Fig. 3. Expression of dorsal and ventral markers. Embryos were infected on E3 (HH 15-18) and analyzed on E6. (A-D) PAX2 and PAX6. PAX2 appeared restricted to the optic stalk in control eyes (A), but in noggin-infected embryos (B) it was detectable in the optic stalk, ventral retina (arrow) and a secondary neuroepithelium that replaced the RPE (arrowhead). The region of PAX2 expression in the ventral retina of noggin-infected eyes (C) was completely devoid of PAX6 expression (D), consistent with reports from the literature (Schwarz et al., 2000). (E-H) Expression of ventral retinal markers. VAX expression appeared weak and restricted to the ventral portion of alkaline phosphatase-infected retinas (E), but was much stronger and encompassed both ventral and dorsal regions of the retina in noggin-treated eyes (F). ALDH6 was restricted to the ventral region of the presumptive ciliary body in alkaline phosphatase controls (G), but was also found in the presumptive dorsal ciliary body (arrow, H) and throughout the ventral retina in noggin-infected eyes (arrowhead, H). (I-L) Expression of dorsal retina markers. Two dorsal markers, TBX5 and ALDH1, appeared much more intense in alkaline phosphatase controls (I,K), than in noggin-treated retinas (J,L). TBX5 expression was seen in a subset of ventral ganglion cells (arrowheads) in both control and noggin-treated eyes (boxes in I and J). (M-W) Changes in ventral pigment epithelium. Noggin-infected eyes appeared to be missing the ventral pigment epithelium in the region adjacent to the coloboma (O,P). The region that normally differentiates as ventral RPE in control eyes (arrows, N) was replaced by ectopic neuroepithelium-like tissue in noggin-treated eyes (arrows; P). This secondary neuroepithelial-like structure did not express pigment or the RPE-specific protein MMP115 (arrows, S,T), which are detectable in alkaline phosphatase controls (Q,R), but it was positive for two ventral retina/optic stalk markers, PAX2 and ALDH6 (arrows, U,V). None of these structures was positive for dorsal retina markers such as TBX5 (arrow; W). ALDH6, aldehyde dehrdrogenase 6; ALDH1, aldehyde dehydrogenase 1. Scale bars: A,B, 500 µm; C,D, 500 µm; E-L, 500 µm; N,P, 50 µm; M,O, 50 µm; Q-W, 50 µm.