Fig. 1

Fig. 1

Adgra2^ouchless mislocalizes to the endoplasmic reticulum. (A) Schematic representation of Adgra2, Adgra2^ouchless and Adgra2^ΔLRR3 topology and domain organization. Adgra2^ouchless and Adgra2^ΔLRR3 lack the third LRR motif (red rectangle). The positions of the residue variations resulting from naturally occurring SNPs in adgra2^ouchless are designated by red asterisks. (B) Maximal intensity projection of a confocal z-stack of a WT Tg(kdrl:ras-mCherry) embryo at 36 hpf in lateral view. The red and yellow boxes define, respectively, the magnified areas of the hindbrain vasculature shown in C and the intersegmental vessels shown in E. Scale bar: 100 µm. (C) Maximal intensity projection of a confocal z-stack of WT and adgra2^s984/984 Tg(kdrl:ras-mCherry) embryos at 36 hpf in lateral view after injection of 100 pg of adgra2, adgra2-EGFP, adgra2^ouchless, adgra2^ouchless-EGFP, adgra2^ΔLRR3 or adgra2^ΔLRR3-EGFP mRNA at the one-cell stage. The red arrowheads point to the CtAs invading the hindbrain rhombomeres. Scale bar: 50 µm. (D) Single-plane confocal scans through enveloping layer cells of 5 hpf blastulas injected at the one-cell stage with 50 pg of lyn-RFP mRNA together with 100 pg of adgra2-EGFP, adgra2^ouchless-EGFP or adgra2^ΔLRR3-EGFP mRNA. Scale bar: 50 µm. (E) Single-plane confocal scans through the trunk intersegmental vessels of 30 hpf double-transgenic Tg(kdrl:ras-mCherry); Tg(fliep:Gal4FF) embryos injected at the one-cell stage with 25 pg of Tol2 transposase mRNA and 25 pg of the pTol2-5xUAS:adgra2-EGFP, pTol2-5xUAS:adgra2^ouchless-EGFP and pTol2-5xUAS:adgra2^ΔLRR3-EGFP constructs. Boxes define magnified views of the tip cells presented in the column on the right. Scale bar: 50 µm. (F) Single-plane direct fluorescence confocal scans of non-permeabilized HEK293T cells 48 h after transfection with GPI-RFP, mCherry-SEC61β, Adgra2-EGFP, Adgra2^ouchless-EGFP or Adgra2^ΔLRR3-EGFP encoding constructs. Cells were additionally transfected with reck and Wnt7a (mouse gene) expression constructs. Nuclei were counterstained with Hoechst. Scale bar: 10 μm.