Keratin 8 protection of placental barrier function.
Bottom Line: 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.
Affiliation: The Burnham Institute, La Jolla, CA 92037, USA.
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.
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Mentions: Some K8−/− epithelial cell lines are 100-fold more sensitive to TNF-induced apoptosis (Caulin et al., 2000). To test genetically whether TNF may contribute to the death of K8−/− embryos, we combined the K8 deficiency (FVB/N) with TNF deficiency (C57Bl/6;129). The FVB/N genetic background was chosen for the K8−/− mice to efficiently obtain adequate numbers of progeny for statistical analysis. Interbreeding of the two targeted alleles resulted in mixed background (FVB/N;B6;129) K8+/− parents with either TNF+/− or TNF−/− (Fig. 2, G3). The recovery of K8−/− progeny from three different crosses was measured. If TNF and the maternal immune system participated in the death of K8−/− embryos, a maternal dependence on TNF was expected. Approximately twice as many K8−/− progeny were recovered when the mother was TNF−/− (C1 and C2) than when the mother was TNF+/− (Fig. 3, A and B). The difference in K8−/− recovery from the C1 cross was significantly different from the reciprocal C3 cross in which the father, rather than the mother, was TNF+/− (Fig. 3 B). TNF deficiency of both the mother and father did not yield greater recovery of K8−/− progeny than when only the mother was deficient. Combining the results of both crosses in which the mothers were TNF−/− (C1 and C2) further reinforced the conclusion. However, a full recovery of K8−/− mice was not obtained. The recovery of K8−/− mice increased from ∼25% to >50% of the number expected for Mendelian inheritance (Fig. 2 C). The recovery of K8+/+ and K8+/− mice did not differ significantly from the expected number. Survival of TNF+/− and TNF−/− progeny in the C2 and C3 crosses were not statistically different (46 TNF+/− versus 51 TNF−/−). Thus, TNF deficiency of the embryo does not influence survival. These data indicate that maternally expressed TNF is deleterious to the survival of K8−/− embryos.