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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. |
