Neural retina-specific Aldh1a1 controls dorsal choroidal vascular development via Sox9 expression in retinal pigment epithelial cells
- Akishi Onishi
- Kazuyo Misaki
- Sunao Sugita
- Hiromi Ito
- Yoko Ohigashi
- Masatsugu Ema
- Hirokazu Sakaguchi
- Kohji Nishida
- Masayo Takahashi
- RIKEN Center for Developmental Biology, Japan
- Osaka University Graduate School of Medicine, Japan
- Kobe City Eye Hospital Research Center, Japan
- RIKEN Center for Life Science Technologies, Japan
- Shiga University of Medical Science, Japan
Figures
Figure 1 with 1 supplement
Aldh1a1 is predominantly expressed in the dorsal neural retina during embryonic and adult stages, and Aldh1a1–/– mice have less pigmentation and a thin choroid in the dorsal area.
(A–C) Section immunohistochemistry of the mouse retinas labeled with Aldh1a1, Aldh1a3 and glutamine synthetase (GS) antibodies. (A) At E12.5, Aldh1a1 (green) and Aldh1a3 (red) are expressed in …
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Figure 1—source data 1
Source Data for Figure 1F–H.
- https://doi.org/10.7554/eLife.32358.005
Figure 1—figure supplement 1
In situ hybridization (ISH) with the Aldh1a1 probe in the adult (8-week-old) mouse retina.
(A) Section ISH. Aldh1a1-positive cells were localized in the inner nuclear layer (INL) from the dorsal to the ‘boundary’ regions (black arrow) and the ventral edge (red arrow). Right panels show …
Figure 2
Hypoplasia at the dorsal side of choroid in Aldh1a1–/– mice.
(A) Representative choroidal flat-mount immunohistochemistry of adult (8-week-old) WT (left panel) and Aldh1a1–/– (right panel) posterior eyes stained for endomucin (Emcn, red) and isolectin B4 …
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Figure 2—source data 1
Source Data for Figure 2E,F.
- https://doi.org/10.7554/eLife.32358.007
Figure 3 with 1 supplement
Choroidal vessels of Aldh1a1–/– mice have features of capillaries.
(A) Electron micrographs of the dorsal and ventral areas from adult (8-week-old) WT and Aldh1a1–/– mice. The absence of choroidal pigmentation and a thin choroid layer is observed in the dorsal …
Figure 3—figure supplement 1
Choroidal vessels of the Aldh1a1–/– mice have both Flt1 and Kdr expression.
(A–D) Z-stack images of choroidal flat-mounts of adult (4-week-old) wild type (WT) and Aldh1a1–/–;Flt1-BAC-DsRed;Kdr-BAC-EGFP mice. Merged (A), Kdr-EGFP (C, green) and Flt1-DsRed (D, red) images …
Figure 4 with 2 supplements
Retinoic acids modulate VEGF secretion by RPE cells.
(A) Choroidal flat-mount immunohistochemistry of E16.5 WT and Aldh1a1–/– embryos stained for endomucin (Emcn, red) and ETS-related gene (ERG, green). Note that hypovascularization and fewer vascular …
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Figure 4—source data 1
Source Data for Figure 4B–D,F,H,J.
- https://doi.org/10.7554/eLife.32358.013
Figure 4—figure supplement 1
Developmental flat-mount immunohistochemical analysis of choroid in the Aldh1a1–/– mice.
P0–28 choroidal flat-mounts of WT and Aldh1a1–/– eyes were immunostained with the endomucin antibody (Emcn, red), and images of the dorsal and ventral areas were collected. Poor vascularization was …
Figure 4—figure supplement 2
Neural retina-specific conditional disruption of Aldh1a3 did not show choroidal hypoplasia in the ventral retina.
Choroidal flat-mount immunohistochemistry of adult (5-week-old) Aldh1a3 L2/L2;Pax6-αCre mice labeled with endomucin (Emcn, red) and ETS-related gene antibodies. Both dorsal and ventral choroids show …
Figure 5 with 1 supplement
Sox9 expression is downregulated in RPE cells of Aldh1a1–/– mice.
(A) Sox9 (green) and Aldh1a1 (red) staining of WT and Aldh1a1–/– eyes at E12.5. (B) Sox9 (white) expression in neural retina and RPE (white arrowheads) of WT and Aldh1a1–/– eyes at E12.5 (upper …
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Figure 5—source data 1
Source Data for Figure 5C–K.
- https://doi.org/10.7554/eLife.32358.016
Figure 5—figure supplement 1
Pax6 expression in the developing RPE cells of WT and Aldh1a1–/– mice.
(A) Section immunohistochemistry labeled with the Pax6 antibody (white) in neural retina and RPE cells of WT and Aldh1a1–/– eyes at E12.5 (upper panels) and E14.5 (lower panels). NR, neural retina; …
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Figure 5—figure supplement 1—source data 1
Source Data for Figure 5—figure supplement 1B and C.
- https://doi.org/10.7554/eLife.32358.017
Figure 6 with 1 supplement
RPE-derived Sox9 controls choroidal vasculature development.
(A) Endomucin expression (Emcn, red) in 5-week-old choroidal flat-mounts. RPE-specific conditional knockout of Sox9 (Sox9 RPE-cKO) mimicked the choroidal hypoplasia seen in Aldh1a1–/– mice (middle …
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Figure 6—source data 1
Source Data for Figure 6B and D.
- https://doi.org/10.7554/eLife.32358.020
Figure 6—figure supplement 1
Cre reporter assay of Tyr-Cre and Pax6-α-Cre mice using albinized R26R-H2B-mCherry mice.
(A) Section immunohistochemistry of E16.5 Tyr-Cre eyes labeled with the Sox9 (green, middle panel) antibody and ETS-related gene (ERG, green, right panel) antibody. Cre activity (mCherry, red) could …
Author response image 1
Representative choroidal flat-mount IHC of the central region of adult (8-week-old) WT (left panel) and Aldh1a1–/– (right panel) eyes immunostained with FITC-labeled isolectin B4 (IB4, green) and anti-endomucin antibody (Emcn, red).
There was no difference in choroidal vascular density between WT and Aldh1a1–/– mice.
Author response image 2
The frequency of appearance of pigmentation loss did not correspond with the principles of Mendelian inheritance.
https://doi.org/10.7554/eLife.32358.025
Author response image 3
Section IHCs of E17.5 mouse eyes labeled with anti-Aldh1a3 (green) and anti-Aldh1a1 (red) antibodies.
The region of Aldh1a3 expression did not expand.
Author response image 4
The representative dorsal region of choroidal flat-mounts of 8-week-old WT and Aldh1a1–/– mice stained with FITC-isolectin B4 (IB4, green), anti-endomucin (Emcn, red; upper panels), and anti-CD-31 (red; lower panels).
Emcn specifically visualized choriocapillaris, whereas CD-31 visualized medium-sized/large vessels that were IB4-positive.
Author response image 5
Representative dorsal choroidal flat-mounts of adult (8-week-old) WT (left panel) and Aldh1a1–/– (right panel) eyes immunostained with anti-endomucin (Emcn, red) and anti-ZO-1 (green) antibodies.
There is no difference in the size of RPE between WT and Aldh1a1–/– mice.
Tables
Table 1
Oligonucleotides used in this study
https://doi.org/10.7554/eLife.32358.021| Mouse primers | Oligonucleotide sequences (5’−3’) | |
|---|---|---|
| Aldh1a1-/- | Forward | TGAGCAAATCCTCCACAGCCCTGTTC |
| Reverse | CTGCTAAAGCGCATGCTCCAGACTG | |
| Aldh1a3 flox | Forward | TCTCTGACCAGCTTTCCAACCTTCAG |
| Reverse | CTCAAACCAGCACCACCTCCATATTG | |
| Sox9 flox | Forward | TCAGCAAGACTCTGGGCAAGCTCT |
| Reverse | CTCAAAATCTGAGCCACTCCCTC | |
| Tyr-Cre, Pax6-α-Cre | Forward | CCTGGAAAATGCTTCTGTCCGT |
| Reverse | GTGTCCACATAGTCATTGGCAGAGTG | |
| Sox9-Tg, Kdr-BAC-EGFP | Forward | AGCTGACCCTGAAGTTCATCTG |
| Reverse | GTCGTCCTTGAAGAAGATGGTG | |
| Flt1-BAC-tdsRed | Forward | GCTGCAGGCGCGGAGAAGGGCTCTC |
| Reverse | CTTCACGTACACCTTGGAGC | |
| IL2 (internal control) | Forward | GCCTAGAAGATGAACTTGGACCTCTG |
| Reverse | GTGGAAGGATTCACTTGCACAGTGAC | |
| Human RPE primers | ||
| Sox9 | Forward | CGTACCCGCACTTGCACAAC |
| Reverse | TCTCGCTCTCGTTCAGAAGTC | |
| Vegfa | Forward | TGCCCGCTGCTGTCTAAT |
| Reverse | TCTCCGCTCTGAGCAAGG | |
| β-actin | Forward | CCAACCGCGAGAAGATGA |
| Reverse | CCAGAGGCGTACAGGGATAG |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.32358.022
