The LIM protein complex establishes a retinal circuitry of visual adaptation by regulating Pax6 α-enhancer activity
- Korea Advanced Institute of Science and Technology (KAIST), South Korea
- Chungbuk National University School of Medicine, South Korea
- The Catholic University of Korea, South Korea
- Showa University School of Medicine, Japan
- Gwangju Institute of Science and Technology (GIST), South Korea
- Vanderbilt University, United States
- KAIST Institute of BioCentury, South Korea
Figures
Figure 1 with 3 supplements
Identification of Lhx3 and Tgfb1i1 as Pax6 α-enhancer binding proteins.
(A) (Top) The genomic structure of the mouse Pax6 gene. Exons are shown as boxes, and arrows denote transcription initiation sites. (Bottom) The DF3, PBS, and DF4 sequences in the retina-specific …
Figure 1—figure supplement 1
Lhx3 and Tgfb1i1 expression in embryonic and mature mouse retinas.
E14.5 and P30 P6α-CreiGFP mouse retinas stained with anti-Lhx3 (A) and anti-Tgfb1i1 (B) antibodies. Lhx3 is absent in E14.5 mouse retinas but expressed in bipolar cell subsets in post-natal (P8, Figu…
Figure 1—figure supplement 2
Binding abilities of Lhx3 and Tgfb1i1 to Pax6 α-enhancer sequence.
(A) P7 retinal nuclear extracts were incubated with either the wild-type DF4 (DF4-WT) dsDNA oligomers used in Figure 1B or mutant DF4 dsDNA oligomers (DF4-Mut) in which the homeobox core binding …
Figure 1—figure supplement 3
Relationship between LIM domain transcription factor expression and Pax6 α-enhancer activity in mouse retina.
(A) P14 P6α-CreiGFP mouse retinas stained with rabbit antibodies recognizing LIM domain transcription factors (LIM-TF), Isl1, Lhx2, Lhx3, and Lhx9, and a mouse antibody recognizing GFP, which …
Figure 2
Lhx3 and Isl1 inhibit Pax6 α-enhancer activity in a Tgfb1i1-sensitive manner.
(A) The effects of LIM domain transcription factors on Pax6 α-enhancer activity were measured with a Pax6 α-enhancer luciferase reporter in HEK293T cells. These cells were co-transfected with DNA …
Figure 3 with 1 supplement
Pax6 and Tgfb1i1 antagonistically regulate Isl1-Lhx3 complex formation.
(A) Interactions between endogenous Isl1, Lhx3, and Tgfb1i1 in P7 mouse retinas measured by reciprocal co-immunoprecipitation (co-IP) and subsequent Western blotting (WB) with the indicated …
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Figure 3—source data 1
Protein-protein interaction between LIM proteins.
- https://doi.org/10.7554/eLife.21303.009
Figure 3—figure supplement 1
Pax6 and Tgfb1i1 antagonistically regulate Isl1-Lhx3 complex formation.
(A) Schematics for the full-length and deletion mutants of Isl1, Lhx3, Pax6, and Tgfb1i1 used in these experiments. HD, homeodomain; LIM, LIM domain; LBD, LIM binding domain; LD, leucin-rich domain; …
Figure 4 with 2 supplements
Elevated GABAergic amacrine cell number in Tgfb1i1−/−mouse retinas.
(A) Pax6 α-enhancer-active cells in P14 Tgfb1i1+/+ and Tgfb1i1−/−littermate mouse retinas were visualized by immunodetection of GFP expressed from the P6α-CreiGFP transgene. ONL, outer nuclear layer;…
Figure 4—figure supplement 1
Elevation of Pax6 α-enhancer-active GABAergic amacrine cells in Tgfb1i1−/−mouse retinas.
(A) P14 Tgfb1i1+/+;P6α-CreiGFP and Tgfb1i1−/−;P6α-CreiGFP littermate mouse retinas co-stained with antibodies against amacrine cell subtype markers and GFP. Pax6, pan-amacrine cell marker; ChAT, …
Figure 4—figure supplement 2
Deletion of Lhx3 in the post-natal mouse retina.
(A) To delete Lhx3 in the post-natal mouse retina, we designed two independent sgRNAs complementary to the sequences near the translation initiation site in the exon2 (highlighted in red), following …
Figure 5 with 2 supplements
Pax6 α-enhancer-induced Pax6ΔPD isoform supports GABAergic amacrine cell fate.
(A) Reciprocal co-IP and WB analyses with the indicated antibodies reveal a reduced interaction between Isl1 and Lhx3 in P7 Tgfb1i1−/−mouse retinas compared with littermate Tgfb1i1+/+retinas (top …
Figure 5—figure supplement 1
Distribution of Isl1- and Lhx3-expressing cells in Tgfb1i1+/+ and Tgfb1i1−/− mouse retinas.
P7 (A) and P14 (B) Tgfb1i1+/+ and Tgfb1i1−/− littermate mouse retinas stained with a guinea pig anti-Isl1 antibody (green) and a rabbit anti-Lhx3 antibody (red). Images in the right columns are …
Figure 5—figure supplement 2
Ectopic expression of Pax6 isoforms in the post-natal mouse retinas.
(A) P14 mouse retinas, which had been electroporated with the indicated DNA constructs at P0, were stained for the detection of various amacrine cell markers, including Syntaxin (pan-amacrine), …
Figure 6 with 3 supplements
Pax6-dependent Pax6 α-enhancer activation is positively correlated with GABAergic amacrine cell number.
(A) Genomic DNA was isolated from the tails of Pax6+/+(left) and Pax6ΔPBS/ΔPBS (right) mice for sequencing the Pax6 α-enhancer region. The Pax6 binding sequence (PBS) in the α-enhancer is colored …
Figure 6—figure supplement 1
Impaired response of the Pax6ΔPBS α-enhancer to Pax6.
(A) Luciferase expression at downstream of a Pax6 α-enhancer mutant lacking its PBS (Pax6-αΔPBS) was measured by detecting chemiluminescence emitted from the lysates of HEK293T cells combinatorially …
Figure 6—figure supplement 2
Distribution of amacrine and bipolar cell subsets in Pax6+/+ and Pax6ΔPBS/ΔPBS mouse retina.
P14 Pax6+/+ and Pax6ΔPBS/ΔPBS littermate retinas co-stained with amacrine cell and bipolar cell subtype marker-specific antibodies. Gad67, GABAergic amacrine cells; ChAT, cholinergic amacrine cells; …
Figure 6—figure supplement 3
Fate determination of GABAergic amacrine cells and OFF bipolar cells in the post-natal mouse retinas.
(A) The effects of deletions of Tgfb1i1 (Tgfb1i1−/−) and PBS sequence of Pax6 α-enhancer (Pax6ΔPBS/ΔPBS) on GABAergic amacrine cell development were investigated by immunostaining of various …
Figure 7 with 3 supplements
Pax6 α-enhancer-active amacrine cells are important for visual adaptation.
(A) Visual acuity was measured in P60 mice using the OptoMotry system as previously described (Prusky et al., 2004) (for details, see the Materials and Methods). Error bars indicate STD (n = 6). **p<…
Figure 7—figure supplement 1
ERGs of mouse retinas.
P60 WT, Tgfb1i1−/−, and Pax6ΔPBS/ΔPBS mice were dark-adapted for 16 hr. Then, their scotopic ERG responses were assessed at a light intensity of 2.5 cds (left). Average amplitudes of scotopic ERG …
Figure 7—figure supplement 2
Cell composition of P60 WT, Tgfb1i1−/−, and Pax6ΔPBS/ΔPBS mouse retinas.
(A) Composition of P60 WT, Tgfb1i1−/−, and Pax6ΔPBS/ΔPBS mouse retinas were determined by examining cell type-specific markers. Rhodopsin, rod photoreceptors; M-opsin, M-cone photoreceptors; …
Figure 7—figure supplement 3
Experimental scheme assessing mouse visual responses.
(A) P60 mice were trained to associate water rewards with flashing light stimuli. Correct and incorrect lick rates were used to measure visual detection. (B) Lick rates during the learning period …
Tables
Table 1
Antibody used in this study.
Antigen | Species | Producer | Dilution |
|---|---|---|---|
Bhlhb5 | Goat | Santa Cruz | 1:100 |
Brn3b | Goat | Santa Cruz | 1:200 |
Calbindin | Mouse | Sigma | 1:200 |
Calretinin | Mouse | Millipore | 1:1000 |
ChAT | Goat | Millipore | 1:200 |
Isl1 | Rabbit | gift from Dr. Mi-Ryoung Song | 1:500 |
Isl1 | Guinea Pig | gift from Dr. Mi-Ryoung Song | 1:10,000 |
Gad67 | Mouse | Millipore | 1:500 |
GABA | Guinea Pig | Millipore | 1:300 |
GFAP | Rabbit | Abcam | 1:500 |
GFP | Chick | Abcam | 1:200 |
GFP | Rabbit | Santa Cruz | 1:500 |
GlyT1 | Rabbit | Abcam | 1:200 |
G0α | Mouse | Millipore | 1:300 |
G/R opsin | Rabbit | Millipore | 1:200 |
Lhx2 | Goat | Santa Cruz | 1:200 |
Lhx3 | Rabbit | Abcam | 1:1000 |
Lhx9 | Rabbit | Santa Cruz | 1:500 |
Pax6 | Rabbit | Abcam | 1:200 |
Pax6 | Rabbit | Covance | 1:300 |
PKCα | Mouse | Sigma | 1:200 |
Recoverin | Rabbit | Chemicon | 1:200 |
Rhodopsin | Mouse | Millipore | 1:500 |
Sox2 | Goat | Santa Cruz | 1:100 |
Sox9 | Rabbit | Santa Cruz | 1:200 |
Tgfb1i1(Hic-5) | Mouse | BD | 1:100 |
Tgfb1i1(Hic-5) | Rabbit | Abcam | 1:100 |
Vsx1 | Goat | Santa Cruz | 1:50 |
Vsx2(Chx10) | Mouse | Santa Cruz | 1:200 |
V5 | Mouse | Genway Biotech | 1:1000 |
