Essential requirement of CCAAT/enhancer binding proteins in embryogenesis

Abstract

The CCAAT/enhancer binding proteins C/EBPalpha and C/EBPbeta are related transcription factors that are important for the function of various organs in the postnatal mouse. Gene replacement and tissue culture experiments have suggested partial redundancy of both transcription factors. Here we show that mouse embryos deficient of both C/EBPalpha and C/EBPbeta (C/EBPalphabeta(-/-)) die between embryonic day 10 (E10) and E11 and display defective placentas. In situ hybridization revealed that C/EBPalpha and C/EBPbeta are coexpressed in the chorionic plate at E9.5 and later in the trophoblasts of the labyrinthine layer. In C/EBPalphabeta(-/-) placentas, allantoic blood vessels invaded the chorion; however, vessel expansion and development of the labyrinthine layer was impaired. Furthermore, a single copy of either C/EBPalpha in the absence of C/EBPbeta or C/EBPbeta in the absence of C/EBPalpha is sufficient to complete development, suggesting complementation of these C/EBPs during embryogenesis. A single copy of C/EBPalpha in the absence of C/EBPbeta, however, fails to rescue survival after birth, suggesting haploinsufficiency of C/EBPalpha in newborns. Our data thus reveal novel essential, redundant, and dosage dependent functions of C/EBPs.

FIG. 1.

Expression of C/EBPα, C/EBPβ, and C/EBPδ in wild-type placenta at E9.5 (A to C), E10.5 (D to F) and E11.5 (G to I). (A to C) C/EBPα expression is restricted to the chorionic plate (arrowheads) (A), whereas C/EBPβ is strongly expressed in the ectoplacental cone (EC) and the chorionic plate (arrowheads) (B). In panels D to I, C/EBPα and C/EBPβ are coexpressed in the labyrinthine layer (L). C/EBPβ mRNA is also found in spongiotrophoblasts (Sp). Note the absence of C/EBPδ expression in the placenta at all stages (C, F, and I). Al, allantoic cavity; d, maternal decidua. Scale bar, 10 μm.

FIG. 2.

Histological analysis of controls and C/EBPαβ^−/− placentas at E10. H&E-stained sagittal sections of the labyrinthine layer of +/+ (A), C/EBPα^−/− (B), C/EBPβ^−/− (C), and C/EBPαβ^−/− (D) placentas. In panel D, note the abnormal thickening of the C/EBPαβ^−/− chorionic plate (CP) and poor impregnation of the C/EBPαβ^−/− presumptive labyrinth (L) by fetal vessels (arrowhead, containing blue-stained nucleated fetal erythrocytes). Al, allantoic cavity; arrow, maternal erythrocyte sinus; Sp, spongiotrophoblast; L, labyrinthine layer. Scale bar, 10 μm.

FIG. 3.

Placental abnormalities in C/EBPαβ^−/− conceptus at E10. (A and B) H&E-stained sagittal sections of +/+ (A) and C/EBPαβ^−/− (B) placentas. Giant cells (Gi) are indistinguishable between C/EBPαβ^−/− and wild-type placentas. Fetal blood vessels (arrowhead) were rarely found in the labyrinthine layer (L) of the C/EBPαβ^−/− placenta compared to the wild-type placenta. Maternal blood sinuses (arrow) were present in normal numbers but were never close to embryonal vessels as observed for the wild type. (C and D) Isolectin B4-stained sagittal sections of +/+ (C) and C/EBPαβ^−/− (D) placentas at E10. Isolectin B4 labeled the matrix surrounding the fetal blood vessels. Note the poor labeling of the labyrinthine layer (L) in C/EBPαβ^−/− placentas compared to the wild-type placentas. The staining of the yolk sac (YS) and of the chorionic plate (CP) were indistinguishable between C/EBPαβ^−/− and wild-type placentas. Al, allantoic cavity; d, maternal decidua; Sp, spongiotrophoblast; arrow, maternal erythrocyte sinus. Scale bar, 10 μm.

FIG. 4.

Expression of cell-type-specific markers analyzed by in situ hybridization of wild-type (+/+) and C/EBPαβ^−/− placentas at E10. Sagittal sections (70 μm) of paraformaldehyde-fixed placentas were hybridized with antisense probes for Flt-1 (spongiotrophoblast cells); PL-1 (trophoblast giant cells); PPARγ (diploid trophoblasts); and Gcm1, Dlx3, and VEGF (labyrinthine layer). Note that the expression of Gcm1, Dlx3, and VEGF is lower in C/EBPαβ^−/− placentas than in +/+ placentas. Scale bar, 15 μm.