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This paper reports the cloning of Xenopus laevis, cyclophilin A gene, X-CypA. This study is the first developmental and functional characterisation in vivo of cyclophilin A in a vertebrate. X-CypA belongs to the superfamily of the immunophilin/PPIase proteins that can bind the immunosuppressant drug Cyclosporin A. Sequence analysis showed that X-CypA is highly conserved during evolution. RT-PCR and in situ hybridisation analysis showed that X-CypA expression is regulated during development and its transcripts are found in three major expression domains: nervous system, sensory organs and pronephros. Over-expression of X-CypA in embryos, analysed by in situ hybridisation and RT-PCR, leads to an expansion and disorganisation of the neural crest domain.
Fig. 4. Spatial expression profile of X-CypA during development. Whole-mount in situ hybridisation with an X-CypA DIG-labelled antisense RNA probe was performed on embryos from stages 9–39 as indicated in the figure. Expression was first detected in the nervous system from stage 15 (B), with apparent highest expression in the anteriorforebrain. This distribution persists throughout neurulation and early tailbud stages (C–E). At stage 26, X-CypA expression appears in the sensory organs including the eyes and otic vesicles and is maintained throughout their organogenesis (F–L). From stage 28, the gene is expressed in the pronephric kidney (H, I and M). At stage 39, expression can be clearly detected in the developing lateral line system (F). Panels A–C show embryos cleared in Murrays, panels D, K–M are uncleared embryos and panels F–J uncleared embryos after treatment in 70% ethanol. The sense probe showed no staining pattern (data not shown). ans, anterior nervous system; pn, pronephros; pd, pronephric duct; cg, cement gland; ey, eyes; ov, otic vesicle; ba, branchial arches; ll, lateral line.
Fig. 5. Cryostat sections showing the internal localisation of transcripts of X-CypA. Embryos at stages 26 (A), 33 (Bâ D) and 39 (E) were subjected to whole-mount in situ hybridisation, allowed to over-stain in developing solution, and then embedded in acrylamide. Blocks were frozen in isopentane and sectioned at 20â î¼m. Clear staining is observed in the sensory organs (eyes, otic vesicle) (panels Aâ C) and in the branchial arches (B). Some staining is observed in the central nervous system (A) and also in the peripheral nervous system (ganglion and spinal cord) (panels Câ E). The developing somites are also stained (panels D and E) as are the pronephric tubules (C) and duct (E). ba, branchial arches; c, cornea; df, dorsal fin; ed, epaxial dermomyotome; ev, eye vesicle; gl, ganglion; hd, hypaxial dermomyotome; l, lens; m, mesencephalon; n, neurons; no, notochord; ov, otic vesicle; pd, pronephric duct; pt, pronephric tubules; r, retina; rl, retinal layer; sc, spinal cord.
Fig. 6. Analysis of the effects of X-CypA over-expression on the expression of neural markers by in situ hybridisation. Embryos were co-injected into one cell of the 2-cell stage with 0.152, 0.076 or 0.038â ng X-CypA and 0.8â ng GFP mRNAs and cultured to stage 19. The embryos were then subjected to in situ hybridisation using En2, HoxB9, Krox20, Slug probes. No obvious effects on the expression of En2 or HoxB9 were observed. However, the expression of Krox20 and Slug were affected by the misregulation of X-CypA. Extension and disorganisation of the neural crest domain of Krox 20 is noticeable (compare Jâ L to I), the expression of Slug mRNA is increased on the injected side (compare M to Nâ P). An asterisk indicates the injected side. Arrowheads indicate altered expression pattern.
