Keratin 8 protection of placental barrier function

Abstract

The intermediate filament protein keratin 8 (K8) is critical for the development of most mouse embryos beyond midgestation. We find that 68% of K8-/- embryos, in a sensitive genetic background, are rescued from placental bleeding and subsequent death by cellular complementation with wild-type tetraploid extraembryonic cells. This indicates that the primary defect responsible for K8-/- lethality is trophoblast giant cell layer failure. Furthermore, the genetic absence of maternal but not paternal TNF doubles the number of viable K8-/- embryos. Finally, we show that K8-/- concepti are more sensitive to a TNF-dependent epithelial apoptosis induced by the administration of concanavalin A (ConA) to pregnant mothers. The ConA-induced failure of the trophoblast giant cell barrier results in hematoma formation between the trophoblast giant cell layer and the embryonic yolk sac in a phenocopy of dying K8-deficient concepti in a sensitive genetic background. We conclude the lethality of K8-/- embryos is due to a TNF-sensitive failure of trophoblast giant cell barrier function. The keratin-dependent protection of trophoblast giant cells from a maternal TNF-dependent apoptotic challenge may be a key function of simple epithelial keratins.

Figure 1.

Morphological and histological analysis of (B6;129) wild-type and K8 ^{− / −} extraembryonic tissues. Wild-type (A) and K8 ^−/− (B) E10.5 concepti from which the uterine wall and part of decidua are removed. Note the hematoma (h) located between the decidual tissue (dec) and the yolk sac (ys) in the K8 ^−/− conceptus. In mutant conceptus, the placenta (p) is hidden behind the hematoma. (C and D) Sagittal histological sections of E10.5 wild-type (C) and K8 ^−/− (D) concepti. In the wild-type conceptus, the embryo proper (em) is surrounded by the amnion (am), visceral yolk sac (vys), parietal yolk sac (pys), the layer of trophoblast giant cells (arrowheads), and maternal decidual tissue (dec). In the K8 ^−/− conceptus, the hematoma (h) is located between the decidual tissue (dec) and parietal yolk sac (pys). p, placenta. Bar, 200 μm. (E and F) Trophoblast giant cells in E10.5 wild-type (E) and K8 ^{− / −} (F) concepti at higher magnification. In wild-type conceptus, the continuous layer of trophoblast giant cells (gc) divides maternal decidua (dec) from the parietal yolk sac (pys). Note the disrupted layer of trophoblast giant cells (gc), which undergo degeneration, maternal erythrocytes (er), and fibrin (fb), infiltrated with granulocytes in K8 ^{− / −} conceptus. Bar, 50 μm.

Figure 2.

Breeding strategy to test the influence of TNF on K8 ^−/− survival. Two rounds of matings (G1 and G2) were performed to obtain the mouse genotypes needed for the experimental matings (G3) that produced offspring of experimental matings C1, C2, and C3. All parents were K8^+/−.

Figure 3.

The absence of maternal TNF increases the recovery of K8 ^{− / −} mice. (A) The number of mice of each genotype from the crosses C1, C2, and C3 (Fig. 2) are shown. (B) Statistical analysis using the Chi squared test is presented. The observed number of K8^+/+, K8^+/−, and K8^−/− mice from crosses C1 and C1 + C2 are compared with the numbers expected using the percentages from cross C3 as standard. (C) Graphic representation of the recovery of K8^+/+, K8^+/−, and K8^−/− mice compared with expected Mendelian inheritance.

Figure 4.

More K8 ^{− / −} mice are recovered in the absence of maternal TNFR2. The same strategy shown in Fig. 2 was used for the K8 × TNFR2 crosses except that cross C2 was not performed. (A) The number of mice obtained from the K8 × TNFR2 cross as determined by tail DNA PCR. (B) The observed number of K8 wild-type, K8 heterozygote, and K8 knockout mice from crosses C1 are compared with the percentage of recovery from cross C3 as standard using the Chi squared analysis. (C) Graphic representation of the recovery of K8 genotypes to that expected for Mendelian inheritance.

Figure 5.

K8-deficient concepti are more sensitive to ConA-induced hematoma formation. Three K8^+/−TNF^+/− females were bred to a K8^−/−TNF^−/− male. 10 d postcoitus, the mothers were subjected to ConA treatment. 5 h after injection, the mice were killed, and the concepti were removed for histological analysis. Hematoxylin and eosin–stained sections revealed different degrees of bleeding. (A–F) Red blood cells are rendered in black by digital color replacement. Note that most of the hematoma occurs opposite the placenta (E and F). The identity of each specimen is indicated by the litter number and embryo number. Bar, 1 mm. Quantitative measurements of the hemorrhage size for every embryo from the three litters are shown in G, H, and I. The numbers above the horizontal bar of each panel indicate the embryo identification number of that litter. A statistical analysis of these numbers confirms that there is a statistical significant difference in the amount of hemorrhage in K8^+/− versus K8^−/− embryos (P = 0.006), but there is no such difference between TNF^+/− (black bars) and TNF^−/− (gray bars) embryos (P = 0.08).

Figure 6.

Apoptotic trophoblast giant cells in ConA-treated mice. Sections of the placenta (A and B) and uterine wall (C–F) were stained for K8 (A and B) and apoptotic nuclei by the TUNEL method (C–E). F shows a neighboring section of E stained with hematoxylin and eosin. Tissues with K8^−/− concepti are shown in B–F. The tissues are labeled: d, decidua; eb, embryonic blood; gtc, giant trophoblast cell; h, hemorrhage; L, labyrinth region; stc, spongiotrophoblast cell; r, Reicharts membrane; ys, yolk sac. The black arrows in C–F indicate apoptotic giant cells. Bar, 100 μm. The tissue shown in E and F represent an adjacent uterine wall region which has folded up against the placental region as revealed by tracing the folded yolk sac basement membrane. This results in the atypical juxtaposition of the failed trophoblast giant cell layer against the labyrinth region of the placenta. Note the apoptotic giant cell nuclei associate with the accumulated hematomas.