Figure S1. Fz4 expression in adult vasculature.
(A,B) 100 um vibratome sections of a Fz4CKOAP/+;Tie2-Cre adult brain stained histochemically for AP. The Fz4AP allele is expressed throughout the vasculature. Scale bar, 200 um.
(C) Flat mount of Fz4CKOAP/+;PDGFRB-Cre adult retina stained histochemically for AP. Cre-mediated recombination is incomplete and expression of Fz4AP is limited to MCs. Circumferentially oriented vSMCs encircle the artery shown at the center-right of the image. Scale bar, 50 um.
(D) Cross sections of adult Fz4CKOAP/+;Sox2-Cre retina showing AP expression in photoreceptors, inner retinal neurons, and vasculature. Note that AP is enriched in regions with high membrane content: photoreceptor outer segments and inner and outer plexiform layers. OS, outer segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Red arrowheads, vasculature. Scale bar, 50 um.
(E) Cross sections of adult Fz4CKOAP/CKOAP;Sox2-Cre retina showing AP expression in photoreceptors, inner retinal neurons, and vasculature. OS, outer segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. A tortuous vasculature is seen at the vitreal face of the retina and the intra-retinal capillaries are absent. Red arrowheads, vasculature.
Figure S2. Cross sections of Fz4CKOAP/−;Rx-Cre and Fz4CKOAP/−;PDGFRB-Cre adult retinas show vascular patterns indistinguishable from WT.
Cross sections of adult retina show the normal two tiers of capillaries flanking the inner nuclear layer. CC, choriocapillaris; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar, 50 um.
Figure S3. The absence of Fz4 signaling has little effect on retinal cell death, horizontal or amacrine cell abundance, or inner plexiform layer lamination.
(A) TUNEL labeling at P21-P23 shows only occasional apoptotic cells in WT or mutant retinas. DNase treated retinal section serves as a positive control for the TUNEL method.
(B) Quantification of TUNEL+ nuclei at P21-P23 in the three principal retinal layers and in the intraretinal vascular clusters in the mutant retinas. For each genotype, nine retinal sections were scored. None of the comparisons between pairs of mutant lines or between mutant and WT lines are statistically significant.
(C–D) Immunostaining of WT retinas at one month of age and Fz4−/−retinas at one and two months of age with antibodies to calbindin (horizontal cells and amacrine cells), calretinin (amacrine cells), and Thy-1 (RGCs). Among the labeled cells, the abundance, overall morphology, and lamination appear roughly normal in the Fz4−/− retina at these ages.
Figure S4. Construction of an Ndp knockout allele and loss of activity in the resulting C-terminally truncated Norrin protein.
(A) The targeted Ndp
allele is missing the C-terminal 12 amino acids as shown in the amino acid sequence alignment at the bottom of this panel. The deleted region includes cysteines predicted to participate in three conserved disulfide bonds (Berger and Ropers, 2001
(B) Canonical Wnt signaling activity of WT vs. the C-terminally truncated Norrin encoded by the Ndp−
allele. Cotransfection of a luciferase reporter cell line (Xu et al., 2004
) with expression plasmids coding for Fz4, Norrin, and Lrp5 show that the C-terminal Norrin mutant (mNorrin delta-C) is inactive. Bars, SD.
Figure S5. Ultrastructural differences between WT and Fz4−/− cerebellar capillaries.
(A,B) WT cerebellar capillaries show smooth oval cross-sectional profiles and are largely covered by pericytes. WT ECs exhibit tight junctions and have smooth luminal faces. Black arrows, pericyte nuclei. Black arrowhead, endothelial tight junctions.
(C–H) Fz4−/− cerebellar capillaries show variable cross-sectional profiles, ranging from relatively smooth ovals (panel C) to irregular and eccentric cross sections (panels E and G). ECs were observed to have tight junctions (panels D and F) and in some cases numerous small protrusions into the vessel lumen (panel F). Extensive regions devoid of pericyte coverage (panel E) and local detachments between ECs and pericytes were occasionally observed (red arrowhead, panel H). Black arrows, pericyte nuclei; black arrowheads, tight junctions between ECs.
Figure S6. Presence and relative abundances of Lrp5 and Lrp6 transcripts in REC lines and in E10.5 embryos and yolk sacs, as determined by RT-PCR. In WT and Fz4−/− REC lines, Lrp5 transcripts are ~2-fold more abundant than Lrp6 transcripts, and, as expected, Lrp5 transcripts are missing in Lrp5−/− RECs. At E10.5, Lrp5 transcripts are more abundant in vascular cells in the embryo (purified with anti-PECAM magnetic beads) relative to nonvascular cells. Both vascular and nonvascular cells within the yolk sac have similar levels of Lrp5 and Lrp6 transcripts. See Experimental Procedures for a description of the RT-PCR analysis.
Figure S7. Transcriptional profiles of acutely isolated and immunoaffinity-purified WT and mutant ECs.
(A) Acutely isolated adult Fz4−/−
, and Ndp−
RECs show nearly identical changes in transcript abundances compared to acutely isolated WT RECs. Adult retinas were gently dissociated and vascular fragments, consisting of RECs and MCs, were immuno-affinity purified using an anti-PECAM mAb (; Matsubara et al., 2000; Su et al., 2003
). By visual inspection, this purification procedure generates a nearly pure population of vascular fragments. Data points corresponding to the Xist
transcript (green arrows) reflect different male:female ratios in the various pooled samples. As expected, hybridization signals corresponding to the Fz4
transcript are extremely low in Fz4−/−
RECs (red arrows). In the three mutant samples, the many differences from the WT retinal vascular transcriptome presumably reflect some combination of (1) the ongoing absence of Norrin/Fz4/Lrp signaling, (2) vascular responses to chronic retinal hypoxia and/or stress, and (3) a nearly complete absence of intra-retinal capillaries.
(B) Acutely isolated adult WT vs. the average of Fz4−/−, Lrp5−/−, and Ndp− RECs (upper left panel): many transcriptional changes are seen that have high statistical significance. P16 WT and Fz4−/− cerebellar vascular transcriptomes show relatively few differences and they are largely uncorrelated with the differences between WT and Fz4−/− RECs (lower left panel), consistent with the relatively subtle defects in vascular structure in the Fz4−/− cerebellum. The clustered heat map (right) shows the relative abundances of transcripts that changed by more than 4-fold with a P-value <10−6 in the WT vs. mutant REC comparison (delimited by the red borders in the upper plot). Except for the WT data set in the upper plot, which is derived from six replicates, all micro-array data shown in this Figure represent the average of three independent biological replicates; each replicate was prepared from 6–10 mice.