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Research ArticleNew Research, Development

The Isl1-Lhx3 Complex Promotes Motor Neuron Specification by Activating Transcriptional Pathways that Enhance Its Own Expression and Formation

Madalynn Erb, Bora Lee, So Yeon Seo, Jae W. Lee, Seunghee Lee and Soo-Kyung Lee
eNeuro 28 March 2017, 4 (2) ENEURO.0349-16.2017; DOI: https://doi.org/10.1523/ENEURO.0349-16.2017
Madalynn Erb
1Papé Family Pediatric Research Institute, Department of Pediatrics, Oregon Health and Science University, Portland, OR 97239
2Vollum Institute, Oregon Health & Science University, Portland, OR 97239
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Bora Lee
1Papé Family Pediatric Research Institute, Department of Pediatrics, Oregon Health and Science University, Portland, OR 97239
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So Yeon Seo
3College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, Korea
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Jae W. Lee
1Papé Family Pediatric Research Institute, Department of Pediatrics, Oregon Health and Science University, Portland, OR 97239
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Seunghee Lee
3College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, Korea
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Soo-Kyung Lee
1Papé Family Pediatric Research Institute, Department of Pediatrics, Oregon Health and Science University, Portland, OR 97239
3College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, Korea
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  • Figure 1.
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    Figure 1.

    The Isl1-Lhx3 complex binds genomic loci associated with Isl1, Lhx3, and Lmo4. A, Isl1-Lhx3 complex binding sites, identified via ChIP-Seq, in association with Lhx3, Isl1, and Lmo4. B, A close-up of each ChIP-Seq peak. C–G, E12.5 mouse spinal cord ChIP performed with Isl1 or Lhx3 antibodies. qPCR was performed for Lhx3-Peak-A, Lhx3-Peak-B, the Isl1-Peak, the LMO4-Peak, and the negative control region, Untr6. Experiments were performed independently three times. Results shown are from a single representative experiment; n = 3 technical replicates. Results were analyzed with a one-way ANOVA followed by Holm multiple comparison analysis. **p < 0.01, compared to nonspecific IgG controls. Error bars represent the SEM.

  • Figure 2.
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    Figure 2.

    Lhx3-Peak-A is activated by the Isl1-Lhx3 complex. A, HxRE-Long and HxRE-Short sequences identified by ChIP-Seq de novo motif analysis, and the HxRE-Long and HxRE-Short sequences in Lhx3-Peak-A. B, Lhx3-Peak-A HxRE-Long and HxRE-Short mutants used for GFP-reporter experiments. C, GFP-reporter experiments for Lhx3-Peak-A variants, embryos were electroporated with Lhx3-Peak-A-GFP reporter constructs plus ubiquitously expressing LacZ to mark electroporated cells. Sections were immunostained for GFP and Mnr2 to mark MNs. Images are representative of electroporations from multiple embryos. Lhx3-Peak-A-wt: n = 5, Lhx3-Peak-A-ΔHx-L: n = 14, Lhx3-Peak-A-ΔHx-S: n = 12, Lhx3-Peak-A-ΔHx-LΔHx-S: n = 15. D, Embryos electroporated with Lhx3-Peak-A-GFP reporter construct plus Isl1-Lhx3 fusion protein construct. Sections were immunostained for GFP and Mnr2. Images are representative of electroporations from multiple embryos. Lhx3-Peak-A-wt: n = 25, Lhx3-Peak-A-ΔHx-L: n = 5, Lhx3-Peak-A-ΔHx-S: n = 13, Lhx3-Peak-A-ΔHx-LΔHx-S: n = 15. E, Luciferase assays testing Lhx3-Peak-A wt and mutants. Lhx3-Peak variants are the same as those used in GFP-reporter experiments. Luciferase assays performed in cultured P19 cells. Results show the luciferase activation fold upon the addition of Isl1 plus Lhx3, compared to empty vector; n = 5 independent experiments. Results were analyzed with a one-way ANOVA followed by Holm multiple comparison analysis, comparing each reporter construct to control reporter (no enhancer). *p < 0.05, **p < 0.01. Error bars represent the SEM.

  • Figure 3.
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    Figure 3.

    Lhx3-Peak-B is activated by the Isl1-Lhx3 complex. A, HxRE-Long sequence identified by ChIP-Seq de novo motif analysis, and the HxRE-Long sequences in Lhx3-Peak-B. B, Lhx3-Peak-B-wt and HxRE-Long mutant used for GFP-reporter experiments. C, GFP-reporter experiments for Lhx3-Peak-B variants. Embryos were electroporated with Lhx3-Peak-B-GFP reporter constructs plus ubiquitously expressing LacZ to mark electroporated cells. Sections were immunostained for GFP and Mnr2 to mark MNs. Images are representative of electroporations from multiple embryos. Lhx3-Peak-B-wt: n = 20, Lhx3-Peak-B-ΔHx-L: n = 4. D, Embryos electroporated with an Lhx3-Peak-B-GFP reporter construct plus Isl1-Lhx3 fusion protein construct. Sections were immunostained for GFP and Mnr2. Images are representative of electroporations from multiple embryos. Lhx3-Peak-B-wt: n = 7, Lhx3-Peak-B-ΔHx-L: n = 5. E, Luciferase assays testing Lhx3-Peak-B wt and HxRE-Long mutant. Luciferase assays performed in cultured P19 cells. Results show the luciferase activation fold upon the addition of Isl1 plus Lhx3, compared to empty vector; n = 4 independent experiments. Results were analyzed with a one-way ANOVA followed by Holm multiple comparison analysis, comparing each reporter construct to control reporter (no enhancer). Error bars represent the SEM.

  • Figure 4.
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    Figure 4.

    The Isl1-Peak is activated by the Isl1-Lhx3 complex. A, Schematic representation of HxRE-S1, HxRE-S2, 13 TAAT motifs, and A/T-rich motif within the Isl1-Peak. Yellow shading indicates the sequences included in the shortened Isl1-Peak-(1-409). B, HxRE-Short sequence identified by ChIP-Seq de novo motif analysis, and the HxRE-S1 sequences in Isl1-Peak. Isl1-Peak mutants used for GFP-reporter experiments. C, GFP-reporter experiments for Isl1-Peak variants. Embryos were electroporated with Isl1-Peak-GFP reporter constructs plus ubiquitously expressing LacZ to mark electroporated cells. Sections were immunostained for Mnr2 to mark MNs. Images are representative of electroporations from multiple embryos. Isl1-Peak-wt: n = 5, Isl1-Peak-ΔHx-S1: n = 6, Isl1-Peak-ΔH2: n = 5, Isl1-Peak-ΔA/T: n = 16, Isl1-Peak-ΔHx-S1ΔH2: n = 6. Isl1-Peak-(1-409)-wt: n = 17, Isl1-Peak-(1-409)-ΔHx-S1: n = 10, Isl1-Peak-(1-409)-ΔH2: n = 9, Isl1-Peak-(1-409)-ΔHx-S1ΔH2: n = 20. D, Embryos electroporated with an Isl1-Peak-GFP reporter construct plus Isl1-Lhx3 fusion protein construct. Sections immunostained for GFP and Mnr2. Images are representative of electroporations from multiple embryos. Isl1-Peak-wt: n = 6, Isl1-Peak-ΔHx-S1: n = 2, Isl1-Peak-ΔH2: n = 5, Isl1-Peak-ΔA/T: n = 3, Isl1-Peak-ΔHx-S1ΔH2: n = 4 . Isl1-Peak-(1-409)-wt: n = 5, Isl1-Peak-(1-409)-ΔHx-S1: n = 4, Isl1-Peak-(1-409)-ΔH2: n = 6, Isl1-Peak-(1-409)-ΔHx-S1ΔH2: n = 6. E, F, GFP fluorescence intensity for embryos electroporated with Isl1-Peak-GFP reporter constructs + LacZ; n = 4-12 embryos per condition. Results were analyzed with a one-way ANOVA followed by Holm multiple comparison analysis, comparing each mutant reporter construct to full-length wt-Isl1-Peak-GFP reporter or (E) wt-(1-409)-Isl1-Peak (F), *p < 0.05, **p < 0.01. Error bars represent the SEM.

  • Figure 5.
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    Figure 5.

    The LMO4-Peak is activated by the Isl1-Lhx3 complex. A, HxRE-Long and HxRE-Short sequences identified by ChIP-Seq de novo motif analysis, and the HxRE-Long and HxRE-Short sequences in the LMO4-Peak. B, LMO4-Peak HxRE-Long mutant used for GFP-reporter experiments. C, GFP-reporter experiments for LMO4-Peak variants. Embryos were electroporated with LMO4-Peak-GFP reporter constructs plus ubiquitously expressing LacZ to mark electroporated cells. Sections were immunostained for GFP and Mnr2 to mark MNs. Images are representative of electroporations from multiple embryos. LMO4-Peak-wt: n = 13, LMO4-Peak-ΔHx-L: n = 12. D, Embryos electroporated with LMO4-Peak-GFP reporter construct plus Isl1-Lhx3 fusion protein construct. Sections were immunostained for GFP and Mnr2. Images are representative of electroporations from multiple embryos. LMO4-Peak-wt: n = 4, LMO4-Peak-ΔHx-L: n = 5. E, Luciferase assays testing LMO4-Peak-wt and mutants. Luciferase assays performed in cultured P19 cells. Results show the luciferase activation fold upon the addition of Isl1 plus Lhx3, compared to empty vector; n = 5 independent experiments. Results were analyzed with a one-way ANOVA followed by Holm multiple comparison analysis, comparing each reporter construct to control reporter (no enhancer). **p < 0.01. Error bars represent the SEM.

  • Figure 6.
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    Figure 6.

    The Isl1-Lhx3 complex activates transcription of endogenous Isl1, Lhx3, and Lmo4. A–I, Embryos were electroporated with Isl1, Lhx3, or Isl1-Lhx3. In situ hybridization shows the transcription of endogenous Isl1, Lhx3, or Lmo4. Lightning bolts indicate the electroporated side of the embryo (right side), compared to the unelectroproated, control side (left side).

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March/April 2017
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The Isl1-Lhx3 Complex Promotes Motor Neuron Specification by Activating Transcriptional Pathways that Enhance Its Own Expression and Formation
Madalynn Erb, Bora Lee, So Yeon Seo, Jae W. Lee, Seunghee Lee, Soo-Kyung Lee
eNeuro 28 March 2017, 4 (2) ENEURO.0349-16.2017; DOI: 10.1523/ENEURO.0349-16.2017

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The Isl1-Lhx3 Complex Promotes Motor Neuron Specification by Activating Transcriptional Pathways that Enhance Its Own Expression and Formation
Madalynn Erb, Bora Lee, So Yeon Seo, Jae W. Lee, Seunghee Lee, Soo-Kyung Lee
eNeuro 28 March 2017, 4 (2) ENEURO.0349-16.2017; DOI: 10.1523/ENEURO.0349-16.2017
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Keywords

  • Isl1
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