Snippet: The preparations were then permeabilized for 30 min in PBS with 0.5% Triton X-100, blocked for 1 h at RT in PBS containing 1% BSA and 10% donkey serum, then incubated with the primary antibodies diluted in the same solution for 24 h at 4 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$^\circ{\rm C}$$\end{document} ∘ C . Rabbit monoclonal anti-CHD7 (D3F5, #6505S, LOT: 1, Cell signaling Technology, Danvers, MA, USA, raised against a recombinant protein corresponding to the N-terminal region of human CHD7 protein), Alexa Fluor 594-conjugated rabbit anti-CHD7 (#NBP1-77393AF594, LOT: A-030520-AF594, Novus, Littleton, CO, USA, raised against the N-terminal region of the human CHD7 protein (within residues 25–200) [Swiss-Prot Q9P2D1]), and goat anti-FOXI1 (RRID:AB_732416, ab20454, Abcam, Cambridge, MA, USA) antibodies were used as primary antibodies at 1:50, 1:200 and 1:200 dilutions, respectively.

Techniques: Variant Assay

Journal: Human Genetics

Article Title: CHD7 variants associated with hearing loss and enlargement of the vestibular aqueduct

doi: 10.1007/s00439-023-02581-x

Figure Lengend Snippet: Chd7 expression in the developing mouse endolymphatic sac and duct. A , B Single cell expression data (scRNA-seq (Honda et al. )) showing Chd7 expression in the different cell types of the developing mouse endolymphatic sac at E12.5, E16.5, P5 and P30 visualized in the gEAR portal ( https://umgear.org ) ( A ). Violin plots of Chd7 and Foxi1 expression in the cells of the endolymphatic sac and their precursor cells ( B ). While the transcription factor Foxi1 is expressed preferentially in MRCs and their precursor cells, Chd7 is expressed in all cell types of the endolymphatic sac. Each cell group is labelled based on expression patterns of canonical cell markers and results of gene ontology enrichment analysis. ProlC, proliferating cells (representative differentially expressed genes are Mki67, Birc5, Tpx2 ); ProgC, progenitor cells ( Lmx1a, Bmp3, Col11a1 ); MRC, mitochondria-rich cells ( Slc26a4, Foxi1, Atp6v0a4, Atp6v1b1 ); RRC, ribosome-rich cells ( Agt, Cav1, Clu, Dmkn ); Values: TPM transcript per million. C , D Developmental expression pattern of Chd7 in the endolymphatic sac and duct of Chd7 Gt /+ mice revealed by X-gal staining. Low magnification view of whole-mount X-gal staining of Chd7 Gt /+ endolymphatic sac and duct preparations show Chd7 expression at E14.5, E16.5 and P0 ( C ). Close-up views of the endolymphatic sac at higher magnification at E16.5, P5 and P30 ( D ). X-gal staining (blue dots) was detected in the different regions of the endolymphatic sac and along the endolymphatic duct at E14.5, E16.5, P0. Staining seemed to be more intense in the endolymphatic duct at E14.5, E16.5, as compared to the rest of the endolymphatic sac, whereas the staining seemed more homogenous at P0. At P5 and P30, staining was still detected in the endolymphatic sac. No staining was detected in endolymphatic sac and duct tissue from wild-type (WT) littermates processed in parallel. Three to four litters were studied for each datapoint. Whenever possible, animals of both sexes were studied and observed to have no obvious differences. Scale bars: 100 μm ( C ), 20 μm ( D )

Article Snippet: The preparations were then permeabilized for 30 min in PBS with 0.5% Triton X-100, blocked for 1 h at RT in PBS containing 1% BSA and 10% donkey serum, then incubated with the primary antibodies diluted in the same solution for 24 h at 4 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$^\circ{\rm C}$$\end{document} ∘ C . Rabbit monoclonal anti-CHD7 (D3F5, #6505S, LOT: 1, Cell signaling Technology, Danvers, MA, USA, raised against a recombinant protein corresponding to the N-terminal region of human CHD7 protein), Alexa Fluor 594-conjugated rabbit anti-CHD7 (#NBP1-77393AF594, LOT: A-030520-AF594, Novus, Littleton, CO, USA, raised against the N-terminal region of the human CHD7 protein (within residues 25–200) [Swiss-Prot Q9P2D1]), and goat anti-FOXI1 (RRID:AB_732416, ab20454, Abcam, Cambridge, MA, USA) antibodies were used as primary antibodies at 1:50, 1:200 and 1:200 dilutions, respectively.

Techniques: Expressing, Staining

Journal: Human Genetics

Article Title: CHD7 variants associated with hearing loss and enlargement of the vestibular aqueduct

doi: 10.1007/s00439-023-02581-x

Figure Lengend Snippet: Immunolocalization of CHD7 in the developing mouse endolymphatic sac and duct. Confocal microscopy analysis of whole-mount endolymphatic sac and duct preparations from E16.5 mice labelled with antibodies directed against CHD7 (red) and FOXI1 (a mitochondria-rich cell marker, green). Hoechst 33342 (blue) was used to label cell nuclei. Experiments were conducted with two different antibodies directed against CHD7 N-terminal region, with similar results. Here are shown the results with Alexa Fluor 594 conjugated anti-CHD7 from Novus. Maximal intensity projection images at E16.5. A Low magnification images of the endolymphatic sac and duct. B Higher magnification images of the open endolymphatic sac. No labeling was detected in the absence of primary antibodies, or in skin tissue used as a negative control. CHD7 was detected in the nuclei of the cells all along the endolymphatic duct and in both mitochondria-rich cells and ribosome-rich cells in the endolymphatic sac. Scale bars: 100 μm ( A ), 10 μm ( B )

Article Snippet: The preparations were then permeabilized for 30 min in PBS with 0.5% Triton X-100, blocked for 1 h at RT in PBS containing 1% BSA and 10% donkey serum, then incubated with the primary antibodies diluted in the same solution for 24 h at 4 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$^\circ{\rm C}$$\end{document} ∘ C . Rabbit monoclonal anti-CHD7 (D3F5, #6505S, LOT: 1, Cell signaling Technology, Danvers, MA, USA, raised against a recombinant protein corresponding to the N-terminal region of human CHD7 protein), Alexa Fluor 594-conjugated rabbit anti-CHD7 (#NBP1-77393AF594, LOT: A-030520-AF594, Novus, Littleton, CO, USA, raised against the N-terminal region of the human CHD7 protein (within residues 25–200) [Swiss-Prot Q9P2D1]), and goat anti-FOXI1 (RRID:AB_732416, ab20454, Abcam, Cambridge, MA, USA) antibodies were used as primary antibodies at 1:50, 1:200 and 1:200 dilutions, respectively.

Techniques: Confocal Microscopy, Marker, Labeling, Negative Control

Journal: bioRxiv

Article Title: Perturb-tracing enables high-content screening of multiscale 3D genome regulators

doi: 10.1101/2023.01.31.525983

Figure Lengend Snippet: a , Log2 fold change (log2fc) of spatial distance between adjacent TADs versus −log10 false discovery rate (FDR) for each perturbation. Each dot represents a perturbation in the screen library. In all volcano plots, the top hits (nuclear proteins with the largest log2fc and FDRs<0.1) in both directions are indicated with blue (knockout leads to upregulation) and red dots (knockout leads to downregulation), respectively. The top candidate genes which when knocked out led to increased adjacent TAD distances are: RB1, MRVI1 and PIP5K1B; the top candidate genes which when knocked out caused decreased adjacent TAD distances are: GLDC, NR4A1 and ZNF114. Positive controls (NIPBL and CTCF) are marked in black. b , Log2 fold change of adjacent TAD distance across chr22 for selected hits. c , Spatial distances between adjacent TADs for non-targeting control and selected hits. d , Log2 fold change of long-range A-A contact frequency versus −log10 FDR for each perturbation. Top three hits in both directions including NR4A1, PDE1A, HOXB9, RB1, PCBP1 and LRRC10B are labeled. e , Long-range A-A contact frequencies for non-targeting control and selected hits. f , Log2 fold change of long-range A-B contact frequency versus −log10 FDR for each perturbation. Top three hits in both directions, including RFESD, HOXB9, FAM69B, C2CD2, CHD7 and FAM13C, are labeled. g , Long-range A-B contact frequencies for non-targeting control and selected hits. h , Log2 fold change of long-range B-B contact frequency versus −log10 FDR for each perturbation. Top hits in both directions, including FOS, NR4A1, DDX24 and MYBPH, are labeled. i , Long-range B-B contact frequencies for non-targeting control and selected hits. j , Log2 fold change of overall inter-TAD distances versus −log10 FDR for each perturbation. Top three hits in both directions, including PCBP1, RB1, CHD7, GLDC, HOXB9 and CUL1, are labeled. k , Overall inter-TAD distances for non-targeting control and selected hits. l , Log2 fold change of individual overall inter-TAD distances in chr22 for selected hits. P values in c and k were calculated by two-sided Wilcoxon signed rank test. P values in e , g and i were calculated by two-sided Wilcoxon rank sum test. In all box plots throughout the manuscript, the boxes cover the 25 th to 75 th percentiles, the whiskers cover the 10 th to 90 th percentiles, and the line in the middle of the boxes represents the median value. For all relevant panels, significance is represented as *p<0.1. **p<0.05. ***p<0.01.

Article Snippet: Primary antibodies against human CHD7 (Thermo Fisher Scientific, PA5–72964), CTCF (Millipore Sigma, 07–729), HSP90 (CST 4874S), Sox10 (CST 89356S) and Actin (Abcam, ab179467) were incubated with the membrane at recommended concentrations at 4°C overnight with gentle shaking.

Techniques: Knock-Out, Control, Labeling

Journal: bioRxiv

Article Title: Perturb-tracing enables high-content screening of multiscale 3D genome regulators

doi: 10.1101/2023.01.31.525983

Figure Lengend Snippet: a , Western blot of siCtrl- and siCHD7-treated A549-Cas9 nuclear extracts. Top: anti-CHD7 antibody; bottom: anti-Actin B antibody. b , A-B compartment profile of chr22 in siCtrl cells. c , A-B compartment profile of chr22 in siCHD7 cells. d , Polarization indices of chr22 A-B compartments of siCtrl (white) and siCHD7 (orange). Shadowed boxes show the polarization indices from randomized controls, where the compartment identities of TADs are scrambled. e , Compartmental contact frequencies of siCtrl and siCHD7 (shadowed) among A compartment regions (red), between A and B compartment regions (purple), and among B compartment regions (blue). f , Overall inter-TAD distance of siCtrl and siCHD7. g , Radii of gyration of siCtrl and siCHD7. P values in d , e and g were calculated by two-sided Wilcoxon rank sum test. P value in f was calculated by two-sided Wilcoxon signed rank test.

Article Snippet: Primary antibodies against human CHD7 (Thermo Fisher Scientific, PA5–72964), CTCF (Millipore Sigma, 07–729), HSP90 (CST 4874S), Sox10 (CST 89356S) and Actin (Abcam, ab179467) were incubated with the membrane at recommended concentrations at 4°C overnight with gentle shaking.

Techniques: Western Blot

Journal: bioRxiv

Article Title: Perturb-tracing enables high-content screening of multiscale 3D genome regulators

doi: 10.1101/2023.01.31.525983

Figure Lengend Snippet: a , Log2 fold change of inter-TAD distance of siCHD7 compared to siCtrl. Number of traces analyzed: 3,558 (siCtrl) and 4,134 (siCHD7). b , Log2 fold change of short-range (defined as spatial distances between genomic regions that are less than 3Mb apart) and long-range (defined as spatial distances between genomic regions that are more than 3Mb apart) inter-TAD distances between siCHD7 and siCtrl. c , Log2 fold change of inter-TAD distance of CHD7 overexpression compared to GFP overexpression. Number of traces analyzed: 3,157 (GFP OE) and 1,174 (CHD7 OE). d , Log2 fold change of short-range and long-range inter-TAD distances between CHD7 and GFP overexpression. e , Log2 fold change of inter-TAD distance of TSA-treated cells compared to DMSO-treated cells. Number of traces analyzed: 1,214 (DMSO) and 2,223 (TSA). f , Log2 fold change of short-range and long-range inter-TAD distances between cells with TSA and DMSO treatment. g , Log2 fold change of inter-TAD distance of CHD7-ΔBRK (BRK domain deletion) overexpression compared to GFP overexpression. Number of traces analyzed: 3,415 (CHD7-ΔBRK OE) and 2,164 (GFP OE). h , Log2 fold change of short-range and long-range inter-TAD distances between CHD7-ΔBRK OE and GFP OE. i , Log2 fold change of inter-TAD distance of CHD7-K999R overexpression compared to GFP overexpression. Number of traces analyzed: 2,045 (CHD7-K999R) and 2,164 (GFP OE) j , Log2 fold change of short-range and long-range inter-TAD distances between CHD7-K999R OE and GFP OE. All chromatin tracing experiments in this figure were done in the A549 cell background, targeting chr22. P values were calculated by two-sided Wilcoxon signed rank test. k , Representative images of dye-labeled lambda DNA without/with purified CHD7. Scale bar: 500 μm. l , Spatial distribution of 130 genes decoded by RNA MERFISH in siCtrl and siCHD7 cells. Two representative cells are shown for each condition. Scale bar: 10 μm. m , Average RNA counts per cell for each gene in siCHD7 versus siCtrl cells. The red dashed line represents the x=y line. n , −Log10 false discovery rate (FDR) versus log2 fold change (log2fc) of average RNA counts per cell for each gene from siCtrl to siCHD7. Number of cells analyzed: 1,979 (siCtrl) and 1,186 (siCHD7) in m and n . o , Representative cell images of poly-A stain for siCtrl and siCHD7 cells. Scale bar: 20 μm. p , Mean fluorescent intensity of poly-A stain in individual nuclei of siCtrl and siCHD7 cells. P value was calculated by two-sided Wilcoxon rank sum test. Number of nuclei analyzed: 666 (siCtrl) and 594 (siCHD7).

Article Snippet: Primary antibodies against human CHD7 (Thermo Fisher Scientific, PA5–72964), CTCF (Millipore Sigma, 07–729), HSP90 (CST 4874S), Sox10 (CST 89356S) and Actin (Abcam, ab179467) were incubated with the membrane at recommended concentrations at 4°C overnight with gentle shaking.

Techniques: Over Expression, Labeling, Lambda DNA Preparation, Purification, Staining

Journal: bioRxiv

Article Title: Perturb-tracing enables high-content screening of multiscale 3D genome regulators

doi: 10.1101/2023.01.31.525983

Figure Lengend Snippet: a , A-B compartment profile of chr22 in A549-Cas9 cells with GFP overexpression. b , A-B compartment profile of chr22 in A549-Cas9 cells with CHD7 overexpression. c , Polarization indices of cells with GFP (white) and CHD7 (orange) overexpression and the corresponding randomized controls (shadowed). d , Compartmental contact frequencies of cells with GFP of CHD7 (shadowed) overexpression in A compartments (red), across A and B compartments (purple) and in B compartments (blue). e , Overall inter-TAD distance of chr22 in cells with GFP and CHD7 overexpression. f , Radii of gyration of chr22 in cells with GFP and CHD7 overexpression. P values in c , d and f were calculated by two-sided Wilcoxon rank sum test. P value in e were calculated by two-sided Wilcoxon signed rank test.

Article Snippet: Primary antibodies against human CHD7 (Thermo Fisher Scientific, PA5–72964), CTCF (Millipore Sigma, 07–729), HSP90 (CST 4874S), Sox10 (CST 89356S) and Actin (Abcam, ab179467) were incubated with the membrane at recommended concentrations at 4°C overnight with gentle shaking.

Techniques: Over Expression

Journal: bioRxiv

Article Title: Perturb-tracing enables high-content screening of multiscale 3D genome regulators

doi: 10.1101/2023.01.31.525983

Figure Lengend Snippet: a , Western blot of shControl- and shCHD7-transduced human embryonic stem cells (hESC) and human neural crest progenitors (hNCP). Top: anti-CHD7 antibody; middle: anti-SOX10 antibody; bottom: anti-HSP90 antibody. CHD7 increased upon neural crest induction, and reduced in shCHD7 hNCP cells compared to shControl. Sox10, the neural crest marker, was expressed at similar levels in shControl and shCHD7 hNCP cells. HSP90 is a loading control. b , Log2 fold change of overall inter-TAD distance of chr22 between shCHD7 and shControl hNCP cells. Number of traces analyzed: 4,657 (shControl) and 2,796 (shCHD7). c , Log2 fold change of short-range and long-range inter-TAD distances of chr22 between shCHD7 and shContrl hNCP cells. P values were calculated by two-sided Wilcoxon signed rank test.

Article Snippet: Primary antibodies against human CHD7 (Thermo Fisher Scientific, PA5–72964), CTCF (Millipore Sigma, 07–729), HSP90 (CST 4874S), Sox10 (CST 89356S) and Actin (Abcam, ab179467) were incubated with the membrane at recommended concentrations at 4°C overnight with gentle shaking.

Techniques: Western Blot, Marker, Control

Journal: bioRxiv

Article Title: Perturb-tracing enables high-content screening of multiscale 3D genome regulators

doi: 10.1101/2023.01.31.525983

Figure Lengend Snippet: a , Example tracks of CUT&RUN peak profiles of CHD7 and other proteins/epigenetic mark over different genomic regions. b , Heat map of other proteins/epigenetic mark localized to CHD7 peaks by CUT&RUN. c , Peak annotation for all CHD7 CUT&RUN peaks. d , Overlap of CUT&RUN peaks of CTCF, RAD21, and H3K4me3 with CHD7 peaks. e , Top 10 gene ontology terms up and down in siCHD7 cells versus siControl cells based on bulk RNA-seq analyses. Gene ontology was performed using Enrichr. f , Volcano plot of RNA-seq comparing siCHD7 and siControl cells (siCHD7/siControl). Top differentially expressed genes are displayed on the graph as labels. CHD7 is highlighted and is a top differentially downregulated gene in the siCHD7 cells, validating the knockdown.

Article Snippet: Primary antibodies against human CHD7 (Thermo Fisher Scientific, PA5–72964), CTCF (Millipore Sigma, 07–729), HSP90 (CST 4874S), Sox10 (CST 89356S) and Actin (Abcam, ab179467) were incubated with the membrane at recommended concentrations at 4°C overnight with gentle shaking.

Techniques: RNA Sequencing, Knockdown

Journal: Cardiovascular Research

Article Title: CHARGE syndrome-associated CHD7 acts at ISL1-regulated enhancers to modulate second heart field gene expression

doi: 10.1093/cvr/cvad059

Figure Lengend Snippet: Direct targets of CHD7 in the CPM, identified by CUT&RUN. ( A ) PCA of in vivo control ‘SHF’ and ‘HEART’ samples compared with in vitro CM differentiation time points from Wamstad et al. based on RNA-seq data. ( B ) Read density heatmaps (+/−2 kb relative to the centre of CHD7 peaks) and density profiles of CUT&RUN-seq in 3 biological replicates. Orange, CHD7 antibody; blue, IgG antibody; grey, input. ( C ) Distribution of distances from CHD7 peaks to the nearest TSS. ( D ) GO biological processes enriched in genes associated with CHD7 peaks. Terms are ranked by P -value from the Fisher exact test, by Enrichr. ( E ) Venn diagram of DEGs in Chd7 cKO ‘SHF’ with CHD7 binding, and GO terms enriched in these genes ( F ). ( G ) DEGs in Chd7 cKO ‘HEART’ with CHD7 binding, and GO terms enriched ( H ). P -values of Venn diagram overlaps from hypergeometric test and P -values of GO analysis from Fisher exact test.

Article Snippet: The immunoprecipitated samples were analysed by western blot, using antibodies against CHD7 (CST 6505) and ISL1 (DSHB 39.05D).

Techniques: In Vivo, Control, In Vitro, RNA Sequencing, Binding Assay

Journal: Cardiovascular Research

Article Title: CHARGE syndrome-associated CHD7 acts at ISL1-regulated enhancers to modulate second heart field gene expression

doi: 10.1093/cvr/cvad059

Figure Lengend Snippet: Loss of Chd7 in CPM dysregulates the cardiogenic GRN. ( A ) Whole mount E9.5 embryo showing the regions micro-dissected for the ‘SHF’ and ‘HEART’ RNA-seq experiments. ( B–C ) MA plots showing global gene expression changes in ‘SHF’ ( B ) and ‘HEART’ ( C ) in Chd7 cKO embryos relative to controls. Significantly changed genes with FDR < 0.1 are highlighted in red (up-regulated) or blue (down-regulated). ( D ) GO enrichment and ( E ) Jensen disease analysis for the 222 genes down-regulated in Chd7 cKO compared to control ‘SHF’. ( F ) GO terms and ( G ) OMIM disease terms enriched in the 426 genes down-regulated in Chd7 cKO ‘HEART’. Terms are ranked by P -values from the Fisher exact test, by Enrichr. ( H–J ) Venn diagrams showing the overlap between DEGs in Chd7 cKO ‘SHF’ and ‘HEART’ samples ( H ), ‘HEART’ and E11.5 hearts ( I ), and between ‘HEART’ and E13.5 hearts ( J ). DEGs in E11.5 and E13.5 hearts were obtained from previously published data. P -values based on hypergeometric test.

Article Snippet: The immunoprecipitated samples were analysed by western blot, using antibodies against CHD7 (CST 6505) and ISL1 (DSHB 39.05D).

Techniques: RNA Sequencing, Gene Expression, Control

Journal: Cardiovascular Research

Article Title: CHARGE syndrome-associated CHD7 acts at ISL1-regulated enhancers to modulate second heart field gene expression

doi: 10.1093/cvr/cvad059

Figure Lengend Snippet: Loss of CHD7 in the Meps1 lineage leads to reduced expression of anterior SHF markers. Whole mount in situ HCR staining of control and Chd7 cKO ( Mesp1-Cre; Chd7 fl/fl ) embryos at E9–9.5 for Fgf10 ( A–B’’’ ), Isl1 ( C–D’’’ ) and Mef2c ( E–F’’’ ). Boxed regions in ( A ), ( B ), ( C ), ( D ), ( E ), ( F ) are shown in neighbouring panels. Confocal maximum projection of the entire embryo and selected lateral or medial z-stacks are displayed. n = 4. See , for selected stacks highlighting domain differences. Arrowheads indicate cells in branchial arches; green bracket shows the SHF region. Scale bars represent 100 μm. ov, otic vesicle; SHF, second heart field; RV, right ventricle; FL, forelimb; DPW, dorsal pericardial wall; oft, outflow tract; mn, motor neurons.

Article Snippet: The immunoprecipitated samples were analysed by western blot, using antibodies against CHD7 (CST 6505) and ISL1 (DSHB 39.05D).

Techniques: Expressing, In Situ, Staining, Control

Journal: Cardiovascular Research

Article Title: CHARGE syndrome-associated CHD7 acts at ISL1-regulated enhancers to modulate second heart field gene expression

doi: 10.1093/cvr/cvad059

Figure Lengend Snippet: Genes expressed in the venous pole progenitor cells (pSHF) are upregulated in Chd7 cKO embryos. Maximum intensity projections of whole mount in situ HCR of control and Chd7 cKO embryos for Tbx5 ( A–B’ ), Osr1 ( C–D’ ), Foxf1 ( E–F’ ) and Wnt4 ( G–H’ ). Boxed regions in ( A ), ( B ), ( C ), ( D ), ( E ), ( F ), ( G ), ( H ) are shown in the neighbouring panel. Arrows in ( A’ ) and ( B ’) indicate the expanded expression of Tbx5 in the cKO embryos. n = 4. Scale bars, 100 μm. FL, forelimb; pSHF, posterior second heart field.

Article Snippet: The immunoprecipitated samples were analysed by western blot, using antibodies against CHD7 (CST 6505) and ISL1 (DSHB 39.05D).

Techniques: In Situ, Control, Expressing

Journal: Cardiovascular Research

Article Title: CHARGE syndrome-associated CHD7 acts at ISL1-regulated enhancers to modulate second heart field gene expression

doi: 10.1093/cvr/cvad059

Figure Lengend Snippet: CHD7 binds cardiac enhancers in CP cells. ( A ) Overlaps between CHD7 CUT&RUN peaks and active and poised cardiac enhancers. Odds ratio (OR) and P -value by Fisher’s test from each stage is presented, with the overlap defined by BEDTools. ( B ) Overlap of CHD7 peaks with enhancers during MES to CP transition, based on their activity. OR and P -value by Fisher’s test is presented. Genome browser snapshots at Isl1 ( C ) and Fgf10 ( D ) loci. Snapshots include tracks from CHD7 peaks (mid blue), ISL1 peaks (purple), CP enhancers from Wamstad et al. (dark blue), H3K4me1 from E10.5 hearts (light blue) and H3K27ac from E10.5 hearts (green). In ( C ), CP enhancers are expanded and their activity at each stage is indicated as absent (−1), poised (0) or active (1). Details on the tracks used can be found in the methods section.

Article Snippet: The immunoprecipitated samples were analysed by western blot, using antibodies against CHD7 (CST 6505) and ISL1 (DSHB 39.05D).

Techniques: Activity Assay

Journal: Cardiovascular Research

Article Title: CHARGE syndrome-associated CHD7 acts at ISL1-regulated enhancers to modulate second heart field gene expression

doi: 10.1093/cvr/cvad059

Figure Lengend Snippet: CHD7 binding sites co-localize with a subset of ISL1-bound sites and are enriched for cardiac TFs. ( A–C ) TF motifs enriched in all CHD7 peaks ( A ) and in CHD7 peaks associated with ‘SHF’ DEGs ( B ) or ‘HEART’ DEGs ( C ), as discovered by HOMER. ( D ) GO:biological processes enriched in CHD7-ISL1 common peaks, ranked by P -values from the Fisher exact test, by Enrichr. ( E ) Distribution of distances from CHD7-ISL1 common peaks to the nearest TSS. ( F ) Top ten transcription factors bound at the promoters of genes associated with CHD7 peaks, based on the ChEA function of Enrichr ( P -value from Fisher exact test). HS, human; MM, mouse. ( G ) Co-immunoprecipitation with ISL1 antibody and western blot analysis for CHD7 (top) or ISL1 (bottom) in day 5 CP cells. 10% of each IP and 2.5% of input was run on 10% SDS-PAGE gel; arrow indicates CHD7 band at ∼340kD.

Article Snippet: The immunoprecipitated samples were analysed by western blot, using antibodies against CHD7 (CST 6505) and ISL1 (DSHB 39.05D).

Techniques: Binding Assay, Immunoprecipitation, Western Blot, SDS Page

Journal: Cardiovascular Research

Article Title: CHARGE syndrome-associated CHD7 acts at ISL1-regulated enhancers to modulate second heart field gene expression

doi: 10.1093/cvr/cvad059

Figure Lengend Snippet: CHD7—proposed modes of action using Fgf10 enhancer as example. (An extended version of the model presented by Watanabe et al . ) As mentioned above, while CHD7 may participate in recruitment of histone modifiers and splicing the model focuses on its ATPase dependent nucleosome remodelling activity. Depending upon additional specific transcriptional regulators, opening of chromatin could be associated with either up or down regulation, and conversely closure could again be associated with increased or decreased target expression. In the context of the regulation of Fgf10 expression specifically via the intragenic enhancer bound by ISL1 (and TBX1), and its repression via NKX2.5, absence of CHD7 would be predicted to interfere with this balanced control as cells progress from a progenitor to differentiated state.

Article Snippet: The immunoprecipitated samples were analysed by western blot, using antibodies against CHD7 (CST 6505) and ISL1 (DSHB 39.05D).

Techniques: Activity Assay, Expressing, Control

Journal: Experimental eye research

Article Title: Chromatin remodeler Chd7 regulates photoreceptor development and outer segment length

doi: 10.1016/j.exer.2022.109299

Figure Lengend Snippet: (A-B′) Broad expression of Chd7 is apparent in the developing retina at 24 and 48 h post fertilization (hpf). (C–C′) At 72 hpf, Chd7 is expressed throughout ganglion cell layer (GCL), some cells of inner nuclear layer (INL), sporadic cells of outer nuclear layer (ONL), and the ciliary marginal zone (CMZ). (D-E′) Chd7 expression at 4 and 5 days post fertilization (dpf) shows reduction in GCL and INL expression and strong ONL expression. (F) No Chd7 antibody signal is detected at 5 dpf in chd7−/− mutant retina, confirming antibody specificity. Panels A–F show nuclear counterstain with DAPI. (G–H) At 72 hpf (G) and 5 dpf (H) Chd7 is co-expressed in GFP-positive cone photoreceptors marked by the TαC: GFP transgene. IHC for Chd7 in the XOPs:GFP transgenic line which fluorescently labels rod photoreceptors demonstrates that Chd7 has minimal co-expression with rod photoreceptors at 72 hpf (I) and increased co-expression at 5 dpf (I). ONL, outer nuclear layer; INL, Inner nuclear layer; GCL, ganglion cell layer; L, lens; CMZ, ciliary marginal zone; D, dorsal; V, ventral; R, retinal neuroepithelium; hpf, hours post fertilization; dpf, days post fertilization. Scale bars, 30 μm in A-F and 10 μm in J.

Article Snippet: Immunohistochemistry was performed on sections as previously described ( Forbes-Osborne et al., 2013 ), using the following primary antibodies: anti-zebrafish Chd7 (rabbit, 1:500; Boster Bio:DZ01533, Pleasanton, CA); anti-mouse CHD7 (rabbit, 1:2500, Cell Signaling Technologies:6505, Danvers, MA); 4C12, which labels zebrafish rod photoreceptors (mouse, 1:100, J. Fadool, FSU, Tallahassee, FL); 1D1, which labels zebrafish rhodopsin (mouse, 1:100, J. Fadool, FSU, Tallahassee, FL); 5E11, which labels zebrafish amacrine cells (1:1000, J. Fadool, FSU, Tallahassee, FL); Zpr-1, which labels zebrafish red-green double cones (mouse, 1:20, ZIRC); Peanut agglutinin (PNA)-lectin conjugated to Cy5 (Vector Labs cl-1075, 1:1000); HuC/D (mouse, 1:20, Invitrogen, Grand Island, NY), which labels retinal ganglion cells and amacrine cells; Prox1 (rabbit, 1:2,000, Millipore, Billerica, MA), which recognizes horizontal cells; anti-PKCα (mouse, 1:100; cat. no. sc-17769, SantaCruz Biotechnology), which labels bipolar cells; glutamine synthetase (mouse, 1:500, BD Biosciences, Franklin Lakes, NJ) which labels Müller glia; anti-Blue and anti-UV opsin antibodies (rabbit, 1:1000), generously provided by D. Hyde (University of Notre Dame); and 1D4, which labels mouse rhodopsin (Santa Cruz, sc-5743).

Techniques: Expressing, Mutagenesis, Transgenic Assay

Journal: Experimental eye research

Article Title: Chromatin remodeler Chd7 regulates photoreceptor development and outer segment length

doi: 10.1016/j.exer.2022.109299

Figure Lengend Snippet: (A) CHD7 expression is strong in the inner neuroblastic layer (INBL) at E15.5 (B) At E18.5, CHD7 expression continues in the INBL and is observed in sporadic cells of the outer neuroblastic layer (ONBL). (C–F) CHD7 expression at postnatal days P1, P6, P10, and P15. At P1 (C) expression is strong in the ganglion cell layer (GCL) and throughout the inner nuclear layer (INL) and ONBL. At P6 and P10 (D–E), CHD7 expression becomes restricted to the GCL and inner cells of the INL, and expression begins in a few cells of outer nuclear layer (ONL). By P15 (F), CHD7 expression remains strong in GCL, cells of the INL, and in a band of cells in the distal ONL. (G–H) CHD7 expression is significantly decreased in Chd7Gt/+ mutant embryos (E15.5 and mice (P15). Arrows indicate expression in presumptive photoreceptor cells of the ONL. ONL, outer nuclear layer; INL, Inner nuclear layer; GCL, ganglion cell layer; ONBL, outer neuroblastic layer; INBL, inner neuroblastic layer. Scale bar is 100 μm for all panels.

Article Snippet: Immunohistochemistry was performed on sections as previously described ( Forbes-Osborne et al., 2013 ), using the following primary antibodies: anti-zebrafish Chd7 (rabbit, 1:500; Boster Bio:DZ01533, Pleasanton, CA); anti-mouse CHD7 (rabbit, 1:2500, Cell Signaling Technologies:6505, Danvers, MA); 4C12, which labels zebrafish rod photoreceptors (mouse, 1:100, J. Fadool, FSU, Tallahassee, FL); 1D1, which labels zebrafish rhodopsin (mouse, 1:100, J. Fadool, FSU, Tallahassee, FL); 5E11, which labels zebrafish amacrine cells (1:1000, J. Fadool, FSU, Tallahassee, FL); Zpr-1, which labels zebrafish red-green double cones (mouse, 1:20, ZIRC); Peanut agglutinin (PNA)-lectin conjugated to Cy5 (Vector Labs cl-1075, 1:1000); HuC/D (mouse, 1:20, Invitrogen, Grand Island, NY), which labels retinal ganglion cells and amacrine cells; Prox1 (rabbit, 1:2,000, Millipore, Billerica, MA), which recognizes horizontal cells; anti-PKCα (mouse, 1:100; cat. no. sc-17769, SantaCruz Biotechnology), which labels bipolar cells; glutamine synthetase (mouse, 1:500, BD Biosciences, Franklin Lakes, NJ) which labels Müller glia; anti-Blue and anti-UV opsin antibodies (rabbit, 1:1000), generously provided by D. Hyde (University of Notre Dame); and 1D4, which labels mouse rhodopsin (Santa Cruz, sc-5743).

Techniques: Expressing, Mutagenesis

Journal: Experimental eye research

Article Title: Chromatin remodeler Chd7 regulates photoreceptor development and outer segment length

doi: 10.1016/j.exer.2022.109299

Figure Lengend Snippet: (A–C) Immunohistochemistry with a rhodopsin antibody (1D1) in chd7+/+ (A), chd7+/− (B), and chd7−/− (C) retinal sections. (D–E) PNA labeling of cone outer segments in chd7+/+(D), chd7+/− (E), and chd7−/− (F) retinal sections. (G) Average rod outer segment length in chd7+/− and chd7−/− larvae is significantly decreased compared to chd7+/+. (H) Cone outer segments also display a significant decrease in length in chd7+/− and chd7+/+ larvae compared to chd7+/+ larvae. Each data point represents the average measurement for an individual eye, with a minimum of 10 cells measured per eye. Brackets represent region of length measurement for individual outer segments. (*p < 0.05, **p < 0.01, ***p < 0.001). ONL, outer nuclear layer. Scale bar, 15 μm.

Article Snippet: Immunohistochemistry was performed on sections as previously described ( Forbes-Osborne et al., 2013 ), using the following primary antibodies: anti-zebrafish Chd7 (rabbit, 1:500; Boster Bio:DZ01533, Pleasanton, CA); anti-mouse CHD7 (rabbit, 1:2500, Cell Signaling Technologies:6505, Danvers, MA); 4C12, which labels zebrafish rod photoreceptors (mouse, 1:100, J. Fadool, FSU, Tallahassee, FL); 1D1, which labels zebrafish rhodopsin (mouse, 1:100, J. Fadool, FSU, Tallahassee, FL); 5E11, which labels zebrafish amacrine cells (1:1000, J. Fadool, FSU, Tallahassee, FL); Zpr-1, which labels zebrafish red-green double cones (mouse, 1:20, ZIRC); Peanut agglutinin (PNA)-lectin conjugated to Cy5 (Vector Labs cl-1075, 1:1000); HuC/D (mouse, 1:20, Invitrogen, Grand Island, NY), which labels retinal ganglion cells and amacrine cells; Prox1 (rabbit, 1:2,000, Millipore, Billerica, MA), which recognizes horizontal cells; anti-PKCα (mouse, 1:100; cat. no. sc-17769, SantaCruz Biotechnology), which labels bipolar cells; glutamine synthetase (mouse, 1:500, BD Biosciences, Franklin Lakes, NJ) which labels Müller glia; anti-Blue and anti-UV opsin antibodies (rabbit, 1:1000), generously provided by D. Hyde (University of Notre Dame); and 1D4, which labels mouse rhodopsin (Santa Cruz, sc-5743).

Techniques: Immunohistochemistry, Labeling

Journal: Experimental eye research

Article Title: Chromatin remodeler Chd7 regulates photoreceptor development and outer segment length

doi: 10.1016/j.exer.2022.109299

Figure Lengend Snippet: (A) Schematic of wildtype and predicted chd7sa19732 and CRISPR mutant proteins with indicated protein domains. The chd7sa19732 mutant protein is predicted to be 1449 aa and CRISPR mutant protein is predicted to be 43 aa compared to wild type length of 3140 aa. (B) chd7 mutants display craniofacial, swim bladder, and pericardial abnormalities at 5 dpf compared to wildtype. (C) Gross morphology of WT and chd7 mutants at 14 dpf; chd7 mutants continue to display morphological defects and are smaller in size. Black asterisks indicate pericardial edema; red asterisks indicate lack of or smaller swim bladder. Red arrows indicate craniofacial abnormalities. (D) Proportion of surviving mutants at two months shows less than the expected 25% of homozygous mutants. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Immunohistochemistry was performed on sections as previously described ( Forbes-Osborne et al., 2013 ), using the following primary antibodies: anti-zebrafish Chd7 (rabbit, 1:500; Boster Bio:DZ01533, Pleasanton, CA); anti-mouse CHD7 (rabbit, 1:2500, Cell Signaling Technologies:6505, Danvers, MA); 4C12, which labels zebrafish rod photoreceptors (mouse, 1:100, J. Fadool, FSU, Tallahassee, FL); 1D1, which labels zebrafish rhodopsin (mouse, 1:100, J. Fadool, FSU, Tallahassee, FL); 5E11, which labels zebrafish amacrine cells (1:1000, J. Fadool, FSU, Tallahassee, FL); Zpr-1, which labels zebrafish red-green double cones (mouse, 1:20, ZIRC); Peanut agglutinin (PNA)-lectin conjugated to Cy5 (Vector Labs cl-1075, 1:1000); HuC/D (mouse, 1:20, Invitrogen, Grand Island, NY), which labels retinal ganglion cells and amacrine cells; Prox1 (rabbit, 1:2,000, Millipore, Billerica, MA), which recognizes horizontal cells; anti-PKCα (mouse, 1:100; cat. no. sc-17769, SantaCruz Biotechnology), which labels bipolar cells; glutamine synthetase (mouse, 1:500, BD Biosciences, Franklin Lakes, NJ) which labels Müller glia; anti-Blue and anti-UV opsin antibodies (rabbit, 1:1000), generously provided by D. Hyde (University of Notre Dame); and 1D4, which labels mouse rhodopsin (Santa Cruz, sc-5743).

Techniques: CRISPR, Mutagenesis

Journal: Experimental eye research

Article Title: Chromatin remodeler Chd7 regulates photoreceptor development and outer segment length

doi: 10.1016/j.exer.2022.109299

Figure Lengend Snippet: (A–C). Chd7 expression in wild type, chd7 heterozygous, and chd7 homozygous mutant retinas of chd7sa19732 mutant line (D–F). Chd7 expression in wild type, chd7 heterozygous, and chd7 homozygous mutant retinas of CRISPR mutant line. Both lines showed decreased Chd7 expression in heterozygotes and a lack of Chd7 expression in homozygotes. (G) Chd7 mutants have microphthalmia compared to wild-type zebrafish. Measurements taken from perimeter of retina normalized to nose-to-otolith length. (*p < 0.05, **p < 0.01, ***p < 0.001). ONL, outer nuclear layer; INL, Inner nuclear layer; GCL, ganglion cell layer; L, lens; D, dorsal; V, ventral. Scale bar 50 μm.

Article Snippet: Immunohistochemistry was performed on sections as previously described ( Forbes-Osborne et al., 2013 ), using the following primary antibodies: anti-zebrafish Chd7 (rabbit, 1:500; Boster Bio:DZ01533, Pleasanton, CA); anti-mouse CHD7 (rabbit, 1:2500, Cell Signaling Technologies:6505, Danvers, MA); 4C12, which labels zebrafish rod photoreceptors (mouse, 1:100, J. Fadool, FSU, Tallahassee, FL); 1D1, which labels zebrafish rhodopsin (mouse, 1:100, J. Fadool, FSU, Tallahassee, FL); 5E11, which labels zebrafish amacrine cells (1:1000, J. Fadool, FSU, Tallahassee, FL); Zpr-1, which labels zebrafish red-green double cones (mouse, 1:20, ZIRC); Peanut agglutinin (PNA)-lectin conjugated to Cy5 (Vector Labs cl-1075, 1:1000); HuC/D (mouse, 1:20, Invitrogen, Grand Island, NY), which labels retinal ganglion cells and amacrine cells; Prox1 (rabbit, 1:2,000, Millipore, Billerica, MA), which recognizes horizontal cells; anti-PKCα (mouse, 1:100; cat. no. sc-17769, SantaCruz Biotechnology), which labels bipolar cells; glutamine synthetase (mouse, 1:500, BD Biosciences, Franklin Lakes, NJ) which labels Müller glia; anti-Blue and anti-UV opsin antibodies (rabbit, 1:1000), generously provided by D. Hyde (University of Notre Dame); and 1D4, which labels mouse rhodopsin (Santa Cruz, sc-5743).

Techniques: Expressing, Mutagenesis, CRISPR

Journal: Experimental eye research

Article Title: Chromatin remodeler Chd7 regulates photoreceptor development and outer segment length

doi: 10.1016/j.exer.2022.109299

Figure Lengend Snippet: (A–C) Immunohistochemistry with a red-green cone antibody (Zpr1) in chd7+/+ (A,A′), chd7+/− (B,B′), and chd7−/− (C,C′) retinal sections. Arrows indicate missing red-green cone photoreceptors. (D–F) Immunohistochemistry with a rod antibody (4C12) in chd7+/+ (D,D′), chd7+/− (E,E′), and chd7−/− (F,F′) retinal sections. (G) Quantification confirms a decrease in red/green cones in chd7+/− and chd7−/− larvae compared to chd7+/+. (Number of cones per 300 μm; ANOVA followed by t-test; ***p < 0.001). (H) No significant difference in rod photoreceptors between in chd7+/+, chd7−/−, and chd7−/− larvae (Number of rods per 300 μm). ONL, outer nuclear layer; INL, Inner nuclear layer; GCL, ganglion cell layer; L, lens; ON, optic nerve; D, dorsal; V, ventral. Scale bars 25 μm in A′-F′ and 50 μm in A-F. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Immunohistochemistry was performed on sections as previously described ( Forbes-Osborne et al., 2013 ), using the following primary antibodies: anti-zebrafish Chd7 (rabbit, 1:500; Boster Bio:DZ01533, Pleasanton, CA); anti-mouse CHD7 (rabbit, 1:2500, Cell Signaling Technologies:6505, Danvers, MA); 4C12, which labels zebrafish rod photoreceptors (mouse, 1:100, J. Fadool, FSU, Tallahassee, FL); 1D1, which labels zebrafish rhodopsin (mouse, 1:100, J. Fadool, FSU, Tallahassee, FL); 5E11, which labels zebrafish amacrine cells (1:1000, J. Fadool, FSU, Tallahassee, FL); Zpr-1, which labels zebrafish red-green double cones (mouse, 1:20, ZIRC); Peanut agglutinin (PNA)-lectin conjugated to Cy5 (Vector Labs cl-1075, 1:1000); HuC/D (mouse, 1:20, Invitrogen, Grand Island, NY), which labels retinal ganglion cells and amacrine cells; Prox1 (rabbit, 1:2,000, Millipore, Billerica, MA), which recognizes horizontal cells; anti-PKCα (mouse, 1:100; cat. no. sc-17769, SantaCruz Biotechnology), which labels bipolar cells; glutamine synthetase (mouse, 1:500, BD Biosciences, Franklin Lakes, NJ) which labels Müller glia; anti-Blue and anti-UV opsin antibodies (rabbit, 1:1000), generously provided by D. Hyde (University of Notre Dame); and 1D4, which labels mouse rhodopsin (Santa Cruz, sc-5743).

Techniques: Immunohistochemistry

Journal: Experimental eye research

Article Title: Chromatin remodeler Chd7 regulates photoreceptor development and outer segment length

doi: 10.1016/j.exer.2022.109299

Figure Lengend Snippet: (A–D) Immunohistochemistry with rhodopsin antibody (1D4) in Chd7+/+ and Chd7Gt/+ P6 and P15 retinal sections. (E–H) PNA labeling in Chd7+/+ and Chd7Gt/+ P6 and P15 retinal sections. Brackets indicate region of outer segments, which are shorter in Chd7Gt/+ retinas. (I) Quantification of cone cell number shows a significant decrease in Chd7Gt/+ compared to Chd7+/+ at P15 (number of cones per 1000 μM, ***p < 0.001). ONL, outer nuclear layer. Scale bar, 25 μm.

Article Snippet: Immunohistochemistry was performed on sections as previously described ( Forbes-Osborne et al., 2013 ), using the following primary antibodies: anti-zebrafish Chd7 (rabbit, 1:500; Boster Bio:DZ01533, Pleasanton, CA); anti-mouse CHD7 (rabbit, 1:2500, Cell Signaling Technologies:6505, Danvers, MA); 4C12, which labels zebrafish rod photoreceptors (mouse, 1:100, J. Fadool, FSU, Tallahassee, FL); 1D1, which labels zebrafish rhodopsin (mouse, 1:100, J. Fadool, FSU, Tallahassee, FL); 5E11, which labels zebrafish amacrine cells (1:1000, J. Fadool, FSU, Tallahassee, FL); Zpr-1, which labels zebrafish red-green double cones (mouse, 1:20, ZIRC); Peanut agglutinin (PNA)-lectin conjugated to Cy5 (Vector Labs cl-1075, 1:1000); HuC/D (mouse, 1:20, Invitrogen, Grand Island, NY), which labels retinal ganglion cells and amacrine cells; Prox1 (rabbit, 1:2,000, Millipore, Billerica, MA), which recognizes horizontal cells; anti-PKCα (mouse, 1:100; cat. no. sc-17769, SantaCruz Biotechnology), which labels bipolar cells; glutamine synthetase (mouse, 1:500, BD Biosciences, Franklin Lakes, NJ) which labels Müller glia; anti-Blue and anti-UV opsin antibodies (rabbit, 1:1000), generously provided by D. Hyde (University of Notre Dame); and 1D4, which labels mouse rhodopsin (Santa Cruz, sc-5743).

Techniques: Immunohistochemistry, Labeling