Article Figures & Data
Figures
- 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.
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.
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.
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.
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.
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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