(A) Representative eye section of a Wnt1-Cre;Ift88fx/+;mT/mG embryo at E14.5. NCC express the mG reporter (green cells) whereas cells from other embryonic origin express the mT reporter (red cells). …
(A) Breeding strategy to generate NCC ciliary mutant and visualize Cre expression. (B) Representative eye sections of control and cKO embryos at E14.5. NCC express the mG reporter (green cells) …
(A) Representative images of control and cKO eyes at E14.5 and E18.5. Enlarged images show the misorientation of the eyeball in the cKO embryos at E14.5 and the reduced size of the cornea at E18.5. …
(A) Representative ex vivo confocal optical sections taken at different levels (Anterior or Posterior) of the corneal stroma of E18.5 cKO and control embryos crossed with the mT/mG reporter. Here, …
1. An enucleated eye was placed with the cornea facing down on a glass-bottom microwell dish.2. A z-stack of confocal optical sections was acquired between the corneal epithelium and the lens …
(A) Gene expression was evaluated in corneas of E18.5 embryos by RT-qPCR. Data are mean ± SEM. Statistical significance was assessed using two-tailed Student’s t-test (n = 3 embryos/group). ns, …
(A) Workflow followed to collect the NCC from the eyeball by cell sorting. (B) Hh, Wnt and Notch target gene expression in NCC at E18.5. Fold change is expressed as mean ± SEM (n = 3 embryos/group, …
Hh activity assessed by Gli1-LacZ staining at adulthood (5 months). Hh signaling remains active in the ciliary body (empty arrow), in the choroid (orange arrows) and around the optic nerve …
(A) Cell proliferation was assessed at E14.5 in the cornea (delimited by the two yellow lines) and in two areas of the POM. The first area (0–20 µm) extended 20 µm from the white solid line, …
Cell proliferation was assessed at E14.5 in the cornea (delimited by the two white lines) and in two areas of the POM. The first area (0–20 μm) extended 20 μm from the yellow solid line, indicating …
(A) Expression of Dhh, Ihh and Shh in the cornea (including the iridocorneal angle), the neural retina and the posterior half of the eyeball without the neural retina (corresponding to the sclera, …
(A) Foxc1, Pax6 and Pitx2 expression in the cornea (including the iridocorneal angle) (n = 3–6 embryos/group). (B) Representative images of whole-mount corneas stained with an anti-endomucin Ab in …
Enucleated eye was placed facing down in a glass-bottom microwell dish. The observed field was centered on the major arterial circle (yellow line). A z-stack was acquired from the corneal epithelium …
(A) Representative images of whole-mount corneas stained with an anti-TUJ1 Ab in control and cKO embryos at E13.5 and E18.5. At E13.5, three to four nerves bundles (asterisk) are present in controls …
Representative maximum intensity projections of the epithelium in the center of the cornea of E18.5 embryos imaged on live tissues using the mT/mG reporter. Corneal epithelial cells are visualized …
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Strain, strain background (Mus musculus) | Ift88(tm1Bky) | (Haycraft et al., 2007), | RRID: MGI:6315331 | |
Strain, strain background (Mus musculus) | Wnt1-Cre | (Danielian et al., 1998), | RRID: IMSR_JAX:009107 | |
Strain, strain background (Mus musculus) | B6.129(Cg)-Gt(Rosa)26Sor(tm4(ACTB-tdTomato,-EGFP)Luo)/J | (Muzumdar et al., 2007) | Jackson laboratories stock No 007676 RRID: IMSR_JAX:007676 | |
Strain, strain background (Mus musculus) | Gli1(tm2Alj)/J | (Bai et al., 2002) | Jackson Laboratories stock No 008211 RRID: IMSR_JAX:008211 | |
Strain, strain background (Mus musculus) | Gt(ROSA)26Sor(tm1(Sstr3/GFP)Bky) | (O'Connor et al., 2013) | RRID: ISMR_JAX:024540 | |
Antibody | Anti-acetylated tubulin (mouse monoclonal) | Sigma-Aldrich | Cat #: 6-11B-1 RRID: AB_527348 | 1:1 |
Antibody | Anti-Arl13b (rabbit polyclonal) | ProteinTech Group | Cat #: 17711–1-AP RRID: AB_2060867 | 1:800 |
Antibody | Anti-BrdU (rat monoclonal) | Abcam | Cat #: ab6326 RRID: AB_305426 | 1:400 |
Antibody | Anti-CD31 (rat monoclonal) | BD Biosciences | Cat #: 550274 RRID: AB_393571 | 1:500 |
Antibody | Anti-endomucin (rat monoclonal) | eBioscience | Cat #: 14-5851-85 RRID: AB_891531 | 1:500 |
Antibody | Anti-Ki67 (rabbit polyclonal) | Abcam | Cat #: ab15580 RRID: AB_443209 | 1:300 |
Antibody | Anti-pSmad2/3 (goat polyclonal) | Santa Cruz | Cat #: sc11769 RRID: AB_2193189 | 1:200 |
Antibody | Anti-SMO (rabbit polyclonal) | MilliporeSigma | Cat #: ABS1001 | 1:100 |
Antibody | Anti-TUJ1 (rabbit monoclonal) | Covance | Cat #: MRB-435P RRID: AB_663339 | 1:500 |
Antibody | Alexa Fluor 594 goat anti-rabbit IgG | Invitrogen | Cat #: A27034 RRID: AB_2536097 | 1:200 |
Antibody | Alexa Fluor 647 donkey anti-rat | Jackson ImmunoResearch | Cat #: 712-605-153 RRID: AB_2340694 | 1:200 |
Antibody | FITC donkey anti-mouse | Jackson ImmunoResearch | Cat #: 715-096-151 RRID: AB_2340796 | 1:200 |
Antibody | FITC donkey anti-rat | Jackson ImmunoResearch | Cat #: 712-097-003 RRID: AB_2340655 | 1:200 |
Antibody | FITC goat anti-rat IgG2a | Bethyl | Cat #: A110-109F RRID: AB_67287 | 1:200 |
Antibody | TRITC donkey anti-goat | Jackson ImmunoResearch | Cat #: 705-025-147 RRID: AB_2340389 | 1:200 |
Antibody | TRITC donkey anti-rabbit | Jackson ImmunoResearch | Cat #: 711-025-152 RRID:AB_2340588 | 1:200 |
Chemical compound, drug | BrdU | Sigma-Aldrich | Cat #: B5002 | 10 mg/mL |
Chemical compound, drug | Collagenase | Sigma | Cat #: C2674 | 8 mg/mL |
Chemical compound, drug | SMO agonist (SAG) | Calbiochem | Cat #: 566660 | 100 nM |
Chemical compound, drug | X-gal | LabScientific | Cat #: X266 | |
Commercial assay or kit | RNeasy microkit | Qiagen | Cat #: 74004 | |
Software, algorithm | Fiji | (Schindelin et al., 2012) | RRID: SCR_002285 | |
Software, algorithm | Ilastik | (Haubold et al., 2016) | RRID: SCR_015246 |
Table: RT-qPCR primers.