April 1, 2000;
Xenopus laevis gelatinase B (Xmmp-9): development, regeneration, and wound healing.
It has been argued that matrix metalloproteinases play important roles in cellular differentiation and regeneration in certain systems. While studying changes in gene expression associated with the phenomena of cornea
transdifferentiation ("lens regeneration"), which takes place in the larva
of Xenopus laevis, we identified the Xenopus gelatinase B gene. The open reading frame is homologous to other gelatinase B genes identified in other species and encodes all of the domains characteristic of this protein. Xenopus gelatinase B (Xmmp-9) is first expressed during early tail
-bud stages in a subset of mesodermal cells scattered throughout the body. Expression is also seen in the peripheral tissues of the developing liver diverticulum
, the hindgut
, and the paired caudal vein
, and its dorsal branch in the larval tail
. Given the significant role of matrix metalloproteinases in degrading components of the extracellular matrix, Xmmp-9 expression may be important in the morphogenesis of these structures. Xmmp-9 expression was also examined during the processes of cornea
transdifferentiation, epithelial wound healing, and limb
regeneration in Xenopus larvae. Although Xmmp-9 is expressed very early during cornea
transdifferentiation, expression is restricted to the site of the peripheral wound created by removal of the original lens
, which triggers transdifferentiation. Expression was not found in the central, uninjured area of the cornea
where transdifferentiation takes place. Therefore, Xmmp-9 does not appear to play an important role in cornea
transdifferentiation. Xmmp-9 expression is associated with other epithelial wounds, indicating that gelatinase B is expressed in the general context of wound healing in Xenopus. Finally, Xmmp-9 is expressed in the ectoderm
at the tip of the amputated limb
, very early during limb
regeneration, where it is argued to play a role in this process.
[+] show captions
Fig. 2. Embryonic expression patterns of Xenopus gelatinase B (Xmmp-9), revealed by digoxygenin-labeled RNA in situ hybridization. A: Lateral view of a stage 26 embryo showing the first detectable Xmmp-9 expression in scattered mesenchymal cells. B: Increased expression in a stage 37 embryo (black arrowhead points to expression associated with the developing liver diverticulum ). C: High magnification view of the tail of a stage 40 larva. D: Stage 42 larvae showing continued expression in the abdominal region and the tail. Faint staining in the head and in the olfactory epithelium is an artifact, which is sometimes seen in sense RNA controls. E: Stage 46 embryo showing decreased expression of Xmmp-9. A small number of pigmented xanthophores (xn), in the head, dorsal tail, and peritoneum normally appear in albino larvae at this stage, which are not expressing Xmmp-9. F: Negative control stage 38 embryo reveals no appreciable hybridization of sense RNA. All specimens are shown as lateral views with the anterior ends located to the left. cv, caudal veins; ey, eye; ms, mesenchyme cells. Scale bar =500 um in A,B and D and 250uminC.
Fig. 3. Sections taken at different levels showing representative Xmmp-9 mRNA expression in specific cells within the stage 37&38 larvae. A: Left lateral view of a stage 37&38 larva. Dotted lines indicate the locations of the cross sections shown in B, as indicated. B: Cross section through the eye. C: More posterior cross section through the neural tube. D: Cross section through the developing heart and liver diverticulum. E: More anterior section through the liver diverticulum. F: Cross section through the tail. Black arrowheads point to the endothelial cell bodies of the paired caudal veins. G: Section through the developing hindgut. Lateral (H), and ventral (I) views showing the cloaca and proctodeum of a stage 38 larva. Anatomical designations within the heart and blood vascular system follow those of Millard (1945, 1949) and Nieuwkoop and Faber (1956). Dorsal is located up in all sections. an, anus; ao, lumen of aorta; cv, lumens of caudal veins; fg, foregut lumen; hg, hindgut lumen; l, lens; ld, liver diverticulum/lumen; ms, mesenchyme expressing Xmmp-9; nc, notochord; nt, neural tube; rt, retina; sv, sinus venosus; tm, tail musculature; vf, ventral fin. Scale bar = 500 um in A. Scale bar in C applies to B and equals 50 um. Scale bar in G applies to D and equals 50 um. Scale bar in H applies to I and equals 100 um
Fig. 5. Expression of Xmmp-9 mRNA associated with corneal wound healing, in various epithelial wounds, and during the earliest stage of limb regeneration following amputation. A: Left superolateral view 24 hr after lens removal. Expression is restricted to the posterior corneal wound. Pigmented spots on the surface of the eye are xanthophores, which normally develop even in many albino embryos. B: Left lateral view showing expression associated with a lateral epithelial wound 24 hr after surgery in a young stage 41 larva. C: Absence of expression in the unoperated control eye of a stage 52 tadpole larva. D: Expression of Xmmp-9 in sham operated specimens 24 hr after incision of the outer cornea epithelium. E: Negative control with no detectable hybridization of sense RNA probe to the wounded cornea 24 hr after incision of the outer corneal epithelium. F: Xmmp-9 expression in the left hindlimb stump 24 hr after distal amputation in a stage 52 tadpole larva. G: Longitudinal section of an amputated stage 52 limb 6 hr after amputation. Xmmp-9 mRNA is present in the apical ectodermal cap (ep) and the underlying mesoderm (ms). H: Absence of expression in the hindlimb on the unop- erated side. cn, cornea; cw, corneal wound; hl, unoperated hindlimb; ln, lens; ls, hindlimb stump; lw, lateral ectodermal wound; uc, uninjured cornea overlying the central pupillary space. Scale bar =500 um in A, 200 um in B. Scale bar in E applies to C and D and equals 100um. Scale bar in G 50um.Scale bar in H applies to F and equals 100um.