August 1, 2007;
Expression profiles of the duplicated matrix metalloproteinase-9 genes suggest their different roles in apoptosis of larval intestinal epithelial cells during Xenopus laevis metamorphosis.
Matrix metalloproteinases (MMPs) play a pivotal role in development and/or pathogenesis through degrading extracellular matrix (ECM
) components. We have previously shown that Xenopus MMP-9
gene is duplicated. To assess possible roles of MMP-9
and MMP-9TH in X. laevis intestinal remodeling, we here analyzed their expression profiles by in situ hybridization and show that their expression is transiently up-regulated during thyroid
hormone-dependent metamorphosis. Of interest, MMP-9TH mRNA is strictly localized in the connective tissue
and most highly expressed just beneath the larval epithelium
that begins to undergo apoptosis. On the other hand
, cells expressing MMP-9
mRNA become first detectable in the connective tissue
and then, after the start of epithelial apoptosis, also in the larval epithelium
. These results strongly suggest that MMP-9TH is responsible in the larval epithelial apoptosis through degrading ECM
components in the basal lamina
, whereas MMP-9
is involved in the removal of dying epithelial cells during amphibian intestinal remodeling.
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Figure 4. Correlation of matrix metalloproteinase-9 (MMP-9) and MMP-9TH mRNA expression with apoptosis. a-h: Cross-sections of the intestine from tadpoles at stages 61 (a,b) and 62 (e,f) and from premetamorphic tadpoles treated with 10 nM T3 for 3 (c,d) and 5 days (g,h) were examined for apoptosis by terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labeling (TUNEL) assay (a,c,e,g) and for the expression of MMP-9 (f,h) and MMP-9TH mRNAs (b,d) by in situ hybridization (ISH). Brown nuclei labeled with TUNEL indicate nuclei of apoptotic cells or bodies (arrowheads), while dark blue deposits indicate the sites of probe binding (arrows). To compare the localization of cells labeled by TUNEL to that by ISH, serial sections were used throughout the experiments. The morphology in each serial section seems slightly different due to different staining procedures. Fibroblasts expressing MMP-9TH (b) in the connective tissue (CT) are localized immediately beneath the larval epithelium (Ep), which undergoes apoptosis (a) at stage 61. Similar results were obtained in the intestine of premetamorphic tadpoles treated with 10 nM T3 for 3 days (c,d). On the other hand, cells expressing MMP-9 mRNA are detectable at stage 62 both in CT and Ep. Some of them in Ep are localized close to apoptotic nuclei labeled by TUNEL (e,f). Similar results were obtained in the intestine of premetamorphic tadpoles treated with 10 nM T3 for 5 days (g,h). L, lumen. Scale bars = 20 mu m.