membrane-type mmps are indispensable for placental - blood

VASCULAR BIOLOGY

Membrane-type MMPs are indispensable for placental labyrinth formationand developmentLudmila Szabova,1 Mee-Young Son,1 Joanne Shi,1 Marek Sramko,1 Susan S. Yamada,1 William D. Swaim,2 Patricia Zerfas,3

Stacie Kahan,1 and Kenn Holmbeck1

1Matrix Metalloproteinase Unit, Craniofacial and Skeletal Diseases Branch and 2Molecular Physiology and Therapeutics Branch, National Institute of Dental andCraniofacial Research, National Institutes of Health, Bethesda MD; and 3Division of Veterinary Resources, Office of Research Services, National Institutes ofHealth, Bethesda MD

The membrane-type matrix metallopro-teinases (MT-MMPs) are essential for peri-cellular matrix remodeling in late stagesof development, as well as in growth andtissue homeostasis in postnatal life. Al-though early morphogenesis is perceivedto involve substantial tissue remodeling,the roles of MT-MMPs in these processesare only partially characterized. Here weexplore the functions of 2 prominentlyexpressed MT-MMPs, MT1-MMP and MT2-MMP, and describe their roles in the pro-cess of placental morphogenesis. The

fetal portion of the placenta, in particularthe labyrinth (LA), displays strong over-lapping expression of MT1-MMP and MT2-MMP, which is critical for syncytiotro-phoblast formation and in turn for fetalvessels. Disruption of trophoblast syncy-tium formation consequently leads to de-velopmental arrest with only a few poorlybranched fetal vessels entering the LAcausing embryonic death at embryonicday 11.5. Through knockdown of MMPexpression, we demonstrate that eitherMT1-MMP or MT2-MMP is crucial specifi-

cally during development of the LA. Incontrast, knockdown of MT-MMP activityafter LA formation is compatible withdevelopment to term and postnatal life.Taken together these data identify essen-tial but interchangeable roles for MT1-MMP or MT2-MMP in placental vasculo-genesis and provide the first example ofselective temporal and spatial MMP activ-ity required for development of the mouseembryo. (Blood. 2010;116(25):5752-5761)

Introduction

After embryo implantation and decidualization, a critical step inplacental development is the formation of the placental labyrinth(LA), which enables nutrient and gas exchange between theembryonic vasculature and the maternal blood supply.1-5 LAformation is associated with substantial tissue remodeling and celldifferentiation, as well as ingrowth of the embryonic vasculaturethrough the chorion to a point of immediate proximity with thematernal blood supply. During this process, chorionic trophoblasts(CHs) differentiate into 2 perivascular cell populations that formdistinct bilaminar envelopes of syncytiotrophoblasts in immediatecontact with the fetal vascular endothelium.6 Multiple transcriptionfactors, growth factors, adhesion molecules, and gap junctionmolecules are known to influence the formation of the LA.7

Interestingly, few if any proteolytic enzymes have so far beenproven essential for LA formation although tissue remodeling isconsidered an integral part of this morphogenetic process. Severalof the matrix metalloproteinases (MMPs), cathepsins, and serineproteinases are expressed in the placenta during development,however to date none have proven indispensable for developmentof the LA and in turn development of the embryo to term.8-12

Among the 6 known membrane-type MMP (MT-MMP) moleculesin the mouse, MT1-MMP and MT3-MMP possess pericellularcollagenase activity and are required for both prenatal and postnatalremodeling of the major fibrillar collagen types, cell surfacereceptors, and signaling molecules.13-15 Ablation of MT1-MMPdeprives cells of the ability to migrate through and process several

both permanent and provisional extracellular matrices, and in vivoleads to severe defects in postnatal remodeling of connectivetissues.16-19 Moreover, MT1-MMP is required in the stromalcompartment for efficient dissemination of malignant epithelialcells in mouse mammary carcinoma.20 MT1-MMP deficiency ispartially mitigated by the activity of the molecular relative,MT3-MMP, which shares at least some overlapping substratespecificity with MT1-MMP. Accordingly, incremental loss ofalleles encoding each molecule markedly exacerbates the cellularmatrix remodeling deficit in a gene dosage dependent manner,and double deficiency results in severe developmental deficits andperinatal death, but importantly, not preterm loss of embryos.15

As expected from these observations, MT1-MMP and MT3-MMPare frequently coexpressed in the same tissue compartments. Theplacental LA however is devoid of MT3-MMP expression butdisplays conspicuous expression of another MT-MMP familymember, MT2-MMP.11,21 So far, the function of MT2-MMP hasbeen determined biochemically and in cell-based assays. Underthese conditions, MT2-MMP displays the ability to process base-ment membrane components and fibrillar collagen matrices whereasits role in vivo has remained unexplored.22,23 Here we demonstratethat MT2-MMP deficiency in the mouse is compatible withdevelopment and postnatal growth, however, the combined lossof 2 MT-MMPs, MT1-MMP and MT2-MMP, leads to arrest ofgestation at embryonic day (E)10.5. The double-deficient micedie in utero due to a failure of trophoblasts to form the syncytial

Submitted October 20, 2009; accepted September 6, 2010. Prepublishedonline as Blood First Edition paper, September 21, 2010; DOI 10.1182/blood-2009-10-249847.

The online version of this article contains a data supplement.

The publication costs of this article were defrayed in part by page chargepayment. Therefore, and solely to indicate this fact, this article is herebymarked ‘‘advertisement’’ in accordance with 18 USC section 1734.

© 2010 by The American Society of Hematology

5752 BLOOD, 16 DECEMBER 2010 � VOLUME 116, NUMBER 25

For personal use only.on April 3, 2019. by guest www.bloodjournal.orgFrom

http://www.bloodjournal.org/

http://www.bloodjournal.org/site/subscriptions/ToS.xhtml

portion of the trilaminar structure that constitute the labyrinthineinterchange between the fetal vasculature and the maternal bloodsupply in the developing placenta.6 By time dependent ablationof MT-MMP activity in vivo, we specify the temporal requirementfor MT-MMPs in LA formation. Loss of either or both MT-MMPsafter placental LA formation is compatible with development toterm as is postnatal loss of both molecules. These observationsdemonstrate that either MT1-MMP or MT2-MMP activity isirreplaceable in an early developmental program responsible forplacental development and shows, for the first time, that MT-MMPactivity is an obligate requirement for development of the mouseembryo to term.

Methods

Animal experiments

Laboratory animal experiments in this study were conducted with theapproval of the National Institute of Dental and Craniofacial Research(NIDCR) animal use and care committee.

In situ hybridization

Formaldehyde-fixed placental tissue sections were deparaffinized andhybridized to [�-33P] uridine-5�-triphosphate radiolabeled antisense andsense probes specific for MT1-MMP and MT2-MMP as previouslydescribed.24

Real-time PCR

Fetal portions of placentas were dissected and DNase-free RNA wasprepared using an RNAqueous-4PCR kit (Ambion) according to themanufacturer’s protocol.

One microgram of total RNA was transcribed into cDNA using aniScript cDNA Synthesis Kit (Bio-Rad). cDNA (0.5 �L) was subsequentlyamplified by real-time polymerase chain reaction (PCR) using SYBR GreenPCR Master Mix (Bio-Rad) on a MyIQ thermocycler (Bio-Rad) initially at95°C for 10 minutes, then 40 cycles of 95°C for 10 seconds and 62°C for30 seconds. Melting curves were established by 80 cycles of heatingfrom 55°C to 95°C for 10 seconds. Each sample was analyzed in triplicatewith threshold levels set automatically. Cycle threshold (Ct) values werenormalized to the Ct values for 29S ribosomal protein mRNA. Relativeexpression values were calculated by formula 2[Ct(29S)-Ct(MT1-MMP)] � 1000.Primer sequences were kindly provided by Dr Matthew Hoffman (NIDCR)and are available upon request.

Tamoxifen treatment of mice

Presence of a copulation plug in the morning was considered E0.5 forpurposes of developmental staging. Females in different stages of preg-nancy were dosed orally with 8 mg of tamoxifen (TMX) and 4 mg ofprogesterone in vegetable oil once daily by gavage from 3 to 5 days.Embryos were collected 24 hours after the final TMX treatment. Placentaswere dissected, fixed in 4% formaldehyde/phosphate-buffered saline (PBS),and processed for histology. Embryos were frozen and later used for mRNAextraction to examine the efficiency of MT1-MMP deletion. For administra-tion of TMX to suckling pups, lactating females were dosed orally with8 mg of TMX in vegetable oil daily for 5 days. The pups were allowedto reach the desired age and then killed. The hearts, normally a site ofabundant MT1-MMP expression, were used for RNA extraction to examinethe efficiency of MT1-MMP deletion. Adult mice (6-10 weeks old) weredosed orally with 8 mg of TMX daily for 5 days. They were observed andkilled 4 to 5 months later for analysis.

Transmission electron microscopy

The placentas were fixed in 2% glutaraldehyde in 0.1M sodium cacodylatebuffer pH 7.4 overnight at 4°C, washed in cacodylate buffer and subse-

quently postfixed with 2% osmium tetroxide for 2 hours. The tissue waswashed again with 0.1M sodium cacodylate buffer, serially dehydrated inethanol and propylene oxide and embedded in EMBed 812 resin (ElectronMicroscopy Sciences). Thin sections, approximately 80 nm, were obtainedusing the Leica Ultracut-UCT ultramicrotome (Leica), placed onto 300 meshcopper grids, and stained with saturated uranyl acetate in 50% methanol andthen with lead citrate. The grids were viewed in a JEM-1200EXII electronmicroscope (JEOL Ltd) at 80 kV and images were recorded on a XR611M,midmounted, 10.5 megapixel, charge-coupled device (CCD) camera (Ad-vanced Microscopy Techniques).

Immunohistochemistry

For immunohistochemical staining, paraffin slides were dewaxed, rehy-drated through graded ethanol and washed in PBS. The antigen wasretrieved by microwaving for 20� in 10mM Na-citrate buffer pH 6.0, exceptfor detection of laminin. The slides were incubated for 30� in blockingsolution (2% goat serum, 1% bovine serum albumin, 0.1% Triton X-100,0.05% Tween 20, and 0.05% sodium azide in PBS), then in primaryantibody at 1:50 dilution for 1 hour and washed in PBS. For 3,3�-diaminobenzidine staining, endogenous peroxidase activity was blocked byincubation in 3% H2O2 in PBS for 10 minutes and the slides were thenreacted with secondary biotinylated antibody diluted 1:400 for 30 minutesand washed, incubated with avidin-biotin complex (Vector Laboratories)for 30 minutes and washed. Bound avidin-biotin complex was detected with3,3�-diaminobenzidine and the slides were rinsed in water and counter-stained briefly in hematoxylin, then dehydrated and mounted. For immuno-fluorescence, the slides were reacted with Alexa 488–conjugated secondaryantibody (Molecular Probes) 1:100 for 1 hour, washed in PBS, and stainedin 0.2% Sudan black in 70% ethanol for 30 minutes as described.25 TypeI collagen was detected using Abcam 34710, Laminin (Sigma-AldrichL9393) and type IV collagen (Chemicon AB 756P).

Microscopy and imaging

Whole mount darkfield images were captured on an Olympus SZHdissection scope equipped with a Q-imaging Micropublisher RTV CCDcamera (Q-Imaging) and a 0.5� PLFL lens at 3.75-32� magnification.Bright and darkfield images of histologic sections were captured on a ZeissAxioplan 2 (Carl Zeiss Microimaging) upright microscope equipped withan Axiocam MRc CCD camera using Zeiss Plan-NEOFLUAR 2.5�/NA0.075; 5�/NA 0.15; 10�/NA 0.30; and 20�/NA 0.5. Immunofluorescencewas imaged as gray scale on the fluorescein isothiocyanate and DAPI(4�,6-diamidino-2-phenylindole) channels and pseudocolored using ZeissAxiovision Version 4.6 imaging software (Carl Zeiss Microimaging).

Results

MT1-MMP and MT2-MMP are coexpressed in the placenta

To dissect the role of MT-MMP activity in early development weinitially analyzed placental tissues for expression of MT-MMPs. Insitu hybridization of placenta sections demonstrated a particularlypronounced expression of both MT1-MMP and MT2-MMP atE10.5 when development of the LA occurs (Figure 1A). MT1-MMP was expressed throughout the fetal portions of the E10.5placenta, including the allantoic mesenchyme (AM), CHs anddifferentiated as well as undifferentiated trophoblasts of theprospective LA. Moreover, a significant MT1-MMP signal wasdetected in the decidual part of the placenta (Figure 1B,D,F).MT2-MMP was likewise detected in the fetal portion of theplacenta with intense signal in the CHs and in the prospective LA.Unlike the signal for MT1-MMP, however, the MT2-MMP signalin the decidua was largely absent (Figure 1C,E,G).

MT-MMPS IN PLACENTAL VASCULOGENESIS 5753BLOOD, 16 DECEMBER 2010 � VOLUME 116, NUMBER 25




Loss of MT2-MMP is compatible with normal development,growth, and reproduction

To establish the biologic role of MT2-MMP in development, wecloned part of the sequence encoding this MT-MMP from a mousegenomic lambda phage library. A targeting vector deleting thesequences between the 3� part of exon 2 through exon 5, includingthe catalytic site, was constructed and used for generation of micecarrying this deletion in its germ line. When mice heterozygous forthis deleted MT2-MMP allele (2-) were interbred, homozygousoffspring henceforth referred to as (2�/�), were recorded with afrequency consistent with unperturbed Mendelian distribution ofthe wild-type and mutated alleles. Homozygous mutant miceexpressed no mRNA for MT2-MMP when tested by reversetranscription PCR (supplemental Figure 1C, available on the BloodWeb site; see the Supplemental Materials link at the top of theonline article) and based on this result we concluded that the(2�/�) locus was functionally a null allele.

Neither female or male mice with the (2�/�) genotypedisplayed any significant difference in bodyweight compared withwild-type and heterozygous littermates. Consistent with the normalbodyweight, (2�/�) mice likewise displayed no overt aberra-tions in size and general appearance. The lifespan, behavior, and

grooming habits were normal and the (2�/�) mice reproducednormally and reared litters to weaning age without impediments.

MT-MMP activity is required for placentogenesis anddevelopment to term

Based on the lack of overt physiologic consequences of MT2-MMP-deficiency, we subsequently tested if MT2-MMP was completelyredundant or if the function of MT2-MMP is wholly or partlycompensated for by other proteases of related molecular structure,expression pattern and substrate specificity. Since MT1-MMP, byvirtue of its overlapping expression pattern, related molecularstructure and relative expression level,11 could be compensating forthe loss of MT2-MMP in the placenta and vice versa, we crossedthe MT2-MMP null allele, (2�), into our MT1-MMP–deficientbackground,16 henceforth referred to as (1�/�). Mice with the(1�/�) genotype do not reproduce and we therefore first estab-lished (1�/�; 2�/�) mice, which appeared healthy and unaffectedby the loss of one MT1-MMP and one MT2-MMP allele. Subse-quent interbreeding of (1�/�; 2�/�) mice surprisingly yielded nooffspring with the (1�/�; 2�/�) genotype while the remaininggenotype combinations appeared with expected Mendelian fre-quency. To eliminate the possibility that the double-deficient mice

Figure 1. Mouse placental structure and expression of MT-MMPs.(A) Schematic representation of mouse placental development betweenE9.5 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. Fetalvessels (FVs) make their way into the prospective LA, and after differentia-tion of CHs, the vessels are ensheathed in 2 layers of syncytiotrophoblaststhat separate the FVs from the adjacent maternal blood sinuses (MSs).This structure facilitates nutrient and gas exchange. (B-C) Hematoxylinand eosin (H&E)–stained cross sections of wild-type mouse placentas atE10.5 with abundant FVs containing nucleated red cells and MSs withenucleated erythrocytes. The yellow dotted lines in panels B-C representthe approximate border between the fetal portion of the placenta and thedecidua (maternal portion). (D) Darkfield image of section serial with thatshown in panel B hybridized to antisense probe (AS) for MT1-MMP. Notethe abundant signal in the embryonic mesenchyme, the LA and thedecidua. (E) Darkfield section serial with that shown in panel C hybridizedto MT2-MMP AS. Note the signal in the trophoblasts and the limited orabsent signal in decidua. (F-G) Brightfield images of sections shown inpanels D-E. Note that the signal shown here is pseudocoloration of thedarkfield signal projected onto the brightfield image. Scale bar (B-G):200 �m.

5754 SZABOVA et al BLOOD, 16 DECEMBER 2010 � VOLUME 116, NUMBER 25




had perished before weaning or that some random skewing of thegenotype distribution could be the cause of the lack of doublemutant pups, we set up breeding between (1�/�; 2�/�) parentsexpecting a 25% frequency of (1�/�; 2�/�) pups. However,among more than 100 offspring not a single (1�/�; 2�/�) pupwas identified and we therefore concluded that (1�/�; 2�/�)embryos were lost in utero. Using timed pregnancies, we initiatedgenotyping of embryos at E9.5. After genotyping, we documentedthe presence of (1�/�; 2�/�) embryos with approximately 25%frequency in 170 embryos and these were indistinguishable insize and appearance from either (1�/�; 2�/�) or (1�/�; 2�/�)siblings. At E10.5 the number of (1�/�; 2�/�) embryos did notdeviate significantly from the expected number in 311 samples, butthey were easily identified by their smaller size, prominent dilationof their vasculature (Figure 2A-B) and enlargement of the pericar-dium. Despite the expected frequency of the (1�/�; 2�/�)genotype in 166 embryos at E11.5, none were found alive and allwere uniformly small and partially involuted. Consistent with thesefindings, only 3 (1�/�; 2�/�) were found in 91 E12.5 ageembryos (�2 � 25.00, P .0001). We concluded based on theseresults that the combined loss of MT1-MMP and MT2-MMPcaused abrogation of embryogenesis shortly after E10.5 andsubsequent involution of the embryo.

Whole mount preparations and histologic sections of the doublemutant E9.5 embryos revealed no overt difference compared withthe control littermates in either embryo or placenta. However, atE10.5 dilated vasculature and an enlarged pericardium in thedouble mutant embryos (Figure 2) suggested placental abnormali-

ties as previously reported in Fra-1 deficient mice.26 We thensought to establish the cause of the premature demise of (1�/�;2�/�) embryos. Analysis of the placentas from double-deficientmice revealed that vascularization of the fetal portion of the tissuesthat eventually establish the labyrinthine layer of the embryonicand maternal vascular interchange was retarded (Figure 3B,D,F,H,J) compared with placentas from control littermates (Figure3A,C,E,G,I). Specifically, the number of FVs extending fromthe AM through the CH layer and into the prospective LA wasdiminished (outline, Figure 3E-F). Moreover, when FVs didpenetrate they invariably displayed a greatly reduced degree ofbranching (Figure 3G-H, LA and asterisks).

Arrest of development in (1�/�; 2�/�) embryos is notassociated with accumulation of extracellular matrixin the placental LA

Because MT1-MMP is essential for remodeling of extracellularmatrix molecules, such as collagen, in postnatal life, we probed if adefect in matrix remodeling leading to matrix accumulation couldbe documented in double-deficient placentas. We therefore ana-lyzed tissue for detectable imbalances in the amount of extracellu-lar matrix proteins present, including basement membrane compo-nents. We performed immunohistochemistry for type IV collagen(Figure 3G-H), laminin (supplemental Figure 4G-H), and collagentype I (supplemental Figure 4C-D). In addition, we assessedthe content of type III collagen by reticulin stain (supplementalFigure 4E-F) as well as fibronectin (supplemental Figure 4I-J). All

Figure 2. Loss of MT1-MMP and MT2-MMP leads to embryonicdemise. (A) Whole mount darkfield images of mouse embryos. Double-deficient embryos (1�/�; 2�/�) at E9.5 are indistinguishable from theircontrol littermates; at E10.5, however, they are easily distinguished bytheir retarded growth. (B) Double-mutant embryos moreover displaydilated vasculature (v) and enlarged pericardia (P). All double-deficientembryos are dead at E11.5. Scale bars: (A) 1 mm; (B) 0.3 mm.





immunohistochemical stains demonstrated that extracellular matrixcomponents were predominantly localized to the AM and fetalvessel basement membrane (collagen type IV and laminin) orassociated perivascular tissue (collagen type I). Aside from thedifference in fetal vessel density, we saw no overt change in thedistribution of these potential MT-MMP substrates that wouldsuggest a defect in processing. When assessed in detail by confocalmicroscopy (supplemental Figures 6-8), there was a slight increaseof fluorescence intensity in the AM of (1�/�; 2�/�) placentas.Importantly, however, the matrix content around vessels or pros-pective vessels in the LA was reduced compared with controllittermates suggesting that matrix accumulation was not takingplace there. Moreover, we recorded no accumulation of extracel-lular matrix components in the prospective LA where FVs indouble-deficient placentas failed to advance. Reticulin fibers werepresent principally in the AM and in the decidua, while none weredetected in the prospective LA irrespective of genotype.

We further analyzed the levels of mRNA of basement mem-brane components by real-time PCR analysis, but in accordancewith the immunohistochemistry for type IV collagen and lamininwe found no significant deviations from control placenta (1�/�;2�/�) mRNA levels for these 2 molecules. Additionally mRNAlevels for nidogen, perlecan, and fibronectin did not deviate fromthose found in the controls (supplemental Figure 2A). Furthermore,we found no significant change in the level of MT1-MMP mRNAin the absence of MT2-MMP and vice versa. Likewise, neitherpartial or complete loss of MT1-MMP and MT2-MMP resulted inany detectable alteration in MT3-MMP expression levels (supple-mental Figure 2B). Moreover, we ascertained if loss of MT1-MMPand MT2-MMP would affect the mRNA expression levels ofseveral collagen types. Consistent with our immunohistochemistryresults, the mRNA levels for type I and type IV collagen wereidentical to that in control tissue and type III and type XVIIIcollagen were likewise unchanged in response to loss of one orboth of the MT-MMPs (supplemental Figure 2C). Finally, tobroaden our search for potential substrates that were unprocessed inthe absence of MT1-MMP and MT2-MMP, we performed expres-sion array analysis and mass-spectrometry analysis on the fetalportion of the placenta isolated from E10.5 embryos. Both analysesrevealed no differences pointing to accumulation of known struc-tural substrates and we concluded that any candidate moleculewas likely to be present in quantities below the detection limit forthese assays.

MT1-MMP or MT2-MMP is required for placentalsyncytium formation

Next we questioned if the observed defect in the placenta wasassociated with decreased viability of cells in the placenta byterminal deoxynucleotidyl transferase deoxyuridine-triphosphatasenick end labeling staining (data not shown), but only recorded celldeath in the immediate vicinity of the few FVs present, therebyconfirming our earlier observations of apoptotic bodies in the fetalvessel perimeter of mutant placentas (Figure 3D inset, outline).Bromodeoxyuridine labeling (data not shown) did not demonstratediminished cell proliferation and we concluded that the placentaldefect observed did not stem from a major deficit in cell prolifera-tion or viability.

We next considered if cell differentiation could be affected inthe absence of MT1-MMP and MT2-MMP. To ascertain if tropho-blasts differentiated from the chorionic state to syncytiotropho-blasts, sections were stained for hepatocyte growth factor activatorinhibitor (HAI-1), which stains only CHs and differentiatedsyncytiotrophoblasts.27 Because these 2 types of cells are morpho-logically distinct in the placenta, HAI-1 very clearly delineatestheir distribution and in turn the degree of tissue differentiation.The staining for HAI-1 demonstrated that trophoblasts in (1�/�;2�/�) were present mostly as compact undifferentiated CHswhereas far fewer were present as syncytiotrophoblasts in the LAcompared with the control samples (Figure 4A-B outline). Thissuggests that the apparent defect in the placenta is the inability oftrophoblasts to differentiate from the chorionic state to the syncy-tiotrophoblast state.

To gain a more detailed understanding of the defect leading tolack of LA formation, we performed transmission electron micros-copy analysis of control (1�/�; 2�/�) and mutant (1�/�; 2�/�)placentas (Figure 4C-F). In control placentas (Figure 4C), theinterchange between maternal blood sinuses (MSs) and FVsdisplayed a morphology of 4 distinct layers consisting of amononuclear sinus trophoblast giant cell (STGC) layer lining the

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 theplacental LA with abundant FVs and MSs. Insets in (A) and (B) show entire placentasin cross section at low magnification. (B) Placenta from (1�/�; 2�/�) embryodisplaying 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 withnucleated red cells and the adjacent MSs with enucleated red cells. (D) Area framedin panel B demonstrating the underdeveloped LA of (1�/�; 2�/�) placentas. FVs aresparse and penetrate only to a shallow depth. Top white frame is enlarged in rightbottom corner and displays apoptotic bodies and dead cells surrounding the fetalvessel (arrowheads). (E-F) Same images as in panels C-D with FVs outlined inyellow. (G) Immunohistochemical localization of collagen type IV outlining FVs of theelaborately 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 inpanels G-H. Scale bars: (A-B) 200 �m; (A-B inset) 1 mm; (B-J) 100 �m.





MSs and 2 peripheral cell compartments (st1 and st2) constitutedby trophoblast syncytia, which enveloped the fetal vascular endo-thelium (ve; Figure 4C,E).6 In the mutant placenta (Figure 4D,F),the MSs and the occasional FVs invariably were farther apart dueto the diminished branching of the FVs. Moreover, the ultrastruc-tural analysis revealed a striking absence of the double layer ofsyncytiotrophoblasts, reducing the fetal perivascular microenviron-ment to the undifferentiated trophoblast (ut) species lining thevascular endothelium (ve, Figure 4D). The major defect associatedwith combined loss of MT1-MMP and MT2-MMP activity thusappeared to be inability of trophoblasts to form the syncytiarequired for the development of a functional LA.

MT-MMP activity is required for LA formation and isdispensable thereafter

To further refine our observations, we addressed the temporalrequirement for the activity of MT1-MMP and MT2-MMP with a

time-dependent inactivation of MT1-MMP using a TMX-sensitiveCre allele28 and a floxed MT1-MMP allele (1loxP). Briefly, the geneproduct of the beta-actin/CreERT allele used here is retained in thecytoplasm in the absence of TMX and only dissociated from heatshock protein 90 and translocated to the nucleus after TMXadministration. This strategy enabled recombination and knock-down of a floxed MT1-MMP allele (1loxP) at will (supplementalFigure 3) and enabled time-dependent analysis of MT1-MMPfunction. We generated (1loxP/�; 2�/�); Cre� mice and (1loxP/�;2�/�); Cre� littermates, and evaluated the requirement forMT1-MMP during different time points of gestation in an MT2-MMP deficient background by TMX administration. Pregnantfemale mice were first administered TMX starting at day 7.5 post-coitum (pc) and repeatedly dosed at 24-hour intervals for 5 days.At day 12.5 pc, the embryos were collected (Figure 5A) andanalyzed for the levels of MT1-MMP message by real-time PCR.In addition, placentas were sectioned and stained to evaluate

Figure 4. MT1-MMP/MT2-MMP deficiency leads todisruption of LA architecture. (A) Control placentafrom E10.5 embryo stained for HAI-1. Note the localiza-tion of brown immunoreactivity in CHs and in the differen-tiated trophoblasts of the LA outlined in yellow. (B) Thestaining pattern in a (1�/�; 2�/�) littermate is confinedto a more restricted area due to the poor development ofthe LA (yellow outline). (C) Ultrastructure of the LA from acontrol placenta demonstrating the trilaminar structureof the fetal vasculature. Pseudocolors show the fetal-maternal interface composed of the fetal vascular endo-thelium (ve, green), 2 layers of syncytial trophoblasts(st2, orange and st1, yellow) and the STGC lining theMSs (stgc, blue). Fetal blood cell (FB, purple), maternalblood cell (MB, red). Note fetal red cells are nucleated incontrast to maternal cells. (D) Pseudocolored electronmicroscope image shows that the 2 syncytial layers aremissing in the double-deficient placenta and the MSs arenot in proximity. The fetal vascular endothelium in green(ve) is surrounded by undifferentiated trophoblasts incyan (ut). Fetal blood cells (FB, purple). (E) Unalteredversion of image shown in panel C. (F) Unaltered versionof image shown in panel D. Scale bars: (A-B) 200 �m;(C-F) 2 �m.





development after induced loss of MT1-MMP in an MT2-MMP-deficient background (Figure 5B-E). As observed earlier, theplacental LA failed to develop in the absence of MT1-MMP andMT2-MMP and accordingly the placentas in (1LoxP/�; 2�/�);Cre� mice displayed gross evidence of cell demise around the fewFVs as previously seen with the unconditional deletion of MT1-MMP and MT2-MMP (Figure 5D-E). Notably, (1LoxP/�; 2�/�);Cre� embryos developed unimpeded thereby demonstrating thatthe observed defects in development of the (1LoxP/�; 2�/�); Cre�embryos were a direct consequence of MT1-MMP deficiencyrather than TMX toxicity (Figure 5B-C).

To evaluate the need for MT1-MMP and MT2-MMP after LAdevelopment we next administered TMX to pregnant femalesstarting at day 12.5 pc when the placental LA is fully establishedand collected the embryos at day 17.5 pc. Despite complete lossof MT1-MMP mRNA after TMX administration in Cre� mice, allembryos regardless of genotype were largely normal in appearanceand displayed only the phenotype described previously for em-bryos deficient for MT1-MMP (Figure 6A-C).16 Notably, when theplacentas were analyzed, they uniformly displayed a normalappearance with extensive and elaborate LAs (Figure 6D-E). Tofurther explore the viability of double-deficient mice, several litterswere brought to term with no evidence of excess mortality. Wefurther probed the postnatal viability of (1LoxP/�; 2�/�); Cre�

mice by inducing double gene deficiency at various time points.These experiments demonstrated that double deficiency was toler-ated at all times after birth although generating a phenotypeequivalent to that described previously for unconditional MT1-MMP deletion when the gene was deleted prenatally or earlypostnatally.16 Finally, to evaluate if a possible defect in basementmembrane remodeling was the root cause of the placental defect,we tested the ability of double-deficient female mice to undergomammary gland involution. A general failure of basement remodel-ing in the involution of the lactating gland was anticipated to causea partial or complete arrest of the process in which the bothMT1-MMP and MT2-MMP are abundantly coexpressed.24 Despiteloss of both proteases after TMX administration, we recorded nodifferences in involution between double-deficient Cre� mice andcontrol Cre� mice and therefore concluded that a systemicinability to remodel basement membrane components was anunlikely cause of the placental defect (supplemental Figure 5).

Taken together our experiments show that either MT1-MMPor MT2-MMP is stringently required during a narrow window ofdevelopment for syncytiotrophoblast formation, coincident withthe establishment of the placental LA, and that a deficiency of oneor both of these molecules after LA formation is compatible withdevelopment to term and postnatal life.

Figure 5. Early conditional loss of MT1-MMP in anMT2-MMP–deficient background replicates uncon-ditional loss of both genes and LA disruption.(A) Whole mount images of embryos collected at E12.5with double-deficiency induced before the formation ofthe LA via TMX-induced Cre-mediated excision fromE7.5 to 11.5. (B-E) Corresponding LA structure of em-bryos in panel A. (B) Normal LA with abundant FVs andMSs juxtaposed. (C) Equivalent LA structure to thatshown in panel B except the embryo is heterozygous forthe unconditional MT1-MMP allele. (D) LA from Cre�placenta displaying a LA morphology equivalent to thatfound in (1�/�; 2�/�) placentas featuring a compactstructure and sparse vessels. Note the many deadcells (arrowheads) and the large distance between FVsand MSs. (E) LA from another Cre� placenta demon-strating multiple dead cells around the FVs (yellowoutline). (F) Relative MT1-MMP mRNA level measuredby real-time PCR of embryonic tissues shown in panelA after TMX treatment. Note that TMX treatment in Cre�mice does not affect development. Scale bars: (A) 1 mm;(B-E) 50 �m.





Discussion

Retardation of placental development, and specifically establish-ment of the LA, is observed in several mouse mutant strains withgenetic alterations affecting matrix molecules, signaling molecules,and molecules important in cell-cell interaction, but interestingly,with very few proteolytic enzymes.7 Based on both well establishedand proposed functions of MT1-MMP and MT2-MMP, we con-sidered the most likely cause of the placental defect in double-deficient mice to be either disrupted processing of known structuralsubstrates such as collagens and other extracellular matrix macro-molecules or a defect in processing of uncharacterized lowabundance substrates.

MT1-MMP is the principal collagenase in mice and is requiredfor sustained collagen remodeling in late development and inpostnatal life.29 In contrast, we demonstrate here that the molecularrelative MT2-MMP is largely dispensable in the presence ofMT1-MMP and offers little in the way of a possible physiologicfunction despite its prominent expression in distinct tissue compart-

ments of diverse species.11,21,24 Notwithstanding the seeminglyinconsequential nature of MT2-MMP deficiency, this moleculebecomes an obligate requirement for placental development and inturn successful gestation to term in the absence of MT1-MMP andvice versa. Based on the interchangeable functions of MT1-MMPand MT2-MMP in the placenta it is difficult to determine whetherone or the other serves a dedicated function here. The LA formationin the presence of only one MT-MMP-type is indistinguishablefrom that found in wild-type mice. Given the inability of MT1-MMP-deficient mice to breed, the selective pressure on germlinenull mutations in this locus precludes propagating such alleles.16,19

Consequently, MT1-MMP is invariably expressed in the placentaand can facilitate LA morphogenesis. One may thus ponder ifMT2-MMP is functionally relevant in the placenta in the presenceof MT1-MMP, or confers competitive advantages in other aspectsof life such as pathogen challenge or behavioral adaptation, whichis not easily mimicked in an experimental environment. Althoughwe demonstrate here that the activity of either MT1-MMP orMT2-MMP is needed for establishment of the placental LA, bothgene products are dispensable for the development of the mouse

Figure 6. Conditional loss of MT1-MMP in an MT2-MMP deficientbackground after LA formation is compatible with development toterm. (A) Whole mount preparation of a Cre� embryo treated with TMXafter the LA was formed (from E12.5 to 16.5). (B) Cre� littermate withidentical gross appearance despite MT1-MMP and MT2-MMP-deficiencyafter TMX treatment. (C) Relative expression level of MT1-MMP mRNAevaluated by real-time PCR on embryonic tissue (from A and B) demon-strates complete ablation of MT1-MMP after treatment with TMX in thepresence of Cre. (D-E) H&E–stained cross-sections of the placentascorresponding to the embryos shown in panels A and B, respectively. Notethe abundant vascularization of both placental LAs despite the conditionalloss of both MT1-MMP and MT2-MMP in (E). Compare with the controlplacenta after formation of the LA in panel D. Scale bars: (A-B) 1 mm;(D-E) 200 �m.





embryo to term after establishment of the LA. These findings pointto a very limited yet essential requirement for at least one of theproteinases, and constitute a novel observation linking the morpho-genetic process of LA development to the 2 MT-MMPs analyzedhere. Imbalance in proteolytic activity after ablation of HAI-1 haspreviously been reported to result in defective LA formation,highlighting the liability of excess proteolytic activity.27 Morphoge-netic defects in placenta have likewise been observed afteroverexpression of cysteine proteinases.30 To our knowledge, thisis the first report of proteolytic activity being required in theformation of the placental LA, and supports the widely held notionthat placental development is associated with substantial tissueremodeling. However, in the evaluation of the specific cause of thisdevelopmental defect, 2 notable observations stand out. First, wedocument no obvious accumulation of either established or po-tential extracellular matrix substrates in the prospective LA, eitherby gross analysis or by universal expression analysis and massspectrometry.33 Nor did ultrastructural analysis reveal aberrantaccumulation of potential substrates in detectable quantities in theLA of double mutants. Second, vasculogenic or angiogenic pro-cesses are unaffected by single deficiencies of either MT1-MMP orMT2-MMP, despite the expression of MT1-MMP in both endothe-lial cells and smooth muscle actin–positive pericytes.31 In furthersupport of this observation, vessel formation was unaffected indevelopmental stages of embryogenesis subsequent to LA forma-tion in the conditional double-deficient embryos. Moreover, preg-nant double-deficient female mice displayed no impairment in theprocesses leading to basement membrane remodeling duringmammary gland involution, which had been considered a possibil-ity based on the coexpression of the 2 MT-MMPs in involutingglands.24 Collectively these observations point to a defect otherthan impaired basement membrane remodeling, but rather dis-rupted vessel formation secondary to absence of syncytium for-mation. This defect in vessel formation, however, is only evidentin the special environment of the prospective LA where a lowabundance substrate may be the target of MT1-MMP and MT2-MMP. This suggestion is supported by the observation that merelyone functional allele out of 4 confers unimpeded LA formation,gestation to term and postnatal viability. In contrast, the processingof high abundance fibrillar collagen substrates requires multiplegene copies. Accordingly, incremental loss of alleles characteristi-cally leads to a proportional reduction of viability in mice deficientfor alleles of MT1-MMP and MT3-MMP.15

Our ultrastructural analysis points to a defect in syncytiotropho-blast formation, which is an integral part of fetal perivasculartissue. This unique structure is the terminal state of an incompletely

characterized process in which CHs differentiate into a bilaminarstructure consisting of an outer (ST-I) and inner (ST-II) syncytium.This anatomical structure envelops the fetal vascular endotheliumand facilitates nutrient, waste and gas exchange with the adjacentMS, which is lined by a STGC.6 The absence of both ST layers inMT1-MMP/MT2-MMP double-deficient mice point to a commondefect in cell fusion, which affects both syncytial layers. Given theunimpeded differentiation and cell migration observed in earlydevelopment of double-deficient embryos and later in conditionallydeficient embryos after LA formation, we infer that the more likelydefect is in the specific process of cell fusion whether related tofusogenic ligands, accessory factors or the presently unknowncognate receptor(s) of the mouse fusogenic proteins.32

In summary, we present here the first evidence for the require-ment of MT-MMP activity in development by demonstrating thatMT-MMP activity is not only critical in late embryonic develop-ment and postnatal life, but also catalyzes essential processes inearly development required for the formation of syncytiotropho-blasts in the placental LA.

Acknowledgments

We thank Larry Fisher, Marian Young, and Pamela Gehron Robeyof NIDCR for critical reading of the manuscript, and IvanRebustini and Matthew Hoffman of NIDCR for technical assistance.

This study was supported by the Division of IntramuralResearch, NIDCR of the Intramural Research Program, NationalInstitutes of Health. M.S. was supported in part by postdoctoralfellowship funds from the Korea Science and EngineeringFoundation.

Authorship

Contribution: L.S. designed and performed the research, analyzedthe data, and wrote the manuscript; M.S. performed the researchand analyzed the data; J.S. performed research; S.Y. performed theresearch, analyzed data, and wrote the manuscript. S.C. and M.S.performed research; P.Z. and W.S. performed research and ana-lyzed data; and K.H. designed and performed the research,analyzed the data, and wrote the manuscript.

Conflict-of-interest disclosure: The authors declare no compet-ing financial interests.

Correspondence: Kenn Holmbeck, National Institutes of Health,Bldg 30, Rm 125, 30 Convent Dr, MSC 4380, Bethesda, MD20892-4380; e-mail: [email protected].

References

1. Abrahamsohn PA, Zorn TM. Implantation and de-cidualization in rodents. J Exp Zool. 1993;266(6):603-628.

2. Cross JC, Nakano H, Natale DR, Simmons DG,Watson ED. Branching morphogenesis duringdevelopment of placental villi. Differentiation.2006;74(7):393-401.

3. Rinkenberger JL, Cross JC, Werb Z. Moleculargenetics of implantation in the mouse. Dev Genet.1997;21(1):6-20.

4. Salamonsen LA, Dimitriadis E, Jones RL, Nie G.Complex regulation of decidualization: a role forcytokines and proteases–a review. Placenta.2003;24 Suppl A:S76-S85.

5. Wang H, Dey SK. Roadmap to embryo implanta-tion: clues from mouse models. Nat Rev Genet.2006;7(3):185-99.

6. Simmons DG, Natale DR, Begay V, Hughes M,Leutz A, Cross JC. Early patterning of the chorionleads to the trilaminar trophoblast cell structurein the placental labyrinth. Development. 2008;135(12):2083-2091.

7. Watson ED, Cross JC. Development of structuresand transport functions in the mouse placenta.Physiology (Bethesda). 2005;20:180-193.

8. Ishida M, Ono K, Taguchi S, et al. Cathepsin geneexpression in mouse placenta during the latterhalf of pregnancy. J Reprod Dev. 2004;50(5):515-523.

9. Varanou A, Withington SL, Lakasing L, WilliamsonC, Burton GJ, Hemberger M. The importance ofcysteine cathepsin proteases for placental devel-opment. J Mol Med. 2006;84(4):305-317.

10. Mason RW. Emerging functions of placentalcathepsins. Placenta. 2008;29(5):385-390.

11. Nuttall RK, Sampieri CL, Pennington CJ, Gill SE,Schultz GA, Edwards DR. Expression analysis ofthe entire MMP and TIMP gene families duringmouse tissue development. FEBS Lett. 2004;563(1-3):129-134.

12. Szabo R, Bugge TH. Type II transmembraneserine proteases in development and disease. IntJ Biochem Cell Biol. 2008;40(6-7):1297-1316.

13. Basile JR, Holmbeck K, Bugge TH, Gutkind JS.MT1-MMP controls tumor-induced angiogenesisthrough the release of semaphorin 4D. J BiolChem. 2007;282(9):6899-6905.

14. Kajita M, Itoh Y, Chiba T, et al. Membrane-type 1matrix metalloproteinase cleaves CD44 and





promotes cell migration. J Cell Biol. 2001;153(5):893-904.

15. Shi J, Son MY, Yamada S, et al. Membrane-typeMMPs enable extracellular matrix permissivenessand mesenchymal cell proliferation during em-bryogenesis. Dev Biol. 2008;313(1):196-209.

16. Holmbeck K, Bianco P, Caterina J, et al. MT1-MMP-deficient mice develop dwarfism, osteope-nia, arthritis, and connective tissue disease dueto inadequate collagen turnover. Cell. 1999;99(1):81-92.

17. Hotary KB, Yana I, Sabeh F, et al. Matrix metallo-proteinases (MMPs) regulate fibrin-invasive activ-ity via MT1-MMP-dependent and -independentprocesses. J Exp Med. 2002;195(3):295-308.

18. Sabeh F, Ota I, Holmbeck K, et al. Tumor cell traf-fic through the extracellular matrix is controlled bythe membrane-anchored collagenase MT1-MMP.J Cell Biol. 2004;167(4):769-781.

19. Zhou Z, Apte SS, Soininen R, et al. Impaired en-dochondral ossification and angiogenesis in micedeficient in membrane-type matrix metallopro-teinase I. Proc Natl Acad Sci U S A. 2000;97(8):4052-4057.

20. Szabova L, Chrysovergis K, Yamada SS,Holmbeck K. MT1-MMP is required for efficienttumor dissemination in experimental metastaticdisease. Oncogene. 2008;27(23):3274-3281.

21. Bjorn SF, Hastrup N, Larsen JF, Lund LR, Pyke

C. Messenger RNA for membrane-type 2 matrixmetalloproteinase, MT2-MMP, is expressed inhuman placenta of first trimester. Placenta. 21(2-3):170-176, 2000.

22. d’Ortho MP, Will H, Atkinson S, et al. Membrane-type matrix metalloproteinases 1 and 2 exhibitbroad-spectrum proteolytic capacities compa-rable to many matrix metalloproteinases. EurJ Biochem. 1997;250(3):751-757.

23. Hotary K, Li XY, Allen E, Stevens SL, Weiss SJ.A cancer cell metalloprotease triad regulates thebasement membrane transmigration program.Genes Dev. 2006;20(19):2673-2686.

24. Szabova L, Yamada SS, Birkedal-Hansen H,Holmbeck K. Expression pattern of four mem-brane-type matrix metalloproteinases in the nor-mal and diseased mouse mammary gland. J CellPhysiol. 2005;205(1):123-132.

25. Romijn HJ, van Uum JF, Breedijk I, Emmering J,Radu I, Pool CW. Double immunolabeling of neu-ropeptides in the human hypothalamus as ana-lyzed by confocal laser scanning fluorescencemicroscopy. J Histochem Cytochem. 1999;47(2):229-236.

26. Schreiber M, Wang ZQ, Jochum W, Fetka I, ElliottC, Wagner EF. Placental vascularisation requiresthe AP-1 component fra1. Development. 2000;127(22):4937-4948.

27. Szabo R, Molinolo A, List K, Bugge TH. Matriptase

inhibition by hepatocyte growth factor activatorinhibitor-1 is essential for placental development.Oncogene. 2007;26(11):1546-1556.

28. Hayashi S, McMahon AP. Efficient recombinationin diverse tissues by a tamoxifen-inducible formof Cre: a tool for temporally regulated gene acti-vation/inactivation in the mouse. Dev Biol. 2002;244(2):305-318.

29. Holmbeck K, Bianco P, Yamada S, Birkedal-Hansen H. MT1-MMP: a tethered collagenase.J Cell Physiol. 2004;200(1):11-19.

30. Screen M, Dean W, Cross JC, Hemberger M.Cathepsin proteases have distinct roles in tropho-blast function and vascular remodelling. Develop-ment. 2008;135(19):3311-3320.

31. Yana I, Sagara H, Takaki S, et al. Crosstalk be-tween neovessels and mural cells directs the site-specific expression of MT1-MMP to endothelialtip cells. J Cell Sci. 2007;120(Pt 9):1607-1614.

32. Dupressoir A, Marceau G, Vernochet C, et al.Syncytin-A and syncytin-B, two fusogenic pla-centa-specific murine envelope genes of retrovi-ral origin conserved in Muridae. Proc Natl AcadSci U S A. 2005;102(3):725-730.

33. Patel V, Hood BL, Molinolo AA, et al. Proteomicanalysis of laser-captured paraffin-embeddedtissues: a molecular portrait of head and neckcancer progression. Clin Cancer Res. 2008;14(4):1002-1014.





online September 21, 2010 originally publisheddoi:10.1182/blood-2009-10-249847

2010 116: 5752-5761

Swaim, Patricia Zerfas, Stacie Kahan and Kenn HolmbeckLudmila Szabova, Mee-Young Son, Joanne Shi, Marek Sramko, Susan S. Yamada, William D. formation and developmentMembrane-type MMPs are indispensable for placental labyrinth

http://www.bloodjournal.org/content/116/25/5752.full.htmlUpdated information and services can be found at:

(524 articles)Vascular Biology Articles on similar topics can be found in the following Blood collections

http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requestsInformation about reproducing this article in parts or in its entirety may be found online at:

http://www.bloodjournal.org/site/misc/rights.xhtml#reprintsInformation about ordering reprints may be found online at:

http://www.bloodjournal.org/site/subscriptions/index.xhtmlInformation about subscriptions and ASH membership may be found online at:

Copyright 2011 by The American Society of Hematology; all rights reserved.of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society


http://www.bloodjournal.org/content/116/25/5752.full.html

http://www.bloodjournal.org/cgi/collection/vascular_biology

http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests

http://www.bloodjournal.org/site/misc/rights.xhtml#reprints

http://www.bloodjournal.org/site/subscriptions/index.xhtml





membrane-type mmps are indispensable for placental - blood

Documents