Pregnant CD-1 mice were purchased from Charles River Breeding Laboratories, Wilmington, MA, USA. Animal experiments were approved by the Johns Hopkins University Institutional Animal Care and Use Committee.
In vivo electroporation
In vivo electroporation of mouse retina was performed as described . Retinas were electroporated at P0.5. Dissociated cells and section immunohistochemistry data shown here were performed at P21. Error bars represent ± standard error of the mean for at least three independent electroporated retinas. A two-tailed Students t-test was performed to determine the P-value. Control and experimental constructs were co-electroporated with 0.2 μg of pCAG-GFP to readily visualize electroporated cells by GFP . For overexpression experiments, 1 μg of pCAG-GFP was injected with either 1 μg of pCAG-Six3 or 1 μg of CAG-Six3OS. For simultaneous overexpression of Six3 and Six3OS, retinas were electroporated with 1 μg of pCAG-Six3 and 1 μg of CAG-Six3OS. For knockdown of Six3 and Six3OS, 1 μg of U6-control was injected with either 1 μg of U6-Six3 or 1 μg of U6-Six3OS. For simultaneous knockdown, 1 μg of U6-Six3 and 1 μg of U6-Six3OS were injected. For overexpression of Six3 in combination with knockdown of Six3OS, retinas were electroporated with 1 μg of pCAG-Six3 and 1 μg of U6-Six3OS were injected. Finally, for overexpression of Six3OS in combination with knockdown of Six3, 1 μg of pCAG-Six3OS and 1 μg of U6-Six3 were injected and electroporated.
In situ hybridization
For cryosections, untimed E14.5 and E16.5 embryos and P0.5 neonates were dissected, fixed overnight in 4% paraformaldehyde in PBS at 4°C and cryoprotected overnight in 30% sucrose/PBS at 4°C before being embedded in OCT compound (Sakura, Torrance, CA, USA) on dry ice. Cryosections (20 μm) were cut on a cryostat. Section ISH methodology was as previously described  with the exception that probe BC065087 was used and a Tyramide Signal Amplification system (TSA Plus, Perkin Elmer, NEL 744, Waltham, MA, USA) combined with an antidigoxigenin-HRP antibody (1:1, 000; Roche, Indianapolis, IN, USA). for fluorescent ISH. Fluorescent ISH sections were counterstained with DAPI. Fluorescent ISH samples were photographed on a Zeiss Meta 510 confocal microscope. Chromogenic images were visualized on a Zeiss Axioskop2 microscope.
For cryosections, electroporated eyes were harvested 21 days after electroporation, fixed for 1 hour in 4% paraformaldehyde in PBS at 4°C and cryoprotected overnight in 30% sucrose/PBS at 4°C before being embedded in OCT compound (Sakura) on dry ice. Cryosections (20 μm) were cut on a cryostat. Retinal cryosections were immunostained as described except that sections were post fixed in 4% paraformaldehyde for 5 minutes prior to blocking . Samples were photographed on a Zeiss Meta 510 confocal microscope.
For cell marker analysis, dissociation and immunostaining were performed as described  except that retinas were harvested at P21. Samples were visualized and quantified on a Zeiss Axioskop2 microscope. To quantify the effects of Six3 knockdown, retinas were electroporated with Six3 shRNA, harvested at P4.5, dissociated, and immunostained as described above with anti-Six3 and detection with Alexa568 goat anti-guinea pig IgG and rabbit anti-GFP and detection with Alexa488 goat anti-rabbit. For Six3 quantification, Six3OS overexpression and SixOS shRNA electroporated retinas were harvested at P5.5, dissociated and immunostained as described above with anti-Six3 and detection with Alexa568 goat anti-guinea pig IgG and rabbit anti-GFP and detection with Alexa488 goat anti-rabbit. Samples were visualized on a Zeiss Axioskop2 microscope and signal intensity was quantified with Velocity 4.0 (Perkin Elmer) software by calculating the average signal intensity per cell and normalized to cell size.
In situ hybridization and immunocytochemistry
For Six3OS knockdown quantification, retinas were harvested at P4.5 and dissociated as described above. FISH was performed as described using TSA Plus Cyanine3 kit (1:125; Perkin Elmer, NEL 744) followed by staining as described (with rabbit anti-GFP and detection with Alexa488 goat anti rabbit). Samples were visualized and quantified as above. For all dissociations, nuclear DNA was visualized with DAPI counterstaining. Cell counts were analyzed using the two-tailed Student's t-test. A minimum of three retinas were counted for each construct examined using dissociated immunocytochemistry, with 100 to 300 GFP-positive cells per retina counted for each marker tested.
HeLa cell FISH followed by immunocytochemistry was performed as previously described  except that 20 cells were counted.
Luciferase reporter assays
NIH 3T3 cells were grown in DMEM supplemented with 10% FCS. Cells were transfected with Fugene6 (Roche) per the manufacturer's instructions. Cells were transfected with 500 ng of luciferase reporter construct and 50 ng of the expression plasmids for Six3 and Six3OS. Cells were harvested 2 days post-transfection. Luciferase was measured per manufacturer's instructions with the Dual-Luciferase Reporter System (Promega, E1910, Madison, WI, USA). pTK-Renilla (50 ng) was co-transfected to control for transfection efficiency.
Synthesis of RNA probes for protein microarray
BC065087 (5 μg) was linearized with DraI, and 5 μg of BM663835, BX115070, and BM451513 were linearized with NotI. Linearized DNA was then phenol-chloroform extracted, ethanol precipitated and resuspended in 10 μl water. Probes were in vitro transcribed with T7 polymerase per manufacturer's instructions using the Riboprobe Combination system kit (Promega, P1405), spiking the reaction with 1 μl of 5 mM Cy5 labeled CTP (GE Healthcare, 25-8010-87, Piscataway, NJ, USA). The RNA probe was ethanol precipitated with LiCl and resuspended in 1 mM EDTA.
Protein microarray analysis
Transcription factor/RNA binding protein chips were generated as previously described . Protein chips were preblocked with Superblock Buffer (Thermo Scientific, 37516, Waltham, MA, USA) supplemented with 10 μg ml-1 sperm DNA, 2 mM MgCl2, 2 mg ml-1 BSA for 1 hour at room temperature. RNA was hybridized at 250 nM concentration in binding buffer (PBS supplemented with 2 mM MgCl2, 2 mg ml-1 BSA) at room temperature for 1 hour. The slides were washed four times in TBST, dried and scanned by a GenePix 400B scanner. Data were analyzed with GenePix Pro 6.10 as previously described .
RNA immunoprecipitation experiments
HEK 293T cells were grown in DMEM supplemented with 10% FCS. Cells were transfected with Fugene6 (Roche) per the manufacturer's instructions. Ten million cells were transfected 24 hours post-plating with 5 μg of pCAGIG-V5, pCAGIG-Six3-V5, pCAGIG Eya1-V5, pCAGIG Eya3-V5, pCAGIG Eya4-V5 or pCAGIG Ezh2-V5, and pCAG-Six3OS. Cells were harvested 2 days post-transfection, lysed and precipitated essentially as previously described , except that 5 μg anti-V5 antibody was used (1:5, 000; Invitrogen, R96025, Carlsbad, CA, USA) with 50 μl protein A-agarose (Invitrogen, 15918-014) in each immunoprecipitation reaction. For each sample, 10% of total volume was set aside after lysis for RNA extraction and 5% set aside for immunoblot analysis. After precipitation, 80% of the beads were resuspended in Trizol and 20% were resuspended in Laemmle buffer. RNA was Trizol extracted and resuspended in 50 μl of nuclease free water. RNA was then DNAse treated with Turbo DNA-free (Ambion, AM1907, Carlsbad, CA, USA) per manufacturer's instructions. RNA was quantified by quantitative RT-PCR using Brilliant II SYBR Green QRT_PCR Master Mix (Agilent, 600825, Santa Clara, CA, USA) on a Roche LightCycler480. No-RT controls were simultaneously performed to demonstrate that signal was not from DNA contamination.
Expression of V5-tagged protein was confirmed by western analysis using anti-V5 antibody (1:10, 000; Invitrogen, R96025) dilution in 5% milk in PBST, and detected with horse radish peroxidase goat anti-mouse IgG (1:10, 000; Santa Cruz, sc-2031, Santa Cruz, CA, USA) and ECL Western Blotting Detection System (GE Healthcare, RPN2132 Piscataway, NJ, USA) per the manufacturers' instructions.
Full details of all plasmids and antibodies used in this study are included in Additional File 9.