Structured Review

Santa Cruz Biotechnology anti islet 1
Green fluorescent protein (GFP) expression detected under a fluorescence microscope. The lentiviral vectors carried the GFP gene; thus, a fluorescence microscope was used to detected GFP expression in the C3H10T1/2 cells transfected with Lenti-N and the C3H10T1/2 cells transfected with <t>Lenti-Islet-1</t> at 3 days after transfection. (A) Cell morphology of the C3H10T1/2 cells transfected with Lenti-N observed under a microscope (magnification, ×20). (B) GFP expression of the C3H10T1/2 cells transfected with Lenti-N observed udner a fluorescence microscope (magnification, ×20). (C) Cell morphology of the C3H10T1/2 cells transfected with Lenti-Islet-1 observed under a microscope (magnification, ×20). (D) Cell morphology of the C3H10T1/2 cells transfected with Lenti-Islet-1 observed under a microscope (magnification, ×20). Scale bar, 20 μm. (E and F) Transfection efficiency of teh C3H10T1/2 cells transfected with Lenti-N and the C3H10T1/2 cells transfected with Lenti-Islet-1 detected by flow cytometry (FCM). The transfection efficiency of the C3H10T1/2 cells transduced with lentiviral vectors with pWPI-GFP plasmid or lentiviral vectors with pWPI-GFP-Islet-1 plasmid was detected by FCM. (E) The transfection efficiency of the C3H10T1/2 cells transfected with Lenti-N was 90.12%. (F) The transfection efficiency of the C3H10T1/2 cells transfected with Lenti-Islet-1 was 88.82%.
Anti Islet 1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 89/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "Islet-1 promotes the cardiac-specific differentiation of mesenchymal stem cells through the regulation of histone acetylation"

Article Title: Islet-1 promotes the cardiac-specific differentiation of mesenchymal stem cells through the regulation of histone acetylation

Journal: International Journal of Molecular Medicine

doi: 10.3892/ijmm.2014.1687

Green fluorescent protein (GFP) expression detected under a fluorescence microscope. The lentiviral vectors carried the GFP gene; thus, a fluorescence microscope was used to detected GFP expression in the C3H10T1/2 cells transfected with Lenti-N and the C3H10T1/2 cells transfected with Lenti-Islet-1 at 3 days after transfection. (A) Cell morphology of the C3H10T1/2 cells transfected with Lenti-N observed under a microscope (magnification, ×20). (B) GFP expression of the C3H10T1/2 cells transfected with Lenti-N observed udner a fluorescence microscope (magnification, ×20). (C) Cell morphology of the C3H10T1/2 cells transfected with Lenti-Islet-1 observed under a microscope (magnification, ×20). (D) Cell morphology of the C3H10T1/2 cells transfected with Lenti-Islet-1 observed under a microscope (magnification, ×20). Scale bar, 20 μm. (E and F) Transfection efficiency of teh C3H10T1/2 cells transfected with Lenti-N and the C3H10T1/2 cells transfected with Lenti-Islet-1 detected by flow cytometry (FCM). The transfection efficiency of the C3H10T1/2 cells transduced with lentiviral vectors with pWPI-GFP plasmid or lentiviral vectors with pWPI-GFP-Islet-1 plasmid was detected by FCM. (E) The transfection efficiency of the C3H10T1/2 cells transfected with Lenti-N was 90.12%. (F) The transfection efficiency of the C3H10T1/2 cells transfected with Lenti-Islet-1 was 88.82%.
Figure Legend Snippet: Green fluorescent protein (GFP) expression detected under a fluorescence microscope. The lentiviral vectors carried the GFP gene; thus, a fluorescence microscope was used to detected GFP expression in the C3H10T1/2 cells transfected with Lenti-N and the C3H10T1/2 cells transfected with Lenti-Islet-1 at 3 days after transfection. (A) Cell morphology of the C3H10T1/2 cells transfected with Lenti-N observed under a microscope (magnification, ×20). (B) GFP expression of the C3H10T1/2 cells transfected with Lenti-N observed udner a fluorescence microscope (magnification, ×20). (C) Cell morphology of the C3H10T1/2 cells transfected with Lenti-Islet-1 observed under a microscope (magnification, ×20). (D) Cell morphology of the C3H10T1/2 cells transfected with Lenti-Islet-1 observed under a microscope (magnification, ×20). Scale bar, 20 μm. (E and F) Transfection efficiency of teh C3H10T1/2 cells transfected with Lenti-N and the C3H10T1/2 cells transfected with Lenti-Islet-1 detected by flow cytometry (FCM). The transfection efficiency of the C3H10T1/2 cells transduced with lentiviral vectors with pWPI-GFP plasmid or lentiviral vectors with pWPI-GFP-Islet-1 plasmid was detected by FCM. (E) The transfection efficiency of the C3H10T1/2 cells transfected with Lenti-N was 90.12%. (F) The transfection efficiency of the C3H10T1/2 cells transfected with Lenti-Islet-1 was 88.82%.

Techniques Used: Expressing, Fluorescence, Microscopy, Transfection, Flow Cytometry, Cytometry, Transduction, Plasmid Preparation

Differences in histone acetylation levels in the untransfected C3H10T1/2 cells, the C3H10T1/2 cells transfected with Lenti-N and the C3H10T1/2 cells transfected with Lenti-Islet-1. (A) Acetylated histone H3 (AcH3) was detected by western blot analysis in the untranstected C3H10T1/2 cells, the C3H10T1/2 cells transfected with Lenti-N and the C3H10T1/2 cells transfected with Lenti-Islet-1. The AcH3 relative amount in the C3H10T1/2 cells transfected with Lenti-Islet-1 was higher than that in the untransfected C3H10T1/2 cells and the C3H10T1/2 cells transfected with Lenti-N ( * P
Figure Legend Snippet: Differences in histone acetylation levels in the untransfected C3H10T1/2 cells, the C3H10T1/2 cells transfected with Lenti-N and the C3H10T1/2 cells transfected with Lenti-Islet-1. (A) Acetylated histone H3 (AcH3) was detected by western blot analysis in the untranstected C3H10T1/2 cells, the C3H10T1/2 cells transfected with Lenti-N and the C3H10T1/2 cells transfected with Lenti-Islet-1. The AcH3 relative amount in the C3H10T1/2 cells transfected with Lenti-Islet-1 was higher than that in the untransfected C3H10T1/2 cells and the C3H10T1/2 cells transfected with Lenti-N ( * P

Techniques Used: Transfection, Western Blot

(A) The expression of Gata4, Nkx2.5 and Mef2c gene at different time points in C3H10T1/2 cells transfected with Lenti-Islet-1. 2d, 2nd day; 1w, 1st week; 2w, 2nd week; 3w, 3rd week. The peak expression of cardiac-specific genes in the C3H10T1/2 cells transfected with Lenti-Islet-1 was significantly higher than that in the untransfected C3H10T1/2 cells and the C3H10T1/2 cells transfected with Lenti-N at 2 weeks after transfection ( * P
Figure Legend Snippet: (A) The expression of Gata4, Nkx2.5 and Mef2c gene at different time points in C3H10T1/2 cells transfected with Lenti-Islet-1. 2d, 2nd day; 1w, 1st week; 2w, 2nd week; 3w, 3rd week. The peak expression of cardiac-specific genes in the C3H10T1/2 cells transfected with Lenti-Islet-1 was significantly higher than that in the untransfected C3H10T1/2 cells and the C3H10T1/2 cells transfected with Lenti-N at 2 weeks after transfection ( * P

Techniques Used: Expressing, Transfection

(A) Islet-1 gene expression in the C3H10T1/2 cells transfected with Lenti-Islet-1 was higher ( * P
Figure Legend Snippet: (A) Islet-1 gene expression in the C3H10T1/2 cells transfected with Lenti-Islet-1 was higher ( * P

Techniques Used: Expressing, Transfection

(A) Detection of lentiviral vector. PCR of random clones. Lane M, DL15000 DNA marker; lanes 1–9, clones that we selected. The 7th lane shows the positive clone. (B and C) Part of the Lenti-Islet-1 plasmid sequencing result. (D) Detection of green fluorescent protein (GFP) expression in 293T cells following transfection with Lenti-Islet-1 vectors (magnification, ×10). Scale bar, 100 μm.
Figure Legend Snippet: (A) Detection of lentiviral vector. PCR of random clones. Lane M, DL15000 DNA marker; lanes 1–9, clones that we selected. The 7th lane shows the positive clone. (B and C) Part of the Lenti-Islet-1 plasmid sequencing result. (D) Detection of green fluorescent protein (GFP) expression in 293T cells following transfection with Lenti-Islet-1 vectors (magnification, ×10). Scale bar, 100 μm.

Techniques Used: Plasmid Preparation, Polymerase Chain Reaction, Clone Assay, Marker, Sequencing, Expressing, Transfection

2) Product Images from "The developmental and genetic basis of ‘clubfoot’ in the peroneal muscular atrophy mutant mouse"

Article Title: The developmental and genetic basis of ‘clubfoot’ in the peroneal muscular atrophy mutant mouse

Journal: Development (Cambridge, England)

doi: 10.1242/dev.160093

Apoptosis and motor neuron survival in lumbar neural tube of pma/pma homozygotes. (A) Immunohistochemistry on cross-sections of lumbar neural tube of E14.5 wild-type (WT; left) and pma/pma (right) foetuses. Islet 1 staining (yellow/magenta) is evident in the dorsal root ganglia and more weakly so in the lateral motor columns of both genotypes. There are rare apoptotic events in both genotypes (TUNEL labelling, green) but quantification was not possible as so few apoptotic cells were detected. (B) Quantification of motor neuron numbers in the neural tube of E16.5 wild-type and pma/pma foetuses. There are significantly fewer surviving motor neurons in pma/pma foetuses specifically at the level of the hindlimb (lumbar) but not more posteriorly (sacral). Error bars represent s.e.m. n/s, not significant. Scale bar: 50 µm.
Figure Legend Snippet: Apoptosis and motor neuron survival in lumbar neural tube of pma/pma homozygotes. (A) Immunohistochemistry on cross-sections of lumbar neural tube of E14.5 wild-type (WT; left) and pma/pma (right) foetuses. Islet 1 staining (yellow/magenta) is evident in the dorsal root ganglia and more weakly so in the lateral motor columns of both genotypes. There are rare apoptotic events in both genotypes (TUNEL labelling, green) but quantification was not possible as so few apoptotic cells were detected. (B) Quantification of motor neuron numbers in the neural tube of E16.5 wild-type and pma/pma foetuses. There are significantly fewer surviving motor neurons in pma/pma foetuses specifically at the level of the hindlimb (lumbar) but not more posteriorly (sacral). Error bars represent s.e.m. n/s, not significant. Scale bar: 50 µm.

Techniques Used: Immunohistochemistry, Staining, TUNEL Assay

Related Articles

Sonication:

Article Title: ISL1 Directly Regulates FGF10 Transcription during Human Cardiac Outflow Formation
Article Snippet: .. An anti-ISL1 (10 µL, Santa Cruz Sc-23590X) or an anti-GFP antibody as negative control (10 µL, Abcam ab1218), were used per 10 µg of sonicated chromatin. .. Immunoprecipitated DNA was analysed by end-point PCR (primers, Supplementary ).

Negative Control:

Article Title: ISL1 Directly Regulates FGF10 Transcription during Human Cardiac Outflow Formation
Article Snippet: .. An anti-ISL1 (10 µL, Santa Cruz Sc-23590X) or an anti-GFP antibody as negative control (10 µL, Abcam ab1218), were used per 10 µg of sonicated chromatin. .. Immunoprecipitated DNA was analysed by end-point PCR (primers, Supplementary ).

Nucleic Acid Electrophoresis:

Article Title: Transduction of Wnt11 Promotes Mesenchymal Stem Cell Transdifferentiation into Cardiac Phenotypes
Article Snippet: .. Denatured proteins (25 and 50 μg) were separated using 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane (Bio-Rad), and immunoblotted overnight at 4°C with a primary antibody: anti-Wnt11 (Abcam), anti-GATA-4 (Abcam), anti-BNP (Abcam), anti-islet-1 (Santa Cruz), or anti-β-actin (Cell Signaling). .. The membranes were then incubated for 1 h with HRP-conjugated secondary antibody at room temperature, washed, and developed with the ECL plus kit (GE Healthcare).

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    Santa Cruz Biotechnology guinea pig anti isl1
    Conditional deletion of Dicer results in the loss of oligodendrocyte progenitors, but normal production of astrocytes and motor neuron progenitors . (A–D) Specification of pMN (Olig2 on ) progenitors and generic motor neurons <t>(Isl1</t> on or Hb9 on ) are not affected in Olig2 Cre/+ ; Dicer flox/flox spinal cord at E11.5. (E) Quantification of ventral pMN and postmitotic motor neurons (MNs; number of positive cells per 15 μm brachial spinal cord hemisection) in WT and conditional KO embryos (mean ± SD) reveals a comparable cell number of pMN progenitors and generic motor neurons ( N = 3 embryos at E11.5). (F,G) Expression of OPC (oligodendrocyte precursors) and astrocyte markers in E16.5 WT and conditional KO spinal cord sections. (F′,G′) Higher magnification images of areas outlined by the dashed rectangles. (H) Quantification of OPC precursors (Sox9 on , Olig2 on ) and astrocyte precursors (Sox9 on , Olig2 off ; number of positive cells per 15 μm brachial spinal cord hemisection) in WT and conditional Dicer KO embryos, mean ± SD reveals a decrease in the number of OPC precursors ( p
    Guinea Pig Anti Isl1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 88/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology mouse anti isl1
    Loss of N-cadherin impairs proliferation and induces premature differentiation of AHF progenitor cells. (A) Representative sagittal section of control and mutant embryos at E9.5 immunostained for <t>Isl1</t> (green) and phospho-Histone 3 (red); arrows point to Isl1/PiH3 double-positive cells in the OFT and AHF (enlarged image). Nuclei are marked by DAPI. Scale bar, 200 μm. (B) Quantification of cell proliferation indicated by mitotic index in the anterior heart field and neural tube regions of wild-type and mutants at E9.5. Error bars indicate SD, n = 4 experiments, ** P = 0.01, evaluated by Student's t -test. (C) Representative transverse sections of control and mutant embryos at E9.0 immunostained for Isl1 (green) and cleaved Caspase 3 (red) in outflow tract (OFT) and neural tube (NT). Nuclei are marked by DAPI. Scale bar, 200 μm. (D) Sagittal sections of control and mutant embryos at E9.0 immunostained for cardiac troponin T (cTnT) (red). Note the presence of ectopic cTnT+ cells (arrows) in the AHF. Nuclei are marked by DAPI. Scale bar, 200 μm. (E) Quantification of cTnT+ cells in the AHF of the control and mutant embryos at E9.0. cTnT+ cells within the AHF, the region between OFT and anterior portion of pharyngeal mesoderm were counted. A total of 7-9 serial sections for each embryo were used. Error bars indicate SD, n = 6 embryos. *** P = 0.001, evaluated by Student's t -test.
    Mouse Anti Isl1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology isl1
    Wnt2+ and Gli1+ cells define a cardiopulmonary progenitor (CPP) and generate mesoderm derivatives of the lung and cardiac inflow tract Wnt2 is expressed in the ventral mesoderm surrounding the anterior foregut and overlapping the posterior pole of the heart (a and b, arrows). Wnt2+ cells tagged at E8.5 can generate cells within the cardiac inflow tract as well as in the lung mesoderm (c and d). Wnt2+ cells tagged at E8.5 generate myocardium of the PV, smooth muscle of the PA, endothelium of the proximal PA, and Pdgfrβ+ pericyte-like cells in the lung by E17.5 (e–h). Gli1+ cells generate derivatives within the inflow tract and early lung mesoderm similar to Wnt2+ cells (i and j) and generate mesoderm lineages within the lung including airway and vascular smooth muscle (k and l), and endothelium of the proximal pulmonary vessels (n) as well as the myocardium of the atria (m). CPPs are located in a region of overlapping Wnt2, Gli1, and <t>Isl1</t> expression between the developing heart and the anterior foregut (o). AP=anterior pole of the heart, PP=posterior pole of the heart, AFG=anterior foregut, AT=atria, OFT=outflow tract, PA=pulmonary artery, PV=pulmonary vein, LB=lung bud, ASM=airway smooth muscle, VSM=vascular smooth muscle, SMA=smooth muscle actin, SAA= sarcomeric α-actinin, VWF=von Willibrand factor. Scale bars b=100 μm, e–h=50 μm, k–n=50 μm.
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    Santa Cruz Biotechnology rabbit anti isl1
    ChIP-seq assays revealed Isl-Lhx3-hexamer-binding sites in a cholinergic gene battery. ( A ) Schematic representation of cholinergic neurotransmission system. Acly, ATP-citrate lyase; CoA, coenzyme A; ChAT, choline acetyltransferase; ACh, acetylcholine; VAChT, vesicular acetylcholine transporter; AChE, Acetylcholine esterase; CHT, high affinity choline transporter; AChRs, Acetylcholine receptors. ( B ) ChIP-seq tag profile of the genomic region surrounding a battery of cholinergic genes ChAT/VAChT , CHT , and Acly loci. Each cholinergic gene is indicated, and the blue arrows represent the direction of transcription. Mam cons., mammalian conservation. The ChIP-seq data was deposited in the GEO database (assession no. GSE50993) [20] . ( C ) Schematic representation of the location of the HxRE motifs in each of the 500 bp-long cholinergic gene peaks. The number shows the relative position within the peak (0, the center position of each peak). ( D ) In vivo ChIP assays in dissected E12.5 embryonic spinal cords to monitor the binding of the <t>Isl1-Lhx3-hexamer</t> to the cholinergic enhancers. Schematic representation of the ChAT gene is shown on the top. The arrows indicate two sets of primers detecting ChAT -enhancer ( ChAT -enh) and a negative control region lacking the Isl1-Lhx3-binding peak ( ChAT -neg). Isl1, Lhx3, and NLI were recruited to the cholinergic enhancers in embryonic spinal cords. Error bars indicate standard deviation.
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    Conditional deletion of Dicer results in the loss of oligodendrocyte progenitors, but normal production of astrocytes and motor neuron progenitors . (A–D) Specification of pMN (Olig2 on ) progenitors and generic motor neurons (Isl1 on or Hb9 on ) are not affected in Olig2 Cre/+ ; Dicer flox/flox spinal cord at E11.5. (E) Quantification of ventral pMN and postmitotic motor neurons (MNs; number of positive cells per 15 μm brachial spinal cord hemisection) in WT and conditional KO embryos (mean ± SD) reveals a comparable cell number of pMN progenitors and generic motor neurons ( N = 3 embryos at E11.5). (F,G) Expression of OPC (oligodendrocyte precursors) and astrocyte markers in E16.5 WT and conditional KO spinal cord sections. (F′,G′) Higher magnification images of areas outlined by the dashed rectangles. (H) Quantification of OPC precursors (Sox9 on , Olig2 on ) and astrocyte precursors (Sox9 on , Olig2 off ; number of positive cells per 15 μm brachial spinal cord hemisection) in WT and conditional Dicer KO embryos, mean ± SD reveals a decrease in the number of OPC precursors ( p

    Journal: Frontiers in Neuroscience

    Article Title: Apoptosis of Limb Innervating Motor Neurons and Erosion of Motor Pool Identity Upon Lineage Specific Dicer Inactivation

    doi: 10.3389/fnins.2012.00069

    Figure Lengend Snippet: Conditional deletion of Dicer results in the loss of oligodendrocyte progenitors, but normal production of astrocytes and motor neuron progenitors . (A–D) Specification of pMN (Olig2 on ) progenitors and generic motor neurons (Isl1 on or Hb9 on ) are not affected in Olig2 Cre/+ ; Dicer flox/flox spinal cord at E11.5. (E) Quantification of ventral pMN and postmitotic motor neurons (MNs; number of positive cells per 15 μm brachial spinal cord hemisection) in WT and conditional KO embryos (mean ± SD) reveals a comparable cell number of pMN progenitors and generic motor neurons ( N = 3 embryos at E11.5). (F,G) Expression of OPC (oligodendrocyte precursors) and astrocyte markers in E16.5 WT and conditional KO spinal cord sections. (F′,G′) Higher magnification images of areas outlined by the dashed rectangles. (H) Quantification of OPC precursors (Sox9 on , Olig2 on ) and astrocyte precursors (Sox9 on , Olig2 off ; number of positive cells per 15 μm brachial spinal cord hemisection) in WT and conditional Dicer KO embryos, mean ± SD reveals a decrease in the number of OPC precursors ( p

    Article Snippet: Immunostaining and antibodies Antibodies used in this study include: rabbit anti-mouse Olig2 (Millipore, AB9610), guinea pig anti-mouse Olig2, mouse anti-Lhx3 and Hb9, guinea pig anti-Isl1, FoxP1, and SCIP (gift from Tom Jessell), rabbit anti-Sox9 and GFAP, rabbit anti-pSmad (gift from Ed Laufer), mouse anti-Hoxc8 (gift from Tom Jessell), goat anti-Hoxc6 (Santa Cruz, sc-66925), and Alexa488-, FITC-, Cy3-, and Cy5-conjugated secondary antibodies were obtained from either Invitrogen or Jackson Immunoresearch.

    Techniques: Expressing

    Increased apoptosis is detected in E14.5 Dicer mutant LMC . (A–F) TUNEL labeling of fragmented apoptotic nuclei in the developing spinal cord at E11.5, 12.5, and E14.5. While the number of TUNEL positive cells in the ventral spinal cord was overall low, we observed consistently higher number of TUNEL positive nuclei within the LMC (Isl1 positive) of conditional Dicer KO embryos on day E14.5 (E,F) .

    Journal: Frontiers in Neuroscience

    Article Title: Apoptosis of Limb Innervating Motor Neurons and Erosion of Motor Pool Identity Upon Lineage Specific Dicer Inactivation

    doi: 10.3389/fnins.2012.00069

    Figure Lengend Snippet: Increased apoptosis is detected in E14.5 Dicer mutant LMC . (A–F) TUNEL labeling of fragmented apoptotic nuclei in the developing spinal cord at E11.5, 12.5, and E14.5. While the number of TUNEL positive cells in the ventral spinal cord was overall low, we observed consistently higher number of TUNEL positive nuclei within the LMC (Isl1 positive) of conditional Dicer KO embryos on day E14.5 (E,F) .

    Article Snippet: Immunostaining and antibodies Antibodies used in this study include: rabbit anti-mouse Olig2 (Millipore, AB9610), guinea pig anti-mouse Olig2, mouse anti-Lhx3 and Hb9, guinea pig anti-Isl1, FoxP1, and SCIP (gift from Tom Jessell), rabbit anti-Sox9 and GFAP, rabbit anti-pSmad (gift from Ed Laufer), mouse anti-Hoxc8 (gift from Tom Jessell), goat anti-Hoxc6 (Santa Cruz, sc-66925), and Alexa488-, FITC-, Cy3-, and Cy5-conjugated secondary antibodies were obtained from either Invitrogen or Jackson Immunoresearch.

    Techniques: Mutagenesis, TUNEL Assay, Labeling

    Efficient and specific disruption miRNA biosynthesis in Olig2 Cre/+ ; Dicer flox/ flox animals . (A) Schematic illustration of Olig2-Crelineage tracing (YFP on cells) in Olig2 Cre/+ ; ROSA26-loxp-STOP-loxp-YFP E13.5 spinal cord sections. (B,C) Cre mediated recombination in Olig2 expressing progenitors results in YFP expression in all cells derived from this progenitor domain. YFP is expressed in all postmitotic motor neurons in the ventral horn of the spinal cord (YFP on and Isl1 on ), but not in dorsal dl3 interneurons (YFP off and Isl1 on ). In addition, nearly all Olig2 + oligodendrocyte precursors express the lineage tracer YFP. (D) Cross between Olig2 Cre/+ ; Dicer flox/ WT ; and Dicer flox/ flox female mouse to get control wild type (WT) and conditional Dicer knock out (conditional KO) embryos. (E–H) Spinal cord sections from E13.5 WT and conditional KO embryos examined for miR-100 and mir-9 expression by in situ hybridization. Ventral horn is marked by the dashed square.

    Journal: Frontiers in Neuroscience

    Article Title: Apoptosis of Limb Innervating Motor Neurons and Erosion of Motor Pool Identity Upon Lineage Specific Dicer Inactivation

    doi: 10.3389/fnins.2012.00069

    Figure Lengend Snippet: Efficient and specific disruption miRNA biosynthesis in Olig2 Cre/+ ; Dicer flox/ flox animals . (A) Schematic illustration of Olig2-Crelineage tracing (YFP on cells) in Olig2 Cre/+ ; ROSA26-loxp-STOP-loxp-YFP E13.5 spinal cord sections. (B,C) Cre mediated recombination in Olig2 expressing progenitors results in YFP expression in all cells derived from this progenitor domain. YFP is expressed in all postmitotic motor neurons in the ventral horn of the spinal cord (YFP on and Isl1 on ), but not in dorsal dl3 interneurons (YFP off and Isl1 on ). In addition, nearly all Olig2 + oligodendrocyte precursors express the lineage tracer YFP. (D) Cross between Olig2 Cre/+ ; Dicer flox/ WT ; and Dicer flox/ flox female mouse to get control wild type (WT) and conditional Dicer knock out (conditional KO) embryos. (E–H) Spinal cord sections from E13.5 WT and conditional KO embryos examined for miR-100 and mir-9 expression by in situ hybridization. Ventral horn is marked by the dashed square.

    Article Snippet: Immunostaining and antibodies Antibodies used in this study include: rabbit anti-mouse Olig2 (Millipore, AB9610), guinea pig anti-mouse Olig2, mouse anti-Lhx3 and Hb9, guinea pig anti-Isl1, FoxP1, and SCIP (gift from Tom Jessell), rabbit anti-Sox9 and GFAP, rabbit anti-pSmad (gift from Ed Laufer), mouse anti-Hoxc8 (gift from Tom Jessell), goat anti-Hoxc6 (Santa Cruz, sc-66925), and Alexa488-, FITC-, Cy3-, and Cy5-conjugated secondary antibodies were obtained from either Invitrogen or Jackson Immunoresearch.

    Techniques: Expressing, Derivative Assay, Knock-Out, In Situ Hybridization

    Loss of N-cadherin impairs proliferation and induces premature differentiation of AHF progenitor cells. (A) Representative sagittal section of control and mutant embryos at E9.5 immunostained for Isl1 (green) and phospho-Histone 3 (red); arrows point to Isl1/PiH3 double-positive cells in the OFT and AHF (enlarged image). Nuclei are marked by DAPI. Scale bar, 200 μm. (B) Quantification of cell proliferation indicated by mitotic index in the anterior heart field and neural tube regions of wild-type and mutants at E9.5. Error bars indicate SD, n = 4 experiments, ** P = 0.01, evaluated by Student's t -test. (C) Representative transverse sections of control and mutant embryos at E9.0 immunostained for Isl1 (green) and cleaved Caspase 3 (red) in outflow tract (OFT) and neural tube (NT). Nuclei are marked by DAPI. Scale bar, 200 μm. (D) Sagittal sections of control and mutant embryos at E9.0 immunostained for cardiac troponin T (cTnT) (red). Note the presence of ectopic cTnT+ cells (arrows) in the AHF. Nuclei are marked by DAPI. Scale bar, 200 μm. (E) Quantification of cTnT+ cells in the AHF of the control and mutant embryos at E9.0. cTnT+ cells within the AHF, the region between OFT and anterior portion of pharyngeal mesoderm were counted. A total of 7-9 serial sections for each embryo were used. Error bars indicate SD, n = 6 embryos. *** P = 0.001, evaluated by Student's t -test.

    Journal: Cell Research

    Article Title: N-cadherin prevents the premature differentiation of anterior heart field progenitors in the pharyngeal mesodermal microenvironment

    doi: 10.1038/cr.2014.142

    Figure Lengend Snippet: Loss of N-cadherin impairs proliferation and induces premature differentiation of AHF progenitor cells. (A) Representative sagittal section of control and mutant embryos at E9.5 immunostained for Isl1 (green) and phospho-Histone 3 (red); arrows point to Isl1/PiH3 double-positive cells in the OFT and AHF (enlarged image). Nuclei are marked by DAPI. Scale bar, 200 μm. (B) Quantification of cell proliferation indicated by mitotic index in the anterior heart field and neural tube regions of wild-type and mutants at E9.5. Error bars indicate SD, n = 4 experiments, ** P = 0.01, evaluated by Student's t -test. (C) Representative transverse sections of control and mutant embryos at E9.0 immunostained for Isl1 (green) and cleaved Caspase 3 (red) in outflow tract (OFT) and neural tube (NT). Nuclei are marked by DAPI. Scale bar, 200 μm. (D) Sagittal sections of control and mutant embryos at E9.0 immunostained for cardiac troponin T (cTnT) (red). Note the presence of ectopic cTnT+ cells (arrows) in the AHF. Nuclei are marked by DAPI. Scale bar, 200 μm. (E) Quantification of cTnT+ cells in the AHF of the control and mutant embryos at E9.0. cTnT+ cells within the AHF, the region between OFT and anterior portion of pharyngeal mesoderm were counted. A total of 7-9 serial sections for each embryo were used. Error bars indicate SD, n = 6 embryos. *** P = 0.001, evaluated by Student's t -test.

    Article Snippet: The primary antibodies used were rabbit anti-N-cadherin (1:100; Millipore 1126), mouse anti-Isl1 (1:200; Hybrodoma Bank 39.4D5), mouse anti-PY654-β-catenin (membrane bound) (Hybridoma bank), mouse anti-PY489-β-catenin (nuclear) (Hybridoma bank), rat anti-E-cadherin (1:100; Santa Cruz sc-59778) and mouse anti-cardiac troponin T (1:400; Abcam ab8295).

    Techniques: Mutagenesis

    Wnt2+ and Gli1+ cells define a cardiopulmonary progenitor (CPP) and generate mesoderm derivatives of the lung and cardiac inflow tract Wnt2 is expressed in the ventral mesoderm surrounding the anterior foregut and overlapping the posterior pole of the heart (a and b, arrows). Wnt2+ cells tagged at E8.5 can generate cells within the cardiac inflow tract as well as in the lung mesoderm (c and d). Wnt2+ cells tagged at E8.5 generate myocardium of the PV, smooth muscle of the PA, endothelium of the proximal PA, and Pdgfrβ+ pericyte-like cells in the lung by E17.5 (e–h). Gli1+ cells generate derivatives within the inflow tract and early lung mesoderm similar to Wnt2+ cells (i and j) and generate mesoderm lineages within the lung including airway and vascular smooth muscle (k and l), and endothelium of the proximal pulmonary vessels (n) as well as the myocardium of the atria (m). CPPs are located in a region of overlapping Wnt2, Gli1, and Isl1 expression between the developing heart and the anterior foregut (o). AP=anterior pole of the heart, PP=posterior pole of the heart, AFG=anterior foregut, AT=atria, OFT=outflow tract, PA=pulmonary artery, PV=pulmonary vein, LB=lung bud, ASM=airway smooth muscle, VSM=vascular smooth muscle, SMA=smooth muscle actin, SAA= sarcomeric α-actinin, VWF=von Willibrand factor. Scale bars b=100 μm, e–h=50 μm, k–n=50 μm.

    Journal: Nature

    Article Title: Coordination of heart and lung co-development by a multipotent cardiopulmonary progenitor

    doi: 10.1038/nature12358

    Figure Lengend Snippet: Wnt2+ and Gli1+ cells define a cardiopulmonary progenitor (CPP) and generate mesoderm derivatives of the lung and cardiac inflow tract Wnt2 is expressed in the ventral mesoderm surrounding the anterior foregut and overlapping the posterior pole of the heart (a and b, arrows). Wnt2+ cells tagged at E8.5 can generate cells within the cardiac inflow tract as well as in the lung mesoderm (c and d). Wnt2+ cells tagged at E8.5 generate myocardium of the PV, smooth muscle of the PA, endothelium of the proximal PA, and Pdgfrβ+ pericyte-like cells in the lung by E17.5 (e–h). Gli1+ cells generate derivatives within the inflow tract and early lung mesoderm similar to Wnt2+ cells (i and j) and generate mesoderm lineages within the lung including airway and vascular smooth muscle (k and l), and endothelium of the proximal pulmonary vessels (n) as well as the myocardium of the atria (m). CPPs are located in a region of overlapping Wnt2, Gli1, and Isl1 expression between the developing heart and the anterior foregut (o). AP=anterior pole of the heart, PP=posterior pole of the heart, AFG=anterior foregut, AT=atria, OFT=outflow tract, PA=pulmonary artery, PV=pulmonary vein, LB=lung bud, ASM=airway smooth muscle, VSM=vascular smooth muscle, SMA=smooth muscle actin, SAA= sarcomeric α-actinin, VWF=von Willibrand factor. Scale bars b=100 μm, e–h=50 μm, k–n=50 μm.

    Article Snippet: Antibodies used are anti-smooth muscle actin (mouse anti-SMA 1:200 Abcam), CD31 (rat anti-CD31 1:500 BD Pharmingen), Von Willibrand Factor (rabbit anti-VWF 1:200 Sigma), MF20 (mouse anti-MF20 1:20 Abcam), Isl1 (mouse anti-Isl1 1:10 HybridomaBank), Nkx2.5 (goat anti-Nkx2.5 1:10 Santa Cruz), Sarcomeric α-actinin (mouse anti-SAA 1:100 Sigma), NG2 (rabbit anti-NG2 1:100 Millipore), and GFP (goat anti-GFP 1:100 Abcam).

    Techniques: Conditioned Place Preference, Expressing

    The pulmonary vasculature develops in the absence of lung specification The paired PAs are observed tracking in a posterior direction in both the control (a–c, arrowheads) and Shh cre :Ctnnb1 flox/flox mutants (f–h, arrowheads) at E10.5 using CD31 whole mount immunostaining and section immunostaining (d and i, arrows). The primitive PVs are observed emerging from the atria and extending towards the region of the foregut where the lung would normally form in both control and Shh cre :Ctnnb1 flox/flox mutants (c and h, arrows). These developmental hallmarks are illustrated in models (e and j). Isl1+ progenitors tagged at E8.5 give rise to the PA and airway smooth muscle (k), endothelial (l), myocardial (m), and Pdgfrβ+ pericyte-like (n) lineages of the pulmonary vasculature and other mesodermal derivatives. This overlapping but distinct origin of lung mesoderm derivatives is diagrammed in (o). PA=pulmonary artery, PV=pulmonary vein, AT=atria, AFG=anterior foregut. OFT=outflow tract, L=lung, T=trachea, E=esophagus. Control for a–h= Shh cre :Ctnnb1 flox/+ . Scale bar (g and h)=100 μm.

    Journal: Nature

    Article Title: Coordination of heart and lung co-development by a multipotent cardiopulmonary progenitor

    doi: 10.1038/nature12358

    Figure Lengend Snippet: The pulmonary vasculature develops in the absence of lung specification The paired PAs are observed tracking in a posterior direction in both the control (a–c, arrowheads) and Shh cre :Ctnnb1 flox/flox mutants (f–h, arrowheads) at E10.5 using CD31 whole mount immunostaining and section immunostaining (d and i, arrows). The primitive PVs are observed emerging from the atria and extending towards the region of the foregut where the lung would normally form in both control and Shh cre :Ctnnb1 flox/flox mutants (c and h, arrows). These developmental hallmarks are illustrated in models (e and j). Isl1+ progenitors tagged at E8.5 give rise to the PA and airway smooth muscle (k), endothelial (l), myocardial (m), and Pdgfrβ+ pericyte-like (n) lineages of the pulmonary vasculature and other mesodermal derivatives. This overlapping but distinct origin of lung mesoderm derivatives is diagrammed in (o). PA=pulmonary artery, PV=pulmonary vein, AT=atria, AFG=anterior foregut. OFT=outflow tract, L=lung, T=trachea, E=esophagus. Control for a–h= Shh cre :Ctnnb1 flox/+ . Scale bar (g and h)=100 μm.

    Article Snippet: Antibodies used are anti-smooth muscle actin (mouse anti-SMA 1:200 Abcam), CD31 (rat anti-CD31 1:500 BD Pharmingen), Von Willibrand Factor (rabbit anti-VWF 1:200 Sigma), MF20 (mouse anti-MF20 1:20 Abcam), Isl1 (mouse anti-Isl1 1:10 HybridomaBank), Nkx2.5 (goat anti-Nkx2.5 1:10 Santa Cruz), Sarcomeric α-actinin (mouse anti-SAA 1:100 Sigma), NG2 (rabbit anti-NG2 1:100 Millipore), and GFP (goat anti-GFP 1:100 Abcam).

    Techniques: Immunostaining

    Hedgehog signaling is required in CPPs to coordinate the vascular connection between the heart and lung Shh expression at E9.5 in the anterior foregut endoderm by in situ hybridization (a). Shh−/− mutants and Isl1 cre :Smo flox/flox mutants display a disrupted vascular plexus between the developing heart and lung at E10.5 (b–d, brackets, control= Shh +/− ). Inactivation of Smo within Isl1+ CPPs inhibits their contribution to airway and vascular smooth muscle at E13.5 (e–h, k, control= Isl1 cre :Smo flox/+ :R26R mTmG ). Inactivation of Smo within Gli1+ CPPs inhibits their contribution to airway and vascular smooth muscle at E13.5 (i, j, l, control= Gli1 creERT2 :Smo flox/+ :R26R mTmG ). CPPs orchestrate lung and cardiac co-development and are regulated in turn by Shh expression from the anterior foregut endoderm (m–p). AT=atria, AFG=anterior foregut, OFT=outflow tract, LB=lung bud, PA=pulmonary artery, Ao=aorta, PV=pulmonary vein, SV=sinus venosus, SVC and IVC=superior and inferior vena cava. *p

    Journal: Nature

    Article Title: Coordination of heart and lung co-development by a multipotent cardiopulmonary progenitor

    doi: 10.1038/nature12358

    Figure Lengend Snippet: Hedgehog signaling is required in CPPs to coordinate the vascular connection between the heart and lung Shh expression at E9.5 in the anterior foregut endoderm by in situ hybridization (a). Shh−/− mutants and Isl1 cre :Smo flox/flox mutants display a disrupted vascular plexus between the developing heart and lung at E10.5 (b–d, brackets, control= Shh +/− ). Inactivation of Smo within Isl1+ CPPs inhibits their contribution to airway and vascular smooth muscle at E13.5 (e–h, k, control= Isl1 cre :Smo flox/+ :R26R mTmG ). Inactivation of Smo within Gli1+ CPPs inhibits their contribution to airway and vascular smooth muscle at E13.5 (i, j, l, control= Gli1 creERT2 :Smo flox/+ :R26R mTmG ). CPPs orchestrate lung and cardiac co-development and are regulated in turn by Shh expression from the anterior foregut endoderm (m–p). AT=atria, AFG=anterior foregut, OFT=outflow tract, LB=lung bud, PA=pulmonary artery, Ao=aorta, PV=pulmonary vein, SV=sinus venosus, SVC and IVC=superior and inferior vena cava. *p

    Article Snippet: Antibodies used are anti-smooth muscle actin (mouse anti-SMA 1:200 Abcam), CD31 (rat anti-CD31 1:500 BD Pharmingen), Von Willibrand Factor (rabbit anti-VWF 1:200 Sigma), MF20 (mouse anti-MF20 1:20 Abcam), Isl1 (mouse anti-Isl1 1:10 HybridomaBank), Nkx2.5 (goat anti-Nkx2.5 1:10 Santa Cruz), Sarcomeric α-actinin (mouse anti-SAA 1:100 Sigma), NG2 (rabbit anti-NG2 1:100 Millipore), and GFP (goat anti-GFP 1:100 Abcam).

    Techniques: Expressing, In Situ Hybridization

    ChIP-seq assays revealed Isl-Lhx3-hexamer-binding sites in a cholinergic gene battery. ( A ) Schematic representation of cholinergic neurotransmission system. Acly, ATP-citrate lyase; CoA, coenzyme A; ChAT, choline acetyltransferase; ACh, acetylcholine; VAChT, vesicular acetylcholine transporter; AChE, Acetylcholine esterase; CHT, high affinity choline transporter; AChRs, Acetylcholine receptors. ( B ) ChIP-seq tag profile of the genomic region surrounding a battery of cholinergic genes ChAT/VAChT , CHT , and Acly loci. Each cholinergic gene is indicated, and the blue arrows represent the direction of transcription. Mam cons., mammalian conservation. The ChIP-seq data was deposited in the GEO database (assession no. GSE50993) [20] . ( C ) Schematic representation of the location of the HxRE motifs in each of the 500 bp-long cholinergic gene peaks. The number shows the relative position within the peak (0, the center position of each peak). ( D ) In vivo ChIP assays in dissected E12.5 embryonic spinal cords to monitor the binding of the Isl1-Lhx3-hexamer to the cholinergic enhancers. Schematic representation of the ChAT gene is shown on the top. The arrows indicate two sets of primers detecting ChAT -enhancer ( ChAT -enh) and a negative control region lacking the Isl1-Lhx3-binding peak ( ChAT -neg). Isl1, Lhx3, and NLI were recruited to the cholinergic enhancers in embryonic spinal cords. Error bars indicate standard deviation.

    Journal: PLoS Genetics

    Article Title: Isl1 Directly Controls a Cholinergic Neuronal Identity in the Developing Forebrain and Spinal Cord by Forming Cell Type-Specific Complexes

    doi: 10.1371/journal.pgen.1004280

    Figure Lengend Snippet: ChIP-seq assays revealed Isl-Lhx3-hexamer-binding sites in a cholinergic gene battery. ( A ) Schematic representation of cholinergic neurotransmission system. Acly, ATP-citrate lyase; CoA, coenzyme A; ChAT, choline acetyltransferase; ACh, acetylcholine; VAChT, vesicular acetylcholine transporter; AChE, Acetylcholine esterase; CHT, high affinity choline transporter; AChRs, Acetylcholine receptors. ( B ) ChIP-seq tag profile of the genomic region surrounding a battery of cholinergic genes ChAT/VAChT , CHT , and Acly loci. Each cholinergic gene is indicated, and the blue arrows represent the direction of transcription. Mam cons., mammalian conservation. The ChIP-seq data was deposited in the GEO database (assession no. GSE50993) [20] . ( C ) Schematic representation of the location of the HxRE motifs in each of the 500 bp-long cholinergic gene peaks. The number shows the relative position within the peak (0, the center position of each peak). ( D ) In vivo ChIP assays in dissected E12.5 embryonic spinal cords to monitor the binding of the Isl1-Lhx3-hexamer to the cholinergic enhancers. Schematic representation of the ChAT gene is shown on the top. The arrows indicate two sets of primers detecting ChAT -enhancer ( ChAT -enh) and a negative control region lacking the Isl1-Lhx3-binding peak ( ChAT -neg). Isl1, Lhx3, and NLI were recruited to the cholinergic enhancers in embryonic spinal cords. Error bars indicate standard deviation.

    Article Snippet: ChAT -enhancer forward 5′-TAC TAA TTG GAT TAA TTG ATT TGC reverse 5′-GGG AAT TAA TAA CTT AGA ATT TGA ChAT -negative forward 5′- CTG TGG CTC ATA ACG CTC ATT TTG reverse 5′- AGT TTG TGG TGG GCC GAG ATG GCA Acly -enh1 forward 5′- TGA TAG CAC ACT ACT TTG CTC TGG reverse 5′-CAG TGA CGC ACG GCG AGC GGG AAG CHT -enhancer forward 5′-TGA GCA GCC TAT GCC ACA AGG ACA reverse 5′- CAT TAG GAG AGC TTG TTC CAG TGA The following antibodies were used for ChIP-PCR; mouse/rabbit IgG (Santa Cruz), rabbit anti-Isl1 , rabbit anti-Lhx3 , rabbit anti-NLI , and goat anti-Lhx8 (sc-22216, Santa Cruz).

    Techniques: Chromatin Immunoprecipitation, Binding Assay, In Vivo, Negative Control, Standard Deviation

    The formation of the Isl1-Lhx8-hexamer complex. ( A ) Schematic representation of the Isl1-Lhx8-hexamer consisting of Isl1, Lhx8 and NLI. The model depicts that the Isl1-Lhx8-complex regulates the cholinergic genes via binding to HxREs. ( B ) In vitro GST-pull down assays. Lhx8 and Lhx3 bind to both Isl1 and NLI with high affinity, whereas Lhx1 binds to only NLI, but not to Isl1. ( C ) GST-pull down assays in HEK293 cells transfected with Flag- and GST-tagged constructs as indicated above. Lhx8 interacts with both Isl1 and NLI in cells. ( D ) CoIP assays in HEK293 cells transfected with Flag-Lhx8 and HA-tagged NLI DD -Isl1 ΔLIM . Lhx8 interact with NLI DD -Isl1 ΔLIM , forming the FCN-hexamer-mimicking complex. ( E, F ) The SELEX methods revealed the high affinity binding sites for Isl1-Lhx8 fusion (E-value, 2.5e-79) and the mixture of Isl1 and Lhx8 (E-value, 2.8e-65). The bottom sequence logo shows reverse complementary sequences of the upper logo.

    Journal: PLoS Genetics

    Article Title: Isl1 Directly Controls a Cholinergic Neuronal Identity in the Developing Forebrain and Spinal Cord by Forming Cell Type-Specific Complexes

    doi: 10.1371/journal.pgen.1004280

    Figure Lengend Snippet: The formation of the Isl1-Lhx8-hexamer complex. ( A ) Schematic representation of the Isl1-Lhx8-hexamer consisting of Isl1, Lhx8 and NLI. The model depicts that the Isl1-Lhx8-complex regulates the cholinergic genes via binding to HxREs. ( B ) In vitro GST-pull down assays. Lhx8 and Lhx3 bind to both Isl1 and NLI with high affinity, whereas Lhx1 binds to only NLI, but not to Isl1. ( C ) GST-pull down assays in HEK293 cells transfected with Flag- and GST-tagged constructs as indicated above. Lhx8 interacts with both Isl1 and NLI in cells. ( D ) CoIP assays in HEK293 cells transfected with Flag-Lhx8 and HA-tagged NLI DD -Isl1 ΔLIM . Lhx8 interact with NLI DD -Isl1 ΔLIM , forming the FCN-hexamer-mimicking complex. ( E, F ) The SELEX methods revealed the high affinity binding sites for Isl1-Lhx8 fusion (E-value, 2.5e-79) and the mixture of Isl1 and Lhx8 (E-value, 2.8e-65). The bottom sequence logo shows reverse complementary sequences of the upper logo.

    Article Snippet: ChAT -enhancer forward 5′-TAC TAA TTG GAT TAA TTG ATT TGC reverse 5′-GGG AAT TAA TAA CTT AGA ATT TGA ChAT -negative forward 5′- CTG TGG CTC ATA ACG CTC ATT TTG reverse 5′- AGT TTG TGG TGG GCC GAG ATG GCA Acly -enh1 forward 5′- TGA TAG CAC ACT ACT TTG CTC TGG reverse 5′-CAG TGA CGC ACG GCG AGC GGG AAG CHT -enhancer forward 5′-TGA GCA GCC TAT GCC ACA AGG ACA reverse 5′- CAT TAG GAG AGC TTG TTC CAG TGA The following antibodies were used for ChIP-PCR; mouse/rabbit IgG (Santa Cruz), rabbit anti-Isl1 , rabbit anti-Lhx3 , rabbit anti-NLI , and goat anti-Lhx8 (sc-22216, Santa Cruz).

    Techniques: Binding Assay, In Vitro, Transfection, Construct, Co-Immunoprecipitation Assay, Sequencing

    Isl1 is required for the formation of striatal cholinergic interneurons in the developing forebrain. Immunohistochemical analyses on the CPu (A, B) and BMC (C, D) of E16.5 Isl1 f/f ;Nkx2.1Cre and littermate control embryos. VAChT + cholinergic interneurons in the CPu failed to form in the MGE-specific Isl1 -null embryos. (E, F) Quantification of the number of Lhx8 + VAChT + (E) or Nkx2.1 + VAChT + cells in the CPu of E16.5 control and Isl1 mutant embryos. Histogram shows average ± standard deviation. ** p

    Journal: PLoS Genetics

    Article Title: Isl1 Directly Controls a Cholinergic Neuronal Identity in the Developing Forebrain and Spinal Cord by Forming Cell Type-Specific Complexes

    doi: 10.1371/journal.pgen.1004280

    Figure Lengend Snippet: Isl1 is required for the formation of striatal cholinergic interneurons in the developing forebrain. Immunohistochemical analyses on the CPu (A, B) and BMC (C, D) of E16.5 Isl1 f/f ;Nkx2.1Cre and littermate control embryos. VAChT + cholinergic interneurons in the CPu failed to form in the MGE-specific Isl1 -null embryos. (E, F) Quantification of the number of Lhx8 + VAChT + (E) or Nkx2.1 + VAChT + cells in the CPu of E16.5 control and Isl1 mutant embryos. Histogram shows average ± standard deviation. ** p

    Article Snippet: ChAT -enhancer forward 5′-TAC TAA TTG GAT TAA TTG ATT TGC reverse 5′-GGG AAT TAA TAA CTT AGA ATT TGA ChAT -negative forward 5′- CTG TGG CTC ATA ACG CTC ATT TTG reverse 5′- AGT TTG TGG TGG GCC GAG ATG GCA Acly -enh1 forward 5′- TGA TAG CAC ACT ACT TTG CTC TGG reverse 5′-CAG TGA CGC ACG GCG AGC GGG AAG CHT -enhancer forward 5′-TGA GCA GCC TAT GCC ACA AGG ACA reverse 5′- CAT TAG GAG AGC TTG TTC CAG TGA The following antibodies were used for ChIP-PCR; mouse/rabbit IgG (Santa Cruz), rabbit anti-Isl1 , rabbit anti-Lhx3 , rabbit anti-NLI , and goat anti-Lhx8 (sc-22216, Santa Cruz).

    Techniques: Immunohistochemistry, Mutagenesis, Standard Deviation

    Isl1-Lhx8 induces a cholinergic fate in ESC-derived neurons. ( A, B ) In Isl1-Lhx8-ESCs, the expression of Flag-tagged Isl1-Lhx8 was induced by Dox, as detected by western blotting assays with α-Flag antibodies (A) and immunohistochemistry assays with α-Isl1 and α-Lhx8 antibodies (B). ( C, D ) Quantitative RT-PCR analyses in Isl1-Lhx8-ESCs when cultured as a monolayer. Cholinergic genes, but not the MN gene Hb9, were induced by Isl1-Lhx8. ( E–G ) Cell differentiation analyses in floating EBs derived from Isl1-Lhx8-ESCs, cultured with or without Dox, which triggers expression of Isl1-Lhx8. Immunohistochemical analyses show that Isl-Lhx8 expression induces differentiation of VAChT + TuJ + c holinergic neurons (E, F). Quantitative RT-PCR analyses show that cholinergic pathway genes, but not MN genes Hb9 and Isl2, were induced by Isl1-Lhx8 (G). ( H ) Quantitative RT-PCR analyses of Hb9 expression in Isl1-Lhx3-ESCs. Hb9 was induced by Dox treatment, which induces the expression of Isl1-Lhx3 in Isl1-Lhx3-ESCs, when cultured in either monolayer (M) or spinal neuronal differentiation (SN) conditions. Error bars represent the standard deviation in all graphs (C, D, G, H).

    Journal: PLoS Genetics

    Article Title: Isl1 Directly Controls a Cholinergic Neuronal Identity in the Developing Forebrain and Spinal Cord by Forming Cell Type-Specific Complexes

    doi: 10.1371/journal.pgen.1004280

    Figure Lengend Snippet: Isl1-Lhx8 induces a cholinergic fate in ESC-derived neurons. ( A, B ) In Isl1-Lhx8-ESCs, the expression of Flag-tagged Isl1-Lhx8 was induced by Dox, as detected by western blotting assays with α-Flag antibodies (A) and immunohistochemistry assays with α-Isl1 and α-Lhx8 antibodies (B). ( C, D ) Quantitative RT-PCR analyses in Isl1-Lhx8-ESCs when cultured as a monolayer. Cholinergic genes, but not the MN gene Hb9, were induced by Isl1-Lhx8. ( E–G ) Cell differentiation analyses in floating EBs derived from Isl1-Lhx8-ESCs, cultured with or without Dox, which triggers expression of Isl1-Lhx8. Immunohistochemical analyses show that Isl-Lhx8 expression induces differentiation of VAChT + TuJ + c holinergic neurons (E, F). Quantitative RT-PCR analyses show that cholinergic pathway genes, but not MN genes Hb9 and Isl2, were induced by Isl1-Lhx8 (G). ( H ) Quantitative RT-PCR analyses of Hb9 expression in Isl1-Lhx3-ESCs. Hb9 was induced by Dox treatment, which induces the expression of Isl1-Lhx3 in Isl1-Lhx3-ESCs, when cultured in either monolayer (M) or spinal neuronal differentiation (SN) conditions. Error bars represent the standard deviation in all graphs (C, D, G, H).

    Article Snippet: ChAT -enhancer forward 5′-TAC TAA TTG GAT TAA TTG ATT TGC reverse 5′-GGG AAT TAA TAA CTT AGA ATT TGA ChAT -negative forward 5′- CTG TGG CTC ATA ACG CTC ATT TTG reverse 5′- AGT TTG TGG TGG GCC GAG ATG GCA Acly -enh1 forward 5′- TGA TAG CAC ACT ACT TTG CTC TGG reverse 5′-CAG TGA CGC ACG GCG AGC GGG AAG CHT -enhancer forward 5′-TGA GCA GCC TAT GCC ACA AGG ACA reverse 5′- CAT TAG GAG AGC TTG TTC CAG TGA The following antibodies were used for ChIP-PCR; mouse/rabbit IgG (Santa Cruz), rabbit anti-Isl1 , rabbit anti-Lhx3 , rabbit anti-NLI , and goat anti-Lhx8 (sc-22216, Santa Cruz).

    Techniques: Derivative Assay, Expressing, Western Blot, Immunohistochemistry, Quantitative RT-PCR, Cell Culture, Cell Differentiation, Standard Deviation

    The cholinergic enhancers are activated by the Isl1-Lhx3-hexamer in the developing spinal cord. ( A–E ) Luciferase reporter assays in mouse embryonic P19 cells using Acly -enh1-wt:LUC or Acly -enh1-mt:LUC, in which HxRE motifs are mutated (A), ChAT -enh:LUC (B), CHT -enh:LUC (C), Acly -HxRE:LUC (D), and ChAT -HxRE:LUC (E) reporters with expression vectors as indicated below each graph. The co-expression of Lhx3 wild-type and Isl1 wild-type, indicated as w or +, strongly activated the reporters linked to the cholinergic enhancers, but not the LUC vector alone or Acly -enh1-mt:LUC. Lhx3-N211S and Isl1-N230S, the DNA-binding defective missense mutants of Lhx3 or Isl1 that are indicated as m, failed to activate Acly -enh1-wt:LUC (A). (A–E) Error bars represent the standard deviation in all graphs. Error bars indicate standard deviation. ( F–H ) GFP reporter activity was monitored in chick embryos electroporated with Acly -enh1:GFP (F), Acly -enh1-HxRE-mt:GFP (G), and Acly -HxRE:GFP (H) reporters with either LacZ or Isl1 plus Lhx3 as indicated above. Acly -enh1 and Acly -HxRE drove MN-specific GFP expression, and were ectopically activated by co-expression of Isl1 and Lhx3 in the dorsal spinal cord (F, H). Acly -enh1-HxRE-mt:GFP did not display GFP expression in MNs and failed to respond to the co-electroporated Isl1 and Lhx3 (G), indicating that the HxRE motif is required for the MN-specific enhancer activity of Acly -enh1. + indicates the electroporated side. The areas of ectopic Hb9 + MNs, induced by co-expression of Isl1 and Lhx3, are marked by brackets.

    Journal: PLoS Genetics

    Article Title: Isl1 Directly Controls a Cholinergic Neuronal Identity in the Developing Forebrain and Spinal Cord by Forming Cell Type-Specific Complexes

    doi: 10.1371/journal.pgen.1004280

    Figure Lengend Snippet: The cholinergic enhancers are activated by the Isl1-Lhx3-hexamer in the developing spinal cord. ( A–E ) Luciferase reporter assays in mouse embryonic P19 cells using Acly -enh1-wt:LUC or Acly -enh1-mt:LUC, in which HxRE motifs are mutated (A), ChAT -enh:LUC (B), CHT -enh:LUC (C), Acly -HxRE:LUC (D), and ChAT -HxRE:LUC (E) reporters with expression vectors as indicated below each graph. The co-expression of Lhx3 wild-type and Isl1 wild-type, indicated as w or +, strongly activated the reporters linked to the cholinergic enhancers, but not the LUC vector alone or Acly -enh1-mt:LUC. Lhx3-N211S and Isl1-N230S, the DNA-binding defective missense mutants of Lhx3 or Isl1 that are indicated as m, failed to activate Acly -enh1-wt:LUC (A). (A–E) Error bars represent the standard deviation in all graphs. Error bars indicate standard deviation. ( F–H ) GFP reporter activity was monitored in chick embryos electroporated with Acly -enh1:GFP (F), Acly -enh1-HxRE-mt:GFP (G), and Acly -HxRE:GFP (H) reporters with either LacZ or Isl1 plus Lhx3 as indicated above. Acly -enh1 and Acly -HxRE drove MN-specific GFP expression, and were ectopically activated by co-expression of Isl1 and Lhx3 in the dorsal spinal cord (F, H). Acly -enh1-HxRE-mt:GFP did not display GFP expression in MNs and failed to respond to the co-electroporated Isl1 and Lhx3 (G), indicating that the HxRE motif is required for the MN-specific enhancer activity of Acly -enh1. + indicates the electroporated side. The areas of ectopic Hb9 + MNs, induced by co-expression of Isl1 and Lhx3, are marked by brackets.

    Article Snippet: ChAT -enhancer forward 5′-TAC TAA TTG GAT TAA TTG ATT TGC reverse 5′-GGG AAT TAA TAA CTT AGA ATT TGA ChAT -negative forward 5′- CTG TGG CTC ATA ACG CTC ATT TTG reverse 5′- AGT TTG TGG TGG GCC GAG ATG GCA Acly -enh1 forward 5′- TGA TAG CAC ACT ACT TTG CTC TGG reverse 5′-CAG TGA CGC ACG GCG AGC GGG AAG CHT -enhancer forward 5′-TGA GCA GCC TAT GCC ACA AGG ACA reverse 5′- CAT TAG GAG AGC TTG TTC CAG TGA The following antibodies were used for ChIP-PCR; mouse/rabbit IgG (Santa Cruz), rabbit anti-Isl1 , rabbit anti-Lhx3 , rabbit anti-NLI , and goat anti-Lhx8 (sc-22216, Santa Cruz).

    Techniques: Luciferase, Expressing, Plasmid Preparation, Binding Assay, Standard Deviation, Activity Assay

    The Isl1-Lhx8-hexamer activates the cholinergic genes. ( A ) ChIP assays with IgG, α-Isl1, α-Lhx8, and α-NLI antibodies in dissected E15.5 embryonic forebrains. The location of two sets of primers in the ChAT gene is indicated (arrows). The FCN-hexamer is recruited to the cholinergic enhancers. ( B, C ) Luciferase reporter assays in P19 cells using Acly -HxRE:LUC (B), and ChAT -HxRE:LUC (C) reporters with vectors indicated below each graph. The co-expression of Lhx8 and Isl1 strongly activated these reporters. Error bars represent the standard deviation in all graphs (A–C). ( D ) Schematic representation of ex vivo electroporation of the ventral forebrain. Sections containing the appropriate regions of the ventral forebrain were focally injected with combinations of plasmids and subjected to slice electroporation, followed by slice culture. The area of transfection was indicated in green. Due to the electroporation process with slices, part of cortex was also transfected with plasmids. ( E–H ) Activation of Acly -HxRE:GFP reporter in the ventral forebrains electroporated with constructs, LacZ (E), Lhx8 (F), Isl1 (G), and Isl1 and Lhx8 (H). The co-expression of Isl1 and Lhx8 strongly activated Acly -HxRE in the forebrain.

    Journal: PLoS Genetics

    Article Title: Isl1 Directly Controls a Cholinergic Neuronal Identity in the Developing Forebrain and Spinal Cord by Forming Cell Type-Specific Complexes

    doi: 10.1371/journal.pgen.1004280

    Figure Lengend Snippet: The Isl1-Lhx8-hexamer activates the cholinergic genes. ( A ) ChIP assays with IgG, α-Isl1, α-Lhx8, and α-NLI antibodies in dissected E15.5 embryonic forebrains. The location of two sets of primers in the ChAT gene is indicated (arrows). The FCN-hexamer is recruited to the cholinergic enhancers. ( B, C ) Luciferase reporter assays in P19 cells using Acly -HxRE:LUC (B), and ChAT -HxRE:LUC (C) reporters with vectors indicated below each graph. The co-expression of Lhx8 and Isl1 strongly activated these reporters. Error bars represent the standard deviation in all graphs (A–C). ( D ) Schematic representation of ex vivo electroporation of the ventral forebrain. Sections containing the appropriate regions of the ventral forebrain were focally injected with combinations of plasmids and subjected to slice electroporation, followed by slice culture. The area of transfection was indicated in green. Due to the electroporation process with slices, part of cortex was also transfected with plasmids. ( E–H ) Activation of Acly -HxRE:GFP reporter in the ventral forebrains electroporated with constructs, LacZ (E), Lhx8 (F), Isl1 (G), and Isl1 and Lhx8 (H). The co-expression of Isl1 and Lhx8 strongly activated Acly -HxRE in the forebrain.

    Article Snippet: ChAT -enhancer forward 5′-TAC TAA TTG GAT TAA TTG ATT TGC reverse 5′-GGG AAT TAA TAA CTT AGA ATT TGA ChAT -negative forward 5′- CTG TGG CTC ATA ACG CTC ATT TTG reverse 5′- AGT TTG TGG TGG GCC GAG ATG GCA Acly -enh1 forward 5′- TGA TAG CAC ACT ACT TTG CTC TGG reverse 5′-CAG TGA CGC ACG GCG AGC GGG AAG CHT -enhancer forward 5′-TGA GCA GCC TAT GCC ACA AGG ACA reverse 5′- CAT TAG GAG AGC TTG TTC CAG TGA The following antibodies were used for ChIP-PCR; mouse/rabbit IgG (Santa Cruz), rabbit anti-Isl1 , rabbit anti-Lhx3 , rabbit anti-NLI , and goat anti-Lhx8 (sc-22216, Santa Cruz).

    Techniques: Chromatin Immunoprecipitation, Luciferase, Expressing, Standard Deviation, Ex Vivo, Electroporation, Injection, Transfection, Activation Assay, Construct

    Isl1-Lhx8 induces the expression of cholinergic gene battery in the forebrain, but not in the spinal cord. ( A ) Schematic representation of in utero electroporation of the cortex, followed by quantitative RT-PCR (qRT-PCR) analyses. E13.5 brains were subjected to electroporation after each combination of constructs was injected into the lateral ventricle. The GFP + region of electroporated (+) cortex and the comparable region of unelectroporated (−) cortex were micro-dissected and analyzed for gene expression. ( B ) Expression analyses of the cholinergic pathway genes, ChAT, VAChT and CHT , in mouse cortices electroporated with constructs, as indicated by color bars. Y-axis indicates the relative expression levels of each cholinergic gene on the electropoated side over the control side. The expression of Isl1-Lhx8 led to upregulation of the cholinergic genes in the cortex, whereas that of Isl1-Lhx3 failed to induce cholinergic genes in this context. Error bars indicate standard deviation. ** p

    Journal: PLoS Genetics

    Article Title: Isl1 Directly Controls a Cholinergic Neuronal Identity in the Developing Forebrain and Spinal Cord by Forming Cell Type-Specific Complexes

    doi: 10.1371/journal.pgen.1004280

    Figure Lengend Snippet: Isl1-Lhx8 induces the expression of cholinergic gene battery in the forebrain, but not in the spinal cord. ( A ) Schematic representation of in utero electroporation of the cortex, followed by quantitative RT-PCR (qRT-PCR) analyses. E13.5 brains were subjected to electroporation after each combination of constructs was injected into the lateral ventricle. The GFP + region of electroporated (+) cortex and the comparable region of unelectroporated (−) cortex were micro-dissected and analyzed for gene expression. ( B ) Expression analyses of the cholinergic pathway genes, ChAT, VAChT and CHT , in mouse cortices electroporated with constructs, as indicated by color bars. Y-axis indicates the relative expression levels of each cholinergic gene on the electropoated side over the control side. The expression of Isl1-Lhx8 led to upregulation of the cholinergic genes in the cortex, whereas that of Isl1-Lhx3 failed to induce cholinergic genes in this context. Error bars indicate standard deviation. ** p

    Article Snippet: ChAT -enhancer forward 5′-TAC TAA TTG GAT TAA TTG ATT TGC reverse 5′-GGG AAT TAA TAA CTT AGA ATT TGA ChAT -negative forward 5′- CTG TGG CTC ATA ACG CTC ATT TTG reverse 5′- AGT TTG TGG TGG GCC GAG ATG GCA Acly -enh1 forward 5′- TGA TAG CAC ACT ACT TTG CTC TGG reverse 5′-CAG TGA CGC ACG GCG AGC GGG AAG CHT -enhancer forward 5′-TGA GCA GCC TAT GCC ACA AGG ACA reverse 5′- CAT TAG GAG AGC TTG TTC CAG TGA The following antibodies were used for ChIP-PCR; mouse/rabbit IgG (Santa Cruz), rabbit anti-Isl1 , rabbit anti-Lhx3 , rabbit anti-NLI , and goat anti-Lhx8 (sc-22216, Santa Cruz).

    Techniques: Expressing, In Utero, Electroporation, Quantitative RT-PCR, Construct, Injection, Standard Deviation

    Isl1 is co-expressed with Nkx2.1 and Lhx8 in the CPu and BMC and is important for the formation of Nkx2.1/Lhx8-expressing striatal interneurons. ( A ) Schematic representation of the coronal section of E16.5 forebrain. CPu, Caudate-putamen; BMC, basal meganocellular complex. ( B–I ) Immunohistochemical analyses on the CPu and BMC of E16.5 Isl1 f/f ;Nkx2.1Cre and littermate control embryos. Isl1 is co-expressed with Nkx2.1 and Lhx8 in subsets of neurons in the CPu and the BMC (B, C, F, G). The dotted circles depict Isl1/Nkx2.1-double positive cells (D, F). In Isl1 f/f ;Nkx2.1Cre embryos, the number of Nkx2.1 or Lhx8-expressing interneurons in the CPu is reduced (B–E), and Isl1 + cells in the BMC drastically decreased (F–I). ( J, K ) Quantification of the number of Lhx8- and Nkx2.1-expressing cells in the CPu of E16.5 control and Isl1 mutant embryos. Histogram shows average ± standard deviation. ** p

    Journal: PLoS Genetics

    Article Title: Isl1 Directly Controls a Cholinergic Neuronal Identity in the Developing Forebrain and Spinal Cord by Forming Cell Type-Specific Complexes

    doi: 10.1371/journal.pgen.1004280

    Figure Lengend Snippet: Isl1 is co-expressed with Nkx2.1 and Lhx8 in the CPu and BMC and is important for the formation of Nkx2.1/Lhx8-expressing striatal interneurons. ( A ) Schematic representation of the coronal section of E16.5 forebrain. CPu, Caudate-putamen; BMC, basal meganocellular complex. ( B–I ) Immunohistochemical analyses on the CPu and BMC of E16.5 Isl1 f/f ;Nkx2.1Cre and littermate control embryos. Isl1 is co-expressed with Nkx2.1 and Lhx8 in subsets of neurons in the CPu and the BMC (B, C, F, G). The dotted circles depict Isl1/Nkx2.1-double positive cells (D, F). In Isl1 f/f ;Nkx2.1Cre embryos, the number of Nkx2.1 or Lhx8-expressing interneurons in the CPu is reduced (B–E), and Isl1 + cells in the BMC drastically decreased (F–I). ( J, K ) Quantification of the number of Lhx8- and Nkx2.1-expressing cells in the CPu of E16.5 control and Isl1 mutant embryos. Histogram shows average ± standard deviation. ** p

    Article Snippet: ChAT -enhancer forward 5′-TAC TAA TTG GAT TAA TTG ATT TGC reverse 5′-GGG AAT TAA TAA CTT AGA ATT TGA ChAT -negative forward 5′- CTG TGG CTC ATA ACG CTC ATT TTG reverse 5′- AGT TTG TGG TGG GCC GAG ATG GCA Acly -enh1 forward 5′- TGA TAG CAC ACT ACT TTG CTC TGG reverse 5′-CAG TGA CGC ACG GCG AGC GGG AAG CHT -enhancer forward 5′-TGA GCA GCC TAT GCC ACA AGG ACA reverse 5′- CAT TAG GAG AGC TTG TTC CAG TGA The following antibodies were used for ChIP-PCR; mouse/rabbit IgG (Santa Cruz), rabbit anti-Isl1 , rabbit anti-Lhx3 , rabbit anti-NLI , and goat anti-Lhx8 (sc-22216, Santa Cruz).

    Techniques: Expressing, Immunohistochemistry, Mutagenesis, Standard Deviation

    The Isl1-Lhx3-hexamer plays a crucial role in inducing the expression of cholinergic pathway genes in the developing spinal MNs. ( A ) Expression analyses of the cholinergic pathway genes in chick embryos electroporated with Isl1 and Lhx3 using in situ hybridization. The co-electroporation of Isl1 and Lhx3 triggered the ectopic expression of cholinergic genes in the dorsal spinal cord, as marked by brackets. + indicates the electroporated side. ( B ) Expression analyses of the cholinergic pathway genes using either immunohistochemistry or in situ hybridization on the spinal cord of E12.5 Isl1 f/f ;nestinCre and littermate control embryos. The ventral quadrant spinal cord is shown. The cholinergic genes are markedly downregulated in Isl1 f/f ;nestinCre mice. The remaining VAChT expression is correlated with the residual Isl1 expression in Isl1 f/f ;nestinCre mice, as determined by immunostaining assays.

    Journal: PLoS Genetics

    Article Title: Isl1 Directly Controls a Cholinergic Neuronal Identity in the Developing Forebrain and Spinal Cord by Forming Cell Type-Specific Complexes

    doi: 10.1371/journal.pgen.1004280

    Figure Lengend Snippet: The Isl1-Lhx3-hexamer plays a crucial role in inducing the expression of cholinergic pathway genes in the developing spinal MNs. ( A ) Expression analyses of the cholinergic pathway genes in chick embryos electroporated with Isl1 and Lhx3 using in situ hybridization. The co-electroporation of Isl1 and Lhx3 triggered the ectopic expression of cholinergic genes in the dorsal spinal cord, as marked by brackets. + indicates the electroporated side. ( B ) Expression analyses of the cholinergic pathway genes using either immunohistochemistry or in situ hybridization on the spinal cord of E12.5 Isl1 f/f ;nestinCre and littermate control embryos. The ventral quadrant spinal cord is shown. The cholinergic genes are markedly downregulated in Isl1 f/f ;nestinCre mice. The remaining VAChT expression is correlated with the residual Isl1 expression in Isl1 f/f ;nestinCre mice, as determined by immunostaining assays.

    Article Snippet: ChAT -enhancer forward 5′-TAC TAA TTG GAT TAA TTG ATT TGC reverse 5′-GGG AAT TAA TAA CTT AGA ATT TGA ChAT -negative forward 5′- CTG TGG CTC ATA ACG CTC ATT TTG reverse 5′- AGT TTG TGG TGG GCC GAG ATG GCA Acly -enh1 forward 5′- TGA TAG CAC ACT ACT TTG CTC TGG reverse 5′-CAG TGA CGC ACG GCG AGC GGG AAG CHT -enhancer forward 5′-TGA GCA GCC TAT GCC ACA AGG ACA reverse 5′- CAT TAG GAG AGC TTG TTC CAG TGA The following antibodies were used for ChIP-PCR; mouse/rabbit IgG (Santa Cruz), rabbit anti-Isl1 , rabbit anti-Lhx3 , rabbit anti-NLI , and goat anti-Lhx8 (sc-22216, Santa Cruz).

    Techniques: Expressing, In Situ Hybridization, Electroporation, Immunohistochemistry, Mouse Assay, Immunostaining

    Isl1-Lhx8-hexamer and Isl1-Lhx3-hexamer complexes establish a cholinergic neuronal identity in FCNs and spinal MNs, respectively, by directly upregulating cholinergic gene battery. During CNS development, the cholinergic genes recruit the Isl1-Lhx8-hexamer in the forebrain and the Isl1-Lhx3-hexamer in spinal cord via hexamer-response elements. This recruitment leads to concerted induction of the cholinergic genes, therefore enabling MNs and FCNs to acquire the cholinergic neuronal identity. The Isl1-Lhx8-hexamer and Isl1-Lhx3-hexamer likely induce unique sets of target genes in FCNs and MNs. These hexamers may cooperate with other transcription factors (TFs) in establishing cell type-specific gene expression patterns.

    Journal: PLoS Genetics

    Article Title: Isl1 Directly Controls a Cholinergic Neuronal Identity in the Developing Forebrain and Spinal Cord by Forming Cell Type-Specific Complexes

    doi: 10.1371/journal.pgen.1004280

    Figure Lengend Snippet: Isl1-Lhx8-hexamer and Isl1-Lhx3-hexamer complexes establish a cholinergic neuronal identity in FCNs and spinal MNs, respectively, by directly upregulating cholinergic gene battery. During CNS development, the cholinergic genes recruit the Isl1-Lhx8-hexamer in the forebrain and the Isl1-Lhx3-hexamer in spinal cord via hexamer-response elements. This recruitment leads to concerted induction of the cholinergic genes, therefore enabling MNs and FCNs to acquire the cholinergic neuronal identity. The Isl1-Lhx8-hexamer and Isl1-Lhx3-hexamer likely induce unique sets of target genes in FCNs and MNs. These hexamers may cooperate with other transcription factors (TFs) in establishing cell type-specific gene expression patterns.

    Article Snippet: ChAT -enhancer forward 5′-TAC TAA TTG GAT TAA TTG ATT TGC reverse 5′-GGG AAT TAA TAA CTT AGA ATT TGA ChAT -negative forward 5′- CTG TGG CTC ATA ACG CTC ATT TTG reverse 5′- AGT TTG TGG TGG GCC GAG ATG GCA Acly -enh1 forward 5′- TGA TAG CAC ACT ACT TTG CTC TGG reverse 5′-CAG TGA CGC ACG GCG AGC GGG AAG CHT -enhancer forward 5′-TGA GCA GCC TAT GCC ACA AGG ACA reverse 5′- CAT TAG GAG AGC TTG TTC CAG TGA The following antibodies were used for ChIP-PCR; mouse/rabbit IgG (Santa Cruz), rabbit anti-Isl1 , rabbit anti-Lhx3 , rabbit anti-NLI , and goat anti-Lhx8 (sc-22216, Santa Cruz).

    Techniques: Expressing

    Co-expression of Isl1, Lhx8 and NLI in the developing ventral forebrain. ( A ) Schematic representation of the coronal section of E12.5 forebrain. The MGE produces striatal cholinergic interneurons in the CPu and cholinergic projection neurons in the BMC, which take different migratory paths. NCx, neocortex; MGE, medial ganaglionic eminence; LGE, lateral ganglionic eminence; CPu, Caudate-putamen; BMC, basal meganocellular complex. ( B–E ) Immunohistochemical analyses of expression of Nkx2.1, Isl1, Lhx8, and NLI on coronal sections of E12.5 mouse forebrains. Isl1 is co-expressed with Lhx8 and NLI in the mantle zone of the MGE (yellow asterisk) and LGE (red asterisk).

    Journal: PLoS Genetics

    Article Title: Isl1 Directly Controls a Cholinergic Neuronal Identity in the Developing Forebrain and Spinal Cord by Forming Cell Type-Specific Complexes

    doi: 10.1371/journal.pgen.1004280

    Figure Lengend Snippet: Co-expression of Isl1, Lhx8 and NLI in the developing ventral forebrain. ( A ) Schematic representation of the coronal section of E12.5 forebrain. The MGE produces striatal cholinergic interneurons in the CPu and cholinergic projection neurons in the BMC, which take different migratory paths. NCx, neocortex; MGE, medial ganaglionic eminence; LGE, lateral ganglionic eminence; CPu, Caudate-putamen; BMC, basal meganocellular complex. ( B–E ) Immunohistochemical analyses of expression of Nkx2.1, Isl1, Lhx8, and NLI on coronal sections of E12.5 mouse forebrains. Isl1 is co-expressed with Lhx8 and NLI in the mantle zone of the MGE (yellow asterisk) and LGE (red asterisk).

    Article Snippet: ChAT -enhancer forward 5′-TAC TAA TTG GAT TAA TTG ATT TGC reverse 5′-GGG AAT TAA TAA CTT AGA ATT TGA ChAT -negative forward 5′- CTG TGG CTC ATA ACG CTC ATT TTG reverse 5′- AGT TTG TGG TGG GCC GAG ATG GCA Acly -enh1 forward 5′- TGA TAG CAC ACT ACT TTG CTC TGG reverse 5′-CAG TGA CGC ACG GCG AGC GGG AAG CHT -enhancer forward 5′-TGA GCA GCC TAT GCC ACA AGG ACA reverse 5′- CAT TAG GAG AGC TTG TTC CAG TGA The following antibodies were used for ChIP-PCR; mouse/rabbit IgG (Santa Cruz), rabbit anti-Isl1 , rabbit anti-Lhx3 , rabbit anti-NLI , and goat anti-Lhx8 (sc-22216, Santa Cruz).

    Techniques: Expressing, Immunohistochemistry