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A Hyal2-type hyaluronidase of Xenopus laevis (Xhyal2) was characterized by molecular cloning, biochemical analysis and ectopic overexpression in embryos. When expressed in Xenopus oocytes, Xhyal2 exists as a soluble protein in the extracellular space and in intercellular compartments as well as being attached to the cell surface through a glycosyl-phosphatidyl-inositol anchor. This enzyme specifically degrades hyaluronan not only at acidic pH values but more slowly also under physiological conditions. Xhyal2 is differentially expressed during embryogenesis. Particularly striking is the high level of expression in the developing brain, the headmesenchyme and the pronephros. Elevated levels of mRNA were also found in endothelial cells which will later form vascular structures. Ectopic overexpression of Xhyal2 in frog embryos causes loss of hyaluronan in the cellular environment. This causes severe defects in the assembly of the highly structured plexus of the vitelline vessels from prevascular endothelial cells. Our data support the notion that the level of Xhyal2 expression determines the organization of the extracellular environment so that cells can merge and/or migrate within an originally impenetrable matrix.
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11804776
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Fig. 2. Whole-mount in situ hybridization. Expression of Xhyal2 during Xenopus embryogenesis. (A) Mid-sagittal section of a gastrula stage embryo, animal side up, dorsal side to the right; bc, blastocoel; bp, dorsal blastopore. (B) Neurula stage 17 embryo, dorsolateral view, anterior side to the right; hf, head folds. (C) Longitudinal section of neurula stage 17 embryo, anterior side up; a, archenteron; e, epidermis; n, notochord; nf, neural fold; pm, prechordal mesoderm. (D) Tailbud stage 28 embryo, lateral view, anterior side to the left; cg, cement gland; pn, pronephros. (E) Tadpole stage 37, lateral view; pcv, posterior cardinal vein. (F) Close-up view of the heart and ear region of a stage 30 embryo rendered transparent by mounting in tetrahydro-naphthaline; ea, eye anlagen; ov, otic vesicle; black triangles mark precursors of the common cardinal veins. (G,H) Close-up views of embryo shown in (E). (G) Nephric region; white triangles mark the forming ureteric duct originating from the pronephros (pn); black triangles mark the intersomitic vessels that end in the posterior cardinal vein; pv, prevascular endothelial cell. (H) Specimen rendered transparent by mounting in tetrahydro-naphthaline; trunk region; black triangles mark the medial part of the notochord.
Fig. 6. In situ hybridization of Xmsr and Xfer. Xmsr and Xfer whole-mount in situ hybridization of XHyal2-injected and control embryos. Xmsr expression was used as a marker for PVEC of the primordial vitelline veins. Very little PVEC were detected in the posterior-ventral part of Xhyal2-injected embryos (D,E) when compared to control embryos (A,B). Dashed lines show the boundary on the flanking position of the PVEC pattern. In (B,E) close-up views are presented. Arrowheads indicate the posterior-ventral border of migrating PVEC as indicated by a dashed line in (A,D). Hyperfusion of merging PVEC in Xhyal2-injected embryos is indicated by a white arrow (E). The expression of Xfer, a marker for red blood cells, appeared largely equivalent in XHyal2-injected embryos and controls with subtle individual differences that were also observed in uninjected siblings (C,F).
