Membrane-type MMPs are indispensable for placental labyrinth formation and development

Abstract

The membrane-type matrix metalloproteinases (MT-MMPs) are essential for pericellular matrix remodeling in late stages of development, as well as in growth and tissue homeostasis in postnatal life. Although early morphogenesis is perceived to involve substantial tissue remodeling, the roles of MT-MMPs in these processes are only partially characterized. Here we explore the functions of 2 prominently expressed MT-MMPs, MT1-MMP and MT2-MMP, and describe their roles in the process of placental morphogenesis. The fetal portion of the placenta, in particular the labyrinth (LA), displays strong overlapping expression of MT1-MMP and MT2-MMP, which is critical for syncytiotrophoblast formation and in turn for fetal vessels. Disruption of trophoblast syncytium formation consequently leads to developmental arrest with only a few poorly branched fetal vessels entering the LA causing embryonic death at embryonic day 11.5. Through knockdown of MMP expression, we demonstrate that either MT1-MMP or MT2-MMP is crucial specifically during development of the LA. In contrast, knockdown of MT-MMP activity after LA formation is compatible with development to term and postnatal life. Taken together these data identify essential but interchangeable roles for MT1-MMP or MT2-MMP in placental vasculogenesis and provide the first example of selective temporal and spatial MMP activity required for development of the mouse embryo.

Figure 1

Mouse placental structure and expression of MT-MMPs. (A) Schematic representation of mouse placental development between E9.5 and E10.5. At E9.5, the fetal portion of the placenta is of limited size, but CHs rapidly expand the fetal portion of the placenta at this time. Fetal vessels (FVs) make their way into the prospective LA, and after differentiation of CHs, the vessels are ensheathed in 2 layers of syncytiotrophoblasts that separate the FVs from the adjacent maternal blood sinuses (MSs). This structure facilitates nutrient and gas exchange. (B-C) Hematoxylin and eosin (H&E)–stained cross sections of wild-type mouse placentas at E10.5 with abundant FVs containing nucleated red cells and MSs with enucleated erythrocytes. The yellow dotted lines in panels B-C represent the approximate border between the fetal portion of the placenta and the decidua (maternal portion). (D) Darkfield image of section serial with that shown in panel B hybridized to antisense probe (AS) for MT1-MMP. Note the abundant signal in the embryonic mesenchyme, the LA and the decidua. (E) Darkfield section serial with that shown in panel C hybridized to MT2-MMP AS. Note the signal in the trophoblasts and the limited or absent signal in decidua. (F-G) Brightfield images of sections shown in panels D-E. Note that the signal shown here is pseudocoloration of the darkfield signal projected onto the brightfield image. Scale bar (B-G): 200 μm.

Figure 2

Loss of MT1-MMP and MT2-MMP leads to embryonic demise. (A) Whole mount darkfield images of mouse embryos. Double-deficient embryos (1−/−; 2−/−) at E9.5 are indistinguishable from their control littermates; at E10.5, however, they are easily distinguished by their retarded growth. (B) Double-mutant embryos moreover display dilated vasculature (v) and enlarged pericardia (P). All double-deficient embryos are dead at E11.5. Scale bars: (A) 1 mm; (B) 0.3 mm.

Figure 3

Loss of MT1-MMP and MT2-MMP leads to defective LA formation. (A) Cross section of H&E-stained normal placentas at E10.5 showing the AM and the placental LA with abundant FVs and MSs. Insets in (A) and (B) show entire placentas in cross section at low magnification. (B) Placenta from (1−/−; 2−/−) embryo displaying a more compact structure of the labyrinth with sparse FVs and MSs. (C) Area framed in panel A shown at high magnification. Note the abundant FVs with nucleated red cells and the adjacent MSs with enucleated red cells. (D) Area framed in panel B demonstrating the underdeveloped LA of (1−/−; 2−/−) placentas. FVs are sparse and penetrate only to a shallow depth. Top white frame is enlarged in right bottom corner and displays apoptotic bodies and dead cells surrounding the fetal vessel (arrowheads). (E-F) Same images as in panels C-D with FVs outlined in yellow. (G) Immunohistochemical localization of collagen type IV outlining FVs of the elaborately branched LA. (H) Collagen type IV–specific stain of placenta from (1−/−; 2−/−) littermate showing diminished vessel branching and penetration (asterisks) into the prospective LA. (I-J) H&E stains of sections serial with sections shown in panels G-H. Scale bars: (A-B) 200 μm; (A-B inset) 1 mm; (B-J) 100 μm.

Figure 4

MT1-MMP/MT2-MMP deficiency leads to disruption of LA architecture. (A) Control placenta from E10.5 embryo stained for HAI-1. Note the localization of brown immunoreactivity in CHs and in the differentiated trophoblasts of the LA outlined in yellow. (B) The staining pattern in a (1−/−; 2−/−) littermate is confined to a more restricted area due to the poor development of the LA (yellow outline). (C) Ultrastructure of the LA from a control placenta demonstrating the trilaminar structure of the fetal vasculature. Pseudocolors show the fetal-maternal interface composed of the fetal vascular endothelium (ve, green), 2 layers of syncytial trophoblasts (st2, orange and st1, yellow) and the STGC lining the MSs (stgc, blue). Fetal blood cell (FB, purple), maternal blood cell (MB, red). Note fetal red cells are nucleated in contrast to maternal cells. (D) Pseudocolored electron microscope image shows that the 2 syncytial layers are missing in the double-deficient placenta and the MSs are not in proximity. The fetal vascular endothelium in green (ve) is surrounded by undifferentiated trophoblasts in cyan (ut). Fetal blood cells (FB, purple). (E) Unaltered version of image shown in panel C. (F) Unaltered version of image shown in panel D. Scale bars: (A-B) 200 μm; (C-F) 2 μm.

Figure 5

Early conditional loss of MT1-MMP in an MT2-MMP–deficient background replicates unconditional loss of both genes and LA disruption. (A) Whole mount images of embryos collected at E12.5 with double-deficiency induced before the formation of the LA via TMX-induced Cre-mediated excision from E7.5 to 11.5. (B-E) Corresponding LA structure of embryos in panel A. (B) Normal LA with abundant FVs and MSs juxtaposed. (C) Equivalent LA structure to that shown in panel B except the embryo is heterozygous for the unconditional MT1-MMP allele. (D) LA from Cre+ placenta displaying a LA morphology equivalent to that found in (1−/−; 2−/−) placentas featuring a compact structure and sparse vessels. Note the many dead cells (arrowheads) and the large distance between FVs and MSs. (E) LA from another Cre+ placenta demonstrating multiple dead cells around the FVs (yellow outline). (F) Relative MT1-MMP mRNA level measured by real-time PCR of embryonic tissues shown in panel A after TMX treatment. Note that TMX treatment in Cre− mice does not affect development. Scale bars: (A) 1 mm; (B-E) 50 μm.

Figure 6

Conditional loss of MT1-MMP in an MT2-MMP deficient background after LA formation is compatible with development to term. (A) Whole mount preparation of a Cre− embryo treated with TMX after the LA was formed (from E12.5 to 16.5). (B) Cre+ littermate with identical gross appearance despite MT1-MMP and MT2-MMP-deficiency after TMX treatment. (C) Relative expression level of MT1-MMP mRNA evaluated by real-time PCR on embryonic tissue (from A and B) demonstrates complete ablation of MT1-MMP after treatment with TMX in the presence of Cre. (D-E) H&E–stained cross-sections of the placentas corresponding to the embryos shown in panels A and B, respectively. Note the abundant vascularization of both placental LAs despite the conditional loss of both MT1-MMP and MT2-MMP in (E). Compare with the control placenta after formation of the LA in panel D. Scale bars: (A-B) 1 mm; (D-E) 200 μm.