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First published online 24 July 2008
doi: 10.1242/dev.021097

Development 135, 2939-2948 (2008)
Published by The Company of Biologists 2008
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Cited2 is required for the proper formation of the hyaloid vasculature and for lens morphogenesis

Yu Chen1, Yong-qiu Doughman2, Shi Gu1, Andrew Jarrell1, Shin-ichi Aota3, Ales Cvekl4, Michiko Watanabe2, Sally L. Dunwoodie5, Randall S. Johnson6, Veronica van Heyningen7, Dirk A. Kleinjan7, David C. Beebe8 and Yu-Chung Yang1,*

1 Department of Biochemistry and Cancer Center, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA.
2 Department of Pediatrics, Rainbow Babies' and Children's Hospital, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA.
3 Developmental Biology, Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan.
4 Departments of Ophthalmology and Visual Sciences and Molecular Genetics, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
5 Developmental Biology Program, The Victor Chang Cardiac Research Institute, 384 Victoria Street, Darlinghurst, NSW 2010, Australia.
6 Molecular Biology Section, Division of Biological Sciences, School of Medicine, UCSD, La Jolla, CA 92093, USA.
7 MRC Human Genetics Unit, Western General Hospital, Edinburgh EH4 2XU, UK.
8 Department of Ophthalmology and Visual Sciences, Department of Cell Biology and Physiology, Washington University, St Louis, MO 63110, USA.


Figure 1
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  		Fig. 1. Cited2 is expressed in the developing mouse lens. Immunostaining for Cited2 on eye sections from various developmental stages. Cited2 expression (red) was detected in the surface ectoderm at 9.5 dpc (A), invaginating lens placode at 10.5 dpc (B), and in lens epithelial cells at 15.5 dpc (C), but not in the negative control (D). Cited2 immunostaining (red) (E-G) and negative control (H) from corresponding stages were merged with DAPI nuclei staining (blue).

 

Figure 2
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  		Fig. 2. Formation of the lens stalk in Cited2-/- eyes. (A-F) Histological examination was performed after Hematoxylin and Eosin (H&E) staining of serial paraffin sections of mouse embryo heads. Compared with wild-type littermate controls at corresponding stages (A,C,E), fusion of the lens to the surface ectoderm was detected at 11.5 dpc (arrow in B) and the resultant lens stalk persisted throughout development as shown in representative pictures from 15.5 (arrow in D) and 18.5 dpc (arrow in F) in Cited2-/- eyes. (G,H) E-cadherin immunostaining was performed on sections from 13.5 dpc. In contrast to the expression in corneal epithelium and lens epithelial cells in the wild-type littermate control (G), positive E-cadherin staining for lens stalk was revealed in Cited2-/- eyes (arrow in H).

 

Figure 3
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  		Fig. 3. Proliferation and cell death in Cited2-/- lens. (A-C) Proliferation of the developing mouse lens at 10.5 dpc was examined by phosphorylated histone H3 immunostaining (green) and counterstained with DAPI (blue). No significant difference in proliferation was detected between Cited2+/+ littermate controls (A) (n=3) and Cited2-/- lens (B) (n=5); the data are summarized in C (P>0.05). (D-F) Cell death of the developing lens at 10.5 dpc was examined by the TUNEL assay. For counting cell number, sections were counterstained with DAPI to reveal nuclei (data not shown). Compared with Cited2+/+ littermate controls (D) (n=3), increased cell death was observed in Cited2-/- lens (E) (n=5); the data are summarized in F (P<0.01).

 

Figure 4
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  		Fig. 4. Hyaloid hypercellularity and aberrant vasculature in Cited2-/- eyes. (A-D) Histological examination was performed after H&E staining of serial paraffin sections of mouse embryo heads. The analysis revealed hyaloid hypercellularity and aberrant vasculature in Cited2-/- eyes at 15.5 (arrow in B) and 18.5 (arrow in D) dpc, in comparison to normal intraocular vasculature in wild-type littermate controls at 15.5 (A) and 18.5 (C) dpc. (E-H) The abnormal vasculature was further confirmed by immunostaining Cited2-/- eye sections at 15.5 dpc for endothelial cells by CD31 (arrow in F) and endothelial and angioblast cells by VEGFR2 (arrow in H), as compared with normal expression patterns for CD31 (E) and VEGFR2 (G) in wild-type controls. (I) Vegf mRNA expression was increased in Cited2-/- lens (n=4) compared with the wild-type littermate control (n=4), as quantified by real-time PCR analysis.

 

Figure 5
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  		Fig. 5. Deletion of Hif1a in the lens specifically eliminates the hyaloid hypercellularity and aberrant vasculature of Cited2-/- eyes. (A-D) H&E-stained serial paraffin sections of mouse embryo heads at 15.5 and 17.5 dpc revealed that at both stages, Cited2-/-;Hif1aflox/flox;Le-Cre- eyes displayed lens stalk formation (arrowhead in A,C) and hyaloid hypercellularity with aberrant vasculature (arrow in A,C) (n=3). In Cited2-/-;Hif1aflox/flox;Le-Cre+ eyes (n=3), only the lens stalk (arrowhead in B,D), but not the hyaloid hypercellularity, was detected. Data shown are representative of three independent litters examined. (E) Le-Cre transgene-mediated deletion of Hif1a was assessed by real-time PCR analysis of Hif1a mRNA expression at 14.5 dpc, which revealed a 5-fold decrease in the Hif1a mRNA level in Cited2-/-;Hif1aflox/flox;Le-Cre+ lens (n=3) compared with the level detected in Cited2-/-;Hif1aflox/flox;Le-Cre- lens (n=3). (F) As a consequence, Vegf mRNA expression decreased about 3-fold in Cited2-/-;Hif1aflox/flox;Le-Cre+ lens (n=3) compared with the level detected in Cited2-/-;Hif1aflox/flox;Le-Cre- lens (n=3).

 

Figure 6
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  		Fig. 6. Cited2 is a positive regulator for Pax6 expression. (A,B) Expression of Pax6 in Cited2-/- lens epithelial cells. Immunostaining revealed an appreciable level of Pax6 expression in Cited2-/- lens epithelial cells at 13.5 dpc (B) compared with that of wild-type littermate controls (A). (C) Pax6 mRNA expression in developing lens at 14.5 dpc was analyzed by real-time PCR, which revealed a 2.5-fold reduction of Pax6 expression in Cited2-/- lens (n=4) as compared with wild-type littermate controls (n=4). (D,E) Effect of Cited2 on Pax6 autoregulation. Transcriptional activation of reporters containing Pax6 enhancer and promoter fragments, LE9-P0, LE0-P0 and P0, in Cited2- or Pax6-overexpressing cells was measured by reporter assays. {alpha}-TN4-1 mouse lens epithelial cells were transfected with a reporter plasmid containing the indicated fragment (270 ng) with different combinations of Cited2 (225 ng) and Pax6 (75 ng) expression plasmids. Cited2 overexpression in Pax6-expressing {alpha}-TN4-1 cells significantly increased the activity of LE9-P0, LE0-P0 and P0 reporters (D). This effect was Pax6-dependent because Cited2 overexpression had no effect on LE9-P0 reporter activity in NMuMG and HEK293 cells, which do not express Pax6, but co-expression of Pax6 and Cited2 significantly increased the reporter activity (E). (F) Cited2 is present on the Pax6 promoter. ChIP assays were performed using 2x106 {alpha}-TN4-1 cells and antibodies against Cited2 and Pax6. The precipitated DNA was analyzed by PCR with primers covering the LE9 ectoderm enhancer and the P0 promoter region. Normal mouse IgG and PCR amplifying the sequence between LE9 and P0 were also included as negative controls. Input was 10% of the chromatin for immunoprecipitation. Representative pictures show occupancy of Cited2 on LE9 (a) and P0 region (b) as compared with the negative control (NC) (c). (G,H) Histological examination was performed after H&E staining of paraffin-embedded eye serial sections collected from Cited2-/-;PAX77- and Cited2-/-;PAX77+ littermates at 14.5 dpc. Abnormal lens stalk formation was consistently detected in Cited2-/-;PAX77- embryonic eyes (n=2) (arrowhead in G). However, no lens stalk was detected in any of the serial sections collected from Cited2-/-;PAX77+ mouse embryos (n=3) (H).

 

Figure 7
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  		Fig. 7. Le-Cre-mediated deletion of Cited2. (A-D) Le-Cre transgene-mediated recombination of floxed Cited2 alleles was revealed by lacZ staining in Cited2flox/flox;Le-Cre+ developing mouse lens at 9.5 (A), 10.5 (B) and 11.5 (C) dpc, which is in contrast to the negative lacZ staining pattern in Cited2flox/flox;Le-Cre- control eyes (D). (E,F) Macroscopic examination revealed smaller eyes and failure to form the anterior chamber in Cited2flox/flox;Le-Cre+ embryos (F) as compared with those from Cited2flox/flox;Le-Cre- embryos (E) at 6 weeks of age. (G-L) H&E staining of cross-sections revealed failed separation of the lens from the cornea in Cited2flox/flox;Le-Cre+ eyes (arrow in H) as compared with the normal histology in Cited2flox/flox;Le-Cre- eyes (G) at 6 weeks of age. In addition, abnormal retrolenticular tissue (boxed) was also detected in Cited2flox/flox;Le-Cre+ eyes (J) as compared with Cited2flox/flox;Le-Cre- controls (I). Higher magnification of boxed retrolenticular tissue from J shows melanocytes and aberrant blood vessels (K). Immunostaining for {alpha}-SMA (red) and counterstaining with DAPI (blue) demonstrates {alpha}-SMA-positive pericytes surrounding the blood vessels in Cited2flox/flox;Le-Cre+ eyes (L). R, retina.

 

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© The Company of Biologists Ltd 2008