Fig. S2

Craniofacial and ocular phenotypes in Nr2f mutants. (A) Nr2f deletion alleles generated in this study. Care was taken to introduce early frame-shift mutations likely abrogating all protein function. The mutation sites in each gene are indicated with arrows. (B) Alcian blue and Alizarin red staining of nr2f1a, nr2f1b, nr2f2, and nr2f5 single mutants at 5 dpf revealed no gross skeletal abnormalities. Combinatorial loss of nr2f1b, nr2f2, and nr2f5 alleles led to fusion of the hyoid joint (black arrow) and reduction of the dorsal hyomandibula cartilage and opercle bone (magenta arrow). Loss of nr2f1a in nr2f5 mutants deficient for nr2f1b and nr2f2 in some cases led to syngnathia (open arrowhead). The full facial skeleton is reduced in quadruple mutants. The probable identities of residual fused cartilages in the quadruple mutant are indicated. M, Meckel’s cartilage; Pq, palatoquadrate; Ch, ceratohyal. (C) First arch origins of the retroarticular process of Meckel’s cartilage in zebrafish. (Left panel) sox10:kikGR+ cells in the center of the first arch were photoconverted from green to red (magenta) fluorescence by selective exposure to UV light at 35 hpf. The right panel shows the same fish at 5 dpf, with residual photoconverted protein (magenta, also see bottom right panel) in cells within the retroarticular process (ra) and jaw joint (jj) region. The boxed area is magnified in the bottom left images. Images are maximum intensity projections except the high magnifications, which are single optical sections. (D) Coloboma in nr2f2; nr2f5 mutants (magenta triangle) was fully penetrant and allowed for straightforward identification of double mutants as early as 30 hpf. Scale bars: B = 50 μm, C = 20 μm, D = 250 μm