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  • 86
    Developmental Studies Hybridoma Bank mouse anti isl1
    Hypothalamic NPY neurons in conditional Nkx2.1 mutant mice. a–f , The number of NPY + neurons is normal in Nkx2.1 KO@E9.5 ( b ), <t>Isl1</t> Nkx2.1 KO ( d ), and Pomc Nkx2.1 KO ( f ) E12.5 mouse embryos compared with their corresponding control littermates ( a , c , e ), as determined by immunofluorescence using anti-NPY (green) antibody on sagittal cryosections. g–j , The signal density of NPY + cells in Pomc Nkx2.1 KO mice is also normal at E15.5 ( g , h ) and in the adult arcuate nucleus ( i , j ).
    Mouse Anti Isl1, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Developmental Studies Hybridoma Bank mouse anti islet1
    Generation of neural cells with specific AP identities from ESCs. (A) Schematic representation of differentiation conditions used for the generation of NPCs with specific Anterior (N A ), Hindbrain (N H ) and Spinal cord (N P ) identities. (B) Relative expression levels of the indicated genes from N A , N H and N P cells at day 5 (D5) of differentiation indicate that N A , N H and N P cells express distinct sets of genes. The standard scores (z-scores) of the indicated genes from mRNA-seq analysis reveals that N A cells express high levels of forebrain markers including Otx1 and Otx2 ; N H cells express genes characteristic of hindbrain including Mafb and Hoxa2 genes; N P cells express high levels of posterior 5′ Hox genes including Hoxc8 and Hoxc9 . The individual Z-score for each replicate is indicated on the graph with circles, triangles and squares. (C) Time course of Hoxb and Hoxc cluster activation in cells cultured in N H and N P conditions showing fold change compared to D1. Posterior Hox genes are selectively activated only in the N P conditions and show temporal colinearity with the induction of anterior Hox genes prior to posterior Hox genes [74] . In N H cells Hoxb1 and Hoxb2 are induced prior to Hoxc4 . However, the more posterior Hox genes are not induced. By contrast, in N P conditions the 5′ Hox genes Hoxc6 , Hoxc8 and Hoxc9 are induced at D4 and their expression is maintained at day 5. (Note log 2 scale). (D) Immunohistochemistry indicates that N H cells analysed at D8 differentiate into MNs of hindbrain identity coexpressing Hoxb4 and Phox2b. (E) N P cells exposed to SAG generate spinal neurons coexpressing Hoxc6 and Hoxc9 with b-tubulin (Tuj1). These were not detected in N H conditions. Coexpression of Hoxc6 and Hoxc9 with <t>Islet1</t> indicates the generation of spinal MNs of forelimb and thoracic identity, respectively. These MNs also expressed Lim3/Raldh2 and HB9. (F) Graph showing the standard scores (z-scores) of Zfp42 (Rex1), Pou5F1 (Oct3/4) and Fgf5 from the mRNA-seq from D1 to D3. The kinetics of gene expression indicate that ESCs progressively lose their stem cell identity and acquire a transient epiblast identity at D2. (G) Hoxc6/Tuj1 and Hoxc9/Tuj1 positive cells were quantified in independent fields of D8 cells differentiated in N P conditions. All data used to generate the plots of Figure 1 can be found in Data S1 .
    Mouse Anti Islet1, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Developmental Studies Hybridoma Bank anti isl1
    Chromatin accessibility in CPCs is shaped by <t>Isl1.</t> a Number of differential chromatin accessibility peaks (log2(FC) > 2, false discovery rate [FDR]
    Anti Isl1, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Developmental Studies Hybridoma Bank isl1
    Several transcription factor dependency genes are marked by extensive H3K27ac and are enriched for dependency in human MYCN -amplified neuroblastoma a. Normalized ChIP-seq tracks for H3K27ac demonstrate a SE at the GATA3 locus compared to a typical enhancer at the RAD23B locus in BE2C; tracks represent a combination of 2 independent experiments. ChIP-seq read densities (y-axis) were normalized to reads per million reads sequenced in each sample. b. Gene ontology classification of terms associated with 77 shared SE-associated genes across five MYCN -amplified neuroblastoma cell lines reveals enrichment for transcription factor activity or binding (p=1.48×10 −6 ), and nucleic acid binding proteins (p=1.40×10 −2 ); n=77 genes, 130 GO-slim molecular class assignments, using 1-sided Fisher exact test (adjusted using Benjamini-Hochberg correction) comparing ontologies in SE-associated genes to all genes in the genome. c. Identification of enhancers by ranked H3K27ac signal across all genes in the MYCN -amplified cell lines Kelly and BE2C. Highlighted are transcription factor dependencies with shared SEs across all five MYCN- amplified cell lines. d. The rank of CRISPR-Cas9 dependencies genome-wide demonstrates selective dependency of shared SE-associated genes. Eleven of 69 SE-associated genes were enriched for selective dependency in neuroblastoma (p=1.11×10 −10 by 2-sided Fisher exact test compared to all genes assayed). Highlighted are 10 of 11 SE-associated dependency genes annotated with transcription factor activity/binding or nucleic acid binding ontologies. Closed circles reflect transcription factors evaluable by ChIP-seq; open circles represent those not evaluated. X-axis shows gene rank in enrichment analysis (modified Kolmogorov-Smirnov test with permutation testing) for MYCN -amplified neuroblastoma versus other cancer cell lines. Y-axis shows the -log 10 (p-value) from enrichment analysis. Enrichment p-value for MYCN, HAND2, <t>ISL1</t> and LDB1 was
    Isl1, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    isl1 - by Bioz Stars, 2021-05
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    N/A
    Mouse monoclonal to Islet 1 Conjugation note Unconjugated Application note WB IHC p IF ELISA Reactivity note Human
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    N/A
    15661 1 AP targets Islet 1 in WB IP IHC IF ELISA applications and shows reactivity with human samples
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    N/A
    Islet 1 Antibody is a Rabbit Polyclonal against Islet 1
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    N/A
    The Islet 1 Antibody from Novus Biologicals is a rabbit polyclonal antibody to Islet 1 This antibody reacts with human The Islet 1 Antibody has been validated for the following
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    Image Search Results


    Hypothalamic NPY neurons in conditional Nkx2.1 mutant mice. a–f , The number of NPY + neurons is normal in Nkx2.1 KO@E9.5 ( b ), Isl1 Nkx2.1 KO ( d ), and Pomc Nkx2.1 KO ( f ) E12.5 mouse embryos compared with their corresponding control littermates ( a , c , e ), as determined by immunofluorescence using anti-NPY (green) antibody on sagittal cryosections. g–j , The signal density of NPY + cells in Pomc Nkx2.1 KO mice is also normal at E15.5 ( g , h ) and in the adult arcuate nucleus ( i , j ).

    Journal: The Journal of Neuroscience

    Article Title: The Homeodomain Transcription Factor NKX2.1 Is Essential for the Early Specification of Melanocortin Neuron Identity and Activates Pomc Expression in the Developing Hypothalamus

    doi: 10.1523/JNEUROSCI.2924-18.2019

    Figure Lengend Snippet: Hypothalamic NPY neurons in conditional Nkx2.1 mutant mice. a–f , The number of NPY + neurons is normal in Nkx2.1 KO@E9.5 ( b ), Isl1 Nkx2.1 KO ( d ), and Pomc Nkx2.1 KO ( f ) E12.5 mouse embryos compared with their corresponding control littermates ( a , c , e ), as determined by immunofluorescence using anti-NPY (green) antibody on sagittal cryosections. g–j , The signal density of NPY + cells in Pomc Nkx2.1 KO mice is also normal at E15.5 ( g , h ) and in the adult arcuate nucleus ( i , j ).

    Article Snippet: Primary antibodies used were as follows: rabbit anti-rACTH (repository adrenocorticotropic hormone; 1:1000; National Hormone and Peptide Program); mouse anti-ISL1 (1:20; catalog #40.2D6, Developmental Studies Hybridoma Bank, University of Iowa); rabbit anti-NKX2.1 (1:1000; catalog #ab40880, Abcam); rabbit anti-NKX2.1 (1:1000; catalog #sc-13040, Santa Cruz Biotechnology); chicken anti-EGFP (1:2000; GFP-1020, Aves Labs); mouse anti-ASCL1 (1:200; catalog #556604, BD PharMingen); mouse anti-NGN3 (1:2000; catalog #F25A1B3, Developmental Studies Hybridoma Bank, University of Iowa); and rabbit anti-NPY (1:1000; T-4070 Peninsula Laboratories).

    Techniques: Mutagenesis, Mouse Assay, Immunofluorescence

    Hypothalamic Pomc expression is impaired in Nkx2.1 conditional knock-out mice. a–h , Immunofluorescence analysis using anti-NKX2.1 (red) and anti-ACTH (green) antibodies in sagittal cryosections of E12.5 mouse embryos injected with tamoxifen at E8.5 ( a–d ) or E9.5 ( e–h ). Cre + and Cre − embryos are littermates. i–n , Immunofluorescence analysis of E12.5 mouse embryos injected with tamoxifen at E9.5 using antibodies against ISL1 ( i , l ) or ACTH ( j , m ); closed-up merged pictures are also shown ( k , n ).

    Journal: The Journal of Neuroscience

    Article Title: The Homeodomain Transcription Factor NKX2.1 Is Essential for the Early Specification of Melanocortin Neuron Identity and Activates Pomc Expression in the Developing Hypothalamus

    doi: 10.1523/JNEUROSCI.2924-18.2019

    Figure Lengend Snippet: Hypothalamic Pomc expression is impaired in Nkx2.1 conditional knock-out mice. a–h , Immunofluorescence analysis using anti-NKX2.1 (red) and anti-ACTH (green) antibodies in sagittal cryosections of E12.5 mouse embryos injected with tamoxifen at E8.5 ( a–d ) or E9.5 ( e–h ). Cre + and Cre − embryos are littermates. i–n , Immunofluorescence analysis of E12.5 mouse embryos injected with tamoxifen at E9.5 using antibodies against ISL1 ( i , l ) or ACTH ( j , m ); closed-up merged pictures are also shown ( k , n ).

    Article Snippet: Primary antibodies used were as follows: rabbit anti-rACTH (repository adrenocorticotropic hormone; 1:1000; National Hormone and Peptide Program); mouse anti-ISL1 (1:20; catalog #40.2D6, Developmental Studies Hybridoma Bank, University of Iowa); rabbit anti-NKX2.1 (1:1000; catalog #ab40880, Abcam); rabbit anti-NKX2.1 (1:1000; catalog #sc-13040, Santa Cruz Biotechnology); chicken anti-EGFP (1:2000; GFP-1020, Aves Labs); mouse anti-ASCL1 (1:200; catalog #556604, BD PharMingen); mouse anti-NGN3 (1:2000; catalog #F25A1B3, Developmental Studies Hybridoma Bank, University of Iowa); and rabbit anti-NPY (1:1000; T-4070 Peninsula Laboratories).

    Techniques: Expressing, Knock-Out, Mouse Assay, Immunofluorescence, Injection

    Specific deletion of Nkx2.1 from ISL1 + neurons impairs the onset of Pomc expression. a–l , Immunofluorescence analysis in sagittal cryosections of E11.5 embryos using anti-ACTH ( a , b ), anti-NKX2.1 ( c , d ), anti-ISL1 ( e , f ), anti-ASCL1 ( i , j ), and anti-NGN3 ( k , l ) antibodies. g , h , Loss of Nkx2.1 in ISL1 + neurons is evident by double labeling. Arrows point to the mantle zone of the mediobasal hypothalamus detailed in the insets above. m–p , E12.5 Isl1 Nkx2.1 KO embryos show reduced numbers of ACTH + neurons compared with controls ( m , n ) and loss of NKX2.1 in the mantle zone ( o , p ). DAPI is shown in blue.

    Journal: The Journal of Neuroscience

    Article Title: The Homeodomain Transcription Factor NKX2.1 Is Essential for the Early Specification of Melanocortin Neuron Identity and Activates Pomc Expression in the Developing Hypothalamus

    doi: 10.1523/JNEUROSCI.2924-18.2019

    Figure Lengend Snippet: Specific deletion of Nkx2.1 from ISL1 + neurons impairs the onset of Pomc expression. a–l , Immunofluorescence analysis in sagittal cryosections of E11.5 embryos using anti-ACTH ( a , b ), anti-NKX2.1 ( c , d ), anti-ISL1 ( e , f ), anti-ASCL1 ( i , j ), and anti-NGN3 ( k , l ) antibodies. g , h , Loss of Nkx2.1 in ISL1 + neurons is evident by double labeling. Arrows point to the mantle zone of the mediobasal hypothalamus detailed in the insets above. m–p , E12.5 Isl1 Nkx2.1 KO embryos show reduced numbers of ACTH + neurons compared with controls ( m , n ) and loss of NKX2.1 in the mantle zone ( o , p ). DAPI is shown in blue.

    Article Snippet: Primary antibodies used were as follows: rabbit anti-rACTH (repository adrenocorticotropic hormone; 1:1000; National Hormone and Peptide Program); mouse anti-ISL1 (1:20; catalog #40.2D6, Developmental Studies Hybridoma Bank, University of Iowa); rabbit anti-NKX2.1 (1:1000; catalog #ab40880, Abcam); rabbit anti-NKX2.1 (1:1000; catalog #sc-13040, Santa Cruz Biotechnology); chicken anti-EGFP (1:2000; GFP-1020, Aves Labs); mouse anti-ASCL1 (1:200; catalog #556604, BD PharMingen); mouse anti-NGN3 (1:2000; catalog #F25A1B3, Developmental Studies Hybridoma Bank, University of Iowa); and rabbit anti-NPY (1:1000; T-4070 Peninsula Laboratories).

    Techniques: Expressing, Immunofluorescence, Labeling

    Overlapping expression patterns of Nkx2.1 , Isl1 , and Pomc in the developing hypothalamus. a–j , Immunofluorescence analysis using anti-NKX2.1 (blue) and anti-ISL1 (red) antibodies in sagittal cryosections of E10.5 ( a–e ) and E12.5 ( f–j ) Pomc -EGFP mouse embryos. Confocal images showing superimposed NKX2.1- and ISL1-immunopositive neurons ( a , f ) and triple merged with Pomc -EGFP-immunoreactive neurons ( e , j ). Arrows denote examples of neurons coexpressing NKX2.1, ISL1, and EGFP.

    Journal: The Journal of Neuroscience

    Article Title: The Homeodomain Transcription Factor NKX2.1 Is Essential for the Early Specification of Melanocortin Neuron Identity and Activates Pomc Expression in the Developing Hypothalamus

    doi: 10.1523/JNEUROSCI.2924-18.2019

    Figure Lengend Snippet: Overlapping expression patterns of Nkx2.1 , Isl1 , and Pomc in the developing hypothalamus. a–j , Immunofluorescence analysis using anti-NKX2.1 (blue) and anti-ISL1 (red) antibodies in sagittal cryosections of E10.5 ( a–e ) and E12.5 ( f–j ) Pomc -EGFP mouse embryos. Confocal images showing superimposed NKX2.1- and ISL1-immunopositive neurons ( a , f ) and triple merged with Pomc -EGFP-immunoreactive neurons ( e , j ). Arrows denote examples of neurons coexpressing NKX2.1, ISL1, and EGFP.

    Article Snippet: Primary antibodies used were as follows: rabbit anti-rACTH (repository adrenocorticotropic hormone; 1:1000; National Hormone and Peptide Program); mouse anti-ISL1 (1:20; catalog #40.2D6, Developmental Studies Hybridoma Bank, University of Iowa); rabbit anti-NKX2.1 (1:1000; catalog #ab40880, Abcam); rabbit anti-NKX2.1 (1:1000; catalog #sc-13040, Santa Cruz Biotechnology); chicken anti-EGFP (1:2000; GFP-1020, Aves Labs); mouse anti-ASCL1 (1:200; catalog #556604, BD PharMingen); mouse anti-NGN3 (1:2000; catalog #F25A1B3, Developmental Studies Hybridoma Bank, University of Iowa); and rabbit anti-NPY (1:1000; T-4070 Peninsula Laboratories).

    Techniques: Expressing, Immunofluorescence

    Generation of neural cells with specific AP identities from ESCs. (A) Schematic representation of differentiation conditions used for the generation of NPCs with specific Anterior (N A ), Hindbrain (N H ) and Spinal cord (N P ) identities. (B) Relative expression levels of the indicated genes from N A , N H and N P cells at day 5 (D5) of differentiation indicate that N A , N H and N P cells express distinct sets of genes. The standard scores (z-scores) of the indicated genes from mRNA-seq analysis reveals that N A cells express high levels of forebrain markers including Otx1 and Otx2 ; N H cells express genes characteristic of hindbrain including Mafb and Hoxa2 genes; N P cells express high levels of posterior 5′ Hox genes including Hoxc8 and Hoxc9 . The individual Z-score for each replicate is indicated on the graph with circles, triangles and squares. (C) Time course of Hoxb and Hoxc cluster activation in cells cultured in N H and N P conditions showing fold change compared to D1. Posterior Hox genes are selectively activated only in the N P conditions and show temporal colinearity with the induction of anterior Hox genes prior to posterior Hox genes [74] . In N H cells Hoxb1 and Hoxb2 are induced prior to Hoxc4 . However, the more posterior Hox genes are not induced. By contrast, in N P conditions the 5′ Hox genes Hoxc6 , Hoxc8 and Hoxc9 are induced at D4 and their expression is maintained at day 5. (Note log 2 scale). (D) Immunohistochemistry indicates that N H cells analysed at D8 differentiate into MNs of hindbrain identity coexpressing Hoxb4 and Phox2b. (E) N P cells exposed to SAG generate spinal neurons coexpressing Hoxc6 and Hoxc9 with b-tubulin (Tuj1). These were not detected in N H conditions. Coexpression of Hoxc6 and Hoxc9 with Islet1 indicates the generation of spinal MNs of forelimb and thoracic identity, respectively. These MNs also expressed Lim3/Raldh2 and HB9. (F) Graph showing the standard scores (z-scores) of Zfp42 (Rex1), Pou5F1 (Oct3/4) and Fgf5 from the mRNA-seq from D1 to D3. The kinetics of gene expression indicate that ESCs progressively lose their stem cell identity and acquire a transient epiblast identity at D2. (G) Hoxc6/Tuj1 and Hoxc9/Tuj1 positive cells were quantified in independent fields of D8 cells differentiated in N P conditions. All data used to generate the plots of Figure 1 can be found in Data S1 .

    Journal: PLoS Biology

    Article Title: In Vitro Generation of Neuromesodermal Progenitors Reveals Distinct Roles for Wnt Signalling in the Specification of Spinal Cord and Paraxial Mesoderm IdentityDevelopment of Spinal Cord Neurons in Delicate Balance

    doi: 10.1371/journal.pbio.1001937

    Figure Lengend Snippet: Generation of neural cells with specific AP identities from ESCs. (A) Schematic representation of differentiation conditions used for the generation of NPCs with specific Anterior (N A ), Hindbrain (N H ) and Spinal cord (N P ) identities. (B) Relative expression levels of the indicated genes from N A , N H and N P cells at day 5 (D5) of differentiation indicate that N A , N H and N P cells express distinct sets of genes. The standard scores (z-scores) of the indicated genes from mRNA-seq analysis reveals that N A cells express high levels of forebrain markers including Otx1 and Otx2 ; N H cells express genes characteristic of hindbrain including Mafb and Hoxa2 genes; N P cells express high levels of posterior 5′ Hox genes including Hoxc8 and Hoxc9 . The individual Z-score for each replicate is indicated on the graph with circles, triangles and squares. (C) Time course of Hoxb and Hoxc cluster activation in cells cultured in N H and N P conditions showing fold change compared to D1. Posterior Hox genes are selectively activated only in the N P conditions and show temporal colinearity with the induction of anterior Hox genes prior to posterior Hox genes [74] . In N H cells Hoxb1 and Hoxb2 are induced prior to Hoxc4 . However, the more posterior Hox genes are not induced. By contrast, in N P conditions the 5′ Hox genes Hoxc6 , Hoxc8 and Hoxc9 are induced at D4 and their expression is maintained at day 5. (Note log 2 scale). (D) Immunohistochemistry indicates that N H cells analysed at D8 differentiate into MNs of hindbrain identity coexpressing Hoxb4 and Phox2b. (E) N P cells exposed to SAG generate spinal neurons coexpressing Hoxc6 and Hoxc9 with b-tubulin (Tuj1). These were not detected in N H conditions. Coexpression of Hoxc6 and Hoxc9 with Islet1 indicates the generation of spinal MNs of forelimb and thoracic identity, respectively. These MNs also expressed Lim3/Raldh2 and HB9. (F) Graph showing the standard scores (z-scores) of Zfp42 (Rex1), Pou5F1 (Oct3/4) and Fgf5 from the mRNA-seq from D1 to D3. The kinetics of gene expression indicate that ESCs progressively lose their stem cell identity and acquire a transient epiblast identity at D2. (G) Hoxc6/Tuj1 and Hoxc9/Tuj1 positive cells were quantified in independent fields of D8 cells differentiated in N P conditions. All data used to generate the plots of Figure 1 can be found in Data S1 .

    Article Snippet: The following primary antibodies were used: mouse anti-Hoxc6 (1∶10) (DSHB), mouse anti-Hoxc9 (1∶10) (gift of T. Jessell), mouse anti-Hoxc10 (1∶50) (DSHB), rat anti-Hoxb4 (1∶100) (gift of A. Gould), rabbit anti-Phox2b (1∶200), mouse anti-Tuj1 (1∶1000) (Covance), rabbit anti-Tuj1 (1∶500) (Covance), rabbit anti-Olig2 (1∶500) (Chemicon), mouse anti-Sox2 (1∶200) (ab92494, Abcam), rabbit anti-Sox2 (1∶200) (Millipore), goat anti-Sox2 (1∶100) (R & D), goat anti-Tbx6 (1∶200) (R & D) or rabbit anti-Tbx6 (0.6 µg/ml) (ab38883, Abcam), goat anti-Brachyury (1∶500) (R & D), rabbit anti-RALDH2 (1∶500) (Sigma), rabbit anti-Desmin (1∶500) (Abcam), mouse anti-MyoD1 (1∶200) (DAKO), mouse anti-Islet1 (1∶2000) (gift of T. Jessell), mouse anti-Lim3 (1∶10) (DSHB), mouse anti-HB9 (1∶100) (DSHB), anti-Foxa2 (1 mg/ml) (Santa Cruz; sc-6554), anti-GFP (10 µg/ml) (Abcam; ab13970), anti-Pax3 (1∶20) (DSHB);. anti-Nanog (2.5 µg/ml) (14-5761-80, eBioscience); anti-Oct4 (1 µg/ml) (N-19, Santa Cruz), HoxC8 (5 µg/ml) (Abcam).

    Techniques: Expressing, Activation Assay, Cell Culture, Immunohistochemistry

    Chromatin accessibility in CPCs is shaped by Isl1. a Number of differential chromatin accessibility peaks (log2(FC) > 2, false discovery rate [FDR]

    Journal: Nature Communications

    Article Title: Single cell RNA-seq and ATAC-seq analysis of cardiac progenitor cell transition states and lineage settlement

    doi: 10.1038/s41467-018-07307-6

    Figure Lengend Snippet: Chromatin accessibility in CPCs is shaped by Isl1. a Number of differential chromatin accessibility peaks (log2(FC) > 2, false discovery rate [FDR]

    Article Snippet: The following antibodies with indicated concentration were used: anti-Nkx2-5 (ThermoScientific Cat# PA5-49431, 1:1,000) and anti-Isl1 (DSHB 39.4D5, 1:100).

    Techniques:

    Inactivation of Isl1 prevents CPC fate bifurcation. a Schematic illustration depicting generation of Isl1 embryos and scRNA-seq. b t-SNE plots showing the predicted diffusion pseudotime of Isl1 knockout CPCs projected on Isl1 + cells (left), and clustering with Isl1 + cells (right). c Ratios of cycling and non-cycling Isl1 knockout and wild type Isl1 + CPCs. χ 2 test: p = 0.062. n indicates cells numbers. d Heatmap showing expression of deregulated genes in Isl1 + cells at E8.5 and E9.5 (cluster 1, 2, and 5) isolated from Isl1 knockout and control embryos. Source data are provided in the Source Data file

    Journal: Nature Communications

    Article Title: Single cell RNA-seq and ATAC-seq analysis of cardiac progenitor cell transition states and lineage settlement

    doi: 10.1038/s41467-018-07307-6

    Figure Lengend Snippet: Inactivation of Isl1 prevents CPC fate bifurcation. a Schematic illustration depicting generation of Isl1 embryos and scRNA-seq. b t-SNE plots showing the predicted diffusion pseudotime of Isl1 knockout CPCs projected on Isl1 + cells (left), and clustering with Isl1 + cells (right). c Ratios of cycling and non-cycling Isl1 knockout and wild type Isl1 + CPCs. χ 2 test: p = 0.062. n indicates cells numbers. d Heatmap showing expression of deregulated genes in Isl1 + cells at E8.5 and E9.5 (cluster 1, 2, and 5) isolated from Isl1 knockout and control embryos. Source data are provided in the Source Data file

    Article Snippet: The following antibodies with indicated concentration were used: anti-Nkx2-5 (ThermoScientific Cat# PA5-49431, 1:1,000) and anti-Isl1 (DSHB 39.4D5, 1:100).

    Techniques: Diffusion-based Assay, Knock-Out, Expressing, Isolation

    Single cell chromatin accessibility profiles of Isl1 + CPCs. a Representative genomic region showing ATAC-seq tracks of single, aggregate and bulk cells. b, c t-SNE visualization of individual Nkx2-5 + and Isl1 + CPCs to identify subpopulations based on chromatin accessibility. Colors denote corresponding clusters ( b ), and ( c ) development stages. d Gene ontology (GO) enrichment analyses of scATAC-seq clusters 1, 2, 5 of Isl1 + CPCs. Each bubble represents one of the top enriched GO terms. The relevant GO terms ( p

    Journal: Nature Communications

    Article Title: Single cell RNA-seq and ATAC-seq analysis of cardiac progenitor cell transition states and lineage settlement

    doi: 10.1038/s41467-018-07307-6

    Figure Lengend Snippet: Single cell chromatin accessibility profiles of Isl1 + CPCs. a Representative genomic region showing ATAC-seq tracks of single, aggregate and bulk cells. b, c t-SNE visualization of individual Nkx2-5 + and Isl1 + CPCs to identify subpopulations based on chromatin accessibility. Colors denote corresponding clusters ( b ), and ( c ) development stages. d Gene ontology (GO) enrichment analyses of scATAC-seq clusters 1, 2, 5 of Isl1 + CPCs. Each bubble represents one of the top enriched GO terms. The relevant GO terms ( p

    Article Snippet: The following antibodies with indicated concentration were used: anti-Nkx2-5 (ThermoScientific Cat# PA5-49431, 1:1,000) and anti-Isl1 (DSHB 39.4D5, 1:100).

    Techniques:

    Identification of CPC subpopulations by single-cell RNA-seq. a Schematic representation of the Nkx2-5-emGFP transgenic reporter and Isl1 nGFP/+ allele (top). Expression of Nkx2-5-emGFP and Isl1-nGFP at E8.5 in mouse embryonic hearts. (bottom). b Sampling time points for scRNA-seq, bulk RNA-seq, scATAC-seq, and bulk ATAC-seq. The table shows numbers of cells used for scRNA-seq. QC: quality control. c , d t-SNE visualization of individual Nkx2-5 + and Isl1 + CPCs to identify subpopulations. Colors denote corresponding clusters, and ( d ) development stages. Outlier cells are indicated by gray crosses. e Hierarchical clustering of expression heatmaps showing differentially expressed marker genes (AUROC > 0.8, FDR

    Journal: Nature Communications

    Article Title: Single cell RNA-seq and ATAC-seq analysis of cardiac progenitor cell transition states and lineage settlement

    doi: 10.1038/s41467-018-07307-6

    Figure Lengend Snippet: Identification of CPC subpopulations by single-cell RNA-seq. a Schematic representation of the Nkx2-5-emGFP transgenic reporter and Isl1 nGFP/+ allele (top). Expression of Nkx2-5-emGFP and Isl1-nGFP at E8.5 in mouse embryonic hearts. (bottom). b Sampling time points for scRNA-seq, bulk RNA-seq, scATAC-seq, and bulk ATAC-seq. The table shows numbers of cells used for scRNA-seq. QC: quality control. c , d t-SNE visualization of individual Nkx2-5 + and Isl1 + CPCs to identify subpopulations. Colors denote corresponding clusters, and ( d ) development stages. Outlier cells are indicated by gray crosses. e Hierarchical clustering of expression heatmaps showing differentially expressed marker genes (AUROC > 0.8, FDR

    Article Snippet: The following antibodies with indicated concentration were used: anti-Nkx2-5 (ThermoScientific Cat# PA5-49431, 1:1,000) and anti-Isl1 (DSHB 39.4D5, 1:100).

    Techniques: RNA Sequencing Assay, Transgenic Assay, Expressing, Sampling, Marker

    Reconstruction of trajectories and transition states of CPCs. a t-SNE plots showing diffusion pseudotimes (gray arrows) of Nkx2-5+ and b Isl1+ CPCs. Clusters and development stages of individual cells are color-coded as indicated. c Boxplots representing the distribution of I C (C) values from all marker genes for each cluster of Nkx2-5 + (left) and Isl1 + (right) cells. Lower and upper hinges correspond to the first and third quantile (25th and 75th percentile), while whiskers extend from the hinge to the smallest (largest) datum not further than 1.5 times the interquartile range. Outliers are plotted individually. d Violin plots showing the distribution of pairwise cell-to-cell distances across each cluster of Nkx2-5 + (left) and Isl1 + (right) cells. Inset boxplots show the median, lower and upper hinges as well as whiskers and outliers as in ( c ). e, f Expression levels of different transcription factors and key marker genes on the pseudotime axis in Nkx2-5 + ( e ) and Isl1 + ( f ) cells. Trend lines calculated by Loess regression are indicated in gray. Source data for ( c – f ) are provided in the Source Data file

    Journal: Nature Communications

    Article Title: Single cell RNA-seq and ATAC-seq analysis of cardiac progenitor cell transition states and lineage settlement

    doi: 10.1038/s41467-018-07307-6

    Figure Lengend Snippet: Reconstruction of trajectories and transition states of CPCs. a t-SNE plots showing diffusion pseudotimes (gray arrows) of Nkx2-5+ and b Isl1+ CPCs. Clusters and development stages of individual cells are color-coded as indicated. c Boxplots representing the distribution of I C (C) values from all marker genes for each cluster of Nkx2-5 + (left) and Isl1 + (right) cells. Lower and upper hinges correspond to the first and third quantile (25th and 75th percentile), while whiskers extend from the hinge to the smallest (largest) datum not further than 1.5 times the interquartile range. Outliers are plotted individually. d Violin plots showing the distribution of pairwise cell-to-cell distances across each cluster of Nkx2-5 + (left) and Isl1 + (right) cells. Inset boxplots show the median, lower and upper hinges as well as whiskers and outliers as in ( c ). e, f Expression levels of different transcription factors and key marker genes on the pseudotime axis in Nkx2-5 + ( e ) and Isl1 + ( f ) cells. Trend lines calculated by Loess regression are indicated in gray. Source data for ( c – f ) are provided in the Source Data file

    Article Snippet: The following antibodies with indicated concentration were used: anti-Nkx2-5 (ThermoScientific Cat# PA5-49431, 1:1,000) and anti-Isl1 (DSHB 39.4D5, 1:100).

    Techniques: Diffusion-based Assay, Marker, Expressing

    Spatial expression pattern of genes identified by scRNA-seq of CPCs. a Heatmap showing expression of selected genes in Isl1 + and Nkx2-5 + CPCs at E8.5. b – d In situ hybridization of sections from E8.5 embryos to reveal spatial expression profiles of genes identified by scRNA-seq. Scale bar: 100 μm for ( b ), 50 μm for ( c , d ). V: ventricle. PA: primitive atria. PhA: pharyngeal arches. OFT: outflow tract. Arrows indicate positive cells

    Journal: Nature Communications

    Article Title: Single cell RNA-seq and ATAC-seq analysis of cardiac progenitor cell transition states and lineage settlement

    doi: 10.1038/s41467-018-07307-6

    Figure Lengend Snippet: Spatial expression pattern of genes identified by scRNA-seq of CPCs. a Heatmap showing expression of selected genes in Isl1 + and Nkx2-5 + CPCs at E8.5. b – d In situ hybridization of sections from E8.5 embryos to reveal spatial expression profiles of genes identified by scRNA-seq. Scale bar: 100 μm for ( b ), 50 μm for ( c , d ). V: ventricle. PA: primitive atria. PhA: pharyngeal arches. OFT: outflow tract. Arrows indicate positive cells

    Article Snippet: The following antibodies with indicated concentration were used: anti-Nkx2-5 (ThermoScientific Cat# PA5-49431, 1:1,000) and anti-Isl1 (DSHB 39.4D5, 1:100).

    Techniques: Expressing, In Situ Hybridization

    Comparison of Isl1 + and Nkx2-5 + cardiac progenitor cells. a Confocal images showing nuclear-, cytoplasmic- and co-localization of GFP in CPCs FACS-sorted from Isl1 +/nGFP /Nkx2-5-emGFP + embryos. Nuclei were stained with DAPI (blue). b Immunofluorescence-based quantification of ( a ). Isl1 + Nkx2-5 − , Isl1 + Nkx2-5 + and Isl1 − Nkx2-5 + cells were FACS-sorted from Isl1 +/nGFP /Nkx2-5-emGFP + embryos at E8.5 and E9.5. Quantification of different cell populations was achieved by counting all immunostained cells in a multiwell dish. Mean ± s.d. are shown. Circles represent results from different biological replicates [ n = 3; Σ (cell number) of E8.5 = 225, 260, 100; Σ (cell number) of E9.5 = 175, 180, 100]. c Clustering of Isl1 and Nkx2-5 co-expressing cells in Nkx2-5 + and Isl1 + CPC subpopulations. Cells that are not double-positive are labeled in gray, and clusters are indicated by colored circles. d , e Plots showing the predicted diffusion pseudotime of Nkx2-5 + cells projected on t-SNE plots of Isl1 + cells, and the expression of Isl1 + ( d ) and Nkx2-5 + ( e ). Expression levels of Isl1 and Nkx2-5 in CPCs are represented by a color spectrum as indicated. EC, endothelial cell. CM, cardiomyocyte

    Journal: Nature Communications

    Article Title: Single cell RNA-seq and ATAC-seq analysis of cardiac progenitor cell transition states and lineage settlement

    doi: 10.1038/s41467-018-07307-6

    Figure Lengend Snippet: Comparison of Isl1 + and Nkx2-5 + cardiac progenitor cells. a Confocal images showing nuclear-, cytoplasmic- and co-localization of GFP in CPCs FACS-sorted from Isl1 +/nGFP /Nkx2-5-emGFP + embryos. Nuclei were stained with DAPI (blue). b Immunofluorescence-based quantification of ( a ). Isl1 + Nkx2-5 − , Isl1 + Nkx2-5 + and Isl1 − Nkx2-5 + cells were FACS-sorted from Isl1 +/nGFP /Nkx2-5-emGFP + embryos at E8.5 and E9.5. Quantification of different cell populations was achieved by counting all immunostained cells in a multiwell dish. Mean ± s.d. are shown. Circles represent results from different biological replicates [ n = 3; Σ (cell number) of E8.5 = 225, 260, 100; Σ (cell number) of E9.5 = 175, 180, 100]. c Clustering of Isl1 and Nkx2-5 co-expressing cells in Nkx2-5 + and Isl1 + CPC subpopulations. Cells that are not double-positive are labeled in gray, and clusters are indicated by colored circles. d , e Plots showing the predicted diffusion pseudotime of Nkx2-5 + cells projected on t-SNE plots of Isl1 + cells, and the expression of Isl1 + ( d ) and Nkx2-5 + ( e ). Expression levels of Isl1 and Nkx2-5 in CPCs are represented by a color spectrum as indicated. EC, endothelial cell. CM, cardiomyocyte

    Article Snippet: The following antibodies with indicated concentration were used: anti-Nkx2-5 (ThermoScientific Cat# PA5-49431, 1:1,000) and anti-Isl1 (DSHB 39.4D5, 1:100).

    Techniques: FACS, Staining, Immunofluorescence, Expressing, Labeling, Diffusion-based Assay

    Several transcription factor dependency genes are marked by extensive H3K27ac and are enriched for dependency in human MYCN -amplified neuroblastoma a. Normalized ChIP-seq tracks for H3K27ac demonstrate a SE at the GATA3 locus compared to a typical enhancer at the RAD23B locus in BE2C; tracks represent a combination of 2 independent experiments. ChIP-seq read densities (y-axis) were normalized to reads per million reads sequenced in each sample. b. Gene ontology classification of terms associated with 77 shared SE-associated genes across five MYCN -amplified neuroblastoma cell lines reveals enrichment for transcription factor activity or binding (p=1.48×10 −6 ), and nucleic acid binding proteins (p=1.40×10 −2 ); n=77 genes, 130 GO-slim molecular class assignments, using 1-sided Fisher exact test (adjusted using Benjamini-Hochberg correction) comparing ontologies in SE-associated genes to all genes in the genome. c. Identification of enhancers by ranked H3K27ac signal across all genes in the MYCN -amplified cell lines Kelly and BE2C. Highlighted are transcription factor dependencies with shared SEs across all five MYCN- amplified cell lines. d. The rank of CRISPR-Cas9 dependencies genome-wide demonstrates selective dependency of shared SE-associated genes. Eleven of 69 SE-associated genes were enriched for selective dependency in neuroblastoma (p=1.11×10 −10 by 2-sided Fisher exact test compared to all genes assayed). Highlighted are 10 of 11 SE-associated dependency genes annotated with transcription factor activity/binding or nucleic acid binding ontologies. Closed circles reflect transcription factors evaluable by ChIP-seq; open circles represent those not evaluated. X-axis shows gene rank in enrichment analysis (modified Kolmogorov-Smirnov test with permutation testing) for MYCN -amplified neuroblastoma versus other cancer cell lines. Y-axis shows the -log 10 (p-value) from enrichment analysis. Enrichment p-value for MYCN, HAND2, ISL1 and LDB1 was

    Journal: Nature genetics

    Article Title: Selective gene dependencies in MYCN-amplified neuroblastoma include the core transcriptional regulatory circuitry

    doi: 10.1038/s41588-018-0191-z

    Figure Lengend Snippet: Several transcription factor dependency genes are marked by extensive H3K27ac and are enriched for dependency in human MYCN -amplified neuroblastoma a. Normalized ChIP-seq tracks for H3K27ac demonstrate a SE at the GATA3 locus compared to a typical enhancer at the RAD23B locus in BE2C; tracks represent a combination of 2 independent experiments. ChIP-seq read densities (y-axis) were normalized to reads per million reads sequenced in each sample. b. Gene ontology classification of terms associated with 77 shared SE-associated genes across five MYCN -amplified neuroblastoma cell lines reveals enrichment for transcription factor activity or binding (p=1.48×10 −6 ), and nucleic acid binding proteins (p=1.40×10 −2 ); n=77 genes, 130 GO-slim molecular class assignments, using 1-sided Fisher exact test (adjusted using Benjamini-Hochberg correction) comparing ontologies in SE-associated genes to all genes in the genome. c. Identification of enhancers by ranked H3K27ac signal across all genes in the MYCN -amplified cell lines Kelly and BE2C. Highlighted are transcription factor dependencies with shared SEs across all five MYCN- amplified cell lines. d. The rank of CRISPR-Cas9 dependencies genome-wide demonstrates selective dependency of shared SE-associated genes. Eleven of 69 SE-associated genes were enriched for selective dependency in neuroblastoma (p=1.11×10 −10 by 2-sided Fisher exact test compared to all genes assayed). Highlighted are 10 of 11 SE-associated dependency genes annotated with transcription factor activity/binding or nucleic acid binding ontologies. Closed circles reflect transcription factors evaluable by ChIP-seq; open circles represent those not evaluated. X-axis shows gene rank in enrichment analysis (modified Kolmogorov-Smirnov test with permutation testing) for MYCN -amplified neuroblastoma versus other cancer cell lines. Y-axis shows the -log 10 (p-value) from enrichment analysis. Enrichment p-value for MYCN, HAND2, ISL1 and LDB1 was

    Article Snippet: The following antibodies were used for ChIP: GATA3 (Thermo Fisher MA1028), HAND2 (Santa Cruz sc9409X), ISL1 (Developmental Hybridoma Studies Bank 40.3A4), MYCN (Invitrogen Biotechnology, MA-1-16638), PHOX2B (Santa Cruz sc376997X), TBX2 (Abcam ab33298), and H3K27ac (Abcam ab4729).

    Techniques: Amplification, Chromatin Immunoprecipitation, Activity Assay, Binding Assay, CRISPR, Genome Wide, Modification, Significance Assay

    Genome-scale CRISPR-Cas9 screening identifies selective neuroblastoma gene dependencies enriched for transcription factors a. Gene ontology classification of terms associated with selective dependencies in neuroblastoma reveals enrichment for transcription factor activity or binding and nucleic acid binding proteins. n=147 genes, 252 GO-slim molecular class assignments. p=8.48×10 −3 nucleic acid binding, p=6.78×10 −3 transcription factor activity or binding, by 1-sided Fisher exact test (adjusted using Benjamini-Hochberg correction) comparing the ontologie s of dependencies to all assayed genes in the genome. b. STRING database analysis demonstrates 25 of 30 transcription factor dependencies have putative protein-protein interactions. Edge widths correspond to the level of confidence in interactions (medium confidence STRING score 0.4; high confidence STRING score 0.7; highest confidence STRING score 0.9). Red nodes indicate transcription factors previously co-reported with neuroblastoma in a literature search. c and d. Representative scatter plots showing neuroblastoma relative dependency on HAND2 (c) and ISL1 (d) with p

    Journal: Nature genetics

    Article Title: Selective gene dependencies in MYCN-amplified neuroblastoma include the core transcriptional regulatory circuitry

    doi: 10.1038/s41588-018-0191-z

    Figure Lengend Snippet: Genome-scale CRISPR-Cas9 screening identifies selective neuroblastoma gene dependencies enriched for transcription factors a. Gene ontology classification of terms associated with selective dependencies in neuroblastoma reveals enrichment for transcription factor activity or binding and nucleic acid binding proteins. n=147 genes, 252 GO-slim molecular class assignments. p=8.48×10 −3 nucleic acid binding, p=6.78×10 −3 transcription factor activity or binding, by 1-sided Fisher exact test (adjusted using Benjamini-Hochberg correction) comparing the ontologie s of dependencies to all assayed genes in the genome. b. STRING database analysis demonstrates 25 of 30 transcription factor dependencies have putative protein-protein interactions. Edge widths correspond to the level of confidence in interactions (medium confidence STRING score 0.4; high confidence STRING score 0.7; highest confidence STRING score 0.9). Red nodes indicate transcription factors previously co-reported with neuroblastoma in a literature search. c and d. Representative scatter plots showing neuroblastoma relative dependency on HAND2 (c) and ISL1 (d) with p

    Article Snippet: The following antibodies were used for ChIP: GATA3 (Thermo Fisher MA1028), HAND2 (Santa Cruz sc9409X), ISL1 (Developmental Hybridoma Studies Bank 40.3A4), MYCN (Invitrogen Biotechnology, MA-1-16638), PHOX2B (Santa Cruz sc376997X), TBX2 (Abcam ab33298), and H3K27ac (Abcam ab4729).

    Techniques: CRISPR, Activity Assay, Binding Assay

    Dependency transcription factors form the core regulatory circuitry in MYCN -amplified neuroblastoma a. Normalized ChIP-seq tracks for H3K27ac, MYCN, and five transcription factors at the HAND2 , ISL1 and PHOX2B loci in BE2C cells; H3K27ac track represents a combination of 2 independent experiments in BE2C and other tracks are representative of an independent experiment performed in BE2C and Kelly cells. ChIP-seq read densities (y-axis) were normalized to reads per million reads sequenced in each sample. SEs are noted as red bars and arrows indicate epicentres. b. Genome-wide co-occupancy for CRC transcription factors as determined by ChIP-seq. Regions (rows) were defined as those enriched in ChIP-seq reads for at least one transcription factor and are ranked by the MYCN signal therein. Color keys for reads-per-million-normalized signal are displayed below each heatmap. c. Quantitative RT-PCR of BE2C cells treated with transient siRNA to each CRC gene - HAND2 , ISL1 , PHOX2B , GATA3 and TBX2 - results in decreased expression of mRNA transcripts for all of the CRC members, which was not observed for non-targeting control siRNA transfection (n=3 independent biological experiments, all siRNA-treated transcription factor gene expression are significantly different from both control siRNAs with p

    Journal: Nature genetics

    Article Title: Selective gene dependencies in MYCN-amplified neuroblastoma include the core transcriptional regulatory circuitry

    doi: 10.1038/s41588-018-0191-z

    Figure Lengend Snippet: Dependency transcription factors form the core regulatory circuitry in MYCN -amplified neuroblastoma a. Normalized ChIP-seq tracks for H3K27ac, MYCN, and five transcription factors at the HAND2 , ISL1 and PHOX2B loci in BE2C cells; H3K27ac track represents a combination of 2 independent experiments in BE2C and other tracks are representative of an independent experiment performed in BE2C and Kelly cells. ChIP-seq read densities (y-axis) were normalized to reads per million reads sequenced in each sample. SEs are noted as red bars and arrows indicate epicentres. b. Genome-wide co-occupancy for CRC transcription factors as determined by ChIP-seq. Regions (rows) were defined as those enriched in ChIP-seq reads for at least one transcription factor and are ranked by the MYCN signal therein. Color keys for reads-per-million-normalized signal are displayed below each heatmap. c. Quantitative RT-PCR of BE2C cells treated with transient siRNA to each CRC gene - HAND2 , ISL1 , PHOX2B , GATA3 and TBX2 - results in decreased expression of mRNA transcripts for all of the CRC members, which was not observed for non-targeting control siRNA transfection (n=3 independent biological experiments, all siRNA-treated transcription factor gene expression are significantly different from both control siRNAs with p

    Article Snippet: The following antibodies were used for ChIP: GATA3 (Thermo Fisher MA1028), HAND2 (Santa Cruz sc9409X), ISL1 (Developmental Hybridoma Studies Bank 40.3A4), MYCN (Invitrogen Biotechnology, MA-1-16638), PHOX2B (Santa Cruz sc376997X), TBX2 (Abcam ab33298), and H3K27ac (Abcam ab4729).

    Techniques: Amplification, Chromatin Immunoprecipitation, Genome Wide, Quantitative RT-PCR, Expressing, Transfection