(A) Phylogenetic tree depicting the evolutionary relationships among PAR proteins, as established by the Neighbor-Joining (NJ) method (http://www.ebi.ac.uk/Tools/clustalw2), in which proteins cluster together based on sequence similarity. Bootstrap values indicated at each branch point were calculated from 1,000 replicate runs. (B) Schematic diagrams of the synteny in the regions of zebrafish chromosomes 5 and 21 (site of par genes) and human chromosome 5 (site of PAR genes).

Whole-mount in situ hybridization was performed on embryos at various stages: 8-cell (A); sphere (B); gastrula (C-E); 10-somite (F-H, arrows indicate posterior and ventral mesoderm); 1 dpf (I-N; I: lateral view of a embryo showing par1 expression in the heart, posterior cardinal vein (PCV), cardinal vein (CV), intermediate cell mass of mesoderm (ICM); J: high-magnification image of anterior region of I; K: dorsal view of the anterior region of the embryo showing the par1 expression in common cardinal vein (CCV); L: high-magnification image of the trunk region of I, location of the notochord (nc) is indicated; M-N: sagittal and transverse sections of the trunk area indicated by the black line shown in I); day 2 (O-S; O: Lateral view showing par1 expression in the heart, branchial arches, caudal hematopoietic tissue (CHT); P: ventral view of the anterior region of O at high magnification; Q-R: lateral view of the trunk region of O at high magnification; S: transverse section at the trunk region showing par1 expression in notochord (nc), vein, pronephric duct (pd) and gut; 3 dpf (T-V; T: lateral view showing par1 expression in the heart, pharynx, CHT; V: ventral view of the anterior region at high magnification; par1 expression in pancreas is indicated). Most embryos are shown in lateral view, with the animal pole up. Exceptions include G (dorsal view), H (dorsal-posterior view), P (ventral view), and K, P, V (dorsal view). Scale bars: 100 μm.

Amino acid alignments of zebrafish (Danio rerio, dr), human (Homo sapiens, hs) and mouse (Mus musculus, mm) Par1 (A) and Par2 (B), generated using the ESpript 2.2 program (http://espript.ibcp.fr/ESPript/ESPript). Amino acids are numbered to the right of each line. The transmembrane (TM) domains are indicated by solid lines above the sequences. The putative protease cleavage sites and the tethered ligands are indicated by the arrow or arrowhead and dashed line boxes, respectively. The CHD motifs are indicated by asterisks above the sequences. Identical amino acids are highlighted in black boxes; similar amino acids are shown in black bolded characters in black frames.

HEK (human embryonic kidney) 293 cells transfected with cDNA encoding GFP-tagged zebrafish Par1 or Par2b were stimulated with either thrombin (50 nM, B, E) or trypsin (10 μM, C, F) for 30-60 min. Untreated cells served as control (A, D). Scale bar: 10 μm.

(A-E) 1 dpf. A: Lateral view; B: high-magnification view of the trunk region of A; C-D: transverse and sagittal sections, respectively, of the trunk area indicated by the black line shown in A, with black arrows denoting par2b expression in the pronephric duct (pd). Note that par2b is highly expressed on the apical side of the epithelial cells of the pronephric duct; E: dorsal view of anterior region in A, showing par2b expression in the otic vesicle. (F-J) 2 dpf. F: Lateral view; G-H: lateral and dorsal views, respectively, of the anterior region of F at high magnification, showing par2b expression in the gut and notochord (nc); I: lateral view of the trunk region of F at high magnification, showing par2b expression in the gut and notochord; J: sagittal section of the trunk area. (K-P): 3 dpf. K-L: Lateral view, arrows denote pharynx and liver; M: ventral view, showing par2b expression in branchial arches and the liver; N: lateral view of the trunk region at high magnification, showing par2b expression in the gut and pronephric duct (pd); O-P: transverse (O) and sagittal (P) sections of the trunk area. Note that par2b transcript was enriched on the apical side of the epithelial cells of both the pronephric duct and gut. Scale bars: 100 μm unless specifically labeled.