Article Figures & Data
Figures
- FIG. 1.
Generation of PLCδ3 KO and PLCδ1/PLCδ3 DKO mice. (A) Genetic map of the genomic structure of the PLCδ3 gene. Exons are indicated by numbered boxes. Introns are lettered (a through n), and their approximate lengths in kbp are given. The structure of the retroviral trap vector to disrupt the mouse PLCδ3 is also shown. LTR, long terminal repeat; SA, splice acceptor site; geo, galactosidase-neomycin resistance fusion gene; pA, polyadenylation consensus site; PGK, PGK promoter; puro, puromycin resistance cassette; SD, splice donor site. The gene trap is inserted into intron a of the PLCδ3 gene as indicated by an arrow. (B) PCR genotyping. Genomic DNAs were isolated from tails of wild-type (+/+), heterozygous (+/−), and PLCδ3 KO (−/−) mice. W, wild type; M, mutant. (C) Western blot analysis of expression of PLCδ3 in heart from wild-type (+/+) and PLCδ3 KO (−/−) mice. (D) Dorsal view of PLCδ3 +/+ and PLCδ3 −/− mice. (E) Western blot analysis of the tissue distribution of PLCδ1 and PLCδ3. Protein (40 μg) was subjected to SDS-polyacrylamide gel electrophoresis, and Western blot analysis was performed. (F) Amino acid homology between PLCδ1 and PLCδ3. Numbers indicate the percentage of similar amino acids. PH, pleckstrin homology domain; EF, EF-hand motif; X, X domain; Y, Y domain; C2, C2 domain. (G) Genotyping of embryos from PLCδ1 + / − PLCδ3 +/ − intercrosses at E11.5. Lane 5, PLCδ1/PLCδ3 DKO embryos. W, wild type; M, mutant.
- FIG. 2.
Macroscopic and vascular abnormalities of PLCδ1/PLCδ3 DKO embryos and extraembryonic tissues. (A to F) Macroscopic views of embryos and yolk sacs. Views of E11.5 PLCδ1 − / − PLCδ3 +/+ (A) and E11.5 PLCδ1/PLCδ3 DKO (B) embryos and E12.5 PLCδ1 − / − PLCδ3 +/+ (C) and E12.5 PLCδ1/PLCδ3 DKO (D) embryos. Yolk sacs of E12.5 PLCδ1 − / − PLCδ3 +/+ (E) and E12.5 PLCδ1/PLCδ3 DKO (F) embryos are also shown. At E12.5, PLCδ1/PLCδ3 DKO embryos exhibit hemorrhaging in the cardiac and ventral body wall regions (arrow in panel D). (G to J) Whole-mount CD31 staining of E12.5 heads and yolk sacs. Vessels of the head regions in PLCδ1 − / − PLCδ3 +/+ embryos were well remodeled (arrow in panel G), whereas in PLCδ1/PLCδ3 DKO embryos, the vasculature of the head region was irregularly shaped (arrow in panel H). Yolk sac vessels of PLCδ1 − / − PLCδ3 +/+ embryos were well remodeled with both large (arrows in panel I) and small (arrowheads in panel I) vessels, whereas those of PLCδ1/PLCδ3 DKO embryos contained mostly equal-diameter vessels (arrowheads in panel J).
- FIG. 3.
Expression of PLCδ1 and PLCδ3 in placenta and trophoblast. (A) Western blot analysis of expression of PLCδ1 and PLCδ3 in embryonic and extraembryonic tissues at E11.5. (B) Western blot analysis of expression of PLCδ1 and PLCδ3 in fetal and maternal parts of the placenta. (C) PLCδ1 and PLCδ3 are expressed at high levels in TS cells. MEF, mouse embryonic fibroblast; Stem, undifferentiated TS cells; Day 2, Day 4, and Day 6, TS cells were induced to differentiate for 2 days, 4 days, and 6 days, respectively. β-Actin (A to C) was included as a loading control. (D to F) Immunohistochemical detection of PLCδ1 in the placenta. Immunohistochemical staining of wild-type (D and F) and PLCδ1 KO (E) placentas at E11.5 with a polyclonal anti-PLCδ1 antibody. (F) Magnified view of panel D. Bars: D and E (shown in panel D) = 100 μm; F= 50 μm.
- FIG. 4.
Histological abnormalities in placentas of PLCδ1/PLCδ3 DKO mice. (A to H) HE staining of placenta sections from PLCδ1 − / − PLCδ3 +/+ (A, C, E, and G) and PLCδ1/PLCδ3 DKO (B, D, F, and H) embryos at E10.5 (A to D) and E11.5 (E to H). The arrows indicate embryonal vessels, and the arrowheads indicate maternal vessels. la, labyrinth area. Note that vascularization is reduced in the labyrinth area of the PLCδ1/PLCδ3 DKO placenta (F and H). (I) For quantification, the ratio of the vascularized area to the total labyrinth area was calculated with NIH Image software. Values represent the average ratios in five fields from five sections from different mice. Statistical significance was determined by Student's t test; the error bars indicate standard deviations. Bars: E and F (shown in panel E) = 100 μm; G and H (shown in panel G) = 50 μm.
- FIG. 5.
Apoptotic and proliferative defects in trophoblasts of PLCδ1/PLCδ3 DKO placenta. (A to D) Proliferative activity was determined by BrdU incorporation analysis of labyrinth layer cells in PLCδ1 − / − PLCδ3 +/+ (A and C) and PLCδ1/PLCδ3 DKO (B and D) placentas. (C and D) Higher magnifications of the fields in panels A and B, respectively. HE (E) and TUNEL staining (F to H) of PLCδ1 − / − PLCδ3 +/+ (F) and PLCδ1/PLCδ3 DKO (E, G, and H) placentas. Apoptotic or necrotic cells were observed in the labyrinth area of the PLCδ1/PLCδ3 DKO placenta (arrows in panel E). (H) Higher magnification of the field in panel G. TUNEL-positive cells were observed in the labyrinth area of the PLCδ1/PLCδ3 DKO placenta (arrows in panel H). (I to P) Ultrastructural analysis of cells in PLCδ1 − / − PLCδ3 +/+ (I) and DKO (J to P) labyrinth layers. Note that the structure composed of mononuclear trophoblast (Mono), syncytiotrophoblast (SynT), and endothelial cell (Endo) is intact in the PLCδ1 − / − PLCδ3 +/+ labyrinth (I). Arrows (J to M) indicate vacuolation of nuclei in apoptotic trophoblasts. Asterisks (J, K, N, O, and P) indicate fragmented trophoblasts. Arrowheads (J, K, O, and P) indicate endothelial cells. e, embryonic vessels; m, maternal vessels. Bars: A and B (shown in panel A) = 100 μm; C and D (shown in panel C) = 100 μm; E = 50 μm; F and G (shown in panel F) = 100 μm; H = 50 μm; I = 2 μm; J = 2 μm; K = 2 μm; L = 1 μm; M = 1 μm; N = 2 μm; O = 2 μm; P = 1 μm.
- FIG. 6.
Rescue of PLCδ1/PLCδ3 DKO placenta and embryo by tetraploid aggregation method. (A) Strategy of tetraploid chimera generation. 4N wild-type cells (+/+) were generated by electrofusion of 2N wild-type cells, and then 4N wild-type cells and 2N cells from intercrosses between PLCδ1 − / − PLCδ3 +/ − (DKO; δ1 − / − δ3 +/ − and δ1 − / − δ3 +/+) were aggregated. Blastocysts resulting from the aggregations were transferred into the uteri of pseudopregnant recipient mice. (B) Number of embryos generated by 4N aggregation method at E14.5. Aggregation of 4N wild-type cells and 2N cells from PLCδ1 − / − PLCδ3 +/ − intercrosses was carried out. (C and D) Macroscopic views of rescued PLCδ1 − / − PLCδ3 +/+ (C) and PLCδ1/PLCδ3 DKO (D) embryos at E14.5. No obvious abnormalities were observed in the rescued PLCδ1/PLCδ3 DKO embryos. (E and F) HE staining of sections of placentas from rescued PLCδ1 − / − PLCδ3 +/+ (E) and PLCδ1/PLCδ3 DKO (F) embryos at E14.5. (G) Quantification of the ratio of vascularized area to total labyrinth area in nonrescued or 4N aggregation-rescued placenta. The values represent the average ratios in four fields from two sections from different tetraploid rescued mice (4N) and in five fields from five sections from different nonrescued mice (non). Statistical significance was determined by Student's t test. n.s., not significant. The error bars represent standard deviations.
Tables
- TABLE 1.
Genotypes of offspring from PLCδ1 −/− PLCδ3 +/− × PLCδ1 −/− PLCδ3 +/− mating
Day of analysis No. with genotype a: Total δ1−/− δ3+/+ δ1−/− δ3+/− δ1−/− δ3−/− E9.5 16 24 8 48 E10.5 14 34 23 71 E11.5 56 121 51 (22) 228 E12.5 38 73 32 (18) 143 E13.5 20 30 17 (17) 67 P0 b 12 21 0 33
