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The role of bone morphogenetic proteins in the differentiation of the ventral optic cup

¹ The Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA
² Department of Biology, Indiana University Purdue University Indianapolis, Indianapolis, IN 46202, USA

View larger version (138K): [in a new window]   Fig. 1. Lack of BMP signaling leads to microphthalmia. Embryos were infected at HH stage 9-11 with retrovirus expressing either alkaline phosphatase (AP; A,C,F,H,I,L,N,P,R,T) or the BMP-binding protein noggin (B,D,E,G,J,K,M,O,Q,S,U), and studied at E6. (A-E) Gross morphology. Noggin-infected eyes (B) appeared much smaller and less pigmented than alkaline phosphatase controls (A). In histological sections, alkaline phosphatase-infected controls (C) showed no gross abnormalities, while noggin-infected eyes contained rudimentary lens (D, arrow) and neural retina, pigment epithelium, and optic stalk (E, arrowhead). C and D are the same magnification. (F,G) Expression of PROX1 in the lens. Few irregularly distributed PROX1-positive nuclei are scattered throughout the lens of noggin-infected eyes (G), but are more abundant and regularly arranged in alkaline phosphatase-infected controls (F). (H-K) Expression of the pigment-epithelial marker MMP115. Despite the abnormal configuration of the eye in noggin-infected embryos, some pigment epithelium differentiation was apparent both in the presence of pigmented cells (K) and immunoreactivity for MMP115 (J). However, these cells appear less well organized than their counterparts in alkaline phosphatase-infected controls (H,I). (L-U) Expression of neural retina-specific markers. Retinal-specific markers, such as PAX6, islet 1, visinin, and BRN3A, were found in both alkaline phosphatase- (L,N,P,R) and noggin-infected eyes (M,O,Q,S). Positive cells were scarcer and less regularly distributed in noggin-infected eyes. (T,U) Ganglion cell axons. Fibers immunoreactive with the NAPA73 antibody, which is specific for ganglion cell axons, appeared less abundant in noggin-infected eyes, in which they followed abnormal trajectories into the neuroepithelium (arrow U). AP, alkaline phosphatase-infected; Nog, noggin-infected; MMP, MMP115, ISL1; islet 1; VIS, visinin; NAPA, NAPA73. Scale bars: A,B, 50 µm; C,D, 500 µm; E, 50 µm; F-U, 50 µm.

View larger version (97K): [in a new window]   Fig. 2. Ventral eye abnormalities in embryos injected with RCAS-noggin at HH stages 15-18. (A-D) Expression of viral gag proteins in eyes infected with RCAS-alkaline phosphatase or RCAS-noggin. Immunocytochemistry with AMV 3C2 antibody, which recognizes retroviral antigens, was carried out to monitor the extent of infection within each treated eye. Sections through eyes infected with alkaline phosphatase (A,B) or noggin (C,D) retrovirus showed infection throughout the retina, indicating that both retroviral stocks contained infective viral capsids. (E-J) Anatomical and histological features. E8, alkaline phosphatase controls (E-G) show a normally closed choroid fissure in the anterior segment of the eye (arrow, E), normal retina (F), and the presence of the pecten, depicted at both low (F, arrow) and high magnification (G, corresponding to boxed area in F). (H-J) Noggin-infected eyes showed lack of choroid fissure closure (colobomas; H, arrows). The pecten was undetectable in histological sections, and a large mass of mesenchymal-like tissue was noted in the ventral region of the optic cup (I, arrow; J is a higher magnification of the boxed region from I). (K,M) Internal aspect of the fundal region of eyes infected with alkaline phosphatase (K) or noggin (M), as seen after dissecting away the anterior region of the eye. An enlarged choroid fissure was found in noggin-infected embryos in the posterior chamber (M). The fissure was still present, but diminished, in the posterior pole of alkaline phosphatase controls (K). Noggin-infected embryos also showed a white fibrous material, projecting into the vitreal cavity of the eye (arrows, M). (L,N) Sections through the fundal region of alkaline phosphatase- (L) and noggin-infected (N) eyes, labeled with antibodies specific for class III ß-tubulin. Controls show darkly labeled ganglion cell axons at the vitreal edge of the retina, and into the optic nerve head (arrow, L). In noggin-infected eyes, in contrast, fibers were generally undetectable in the optic nerve but could be seen projecting into the vitreal chamber (arrow, N), corresponding to those in M. Scale bars: A-D, 50 µm; E,H, 500 µm; F,I, 500 µm; G,J, 50 µm; K,M, 500 µm; L,N, 500 µm.

View larger version (92K): [in a new window]   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.

View larger version (90K): [in a new window]   Fig. 4. Expression of SHH and FGF8 in noggin-infected and control eyes. Embryos were infected at stage 15-18 with RCAS-noggin or RCAS-alkaline phosphatase and were analyzed on E6. (A-F) SHH expression. Both alkaline phosphatase- and noggin-infected eyes expressed SHH very heavily in the ventral neural tube (A,B and D,E), and weakly in the neural retina (arrows, C,F). No apparent changes in either the relative intensity or pattern of distribution of the signals were noted in noggin-infected eyes (D-F) in comparison to controls (A-C). (G-L) PTC expression. Intensity of signals for the transcripts of the SHH receptor patched (PTC) provides an indication of levels of SHH signaling because PTC is upregulated by SHH (Marigo and Tabin, 1996; Goodrich et al., 1996). PTC expression was conspicuous in the neural tube (G; top box enlarged in H); there was very little expression in the neural retina, restricted to the optic nerve head region (G; lower box enlarged in I; arrow points to positive cells). Embryos infected with noggin showed increases in signal in the neural tube (arrows in J; box enlarged in K), likely due to seepage of intravitreally injected virus into the neural tube through the optic stalk. No such increases in PTC signal were observed in the retinae of the same noggin-infected embryos (J, box enlarged in L). (M,N) FGF8 expression. FGF8 was weakly expressed in a region just dorsal to the optic stalk in alkaline phosphatase-infected controls (arrow, M), as well as the optic stalk itself (arrowhead, M). In noggin-infected eyes, however, FGF8 signal was much more intense and found in a larger region than in controls (arrow, N). Scale bars: A,D,G,J,M,N, 500 µm; B,C,E,F,H,I,K,L, 50 µm.

View larger version (88K): [in a new window]   Fig. 5. Laminin immunoreactivity in colobomatous eyes. Embryos infected at stage 15-18 (E3) were processed immunohistochemically at stage 29 (E6) with antibodies against laminin. (A,B) Alkaline phosphatase-infected eye. In the area where the choroid fissure is closing, laminin immunoreactivity is present only at the vitreal edge of the retina (top) and at the choroidal surface of the pigmented epithelium (bottom). (C,D) Noggin-infected retinas. The choroid fissure has not closed in an area similar to that depicted in A, B. Laminin immunoreactivity is observed in the region between the two opposing surfaces of the retina and RPE in this area (arrow, C). Scale bars: 50 µm.

View larger version (118K): [in a new window]   Fig. 6. Expression of axonal guidance molecules. Embryos infected at E3 were analyzed at either E6 (A,B,I-T) or E8 (C-H). (A-D) Expression of agrin (A,B) and chondroitin sulfate proteoglycan (C,D). No differences were observed between alkaline phosphatase- (A,C) and noggin- (B,D) infected embryos. (E-H) R-cadherin immunoreactivity. R-cadherin appeared localized to the pecten, optic nerve head and adjacent RPE in alkaline phosphatase-infected embryos (E,F), but was present in a much wider and more diffuse region in noggin-infected eyes (G,H). (I-P) Netrin immunoreactivity. Signals were localized to the optic nerve and ventral region of the presumptive ciliary body of alkaline phosphatase controls (I-L), but encompassed the entire ventral retina and secondary neuroepithelium of noggin-infected eyes (M-P). (Q-T) Netrin 1 mRNA expression. Netrin 1-positive cells were localized to the optic stalk in both alkaline phosphatase- (Q) and noggin- (S) infected embryos, but the latter also showed positive cells in the secondary neuroepithelium (arrows, T). AP, alkaline phosphatase infected; Nog, noggin infected; CSPG, chondroitan sulfate proteoglycan; IHC, immunohistochemistry; ISH, in situ hybridization. Scale bars: A-D, 50 µm; F,H, 100 µm; E,G, 500 µm; I,M, 500 µm; J-L, N-P, 50 µm; Q-T, 50 µm.