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Mech Dev
2008 Jan 01;12511-12:1059-70. doi: 10.1016/j.mod.2008.07.005.
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Three matrix metalloproteinases are required in vivo for macrophage migration during embryonic development.
Tomlinson ML
,
Garcia-Morales C
,
Abu-Elmagd M
,
Wheeler GN
.
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Macrophages are essential in development, repair and pathology of a variety of tissues via their roles in tissue remodelling, wound healing and inflammation. These biological functions are also associated with a number of human diseases, for example tumour associated macrophages have well defined functions in cancer progression. Xenopus embryonic macrophages arise from a haematopoietic stem cell population by direct differentiation and act as the main mechanism of host defence, before lymphoid cells and a circulatory system have developed. This function is conserved in mouse and human development. Macrophages express a number of matrix metalloproteinases (MMPs), which are central to their function. MMPs are a large family of zinc-dependent endoproteases with multiple roles in extracellular matrix remodelling and the modulation of signalling pathways. We have previously shown MMP-7 to be expressed by Xenopus embryonic macrophages. Here we investigate the role of MMP-7 and two other MMPs (MMP-18 and MMP-9) that are also expressed in the migrating macrophages. Using morpholino (MO) mediated knockdown of each of the MMPs we demonstrate that they are necessary for normal macrophage migration in vivo. The loss-of-function effect can be rescued using the specific MMPs, altered to be resistant to morpholinos but not by overexpression of the other MMPs. Double and triple morpholino knockdowns further suggest that these MMPs act combinatorily to promote embryonic macrophage migration. Thus, our results imply that these three MMPs have distinct functions, which together are crucial to mediate macrophage migration in the developing embryo. This demonstrates conclusively that MMPs are required for normal macrophage cell migration in the whole organism.
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18684398
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Fig. 1. Whole-mount in situ hybridisations of Xenopus embryos showing specific expression patterns of the MMPs-7, -9 and -18 in migrating macrophages (stage 24). Representative examples of the 10 embryos assayed. (A) MMP-7, (B) MMP-9 and (C) MMP-18. (D–F) Double whole-mount in situ hybridisation with POX2 (Di, Ei, Fi) developed with fast red and MMP-7 (Dii), MMP-9 (Eii) and MMP-18 (Fii) developed with NBT/BCIP. Arrows indicate single macrophages stained for both POX2 and an MMP. All embryos are shown in lateral view, with anterior to the left.
Fig. 2. Loss-of-function studies reveal a separate functional contribution for all three MMPs in promoting macrophage migration. (A–E) Control experiments; (A and E) non-injected embryos (Ni), (B and E) control morpholino (CMO) injected embryos at 100 ng (non-specific scrambled sequence oligonucleotide). (C–E) MMP-14 morpholino (MO) at 100 ng in both VMZ (C) and DMZ (D), MMP-14 is not expressed in the migrating macrophages. (E) Bar chart showing the effect of morpholino injection on macrophage migration. (F–J) MMP-7 morpholino DMZ injections (F) 30 ng, (G) 50 ng, (H) 80 ng, (I) 100 ng; shows a dose-dependent effect on the extent of macrophage migration. (K–O) MMP-9 morpholino injections to both the VMZ and DMZ, (K) 30 ng, (L) 50 ng, (M) 80 ng, (N) 100 ng. Only with the 100 ng injection (N + O, DMZ) is a partial delay on migration observed. (P–T) MMP-18 morpholino injections to both the VMZ and DMZ, MMP-18 morpholino injections to the DMZ show the strongest effect on macrophage migration, (P) 10 ng, (Q) 50 ng, (R) 80 ng, (S) 100 ng. All macrophages were visualised by in situ hybridisation with POX2. Graphs illustrate the mean values, with standard deviation plotted on the error bars. All embryos are stage 26 and are shown in lateral view, with anterior to the left.
Fig. 3. Rescue of normal macrophage migration using an MMP morpholino and the co-injection of mutated RNA. (A–E) MMP-7 rescue experiments. (A) Non-injected control embryo (Ni). (B) Embryos injected with 750 pg of mutated MMP-7m RNA. (C) MMP-7 morpholino injected at 40 ng in the DMZ. (D) Co-injection of 750 pg of MMP-7m RNA and 40 ng of MMP-7 morpholino restores normal migration. (E) Quantification of results from A–D plus co-injection of 750 pg of MMP-9 or 18 RNA with 40 ng MMP-7 morpholino fails to rescue normal migration. (F–J) MMP-9 rescue experiments. (F) Non-injected control embryo (Ni). (G) Embryos injected with 750 pg of mutated MMP-9m RNA. (H) MMP-9 morpholino injected at 100 ng in the DMZ. (I) Co-injection of 750 pg of MMP-9m RNA and 100 ng of MMP-9 morpholino restores normal migration. (J) Quantification of results from F–I plus co-injection of 750 pg of MMP-7 or 18 RNA with 100 ng MMP-9 morpholino fails to rescue normal migration. (K–O) MMP-18 rescue experiments. (K) Non-injected control embryo (Ni). (L) MMP-18m RNA. (M) MMP-18 morpholino 20 ng. (N) Co-injection of 750 pg of MMP-18m RNA and 20 ng of MMP-18 morpholino restores macrophage migration. (O) Quantification of results from K–N plus co-injection of 750 pg of 7 or 9 RNA with 20 ng MMP-18 morpholino fails to rescue normal migration. All embryos are stage 26 and are shown in lateral view, with anterior to the left. All macrophages were visualised by in situ hybridisation with POX2. Significant difference from the morpholino injected embryos and the co-injected rescue embryos (asterisks indicate values compared) using a Student’s t-test were found for MMP-7 (P < 0.001), MMP-9 (P < 0.001) and MMP-18 (P < 0.05).
Fig. 4. Multiple loss-of-function experiments demonstrate that the MMPs are acting combinatorily to promote macrophage migration. (A) Bar chart showing result of double morpholino injection loss-of-function experiments. (B–F) Triple loss-of-function experiments. (B) Non-injected embryo (Ni). (C) All three morpholinos injected into the DMZ at 10 ng each. (D) All three morpholinos injected into the DMZ at 20 ng. (E) All three morpholinos injected into the DMZ at 30 ng, showing a complete block on migration. (F) Graph illustrates the control VMZ injections and the DMZ injections, acting in a dose-dependent manner. All embryos are stage 26 and are shown in lateral view, with anterior to the left. All macrophages were visualised by in situ hybridisation with POX2.
Fig. 5. Macrophage localisation and morphology. In the triple loss-of-function embryos the macrophages are rounded in shape and stay in the mesenchyme and between the epidermis and mesenchyme. (A) 30Â ng MMP-7/9/18 injected into the VMZ. (B) 30Â ng MMP-7/9/18 morpholino injections into the DMZ. Ai and Bi show in situ hybridisation of macrophages with POX2 as a probe, viewing the ventrallateral side of the embryo. Insert boxes show close ups of the macrophages. Aii and Bii show transverse sections of the embryos. Arrows point to individual macrophages.
Fig. 6. Knockdown of the three MMPs does not block macrophage migration through the presumptive heart field. In triple loss-of-function assays (30Â ng MMP-7/9/18) a distinct group of macrophages migrates through the presumptive cardiac field (7/10 embryos). Whereas the remaining macrophages are constrained from their normal radial migration route. (A) 30Â ng MMP-7/9/18 into the VMZ. (B) 30Â ng MMP-7/9/18 morpholino injections into the DMZ. The arrows mark the location of the migrating heart fields at this stage of development. The embryos are viewed from the ventral side with anterior to the top. All macrophages were visualised by in situ hybridisation with POX2.
mmp7 (matrix metallopeptidase 7) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 24, lateral view, anteriorleft, dorsal up.
mmp9.1 (matrix metallopeptidase 9, gene 1) gene expression in Xenopus laevis embryo via in situ hybridization, NF stage 24, lateral view, anteriorleft, dorsal up.
mmp1 (matrix metallopeptidase 1) gene expression in Xenopus laevis embryo via in situ hybridization, NF stage 24, lateral view, anteriorleft.
