Heads and eyes were fixed in 4% paraformaldehyde, cryoembedded, and sectioned, usually at 12 μm. If necessary, cryostat sections were treated for antigen retrieval with hot citrate buffer (pH 6) or with 1% Triton X-100. The following primary antibodies or markers were used: BRN3A/POU4F1 (1:50, #AB1585; EMD Millipore, Billerica, MA, USA), BRN3 (1:50, sc-6026; Santa Cruz Biotechnology, Dallas, TX, USA), CALB1 (1:500, #AB1778; EMD Millipore), F-actin/Phalloidin (1:500, #A12379; Life Technologies, Carlsbad, CA, USA), β-galactosidase (1:5000, #55976; Cappel/MP Biomedicals, Aurora, OH, USA), β-galactosidase (1:750, generous gift from Nadean Brown, University of California, Davis, CA, USA), Laminin (1:2000, #ab30320; Abcam, Cambridge, MA, USA), LEF1 (1:100, #C12A5; Cell Signaling, Danvers, MA, USA), MITF (1:400, #X1405M; Exalpha, Exalpha Biologicals, Shirley, MA, USA), OTX1/2 (1:1500, #NG1734326; EMD Millipore), PAX2 (1:100, #PRB-276P; Covance/BioLegend, Dedham, MA, USA), PAX6 (1:300, #AB2237; EMD Millipore), PITX2 (1:1000, #PA1020-100; Capra Science, Angelholm, Sweden), PROX1 (1:1000, #11-002; AngioBio Co., San Diego, CA, USA), SOX9 (1:50, #AB5535; EMD Millipore), TCF4 (1:100, #2569; Cell Signaling Technology), and VSX2 (1:300, #X1180P; Exalpha Biologicals). These antibodies were used in combination with the following secondary antibodies: Alexa 488/568/647–conjugated secondary antibodies (1:1000; Life Technologies), donkey-anti-goat TRITC (1:500, #705-025-147; Jackson ImmunoResearch, West Grove, PA, USA), and donkey-anti-sheep TRITC (1:500, #713-165-003; Jackson ImmmunoResearch). We analyzed 5 and 4 Axin2lacZ/lacZ embryos with uni- or bilateral ocular defects at E12.5 and E15.5, respectively (see also Results for further details). Each marker was analyzed in two to four tissue sections in central regions of each eye, and several markers were analyzed repeatedly.