anti islet 1  (R&D Systems)

 
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    Name:
    Human Islet 1 Antibody
    Description:
    The Human Islet 1 Antibody from R D Systems is a goat polyclonal antibody to Islet 1 This antibody reacts with human The Human Islet 1 Antibody has been validated for the following applications Western Blot Simple Western Immunocytochemistry
    Catalog Number:
    af1837
    Price:
    369
    Applications:
    Western Blot, Simple Western, Immunocytochemistry
    Host:
    Goat
    Purity:
    Antigen Affinity-purified
    Conjugate:
    Unconjugated
    Immunogen:
    E. coli-derived recombinant human Islet-1, Met4-Ala349, Accession # P61371
    Size:
    100 ug
    Category:
    Primary Antibodies
    Isotype:
    IgG
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    Structured Review

    R&D Systems anti islet 1
    Human Islet 1 Antibody
    The Human Islet 1 Antibody from R D Systems is a goat polyclonal antibody to Islet 1 This antibody reacts with human The Human Islet 1 Antibody has been validated for the following applications Western Blot Simple Western Immunocytochemistry
    https://www.bioz.com/result/anti islet 1/product/R&D Systems
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti islet 1 - by Bioz Stars, 2020-09
    93/100 stars

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    Related Articles

    Western Blot:

    Article Title: Isl1 and Pou4f2 Form a Complex to Regulate Target Genes in Developing Retinal Ganglion Cells
    Article Snippet: .. Antibodies Antibodies used for Western blotting in this study include: monoclonal mouse anti-Isl1 (DSHB, 39.4D5, 1∶1000); polyclonal goat-anti-Isl1 (R & D systems, AF1837 1∶1000); polyclonal rabbit anti-Lhx1 (Santa-Cruz, sc-133735 1∶500); polyclonal goat anti-Lhx2 (Santa-Cruz, sc-19342, 1∶1000); polyclonal goat anti-Pou4f2 (Santa-Cruz, sc-6026, 1∶1000); polyclonal rabbit anti-GST (Sigma, G7781, 1∶1000); and monoclonal mouse anti-His (Santa-Cruz, sc-8036, 1∶500). .. Antibodies used for immunoprecipitation (IP) ChIP include polyclonal rabbit anti-Is1 (Millipore, NG1897037) and polyclonal goat anti-Pou4f2 (Santa-Cruz, sc-6026).

    Incubation:

    Article Title: Nociceptor Deletion of Tsc2 Enhances Axon Regeneration by Inducing a Conditioning Injury Response in Dorsal Root Ganglia
    Article Snippet: .. Subsequently, sections were incubated overnight at 4°C in primary antibodies diluted in blocking reagent: Tsc2 (D93F12, 1:200; Cell Signaling catalog #4308, RRID: AB_10547134 ), SCG10/Stmn2 (1:1000; Novus catalog #NBP1-49461, RRID: AB_10011569 ), cJun (1:250; Cell Signaling catalog #9165, RRID: AB_2130165 ), Atf3 (1:250; Novus catalog #NBP1-85816, RRID: AB_11014863 ), Islet1 (1:500; R and D Systems catalog #AF1837, RRID: AB_2126324 ), Gfap (1:1000; Agilent catalog #Z0334, RRID: AB_10013382 ), and Iba1 (1:1000; Wako catalog #019-19741, RRID: AB_839504 ). .. Griffonia simplicifolia isolectin B4 (IB4) directly conjugated to Alexa Fluor 488 or Alexa Fluor 594 (1:250; Thermo Fisher Scientific catalog #I21411 and #I21413) was incubated with primary antibodies.

    Article Title: Cell freezing protocol suitable for ATAC-Seq on motor neurons derived from human induced pluripotent stem cells
    Article Snippet: .. We incubated the cells overnight at 4 °C with the following primary antibodies: anti-SMI32 (mouse monoclonal, 1:1000, BioLegend, cat. no. SMI-32R) and anti-ISL1 (goat polyclonal, 1:250, R & D Systems, cat. no. AF1837). .. We subsequently rinsed the cells and incubated them with species-specific Alexa Fluor 488-conjugated secondary antibody (donkey anti-mouse immunoglobulin G (IgG), 1:1000, Life Technologies, cat. no. A-21202) and Alexa Fluor 594-conjugated secondary antibody (donkey anti-goat IgG, 1:1000, Life Technologies, cat. no. A-11058).

    Blocking Assay:

    Article Title: Nociceptor Deletion of Tsc2 Enhances Axon Regeneration by Inducing a Conditioning Injury Response in Dorsal Root Ganglia
    Article Snippet: .. Subsequently, sections were incubated overnight at 4°C in primary antibodies diluted in blocking reagent: Tsc2 (D93F12, 1:200; Cell Signaling catalog #4308, RRID: AB_10547134 ), SCG10/Stmn2 (1:1000; Novus catalog #NBP1-49461, RRID: AB_10011569 ), cJun (1:250; Cell Signaling catalog #9165, RRID: AB_2130165 ), Atf3 (1:250; Novus catalog #NBP1-85816, RRID: AB_11014863 ), Islet1 (1:500; R and D Systems catalog #AF1837, RRID: AB_2126324 ), Gfap (1:1000; Agilent catalog #Z0334, RRID: AB_10013382 ), and Iba1 (1:1000; Wako catalog #019-19741, RRID: AB_839504 ). .. Griffonia simplicifolia isolectin B4 (IB4) directly conjugated to Alexa Fluor 488 or Alexa Fluor 594 (1:250; Thermo Fisher Scientific catalog #I21411 and #I21413) was incubated with primary antibodies.

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  • 93
    R&D Systems human islet 1 antibody
    Hb9::Foxp1 ESCs generate LMCm and LMCl MNs. ( a ) Newly derived ESC lines displayed characteristic ESC morphology and expressed pluripotency markers. Both Hb9::GFP and Hb9::Foxp1 ESC lines differentiated into GFP + /Hb9 + MNs, but only Hb9::Foxp1 ESC-derived MNs expressed high levels of Foxp1 protein. Scale bars, 50 μm. ( b ) Analysis of MN columnar marker expression in ESC-derived MNs. The majority of control Hb9::GFP ESC-derived MNs expressed Lhx3 and low levels of Foxp1, Lhx1 and Aldh1a2. Hb9::Foxp1 ESC-derived MNs expressed reduced levels of Lhx3 and increased levels of LMCm <t>(Isl1</t> + /Foxp1 + ) and LMCl (Hb9 + /Lhx1 + ) MN markers. Inset shows a single Aldh1a2 + MN. Scale bars, 50 μm. ( c ) Quantification of MN subtypes generated by each ESC line (mean±s.e.m.; n =3 independent experiments, 373 Hb9::GFP MNs and 403 Hb9::Foxp1 MNs total; Student's t -test). Lhx3: *** P =0.0002. Foxp1: *** P =0.0008. Lhx3/Foxp1: ** P =0.002. NS, not significant, P > 0.5. ( d ) Quantification of the percentages of Hb9::Foxp1 ESC-derived Foxp1 + MNs that express markers of LMCm (GFP + /Foxp1 + ) and LMCl (GFP + /Foxp1 + /Lhx1 + ) MNs (mean±s.e.m.; n =3 independent experiments, 222 Hb9::Foxp1 MNs total). ( e ) Endogenous Foxp1 mRNA was expressed approximately threefold higher in Hb9::Foxp1 ESC-derived MNs, compared with Hb9::GFP ESC-derived MNs. E11.5 Foxp1 +/− and Foxp1 −/− purified MNs were used as controls (mean±s.e.m.; n =3 Foxp1 +/− embryos, 3 Foxp1 −/− embryos, 3 independent RNA collections of Hb9::GFP and Hb9::Foxp1 MNs; one-way analysis of variance with Bonferroni adjustment). *** P =0.001. ( f ) Aldh1a2 mRNA levels were elevated approximately sixfold higher in Hb9::Foxp1 ESC-derived MNs, compared with Hb9::GFP controls (mean±s.e.m.; n =5 independent RNA collections of Hb9::GFP and Hb9::Foxp1 MNs; paired two-tailed t -test). * P =0.017.
    Human Islet 1 Antibody, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human islet 1 antibody/product/R&D Systems
    Average 93 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    human islet 1 antibody - by Bioz Stars, 2020-09
    93/100 stars
      Buy from Supplier

    88
    R&D Systems goat anti isl1
    Ventral forebrain inactivation of <t>Isl1</t> reduces Isl1 expression in the striatum as well as the reticular thalamus and leads to a disorganization of the striatonigral output pathway. At adult stages the ventral forebrain, inactivation of Isl1 causes a deletion
    Goat Anti Isl1, supplied by R&D Systems, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/goat anti isl1/product/R&D Systems
    Average 88 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    goat anti isl1 - by Bioz Stars, 2020-09
    88/100 stars
      Buy from Supplier

    Image Search Results


    Hb9::Foxp1 ESCs generate LMCm and LMCl MNs. ( a ) Newly derived ESC lines displayed characteristic ESC morphology and expressed pluripotency markers. Both Hb9::GFP and Hb9::Foxp1 ESC lines differentiated into GFP + /Hb9 + MNs, but only Hb9::Foxp1 ESC-derived MNs expressed high levels of Foxp1 protein. Scale bars, 50 μm. ( b ) Analysis of MN columnar marker expression in ESC-derived MNs. The majority of control Hb9::GFP ESC-derived MNs expressed Lhx3 and low levels of Foxp1, Lhx1 and Aldh1a2. Hb9::Foxp1 ESC-derived MNs expressed reduced levels of Lhx3 and increased levels of LMCm (Isl1 + /Foxp1 + ) and LMCl (Hb9 + /Lhx1 + ) MN markers. Inset shows a single Aldh1a2 + MN. Scale bars, 50 μm. ( c ) Quantification of MN subtypes generated by each ESC line (mean±s.e.m.; n =3 independent experiments, 373 Hb9::GFP MNs and 403 Hb9::Foxp1 MNs total; Student's t -test). Lhx3: *** P =0.0002. Foxp1: *** P =0.0008. Lhx3/Foxp1: ** P =0.002. NS, not significant, P > 0.5. ( d ) Quantification of the percentages of Hb9::Foxp1 ESC-derived Foxp1 + MNs that express markers of LMCm (GFP + /Foxp1 + ) and LMCl (GFP + /Foxp1 + /Lhx1 + ) MNs (mean±s.e.m.; n =3 independent experiments, 222 Hb9::Foxp1 MNs total). ( e ) Endogenous Foxp1 mRNA was expressed approximately threefold higher in Hb9::Foxp1 ESC-derived MNs, compared with Hb9::GFP ESC-derived MNs. E11.5 Foxp1 +/− and Foxp1 −/− purified MNs were used as controls (mean±s.e.m.; n =3 Foxp1 +/− embryos, 3 Foxp1 −/− embryos, 3 independent RNA collections of Hb9::GFP and Hb9::Foxp1 MNs; one-way analysis of variance with Bonferroni adjustment). *** P =0.001. ( f ) Aldh1a2 mRNA levels were elevated approximately sixfold higher in Hb9::Foxp1 ESC-derived MNs, compared with Hb9::GFP controls (mean±s.e.m.; n =5 independent RNA collections of Hb9::GFP and Hb9::Foxp1 MNs; paired two-tailed t -test). * P =0.017.

    Journal: Nature Communications

    Article Title: Foxp1-mediated programming of limb-innervating motor neurons from mouse and human embryonic stem cells

    doi: 10.1038/ncomms7778

    Figure Lengend Snippet: Hb9::Foxp1 ESCs generate LMCm and LMCl MNs. ( a ) Newly derived ESC lines displayed characteristic ESC morphology and expressed pluripotency markers. Both Hb9::GFP and Hb9::Foxp1 ESC lines differentiated into GFP + /Hb9 + MNs, but only Hb9::Foxp1 ESC-derived MNs expressed high levels of Foxp1 protein. Scale bars, 50 μm. ( b ) Analysis of MN columnar marker expression in ESC-derived MNs. The majority of control Hb9::GFP ESC-derived MNs expressed Lhx3 and low levels of Foxp1, Lhx1 and Aldh1a2. Hb9::Foxp1 ESC-derived MNs expressed reduced levels of Lhx3 and increased levels of LMCm (Isl1 + /Foxp1 + ) and LMCl (Hb9 + /Lhx1 + ) MN markers. Inset shows a single Aldh1a2 + MN. Scale bars, 50 μm. ( c ) Quantification of MN subtypes generated by each ESC line (mean±s.e.m.; n =3 independent experiments, 373 Hb9::GFP MNs and 403 Hb9::Foxp1 MNs total; Student's t -test). Lhx3: *** P =0.0002. Foxp1: *** P =0.0008. Lhx3/Foxp1: ** P =0.002. NS, not significant, P > 0.5. ( d ) Quantification of the percentages of Hb9::Foxp1 ESC-derived Foxp1 + MNs that express markers of LMCm (GFP + /Foxp1 + ) and LMCl (GFP + /Foxp1 + /Lhx1 + ) MNs (mean±s.e.m.; n =3 independent experiments, 222 Hb9::Foxp1 MNs total). ( e ) Endogenous Foxp1 mRNA was expressed approximately threefold higher in Hb9::Foxp1 ESC-derived MNs, compared with Hb9::GFP ESC-derived MNs. E11.5 Foxp1 +/− and Foxp1 −/− purified MNs were used as controls (mean±s.e.m.; n =3 Foxp1 +/− embryos, 3 Foxp1 −/− embryos, 3 independent RNA collections of Hb9::GFP and Hb9::Foxp1 MNs; one-way analysis of variance with Bonferroni adjustment). *** P =0.001. ( f ) Aldh1a2 mRNA levels were elevated approximately sixfold higher in Hb9::Foxp1 ESC-derived MNs, compared with Hb9::GFP controls (mean±s.e.m.; n =5 independent RNA collections of Hb9::GFP and Hb9::Foxp1 MNs; paired two-tailed t -test). * P =0.017.

    Article Snippet: The following primary antibodies were used: guinea pig anti-Aldh1a2 (1:20,000) , goat anti-ChAT (1:200; Millipore AB144P) , rabbit anti-EphA4 (1:500; Santa Cruz Sc-291) , rabbit anti-Etv4 (1:1,000) , guinea pig anti-Foxp1 (1:16,000) , sheep anti-GFP (1:800; AbD Serotec 4745-1051), rabbit anti-GFP (1:3,000; Life Technologies A6455) , chick anti-GFP (1:1,000; Aves Labs GFP-1020), rabbit anti-Hb9 (1:8,000) , guinea pig anti-Hoxa5 (1:8,000) , goat anti-Hoxa9 (1:1,000; Santa Cruz SC-17155) , rabbit anti-Hoxc6 (1:1,000; Santa Cruz SC-66925) , mouse anti-Hoxc8 (1:2,000; Covance MMS-266R) , goat anti-Isl1 (1:8,000, R & D Systems AF1837) , mouse anti-Isl1/2 (39.4D5; 1:100; Developmental Studies Hybridoma Bank) , mouse anti-Lhx1 (4F2; 1:100; Developmental Studies Hybridoma Bank) , rabbit anti-Lhx3 (1:1,000; Abcam Ab14555), mouse anti-Lhx3 (67.4E12; 1:100; Developmental Studies Hybridoma Bank) , mouse anti-myosin heavy chain (MF20; 1:100; Developmental Studies Hybridoma Bank) , rabbit anti-Nkx6.1 (1:2,500) ; rabbit anti-nNOS (1:10,000; ImmunoStar 24287) , rabbit anti-Pou3f1 (1:2,000) ; mouse anti-Pou5f1 (1:1,000; Santa Cruz SC-5279), goat anti-Sox2 (1:2,000, Santa Cruz SC-17320) , mouse anti-Synaptotagmin (mAb 30; 1:100; Developmental Studies Hybridoma Bank) , mouse anti-Synaptotagmin 2 (znp-1; 1:100; Developmental Studies Hybridoma Bank) , rabbit anti-βIII Tubulin (1:3,000; Covance MRB-435P) and mouse anti-βIII Tubulin (1:3,000; Covance MMS-435P).

    Techniques: Derivative Assay, Marker, Expressing, Generated, Purification, Two Tailed Test

    FOXP1 promotes LMC MN formation from human ESCs. ( a ) Control RFP-transduced human ESC-derived MNs (identified by CHAT expression) expressed high levels of LHX3 and low levels of FOXP1 and LHX1. FOXP1-transduced human ESC-derived MNs expressed increased levels of LHX1 and decreased levels of LHX3. ( b ) Control RFP-transduced human ESC-derived MNs did not express high levels of known LMC motor pool markers such as ETV4, POU3F1 and NKX6.1. By contrast, many FOXP1-transduced human ESC-derived MNs expressed ETV4 or POU3F1, with a smaller number expressing NKX6.1. ( c ) Quantification of MN subtype markers in transduced human ESC-derived MNs (mean±s.e.m.; n =2 independent experiments, 700 RFP-transduced MNs and 655 FOXP1-transduced MNs total; Student's t -test). LHX3: ** P =0.0055. FOXP1: ** P =0.0056. LHX1: ** P =0.0035. ( d ) Quantification of LMC motor pool markers in transduced human ESC-derived MNs (mean±s.e.m.; n =3 independent experiments, 2342 RFP-transduced MNs and 1624 FOXP1-transduced MNs total; Student's t -test). NKX6.1: * P =0.0108. ETV4 (LMCm): ** P =0.0042. ETV4 (LMCl): * P =0.0263. POU3F1: ** P =0.0053. NKX6.1, ETV4 (LMCm) and POU3F1 motor pools were identified by co-localization of ISL1, FOXP1 and the respective motor pool marker. The ETV4 (LMCl) motor pool was identified by co-localization of FOXP1 and ETV4.

    Journal: Nature Communications

    Article Title: Foxp1-mediated programming of limb-innervating motor neurons from mouse and human embryonic stem cells

    doi: 10.1038/ncomms7778

    Figure Lengend Snippet: FOXP1 promotes LMC MN formation from human ESCs. ( a ) Control RFP-transduced human ESC-derived MNs (identified by CHAT expression) expressed high levels of LHX3 and low levels of FOXP1 and LHX1. FOXP1-transduced human ESC-derived MNs expressed increased levels of LHX1 and decreased levels of LHX3. ( b ) Control RFP-transduced human ESC-derived MNs did not express high levels of known LMC motor pool markers such as ETV4, POU3F1 and NKX6.1. By contrast, many FOXP1-transduced human ESC-derived MNs expressed ETV4 or POU3F1, with a smaller number expressing NKX6.1. ( c ) Quantification of MN subtype markers in transduced human ESC-derived MNs (mean±s.e.m.; n =2 independent experiments, 700 RFP-transduced MNs and 655 FOXP1-transduced MNs total; Student's t -test). LHX3: ** P =0.0055. FOXP1: ** P =0.0056. LHX1: ** P =0.0035. ( d ) Quantification of LMC motor pool markers in transduced human ESC-derived MNs (mean±s.e.m.; n =3 independent experiments, 2342 RFP-transduced MNs and 1624 FOXP1-transduced MNs total; Student's t -test). NKX6.1: * P =0.0108. ETV4 (LMCm): ** P =0.0042. ETV4 (LMCl): * P =0.0263. POU3F1: ** P =0.0053. NKX6.1, ETV4 (LMCm) and POU3F1 motor pools were identified by co-localization of ISL1, FOXP1 and the respective motor pool marker. The ETV4 (LMCl) motor pool was identified by co-localization of FOXP1 and ETV4.

    Article Snippet: The following primary antibodies were used: guinea pig anti-Aldh1a2 (1:20,000) , goat anti-ChAT (1:200; Millipore AB144P) , rabbit anti-EphA4 (1:500; Santa Cruz Sc-291) , rabbit anti-Etv4 (1:1,000) , guinea pig anti-Foxp1 (1:16,000) , sheep anti-GFP (1:800; AbD Serotec 4745-1051), rabbit anti-GFP (1:3,000; Life Technologies A6455) , chick anti-GFP (1:1,000; Aves Labs GFP-1020), rabbit anti-Hb9 (1:8,000) , guinea pig anti-Hoxa5 (1:8,000) , goat anti-Hoxa9 (1:1,000; Santa Cruz SC-17155) , rabbit anti-Hoxc6 (1:1,000; Santa Cruz SC-66925) , mouse anti-Hoxc8 (1:2,000; Covance MMS-266R) , goat anti-Isl1 (1:8,000, R & D Systems AF1837) , mouse anti-Isl1/2 (39.4D5; 1:100; Developmental Studies Hybridoma Bank) , mouse anti-Lhx1 (4F2; 1:100; Developmental Studies Hybridoma Bank) , rabbit anti-Lhx3 (1:1,000; Abcam Ab14555), mouse anti-Lhx3 (67.4E12; 1:100; Developmental Studies Hybridoma Bank) , mouse anti-myosin heavy chain (MF20; 1:100; Developmental Studies Hybridoma Bank) , rabbit anti-Nkx6.1 (1:2,500) ; rabbit anti-nNOS (1:10,000; ImmunoStar 24287) , rabbit anti-Pou3f1 (1:2,000) ; mouse anti-Pou5f1 (1:1,000; Santa Cruz SC-5279), goat anti-Sox2 (1:2,000, Santa Cruz SC-17320) , mouse anti-Synaptotagmin (mAb 30; 1:100; Developmental Studies Hybridoma Bank) , mouse anti-Synaptotagmin 2 (znp-1; 1:100; Developmental Studies Hybridoma Bank) , rabbit anti-βIII Tubulin (1:3,000; Covance MRB-435P) and mouse anti-βIII Tubulin (1:3,000; Covance MMS-435P).

    Techniques: Derivative Assay, Expressing, Marker

    Foxp1 misexpression rescues the Foxp1 -mutant phenotype. Analysis of MN subtypes in the ventral horn of E12.5 Foxp +/+ , Foxp −/− and Hb9::Foxp1; Foxp1 −/− littermate embryo spinal cords. Scale bar, 50 μm. ( a ) MN subtypes at cervical levels. Foxp1 −/− embryos had reduced numbers of LMCm (Isl1 + /Foxp1 + ) and LMCl (Hb9 + /Lhx1 + ) MNs, while HMC (Hb9 + /Isl1 + ) and MMC-rhomboideus (Isl1 + /Lhx3 low ) MNs were expanded compared with Foxp1 +/+ embryos 13 . Hb9::Foxp1; Foxp1 −/− embryos had partially restored LMCm and LMCl MNs that resided at the correct lateral positions, and reduced numbers of ectopic HMC and MMC-Rb MNs, compared with Foxp1 −/− embryos. Rb, rhomboideus. ( b ) MN subtypes at thoracic levels. The loss of PGC (Isl1 + /nNOS + ) MNs in Foxp1 −/− embryos was rescued in Hb9::Foxp1; Foxp1 −/− embryos. ( c ) Quantification of MN percentages at cervical levels (mean±s.e.m.; n =3 sections averaged per embryo, 3 embryos per genotype; two-way analysis of variance (ANOVA) with Bonferroni adjustment). MMC MNs: *** P

    Journal: Nature Communications

    Article Title: Foxp1-mediated programming of limb-innervating motor neurons from mouse and human embryonic stem cells

    doi: 10.1038/ncomms7778

    Figure Lengend Snippet: Foxp1 misexpression rescues the Foxp1 -mutant phenotype. Analysis of MN subtypes in the ventral horn of E12.5 Foxp +/+ , Foxp −/− and Hb9::Foxp1; Foxp1 −/− littermate embryo spinal cords. Scale bar, 50 μm. ( a ) MN subtypes at cervical levels. Foxp1 −/− embryos had reduced numbers of LMCm (Isl1 + /Foxp1 + ) and LMCl (Hb9 + /Lhx1 + ) MNs, while HMC (Hb9 + /Isl1 + ) and MMC-rhomboideus (Isl1 + /Lhx3 low ) MNs were expanded compared with Foxp1 +/+ embryos 13 . Hb9::Foxp1; Foxp1 −/− embryos had partially restored LMCm and LMCl MNs that resided at the correct lateral positions, and reduced numbers of ectopic HMC and MMC-Rb MNs, compared with Foxp1 −/− embryos. Rb, rhomboideus. ( b ) MN subtypes at thoracic levels. The loss of PGC (Isl1 + /nNOS + ) MNs in Foxp1 −/− embryos was rescued in Hb9::Foxp1; Foxp1 −/− embryos. ( c ) Quantification of MN percentages at cervical levels (mean±s.e.m.; n =3 sections averaged per embryo, 3 embryos per genotype; two-way analysis of variance (ANOVA) with Bonferroni adjustment). MMC MNs: *** P

    Article Snippet: The following primary antibodies were used: guinea pig anti-Aldh1a2 (1:20,000) , goat anti-ChAT (1:200; Millipore AB144P) , rabbit anti-EphA4 (1:500; Santa Cruz Sc-291) , rabbit anti-Etv4 (1:1,000) , guinea pig anti-Foxp1 (1:16,000) , sheep anti-GFP (1:800; AbD Serotec 4745-1051), rabbit anti-GFP (1:3,000; Life Technologies A6455) , chick anti-GFP (1:1,000; Aves Labs GFP-1020), rabbit anti-Hb9 (1:8,000) , guinea pig anti-Hoxa5 (1:8,000) , goat anti-Hoxa9 (1:1,000; Santa Cruz SC-17155) , rabbit anti-Hoxc6 (1:1,000; Santa Cruz SC-66925) , mouse anti-Hoxc8 (1:2,000; Covance MMS-266R) , goat anti-Isl1 (1:8,000, R & D Systems AF1837) , mouse anti-Isl1/2 (39.4D5; 1:100; Developmental Studies Hybridoma Bank) , mouse anti-Lhx1 (4F2; 1:100; Developmental Studies Hybridoma Bank) , rabbit anti-Lhx3 (1:1,000; Abcam Ab14555), mouse anti-Lhx3 (67.4E12; 1:100; Developmental Studies Hybridoma Bank) , mouse anti-myosin heavy chain (MF20; 1:100; Developmental Studies Hybridoma Bank) , rabbit anti-Nkx6.1 (1:2,500) ; rabbit anti-nNOS (1:10,000; ImmunoStar 24287) , rabbit anti-Pou3f1 (1:2,000) ; mouse anti-Pou5f1 (1:1,000; Santa Cruz SC-5279), goat anti-Sox2 (1:2,000, Santa Cruz SC-17320) , mouse anti-Synaptotagmin (mAb 30; 1:100; Developmental Studies Hybridoma Bank) , mouse anti-Synaptotagmin 2 (znp-1; 1:100; Developmental Studies Hybridoma Bank) , rabbit anti-βIII Tubulin (1:3,000; Covance MRB-435P) and mouse anti-βIII Tubulin (1:3,000; Covance MMS-435P).

    Techniques: Mutagenesis, Pyrolysis Gas Chromatography

    iPSC-MN cultures recapitulate developmental gene expression patterns. A. Immunostaining day 18 cultures from three individual subject lines for the expression of MN markers ISL1 and SMI-32. B. Experiment schematic for analyzing the time course of MN differentiation over the course of 18 days. C - D. Violin plots indicate refinement of gene expression programs during MN differentiation. C depicts the number of unique molecular identifiers (nUMI), and D depicts the number of detectable genes per time point. E. tSNE of spinal MN differentiation time course samples. F. Monocle analysis projects samples into a pseudo-time axis consistent with the order of time points. G. Histogram displays meta data for all samples profiled in this study. This data is also presented in Figure S4 . H. UMI counts were summed for each gene across the expression matrix for each sample, thereby simulating bulk RNA-seq data. Simulated bulk gene expression profiles for each sample were correlated to bulk RNA-seq gene expression profiles from fetal hindbrain and spinal cord at various Carnegie stages analyzed in de Kovel et al., 2017 . The median Spearman correlation was calculated for each column of day 18 samples and displayed along the bottom row. For each row of pairwise correlations, the top three ranked correlations are outlined, indicating which sample from de Kovel et al., 2017 most globally similar to each sample analyzed by single-cell RNA-seq.

    Journal: bioRxiv

    Article Title: Single-cell RNA-seq analysis of human iPSC-derived motor neurons resolves early and predictive ALS signatures

    doi: 10.1101/2020.04.27.064584

    Figure Lengend Snippet: iPSC-MN cultures recapitulate developmental gene expression patterns. A. Immunostaining day 18 cultures from three individual subject lines for the expression of MN markers ISL1 and SMI-32. B. Experiment schematic for analyzing the time course of MN differentiation over the course of 18 days. C - D. Violin plots indicate refinement of gene expression programs during MN differentiation. C depicts the number of unique molecular identifiers (nUMI), and D depicts the number of detectable genes per time point. E. tSNE of spinal MN differentiation time course samples. F. Monocle analysis projects samples into a pseudo-time axis consistent with the order of time points. G. Histogram displays meta data for all samples profiled in this study. This data is also presented in Figure S4 . H. UMI counts were summed for each gene across the expression matrix for each sample, thereby simulating bulk RNA-seq data. Simulated bulk gene expression profiles for each sample were correlated to bulk RNA-seq gene expression profiles from fetal hindbrain and spinal cord at various Carnegie stages analyzed in de Kovel et al., 2017 . The median Spearman correlation was calculated for each column of day 18 samples and displayed along the bottom row. For each row of pairwise correlations, the top three ranked correlations are outlined, indicating which sample from de Kovel et al., 2017 most globally similar to each sample analyzed by single-cell RNA-seq.

    Article Snippet: Primary antibody solution in blocking solution containing various combinations of goat polyclonal IgG anti-Human ISL1 (1:200) (R & D Systems AF1837, RRID: AB_2126324), mouse monoclonal IgG1 anti-NF-H (SMI-32) (1:200) (BioLegend 801701, RRID: AB_2564642), goat polyclonal anti-ChAT (1:200) (Millipore AB144P, RRID: AB_2079751), rabbit polyclonal IgG anti-PHOX2B (1:200) (GeneTex GTX109677, RRID: AB_1951223), mouse monoclonal IgG2b, rabbit polyclonal IgG anti-CHX10 (VSX2) (1:200) (Novus NBP1-84476, RRID: AB_11022841), and rabbit polyclonal IgG anti-SOX1 [EPR4766] 1:200) (GeneTex GTX62974) were incubated, rinsed with 0.2% Tween-20 in PBS, and incubated in species-specific Alexa-fluor secondary antibodies (1:2,000), and rinsed with 0.2% Tween-20 in PBS with DAPI staining.

    Techniques: Expressing, Immunostaining, RNA Sequencing Assay

    iPSC differentiated spinal MN cultures globally resemble fetal spinal tissue. Related to Figure 1 . A. Pseudo-time plots of MN development and maturation markers ( Ho et al., 2016 ) indicate iPSC-MNs recapitulate downregulation of pluripotent stem cell and cell cycle markers and upregulate early spinal MN patterning and progenitor markers with modest expression of maturation markers. B. Quantification of immunostained day 18 cultures from several subject lines (three control, one sporadic ALS, one C9orf72 repeat expansion, and one C9orf72 repeat expansion-corrected isogenic control) for the double positive expression of spinal MN markers ISL1 and SMI-32. Cultures were fixed and stained at day 18, and parallel differentiation cultures were processed on the same day for scRs. Color of bar graphs indicate which differentiation experiments were processed on either the DDSEQ or TENEX scRs technology platform. Three fields of view were quantified and averaged per sample, and error bars indicate standard error.

    Journal: bioRxiv

    Article Title: Single-cell RNA-seq analysis of human iPSC-derived motor neurons resolves early and predictive ALS signatures

    doi: 10.1101/2020.04.27.064584

    Figure Lengend Snippet: iPSC differentiated spinal MN cultures globally resemble fetal spinal tissue. Related to Figure 1 . A. Pseudo-time plots of MN development and maturation markers ( Ho et al., 2016 ) indicate iPSC-MNs recapitulate downregulation of pluripotent stem cell and cell cycle markers and upregulate early spinal MN patterning and progenitor markers with modest expression of maturation markers. B. Quantification of immunostained day 18 cultures from several subject lines (three control, one sporadic ALS, one C9orf72 repeat expansion, and one C9orf72 repeat expansion-corrected isogenic control) for the double positive expression of spinal MN markers ISL1 and SMI-32. Cultures were fixed and stained at day 18, and parallel differentiation cultures were processed on the same day for scRs. Color of bar graphs indicate which differentiation experiments were processed on either the DDSEQ or TENEX scRs technology platform. Three fields of view were quantified and averaged per sample, and error bars indicate standard error.

    Article Snippet: Primary antibody solution in blocking solution containing various combinations of goat polyclonal IgG anti-Human ISL1 (1:200) (R & D Systems AF1837, RRID: AB_2126324), mouse monoclonal IgG1 anti-NF-H (SMI-32) (1:200) (BioLegend 801701, RRID: AB_2564642), goat polyclonal anti-ChAT (1:200) (Millipore AB144P, RRID: AB_2079751), rabbit polyclonal IgG anti-PHOX2B (1:200) (GeneTex GTX109677, RRID: AB_1951223), mouse monoclonal IgG2b, rabbit polyclonal IgG anti-CHX10 (VSX2) (1:200) (Novus NBP1-84476, RRID: AB_11022841), and rabbit polyclonal IgG anti-SOX1 [EPR4766] 1:200) (GeneTex GTX62974) were incubated, rinsed with 0.2% Tween-20 in PBS, and incubated in species-specific Alexa-fluor secondary antibodies (1:2,000), and rinsed with 0.2% Tween-20 in PBS with DAPI staining.

    Techniques: Expressing, Staining

    Distinct markers of neuronal subtypes are expressed as proteins in iPSC differentiated spinal MN cultures and enact distinct pathways in ALS conditions. Related to Figures 3 , 4 , and 5. A. Immunostaining day 18 cultures from one control subject line, 1034 for the expression of spinal MN marker, ISL1, a cranial MN marker, PHOX2B, a V2a interneuron marker, VSX2, and a V2c interneuron marker, SOX1. DAPI and SMI-32 are also used as counter stains. Scale bar, 50 um. B. Venn diagrams of overlapping gene sets between 1) sporadic ALS to CTR and 2) C9orf72 HRE ALS to CTR comparisons within the neuronal subtypes MN, V1 Renshaw, and V2a. Upregulated gene sets were only compared each other, and downregulated gene sets were only compared to each other. P-values of each intersection was calculated by a hypergeometric test. C. Enriched GO terms among genes significantly upregulated or downregulated in ALS compared to control neuronal subtypes are intersect in Venn diagrams. Representative GO terms are displayed from each overlapping or unique set. Full set of GO terms are shown in Table S7B-G. D. Left: measure of how well 52 modules defined in data set A are preserved in the modules defined in data set B. The Z-summary statistic (y-axis) for is plotted against module size (x-axis). Data points reflect module color. Dashed green line indicate threshold at Z = 10, and dashed blue line indicate threshold at Z = 2. For the likelihood of module preservation, Z-summary > 10 indicates strong evidence; 10 > Z-summary > 2 indicates moderate to weak evidence, and Z-summary

    Journal: bioRxiv

    Article Title: Single-cell RNA-seq analysis of human iPSC-derived motor neurons resolves early and predictive ALS signatures

    doi: 10.1101/2020.04.27.064584

    Figure Lengend Snippet: Distinct markers of neuronal subtypes are expressed as proteins in iPSC differentiated spinal MN cultures and enact distinct pathways in ALS conditions. Related to Figures 3 , 4 , and 5. A. Immunostaining day 18 cultures from one control subject line, 1034 for the expression of spinal MN marker, ISL1, a cranial MN marker, PHOX2B, a V2a interneuron marker, VSX2, and a V2c interneuron marker, SOX1. DAPI and SMI-32 are also used as counter stains. Scale bar, 50 um. B. Venn diagrams of overlapping gene sets between 1) sporadic ALS to CTR and 2) C9orf72 HRE ALS to CTR comparisons within the neuronal subtypes MN, V1 Renshaw, and V2a. Upregulated gene sets were only compared each other, and downregulated gene sets were only compared to each other. P-values of each intersection was calculated by a hypergeometric test. C. Enriched GO terms among genes significantly upregulated or downregulated in ALS compared to control neuronal subtypes are intersect in Venn diagrams. Representative GO terms are displayed from each overlapping or unique set. Full set of GO terms are shown in Table S7B-G. D. Left: measure of how well 52 modules defined in data set A are preserved in the modules defined in data set B. The Z-summary statistic (y-axis) for is plotted against module size (x-axis). Data points reflect module color. Dashed green line indicate threshold at Z = 10, and dashed blue line indicate threshold at Z = 2. For the likelihood of module preservation, Z-summary > 10 indicates strong evidence; 10 > Z-summary > 2 indicates moderate to weak evidence, and Z-summary

    Article Snippet: Primary antibody solution in blocking solution containing various combinations of goat polyclonal IgG anti-Human ISL1 (1:200) (R & D Systems AF1837, RRID: AB_2126324), mouse monoclonal IgG1 anti-NF-H (SMI-32) (1:200) (BioLegend 801701, RRID: AB_2564642), goat polyclonal anti-ChAT (1:200) (Millipore AB144P, RRID: AB_2079751), rabbit polyclonal IgG anti-PHOX2B (1:200) (GeneTex GTX109677, RRID: AB_1951223), mouse monoclonal IgG2b, rabbit polyclonal IgG anti-CHX10 (VSX2) (1:200) (Novus NBP1-84476, RRID: AB_11022841), and rabbit polyclonal IgG anti-SOX1 [EPR4766] 1:200) (GeneTex GTX62974) were incubated, rinsed with 0.2% Tween-20 in PBS, and incubated in species-specific Alexa-fluor secondary antibodies (1:2,000), and rinsed with 0.2% Tween-20 in PBS with DAPI staining.

    Techniques: Immunostaining, Expressing, Marker, Preserving

    Isl1 and Pou4f2 contribute quantitatively to the full levels of expression of downstream genes. A. Luciferase assays indicating that whereas Pou4f2 and Isl1 can each activate transcription from the reporter alone, together they promote transcription to a higher level. N = 3 for all transfections. Significance of differences was evaluated by student t test. Double black asterisks indicate p

    Journal: PLoS ONE

    Article Title: Isl1 and Pou4f2 Form a Complex to Regulate Target Genes in Developing Retinal Ganglion Cells

    doi: 10.1371/journal.pone.0092105

    Figure Lengend Snippet: Isl1 and Pou4f2 contribute quantitatively to the full levels of expression of downstream genes. A. Luciferase assays indicating that whereas Pou4f2 and Isl1 can each activate transcription from the reporter alone, together they promote transcription to a higher level. N = 3 for all transfections. Significance of differences was evaluated by student t test. Double black asterisks indicate p

    Article Snippet: Antibodies Antibodies used for Western blotting in this study include: monoclonal mouse anti-Isl1 (DSHB, 39.4D5, 1∶1000); polyclonal goat-anti-Isl1 (R & D systems, AF1837 1∶1000); polyclonal rabbit anti-Lhx1 (Santa-Cruz, sc-133735 1∶500); polyclonal goat anti-Lhx2 (Santa-Cruz, sc-19342, 1∶1000); polyclonal goat anti-Pou4f2 (Santa-Cruz, sc-6026, 1∶1000); polyclonal rabbit anti-GST (Sigma, G7781, 1∶1000); and monoclonal mouse anti-His (Santa-Cruz, sc-8036, 1∶500).

    Techniques: Expressing, Luciferase, Transfection

    EMSA with Isl1 truncations and Pou4f2. Left: Pou4f2 forms complexes with Isl1D3, Isl1D4 and Isl1D5 on SBRN3(W), but not with Isl1D1 and Isl1D2. Right: Pou4f2 forms complexes with Isl1D3, Isl1D4 and Isl1D5 on CBRN3(W), but not with Isl1D1 and Isl1D2. F indicates free probes.

    Journal: PLoS ONE

    Article Title: Isl1 and Pou4f2 Form a Complex to Regulate Target Genes in Developing Retinal Ganglion Cells

    doi: 10.1371/journal.pone.0092105

    Figure Lengend Snippet: EMSA with Isl1 truncations and Pou4f2. Left: Pou4f2 forms complexes with Isl1D3, Isl1D4 and Isl1D5 on SBRN3(W), but not with Isl1D1 and Isl1D2. Right: Pou4f2 forms complexes with Isl1D3, Isl1D4 and Isl1D5 on CBRN3(W), but not with Isl1D1 and Isl1D2. F indicates free probes.

    Article Snippet: Antibodies Antibodies used for Western blotting in this study include: monoclonal mouse anti-Isl1 (DSHB, 39.4D5, 1∶1000); polyclonal goat-anti-Isl1 (R & D systems, AF1837 1∶1000); polyclonal rabbit anti-Lhx1 (Santa-Cruz, sc-133735 1∶500); polyclonal goat anti-Lhx2 (Santa-Cruz, sc-19342, 1∶1000); polyclonal goat anti-Pou4f2 (Santa-Cruz, sc-6026, 1∶1000); polyclonal rabbit anti-GST (Sigma, G7781, 1∶1000); and monoclonal mouse anti-His (Santa-Cruz, sc-8036, 1∶500).

    Techniques:

    Pou4f2 and Isl1 bind to DNA elements individually and as a complex in EMSA. A: Wild-type (SBRN3(W)) sequence from the conserved element in the Sonic Hedgehog gene and its mutant sequence SBRN3(M) are shown on the top. Lines on top and beneath the SBRN3(W) sequence indicate the ATTA cores and Pou4f2 binding motif respectively. At Bottom, EMSA shows that either Pou4f2 or Isl1 alone can bind to SBRN3(W) as indicated by the slow migrating DNA-protein complexes, but not to SBRN3(M). When both Pou4f2 and Isl1 are present, they form a further slow-migrating DNA-protein complex with SBRN3(W), but not with SBRN3(M). B: Similarly, Pou4f2 or Isl1 alone binds to CBRN3(W), but not to CBRN3(M); Pou4f2 and Isl1 form a complex on CBRN3(W), but not on CBRN3(M). F indicates free probes. Lines on top and beneath the CBRN3(W) sequence indicate the ATTA cores and Pou4f2 binding motif respectively.

    Journal: PLoS ONE

    Article Title: Isl1 and Pou4f2 Form a Complex to Regulate Target Genes in Developing Retinal Ganglion Cells

    doi: 10.1371/journal.pone.0092105

    Figure Lengend Snippet: Pou4f2 and Isl1 bind to DNA elements individually and as a complex in EMSA. A: Wild-type (SBRN3(W)) sequence from the conserved element in the Sonic Hedgehog gene and its mutant sequence SBRN3(M) are shown on the top. Lines on top and beneath the SBRN3(W) sequence indicate the ATTA cores and Pou4f2 binding motif respectively. At Bottom, EMSA shows that either Pou4f2 or Isl1 alone can bind to SBRN3(W) as indicated by the slow migrating DNA-protein complexes, but not to SBRN3(M). When both Pou4f2 and Isl1 are present, they form a further slow-migrating DNA-protein complex with SBRN3(W), but not with SBRN3(M). B: Similarly, Pou4f2 or Isl1 alone binds to CBRN3(W), but not to CBRN3(M); Pou4f2 and Isl1 form a complex on CBRN3(W), but not on CBRN3(M). F indicates free probes. Lines on top and beneath the CBRN3(W) sequence indicate the ATTA cores and Pou4f2 binding motif respectively.

    Article Snippet: Antibodies Antibodies used for Western blotting in this study include: monoclonal mouse anti-Isl1 (DSHB, 39.4D5, 1∶1000); polyclonal goat-anti-Isl1 (R & D systems, AF1837 1∶1000); polyclonal rabbit anti-Lhx1 (Santa-Cruz, sc-133735 1∶500); polyclonal goat anti-Lhx2 (Santa-Cruz, sc-19342, 1∶1000); polyclonal goat anti-Pou4f2 (Santa-Cruz, sc-6026, 1∶1000); polyclonal rabbit anti-GST (Sigma, G7781, 1∶1000); and monoclonal mouse anti-His (Santa-Cruz, sc-8036, 1∶500).

    Techniques: Sequencing, Mutagenesis, Binding Assay

    Identification of domains within Pou4f2 and Isl1 responsible for their mutual interaction. A. Top: GST-Isl1 could interact with Pou4f2D4 and Pou4f2D5, but not Pou4f2D1, Pou4f2D2 or Pou4f2D3, as detected by anti-His. Full-length Pou4f2 serves as positive control. Middle: Input GST or GST-Isl1 detected by anti-GST. Bottom: Input Pou4f2 and its deletions could all be detected by anti-His. B: Top: GST-Pou4f2 (GST-P), but not GST, could interact with Isl1, Isl1D3, Isl1D4 and Isl1D5, but not with Isl1D1 and Isl1D2. Middle: Input GST or GST-Pou4f2 as detected by anti-GST. Bottom: Input Isl1 or its deletions as detected by a rabbit polyclonal anti-Isl1. Sizes (Kda) of protein markers are indicated on the side. Antibodies used for each blot are indicated at the bottom of each panel.

    Journal: PLoS ONE

    Article Title: Isl1 and Pou4f2 Form a Complex to Regulate Target Genes in Developing Retinal Ganglion Cells

    doi: 10.1371/journal.pone.0092105

    Figure Lengend Snippet: Identification of domains within Pou4f2 and Isl1 responsible for their mutual interaction. A. Top: GST-Isl1 could interact with Pou4f2D4 and Pou4f2D5, but not Pou4f2D1, Pou4f2D2 or Pou4f2D3, as detected by anti-His. Full-length Pou4f2 serves as positive control. Middle: Input GST or GST-Isl1 detected by anti-GST. Bottom: Input Pou4f2 and its deletions could all be detected by anti-His. B: Top: GST-Pou4f2 (GST-P), but not GST, could interact with Isl1, Isl1D3, Isl1D4 and Isl1D5, but not with Isl1D1 and Isl1D2. Middle: Input GST or GST-Pou4f2 as detected by anti-GST. Bottom: Input Isl1 or its deletions as detected by a rabbit polyclonal anti-Isl1. Sizes (Kda) of protein markers are indicated on the side. Antibodies used for each blot are indicated at the bottom of each panel.

    Article Snippet: Antibodies Antibodies used for Western blotting in this study include: monoclonal mouse anti-Isl1 (DSHB, 39.4D5, 1∶1000); polyclonal goat-anti-Isl1 (R & D systems, AF1837 1∶1000); polyclonal rabbit anti-Lhx1 (Santa-Cruz, sc-133735 1∶500); polyclonal goat anti-Lhx2 (Santa-Cruz, sc-19342, 1∶1000); polyclonal goat anti-Pou4f2 (Santa-Cruz, sc-6026, 1∶1000); polyclonal rabbit anti-GST (Sigma, G7781, 1∶1000); and monoclonal mouse anti-His (Santa-Cruz, sc-8036, 1∶500).

    Techniques: Positive Control

    Conserved DNA motifs in Ebf3 and Irx6 are recognized by Isl1/Pou4f2 complex. A: Mouse-human comparison to identify conserved regions in Ebf3 and Irx6 by VISTA. Red marks conserved non-coding region, light-blue is UTR, and dark-blue indicates exons. Heights of peaks indicate percentage of identify. The regions where the potential Isl1/Pou4f2 binding sites were identified are underlined. B: EMSA with wild-type and mutant probes derived from Ebf3 . The sequences of wild-type and mutant probes are shown on the top. Lines on top and underneath the wild-type sequences indicate the ATTA cores and Pou4f2 binding motif respectively. The bottom is the EMSA results. Both Isl1 and Pou4f2 could independently bind to the wild-type probe, but not the mutant probe. The Isl1/Pou4f2 complex can bind the wild-type probe, but not the mutant, as indicated by the slow-migrating complex. C: The same experiment as that of B, except that the wild-type and mutant probes were derived from Irx6 . Both proteins as well as the complex could bind to the wild-type probe, but not the mutant one. D: ChIP analysis of Isl1 and Pou4f2 binding in vivo. Genomic DNA from ChIP by anti-Pou4f2 (P4f2) and anti-Isl1 were amplified by PCR with primers flanking the Isl1/Pou4f2-binding regions of Ebf3 and Isl1 and a control region of β-actin. Non-specific IgG was used as control in both ChIP experiments.

    Journal: PLoS ONE

    Article Title: Isl1 and Pou4f2 Form a Complex to Regulate Target Genes in Developing Retinal Ganglion Cells

    doi: 10.1371/journal.pone.0092105

    Figure Lengend Snippet: Conserved DNA motifs in Ebf3 and Irx6 are recognized by Isl1/Pou4f2 complex. A: Mouse-human comparison to identify conserved regions in Ebf3 and Irx6 by VISTA. Red marks conserved non-coding region, light-blue is UTR, and dark-blue indicates exons. Heights of peaks indicate percentage of identify. The regions where the potential Isl1/Pou4f2 binding sites were identified are underlined. B: EMSA with wild-type and mutant probes derived from Ebf3 . The sequences of wild-type and mutant probes are shown on the top. Lines on top and underneath the wild-type sequences indicate the ATTA cores and Pou4f2 binding motif respectively. The bottom is the EMSA results. Both Isl1 and Pou4f2 could independently bind to the wild-type probe, but not the mutant probe. The Isl1/Pou4f2 complex can bind the wild-type probe, but not the mutant, as indicated by the slow-migrating complex. C: The same experiment as that of B, except that the wild-type and mutant probes were derived from Irx6 . Both proteins as well as the complex could bind to the wild-type probe, but not the mutant one. D: ChIP analysis of Isl1 and Pou4f2 binding in vivo. Genomic DNA from ChIP by anti-Pou4f2 (P4f2) and anti-Isl1 were amplified by PCR with primers flanking the Isl1/Pou4f2-binding regions of Ebf3 and Isl1 and a control region of β-actin. Non-specific IgG was used as control in both ChIP experiments.

    Article Snippet: Antibodies Antibodies used for Western blotting in this study include: monoclonal mouse anti-Isl1 (DSHB, 39.4D5, 1∶1000); polyclonal goat-anti-Isl1 (R & D systems, AF1837 1∶1000); polyclonal rabbit anti-Lhx1 (Santa-Cruz, sc-133735 1∶500); polyclonal goat anti-Lhx2 (Santa-Cruz, sc-19342, 1∶1000); polyclonal goat anti-Pou4f2 (Santa-Cruz, sc-6026, 1∶1000); polyclonal rabbit anti-GST (Sigma, G7781, 1∶1000); and monoclonal mouse anti-His (Santa-Cruz, sc-8036, 1∶500).

    Techniques: Binding Assay, Mutagenesis, Derivative Assay, Chromatin Immunoprecipitation, In Vivo, Amplification, Polymerase Chain Reaction

    GST pull-down assays show other Lim-Homeodomain and POU-domain proteins interact with Pou4f2 and Isl1 respectively. A: GST-Isl1 (GST-I), but not GST alone, can interact with Pou3f2 and Pou4f3, but not Pou6f1. Pou4f2 was used as positive control. Top: Detection of pulled down proteins by anti-His. Middle: Input GST or GST-Isl1 by anti-GST. Bottom: Input POU-Homeodmain proteins detected by anti-His. B,C,D: GST-Pou4f2 (GST-P), but not GST alone, can interact with Isl2, Lhx2, but not Lhx1. In each panel, the top is detection of pulled down protein by the indicated antibodies, the middle is input GST or GST-Pou4f2 as detected by anti-GST, and the bottom is detection of the input Lim-Homeodomain proteins by the indicated antibodies (anti-Isl1 for Isl1 and Isl2, anti-Lhx2 for Lhx2, and anti-His for Lhx1).

    Journal: PLoS ONE

    Article Title: Isl1 and Pou4f2 Form a Complex to Regulate Target Genes in Developing Retinal Ganglion Cells

    doi: 10.1371/journal.pone.0092105

    Figure Lengend Snippet: GST pull-down assays show other Lim-Homeodomain and POU-domain proteins interact with Pou4f2 and Isl1 respectively. A: GST-Isl1 (GST-I), but not GST alone, can interact with Pou3f2 and Pou4f3, but not Pou6f1. Pou4f2 was used as positive control. Top: Detection of pulled down proteins by anti-His. Middle: Input GST or GST-Isl1 by anti-GST. Bottom: Input POU-Homeodmain proteins detected by anti-His. B,C,D: GST-Pou4f2 (GST-P), but not GST alone, can interact with Isl2, Lhx2, but not Lhx1. In each panel, the top is detection of pulled down protein by the indicated antibodies, the middle is input GST or GST-Pou4f2 as detected by anti-GST, and the bottom is detection of the input Lim-Homeodomain proteins by the indicated antibodies (anti-Isl1 for Isl1 and Isl2, anti-Lhx2 for Lhx2, and anti-His for Lhx1).

    Article Snippet: Antibodies Antibodies used for Western blotting in this study include: monoclonal mouse anti-Isl1 (DSHB, 39.4D5, 1∶1000); polyclonal goat-anti-Isl1 (R & D systems, AF1837 1∶1000); polyclonal rabbit anti-Lhx1 (Santa-Cruz, sc-133735 1∶500); polyclonal goat anti-Lhx2 (Santa-Cruz, sc-19342, 1∶1000); polyclonal goat anti-Pou4f2 (Santa-Cruz, sc-6026, 1∶1000); polyclonal rabbit anti-GST (Sigma, G7781, 1∶1000); and monoclonal mouse anti-His (Santa-Cruz, sc-8036, 1∶500).

    Techniques: Positive Control

    Pou4f2 and Isl1 form a complex in vitro and in vivo. A. Top: GST-Pou4f2 fusion protein (GST-P), but not GST, can interact with Isl1, as detected by anti-Isl1. Bottom: anti-GST demonstrates the GST and GST-Pou4f2 proteins used in the pull-down assays. Smaller bands in the GST-Pou4f2 lane are degradation products. B. Top: GST-Isl1 fusion protein (GST-I), but not GST alone, can interact with Pou4f2 as detected by anti-Pou4f2. Bottom: anti-GST recognizes both GST and GST-Isl1. Like GST-Pou4f2, there was also degradation of GST-Isl1 as indicated by the smaller bands. C. Co-Immunoprecipitation (IP) of Isl1 and Pou4f2 by a rabbit anti-Isl1. Both proteins were expressed in the HEK293 cells and could be detected in the input lysate by a mouse anti-Isl1 and goat anti-Pou4f2. In the rabbit IgG control, neither Isl1 nor Pou4f2 could be detected. When rabbit anti-Isl1 was used for the IP, both Isl1 and Pou4f2 could be detected.

    Journal: PLoS ONE

    Article Title: Isl1 and Pou4f2 Form a Complex to Regulate Target Genes in Developing Retinal Ganglion Cells

    doi: 10.1371/journal.pone.0092105

    Figure Lengend Snippet: Pou4f2 and Isl1 form a complex in vitro and in vivo. A. Top: GST-Pou4f2 fusion protein (GST-P), but not GST, can interact with Isl1, as detected by anti-Isl1. Bottom: anti-GST demonstrates the GST and GST-Pou4f2 proteins used in the pull-down assays. Smaller bands in the GST-Pou4f2 lane are degradation products. B. Top: GST-Isl1 fusion protein (GST-I), but not GST alone, can interact with Pou4f2 as detected by anti-Pou4f2. Bottom: anti-GST recognizes both GST and GST-Isl1. Like GST-Pou4f2, there was also degradation of GST-Isl1 as indicated by the smaller bands. C. Co-Immunoprecipitation (IP) of Isl1 and Pou4f2 by a rabbit anti-Isl1. Both proteins were expressed in the HEK293 cells and could be detected in the input lysate by a mouse anti-Isl1 and goat anti-Pou4f2. In the rabbit IgG control, neither Isl1 nor Pou4f2 could be detected. When rabbit anti-Isl1 was used for the IP, both Isl1 and Pou4f2 could be detected.

    Article Snippet: Antibodies Antibodies used for Western blotting in this study include: monoclonal mouse anti-Isl1 (DSHB, 39.4D5, 1∶1000); polyclonal goat-anti-Isl1 (R & D systems, AF1837 1∶1000); polyclonal rabbit anti-Lhx1 (Santa-Cruz, sc-133735 1∶500); polyclonal goat anti-Lhx2 (Santa-Cruz, sc-19342, 1∶1000); polyclonal goat anti-Pou4f2 (Santa-Cruz, sc-6026, 1∶1000); polyclonal rabbit anti-GST (Sigma, G7781, 1∶1000); and monoclonal mouse anti-His (Santa-Cruz, sc-8036, 1∶500).

    Techniques: In Vitro, In Vivo, Immunoprecipitation

    Diagram of serial deletions of Pou4f2 and Isl1. Diagram of the Pou4f2 (Left) and Isl1 (right) protein structures and the truncated deletions we expressed in E. Coli . The amino acid residues for each deletion and their estimated molecular weights are indicated. All proteins are His-tagged at the C terminus for easy detection by Western Blotting, although the Isl1 protein and its truncates are detected by a polyclonal rabbit anti-Isl1. * indicates constructs capable of protein-protein interaction (see Figs. 3 5 ).

    Journal: PLoS ONE

    Article Title: Isl1 and Pou4f2 Form a Complex to Regulate Target Genes in Developing Retinal Ganglion Cells

    doi: 10.1371/journal.pone.0092105

    Figure Lengend Snippet: Diagram of serial deletions of Pou4f2 and Isl1. Diagram of the Pou4f2 (Left) and Isl1 (right) protein structures and the truncated deletions we expressed in E. Coli . The amino acid residues for each deletion and their estimated molecular weights are indicated. All proteins are His-tagged at the C terminus for easy detection by Western Blotting, although the Isl1 protein and its truncates are detected by a polyclonal rabbit anti-Isl1. * indicates constructs capable of protein-protein interaction (see Figs. 3 5 ).

    Article Snippet: Antibodies Antibodies used for Western blotting in this study include: monoclonal mouse anti-Isl1 (DSHB, 39.4D5, 1∶1000); polyclonal goat-anti-Isl1 (R & D systems, AF1837 1∶1000); polyclonal rabbit anti-Lhx1 (Santa-Cruz, sc-133735 1∶500); polyclonal goat anti-Lhx2 (Santa-Cruz, sc-19342, 1∶1000); polyclonal goat anti-Pou4f2 (Santa-Cruz, sc-6026, 1∶1000); polyclonal rabbit anti-GST (Sigma, G7781, 1∶1000); and monoclonal mouse anti-His (Santa-Cruz, sc-8036, 1∶500).

    Techniques: Western Blot, Construct

    EMSA with Pou4f2 truncations and Isl1. Left: Isl1 forms complexes with Pou4f2D4 and Pou4f2D5 on SBRN3(W), but not with Pou4f2D1, Pou4f2D2 and Pou4f2D3. Right: Isl1 forms complexes with Pou4f2 D4 and Pou4f2D5, but not with Pou4f2D1, Pou4f2D2 and Pou4f2D3 on CBRN(W). F is free probe. These results are consistent with those from the pull-down assays.

    Journal: PLoS ONE

    Article Title: Isl1 and Pou4f2 Form a Complex to Regulate Target Genes in Developing Retinal Ganglion Cells

    doi: 10.1371/journal.pone.0092105

    Figure Lengend Snippet: EMSA with Pou4f2 truncations and Isl1. Left: Isl1 forms complexes with Pou4f2D4 and Pou4f2D5 on SBRN3(W), but not with Pou4f2D1, Pou4f2D2 and Pou4f2D3. Right: Isl1 forms complexes with Pou4f2 D4 and Pou4f2D5, but not with Pou4f2D1, Pou4f2D2 and Pou4f2D3 on CBRN(W). F is free probe. These results are consistent with those from the pull-down assays.

    Article Snippet: Antibodies Antibodies used for Western blotting in this study include: monoclonal mouse anti-Isl1 (DSHB, 39.4D5, 1∶1000); polyclonal goat-anti-Isl1 (R & D systems, AF1837 1∶1000); polyclonal rabbit anti-Lhx1 (Santa-Cruz, sc-133735 1∶500); polyclonal goat anti-Lhx2 (Santa-Cruz, sc-19342, 1∶1000); polyclonal goat anti-Pou4f2 (Santa-Cruz, sc-6026, 1∶1000); polyclonal rabbit anti-GST (Sigma, G7781, 1∶1000); and monoclonal mouse anti-His (Santa-Cruz, sc-8036, 1∶500).

    Techniques:

    Ventral forebrain inactivation of Isl1 reduces Isl1 expression in the striatum as well as the reticular thalamus and leads to a disorganization of the striatonigral output pathway. At adult stages the ventral forebrain, inactivation of Isl1 causes a deletion

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: The LIM homeobox gene Isl1 is required for the correct development of the striatonigral pathway in the mouse

    doi: 10.1073/pnas.1308275110

    Figure Lengend Snippet: Ventral forebrain inactivation of Isl1 reduces Isl1 expression in the striatum as well as the reticular thalamus and leads to a disorganization of the striatonigral output pathway. At adult stages the ventral forebrain, inactivation of Isl1 causes a deletion

    Article Snippet: Primary antibodies were used at the following concentrations: rabbit anti-EGFP (1:500; Invitrogen), goat anti-EGFP (1:5,000; Abcam), guinea pig anti-µ opioid receptor (1:3,000; Millipore), rabbit anti-synapsin 1 (1:1,000; Millipore), rabbit anti-enkephalin (1:500; Millipore), rabbit anti-substance P (1:5,000; Millipore), rabbit anti-activated caspase 3 (1:250; Cell Signaling), rabbit anti-DARPP-32 (1:1,000; Millipore), goat anti-DARPP-32 (1:200; Santa Cruz), goat anti-Helios (1:200; Santa Cruz), goat anti-Ikaros (1:200; Santa Cruz), goat anti-Isl1 (1:2,000; R & D Systems), rabbit anti-phospoHistone 3 (pH3, 1:200; Millipore), guinea pig anti-Ascl1 (1:10,000; gift from J. Johnson, UT Southwestern, Dallas, TX), rabbit anti-DL X (1:500; gift from J. Kohtz, Northwestern University, Chicago, IL), rabbit anti-Gsx2 (1:5,000; Campbell Laboratory), rabbit anti-Isl1 (1:2,000; gift from T. Edlund, Umeå University, Umeå, Sweden), rabbit anti-Meis2 (1:2,000; gift from A. Buchberg, Thomas Jefferson University, Philadelphia, PA), rabbit anti-FoxP1 (1:4,000; gift from E. Morrisey, University of Pennsylvania, Philadelphia, PA), mouse anti-neurofilament 1 2H3 (1:200; developed by T. Jessell and J. Dodd and obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development and maintained by the University of Iowa, Department of Biology, Iowa City, IA 52242).

    Techniques: Expressing

    Striatal neuron differentiation and patch-matrix organization of Isl1 conditional mutant mice are disrupted. Ikaros is expressed in the developing cortical plate and in scattered cells of the striatum ( A ). Inset in A is a 25× magnification of

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: The LIM homeobox gene Isl1 is required for the correct development of the striatonigral pathway in the mouse

    doi: 10.1073/pnas.1308275110

    Figure Lengend Snippet: Striatal neuron differentiation and patch-matrix organization of Isl1 conditional mutant mice are disrupted. Ikaros is expressed in the developing cortical plate and in scattered cells of the striatum ( A ). Inset in A is a 25× magnification of

    Article Snippet: Primary antibodies were used at the following concentrations: rabbit anti-EGFP (1:500; Invitrogen), goat anti-EGFP (1:5,000; Abcam), guinea pig anti-µ opioid receptor (1:3,000; Millipore), rabbit anti-synapsin 1 (1:1,000; Millipore), rabbit anti-enkephalin (1:500; Millipore), rabbit anti-substance P (1:5,000; Millipore), rabbit anti-activated caspase 3 (1:250; Cell Signaling), rabbit anti-DARPP-32 (1:1,000; Millipore), goat anti-DARPP-32 (1:200; Santa Cruz), goat anti-Helios (1:200; Santa Cruz), goat anti-Ikaros (1:200; Santa Cruz), goat anti-Isl1 (1:2,000; R & D Systems), rabbit anti-phospoHistone 3 (pH3, 1:200; Millipore), guinea pig anti-Ascl1 (1:10,000; gift from J. Johnson, UT Southwestern, Dallas, TX), rabbit anti-DL X (1:500; gift from J. Kohtz, Northwestern University, Chicago, IL), rabbit anti-Gsx2 (1:5,000; Campbell Laboratory), rabbit anti-Isl1 (1:2,000; gift from T. Edlund, Umeå University, Umeå, Sweden), rabbit anti-Meis2 (1:2,000; gift from A. Buchberg, Thomas Jefferson University, Philadelphia, PA), rabbit anti-FoxP1 (1:4,000; gift from E. Morrisey, University of Pennsylvania, Philadelphia, PA), mouse anti-neurofilament 1 2H3 (1:200; developed by T. Jessell and J. Dodd and obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development and maintained by the University of Iowa, Department of Biology, Iowa City, IA 52242).

    Techniques: Mutagenesis, Mouse Assay

    In situ hybridization of E18.5 sections indicates that inactivation of Isl1 has a differential affect on factors implicated in striatonigral projection neuron development and axon pathfinding. The secreted ligand for the PlexinD1 receptor, Sema3e, is

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: The LIM homeobox gene Isl1 is required for the correct development of the striatonigral pathway in the mouse

    doi: 10.1073/pnas.1308275110

    Figure Lengend Snippet: In situ hybridization of E18.5 sections indicates that inactivation of Isl1 has a differential affect on factors implicated in striatonigral projection neuron development and axon pathfinding. The secreted ligand for the PlexinD1 receptor, Sema3e, is

    Article Snippet: Primary antibodies were used at the following concentrations: rabbit anti-EGFP (1:500; Invitrogen), goat anti-EGFP (1:5,000; Abcam), guinea pig anti-µ opioid receptor (1:3,000; Millipore), rabbit anti-synapsin 1 (1:1,000; Millipore), rabbit anti-enkephalin (1:500; Millipore), rabbit anti-substance P (1:5,000; Millipore), rabbit anti-activated caspase 3 (1:250; Cell Signaling), rabbit anti-DARPP-32 (1:1,000; Millipore), goat anti-DARPP-32 (1:200; Santa Cruz), goat anti-Helios (1:200; Santa Cruz), goat anti-Ikaros (1:200; Santa Cruz), goat anti-Isl1 (1:2,000; R & D Systems), rabbit anti-phospoHistone 3 (pH3, 1:200; Millipore), guinea pig anti-Ascl1 (1:10,000; gift from J. Johnson, UT Southwestern, Dallas, TX), rabbit anti-DL X (1:500; gift from J. Kohtz, Northwestern University, Chicago, IL), rabbit anti-Gsx2 (1:5,000; Campbell Laboratory), rabbit anti-Isl1 (1:2,000; gift from T. Edlund, Umeå University, Umeå, Sweden), rabbit anti-Meis2 (1:2,000; gift from A. Buchberg, Thomas Jefferson University, Philadelphia, PA), rabbit anti-FoxP1 (1:4,000; gift from E. Morrisey, University of Pennsylvania, Philadelphia, PA), mouse anti-neurofilament 1 2H3 (1:200; developed by T. Jessell and J. Dodd and obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development and maintained by the University of Iowa, Department of Biology, Iowa City, IA 52242).

    Techniques: In Situ Hybridization

    Isl1 fate-mapped synaptic terminals are found in the SNr and not the GP. ( A ) Recombined cells (EGFP+) are distributed throughout the telencephalon and diencephalon of a fate-mapped brain at P30 with synaptic terminal staining in the EP and SNr and not

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: The LIM homeobox gene Isl1 is required for the correct development of the striatonigral pathway in the mouse

    doi: 10.1073/pnas.1308275110

    Figure Lengend Snippet: Isl1 fate-mapped synaptic terminals are found in the SNr and not the GP. ( A ) Recombined cells (EGFP+) are distributed throughout the telencephalon and diencephalon of a fate-mapped brain at P30 with synaptic terminal staining in the EP and SNr and not

    Article Snippet: Primary antibodies were used at the following concentrations: rabbit anti-EGFP (1:500; Invitrogen), goat anti-EGFP (1:5,000; Abcam), guinea pig anti-µ opioid receptor (1:3,000; Millipore), rabbit anti-synapsin 1 (1:1,000; Millipore), rabbit anti-enkephalin (1:500; Millipore), rabbit anti-substance P (1:5,000; Millipore), rabbit anti-activated caspase 3 (1:250; Cell Signaling), rabbit anti-DARPP-32 (1:1,000; Millipore), goat anti-DARPP-32 (1:200; Santa Cruz), goat anti-Helios (1:200; Santa Cruz), goat anti-Ikaros (1:200; Santa Cruz), goat anti-Isl1 (1:2,000; R & D Systems), rabbit anti-phospoHistone 3 (pH3, 1:200; Millipore), guinea pig anti-Ascl1 (1:10,000; gift from J. Johnson, UT Southwestern, Dallas, TX), rabbit anti-DL X (1:500; gift from J. Kohtz, Northwestern University, Chicago, IL), rabbit anti-Gsx2 (1:5,000; Campbell Laboratory), rabbit anti-Isl1 (1:2,000; gift from T. Edlund, Umeå University, Umeå, Sweden), rabbit anti-Meis2 (1:2,000; gift from A. Buchberg, Thomas Jefferson University, Philadelphia, PA), rabbit anti-FoxP1 (1:4,000; gift from E. Morrisey, University of Pennsylvania, Philadelphia, PA), mouse anti-neurofilament 1 2H3 (1:200; developed by T. Jessell and J. Dodd and obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development and maintained by the University of Iowa, Department of Biology, Iowa City, IA 52242).

    Techniques: Staining

    Isl1 progenitors generate striatal-projection neurons. ( A ) Isl1 fate-mapped cells are distributed throughout the striatum and found in scattered cells of the septum at P30. ( B ) Majority of recombined neurons are medium-sized spiny striatal-projection

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: The LIM homeobox gene Isl1 is required for the correct development of the striatonigral pathway in the mouse

    doi: 10.1073/pnas.1308275110

    Figure Lengend Snippet: Isl1 progenitors generate striatal-projection neurons. ( A ) Isl1 fate-mapped cells are distributed throughout the striatum and found in scattered cells of the septum at P30. ( B ) Majority of recombined neurons are medium-sized spiny striatal-projection

    Article Snippet: Primary antibodies were used at the following concentrations: rabbit anti-EGFP (1:500; Invitrogen), goat anti-EGFP (1:5,000; Abcam), guinea pig anti-µ opioid receptor (1:3,000; Millipore), rabbit anti-synapsin 1 (1:1,000; Millipore), rabbit anti-enkephalin (1:500; Millipore), rabbit anti-substance P (1:5,000; Millipore), rabbit anti-activated caspase 3 (1:250; Cell Signaling), rabbit anti-DARPP-32 (1:1,000; Millipore), goat anti-DARPP-32 (1:200; Santa Cruz), goat anti-Helios (1:200; Santa Cruz), goat anti-Ikaros (1:200; Santa Cruz), goat anti-Isl1 (1:2,000; R & D Systems), rabbit anti-phospoHistone 3 (pH3, 1:200; Millipore), guinea pig anti-Ascl1 (1:10,000; gift from J. Johnson, UT Southwestern, Dallas, TX), rabbit anti-DL X (1:500; gift from J. Kohtz, Northwestern University, Chicago, IL), rabbit anti-Gsx2 (1:5,000; Campbell Laboratory), rabbit anti-Isl1 (1:2,000; gift from T. Edlund, Umeå University, Umeå, Sweden), rabbit anti-Meis2 (1:2,000; gift from A. Buchberg, Thomas Jefferson University, Philadelphia, PA), rabbit anti-FoxP1 (1:4,000; gift from E. Morrisey, University of Pennsylvania, Philadelphia, PA), mouse anti-neurofilament 1 2H3 (1:200; developed by T. Jessell and J. Dodd and obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development and maintained by the University of Iowa, Department of Biology, Iowa City, IA 52242).

    Techniques:

    Telencephalic specific deletion of Isl1 leads to a reduced striatonigral pathway. The striatonigral pathway as visualized by breeding the mice onto a DRD1-EGFP background is reduced in Isl1 conditional mice ( B ) compared with control mice ( A ). The striatopallidal

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: The LIM homeobox gene Isl1 is required for the correct development of the striatonigral pathway in the mouse

    doi: 10.1073/pnas.1308275110

    Figure Lengend Snippet: Telencephalic specific deletion of Isl1 leads to a reduced striatonigral pathway. The striatonigral pathway as visualized by breeding the mice onto a DRD1-EGFP background is reduced in Isl1 conditional mice ( B ) compared with control mice ( A ). The striatopallidal

    Article Snippet: Primary antibodies were used at the following concentrations: rabbit anti-EGFP (1:500; Invitrogen), goat anti-EGFP (1:5,000; Abcam), guinea pig anti-µ opioid receptor (1:3,000; Millipore), rabbit anti-synapsin 1 (1:1,000; Millipore), rabbit anti-enkephalin (1:500; Millipore), rabbit anti-substance P (1:5,000; Millipore), rabbit anti-activated caspase 3 (1:250; Cell Signaling), rabbit anti-DARPP-32 (1:1,000; Millipore), goat anti-DARPP-32 (1:200; Santa Cruz), goat anti-Helios (1:200; Santa Cruz), goat anti-Ikaros (1:200; Santa Cruz), goat anti-Isl1 (1:2,000; R & D Systems), rabbit anti-phospoHistone 3 (pH3, 1:200; Millipore), guinea pig anti-Ascl1 (1:10,000; gift from J. Johnson, UT Southwestern, Dallas, TX), rabbit anti-DL X (1:500; gift from J. Kohtz, Northwestern University, Chicago, IL), rabbit anti-Gsx2 (1:5,000; Campbell Laboratory), rabbit anti-Isl1 (1:2,000; gift from T. Edlund, Umeå University, Umeå, Sweden), rabbit anti-Meis2 (1:2,000; gift from A. Buchberg, Thomas Jefferson University, Philadelphia, PA), rabbit anti-FoxP1 (1:4,000; gift from E. Morrisey, University of Pennsylvania, Philadelphia, PA), mouse anti-neurofilament 1 2H3 (1:200; developed by T. Jessell and J. Dodd and obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development and maintained by the University of Iowa, Department of Biology, Iowa City, IA 52242).

    Techniques: Mouse Assay

    Telencephalic-specific deletion of Isl1 leads to a reduced striatonigral pathway. Immunostaining with DARPP-32 shows reduced striatal size and reduced innervation of SNr in the Isl1 conditional knockout ( B ) versus control mice ( A ) whereas innervation

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: The LIM homeobox gene Isl1 is required for the correct development of the striatonigral pathway in the mouse

    doi: 10.1073/pnas.1308275110

    Figure Lengend Snippet: Telencephalic-specific deletion of Isl1 leads to a reduced striatonigral pathway. Immunostaining with DARPP-32 shows reduced striatal size and reduced innervation of SNr in the Isl1 conditional knockout ( B ) versus control mice ( A ) whereas innervation

    Article Snippet: Primary antibodies were used at the following concentrations: rabbit anti-EGFP (1:500; Invitrogen), goat anti-EGFP (1:5,000; Abcam), guinea pig anti-µ opioid receptor (1:3,000; Millipore), rabbit anti-synapsin 1 (1:1,000; Millipore), rabbit anti-enkephalin (1:500; Millipore), rabbit anti-substance P (1:5,000; Millipore), rabbit anti-activated caspase 3 (1:250; Cell Signaling), rabbit anti-DARPP-32 (1:1,000; Millipore), goat anti-DARPP-32 (1:200; Santa Cruz), goat anti-Helios (1:200; Santa Cruz), goat anti-Ikaros (1:200; Santa Cruz), goat anti-Isl1 (1:2,000; R & D Systems), rabbit anti-phospoHistone 3 (pH3, 1:200; Millipore), guinea pig anti-Ascl1 (1:10,000; gift from J. Johnson, UT Southwestern, Dallas, TX), rabbit anti-DL X (1:500; gift from J. Kohtz, Northwestern University, Chicago, IL), rabbit anti-Gsx2 (1:5,000; Campbell Laboratory), rabbit anti-Isl1 (1:2,000; gift from T. Edlund, Umeå University, Umeå, Sweden), rabbit anti-Meis2 (1:2,000; gift from A. Buchberg, Thomas Jefferson University, Philadelphia, PA), rabbit anti-FoxP1 (1:4,000; gift from E. Morrisey, University of Pennsylvania, Philadelphia, PA), mouse anti-neurofilament 1 2H3 (1:200; developed by T. Jessell and J. Dodd and obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development and maintained by the University of Iowa, Department of Biology, Iowa City, IA 52242).

    Techniques: Immunostaining, Knock-Out, Mouse Assay

    Isl1 conditional mice are hyperactive and have altered responses to pharmacological reagents that stimulate the dopamine D1 receptor pathway. Horizontal locomotor activity of Isl1 fl/fl ; Dlx 5/6-CIE and control ( Isl1 fl/fl or Isl1 fl/+ ) mice is expressed

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: The LIM homeobox gene Isl1 is required for the correct development of the striatonigral pathway in the mouse

    doi: 10.1073/pnas.1308275110

    Figure Lengend Snippet: Isl1 conditional mice are hyperactive and have altered responses to pharmacological reagents that stimulate the dopamine D1 receptor pathway. Horizontal locomotor activity of Isl1 fl/fl ; Dlx 5/6-CIE and control ( Isl1 fl/fl or Isl1 fl/+ ) mice is expressed

    Article Snippet: Primary antibodies were used at the following concentrations: rabbit anti-EGFP (1:500; Invitrogen), goat anti-EGFP (1:5,000; Abcam), guinea pig anti-µ opioid receptor (1:3,000; Millipore), rabbit anti-synapsin 1 (1:1,000; Millipore), rabbit anti-enkephalin (1:500; Millipore), rabbit anti-substance P (1:5,000; Millipore), rabbit anti-activated caspase 3 (1:250; Cell Signaling), rabbit anti-DARPP-32 (1:1,000; Millipore), goat anti-DARPP-32 (1:200; Santa Cruz), goat anti-Helios (1:200; Santa Cruz), goat anti-Ikaros (1:200; Santa Cruz), goat anti-Isl1 (1:2,000; R & D Systems), rabbit anti-phospoHistone 3 (pH3, 1:200; Millipore), guinea pig anti-Ascl1 (1:10,000; gift from J. Johnson, UT Southwestern, Dallas, TX), rabbit anti-DL X (1:500; gift from J. Kohtz, Northwestern University, Chicago, IL), rabbit anti-Gsx2 (1:5,000; Campbell Laboratory), rabbit anti-Isl1 (1:2,000; gift from T. Edlund, Umeå University, Umeå, Sweden), rabbit anti-Meis2 (1:2,000; gift from A. Buchberg, Thomas Jefferson University, Philadelphia, PA), rabbit anti-FoxP1 (1:4,000; gift from E. Morrisey, University of Pennsylvania, Philadelphia, PA), mouse anti-neurofilament 1 2H3 (1:200; developed by T. Jessell and J. Dodd and obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development and maintained by the University of Iowa, Department of Biology, Iowa City, IA 52242).

    Techniques: Mouse Assay, Activity Assay