Fig 1: The Fz4 C-terminal PDZ-binding motif binds to PDZ domains 1 and 2 of Dlg1.(A) Co-immunoprecipitation followed by immunoblotting to assess the interactions between Myc-Dlg1 and Rim-Fz4. HEK/293T cells were co-transfected with Myc-Dlg1 and WT Rim-Fz4, Rim-Fz4(4A), or an empty expression vector. Co-immunoprecipitation was performed with anti-Myc antibody (left) or anti-Rim antibody (right). (B) Schematic summarizing the results in (A). Rim-Fz4 is depicted in the plasma membrane (horizontal black lines) with its Rim-tagged N-terminus in the extracellular space (top) and its C-terminus in the cytoplasm (bottom). (C) HEK/293T cells were co-transfected with Rim-Fz4 and either WT Myc-Dlg1, the indicated PDZ deletion derivatives (?PDZ), or an empty expression vector. Co-immunoprecipitation was performed with anti-Myc antibody (left) or anti-Rim antibody (right). In (C) and (E), the red dot indicates a contaminant, which is variably present. (D,E) HEK/293T cells were transfected with WT Myc-Dlg1, the indicated PDZ deletion derivatives (?PDZ), or an empty expression vector. Soluble cytosolic proteins from the transfected cells were captured with an N-terminally biotinylated synthetic peptide corresponding to the C-terminal 12 amino acids of Fz4. (F) MBP was fused at its C-terminus to PDZ1, PDZ2, or PDZ1+2 of Dlg1 and the resulting fusion proteins were expressed in E. coli. The MBP fusion proteins were purified and then tested for binding to an N-terminally biotinylated synthetic peptide corresponding to the C-terminal 12 amino acids of Fz4 or the C-terminal 11 amino acids of bovine rhodopsin, which lacks a PDZ-binding motif. (G) Summary of the binding results between Dlg1 and Fz4. For each interaction examined, a score of - (no binding), +/- (little to no binding), + (minimal binding), or +++ (robust binding) was assigned. Blank spaces signify that binding was not assessed.
Fig 2: Stabilizing beta-catenin in endothelial cells corrects the retinal vascular defect caused by loss of Dlg1.(A–D) P7 retinas from the indicated genotypes, with the boxed region in column 1 shown at higher magnification in columns 2 and 3, and the boxed region in column 4 shown at higher magnification in column 5. Mice were injected IP with 2–3 mg of sulfo-NHS-biotin 1–2 hr before sacrifice. Dashed white lines indicate the edge of the retina. (E–H) Quantification of vascular density (E), vascular coverage (F), the fraction of vessels that immunostain for Claudin5 (G), and the fraction of vessels that immunostain for PLVAP (H), for the genotypes shown in (A–D). Scale bars for columns 1 and 4, 400 µm. Scale bars for columns 2, 3, and 5, 200 µm.
Fig 3: Mutation of the Fz4 C-terminal PDZ-binding domain enhances the severity of retinal vascular defects in a Dlg1 mutant background.(A) Diagram of the Fz4 allele produced by CRISPR/Cas9 editing (Fz4MGK). Numbers indicate exons. TVV, the C-terminal three amino acids, were substituted with MGK. Right, sequencing chromatograms from mouse tail PCR products. Boxes encompass the three altered codons. Top, WT allele. Bottom, Fz4MGK allele. (B) Protein extracts from HEK/293T cells transfected with WT Myc-Dlg1 (+) or an empty expression vector (–) were subjected to affinity capture using a biotin-Fz4 WT C-terminal peptide, a biotin-Fz4 peptide with the C-terminal three amino acids substituted by MGK, or a biotin-rhodopsin C-terminal peptide. Myc-Dlg1 binds only to the Fz4 WT C-terminal peptide. (C) Immunoblot of protein extracts from HEK/293T cells transfected with Rim-Fz4 WT, Rim-Fz4(4A) or Rim-Fz4(MGK), i.e., Fz4 with the C-terminal three amino acids changed to MGK. (D–F) Maximum projection of the superficial, intermediate, and deep vascular plexuses of P18 retinas from the indicated genotypes (column 1) with boxed regions enlarged in columns 2–5. Mice were injected IP with 2–3 mg of sulfo-NHS-biotin 1–2 hr before sacrifice. Arrow in (E) indicates biotin leakage. Scale bar for column 1, 400 µm. Scale bar for columns 2–5, 200 µm. (G–I) Quantification of summed vascular density (G), the fraction of vessels that immunostain for Claudin5 (H), and the fraction of vessels that immunostain for PLVAP (I), for the genotypes shown in (D–F).
Fig 4: Dlg1 genetically interacts with Tspan12 in the developing retinal vasculature.(A–E) Maximum projection of the superficial, intermediate, and deep vascular plexuses of P18 retinas from the indicated genotypes (column 1), with boxed regions displayed at higher magnification in columns 2–5. Mice were injected IP with 2–3 mg of sulfo-NHS-biotin 1–2 hr before sacrifice. Scale bar for column 1, 400 µm. Scale bar for columns 2–5, 200 µm. (F–H) Quantification of vascular density (F), the fraction of vessels that immunostain for Claudin5 (G), and the fraction of vessels that immunostain for PLVAP (H), for the genotypes shown in (A–E) and Figure 1J.
Fig 5: Dlg1 promotes angiogenesis in the mammalian retina.(A–B) The superficial vascular plexus of P7 retinas from the indicated genotypes (column 1), with boxed regions shown at higher magnification (columns 2 and 3). Dashed white lines indicate the edge of the retina. (C) Quantification of vascular density (left) and vascular coverage (right) in P7 retinas, for the genotypes in (A) and (B). Quantification methodology in this and other figures is described in the Materials and methods section. In this and all other figures, bars represent mean ± SD. Statistical significance, determined by the unpaired t-test, is represented by * (p<0.05), ** (p<0.01), *** (p<0.001), and **** (p<0.0001). (D) Quantification of the fraction of vessel length that is Claudin5+ (left) and PLVAP+ (right) in P7 retinas, for the genotypes in (A) and (B). (E–F) The deep vascular plexus of P14 retinas (column 1) with boxed regions shown at higher magnification (columns 2 and 3). (G) Quantification of vascular density in the superficial plexus (left) and deep plexus (right) in P14 retinas for the genotypes in (E) and (F). (H) As in (D), except with P14 retinas. (I–J) Maximum projection of superficial, intermediate, and deep vascular plexuses of P18 retinas (column 1) with boxed regions shown at higher magnification (columns 2 and 3). (K) As in (G), except with P18 retinas. (L) As in (D), except with P18 retinas. Scale bar for column 1, 400 µm. Scale bar for columns 2 and 3, 200 µm.
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