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BMP signaling is required for development of the ciliary body

Shulei Zhao1,*,{dagger}, Qin Chen1, Fang-Cheng Hung2 and Paul A. Overbeek1,2

1 Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
2 Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
* Present address: Lexicon Genetics, 8800 Technology Forest Place, The Woodlands, TX 77381, USA



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  		Fig. 1. Expression of transgenic (TG) (A-C) and endogenous (D-F) Noggin in developing eyes. In situ hybridization using a 35S-labeled SV40 riboprobe show that Noggin transgene was specifically expressed in the lens but at different levels in different families (A-C). Three representative families, OVE1198 (A), OVE1195 (B) and OVE1194 (C) are shown here. In wild-type (WT) developing eyes, endogenous Noggin transcripts were not detected in E13 eyes by in situ hybridization (data not shown). In E18 (D), P1 (E) and P7 (F) eyes, endogenous Noggin transcripts were present in most of the RPE except for the anterior region. The gaps between the neural retina (NR) and the RPE indicated by asterisks (D,F) are processing artifacts. Abbreviations: CE, ciliary epithelium; L, lens. Scale bars: 100 µm.

 


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  		Fig. 2. Altered morphogenesis of the ciliary epithelium in CPV2-Noggin transgenic mice. In P1 wild-type (WT) eyes (A), the ciliary epithelium (CE) had become a thin monolayer of cells distinct from the neural retina (NR). A clear boundary had formed between these two tissues (arrow in A). In CPV2-Noggin transgenic (TG) mice at P1, the CE had either become thickened (B) or was completed disrupted (C). In the latter case, a lump of cells resembling retinal ganglion cells replaced the presumptive CE (C). The boundary between the retinal cells and those of the presumptive ciliary epithelium became less distinct (arrows in B,C). In P7 wild-type eyes (D), the CE became thinner and had started to fold. The presumptive CE in P7 transgenic mice had defects similar to those in the P1 eyes, either thickened (E) or totally altered (F). Abbreviations: L, lens; IE, iris epithelium. Scale bars: 100 µm.

 


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  		Fig. 3. Expression of Brn3b (A,B) and {alpha}SMA (C,D) in developing eyes. In situ hybridization using a 35S-labeled riboprobe shows that Brn3b was specifically expressed in retinal ganglion cells in P1 wild type eyes (A). In P1 transgenic eyes, Brn3b transcripts were also detected in the region of the presumptive ciliary epithelium (B). Immunohistochemistry (C,D) shows that {alpha}SMA was present in the wild-type developing iris (arrow in C) but its level was reduced in transgenic eyes (arrow in D). Morphologies of the inner (red arrowheads) and outer (blue arrowheads) layers of iris epithelia were altered in transgenic mice (D) when compared with the wild-type controls (C). Abbreviation: L, lens. Scale bars: 100 µm in A,B; 50 µm in C,D.

 


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  		Fig. 4. Downregulation of BMP expression by transgenic Noggin. In wild-type eyes, Bmp4 was highly expressed in E18 (A), P1 (C) and P7 (E) ciliary epithelia (CE). No apparent change in Bmp4 expression was detected in the CE of E18 transgenic eyes (B) but its transcripts were significantly downregulated in P1 (D) and P7 (F) eyes. Similarly, transgenic Noggin had no apparent effect on Bmp7 expression in the CE of E18 embryos (data not shown) but drastically downregulated its expression in the postnatal presumptive CE (compare H with G). Abbreviations: IE, iris epithelium; L, lens; NR, neural retina. Scale bars: 100 µm.

 


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  		Fig. 5. Phosphorylation of Smad1 protein in the developing eyes. Nuclei were visualized by blue labeling with DAPI (A,C,E,G,I,K,M,O). Phosphorylated Smad1 (pSmad1) was detected by immunohistochemistry (green fluorescence) (B,D,F,H,J,L,N,P). At E12, pSmad1 was detected in the cytoplasm and the nucleus of most of the cells in wild-type (B) and transgenic eyes (D). At E15, pSmad1 levels decreased in corneal stroma cells in both wild type (F) and transgenic eyes (H). In transgenic P1 and P7 eyes (L,P), pSmad1 levels were significantly reduced in the lens epithelium, the iris, the ciliary body and the corneal endothelium compared with the wild-type eyes (J,N). However, pSmad1 labeling in the corneal epithelial cells was not significantly affected in transgenic eyes (compare white arrowheads in N and P). Arrowheads in A-H indicate the anterior margin of the optic cup. Arrows in I,J,M,N indicate the infolding ciliary epithelium, which was absent in the transgenic eyes. The asterisk in P indicates nonspecific fluorescent labeling. Abbreviations: L, lens; NR, neural retina. Scale bars: 100 µm.

 


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  		Fig. 6. Downregulation of Msx1 expression. In situ hybridization shows that Msx1 was expressed in the developing ciliary epithelium (CE) at E18 (A) and P1 (B), but at a reduced level by P7 (C). In CPV2-Noggin transgenic mice, Msx1 expression was significantly downregulated in E18 (D), P1 (E) and P7 (F) eyes. Scale bars: 100 µm.

 


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  		Fig. 7. Downregulation of Otx1 expression. In wild-type mice, Otx1 transcripts were detected by in situ hybridization in the developing ciliary and iris epithelia at E18 (A), P1 (B) and P7 (C). In CPV2-Noggin transgenic mice, no significant change in expression was observed in E18 eyes (D), but Otx1 appeared to be switched off (arrowheads) in the presumptive ciliary epithelium of P1 (E) and P7 (F) eyes. Levels of Otx1 transcripts remained essentially unchanged in the iris of transgenic mice. Scale bars: 100 µm.

 


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  		Fig. 8. Rescue of the ciliary defects by co-expression of BMP7. In CPV2-BMP7 transgenic mice (OVE1342B), the ciliary epithelium appeared normal (B) compared with the wild-type control (A). In CPV-Noggin transgenic mice (OVE1195), ciliary body development was disrupted (C). Co-expression of BMP7 and Noggin (OVE1342B x OVE1195) rescued the Noggin-induced defects in the developing ciliary epithelium (D). Tissue sections were obtained from mice at P1. Scale bars: 100 µm.

 

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