Livne-bar et al. 10.1073/pnas.0600083103. |
Supporting Figure 6
Supporting Methods
Supporting Figure 7
Supporting Figure 8
Supporting Figure 9
Fig. 6. (A) An illustration of the morphological and positional features used to establish retinal cell types in Figs. 1, 2, and 5. These images are from electroporation experiments described in Fig. 2. In all panels, arrows point to the soma and arrowheads to processes. (Top Left) A photoreceptor in the ONL. (Top Right) An amacrine cell positioned at the lower half of the INL with a process descending to the IPL. (Bottom Left) A bipolar cell at the upper half of the INL with ascending and descending processes, and an amacrine cell at the lower half of the INL with a descending process. (Bottom Right) Two Müller glia with soma in the INL and processes spanning the entire retina. O, ONL; I, INL. (B) Increased proportion of bipolar cells in clones derived from neonatal retinal progenitors infected with Chx10 retrovirus (yellow) relative to control retrovirus (blue). Error bars represent SD. Asterisks indicate significant difference (P < 0.02). (C) Reciprocal expression of photoreceptor markers (green) and Chx10 (red) during separation of the ONL and INL.
Fig. 7. (A) Quantification of cell-type proportions using cell-type specific markers. Newborn mouse retina was electroporated with the vectors indicated and cell-types scored at P21 using cell-specific molecular markers; photoreceptors: recoverin and RetP1; bipolar cells: PKC; Müller glia: CRALBP; amacrine cells: bright Pax6 staining in the inner INL. The graph represent the fraction of each cell type of total transfected (GFP+) cells. Error bars represent SD. Asterisks indicate significant difference from MXIE control (P < 0.05). Chx10 promotes bipolar cells and reduces photoreceptors, whereas Chx10-VP16 has the reverse effect. Am, amacrine cells; BiP, bipolar cells; GCL, ganglion cell layer; INL, unidentified INL cells; Mü, Müller cells; PR, photoreceptor cells. (B) A retinal section electroporated with MXIE vector and stained with PKC. An arrow points to a GFP/PKC+ bipolar cell. (Scale bar: 10 mm.)
Fig. 8. Electroporation of newborn mouse retina with Chx10 or Chx10-VP16 vectors. (A) Typical morphology of a bipolar cell expressing Chx10 includes a cell body, upward process terminating in the OPL, and downward process terminating in the IPL (Left), whereas an unidentified INL cell lacks these processes (Right). (B) Neither Chx10 nor Chx10-VP16 affect apoptosis visualized by TUNEL at P3 or P8 (P > 0.05).
Fig. 9. Design of Chx10 shRNAs. (A) The region targeted by each Chx10 shRNA is depicted, and the target sequences are shown below. (B) Phoenix-Eco cells were transfected with a dsRed vector, MXIE-Chx10, and the indicated shRNA vector. In transfected cells (red), Chx10-shRNA reduced expression of the Chx10-IRES-eGFP cassette, as seen by the absence of eGFP (green), whereas a scrambled control had no effect. (C) The fraction of Chx10 expressing (GFP+) cells was plotted relative to total transfected (ds-Red+) cells. (D) P0 mouse retina was electroporated with the indicated shRNA vector together with a GFP plasmid to mark transfected cells. Chx10+/GFP+ cells were detected in the INL by immunostaining when the retina was transfected with scrambled shRNA (Top) but not Chx10 shRNA (Bottom).
Supporting Methods
Plasmids.
pMXIE viral vectors were built to contain WT and altered versions of Chx10. For pMXIE-ChxV, Chx10-VP16, which has a single C-terminal Flag tag, was excised from SVChxV (1) and cloned into the EcoRI site of pMXIE. pMXIEF-Chx10 was made by first removing the Chx10-VP16 sequences from pMXIE-ChxV with EcoRI/BamHI. The backbone was ligated to a full-length human EcoRI/NheI Chx10 fragment from Chx-ABCD (2) and synthetic triple FLAG-tagged oligonucleotides (DYKDHDG-DYKDHD-IDYKDDDDK-Stop) containing NheI/BamHI overhangs. The control vector pMXIEF-VP16 was generated by cloning a PCR fragment encoding the VP16 activation domain (amino acids 410-490) into the pMXIE EcoRI site. Chx10-VP16 is a fusion of full-length human Chx10 with the activation domain of VP16 at the C terminus. The FLAG tags in the pMXIE vectors do not alter Chx10 DNA binding activity in gel shifts or transcriptional activity of numerous targets (1, 2).shRNA vectors were generated by inserting annealed oligonucleotides into pSilencer 1.0-U6 (Ambion, , Austin, TX) cut with ApaI and EcoRI. Knockdown efficiency was tested by transfecting Phoenix-Eco cells with dsRed (transfection control, 1 mg per well), pMXIE-Chx10-GFP (target construct, 2 mg/well), one of the pSilencer-Chx10 constructs (4 mg/well), or pSilencer-scrambled (4 mg/well). Two days posttransfection, wells were scored for cells expressing Chx10-GFP as a ratio of transfected (dsRed+) cells.
Morphological Identification of GFP+ Cell Types.
Cell-type quantification was performed on 18 mM sections at the optic nerve level stained for eGFPv (see below) and DAPI. At least three sections per eye and three retinas for each electroporated construct were scored. Only cells in which the nucleus was clearly visible were included. Cell types were identified on the basis of their position and morphology (see Fig. 6A). Photoreceptors have their cell body in the ONL, an upward process ending in inner and outer segments, and a downward process terminating in the OPL. Cells located in the upper half of the INL with an upward process that ends in the OPL and a process descending to the IPL were identified as bipolar neurons. Müller cells have a cell body in the middle of the INL and long bidirectional processes that span the entire width of the retina (see Fig. 6A). Cells in the lower half on the INL with a process descending to the IPL are amacrine neurons.Immunofluorescence.
Retinas were fixed in 4% paraformaldehyde for 1 h at 4°C, cryoprotected in 30% sucrose, embedded in OCT (TissueTek 4583), frozen on dry ice, and cut into 14-20-mm sections. For S-phase analysis, BrdU (100 mg/g of body weight) was injected i.p. 2 h before death and fixation. BrdU+ cells were detected by using a biotin-conjugated sheep polyclonal antibody (1:500, Maine Biotechnology Services). For double labeling of BrdU and eGFP, sections were first stained for eGFP (rabbit anti-GFP, Molecular Probes) and refixed with 70% ethanol/ 10% acetic acid for 5 min. Sections were washed, treated with 2N HCl, and stained for BrdU. TUNEL was performed as described in ref. 3. Primary antibodies were visualized with goat anti-mouse Alexa Fluor-488 or Alexa Fluor-568, goat anti-rabbit Alexa Fluor-488 or Alexa Fluor-568 (1:300; Molecular Probes). Nuclei were stained with DAPI (Sigma).For Fig. 7, cell types were scored on the basis of the following molecular markers; photoreceptors: recoverin and RetP1; bipolar cells: PKC; Müller glia: CRALBP; and amacrine cells: bright Pax6 staining in the inner INL using antibodies as described in ref. 3. A minimum of three sections per eye and three eyes per expressed construct were scored. Statistical significance was established by Student’s t test or ANOVA followed by ad hoc Tukey’s analysis. All P values are based on two-sided hypothesis testing.
In Situ
Hybridization. Tissue sections for in situ hybridization were obtained as above. The procedure was performed as described in ref. 4, except that digoxigenin-labeled probes were visualized with Fast Red (Roche). Slides were then washed in PBS and processed for immunostaining. Cells were visualized using a Zeiss Axioplan-2. Double-labeled images were obtained with a Zeiss LSM 510 laser scanning microscope.1. Dorval, K. M., Bobechko, B. P., Fujieda, H., Chen, S., Zack, D. J. & Bremner, R. (2006) J. Biol. Chem. 281, 744-751.
2. Dorval, K. M., Bobechko, B. P., Ahmad, K. F. & Bremner, R. (2005) J. Biol. Chem. 280, 10100-10108.
3. Chen, D., Livne-Bar, I., Vanderluit, J. L., Slack, R. S., Agochiya, M. & Bremner, R. (2004) Cancer Cell 5, 539-551.
4. Schaeren-Wiemers, N. & Gerfin-Moser, A. (1993) Histochemistry 100, 431-440.