Internalization of zebrafish Par1 and Par2b upon thrombin or trypsin stimulation. 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.

Expression of par1 during zebrafish embryogenesis. A–V: Whole-mount in situ hybridization was performed on embryos at various stages: eight-cell (A); sphere (B); gastrula (C–E); 10-somite (F–H, arrows indicate posterior and ventral mesoderm); 1 days postfertilization (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: dorsal 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–U; T: lateral view showing par1 expression in the heart, pharynx, CHT; U: dorsal 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), and K, P, U (dorsal view). Scale bars = 100 μm.

Expression of par2b in embryos at 1–3 days postfertilization (dpf), as detected by whole-mount in situ hybridization. A–E: At 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: At 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: At 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.

Expression of par2a and par2b at early stages of development. Expression of par2a and par2b was detected by whole-mount in situ hybridization, in embryos at various stages: eight-cell, sphere, shield, 80%E, 2-somite and 10-somite. Lateral view, animal pole is up, dorsal toward the right. Scale bar = 100 μm.

Expression of zebrafish par3 detected by reverse transcriptase-polymerase chain reaction (RT-PCR). RT-PCR was performed on RNA from embryos at different stages, as indicated using primers flanked the region that encodes the first 258 amino acids of the putative Par3 protein. β-actin was used as a loading control.