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We report the isolation and expression pattern of a novel gene, Ami in Xenopus laevis. Ami was initially isolated as a highly expressed gene in cardiovascular tissues. The deduced amino acid sequence of Ami was most closely similar to human complement factor D and mouse adipsin in mammals. In adult Xenopus tissues, the transcript of Ami was detected in liver, fat body, lung, gut, vessel, heart, muscle, testis, and ovary, but expression in blood cells or skin was hardly detected. This expression profile was significantly different from that observed for mammalian homologues. Ami transcripts in Xenopus laevis were expressed from the late neurula stage, remained constant until the tadpole stage. The mRNA localized to paraxial regions at the neurula stage and anteriorventral regions at the tailbud stage. From the late tailbud to tadpole stage, expression was detected along the forming blood vessels, including the anterior cardinal veins, posterior cardinal veins, intersomitic veins, dorsal longitudinal anastomosing vessel, dorsal aorta, pronephric sinus, and most prominently around the vascular vitelline network. The expression around the vascular vitelline network demonstrated left-right asymmetry in stage 42 embryo. Comparison with the endothelium marker, Xmsr, suggested that Ami is expressed in endothelial cells.
Fig. 3. Spatial expression pattern of Ami mRNA in developing embryos. Whole-mount in situ hybridization was performed on embryos at various developmental stages. Ami mRNA localize in regions lateral and posterior to the cement gland (A) and in the paraxial region (B and C) of the embryo at stage 17. (A) anterior view; (B) dorsal view; (C), posterior view. Dorsal is upward in (A) and (C), and anterior is upward in (B). The signal was detected in paraxial region including somites (arrowhead) in the transverse section of stage 17 embryo (D). Dorsal is upward in (D). The mRNA localized in regions lateral and posterior to the cement gland in stage 23 embryo (E and F). (E) lateral view; (F) ventral view. Anterior is upward in (F). The signal was seen in mesodermal (H, arrowhead) and endodermal (H, double arrowhead) layers in the transverse section of stage 23 embryo (G and H). H is a magnified view of G. The mRNA was detected in the anteriorventral region of stage 28 embryo (I). Strong signal was detected in endodermal layer of cells (J and K, double arrowhead). K is a magnified view of J. Pigments was seen around the edge of the sections of G, H, J, and K due to the use of wild type embryos for the hybridization with sections instead of albino embryos, and is believed not to be the signal of the hybridization. The mRNA was localized around the posterior cardinal vein (pcv, arrowhead), pronephric sinus (k, arrowhead), and the vascular vitelline network (vvn, arrowhead) at stage 30 (L). The expression around the vascular vitelline network was restricted to the anterior part of the trunk (L and M). The expression around the intersomitic veins (isv, arrowhead) became visible in stage 32 and 34 (N and P). The expression around the vascular vitelline network spread to the anterior one-third (N and O) and anterior half (P and Q) of the trunk at stage 32 and 34, respectively, and the ventral-most region showed no expression of the mRNA. At stage 42, the expression became evident in the anterior cardinal vein (acv, arrowhead), dorsal longitudinal anastomosing vessel (dlav, arrowhead), and dorsal aorta (da, arrowhead) (R). The expression around the vascular vitelline network covered the whole trunk region, except for the most posterior region around the cloaca (S). M, O, Q, and S are magnified views of L, N, P, and R, respectively. Black lines in (F) and (I) indicate the positions of the sections shown in (G and H), and (J and K), respectively. Bar: 100î¼m.
Fig. 4. Ami mRNA expression in transverse sections of stage 42 embryo. Embryo at stage 42 were sectioned and hybridized with the Ami antisense probe. In the section through otic vesicles (Aã¢â â C), expression was observed around the anterior cardinal veins (acv, double arrowheads), notochord (not, double arrows), and ventralaorta (va, arrows), but not around the endocardium (ec, arrow). (B) is the magnified view of (A). In the section through the trunk (Dã¢â â F), expression was observed around the dorsal aorta (da, arrows), posterior cardinal veins (pcv, arrowheads), and vascular vitelline network (vvn, double arrowheads). (E and F) are the magnified views of (D). In the section through the fin (Gã¢â â I), the mRNA was detected around the dorsal longitudinal anastomosing vessel (dlav, G, H arrow) and posterior cardinal veins (pcv, G, I arrowheads). (H and I) are the magnified views of (G). Pigments was seen around the edge of the sections due to the use of wild type embryos for the hybridization with sections instead of albino embryos, and is believed not to be the signal of the hybridization. Bar: 100ã â ã â¼m.
Fig. 5. Left/right asymmetry of the vascular vitelline network. Whole-mount in situ hybridization of Ami mRNA was performed for stage 35 and 42 embryos. At stage 35, areas that were negative for the mRNA were observed on both right (A, white arrowhead) and left (B, white arrowhead) sides of the anteriortrunk region, posterior to the duct of cuvier (dc). The gaps are observed in the line comprising the vascular vitelline network on both sides of the trunk (C, black arrowheads). At stage 42, the signal-less area was seen on the left (E, white arrowhead) trunk region, but not on the right side (D, white arrowhead). The signal digressed from the epidermis, through the endodermal mass specifically on the left side (F, black arrowhead). Black lines in (A, B, D, and E) indicate the positions of the sections shown in (C and F), respectively. K, pronephric sinus; Bar: 100â î¼m.
Fig. 6. Ami mRNA was expressed in monolayer of cells around the lumen of the vessel. In situ hybridization analysis revealed that Xmsr mRNA was present in single layer of cells lining the lumen of the posterior cardinal veins in the fin of the stage 42 embryo (A and B, arrowheads). Ami mRNA was also detected in the single layer of cells surrounding the lumen of the posterior cardinal veins (C and D, arrowheads). Bar: 100μm.
