Disruption of the phosphatidylserine decarboxylase gene in mice causes embryonic lethality and mitochondrial defects - PubMed (original) (raw)

Disruption of the phosphatidylserine decarboxylase gene in mice causes embryonic lethality and mitochondrial defects

Rineke Steenbergen et al. J Biol Chem. 2005.

Abstract

Most of the phosphatidylethanolamine (PE) in mammalian cells is synthesized by two pathways, the CDP-ethanolamine pathway and the phosphatidylserine (PS) decarboxylation pathway, the final steps of which operate at spatially distinct sites, the endoplasmic reticulum and mitochondria, respectively. We investigated the importance of the mitochondrial pathway for PE synthesis in mice by generating mice lacking PS decarboxylase activity. Disruption of Pisd in mice resulted in lethality between days 8 and 10 of embryonic development. Electron microscopy of Pisd-/- embryos revealed large numbers of aberrantly shaped mitochondria. In addition, fluorescence confocal microscopy of Pisd-/- embryonic fibroblasts showed fragmented mitochondria. PS decarboxylase activity and mRNA levels in Pisd+/- tissues were approximately one-half of those in wild-type mice. However, heterozygous mice appeared normal, exhibited normal vitality, and the phospholipid composition of livers, testes, brains, and of mitochondria isolated from livers, was the same as in wild-type littermates. The amount and activity of a key enzyme of the CDP-ethanolamine pathway for PE synthesis, CTP:phosphoethanolamine cytidylyltransferase, were increased by 35-40 and 100%, respectively, in tissues of Pisd+/- mice, as judged by immunoblotting; PE synthesis from [3H]ethanolamine was correspondingly increased in hepatocytes. We conclude that the CDP-ethanolamine pathway in mice cannot substitute for a lack of PS decarboxylase during development. Moreover, elimination of PE production in mitochondria causes fragmented, misshapen mitochondria, an abnormality that likely contributes to the embryonic lethality.

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Figures

FIGURE 1

FIGURE 1. Real-time PCR analysis of Pisd mRNA levels relative to cyclophilin mRNA in mouse tissues

A, tissues from adult mice; B, in livers during development from 5 days (−5) before birth (day 0) to adult (> 50 days old). Data are averages ± S.D. from at least three mice or embryos. Some error bars are too small to be visible.

FIGURE 2

FIGURE 2. Generation of Pisd mutant mice by gene trapping

A, endogenous Pisd gene and transcript. B, gene-trapped Pisd allele. C, PCR analysis of the three Pisd genotypes.

FIGURE 3

FIGURE 3. Analysis of Pisd expression in tissues from Pisd+/− mice as judged by β-galactosidase staining

A, β-galactosidase staining of a Pisd+/− embryo (E13) showing expression of Pisd in the heart. Original magnification, 40×. B, β-galactosidase staining of testis from a 7-week-old Pisd+/− mouse, showing expression of Pisd in Sertoli cells. Original magnification, 40×.

FIGURE 4

FIGURE 4. Histological analysis of 8-day-old Pisd+/+ and _Pisd_−/− embryos

Sagittal sections were made from embryos with surrounding deciduas. Tissues were fixed, embedded in paraffin, and serial sections were stained with hematoxylin/eosin. Size bars in A and C represent 500 nm, whereas for B and D size bars represent 200 nm. A and B, Pisd+/+ embryo. C and D, _Pisd_−/− embryo. A and C show overview of embryos with surrounding deciduas (Dc), placenta (P), and somites (S). B, detailed view of wild-type embryo that has developed the ectoderm (Ec), mesoderm (M), endoderm (En), and the amnion (Am). The extra-embryonic component is clearly defined. D, in _Pisd_-deficient embryos, no differentiation of the three germ layers was observed. The extra-embryonic component is absent, and blood cells (indicated by asterisks) directly surround the embryo.

FIGURE 5

FIGURE 5. Fluorescence confocal microscopy of embryonic fibroblasts isolated from _Pisd_−/−, Pisd+/−, and Pisd+/+ mouse embryos

Fibroblasts were isolated from 8- to 9-day-old embryos. Mitochondria were imaged by confocal microscopy with MitoTracker Green FM. A and B, fibroblasts from wild-type embryos revealing typical tubular-reticular mitochondria concentrated around the nucleus. C and D, fibroblasts from Pisd+/− embryos. E and F, fibroblasts from _Pisd_−/− embryos in which mitochondria are rounded, fragmented, and dispersed throughout the cell. Size bar, 20 nm.

FIGURE 6

FIGURE 6. Electron microscopy of mitochondria in Pisd+/+ and _Pisd_−/− embryos

Embryos (E8) were prepared for electron microscopy. Ultrathin sections were cut and stained with uranyl acetate and osmium tetroxide. A, representative elongated and tubular mitochondrion from a Pisd+/+ embryo. B, typical mitochondria from a _Pisd_−/− embryo; mitochondria are rounded and vesicular but cristae are visible. Size bar, 500 nm. C, length and width of mitochondria in Pisd+/+(open circles) and _Pisd_−/−(closed squares) embryonic tissue were measured from electron micrographs. Each symbol represents a single mitochondrion.

FIGURE 7

FIGURE 7. Pisd mRNA levels in livers from Pisd+/+ and Pisd+/− adult mice (A) and 10-day-old embryos (B)

Pisd mRNA levels relative to cyclophilin mRNA were determined by real-time PCR. Data are means ± S.D. from at least three mice or embryos of each genotype. *, p < 0.005.

FIGURE 8

FIGURE 8. PS decarboxylase activity in tissues of Pisd+/+ and Pisd+/− mice

Homogenates were prepared from livers, brains and testes of adult Pisd+/+ (hatched bars) and Pisd+/− (solid bars) mice. PS decarboxylase activity was measured in vitro. Data are means ± S.D. from at least three mice of each genotype. For liver and brain: *, p < 0.001; for testis: *, p = 0.012.

FIGURE 9

FIGURE 9. Phospholipid composition of tissues of adult Pisd+/+ and Pisd+/− mice

The phospholipid composition was determined in homogenates of testes (A), brains (B), livers (C), and in mitochondria isolated from livers (D) of Pisd+/+ (hatched bars) and Pisd+/− (solid bars) mice. All data are means ± S.D. from analyses of at least four mice of each genotype.

FIGURE 10

FIGURE 10. CTP:phosphoethanolamine cytidylyltransferase protein is increased in Pisd+/− mice

Homogenates were prepared from livers, brains and testes of Pisd+/+ and Pisd+/− mice. Upper panel, representative immunoblot of the cytidylyltransferase (molecular mass ~50 kDa). Lower panel, quantitation of densitometric scanning of immunoblots of tissues from three mice of each genotype. Data for band intensity of the immunoblot from Pisd+/− mice are given relative to that from Pisd+/+ mice. For liver: *, p < 0.05; for testis: *, p < 0.001; for brain: *, p = 0.051.

FIGURE 11

FIGURE 11. Reciprocal regulation of PE synthesis via the CDP-ethanolamine pathway

A, CTP:phosphoethanolamine cytidylyltransferase activity in homogenates of livers from Pisd+/+ and Pisd+/− mice. Data are means ± S.D. of four mice of each genotype. *, p < 0.04. B, incorporation of [3H]ethanolamine into PE in hepatocytes from Pisd+/+(filled symbols) and Pisd+/− (open symbols) mice. Data are means ± S.D. of three independent experiments, each performed in triplicate.

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