Figure 1.

Expression of Fz4 in ECs and Ndp in Muller glia: absence of Fz4 signaling in ECs leads to severe defects in retinal vascular development.

(A) Structure of the Fz4^CKOAP allele. LoxP sites were placed in the 5′ UTR and 3′ of the 3′ UTR. Cre-mediated recombination deletes Fz4 coding sequences and activates AP. E, EcoR I.

(B) Fz4 is expressed in ECs as determined by AP histochemistry of a Fz4^CKOAP/+;Tie2-Cre E11.5 embryo.

(C) AP histochemistry of adult retinas. Fz4^AP/+ ECs produce a WT vasculature, and Fz4^AP/− ECs produce a vascular phenotype identical to that of Fz4^−/− mice. Scale bar, 200 um.

(D) Mosaic analysis of Fz4^CKOAP/+;Tie2-Cre and Fz4^CKOAP/−;Tie2-Cre retinas with incomplete Cre-mediated recombination. a, Fz4^AP/+ ECs populate large vessels and capillaries. b–d, vessels are populated by Fz4^AP/− ECs only if they reside on the vitreal surface (b and c); intraretinal growth of Fz4^AP/− ECs typically ends in a compact ball of cells (c), with some Fz4^AP/− ECs incorporated into the adjacent WT capillary network (white arrows in b). d, rare Tie2-Cre-mediated recombination events generate isolated Fz4^AP/− ECs within mature intra-retinal capillaries. Scale bar, 100 um.

(E) Structure of the Ndp^AP allele. UTR, rabbit beta-globin 3′ UTR; X, Xba I.

(F) AP-stained retinas from Ndp^AP/+ female mice. 40 um cross-sections at P4 and P7 (left) and a flat mount at P7 (right). AP+ Muller glia span the full thickness of the retina. The brown dots in the flat mount are adherent RPE cells. Scale bars, 100 um. RPE, retinal pigment epithelium; OPL, outer plexiform layer; IPL, inner plexiform layer, GCL, ganglion cell layer; OD, optic disc.