Journal: eLife

Article Title: Vascular dimorphism ensured by regulated proteoglycan dynamics favors rapid umbilical artery closure at birth

doi: 10.7554/eLife.60683

Figure Lengend Snippet: ( a ) RNA in situ hybridization shows robust ADAMTS1 and ADAMTS9 expression in umbilical vein endothelium and tunica media (TM) and in outer arterial TM. Robust ADAMTS4 and ADAMTS5 expression was confined to the venous endothelium, with moderate ADAMTS4 expression and minimal ADAMTS5 expression in SMC (n = 3 umbilical cords for each probe). ( b ) ADAMTS-cleaved aggrecan (anti-NITEGE, red) and versican (anti-DPEAAE, red) both showed strong ADAMTS proteolytic activity throughout the venous wall and the outer artery TM. Unlike aggrecan, extensive versican proteolysis is seen in the arterial intima and sub-intima (n = 4 umbilical cords for each antibody). Wj, Wharton’s jelly. The brackets mark TM boundaries. Scale bars in a-b = 100 μm.

Article Snippet: Commercial assay or kit , Adamts1 RNAscope In situ probe , ACD bio , 463361 , Mouse probe.

Techniques: RNA In Situ Hybridization, Expressing, Activity Assay

Journal: eLife

Article Title: Vascular dimorphism ensured by regulated proteoglycan dynamics favors rapid umbilical artery closure at birth

doi: 10.7554/eLife.60683

Figure Lengend Snippet: ( a ) H and E staining of E18.5 wild-type cords showing thicker umbilical arterial (A) and thinner venous (V) wall (n = 6 umbilical cords). ( b ) Aggrecan and versican localization (red, DAPI counterstain blue) in E18.5 wild-type cords showing staining in the arterial inner tunica media (TM) and adventitia but not the vein (n = 3 umbilical cords). ( c ) β-gal (blue) and eosin (red) staining of E18.5 Adamts1 LacZ /+ ( Adamts1 +/- ) cord showing strong Adamts1 expression in venous endothelium and TM and outer artery TM (n = 3 umbilical cords). ( d ) Short umbilical cords in E18.5 Adamts1 -/- and Acan -/- embryos compared to wild type. Red arrowhead indicates an omphalocele in Adamts1 -/- embryos. ( e ) H & E staining of E18.5 wild type, Acan -/- and Adamts1 -/- cord cross-sections showing thinner walls in Acan -/- umbilical vessels and thicker walls in Adamts1 -/- umbilical vessels. ( f–g ) Cord length, TM thickness and vessel luminal area quantifications for Adamts1 -/- ( f ) and Acan -/- mice ( g ) at E18.5 compared to wild-type littermates. Acan -/- umbilical cords show larger lumens and Adamts1 -/- vessels show smaller lumens in (n = 7–11 umbilical cords each, whiskers indicate minimum and maximum values, *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001). ( h ) Phospho-histone H3 (pHH3) staining shows significantly fewer proliferating cells (white arrowheads) in Acan -/- umbilical vessels. Dotted white lines mark the boundaries of vessel lumens (n = 4 cords each, whiskers indicate minimum and maximum values, **, p<0.001; *, p<0.05). Scale bars = 100 μm in ( a ), 25 μm in ( c ), 100 μm and 50 μm in ( e ).

Article Snippet: Commercial assay or kit , Adamts1 RNAscope In situ probe , ACD bio , 463361 , Mouse probe.

Techniques: Staining, Expressing

Journal: eLife

Article Title: Vascular dimorphism ensured by regulated proteoglycan dynamics favors rapid umbilical artery closure at birth

doi: 10.7554/eLife.60683

Figure Lengend Snippet: ( a ) RNA in situ hybridization analysis of E12.5 and E18.5 mouse umbilical cord sections Acan, Vcan and relevant ADAMTS genes (n = 3 umbilical cords for each probe). ( b ) E12.5 and E14.5 wildtype and Acan -/- embryos have a comparable umbilical cord length (n = 2 Acan -/- at E12.5 and n = 3 at E14.5). ( c ) Observed and expected genotype ratios at different embryonic stages from Acan +/- and Adamts1 +/- intercrosses. ( d ) Hematoxylin and eosin staining of E14.5 wild type and Acan -/- umbilical cords shows completion of longitudinal to circumferential reorientation of smooth muscle cells in the mutant arterial tunica media (TM). Upper panels show tangential longitudinal sections whereas the lower panels are taken through the approximate center of each vessel (n = 3 umbilical cords of each genotype). Adv, adventitia; Tm, tunica media. Scale bars in a = 100 μm, 3 mm and 7 mm in ( b ) and 50 μm in ( d ).

Article Snippet: Commercial assay or kit , Adamts1 RNAscope In situ probe , ACD bio , 463361 , Mouse probe.

Techniques: RNA In Situ Hybridization, Staining, Mutagenesis

Journal: eLife

Article Title: Vascular dimorphism ensured by regulated proteoglycan dynamics favors rapid umbilical artery closure at birth

doi: 10.7554/eLife.60683

Figure Lengend Snippet: ( a ) Aggrecan (green) and α-SMA staining (red) in E18.5 umbilical cords show loss of aggrecan and weak α-SMA staining in Acan -/- vessels (n = 3 umbilical cords each genotype). ( b ) Smooth muscle myosin heavy chain (SMMHC, red) and phosphorylated myosin light chain (pMLC, green) staining in E18.5 umbilical cords showing dramatic signal attenuation in the Acan -/- vessels (n = 3 umbilical cords each genotype) ( c ) pMLC (green), endomucin (red), α-SMA (red, center panels) and SMMHC (red, right-hand panels) staining shows blunted dimorphism of Adamts1 -/- umbilical artery and vein with stronger expression of differentiated SMC markers in Adamts1 -/- umbilical vessels and acquisition of endomucin, a venous endothelium marker, by arterial endothelium (n = 3 umbilical cords each genotype) Scale bars = 100 μm in ( a–c ).

Article Snippet: Commercial assay or kit , Adamts1 RNAscope In situ probe , ACD bio , 463361 , Mouse probe.

Techniques: Staining, Expressing, Marker

Journal: eLife

Article Title: Vascular dimorphism ensured by regulated proteoglycan dynamics favors rapid umbilical artery closure at birth

doi: 10.7554/eLife.60683

Figure Lengend Snippet: ( a,b ) E17.5 Adamts1 -/- umbilical vessels show increased aggrecan staining and reduced anti-NITEGE staining in ( a ), quantified in ( b ) (n = 3 cords each genotype, error bars indicate mean ±S.D.*, p<0.05; **, p<0.01; ***, p<0.001). ( c,d ) Adamts1 -/- umbilical vessels show increased versican ( c ) and reduced anti-DPEAAE staining quantified in ( d ) (n = 3 cords each genotype, error bars indicate mean ±S.D. *, p<0.05; **, p<0.01). Scale bars = 50 μm in ( a–c ).

Article Snippet: Commercial assay or kit , Adamts1 RNAscope In situ probe , ACD bio , 463361 , Mouse probe.

Techniques: Staining

Journal: eLife

Article Title: Vascular dimorphism ensured by regulated proteoglycan dynamics favors rapid umbilical artery closure at birth

doi: 10.7554/eLife.60683

Figure Lengend Snippet:

Article Snippet: Commercial assay or kit , Adamts1 RNAscope In situ probe , ACD bio , 463361 , Mouse probe.

Techniques: RNAscope, In Situ, Software, Gene Expression, Microarray

Journal: European Journal of Human Genetics

Article Title: ADAMTS1 , MPDZ, MVD , and SEZ6: candidate genes for autosomal recessive nonsyndromic hearing impairment

doi: 10.1038/s41431-021-00913-x

Figure Lengend Snippet: Information on the variants found in the four candidate NSHI genes.

Article Snippet: The following primary antibodies were used: sheep anti-mouse ADAMTS1 (AF5867-SP; Novus Biologicals, Centennial, CO, USA), rabbit anti-mouse MPDZ (42-2700; Thermofisher Scientific, Waltham, MA, USA), rabbit anti-mouse MVD (PA5-22164; Thermofisher Scientific), and sheep anti-mouse SEZ6 (PA5-47683; Thermofisher Scientific).

Techniques:

Journal: European Journal of Human Genetics

Article Title: ADAMTS1 , MPDZ, MVD , and SEZ6: candidate genes for autosomal recessive nonsyndromic hearing impairment

doi: 10.1038/s41431-021-00913-x

Figure Lengend Snippet: Panel A : Structural model of the wild type ADAMTS1, MPDZ, MVD, and SEZ6 proteins. Panel B : Interacting bond of the wild type proteins for the four candidate genes. Panel C : Mutant type proteins highlighting the change due to the variant. The variants ADAMTS1 [p.(Ser135Ala)] and MPDZ [p.(Pro775Leu)] may result in a difference in interaction pattern with the solvent molecules. As the position of the variant residue is at the loop region, they do not involve a direct interaction, but the native fold of the protein may be affected. MVD [p.Pro379His] mutated protein shows a difference in interacting bond distance in the mutant type from the wild type and may perturb the amino acid side chain. The SEZ6 [p.Val698Ile] mutant protein displayed less interaction distance due to substitution of Val to Ile at position 698, while the SEZ6 wild type protein has a weak H-bond and hydrophobic interaction with Ile635 and Trp609. This variation could possibly change the orientation of the amino acid residue. Structures are displayed as ribbon while residue is represented by stick model. The structure illustrations were created with the PyMOL program. Black dotted lines represent hydrogen bonds.

Article Snippet: The following primary antibodies were used: sheep anti-mouse ADAMTS1 (AF5867-SP; Novus Biologicals, Centennial, CO, USA), rabbit anti-mouse MPDZ (42-2700; Thermofisher Scientific, Waltham, MA, USA), rabbit anti-mouse MVD (PA5-22164; Thermofisher Scientific), and sheep anti-mouse SEZ6 (PA5-47683; Thermofisher Scientific).

Techniques: Mutagenesis, Variant Assay, Solvent, Residue

Journal: European Journal of Human Genetics

Article Title: ADAMTS1 , MPDZ, MVD , and SEZ6: candidate genes for autosomal recessive nonsyndromic hearing impairment

doi: 10.1038/s41431-021-00913-x

Figure Lengend Snippet: A RNA expression of the four candidate genes in the cochlea and utricle during mouse development. RNA-sequencing data of hair cells (GFP+) and surrounding cells (GFP−) from the cochleae and utricles of mice expressing EGFP under the Pou4f3 promoter. Data were collected at four developmental stages: E16, P0, P4, and P7 and RNA expression is represented as normalized counts. Adamts1 , Mpdz , Mvd , and Sez6 were expressed in the hair cells and surrounding cells of the cochlea and utricle during the E16, P0, P4, and P7 stages. B RNA expression of the four candidate genes in the adult inner ear cells. The figure represents expression of the genes of interest in Deiters’ cells (Deiters), Pillar cells (Pillar), Inner Hair Cells (IHC), and Outer Hair Cells (OHC) of adult CBA/J mice. The four candidate genes Adamts1, Mpdz, Mvd , and Sez6 were expressed in the IHC, OHCs, and Deiter’s and Pillar cells. The y-axis represents the gene expression normalized to transcripts per million (TPM). The dataset was obtained from the GEO database (accession number GSE111347).

Article Snippet: The following primary antibodies were used: sheep anti-mouse ADAMTS1 (AF5867-SP; Novus Biologicals, Centennial, CO, USA), rabbit anti-mouse MPDZ (42-2700; Thermofisher Scientific, Waltham, MA, USA), rabbit anti-mouse MVD (PA5-22164; Thermofisher Scientific), and sheep anti-mouse SEZ6 (PA5-47683; Thermofisher Scientific).

Techniques: RNA Expression, RNA Sequencing, Expressing, Gene Expression

Journal: European Journal of Human Genetics

Article Title: ADAMTS1 , MPDZ, MVD , and SEZ6: candidate genes for autosomal recessive nonsyndromic hearing impairment

doi: 10.1038/s41431-021-00913-x

Figure Lengend Snippet: A Whole mount immunostaining of Adamts1, Mpdz, and Sez6 in wildtype mice at P12. Immunoreactivity was visualized with a fluorescently labeled secondary antibody (red) and F-actin was stained with phalloidin 488 nm (green). Immunolabeling of Adamts1 and Sez6 is observed in stereocilia as well as cytoplasm of outer hair cells. Mpdz is observed in the cytoplasmic region of hair cells. B Immunostaining of the organ of Corti at the apical, medial and basal turns of the cochlea at P4. C Immunostaining of Mvd in wildtype mice at P1, P4, and P28. Immunoreactivity of Mvd was visualized with a fluorescently labeled secondary antibody (green), F-actin was stained with rhodamine-phalloidin (red) and nuclear bodies were stained with DAPI (blue). The anti-neurofilament (NF-200, purple) was used to mark the neurons. Immunolabeling of Mvd is observed in spiral ganglion cells (SG). HP Habenula perforata, OC Organ of Corti.

Article Snippet: The following primary antibodies were used: sheep anti-mouse ADAMTS1 (AF5867-SP; Novus Biologicals, Centennial, CO, USA), rabbit anti-mouse MPDZ (42-2700; Thermofisher Scientific, Waltham, MA, USA), rabbit anti-mouse MVD (PA5-22164; Thermofisher Scientific), and sheep anti-mouse SEZ6 (PA5-47683; Thermofisher Scientific).

Techniques: Immunostaining, Labeling, Staining, Immunolabeling

Journal: European Journal of Human Genetics

Article Title: ADAMTS1 , MPDZ, MVD , and SEZ6: candidate genes for autosomal recessive nonsyndromic hearing impairment

doi: 10.1038/s41431-021-00913-x

Figure Lengend Snippet: Functions, expression and animal model phenotypes for the four NSHI candidate genes.

Article Snippet: The following primary antibodies were used: sheep anti-mouse ADAMTS1 (AF5867-SP; Novus Biologicals, Centennial, CO, USA), rabbit anti-mouse MPDZ (42-2700; Thermofisher Scientific, Waltham, MA, USA), rabbit anti-mouse MVD (PA5-22164; Thermofisher Scientific), and sheep anti-mouse SEZ6 (PA5-47683; Thermofisher Scientific).

Techniques: Expressing, Animal Model, Protein-Protein interactions, Membrane