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First published online 12 December 2007
doi: 10.1242/dev.009688

Development 135, 271-280 (2008)
Published by The Company of Biologists 2008
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Eomesodermin, a target gene of Pou4f2, is required for retinal ganglion cell and optic nerve development in the mouse

Chai-An Mao1, Takae Kiyama1, Ping Pan1, Yasuhide Furuta1,2, Anna-Katerina Hadjantonakis3 and William H. Klein1,2,*

1 Department of Biochemistry and Molecular Biology, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA.
2 Graduate Training Program in Genes and Development, The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, TX 77030, USA.
3 Developmental Biology Program, Sloan-Kettering Institute, New York, NY 10021, USA.


Figure 1
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  		Fig. 1. Eomes expression in the developing retina. Retinal sections from the indicated developmental stages were labeled with an Eomes antisense probe (A,B) or anti-Eomes antibody (C-K). Eomes expression starts at E14.5 in the innermost layer of developing retina (A). At E16.5, Eomes expression is mainly localized in the same area (B, black arrowhead) and in the forebrain (white arrowhead). At E13.5, although Eomes expression is detected in the developing forebrain (C, arrowhead), it is undetectable in the retina. (D,E) In Math5+/- retinas (D), Eomes is detected in the GCL (strongly) and NBL (weakly), but in Math5-/- retinas (E), Eomes expression in GCL is undetectable. (F-H) At E14.5, Pou4f2 and Eomes colocalize in RGCs in the innermost layer. Developing RGCs in the NBL with strong Pou4f2 expression are Eomes negative. Weak Eomes expression can be detected in the NBL at E14.5. (I-K) At E16.5, expression of Pou4f2 and Eomes is largely restricted to the GCL. Scale bars: 500 µm in C; 200 µm in D,E; in H, 100 µmfor F-H; in K, 50 µm for I-K.

 

Figure 2
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  		Fig. 2. Eomes expression in the NBL. (A-C) An E18 retina pulse-labeled with BrdU: anti-Eomes (A), anti-BrdU (B) and merged image (C). (D) At P0 in Math5+/- retinas, Eomes is expressed in the GCL and NBL. (E) At P0 in Math5-/- retinas, much of the Eomes expression is absent from the GCL but not the NBL. (F) At P12, some Eomes-positive cells in the GCL co-localize with NFL (arrowheads). Eomes is expressed in the nucleus and NFL is expressed in the cytoplasm. (G) At P30, Eomes is expressed in the GCL and in a subset of amacrine cells that are co-labeled with syntaxin (arrowheads). Syntaxin expression is cytoplasmic. The boxed region in the inset is enlarged to emphasize the co-localization of nuclear Eomes and cytoplasmic syntaxin expression. Scale bars: in C, 100 µm for A-C; in E, 100 µm for D,E; 10 µm in insets in F,G.

 

Figure 3
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  		Fig. 3. Identification of an upstream Eomes retinal enhancer containing Pou4f2-binding sites. (A,B) E14.5 retinal sections from Pou4f2+/lacZ and Pou4f4lacZ/lacZ embryos immunostained with anti-Eomes antibody and counterstained with propidium iodide. (C) 20 kb Vista analysis of Eomes comparing mouse, human, dog and rat genomes. Pou4f2-binding sites A and B are indicated by arrowheads. (D) The DNA sequence of Pou4f2 sites A and B. The 2.8 kb retinal enhancer fragment is indicated in red. (E) Transgenic embryos carrying Eo2.8k-HSP68p-LacZ-pA construct stain positively in the retina and limb (arrowheads). (F) Histological retinal section of Eo2.8k-HSP68p-LacZ-pA E14.5 embryos shows lacZ expression in RGCs within the NBL. lacZ-expressing cells co-localized with Pou4f2-expressing cells, detected using anti-Pou4f2 antibody (G). (H-K) GFP expression in Bac Eomes::GFP retinas. GFP fluorescence in retinas from E14.5 (H) and E16.5 (J) embryos. There is intense auto-fluorescence in retina pigment epithelia layer (RPE). Immunostaining using Alexa-555-anti-GFP antibody on retinal sections from E14.5 (I) and E16.5 (K) embryos. Scale bars: 100 µm in B,G,K for A,B, F,G and H-K, respectively.

 

Figure 4
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  		Fig. 4. EMSA and ChIP of Pou4f2-binding to site A within the Eomes retinal enhancer. (A) Pou4f2 binds to site A in vitro. A probe containing Pou4f2-binding site A forms a Pou4f2-DNA complex. The left-most lane shows the free probe (F.P.). The second lane shows the Pou4f2-DNA complex formed in the presence of in vitro-synthesized Pou4f2. Free probe and complex are indicated by arrows. The remaining lanes show the extent of complex formation in the presence of a 500-fold molar excess of wild-type (WT) and mutated (MT) site A oligonucleotides, site B oligonucleotide, and anti-Pou4f2 antibody. (B) ChIP analysis using retinas from E15.5 embryos shows occupancy of Pou4f2 at site A. Sequences around Eomes site A or Titin were amplified after immunoprecipitation with anti-Pou4f2/Brn3b, anti-acetylated-histone H3 or anti-histone H3 antibodies. The left lanes show the expected size of the amplified products generated using input chromatin extracts. The right lanes show the amplified products resulting from the immunoprecipitation of chromatin extracts. The middle lanes show the amplified products generated using mock immunoprecipitation of chromatin extracts without the primary antibody.

 

Figure 5
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  		Fig. 5. Transcriptional activity of the upstream Eomes retinal enhancer. U2OS cells were transfected using Eo2.8k-HSP68p-LacZ-pA (A,B) or Eo2.8k (AmBm)-HSP68p-LacZ-pA plasmid (C,D) without (A,C) or with (B,D) CMV-Pou4f2 plasmid. Scale bar: 200 µm.

 

Figure 6
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  		Fig. 6. Generation of a floxed conditional Eomes allele and creation of Eomesflox/flox mice. (A) Genomic structure of Eomes, the targeting construct, the targeted Eomes allele (Eomesflox) and the deleted allele (Eomes{Delta}flox). Exons are designated as E1-E6. The black and gray bars indicate the regions that were amplified from genomic DNA to generate the targeting construct. Small arrows below the constructs represent the primers used for PCR genotyping. Red boxes indicate loxP sites, and green bars indicate FRT sites. (B) Southern blot analysis of DNA isolated from ES cells. The 5' probe recognizes 15 kb wild-type and 11.2 kb targeted EcoRV fragments. A targeted ES cell is shown in the middle lane, whereas the left and right lanes are untargeted ES cells. (C) Representative PCR genotyping from tail DNA using a three-primer PCR strategy for the different Eomes alleles and Cre transgene. The top gel represents Eomes allele genotyping, and the bottom gel shows the presence of the Cre transgene. In the top gel, the arrows from top to bottom indicate products amplified from primers 15/18 (303 bp for {Delta}flox), 15/16 (222 bp for flox) and 15/16 (166 bp for wild-type), respectively. The 303 bp fragment for the {Delta}flox allele was often preferentially amplified (lanes 1, 2, 3, 5 and 9) from the tail DNA because of leaky expression from the Six3-Cre transgene.

 

Figure 7
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  		Fig. 7. Reduced numbers of RGCs and RGC axons and cell death in Eomes{Delta}flox/{Delta}flox mice. (A,B) Retinal sections from Eomes+/flox (A) and Eomes{Delta}flox/{Delta}flox (B) retinas at P24 were stained with Hematoxylin and Eosin. (C-F) Immunostaining using anti-NFL antibody on flat-mount Eomes+/flox (C,E) and Eomes{Delta}flox/{Delta}flox (D,F) retinas. NFL staining in the central region of the Eomes{Delta}flox/{Delta}flox retina is less intense and sparser than in the Eomes+/flox control retina (C,D). The difference is more noticeable in the peripheral region (E,F). Many axons are oriented aberrantly (F, arrowheads). OD, optical disc. (G,H) Representative cell death pattern at E18. In Eomes{Delta}flox/{Delta}flox retinas (H), massive cell death can be detected while Eomes+/flox control retinas show little cell death (G). (I,J) Cell death profile from E18 to P24 in the GCL (I), and in the INL and ONL (J).

 

Figure 8
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  		Fig. 8. Defects in optic nerve development and myelin ensheathment in Eomes{Delta}flox/{Delta}flox mice. (A,B) Low-magnification images of cross-sections of optic nerves from Eomes+/flox (A) or Eomes{Delta}flox/{Delta}flox (B) P30 mice. Arrowheads indicate the size of the optic nerve. (C,D) Higher magnification images of cross-sections of optic nerves from Eomes+/flox (C) and Eomes{Delta}flox/{Delta}flox (D) P30 mice. Arrowheads in D, part vi point to abnormal neurites. Each row in C and D represents a higher magnification. Scale bars: 2 µm in C,D, parts i.

 

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© The Company of Biologists Ltd 2008