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First published online 5 November 2008
doi: 10.1242/dev.024620

Development 135, 3923-3933 (2008)
Published by The Company of Biologists 2008
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Temporal requirement of the alternative-splicing factor Sfrs1 for the survival of retinal neurons

Rahul N. Kanadia1,2, Victoria E. Clark1, Claudio Punzo1, Jeffrey M. Trimarchi1 and Constance L. Cepko1,2,3,*

1 Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
2 Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA.
3 Department of Ophthalmology, Harvard Medical School, Boston, MA 02115, USA.


Figure 1
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  		Fig. 1. Sfrs1 expression during mouse retinal development. (A) In situ hybridization detecting Sfrs1 RNA during retinal development. The schematic to the left indicates the location (box) of each row of images in the developing retina. Scale bars: 50 µm. (B) Schematic representation of the Sfrs1 gene, showing exons (blue boxes, 1-5), introns (green lines) and the primers (red arrows) used for RT-PCR analysis. (C) Two RT-PCR products from the Sfrs1 coding sequence. The bottom panel shows Rps17, which was used as a control. The top band for Sfrs1 retains intron 3 (Sfrs1a), whereas the lower band is the canonical isoform with all four exons (Sfrs1b). (D) RT-PCR products of Sfrs1 coding sequence from heart cDNA from different developmental stages are shown along with PCR products for Tnnt2 as assessed by amplifying exon 2 to exon 6. (E) Immunoblot analysis (nuclear and cytoplasmic retinal extracts) to detect production of the new isoform of Sfrs1 at E14.5, P2 and P10. Fractionation and equal protein loading were assayed by probing for Cugbp1. (F) Localization of the two isoforms of Sfrs1 as achieved by fusing Sfrs1a and Sfrs1b with GFP followed by transfection of NIH3T3 cells. The cells are counterstained with phalloidin (red) and DAPI (blue).RPE, retinal pigmented epithelium; GCL, ganglion cell layer; NBL, neuroblastic layer; INBL, inner neuroblastic layer; ONBL, outer neuroblastic layer.

 

Figure 2
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  		Fig. 2. Sfrs1-cKO mice have small eyes at P0. (A) Frontal view of wild-type (WT) and Sfrs1-cKO eyes. (B) Lateral view of the eyes and optic nerve. Insets show cross-sections of the optic nerves stained with DAPI (magenta). (C) Total weight comparison of wild-type (n=4) and mutant (n=4) eyes. (D,E) Hematoxylin and Eosin (H&E) stain of P0 wild-type and mutant retinae. Arrowhead indicates the retinal pigmented epithelium (RPE). (F,G) Higher magnification of D and E, with each ganglion cell nucleus marked with a dot (arrowhead) Scale bars: 100 µm. (H) Quantification of the ganglion cell layer cellularity in wild-type (n=3) and mutant (n=3) retinae. (I-L) Ultrastructural analysis of wild-type and mutant retina, with the basal lamina indicated in the mutant retina (J, arrowhead). Scale bars: 10 µm.

 

Figure 3
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  		Fig. 3. Proliferation and cell death in Sfrs1-cKO mice. (A-H) Proliferation as determined by Ki67 immunofluorescence in wild-type (WT) and Sfrs1-cKO retinae at E12.5, E13.5, E18.0 and P0. (I-P) Cell death as determined by TUNEL analysis in WT and Sfrs1-cKO retina at E12.5, E13.5, E18.0 and P0. (Q-T) Quantification of the number of TUNEL+ cells per section from different animals in the mutant retina as compared with the wild-type at E12.5 (n=4 for WT and Sfrs1-cKO), E13.5 (n=3), E18 (n=3) and P0 (n=5). Scale bars: 50 µm in A,B,I,J; 100 µm in C-H,K-P.

 

Figure 4
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  		Fig. 4. Retinal progenitor cells survive the loss of Sfrs1. (A) Snapshot of a live ultrasound image of an E10.5 mouse embryo imaged through the uterine wall. Inset shows the newly formed optic vesicles and the diencephalon is traced in white. The right-hand panel shows a glass needle that was introduced into the ventricle where the virus was delivered. (B) P14 retinal sections from Sfrs1wt/wt embryos that were injected at E10.5 with a virus expressing Cre and nuclear-GFP. Stained for GFP. The arrowhead shows the position of the amacrine cells. (C) P14 retinal sections from Sfrs1fl/fl embryos injected at E10.5 with the same virus. The arrowhead highlights the absence of the amacrine cells. (D,E) P7 retinal sections from Sfrs1wt/wt and Sfrs1fl/fl mice that were crossed to the RC::PFWE line, which reports the Chx10::Cre-mediated excision event by activating nlacZ expression. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer neuroblastic later. Scale bars: 50 µm.

 

Figure 5
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  		Fig. 5. Cell type composition in the Sfrs1-cKO retina. RNA ISH detecting Sfrs1 (A,B), Brn3b (C,D), Nf68 (E,F), Trβ2 (G,H) RNA in E13.5 wild-type and Sfrs1-cKO mouse retinal sections. (I,J) Higher magnification images of the Trβ2 ISH from G and H. RNA ISH detecting Brn3b (A',B'), Nf68 (C',D'), Rph3a (E',F') and Ap2b (G',H') on P0 retinal sections. RNA ISH on P7 sections detecting Brn3b (A'',B''), Nf68 (C'',D''), S opsin (E'',F''), Ndrg4 (G'',H''), Otx2 (I',J') and Nr2e3 (K,L). Immunofluorescence for Pax6 (green) on P7 retinal sections stained with DAPI (magenta) (M,N). The white arrowhead indicates the horizontal cells, the open arrowhead points to the amacrine cells. Scale bars: 100 µm in A-H,A'-H'; 50 µm in A''-H'',I',J',K,L.

 

Figure 6
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  		Fig. 6. Quantification of the different cell types in the Sfrs1-cKO retina. Quantification by dissociated in situ hybridization on P7 wild-type (n=3) and Sfrs1-cKO (n=3) mouse retinal cells with RNA probes for specific cell types. (A,B) Representative images of cells probed for Nrl (red) and stained with DAPI (blue). (C) The relative percentage of ganglion cells (Nf68), cone photoreceptors (short-wave opsin), and amacrine cells (Pax6) in wild-type and mutant retinae. (D) The relative percentage of bipolar cells (Chx10), rod photoreceptors (rhodopsin) and Müller glia (glutamine synthetase).

 

Figure 7
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  		Fig. 7. Survival and/or production of neurons during embryonic development. (A,B) P7 retinal sections from wild-type and Sfrs1-cKO mice stained for BrdU (red), Pax6 (green) and with DAPI (blue). (C,D) Cells that were heavily labeled for BrdU were identified with an automated spot detection algorithm (IMARIS, Bitplane) utilizing a diameter threshold of 8 µm and pixel intensity threshold of 20.6 and are rendered as white spots. (E,F) The rendered spots shown without the fluorescence. The tabulated data beneath are the quantification of these spots.

 

Figure 8
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  		Fig. 8. Postnatal retinal development in the Sfrs1-cKO mouse. (A-H) Immunofluorescence on P14 wild-type and Sfrs1-cKO retinal sections with anti-glutamine synthetase (Müller glia) (A,B), anti-Chx10 (C,D), anti-rhodopsin (rod photoreceptors, top arrowhead) and anti-Pax6 (amacrine and horizontal cells, bottom arrowhead) (E,F), and anti-red/green opsin (cone photoreceptors) (G,H). (I,J) Wild-type and mutant retinae showing the ciliary margin attached to the mutant retina. (K,L) P30 wild-type and Sfrs1-cKO eyes. (M,N) Retinal sections stained with anti-GFP from P14 Sfrs1-cKO mice that were electroporated with Cre-GFP and LoxP-Stop-LoxP-GFP (M) or GFP alone (N) plasmids at P0. (O-Q) P55 retinal sections stained with anti-GFP (green) and anti-glutamine synthetase (red) antibody from mice that were infected with H2B-GFP-IRES-Cre virus at P0.

 

Figure 9
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  		Fig. 9. Role of Sfrs1 during mouse retinal development. (A) Expression pattern of the two Sfrs1 isoforms (Sfrs1a, Sfrs1b) during development and the requirement (dark gray) and the lack (light gray) of Sfrs1 function. (B) A single progenitor in the wild-type retina is shown undergoing cell division and producing neurons and glia from E10.5 to P14. Mitotic cells are shown as blue ovals, and postmitotic cells as red ovals. The thick black line denotes time, with P0 (dashed line) indicating birth. Below the black line there are boxes depicting the birth order and the duration of each cell type. (C) A progenitor cell in the Sfrs1-cKO retina undergoing cell division and producing neurons, with crosses denoting cell death.

 

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© The Company of Biologists Ltd 2008