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1 Laboratory of Vascular Pathophysiology, Institut National de la Santé et de la Recherche Médicale U882, Commissariat à l'Énergie Atomique (CEA), Grenoble University, CEA, Grenoble, France
2 Laboratory of Informatics and Mathematics for Biology, CEA, Grenoble, France
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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knockout mice; gene profiling; trophoblasts; angiogenesis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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In the mouse, the placenta originates from the ectoplacental cone and the extraembryonic ectoderm. The endothelial cells derive from the allantois (8, 16, 37). From embryonic day (E)10, the placenta is divided in three layers associated with maternal decidual cells. The labyrinth, located on the fetal side, is composed of an intricate array of fetal and maternal vessels that constitute a selective barrier between the two circulation systems (1, 9). The giant cells are located next to the uterine cells and, until E12, constitute the outermost fetal cell layer. Between the labyrinth and the giant cells there is a third layer, the junctional zone, also called the "spongy layer" because of its numerous cavities. The junctional zone has been shown to produce several hormones, but its general function remains elusive. Nevertheless, there is an absolute requirement for this layer, because mutations inducing its disruption are not compatible with embryonic survival (17, 41). The junctional zone is composed of two types of trophoblasts: the spongiotrophoblasts and the glycogen cells, recognizable by their high glycogen contents. The glycogen cells form islets within the junctional zone that migrate from E12.5 into the maternal decidua, beyond the giant cell line (4, 16).
Protocadherins constitute a large family of transmembrane proteins exhibiting calcium-dependent homophilic adhesive properties (15, 22). As opposed to the classical cadherins, protocadherins do not or only weakly associate with the actin cytoskeleton. Although some properties of individual protocadherins have been reported, a comprehensive view of their functions is lacking.
Protocadherin 12 (PCDH12), previously called VE-cadherin 2, was initially identified in mouse endothelial cells (43). We recently showed (35) that its endothelial expression was more specifically detected in angiogenic endothelium. In addition, PCDH12 is abundantly expressed in placental glycogen cells and mesangial cells of renal glomeruli (35).
To gain insight into PCDH12 biological activity in vivo, we produced PCDH12-deficient mice. The mice were alive and fertile. Furthermore, Pcdh12+/– intercrosses produced a normal Mendelian distribution at birth (35). In the present study, we showed that PCDH12-deficient placentas and embryos are smaller than their wild-type counterparts and we observed two major morphological modifications in the mutant placentas, namely, decreased vascular and cell densities in the labyrinth and a missegregation of the labyrinthine and the junctional zone. These general alterations of placental development prompted us to examine whether the placental transcriptional profile is modified in absence of PCDH12. Strikingly, the expression of 2,289 genes was significantly altered in the mutant compared with wild-type placentas. Our data show that transcriptional changes occurred in functional groups of genes involved in tissue morphogenesis, angiogenesis, cell migration, immune response, and chromatin remodeling. Expression changes of specific genes are discussed in relation with the biological alterations observed in PCDH12-deficient placentas.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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/6 x D2 x T. Genotypes were performed on embryonic DNA as previously described (35).
Histology.
For immunolocalization, tissues were snap-frozen in OCT compound and sectioned at 10 µm with a cryomicrotome (Leica Microsystems, Wetzlar, Germany). Sections were permeabilized in 4% paraformaldehyde, 0.5% Triton-PBS for 3 min, fixed in 4% paraformaldehyde-PBS for 20 min, saturated with 2% BSA-PBS, and incubated with anti-CD31 (45) and either peroxidase-conjugated anti-rat immunoglobulin (Bio-Rad, Marnes-la-Coquette, France) or Alexa 488-conjugated anti-rat immunoglobulin (Invitrogen, Cergy-Pontoise, France) antibodies, all at room temperature, by standard procedures. Peroxidase was revealed with diaminobenzidine (DakoCytomation, Trappes, France), followed by nuclear staining with Harris hematoxylin (Sigma-Aldrich, St-Quentin-Fallavier, France). Nuclei in fluorescent images were stained with Hoechst 33258 (Sigma-Aldrich).
For periodic acid Schiff (PAS)-hematoxylin (both from Sigma-Aldrich) staining, paraffin sections were prepared by standard procedures from paraformaldehyde-prefixed tissues. Slides were mounted in Entellan or Aquatex (VWR International, Fontenay-sous-Bois, France) and observed with a Zeiss Axioplan microscope. Pictures were made with a digital camera (Spot-RT, Diagnostic Instruments, Sterling Heights, MI) and were used to calculate the layer surface and cell density with ImageJ software (National Institutes of Health, Bethesda, MD; http://rsb.info.nih.gov/ij/) and the number of the spongiotrophoblast layer islets in the labyrinth.
To quantify capillary length, images were processed for morphometric analysis with ImageJ software. A macrocommand was edited to give the total vessel length after binarization, skeletonization, and pixel count of the CD31-staining fluorescent image.
Extraction of glycogen and measurement of glycogen content.
Glycogen content of placentas was measured by the method of Lo et al. (28). Each placenta was incubated for 30 min at 100°C with 0.5 ml of 30% KOH saturated with Na2SO4. The solution was cooled, and the glycogen was precipitated with 0.55 ml of 100% ethanol at 0°C for 30 min. Glycogen was pelleted by centrifugation at 1,000 g for 30 min at 4°C. The pellet was dissolved in 3 ml of H2O. Aliquots were incubated for 20 min at 30°C with 0.7% phenol and 70% H2SO4, and absorbance was measured at 490 nm. Mussel glycogen (Roche Biochemicals, Meylan, France) was used as standard. Data were expressed as milligrams of glycogen per gram of wet tissue or per total placenta.
Microarray processing and analysis.
Total RNAs were extracted from E12.5 placentas (5 placentas of each genotype, from 3 female and 2 male embryos, were derived from 3 litters) with Tri Reagent (Euromedex, Souffelweyersheim, France), treated with DNase I, and purified with NucleoSpin RNA Clean up (Macherey-Nagel, Hoerdt, France). RNA concentration and integrity were tested with an Agilent 2100 Bioanalyzer (Agilent Technologies, Massy, France).
Affymetrix chip hybridization was performed by the Institute of Genetics and Molecular and Cellular Biology (Strasbourg, France). The amplified RNAs were labeled and hybridized to Affymetrix mouse 430 2.0 gene chips containing >45,000 probe sets and representing >34,000 characterized transcripts. The arrays were scanned with a confocal laser GeneChip scanner 3000 7G System. The resulting images were captured with GeneChip Operating Software (all on Affymetrix instruments). The background adjustment and the data normalization were processed with MAS 5.0 software (Affymetrix). The scaling factors for all arrays formed a homogeneous interval concentrated around 2.33. The exploratory data analysis provided by the Affy package of Bioconductor (www.bioconductor.org) showed no experimental artifacts. Box plots, densities of log intensities, RNA digestion plots, and RNA degradation parameters are provided as supplemental data.1
Full data sets have been deposited in Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo) and are accessible through GEO series accession number GSE7676.
Bioinformatics analysis.
Results were annotated with information provided by Affymetrix. The full data set was reduced to 18,542 genes by discarding genes with "EST" and "unknown" annotation labels. To generate the list of genes classified by Gene Ontology (GO), we used the High-Throughput version of GoMiner (http://discover.nci.nih.gov/gominer/; Refs. 49, 50). We then performed a Gene Set Enrichment Analysis (GSEA) (40) for selected gene sets according to GO biological process categories. The GSEA procedure allowed an interpretation of the gene expression profiles, by using predefined gene sets and ranks of genes to identify significant biological changes in microarray data sets. The familywise error rate (FWER) was used for multiple testing corrections. It provides the probability to get a nonzero false discovery rate. The heat maps obtained from this analysis are presented as supplemental material.
Statistical analysis.
For Figs. 1–5, statistical significance was analyzed with Student's t-test; sample number is indicated in figure legends. We used the significance analysis of the microarray (SAM) score (44) to investigate the differentially expressed genes. The estimation of the false discovery rate was obtained with the Q value package available under the R language (www.r-project.org). The computation of the Q values was done with the default tuning parameters as input, with the exception of the pi0.method entry, for which we chose the "smoother" method. The output value of the pi0—the estimate of the proportion of null hypothesis—was 0.65351.
Real-time RT-PCR.
First-strand cDNAs were generated by reverse transcription from 0.5 µg of total RNA in a total volume of 20 µl with the SuperScript First Strand Synthesis System (Invitrogen) using random primers and SuperScript II reverse transcriptase.
Real-time PCR was performed with LightCycler apparatus (Roche Diagnostics, Meylan, France) and FastStart DNA master SYBR Green I ready-to-use PCR mix according to the manufacturer's protocol (Roche). cDNAs (25 ng) were amplified in a 10-ml total volume PCR reaction with specific primers. Gene expression was normalized with values obtained for the housekeeping gene Gapdh as the reference gene.
Primers used for PCR amplification were Phdal2, 5'-gctgtttttccactccatcc and 5'-gtttcacggacccagagc; Gapdh, 5'-tgtgatgggtgtgaaccacgagaa and 5'-aagcatccaaccacatcaca; Angiopoietin 2, 5'-tacacactgacctcttccccaac and 5'-agtccacaccgccatcttctc; Tek, 5'-gaacaccgaggctatttgtac and 5'-agtgtggaagctgtagtgttgg; Tie1, 5'-ggcagcttccagagtatggt and 5'-tggccagcaatgttaagtca; Gja1, 5'-gagcccgaactctccttttc and 5'-ccatgtctgggcacctct; Angiogenin2, 5'-tcagcactatgatgccaagc and 5'-tcctttgtgtgtgcaagtgg; and Decysin, 5'-aagcatccaaccacatcaca and 5'-tgtataggtgcacaggataggc. Primers were designed by using Integrated DNA Technologies software (available online at http://www.idtdna.com/Scitools/Applications/PrimerQuest) or Primer 3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) and using sequence data from the GenBank database.
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RESULTS |
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We first measured placenta weight and volume at both embryonic ages. At E17.5, but not at E12.5, Pcdh12–/– placenta weights were indeed significantly (P < 0.001) lower than those of wild type (Fig. 1, A and B). Similarly, the volumes of E17.5 PCDH12-deficient placentas were significantly (P < 0.001) reduced, whereas they were similar at E12.5 (Fig. 1, C and D). The volume reduction at E17.5 concerns both the diameter and the width of placentas (not shown). The data show that Pcdh12–/– placentas did not grow significantly after E12.5, while the wild-type placentas were still in an active phase of expansion. Furthermore, Pcdh12–/– embryo weights were significantly lower at both ages (Fig. 1, E and F). Therefore, we conclude that PCDH12 deficiency alters both placenta and embryo growth. However, the average weight of mice of both genotypes at ages from 2 wk to 2 mo was identical (Fig. 1, G and H), thereby indicating that Pcdh12-deficient mice recovered after birth.
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Histological analysis of Pcdh12–/– placentas.
We first examined the histological organization of the different placental layers. E12.5 and E17.5 placenta sections were prepared and labeled with the endothelium-specific anti-CD31 antibody. In the mouse placenta, the labyrinth is highly vascularized, the junctional zone is avascular, and the decidua contains large vessels; the limits of the different placental layers are therefore clearly visualized by this technique, as illustrated in Fig. 2A. The layer surface areas, as well as total placenta area, were measured on parasagittal sections; the average proportions are shown in Fig. 2, B and C. Our results indicate that the proportions were similar between Pcdh12–/– and wild-type placentas at both ages, thereby suggesting that growth of all three layers was reduced in the E17.5 Pcdh12–/– placenta. The giant cell line was unmodified (not shown).
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The labyrinth contains a very dense capillary network. Because the cell density was lower in the labyrinth, we wondered whether this network was altered in the mutant. As illustrated in Fig. 3, A and B, we indeed noted a decrease in vascular density in the mutant labyrinthine layer. We thus measured the total capillary length in CD31 immunofluorescent images. As shown in Fig. 3, C and D, the skeletonized image obtained after processing by ImageJ software superimposed with the CD31-labeled vessels. Our quantitative data show that capillaries were much less developed in the Pcdh12–/– labyrinth at both E12.5 and E17.5 (Fig. 3, F and G). This result might be in direct correlation with the lower cell density of the Pcdh12–/– labyrinth and may also have a functional impact on embryonic development, because optimal maternal-fetal exchanges are required throughout gestation. In contrast, the vascular pattern of maternal deciduas was similar in wild-type and Pcdh12–/– placentas. (Fig. 3, H and I).
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Glycogen metabolism in Pcdh12-deficient placentas.
One of the major roles of the placenta is to behave as an energy supplier for the embryo. Glycogen cells have the capacity to store huge amounts of glycogen, which can be broken down into glucose after glucagon stimulation (6). Glycogen was extracted from whole placentas, and its concentration was measured. Glycogen concentration was significantly higher in Pcdh12–/– placentas at E12.5 and E17.5 (Fig. 5, A and B). Correspondingly, the total amount of glycogen per placenta was increased in E12.5 Pcdh12–/– placenta (Fig. 5C); however, glycogen amounts reached a similar level in placentas of both genotypes at E17.5 (Fig. 5D). This feature can be explained by the weight difference between wild-type and mutant placentas at this age. Thus, although glycogen stores reached a similar level in late gestation, glycogen production was accelerated or its accumulation was more efficient in glycogen cells of Pcdh12–/– placentas.
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Gene expression profiling of PCDH12-deficient placentas.
To understand the molecular events responsible for the phenotypic alterations observed in Pcdh12–/– placentas, we analyzed the genomewide expression profile of E12.5 mutant placentas compared with wild-type placentas. RNAs from five placentas of each genotype were analyzed with Affymetrix chips. The placentas were derived from three litters. RNA quality was examined before hybridization, and expression data were analyzed with several experimental and statistical criteria and gave satisfactory results (see supplemental data).
Remarkably, from the total number of 45,101 probes, 2,289 were assigned as differentially expressed since they present a SAM score superior to 0.75. The SAM threshold of 0.75 used here corresponds to the 0.95 quantile for both upregulated and downregulated genes. A higher proportion of genes were upregulated (1,620 vs. 669). Genes with unknown function were excluded from the initial set of 45,101 probes, and the bioinformatic analysis was performed on a set of 18,542 probes.
Expression profiles obtained with DNA chips were examined by quantitative RT-PCR (qRT-PCR) for some specific genes showing different variation levels and different signal intensities (Table 1). Fold variations may be slightly different between the two technical approaches when the signals were at background levels (e.g., decysin in the wild type); however, the qRT-PCR results broadly confirm the DNA chip data.
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The expression of three classes of cell junction genes was altered in the mutant placenta: the cadherin (N, P, and VE), connexin (Gja1 coding for connexin 43), and claudin (1 and 11) families, whose products are located in adherens, gap, and tight junctions, respectively.
Genes encoding for angiogenic factors and their receptors, such as fibroblast growth factor receptor 1, Tie 1, transforming growth factors beta 1 and 2, and soluble Flt1, were significantly upregulated in the mutant placentas.
Functional grouping of differentially expressed genes revealed that a large number of matrix-related and integrin transcripts were significantly upregulated in the mutant placenta and, for some of these, with a high Pcdh12–/–-to-wild type signal ratio. Table 4 shows selected genes of this category with lowest signal value above 15 and SAM score above 0.50. Genes coding for several collagen chains, or involved in collagen synthesis, for laminin chains, fibrillin 1, and two fibulins are present. Most selected genes encoding matrix proteins are expressed at high levels in the placenta. Six genes encoding integrins or proteins involved in cell-matrix adhesion and cell migration were significantly upregulated. Cell migration also requires matrix and integrin proteases, and indeed a number of metalloproteinase family genes were upregulated. One member, the disintegrin metalloprotease (also called decysin) gene, exhibited a 180-fold overexpression in the mutant placenta.
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DISCUSSION |
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The embryonic growth defect was attributed to placental anomalies because the mutant mice reached the weights of control mice 2 wk after birth. Thus this study supports the close link between placental and embryonic development. Because fetal growth was already retarded at E12.5, it is likely that placental functional defects were present much earlier on.
The catch-up of PCDH12-deficient pups after birth is reminiscent of the phenotype of mice deficient in the homeobox gene Esx1, for which placental defects, including missegregation of labyrinthine and spongiotrophoblast layers, were observed (26). Conversely, mice with other genetic ablations leading to placental anomalies and subsequent embryonic growth retardation did not recover after birth the body weight of their wild-type littermates (10, 47). This observation may have important consequences in pediatrics for the management of children born small because of abnormal placental function: a close examination of placental phenotype and/or genetic alteration may provide indications on child thriving and may impact medical decisions (21).
The macroscopic morphology of PCDH12-deficient placentas was modified in two aspects: placenta size (in late gestation) and segregation of the labyrinthine and the junctional zone. Furthermore, the labyrinthine vasculature was much less developed, a feature that may be related to the lower cell density in the labyrinth. Because the labyrinth is subjected to major development between E12.5 and E17.5, and because vessel and cell densities were lower in the mutant labyrinth, we first suspected that labyrinth growth was specifically reduced during this time period in the mutant placenta. However, the surface proportions of the three layers were not statistically different between the two genotypes at both ages, indicating that placental growth retardation was general and not specific to the labyrinth. Nevertheless, it is possible that labyrinth development influences or coordinates the growth of the junctional zone and decidua. Alternatively, angiogenesis defects in the labyrinth and alteration of glycogen cell behavior in the junctional zone and decidua may independently decrease the growth rate of these layers.
The histological modifications observed here are shared with other mouse genetic models. For example, missegregation of labyrinth and junctional zone was also observed in interspecies hybrids, in cloned conceptuses, and after Esx1 deletion (26, 42, 48). However, this phenotype may be caused by different molecular mechanisms (38).
Considering Pcdh12–/– histomorphological anomalies, it is striking that gene clustering through GoMiner detected that clusters of genes involved in development, tissue morphogenesis, adhesion and migration, as well as angiogenesis are significantly modified by loss of PCDH12. The majority of these genes were upregulated. Interestingly, genes of the chromatin-remodeling cluster, mostly silencing genes, were downregulated in the mutant. This may be connected to the general overexpression of regulated genes in PCDH12-deficient placentas. Upregulation of genes of the immune response cluster is more intriguing; it might reflect a modification in the activity of the uterine natural killer cells of the decidua. More generally, it is noteworthy that loss of a protein exhibiting cell-cell adhesive properties has a significant impact on gene transcription.
Most of the modified genes are expressed by PCDH12-positive cells, i.e., glycogen and endothelial cells. For example, Gja1 (CX43), Psg19, Mmp9, and Cox1 are solely or primarily expressed by glycogen cells (5, 6, 23, 36) and TIE1 and KDR are mainly located on the endothelial cell surface (11, 33). This suggests intracellular signaling mechanisms between the membrane-linked Pcdh12 and the nuclear transcriptional machinery.
Remarkably, the cytoplasmic domains of 
-protocadherins and protocadherin FAT1 may be cleaved and translocated to the nucleus (18, 29). This opens interesting prospects for protocadherin signaling.
Other modified genes are expressed by cell types that do not express PCDH12. For example, PHLDA2 or the EAR family members are specific of labyrinthine trophoblasts (14) or immune response cells (24), respectively. This feature indicates that PCDH12 activity may influence other cell types of the placenta through intercellular signaling mechanisms that remain to be elucidated.
Angiogenesis.
As shown by the histomorphological analysis (Fig. 2), proportions of placenta layers were not altered in Pcdh12-deficient mice. However, labyrinthine vascular density was decreased (Fig. 4), which may be caused by expression variation of several genes: 1) increased levels of the VEGF decoy receptor gene Flt1 and moreover of its soluble form sFlt1; both are known for their antiangiogenic properties and are strongly expressed by the junctional zone (11, 13, 20); similarly, increased expression of Flt1 and sFlt1 was recently shown to be responsible for the vascular defect phenotype observed in Adra2b–/– placentas (30); 2) upregulation of Tie1, an orphan receptor regulating endothelial quiescence (34); 3) upregulation of Tgfbeta1 and 2, which are known promoters of vessel maturation; 4) upregulation of numerous matrix protease genes, which may release cryptic matrix or non-matrix-derived inhibitors of angiogenesis (see Ref. 31 for review); 5) upregulation of fibulin 5, which inhibits the ability of endothelial cells to undergo angiogenic sprouting (2). Some of these antiangiogenic factors may target the maternal vasculature as well (see below).
Placenta morphogenesis and function.
Expression of major placental morphogenetic genes, such as Plac1, Pem, or Ascl2, was not significantly modified by loss of PCDH12, with the exception of the imprinted gene Phlda2 (also called Ipl or Tssc3), which was downregulated. Its protein product acts to limit placental growth in mice. The functional consequences of Phlda2 upregulation are at present unclear.
Genes coding for placental hormones (prolactins) or growth factors (IGF-II, EPO) did not show major alterations in their expression. However, transcription levels of two genes coding for proteins involved in prostaglandin synthesis and uptake, Ptgs1 (cyclooxygenase 1 or Cox1) and Slc21a2 (coding for a prostaglandin transporter), respectively, were upregulated. Prostaglandins are critical molecules for initiation of parturition (reviewed in Ref. 32) and are currently used to trigger delivery in humans. PCDH12 levels may thus influence the timing of birth through regulation of prostaglandin activity. This feature may be related to the upregulation in the mutant placenta of Mmp9 expression, encoding a matrix protease facilitating tissue remodeling in delivery (25).
We observed altered expression of two members of the pregnancy-specific glycoprotein (Psg) gene family, Psg16 and Psg19. PSGs are the most abundant fetal proteins in the maternal bloodstream in late pregnancy (27). In situ hybridization studies showed that Psg19 is exclusively transcribed by the junctional zone in the mouse (23). The importance of PSGs in the maintenance of pregnancy has been demonstrated by injection of anti-PSG antibodies, which induced abortion in mice (19). Most studies on PSGs are linked to the modulation of the maternal immune system, through cytokine secretion induction, preventing rejection of the fetuses. Consistently, expression of several immune response genes was upregulated in Pcdh12–/– placentas, including interleukin and chemokine ligands and receptors.
Matrix protein and proteases.
Various types of collagen, laminin, and fibulin were upregulated in the absence of PCDH12. Once assembled, these secreted proteins are the major constituents of the extracellular matrix, where they have a dual function: they provide tissues with their biomechanical properties, and they support cell guidance during migration. In the placenta, the extracellular matrix enables the attachment of fetal and maternal tissues and provides a path for trophoblast invasion.
Pericellular protease activity further facilitates cell migration and invasion. Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are involved in morphogenesis of many epitheliomesenchymal organs (for review, see Ref. 46). Genes encoding three MMPs, MMP9, -14, and -23, and several members of ADAM (a disintegrin and metalloproteinase) or ADAMT (ADAM with thrombospondin repeats) families were upregulated in the PCDH12-deficient placentas. All are involved in cell adhesion, cell migration, membrane protein shedding, and proteolysis. The most upregulated gene of this category was decysin. This protein is a new member of the ADAM family. In placenta, decysin is mostly expressed in the junctional zone and around maternal vessels (3), i.e., at sites of PCDH12 expression. However, its substrate specificity is unknown.
Altogether, the transcriptional upregulation of these gene families is consistent with increased cell migration and tissue invasion. Therefore, the fact that PCDH12-deficient placentas showed defects in layer segregation is intriguing. It is possible that the orchestration of the various players of the cell migration process is compromised in the mutant placenta, leading to uncoordinated cell movement.
RNases.
Surprisingly, a number of genes coding for secreted RNases, especially those of EAR 1–3 and angiogenin 2, were dramatically upregulated in PCDH12-deficient placentas. Some of these proteins are expressed by neutrophils or macrophages (see Ref. 12 and references therein) and possibly uterine natural killer cells. It has been suggested that these enzymes, as most of the RNase family members, may be involved in host defense. As indicated for the other immune response genes whose expression is modified in the mutant placenta, it is possible that PCDH12 modulates uterine natural killer cell activity by an unknown mechanism.
Conclusions.
Our data show that PCDH12 is a participant in placental morphogenesis. In absence of PCDH12, placentas exhibited growth retardation and histomorphological alterations. The gene profiling study comparing wild-type and Pcdh12–/– placentas shows expression variations of a surprisingly large number of genes. This study constitutes a basis for the investigation of the multiple signaling pathways in which PCDH12 is involved.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 The online version of this article contains supplemental material. 
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REFERENCES |
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2B-adrenoceptor deficient mice. Circ Res 101: 682–691, 2007.
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50 µm) for each placenta at E12.5 (B) and E17.5 (C). Mean values were used to calculate the mean (SD) surface proportion of layers from WT (n = 8 and 12 for E12.5 and E17.5 placentas, respectively) and Pcdh12–/– (n = 8 and 10 for E12.5 and E17.5 placentas, respectively) placentas. D and E: for each placenta at E12.5 (D) and E17.5 (E), nuclei were counted on 3 different areas at high magnification to calculate the cell density. Data are means and SD. A significant difference was observed for labyrinthine cell density in both E12.5 and E17.5 placentas.
