The primary structure of Prdc has been highly conserved during evolution. A: Alignment of the protein related to Dan and Cerberus (PRDC) amino acid sequences from human, dog, mouse, rat, chick, zebrafish, fugu, and tetraodon. The nine conserved cysteine residues are boxed. The CAN domain is shaded. Cysteine residues 1, 3, 4, 6, 8, and 9 form the knot ring structure, 2 and 7 form a cysteine pair to complete the knot domain. Cysteine 5 is unpaired, and it might be involved in intermolecular bonds. Asterisks mark identical amino acids, double dots highly conserved substitutions, and single dots conservative amino acid changes among PRDC orthologues. The percentages of identity and similarity between human, mouse, and zebrafish are indicated below the alignment. The fugu and tetraodon sequences contain a 21-residue insertion between cysteine residues 4 and 5, which is not found in the other species. B: Phylogenetic tree of PRDC sequences from various species. C: Reverse transcriptase-polymerase chain reaction analysis using prdc gene-specific, exon-spanning primers (35 cycles). Gapdh amplification served as a control (28 cycles). The embryonic stages are indicated on top as hours postfertilization (h).

Expression of prdc in the developing eye. A,B: Prdc transcripts appear at 17 hours postfertilization (hpf) in the anterodorsal region of the developing optic lobe and remain in this site till around 23 hpf (black arrowheads). C,D: prdc expression marks the dorsal and ventral outer edges of the optic cup at 26 hpf (black arrowheads). D is dorsal view of C. White arrowheads point to prdc expression in the first pharyngeal arch. E-J: prdc expression in the retinal epithelium (black arrowheads) is localized ventrally at the edges surrounding the choroid fissure (28-42 hpf). E is dorsal view of F. J: At 72 hpf, prdc expression can be seen in a very small ventral region in the marginal zone. K-O: Frontal view (K) of prdc staining in the retina at 28 hpf and sections (L-O) of the corresponding whole-mounts in the adjacent top panels at 32, 36, 42, and 72 hpf illustrate that, after the eye rotates, prdc expression is confined to ventral areas of the developing retina. O: The minute expression at 72 hpf is marked by a black arrowhead. Anterior is to the left (except in K). h, hours postfertilization.

Prdc expression in the pharyngeal arches. A-D: Prdc expression in the pharyngeal arches between 17 and 24 hours postfertilization (hpf) first in arches 1 and 2 and then 3. E-H: prdc and dlx2 expression at 26 hpf. E,F and G,H depict dorsal and lateral views of embryos stained with prdc and dlx2 probes, respectively. I-K: Cryosections (placed in order from anterior [I] to posterior [K] direction) of double-stained embryos with prdc (blue) and dlx2 (red) riboprobes. L-R: prdc and dlx2 expression at 30 hpf. L,M and N,O dorsal and lateral views of prdc and dlx2 whole-mount staining respectively. P-R: Consecutive cryosections (placed in order from anterior [P] to posterior [R]) of double-stained embryos with prdc (blue) and dlx2 (red) riboprobes. S-Y: prdc and dlx2 expression at 36 hpf. S,T and U,V dorsal and lateral views of prdc and dlx2 whole-mount staining, respectively. W-Y: Cryosections (placed in order from anterior [W] to posterior [Y]) of double-stained embryos with prdc (blue) and dlx2 (red) riboprobes. Z-AF: prdc and dlx2 expression at 42 hpf. Z,AA dorsal and lateral views of prdc expression, respectively. Lateral views of dlx2 (AB) and nkx2.3 (AC) expression. AD-AF: Consecutive cryosections (placed in order from anterior [AD] to posterior [AF]) of double-stained embryos with prdc (blue) and dlx2 (red) riboprobes. AG-AI: Dorsal views of 42 hpf embryos hybridized with dlx2, prdc, and nkx2.3 riboprobes. The lines marked a,b depict the planes of plastic sections shown in the adjacent panels. AJ-AO: Plastic cross-sections of dlx2, prdc, and nkx2.3-stained 42 hpf embryos at the level marked with line a (AJ-AL) and line b (AM-AO) in AH. Black arrows point to dlx2 (AJ,AM) and nkx2.3 expression domains (AL,AO) in pharyngeal arch neural crest and endodermal pouches, respectively. Anterior side in whole-mount images is to the left except in AG-AI to the top. h, hours postfertilization.

Prdc expression in somites, swim bladder, and oral epithelium. A-C: prdc is expressed primarily in the ventral part of the developing somites at 24 hours postfertilization (hpf; arrowheads). B and C are plastic sections of the whole-mount prdc in situ hybridization shown in A. The section planes are marked with the lines B and C. D-F: prdc (in blue) and foxa3 (in red) expression at 32, 36, and 42 hpf (D-F). The two probes overlap at the site of the swim bladder bud. The prdc expression in pharyngeal arches is also visible at the top. G: Single foxa3 staining marks the liver (L), swim bladder (S), pancreas (P), and buds and the intestinal bulb (IB). H,I: sections of prdc probe-stained embryos depict expression in the swim bladder mesenchyme (arrowheads) at 42 and 72 hpf. J,K: prdc expression at 72 hpf marks the oral epithelium in whole-mount staining and sections (white arrowheads). Staining in the arches is also seen (black arrowhead in K). Anterior is to the top in panels A, D-G and to the left in J. h, hours postfertilization.