Lateral views of typical wildtype (A) MZtcf7l1a^-/- (B) wildtype injected with control morpholino (C) tcf7l1a morphant (D) and tcf7l1a/tcf7l1b double morphant (E) embryos at 2 days post fertilisation. All conditions n > 100 and over three independent experiments except when specified. Dorsal up, anterior to the left. Scale Bar = 250 µm.

(A) Graph showing RT-qPCR quantification of the mRNA levels of lef1, tcf7, tcf7l1a, tcf7l1b, tcf7l2, otx1b, otx2, six3b and rx3 in Ztcf7l1a^-/- mutants relative to wildtype embryos at 10hpf. Biological and technical triplicates, two independent experiments. (B, C) Quantification of the forebrain domain of the anterior neural plate (B) enclosed by emx3 up to pax2a (D, E) expression by in situ hybridisation (reduction to an average of 76%, n = 11, one experiment, data in Supplementary file 1B), and eye field volume (C) by rx3 fluorescent in situhybridisation confocal volume reconstruction (J–M) (reduction to an average of 55%, n = 10, one experiment, data in Supplementary file 1C). (D–I) Expression of emx3 (arrowhead)/pax2a(D, E), six3b (arrowhead)/pax2a (F, G) and rx3 (arrowhead)/pax2a (H, I) in wildtype (D, F, H) and Ztcf7l1a^-/- (E, G, I) embryos detected by in situ hybridisation at 10hpf. Reduction of six3band rx3 expression 100%, n > 40, three experiments. (J–M) Confocal volume reconstruction of rx3 fluorescent in situ hybridisation in wildtype (J, K) and Ztcf7l1a^-/- (L, M) mutants at 10hpf. (J, L) Dorsal view, anterior to top, and (K, M) transverse view from posterior, dorsal up. Abbreviations: mb, midbrain; pcp, prechordal plate Scale Bars = 250 µm.

(A–F) Dorsal views of confocal images of rx3 mRNA expression (red) detected by fluorescent in situ hybridisation at 10hpf in the anterior neural plates of chimeric embryos containing transplants of (A–C) wildtype (GFP+) donor cells in MZtcf7l1a^-/- host embryos (100%, n = 13), and (D–F) MZtcf7l1a^-/- (GFP+) donor cells in wildtype host embryos (100%, n = 9). Dotted line outlines eye fields; note in A-C that rx3 expression extends considerably caudal to the reduced mutant eye field on the side of the neural plate containing wild-type cells. Dashed line marks the embryo midline. (G–J) In situ hybridisation of rx3 and pax2a in sibling (G, H) and Ztcf7l1a^-/- (I, J) 9hpf embryos, uninjected (G, I) or injected with 50 pg of dkk1 mRNA (H, J). Abbreviations; EF, eyefield; mb, midbrain Scale Bars = 200 µm.

(A) Growth kinetics of the eye in wildtype (blue line) and Ztcf7l1a^-/- (red line) embryos at stages indicated (data in Supplementary file 1F, one experiment, 24hpf, wt n = 12, Ztcf7l1a^-/-n = 14; 28hpf, wt n = 15, Ztcf7l1a^-/- n = 12; 32hpf, wt n = 13, Ztcf7l1a^-/- n = 15; 36hpf, wt n = 16, Ztcf7l1a^-/- n = 14; 48hpf, wt n = 11, Ztcf7l1a^-/- n = 19; 60hpf, wt n = 11, Ztcf7l1a^-/- n = 14; 72hpf, wt n = 13, Ztcf7l1a^-/- n = 19; 96hpf, wt n = 13, Ztcf7l1a^-/- n = 15). (B) Plot showing the ratio of Ztcf7l1a^-/- to wildtype eye volume from data in (A). (C–L) Lateral views (dorsal up, anterior to left) of wildtype (C–G) and Ztcf7l1a^-/- (H–L) eyes at stages indicated above panels. (M–O) Eye development following partial ablation of the optic vesicle in wildtype embryos at five somite stage. (M) Coronal confocal section of evaginating optic vesicles (red) in a wildtype Tg(rx3:RFP) five somite stage embryo. Dashed line indicates the approximate extent of ablations performed. 36hpf (N) and 4dpf (O) eyes in embryos in which cells were unilaterally removed from one optic vesicle (from n = 20). Asterisk indicates the eye that develops from the partially ablated optic vesicle. ZO1, zona ocludens 1. Scale bars = 200 µm.

(A–P) Lateral views of eyes showing atoh7 fluorescent in situ hybridisation in typical wildtype (A–E, M, O), Ztcf7l1a^-/- (F–J), wildtype left-side optic vesicle-ablated (K, L); from n = 5 embryos) and Tg(HS:dkk1)^w32 (N, P) embryos at stages indicated. (M–P) Wildtype (M, O) and heterozygous sibling Tg(HS:dkk1)^w32 embryos (N, P) heat-shocked at 6hpf (M, N); from n = 7/9 embryos) or 24hpf (O, P); from n = 10/10 embryos) for 45’ at 37°C and grown to 28hpf. Anterior is to the left except in (K) in which anterior is to the right. Arrows indicate ventro-nasal retina; arrowheads indicate dorso-temporal retina; dashed line approximate the nasal-temporal division; dashed circle marks lens position. Abbreviations: n, nasal, t, temporal. Scale bar = 100 µm. (Q) Histogram showing the spatial distribution of atoh7 expression in sibling and Ztcf7l1a^-/- retinas at the indicated hours post-fertilisation (data in Supplementary file 1F). VN, ventro nasal expression; VN+, ventro-nasal expression plus a few scattered cells; N+, nasal expression plus scattered cells covering the whole retina; NR, nasal retina expression; WR, whole retina expression; PR, expression localised to the peripheral retina. Numbers in bars represent the number of embryos scored for the particular category of atoh7 expression. (R) Plot showing the growth kinetics of the eye in wildtype (blue line) and Tg(HS:dkk1)^w32 (red line) embryos at times indicated (data in Supplementary file 1K).

(A–B) Immunostaining detecting phosphohistone3 (PH3, green) and RFP (Tg(atoh7:GAP-RFP)^cu2Tg-, red) in wildtype (A) and Ztcf7l1a^-/- (B) eyes at 36hpf . Arrows indicate selected double PH3/RFP positive cells. n, nasal; t, temporal. Scale bar = 100 µm. (C–D) Plot showing the percentage of PH3-positive cells (C) data in Supplementary file 1L) and double PH3/RFP-positive cells (D), data in Supplementary file 1L). Single experiment, wildtype n = 7, Ztcf7l1a^-/-n = 8, figures over the bars show p-values from unpaired t-tests.

(A) Schematic of the genetic strategy to isolate mutations that modify the tcf7l1a^-/- mutant phenotype. (B–C) U910 modifier of the tcf7l1a^-/- mutant phenotype. Lateral views of homozygous U910 embryos that are heterozygous (B) or homozygous (C) for the tcf7l1amutation. (D) Representation of SSLP segregation linkage analysis mapping of U910 modifier of tcf7l1a to a 1.46 megabase (Mb) interval on chromosome 11 (Ch11; rec, recombinants). (E) RT-PCR for hesx1 spanning exons 1–2 (top panel), exons 2–3 (middle panel) and β-actin (bottom panel) on wildtype (lanes 1 and 2) and U910^-/- (lanes 3 and 4) embryo cDNA from 1 cell stage (lanes 1 and 3) and 10hpf (lanes 2 and 4). Single experiment. (F–I) hesx1 in situhybridisation on wildtype (F–H) and U910^-/- (I) embryos at epiboly (ep) stages indicated. Dorsal views, anterior up. (J, K) Lateral views of hesx1^-/- (Δex1/2)/tcf7l1a^+/- (J) and hesx1^-/-(Δex1/2)/Ztcf7l1a^-/- (K) embryos. Four independent experiments, n = 53. Scale bars = 200 µm.

(A–J) Lateral views of wildtype (A, F), Ztcf7l1a^-/- mutant (B, G) cct5^U762/u762 mutants (C, H), double cct5^U762/u762/Ztcf7l1a^-/- mutants (D, I) and double cct5^U762/u762/Ztcf7l1a^-/- mutants injected with 0.8 pmol of cct3 morpholino (E, J) at indicated stages. Scale bar = 100 µm. Full data in Supplementary file 10O, single experiment, 36hpf, wt n = 4, Ztcf7l1a^-/- n = 9, cct5^-/-n = 8, cct5/Ztcf7l1a^-/- n = 3; 52hpf, wt n = 8, Ztcf7l1a^-/- n = 8, cct5^-/- n = 4, cct5/Ztcf7l1a^-/- n = 3; 52hpf + 2 pmol tp53 morpholino, wt n = 6, Ztcf7l1a^-/- n = 13, cct5^-/- n = 13, cct5/Ztcf7l1a^-/-n = 12; 52hpf + 0.8 pmol cct3 morpholino, wt n = 12, Ztcf7l1a^-/- n = 12. (K) Eye volume quantification at the indicated timepoints and conditions shown in A–J) (data in Supplementary file 1O). Unpaired t-test. (L–O) Immunostaining detecting phosphohistone3 (PH3, green) in wildtype (L), Ztcf7l1a^-/- (M), cct5^-/- (N), cct5^-/-/Ztcf7l1a^-/- (O) eyes at 32hpf. (P) Plot showing the percentage of PH3 positive cells in the eyes shown in L–O) (data in Supplementary file 1Q) Single experiment, wildtype n = 10, Ztcf7l1a^-/- n = 10, cct5^-/- n = 9, cct5/Ztcf7l1a^-/-n = 10, unpaired t-tests.

(A–H) Lateral views of eyes in wildtype (A, E), Ztcf7l1a^-/- (B, F), gdf6a^U768/U768 (C, G) and double gdf6a^U768/U768/Ztcf7l1a^-/- (D, H) embryos at 36hpf (A–D) and 52hpf (E–H). Dorsal up, anterior to left. Arrows indicate the lens. Scale bar = 200 µm. (I) Whole mount fluorescent in situ hybridisation for rx3 and pax2a in wildtype (I), Ztcf7l1a^-/- (J), gdf6a^U768/U768(K) and double gdf6a^U768/U768/Ztcf7l1a^-/- (L) embryos at 10hpf. Dorsal view, anterior up. Arrows point to rx3eye field expression. Scale bar = 100 µm. (M) Eye volume quantification in wildtype (n = 7), Ztcf7l1a^-/- (n = 6), gdf6a^-/-(n = 6) and gdf6a^U768/U768/Ztcf7l1a^-/- double mutant siblings (n = 3) at 36hpf (data in Supplementary file 1R). Single experiment, unpaired t-test. (N) Eye field volume quantification from rx3 fluorescent in situ hybridisation shown in I-L in wildtype (n = 7), Ztcf7l1a^-/- (n = 4), gdf6a^-/-(n = 8) and gdf6a^U768/U768/Ztcf7l1a^-/- double mutant siblings (n = 4) at 10hpf (data in Supplementary file 1S). Single experiment, unpaired t-test.