Journal: Development (Cambridge, England)

Article Title: Nephronectin regulates atrioventricular canal differentiation via Bmp4-Has2 signaling in zebrafish

doi: 10.1242/dev.067454

Figure Lengend Snippet: npnt is transiently expressed during rat and zebrafish heart development. ( A ) Npnt expression levels in rat heart tissue from E11 to P10.5 in 12-hour intervals according to microarray analysis (Affymetrix GeneChip Rat Expression Set 230). Blue line, real profile; red line, smoothened profile. ( B ) RT-PCR analysis of Npnt mRNA expression during rat heart development. Gapdh was used as loading control. Ladder indicates a size marker. ( C-L ) Zebrafish npnt expression determined by whole-mount in situ hybridization. (C) Lateral view of a 24 hpf embryo showing npnt expression at tailbud (white arrowhead), head (black arrow) and posterior part of the gut (white arrow). (D) Dorsal view of the anterior part of the trunk demonstrating expression in pharyngeal endoderm (arrows). (E,F) Lateral (E) and dorsal (F) views at 34 hpf showing expression in the pronephric region (arrows) and head (arrowhead). (G,H) Ventrolateral view (G) and parasagittal section (H) at 44 hpf demonstrating npnt expression in heart (arrows) and jaw (arrowheads). (I,J) Paraffin section (4 μm) confirming expression in the myocardium (I, 200×; J, 1000×). (K) Dorsal view showing npnt expression at 53 hpf in pharynx, esophagus (arrow) and pharyngeal endoderm (arrowheads). (L) Transverse section through the trunk confirming expression in the endodermal cells of the anterior gut (arrow). A, atrium; EC, endocardium; MC, myocardium; V, ventricle. Scale bars: 100 μm in C-H,K,L; 50 μm in I; 20 μm in J.

Article Snippet: For in situ probes against npnt and itga8 mRNA, 517 bp ( npnt , 5′-CAATGGTCTGTGTCGGTACG-3′ and 5′-CTGAAGGTCAAAGCCGTCAT-3′) and 713 bp ( itga8 , 5′-GAAAAGCCCACGGTTTACAA-3′ and 5′-TCCCCTGTGAACTCTCCAAC-3′) fragments were amplified from cDNA and cloned into the pGEM-T Easy vector (Promega) to create pGEMTeasy-Z npnt and pGEMTeasy- itga8 .

Techniques: Expressing, Microarray, Reverse Transcription Polymerase Chain Reaction, Control, Marker, In Situ Hybridization, Paraffin Section

Journal: Development (Cambridge, England)

Article Title: Nephronectin regulates atrioventricular canal differentiation via Bmp4-Has2 signaling in zebrafish

doi: 10.1242/dev.067454

Figure Lengend Snippet: Npnt knockdown disrupts heart development and is lethal. ( A ) Scheme of the effect of npnt splice-inhibitory morpholino MO2. Exons are represented by boxes and introns by lines. Dotted lines indicate the region targeted by MO1 or MO2 and arrows indicate RT-PCR primer positions. Exon E3 could not be detected. ( B , C ) RT-PCR analysis of mRNAs from control and MO2-injected zebrafish embryos at 52 hpf using npnt primers as indicated in A and gapdh primers (loading control) ( n =3). MO2 inhibited npnt pre-mRNA splicing, resulting in intron insertion (B) and exon E5 deletion (C). ( D ) Western blot analysis of control and MO1- or MO2-injected embryos at 52 hpf ( n =3). Both morpholinos efficiently knocked down the Npnt protein level. ( E ) Npnt knockdown resulted in 89±7.9% (mean ± s.e.m.) lethality at 7 dpf. ( F ) Lateral view of control, MO1- or MO2-injected embryos at 75 hpf. MO1 or MO2 injection resulted in pericardial edema (arrowheads). ( G ) Quantitative analysis. Valve formation and trabeculation are perturbed in 86±3.9% (mean ± s.e.m.) of morphant hearts at 110 hpf. ( H ) Confocal images of hearts from control and MO2-injected embryos from transgenic Tg(myl7:EGFP-HsHRAS) s883 zebrafish at 80 hpf suggesting that the initiation of trabeculation is perturbed in npnt morphants. Red arrows indicate trabeculae. ( I ) Hematoxylin and Eosin (H&E)-stained sagittal sections of hearts from control and MO2-injected embryos at 110 hpf. In contrast to morphants, control embryos develop proper atrioventricular (AV) valve leaflets (white oval). Black arrowheads indicate the AV boundary; green arrows indicate expanded cardiac jelly. A, atrium; V, ventricle; PC, pericardium. Scale bars: 500 μm in F; 50 μm in H,I.

Article Snippet: For in situ probes against npnt and itga8 mRNA, 517 bp ( npnt , 5′-CAATGGTCTGTGTCGGTACG-3′ and 5′-CTGAAGGTCAAAGCCGTCAT-3′) and 713 bp ( itga8 , 5′-GAAAAGCCCACGGTTTACAA-3′ and 5′-TCCCCTGTGAACTCTCCAAC-3′) fragments were amplified from cDNA and cloned into the pGEM-T Easy vector (Promega) to create pGEMTeasy-Z npnt and pGEMTeasy- itga8 .

Techniques: Knockdown, Reverse Transcription Polymerase Chain Reaction, Control, Injection, Western Blot, Transgenic Assay, Staining

Journal: Development (Cambridge, England)

Article Title: Nephronectin regulates atrioventricular canal differentiation via Bmp4-Has2 signaling in zebrafish

doi: 10.1242/dev.067454

Figure Lengend Snippet: Npnt knockdown causes extension of the AV canal. ( A-D ) Projections of confocal images of hearts from control (A,C) and MO2-injected (B,D) embryos from transgenic Tg(myl7:EGFP-HsHRAS) s883 (A,B) and Tg(–5.1myl7:nDsRed2)f2 (C,D) zebrafish at 52 hpf, suggesting an extension of the AV canal (brackets) in npnt morphants. ( E ) Representative images of hearts from control, MO2-injected and MO2 + npnt mRNA-injected embryos from transgenic Tg(myl7:EGFP-HsHRAS) s883 zebrafish at 52 hpf. Brackets indicate the AV boundary. ( F ) Quantitative analysis ( n >160 from three independent experiments; mean ± s.e.m.). Note that injection of npnt mRNA rescued the MO2-mediated AV canal extension. ( G ) Quantitative analysis scoring of type I (mild extension of the AV canal), type II (obvious extension of the AV canal) and type III (straight heart) AV canal defects (mean ± s.e.m.). A, atrium; V, ventricle. Scale bars: 50 μm.

Article Snippet: For in situ probes against npnt and itga8 mRNA, 517 bp ( npnt , 5′-CAATGGTCTGTGTCGGTACG-3′ and 5′-CTGAAGGTCAAAGCCGTCAT-3′) and 713 bp ( itga8 , 5′-GAAAAGCCCACGGTTTACAA-3′ and 5′-TCCCCTGTGAACTCTCCAAC-3′) fragments were amplified from cDNA and cloned into the pGEM-T Easy vector (Promega) to create pGEMTeasy-Z npnt and pGEMTeasy- itga8 .

Techniques: Knockdown, Control, Injection, Transgenic Assay

Journal: Development (Cambridge, England)

Article Title: Nephronectin regulates atrioventricular canal differentiation via Bmp4-Has2 signaling in zebrafish

doi: 10.1242/dev.067454

Figure Lengend Snippet: Characterization of the extended AV canal. ( A , B ) Confocal sections of whole-mount Alcam-stained (red, indicating myocardium and differentiated endocardial cells) control (A) and MO2-injected (B) embryos from transgenic Tg(kdrl:EGFP) s843 zebrafish at 52 hpf. Npnt depletion caused an increase in differentiated endocardial cells (cuboidal in shape, Alcam positive and EGFP positive) at the AV boundary. Arrowheads indicate the AV boundary; red line, myocardium; green, endocardial cells; black dots, Alcam-positive endocardial cells. ( C ) Quantitative analysis of Alcam-positive endocardial cells (mean ± s.e.m.). SAV, superior AV; IAV, inferior AV. ( D-G ) Sections of 52 hpf control and MO2-injected embryos after whole-mount in situ hybridization for notch1b (D), cspg2 (E), fibulin 1 (F) and bmp4 (G). Expression of these genes was expanded in npnt morphants (brackets). ( H ) In situ hybridization with probes against the chamber-specific marker genes amhc and vmhc . ( I ) Projections of confocal images of hearts from whole-mount control and MO2-injected embryos from double-transgenic [ Tg(–5.1myl7:nDsRed2)f2 × Tg(myl7:EGFP-HsHRAS) s883 ] zebrafish stained for atrial myosin heavy chain (MHC, white) and DsRed (red). Yellow line, AV junction. ( J ) Quantitative analysis of total (chambers + AV boundary) and atrial cardiomyocyte number (mean ± s.e.m.). ns, not significant. A, atrium; V, ventricle. Scale bars: 50 μm.

Article Snippet: For in situ probes against npnt and itga8 mRNA, 517 bp ( npnt , 5′-CAATGGTCTGTGTCGGTACG-3′ and 5′-CTGAAGGTCAAAGCCGTCAT-3′) and 713 bp ( itga8 , 5′-GAAAAGCCCACGGTTTACAA-3′ and 5′-TCCCCTGTGAACTCTCCAAC-3′) fragments were amplified from cDNA and cloned into the pGEM-T Easy vector (Promega) to create pGEMTeasy-Z npnt and pGEMTeasy- itga8 .

Techniques: Staining, Control, Injection, Transgenic Assay, In Situ Hybridization, Expressing, Marker

Journal: Development (Cambridge, England)

Article Title: Nephronectin regulates atrioventricular canal differentiation via Bmp4-Has2 signaling in zebrafish

doi: 10.1242/dev.067454

Figure Lengend Snippet: Npnt knockdown causes cardiac jelly expansion and expanded expression of has2 and tbx2b . ( A , B ) Confocal sections from whole-mount F-actin-stained (red) control (A) and MO2-injected (B) Tg(kdrl:EGFP) s843 zebrafish embryos at 52 hpf. The space between endocardium and myocardium was increased at the AV boundary (arrowheads) and throughout the chambers (red asterisks) in npnt morphants. ( C-F ) Sections of 52 hpf control and MO2-injected embryos after whole-mount in situ hybridization for has2 and tbx2b expression (brackets). Note that the expression of these genes is expanded in npnt morphants. ( G-J ) Sections (4 μm) of control, MO2-, has2 MO-, or has2 MO + MO2-injected zebrafish embryos at 52 hpf stained for hyaluronan (brown) and counterstained with Hematoxylin (blue). ( K-N ) Transmission electron microscopy of hearts (K,L) and endocardial cells (M,N) from control and MO2-injected embryos at 52 hpf. Arrowheads indicate junctional complexes. A, atrium; BC, blood cell; CJ, cardiac jelly; EC, endocardium; MC, myocardium; V, ventricle. Scale bars: 50 μm in A-F; 10 μm in G-J; 5 μm in K,L; 1 μm in M,N.

Article Snippet: For in situ probes against npnt and itga8 mRNA, 517 bp ( npnt , 5′-CAATGGTCTGTGTCGGTACG-3′ and 5′-CTGAAGGTCAAAGCCGTCAT-3′) and 713 bp ( itga8 , 5′-GAAAAGCCCACGGTTTACAA-3′ and 5′-TCCCCTGTGAACTCTCCAAC-3′) fragments were amplified from cDNA and cloned into the pGEM-T Easy vector (Promega) to create pGEMTeasy-Z npnt and pGEMTeasy- itga8 .

Techniques: Knockdown, Expressing, Staining, Control, Injection, In Situ Hybridization, Transmission Assay, Electron Microscopy

Journal: Development (Cambridge, England)

Article Title: Nephronectin regulates atrioventricular canal differentiation via Bmp4-Has2 signaling in zebrafish

doi: 10.1242/dev.067454

Figure Lengend Snippet: Has2 knockdown rescues the endocardial phenotype in npnt morphants. ( A ) Confocal sections and schematics of whole-mount Alcam-stained (red, indicating myocardium and differentiated endocardial cells) MO2-injected, has2 MO + MO2-injected, control-injected and has2 MO-injected embryos from transgenic Tg(kdrl:EGFP) s843 zebrafish at 52 hpf. Red line, myocardium; green, endocardial cells; black dots, Alcam-positive endocardial cells. Npnt depletion caused an increase in differentiated AV endocardial cells (cuboidal in shape, Alcam positive and EGFP positive) (number of tiers ≥8, SAV; number of tiers ≥5, IAV). Knockdown of Has2 in npnt morphants or wild-type embryos reduced the number of tiers to two or fewer in SAV or IAV. Arrowheads indicate the AV boundary. ( B ) Quantitative analysis. Has2 knockdown in npnt morphants rescued the AV canal endocardium extension (mean ± s.e.m.). SAV, superior AV; IAV, inferior AV. ( C ) Bright-field images of 52 hpf MO2-injected and has2 MO + MO2-injected embryos after whole-mount in situ hybridization for notch1b, cspg2 and bmp4 expression (brackets). Note that Has2 knockdown reduced the expanded expression of notch1b but not of cspg2 or bmp4 in npnt morphants (arrows). A, atrium; V, ventricle. Scale bars: 50 μm.

Article Snippet: For in situ probes against npnt and itga8 mRNA, 517 bp ( npnt , 5′-CAATGGTCTGTGTCGGTACG-3′ and 5′-CTGAAGGTCAAAGCCGTCAT-3′) and 713 bp ( itga8 , 5′-GAAAAGCCCACGGTTTACAA-3′ and 5′-TCCCCTGTGAACTCTCCAAC-3′) fragments were amplified from cDNA and cloned into the pGEM-T Easy vector (Promega) to create pGEMTeasy-Z npnt and pGEMTeasy- itga8 .

Techniques: Knockdown, Staining, Injection, Control, Transgenic Assay, In Situ Hybridization, Expressing

Journal: Development (Cambridge, England)

Article Title: Nephronectin regulates atrioventricular canal differentiation via Bmp4-Has2 signaling in zebrafish

doi: 10.1242/dev.067454

Figure Lengend Snippet: Inhibition of BMP signaling reduces AV canal expansion. ( A ) Projections of confocal images and confocal sections of hearts of MO2-injected Tg(kdrl:EGFP) s843 zebrafish embryos at 52 hpf with and without dorsomorphin treatment stained for Alcam (red, indicating myocardium and differentiated endocardial cells). ( B ) Quantitative analysis. Inhibition of BMP signaling by dorsomorphin (Dorso) in the npnt morphants rescued the AV canal endocardium extension (mean ± s.e.m.). SAV, superior AV; IAV, inferior AV. ( C ) Bright-field images of 52 hpf untreated and dorsomorphin-treated npnt morphants after whole-mount in situ hybridization for notch1b, cspg2 and has2 expression. Dorsomorphin treatment reduced the expression of notch1b and has2 at the AV boundary in npnt morphants (brackets). Moreover, ectopic expression of cspg2 at the inflow tract in npnt morphants is abolished (arrowheads) and cspg2 expression at the AV boundary is partially rescued. ( D ) Model of the regulatory role of Npnt. 1 and 2 are two possible mechanisms of how Npnt affects Bmp4 signaling. (1) Npnt activates a receptor that is expressed in the chamber myocardium to repress Bmp4 signaling. (2) Npnt inhibits diffusion of Bmp4, resulting in autocrine signaling. Arrows indicate changes after Npnt depletion in wild type (red) or upon has2 depletion (blue) or dorsomorphin treatment (green) in npnt morphants. Red dashed arrow indicates Bmp4 diffusion in the npnt morphant. Black dashed arrow indicates controlled Bmp4 diffusion in the wild type. A, atrium; V, ventricle; EC, endocardium; MC, myocardium; MO, morpholino. Scale bars: 50 μm.

Article Snippet: For in situ probes against npnt and itga8 mRNA, 517 bp ( npnt , 5′-CAATGGTCTGTGTCGGTACG-3′ and 5′-CTGAAGGTCAAAGCCGTCAT-3′) and 713 bp ( itga8 , 5′-GAAAAGCCCACGGTTTACAA-3′ and 5′-TCCCCTGTGAACTCTCCAAC-3′) fragments were amplified from cDNA and cloned into the pGEM-T Easy vector (Promega) to create pGEMTeasy-Z npnt and pGEMTeasy- itga8 .

Techniques: Inhibition, Injection, Staining, In Situ Hybridization, Expressing, Diffusion-based Assay

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Quantification of designer nuclease induced mutation rates: a direct comparison of different methods

doi: 10.1038/mtm.2016.47

Figure Lengend Snippet: Representative results of T7-endonuclease I (T7E1) assays and gel shift assays performed on gDNA of HEK293 cells transfected or cotransfected with varying amounts of nuclease expression plasmids. (a) T7E1 assay performed after cotransfection of plasmids pAC-CMV-TALE-RM1 and pAC-CMV-TALE-RM2 encoding TALENs binding to the CCR5 locus. (b) T7E1 assay performed after cotransfection of plasmids pTn3 and pTn8 cotransfection cutting at the DMD locus. (c) T7E1 assay performed after cotransfection of plasmids pCMV-FlagAAVS1ELD-T2A-FlagAAVS1KKR transfection specifically binding to the AAVS1 locus. (d) Heteroduplex mobility assay (HMA) after cotransfection of p-CCR5-ZFN-L and p-CCR5-ZFN-R; pAC-CMV-TALE-RM1 and pAC-CMV-TALE-RM2 encoding a ZFN pair and a TALEN pair against the CCR5 locus, respectively. (e) HMA assay after cotransfection of plasmids TN3 and TN8 binding to the DMD locus. (f) Transfection of the plasmid pCMV-FlagAAVS1ELD-T2A-FlagAAVS1KKR expressing a complete ZFN pair from one plasmid. Cleavage products of T7E1 assay a–c and HMA (d-f) indicating heteroduplexes of mutated DNA and wild type DNA are marked by arrows. a–c mutation rates measured for samples treated with designer nucleases are depicted below the respective lanes of the gel pictures. CMV, Cytomegalovirus; EGFP, cells transfected with EGFP only (negative control); UT, untreated cells (negative control); PC, positive control (PCR products from untransfected cells were mixed with equal amounts of PCR products from plasmid with defined deletions at the nuclease binding site).

Article Snippet: For the hAAVS1 locus a PCR product amplified from gDNA of ZFN treated cells having a 2 bp deletion between ZFN binding sites was cloned into the pGEM-Teasy plasmid (Promega).

Techniques: Gel Shift, Transfection, Expressing, Cotransfection, TALENs, Binding Assay, Plasmid Preparation, Mutagenesis, Negative Control, Positive Control

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Quantification of designer nuclease induced mutation rates: a direct comparison of different methods

doi: 10.1038/mtm.2016.47

Figure Lengend Snippet: Quantitative PCR (q-PCR) to quantify different designer nuclease activity after transfection of nuclease expression plasmid. (a) Schematic overview designer nucleases encoded on the plasmids used in this experiment. Transcription activator-like effector nucleases (TALENs) targeting the human DMD - and the human CCR5 locus with respective repeat-variable diresidues (RVD) fused to the Fok I cleavage domain were expressed under the control of the cytomegalovirus (CMV) promoter (CMV-P). Zinc finger nucleases (ZFN) against the CCR5 locus were also expressed under the control of the CMV-P. ZFNs against the AAVS1 site were expressed from a single plasmid under the control of the CMV-P. ZFN domains were separated by a 2A peptide cleavage site. (b) q-PCR mutation detection using gDNA from HEK293 cells transfected with varying amounts (200 ng and 400 ng) of different nuclease expression plasmids. As shown in the mean ± SEM ( n = 3).

Article Snippet: For the hAAVS1 locus a PCR product amplified from gDNA of ZFN treated cells having a 2 bp deletion between ZFN binding sites was cloned into the pGEM-Teasy plasmid (Promega).

Techniques: Real-time Polymerase Chain Reaction, Activity Assay, Transfection, Expressing, Plasmid Preparation, TALENs, Control, Zinc-Fingers, Mutagenesis

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Quantification of designer nuclease induced mutation rates: a direct comparison of different methods

doi: 10.1038/mtm.2016.47

Figure Lengend Snippet: Quantitative PCR (q-PCR) to quantify CCR5 specific zinc finger nuclease (ZFN) activity after transduction of human peripheral blood mononuclear cells (hPBMC) with high capacity adenoviral vector HDAd5/35-CCR5-ZFN. (a) Schematic overview of the genome organization of the high capacity adenoviral vector HDAd5/35-CCR5-ZFN. HDAd5/35-CCR5-ZFN encodes both CCR5 specific ZFN domains fused to the FokI cleavage domain under the control of the elongation factor-1 alpha promoter (EF1α-P). SV40, Simian virus 40; NLS, nuclear localization signal; F1-F4 and Z1-Z4, single fingers in the ZFN protein binding to the target DNA, SV40-pA, SV40 polyadenylation signal; BGH-pA, bovine growth hormone polyadenylation signal; miRNA, microRNA target site suppressing ZFN expression during viral vector production; ITR, adenoviral inverted terminal repeat. (b) q-PCR mutation quantification using gDNA from human peripheral blood mononuclear cells (hPBMC) transduced with high capacity adenoviral vector HDAd5/35-CCR5-ZFN at 1,000 viral particles per cell. As shown in the mean ± SEM ( n = 3).

Article Snippet: For the hAAVS1 locus a PCR product amplified from gDNA of ZFN treated cells having a 2 bp deletion between ZFN binding sites was cloned into the pGEM-Teasy plasmid (Promega).

Techniques: Real-time Polymerase Chain Reaction, Zinc-Fingers, Activity Assay, Transduction, Plasmid Preparation, Control, Virus, Protein Binding, Expressing, Mutagenesis