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Figure 2. Stromelysin-3 (ST3) and collagenase-4 (Col4) have distinct spatiotemporal expression profiles during embryogenesis. Whole-mount in situ localization of ST3 (A,C) and Col4 (B,D) mRNA showed very restricted expression patterns during development at stages 32 (A,B) and 45 (C,D). ST3 was largely restricted to the branchial arches (A, single arrow) and dorsal posterior endoderm (A, double arrow) at stage 32 and to structures in the thorax (C, t) and the dorsal anterior axis (C, d) at stage 45. Col4 could not be detected at stage 32 (B) and was only detectable at approximately stage 45 where it was also present in the thorax (D, t) and the dorsal anterior axis (D, d), although in a somewhat different pattern compared with ST3.
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Figure 3. Stromelysin-3–green fluorescent protein (ST3-GFP) but not GFP is expressed extracellularly and causes developmental defects in transgenic Xenopus laevis. Tadpoles at approximately stage 45 were examined with visible light (A,C,E,G) for morphology or fluorescent light for GFP (B,D,F,H). A,B: Injections using control untreated sperm nuclei resulted in normal embryos (A) that displayed autofluorescence in their intestines (B). C,D: Transgenesis with CMV-GFP and selecting embryos that passed normally through gastrulation resulted in normal embryos (C) that expressed GFP ubiquitously (D). E,F: Some CMV-GFP embryos that did not undergo gastrulation properly (likely due to poor sperm used in transgenesis) resulted in abnormal embryos with large cavities. Although the embryos expressed GFP ubiquitously, these cavities (arrow, E and F) were free of detectable GFP. G,H: Transgenesis with CMV-ST3-GFP and selecting embryos that passed normally through gastrulation resulted in abnormal embryos (G) that expressed GFP ubiquitously (H) (note the presence of a nontransgenic embryo in G, which had normal appearance but no detectable GFP except the auto fluorescence in the intestine in H). Unlike GFP, the ST3-GFP fusion protein could be seen secreted into the extracellular cavities (arrows, G and H).
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Figure 4. Transgenic overexpression of stromelysin-3 (ST3), membrane-type-5–matrix metalloproteinase (MT5-MMP), and collagenase-4 (Col4) leads to embryonic abnormalities and death. Tadpoles at approximately stages 37–42 were examined with visible light (A,C,E,G) for morphology and with fluorescent light for GFP (B,D,F,H). Transgenesis was carried out with CMV–green fluorescent protein (CMV-GFP) or CMV-MMPs, and embryos that passed normally through gastrulation were selected for analysis. A,B: CMV-GFP transgenesis resulted in normal embryos (A) that expressed GFP ubiquitously (B). Note that some GFP was preferentially expressed and localized within the ciliary epithelial cells and results in a dotted staining of the surface (B). C,D: Transgenic animals with CMV-ST3-GFP, which had high levels of GFP fluorescence, were abnormal (C) and expressed ST3-GFP ubiquitously (D). These embryos often developed large edemic cavities that were full of ST3-GFP (D). E,F: Transgenic animals with CMV-MT5-GFP that expressed high levels of GFP were also abnormal (E) and expressed MT5-GFP ubiquitously (F). They also developed large edemic cavities that were full of MT5-GFP and appeared to have a dose-dependent decrease in axis length with embryos expressing the highest level of GFP having the shortest axis (top embryo, E and F). G,H: Transgenesis with CMV-Col4-GFP produced abnormal embryos (G) and expressed Col4-GFP ubiquitously (H). All embryos expressing high levels of MMP-GFP died by stage 50 or shortly after (see also Table 1).
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Figure 6. Wild-type stromelysin-3 (ST3) causes larval death even at low levels, whereas high levels of mutant ST3 has no effect on development in transgenic animals. Transgenic tadpoles at approximately stages 40 (A,B) and stage 46/47 (C–H) were examined with visible light (A,C,E,G) for morphology or fluorescent light for green fluorescent protein (GFP) (B,D,F,H). Transgenesis was carried out with CMV-ST3-GFP, CMV-GFP, and CMV-ST3m-GFP, and embryos that passed normally through gastrulation were selected for analysis. A,B: One animal from among the largely normal embryos within each of the three groups (top, an ST3-GFP embryo; middle, a GFP embryo; and bottom, an ST3m-GFP embryo) expressed GFP ubiquitously (B). However, the ST3-GFP embryo had much lower levels of transgene expression (top embryo) (Note the strong auto-fluorescence in the intestine). Embryos that expressed higher levels of ST3-GFP did not develop normally to this stage. ST3-GFP and ST3m-GFP was often preferentially localized within the hatching gland cells on the head (e.g., bottom embryo, arrow). C,D: The ST3-GFP (top) and GFP (middle) embryos in A/B had noticeable differences when compared later at stage 46/47 (C,D, right and left embryo, respectively). The GFP embryo had bright fluorescence and well-formed gills (triple arrows C and D) compared with the ST3 embryo, which had very low levels of GFP fluorescence but poorly developed gills (C,D, double arrows). E,F: A nontransgenic control embryo (left side) is compared with an ST3 transgenic expressing very low, barely detectable levels of ST3-GFP (right side), which could be seen to have abnormally folded intestines (i in E) and ST3-GFP present in its body cavity (F, the position equivalent to c in E). All embryos expressing detectable levels of ST3-GFP died before metamorphosis (Table 1 and data not shown). G,H: Unlike wild-type Col4, MT5-MMP, and ST3, transgenesis with CMV-ST3m-GFP resulted in normal embryos (G) that expressed GFP ubiquitously (H) throughout development. As with wild-type ST3 expression, ST3m was detected within extracellular cavities (H). i, intestine; c, cavity.
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mmp11 (matrix metallopeptidase 11 (stromelysin 3)) gene expression in Xenopus laevis embryos, NF stage 32, as assayed by in situ hybridization, lateral view, anterior left, dorsal up.
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mmp1 (matrix metallopeptidase 1 (interstitial collagenase)) gene expression in Xenopus laevis embryos, NF stage 45, as assayed by in situ hybridization, lateral view, anterior left, dorsal up.
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