Syncytin-A knockout mice demonstrate the critical role in placentation of a fusogenic, endogenous retrovirus-derived, envelope gene

Abstract

In most mammalian species, a key process of placenta development is the fusion of trophoblast cells into a highly specialized, multinucleated syncytiotrophoblast layer, through which most of the maternofetal exchanges take place. Little is known about this process, despite the recent identification of 2 pairs of envelope genes of retroviral origin, independently acquired by the human (syncytin-1 and syncytin-2) and mouse (syncytin-A and syncytin-B) genomes, specifically expressed in the placenta, and with in vitro cell-cell fusion activity. By generating knockout mice, we show here that homozygous syncytin-A null mouse embryos die in utero between 11.5 and 13.5 days of gestation. Refined cellular and subcellular analyses of the syncytin-A-deficient placentae disclose specific disruption of the architecture of the syncytiotrophoblast-containing labyrinth, with the trophoblast cells failing to fuse into an interhemal syncytial layer. Lack of syncytin-A-mediated trophoblast cell fusion is associated with cell overexpansion at the expense of fetal blood vessel spaces and with apoptosis, adding to the observed maternofetal interface structural defects to provoke decreased vascularization, inhibition of placental transport, and fetal growth retardation, ultimately resulting in death of the embryo. These results demonstrate that syncytin-A is essential for trophoblast cell differentiation and syncytiotrophoblast morphogenesis during placenta development, and they provide evidence that genes captured from ancestral retroviruses have been pivotal in the acquisition of new, important functions in mammalian evolution.

Fig. 1.

Targeted disruption of the syncytin-A gene. (A) Structure of the wild-type (wt) locus, the targeting vector, the targeted recombinant allele (r), the conditional allele, and the deleted, knockout (KO) syncytin-A (single-exon) allele. The loxP and FRT recombination sites (red and empty triangles, respectively), the StuI restriction sites and probe for Southern blot analysis, and the R1–R4 primers for PCR genotyping are indicated. (B) Southern blot analysis of the wild-type (wt) ES cells and of the 2 recombinant (r/wt) clones used to establish independent recombinant mouse lines. StuI-restricted DNA yielded 7.3- and 8.6-kb bands for the wild-type and recombinant alleles, respectively, with the probe in A. (C) PCR-based genotyping of ES cells and mice (wild-type and knockout) using the R3–R4, R1–R2, and R1–R4 primer pairs indicated in A (fragments smaller than 500 bp are shown).

Fig. 2.

Morphological and physiological abnormalities of SynA null embryos and extraembryonic tissues. (A) Mean fetal weight of wild-type, heterozygous, and SynA null embryos at different embryonic ages (E). Values (milligrams) are means ± SEM of a minimum of 7 living embryos (heart beating): 158 ± 5 for wild-type vs. 121 ± 5 for SynA null embryos (P = 0.0005) at E13.5; no difference between wild-type and heterozygous embryos. (B–E) Vascularization of E13.5 wild-type (Left) and living SynA null (Right) embryos and of their extraembryonic annexes. (B) A representative SynA null embryo, smaller and slightly paler than its wild-type littermate. (C and D) Two opposite views of yolk sacs with the blood vessels (arrows) pale and barely visible for the mutant. (E) Fetal side of placentae showing reduced vascularization in the mutant. (Original magnification: B, 12×; C and D, 15×; and E, 25×.) (F) Transplacental passage of rhodamine 123 into E11.5 embryos 2 h after i.p. injection of the fluorescent dye into the mother. (Right) Images of representative wild-type and living SynA null embryos from the same litter. (Left) Quantification of the fluorescence intensity for wild-type and mutant embryos from 9 independent litters. For each litter, the mean fluorescence intensity of the wild-type embryos is set as 100%, and the relative intensity of each littermate is plotted. Two-tailed unpaired t test indicates significant difference (P < 0.0001) between wild-type and mutant embryos (mean fluorescence intensity ± SEM: 85.15 ± 2.2, n = 17 for mutant vs. 100 ± 0.6, n = 16 for wild-type embryos); only living embryos were analyzed.

Fig. 3.

Placental abnormalities in the labyrinth of SynA null embryos. (A) Schematic representation of the mouse placenta (Left) including the labyrinth (lb), the spongiotrophoblast (sp), and the giant cells (gc), adjacent to the maternal decidua (de). In the labyrinth (enlarged view on the Right), maternal blood lacunae—containing maternal red blood cells (mrbc)—are separated from fetal blood vessels (fbv)—containing fetal red blood cells (frbc)—by a trilaminar barrier composed of mononuclear sinusoidal trophoblast giant cell (stgc) and 2 layers of syncytiotrophoblasts (ST-I and ST-II), the latter apposed to the fetal endothelium. (B–H) Light microscopy analysis of wild-type (Left) and mutant (Right) placental tissues. (B and C) Hematoxylin/eosin staining of 5-μm placental sections from living E12.5 embryos with the labyrinth, spongiotrophoblast, and maternal decidua delineated. Enlarged view of the labyrinth (C), with fetal and maternal red blood cells indicated. The labyrinth of SynA null placenta is more compact, with the fetal red blood cells squeezed inside the narrowed vessels. (D–H) Marker analyses by in situ hybridization for 4311 and mPLI (D and E) or by histochemistry for CD34 (F and G) and Ki67 (H) of placenta sections of E11.5 (D, E, and H) and E13.5 (F and G) embryos. CD34-negative nonvascularized areas (in F) are indicated with asterisks; CD34-positive fetal blood vessels (fbv, see enlarged view in G) have a reduced internal space in SynA null labyrinth, with fetal erythrocytes (arrowheads) squeezed. (H) Ki67-positive cells forming aggregates in the SynA null labyrinth. (Scale bars: B and D, 400 μm; E, F, and H, 100 μm; C, 25 μm; and G, 12.5 μm.)

Fig. 4.

Electron micrographs of labyrinth of E11.5 wild-type (A and B) and SynA null (C–G) placentae. (A) Three-layered interhemal barrier of wild-type labyrinth at the maternofetal interface [m indicates maternal blood lacuna with darkly stained erythrocytes, and f indicates fetal blood vessel lined by endothelial cells (ec) with a nucleated erythrocyte] with the mononuclear sinusoidal trophoblast giant cells (stgc) and the 2 syncytiotrophoblast (ST-I and ST-II) layers delineated, and the ST-II-specific lipid inclusions indicated with asterisks. (B) Expanded view of the area boxed in A, showing tight apposition of the ST-I and ST-II layers (outlines of the facing cytoplasmic membranes schematized on the Right). (C and D) same as A and B for the SynA null placenta, with the 3-layered structure still present (C) but with severe disturbance of the putative ST-I (T-I) layer and disrupted interactions with ST-II (D, facing cytoplasmic membranes outlined on the Right); ST-II appears normal (typical lipid inclusions indicated by asterisks). (E and F) Images of unfused T-I cells as observed in less-disturbed SynA null interhemal domains. Apposed cytoplasmic extensions (arrows) are schematized on the Right. (G) Apoptotic cell (arrow) with cup-shaped condensed chromatin, in contact (yellow dotted line) with a sinusoidal trophoblast giant cell (stgc) and the ST-II layer. (Scale bars: 2 μm.)