PAX77 hemizygous mice , designated PAX77+, carry 5 to 7 copies of a 420 Kb human PAX6 YAC (Y593) with all copies integrated at the same locus. We refer to the array of integrated YAC Y593 copies as the PAX77 transgene. PAX77 homozygous mice, designated PAX77+/+, carry 10 to 14 copies of human PAX6 and were genotyped by fluorescent in situ hybridization using the Fat5 probe as described in Schedl et al. (1996) . As no abnormalities are observed in animals carrying the transgene on a Pax6Sey/+ heterozygote background Schedl et al concluded that the ocular abnormalities in PAX77 mice are due to overexpression of PAX6 and not to overexpression of a second gene encoded by the YAC. All mice were maintained on an albino CD1 background. The morning of the vaginal plug was deemed E0.5. The first 24 h after birth was deemed P0. Animal care followed institutional guidelines and UK Home Office regulations.
For each stage and each genotype, at least 2 samples were fixed in 4% paraformaldehyde : 2% glutaraldehyde, dehydrated to 100% ethanol and embedded in cold-polymerizing resin (Technovit 7100, Kulzer Histo-Technik). Sections were cut (5 μm) and stained with cresyl violet. Sections of whole embryos were cut horizontally and sections of postnatal eyes were cut parallel to the optic nerve.
BrdU/IdU analysis of cell-cycle parameters
The BrdU/IdU double labeling protocol is detailed in Martynoga et al.(2005) . In brief, at T = 0 h the pregnant mouse receives an injection of IdU which labels cells going through S-phase from the beginning of the experiment. At T = 1.5 h the female receives an injection of BrdU and the embryos are fixed shortly after (T = 2 h). Thus, the BrdU labels cells which are in S-phase at the end of the experiment; this is the S fraction (Scells). The cells labeled only by the IdU but not by the BrdU are cells which have left S phase in the interval of 1.5 h between the two injections. This is the leaving fraction (Lcells). The cell cycle length (Tc) and the length of S-phase (Ts) can be calculated using the equations of Shibui et al.(1989) : Ts = Ti/(Lcells/Scells) and Tc = (Scells/Pcells), where Ti is the interval between the two injections and Pcells is the total number of proliferating cells. We made the assumption that all progenitor cells of the retina are proliferating. The growth fraction was not determined in this experiment.
Pregnant females were injected intra-peritoneally with 200 ml of 10 mg/ml (in 0.9% NaCl) IdU (Sigma), then 1.5 h later with the same dose of BrdU (Sigma) and they were sacrificed after 30 min. 10 μm wax sections were immunostained with mouse anti-BrdU/IdU (Becton Dickinson, clone B44, 1:100) and rat anti-BrdU (Abcam, clone BU1/75, 1:100). Cells were counted in 100 μm wide sampling boxes in the distal and proximal retina of 3 wild-type and 3 Pax77+/+ E12.5 embryos. Each cell count was repeated on at least 3 non-adjacent sections from each embryo.
Whole embryos or postnatal eyes were fixed in 4% paraformaldehyde and either processed to wax or cryoprotected in 15% and 30% sucrose and frozen.
Immunostaining for Pax6, glial fibrillary acidic protein (GFAP), Brn3a, Islet1, bromodeoxyuridine (BrdU), Syntaxin and N-Cadherin (Ncad) was performed on 10 μm paraffin sections as described in Martynoga et al. (2005) . For studies with BrdU, pregnant females were injected intra-peritoneally with 200 μl of 10 mg/ml BrdU (in 0.9% NaCl; Sigma) and were sacrificed after 1 hour. Immunostaining for Chx10 and Pax2 was performed on 10 μm frozen sections as described in Martynoga et al. (2005) . R-Cadherin (Rcad) immunostaining was performed on frozen sections as described in . Sections of whole embryos were cut horizontally and sections of postnatal eyes were cut parallel to the optic nerve.
For whole-mount immunofluorescence, dissected retinae from E16.5 embryos were incubated in blocking buffer (20% goat serum, 0.2% Triton-X100 in phosphate buffered saline [PBS]) for 20 min, followed by anti-neurofilament or anti-L1 antibody overnight at 4°C. For neurofilament detection, sections were rinsed in 0.2% Triton-X100 in PBS, and incubated with an Alexa488-conjugated anti-rabbit secondary (Molecular Probes, Inc). For L1 detection, a biotinylated secondary antibody was used with Alexa488-conjugated streptavidin (Molecular Probes, Inc).
Primary antibodies were: Pax6 (1:400, DSHB), GFAP (1:50, Dako), Brn3a (1:300, Chemicon; MAB1585), Islet1 (1:50, DSHB), Ncad (1:500, BD), BrdU (1:200, Becton Dickinson), Syntaxin (1:100, Santa-Cruz sc-12736), Chx10 (1:1000, a gift from C. Cepko), Pax2 (1:200, Cambridge Bioscience), Rcad (1:100, a gift from M. Takeichi), Neurofilament (1:100, BIOMOL, USA; NA1297), L1 (1:50, Chemicon; MAB5272).
Quantification of Brn3a-expressing cells in the retina
Retinae were removed from heads previously fixed in 4% paraformaldehyde and an orientating cut made in the ventral retina of E16.5 embryos. Retinae were boiled in 10 mM sodium citrate buffer pH6.0 for 10 minutes and blocked in 5% goat serum, 0.2% BSA, 0.1% Triton-X1000 in PBS at room temperature before incubating overnight at 4°C in primary antibody [Brn3a Chemicon; MAB1585)] diluted 1:300 in blocking buffer. After washing extensively in 0.1% Triton-X1000 in PBS at room temperature retinae were incubated overnight at 4°C with an Alexa488-conjugated anti-mouse secondary (Molecular Probes, Inc), washed extensively, and mounted with the RGC layer uppermost in 90% glycerol containing MOWIOL and DABCO. For E16.5 retinae, a Leica TCS NT confocal microscope was used to acquire (1) a low power image of the entire retina for calculation of retinal area [see Fig 8A], (2) to scan through the entire Brn3a-expressing RGC layer at high power and measure its depth and (3) to collect a series of optical sections for sampling within the RGC layer.
Sections were 160 μm × 160 μm and were collected at 0.16 μm intervals through 8 μm of retinal depth. The boxed area in Fig 8A shows an example of a sampling area in ventrotemporal retina. We used a stereological approach to calculate the density of RGCs within the sampling volume: optical sections comprising the upper and lower 4 μm of the total 8 μm stack were combined separately to generate images like the high power inset shown in Fig 8A. Simply counting the number of Brn3a-expressing nuclei in each image would give an overestimate of nuclear density in each 160 μm × 160 μm × 4 μm volume as nuclei protruding into the sampling volume both from above and from below would be included in the count. To avoid overestimating the nuclear density, we only counted nuclei protruding into the sampling volume from one side while excluding those that protruded from the other. This was done by using Adobe Photoshop to pseudo-colour the nuclei in the upper stack red and those in the lower stack green and then merging the two images. Nuclei appearing yellow [red and green combined] were present in both upper and lower stacks and so were excluded from the count. This gave an accurate number of Brn3a-expressing nuclei present in the sampling volume and was used to calculate their density. For each retina the density was calculated in the dorso-nasal and ventro-temporal retina. The number of Brn3a-expressing cells in the retina was calculated by multiplying the retinal area × depth of RGC layer × density of Brn3a-expressing cells in RGC layer. For E12.5 retinae, where the density of RGCs is much lower, a Leica TCS NT confocal microscope was used to acquire a scan through the whole retina and total numbers of Brn3a positive cells were counted.
Tract-tracing was performed as described in Pratt et al. (2006) . Embryonic heads were fixed at 4°C in 4% paraformaldehyde in PBS overnight. DiI crystals (Molecular probes, USA) were either (1) placed in the optic cup of one eye after removal of the lens or (2) placed in a line over the dorsal thalamus on one side to label axons navigating the optic tract. Heads were returned to 4% paraformaldehyde in PBS in the dark at room temperature for about six weeks to allow tracers to diffuse along axons. In some cases the retinae were removed from the head, cleared in 9:1 glycerol:PBS, and imaged as wholemounts using a Zeiss Axiovert confocal LSM 510 microscope (Zeiss, Germany). In other cases heads were sectioned (200 μm) with a vibratome, cleared in 9:1 glycerol:PBS containing the nuclear counterstain TOPRO3 (1.0 μM, Molecular Probes, USA), mounted in Vectashield (Vector Laboratories, USA), and imaged using an epifluorescence microscope and digital camera (Leica Microsystems, Germany) or a TCS NT confocal microscope (Leica Microsystems, Germany). For quantification of RGC projections the total number of DiI labelled RGCs in each retina was counted in serial vibratome sections using an epifluorescence microscope as follows. Each section was viewed under epifluorescence using a x20 objective at which magnification DiI labelled RGC cell bodies are easily resolved. An eyepiece graticule was used to divide the retina into 50 μm wide bins. Within each bin DiI labelled RGC bodies were counted by focussing through the thickness of the section and counting labelled cell bodies as they came into focus. Individual bin counts were then summed to give totals for RGCs projecting ipsilaterally and contralaterally to the thalamic DiI injection site in each embryo and these totals were used to generate data presented in Fig 7, Fig 8, and Table 3. The numbers of E16.5 embryos used for quantification were as follows: Pax6Sey/+ n = 4; Pax6+/+ n = 9; PAX77+ n = 5; PAX77+/+ n = 4. DiI appears orange and TOPRO3 appears red in epifluorescence images and in confocal images DiI appears red and TOPRO3 appears blue.
Quantitative reverse transcription-PCR (qRT-PCR)
For each sample RNA was extracted from a pool of retinae from a wild-type or PAX77+/+ litter using Qiagen RNeasy kit (Qiagen, USA). cDNA synthesis was performed as described in . qRT-PCR was performed on cDNA from E14.5 and E16.5 retinae with the following primer pairs (n = 3 litters): Ncam (5'-GACCATCAGGAATGTGGA-3' and 5'-AGGCTTCACAGGTCAGAGT-3'; 179 bp product); R-cadherin (5'-CAGTGAAACAGGGGACATC-3' and 5'-ATACGGTTCTCAGGAACCTC-3'; 216 bp product); Ngn2 (5'-CAAACTTTCCCTCTCTGATG-3' and 5'-CATTCAACCCTTACAAAAGC-3' ; 197 bp product);L1 (5'-ACCCTGAGGCATTACACCTG-3' and 5'-CAACTGCTCTTTGCTTTCCC-3'; 140 bp product); Six3 (5'-CTGGAGAACCACAAGTTCAC-3' and 5'-GATCCTGCAGGTACCACTC-3'; 232 bp product); mouse Pax6 (5'-AACACCAACTCCATCAGTTC-3' and 5'-ATCTGGATAATGGGTCCTCT-3'; 153 bp product); human + mouse Pax6 (5'-TAGCGAAAAGCAACAGATG-3' and 5'-TCTATTTCTTTGCAGCTTCC-3'; 250 bp product); Shh (5'-CCCTTTAGCCTACAAGCAGT-3' and 5'-CCACTGGTTCATCACAGAG-3'; 232 bp product) and GAPDH (5'-GGGTGTGAACCACGAGAAAT-3' and 5'-CCTTCCACAATGCCAAAGTT-3'; 121 bp product). Quantitative RT-PCR was performed using Qiagen Quantitect SYBR Green PCR Kit (Qiagen, USA) and a DNA Engine Opticon Continuous Fluorescence Detector (GRI, UK). The abundance of each transcript in the original RNA sample was extrapolated from PCR reaction kinetics using Opticon software and normalised to the level of GAPDH transcript.