The Role of Phospholipid Hydroperoxide Glutathione Peroxidase Isoforms in Murine Embryogenesis
2006; Elsevier BV; Volume: 281; Issue: 28 Linguagem: Inglês
10.1074/jbc.m601195200
ISSN1083-351X
AutoresAstrid Borchert, Chi Chiu Wang, Christoph Ufer, Heike Schiebel, Nicolai Ε. Savaskan, Hartmut Kühn,
Tópico(s)Redox biology and oxidative stress
ResumoPhospholipid hydroperoxide glutathione peroxidase (GPx4) is a selenocysteine-containing enzyme, and three different isoforms (cytosolic, mitochondrial, and nuclear) originate from the GPx4 gene. Homozygous GPx4-deficient mice die in utero at midgestation, since they fail to initiate gastrulation and do not develop embryonic cavities. To investigate the biological basis for embryonic lethality, we first explored expression of the GPx4 in adult murine brain and found expression of the protein in cerebral neurons. Next, we profiled mRNA expression during the time course of embryogenesis (embryonic days 6.5-17.5 (E6.5-17.5)) and detected mitochondrial and cytosolic mRNA species at high concentrations. In contrast, the nuclear isoform was only expressed in small amounts. Cytosolic GPx4 mRNA was present at constant levels (about 100 copies per 1000 copies of glyceraldehyde-3-phosphate dehydrogenase mRNA), whereas nuclear and mitochondrial isoforms were down-regulated between E14.5 and E17.5. In situ hybridization indicated expression of GPx4 isoforms in all developing germ layers during gastrulation and in the somite stage in the developing central nervous system and in the heart. When we silenced expression of GPx4 isoforms during in vitro embryogenesis using short interfering RNA technology, we observed that knockdown of mitochondrial GPx4 strongly impaired segmentation of rhombomeres 5 and 6 during hindbrain development and induced cerebral apoptosis. In contrast, silencing expression of the nuclear isoform led to retardations in atrium formation. Taken together, our data indicate specific expression of GPx4 isoforms in embryonic brain and heart and strongly suggest a role of this enzyme in organogenesis. These findings may explain in part intrauterine lethality of GPx4 knock-out mice. Phospholipid hydroperoxide glutathione peroxidase (GPx4) is a selenocysteine-containing enzyme, and three different isoforms (cytosolic, mitochondrial, and nuclear) originate from the GPx4 gene. Homozygous GPx4-deficient mice die in utero at midgestation, since they fail to initiate gastrulation and do not develop embryonic cavities. To investigate the biological basis for embryonic lethality, we first explored expression of the GPx4 in adult murine brain and found expression of the protein in cerebral neurons. Next, we profiled mRNA expression during the time course of embryogenesis (embryonic days 6.5-17.5 (E6.5-17.5)) and detected mitochondrial and cytosolic mRNA species at high concentrations. In contrast, the nuclear isoform was only expressed in small amounts. Cytosolic GPx4 mRNA was present at constant levels (about 100 copies per 1000 copies of glyceraldehyde-3-phosphate dehydrogenase mRNA), whereas nuclear and mitochondrial isoforms were down-regulated between E14.5 and E17.5. In situ hybridization indicated expression of GPx4 isoforms in all developing germ layers during gastrulation and in the somite stage in the developing central nervous system and in the heart. When we silenced expression of GPx4 isoforms during in vitro embryogenesis using short interfering RNA technology, we observed that knockdown of mitochondrial GPx4 strongly impaired segmentation of rhombomeres 5 and 6 during hindbrain development and induced cerebral apoptosis. In contrast, silencing expression of the nuclear isoform led to retardations in atrium formation. Taken together, our data indicate specific expression of GPx4 isoforms in embryonic brain and heart and strongly suggest a role of this enzyme in organogenesis. These findings may explain in part intrauterine lethality of GPx4 knock-out mice. Phospholipid hydroperoxide glutathione peroxidase (phGPx or GPx4) 2The abbreviations used are: GPx4 or phGPX, glutathione peroxidase-4; c-, m-, and n-GPx4, GPx4 cytosolic, mitochondrial, and nuclear isoform, respectively; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PBS, phosphate-buffered saline; En, embryonic day n;Nn, neonatal day n; RT, reverse transcription; siRNA, short interfering RNA; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; TUNEL, terminal dUTP nick-end labeling; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. is an intracellular antioxidant enzyme (1Imai H. Nakagawa Y. Free Radic. Biol. Med. 2003; 34: 145-169Crossref PubMed Scopus (593) Google Scholar) that directly reduces peroxidized phospholipids even if they are incorporated in biomembranes and lipoproteins (2Ursini F. Bindoli A. Chem. Phys. Lipids. 1987; 44: 255-276Crossref PubMed Scopus (312) Google Scholar, 3Thomas J.P. Maiorino M. Ursini F. Girotti A.W. J. Biol. Chem. 1990; 265: 454-461Abstract Full Text PDF PubMed Google Scholar, 4Sattler W. Maiorino M. Stocker R. Arch. Biochem. Biophys. 1994; 309: 214-221Crossref PubMed Scopus (88) Google Scholar). In addition, the enzyme has been implicated in sperm maturation (5Ursini F. Heim S. Kiess M. Maiorino M. Roveri A. Wissing J. Flohe L. Science. 1999; 285: 1393-1396Crossref PubMed Scopus (723) Google Scholar, 6Roveri A. Flohe L. Maiorino M. Ursini F. Methods Enzymol. 2002; 347: 208-212Crossref PubMed Scopus (12) Google Scholar) and appears to be essential for regular murine embryogenesis (7Yant L.J. Ran Q. Rao L. Van Remmen H. Shibatani T. Belter J.G. Motta L. Richardson A. Prolla T.A. Free Radic. Biol. Med. 2003; 34: 496-502Crossref PubMed Scopus (548) Google Scholar, 8Imai H. Hirao F. Sakamoto T. Sekine K. Mizukura Y. Saito M. Kitamoto T. Hayasaka M. Hanaoka K. Nakagawa Y. Biochem. Biophys. Res. Commun. 2003; 305: 278-286Crossref PubMed Scopus (266) Google Scholar). There are three different isoforms of GPx4 (cytosolic isoform (c-GPx4), mitochondrial isoform (m-GPx4), and nuclear isoform (n-GPx4)), but all of them derive from a single gene, which is located on human chromosome 19 (9Kelner M.J. Montoya M.A. Biochem. Biophys. Res. Commun. 1998; 249: 53-55Crossref PubMed Scopus (44) Google Scholar) and in a sentential region of murine chromosome 10 (10Knopp E.A. Arndt T.L. Eng K.L. Caldwell M. LeBoeuf R.C. Deeb S.S. O'Brien K.D. Mamm. Genome. 1999; 10: 601-605Crossref PubMed Scopus (51) Google Scholar, 11Boschan C. Borchert A. Ufer C. Thiele B.J. Kuhn H. Genomics. 2002; 79: 387-394Crossref PubMed Scopus (21) Google Scholar). The start codons for the m- and c-GPx4 isoforms as well as the targeting sequence that directs the mitochondrial enzyme into the mitochondria are localized in the first exon of the GPx4 gene. Expression of the 34-kDa n-GPx4 (nuclear isoform) involves transcription of an alternative first exon (12Pfeifer H. Conrad M. Roethlein D. Kyriakopoulos A. Brielmeier M. Bornkamm G.W. Behne D. FASEB J. 2001; 15: 1236-1238Crossref PubMed Scopus (102) Google Scholar). The three phGPx isoforms are expressed at low to medium levels in most mammalian cells and have been implicated in expression regulation of redox-sensitive genes (13Brigelius-Flohe R. Free Radic. Biol. Med. 1999; 27: 951-965Crossref PubMed Scopus (882) Google Scholar), in inflammation (14Imai H. Narashima K. Arai M. Sakamoto H. Chiba N. Nakagawa Y. J. Biol. Chem. 1998; 273: 1990-1997Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar), in modulation of programmed cell death (15Arai M. Imai H. Koumura T. Yoshida M. Emoto K. Umeda M. Chiba N. Nakagawa Y. J. Biol. Chem. 1999; 274: 4924-4933Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar), and in oxidative injury. High concentrations of GPx4 were found in testis (16Roveri A. Ursini F. Flohe L. Maiorino M. Biofactors. 2001; 14: 213-222Crossref PubMed Scopus (48) Google Scholar), and low level expression of the protein has been related to male infertility (17Maiorino M. Bosello V. Ursini F. Foresta C. Garolla A. Scapin M. Sztajer H. Flohe L. Biol. Reprod. 2003; 68: 1134-1141Crossref PubMed Scopus (70) Google Scholar). Homozygous GPx4-deficient mice are not viable (7Yant L.J. Ran Q. Rao L. Van Remmen H. Shibatani T. Belter J.G. Motta L. Richardson A. Prolla T.A. Free Radic. Biol. Med. 2003; 34: 496-502Crossref PubMed Scopus (548) Google Scholar, 8Imai H. Hirao F. Sakamoto T. Sekine K. Mizukura Y. Saito M. Kitamoto T. Hayasaka M. Hanaoka K. Nakagawa Y. Biochem. Biophys. Res. Commun. 2003; 305: 278-286Crossref PubMed Scopus (266) Google Scholar), and embryos die in utero at midgestation. In contrast, mice in which expression of the n-GPx4 was selectively silenced were viable and surprisingly also fully fertile (18Conrad M. Moreno S.G. Sinowatz F. Ursini F. Kolle S. Roveri A. Brielmeier M. Wurst W. Maiorino M. Bornkamm G.W. Mol. Cell. Biol. 2005; 25: 7637-7644Crossref PubMed Scopus (203) Google Scholar). These data suggest that m- and/or c-GPx4 are more important for murine embryogenesis than n-GPx4. More detailed studies on intrauterine development of GPx4-/- mice indicated that the embryos fail to develop embryonic cavities but are characterized by enlarged Reichert's membranes. At stage E7.5, when normal embryos have completed gastrulation, homozygous GPx4-deficient mice still resemble pregastrulation embryos showing primitive signs of endo-, meso-, and ectodermic differentiation. At later stages (E8.0-8.5Ursini F. Heim S. Kiess M. Maiorino M. Roveri A. Wissing J. Flohe L. Science. 1999; 285: 1393-1396Crossref PubMed Scopus (723) Google Scholar), when normal embryos undergo organ development, GPx4-deficient individuals enter intrauterine resorption. Heterozygous GPx4-deficient mice are viable and fertile and develop normally (7Yant L.J. Ran Q. Rao L. Van Remmen H. Shibatani T. Belter J.G. Motta L. Richardson A. Prolla T.A. Free Radic. Biol. Med. 2003; 34: 496-502Crossref PubMed Scopus (548) Google Scholar, 8Imai H. Hirao F. Sakamoto T. Sekine K. Mizukura Y. Saito M. Kitamoto T. Hayasaka M. Hanaoka K. Nakagawa Y. Biochem. Biophys. Res. Commun. 2003; 305: 278-286Crossref PubMed Scopus (266) Google Scholar). However, these animals exhibit a reduced survival in response to γ-irradiation, suggesting an impaired antioxidative capacity of the individuals. In fact, cell lines derived from GPx4+/- mice were markedly more sensitive to inducers of oxidative stress as compared with cell lines derived from wild-type control littermates (7Yant L.J. Ran Q. Rao L. Van Remmen H. Shibatani T. Belter J.G. Motta L. Richardson A. Prolla T.A. Free Radic. Biol. Med. 2003; 34: 496-502Crossref PubMed Scopus (548) Google Scholar). Taken together, these data indicated that expression of mitochondrial and/or cytosolic GPx4 isoforms is required for normal embryogenesis, whereas the nuclear isoform may not be essential. Unfortunately, the biological reasons for premature embryonic lethality remain unclear. To address this question, we studied expression of the three GPx4 isoforms during normal embryogenesis and explored the impact of silencing expression of these isoforms during in vitro embryogenesis. At late embryonic stages, we observed abnormal development of hindbrain (m-GPx4 silencing) and heart (n-GPx4 silencing). These data may explain in part the intrauterine lethality of GPx4-deficient mice. Chemicals—The chemicals used were from the following sources: Superscript III reverse transcriptase and RNaseOUT from Invitrogen (Karlsruhe, Germany); BD Advantage 2 Polymerase Mix from BD Biosciences (Pharmingen, Germany); dNTPs from Carl Roth GmbH (Karlsruhe, Germany); DNA molecular weight markers (100 bp, 1 kb) from New England Biolabs GmbH (Schwabach, Germany); and the QuantiTect SYBR Green PCR Kit from Qiagen (Hilden, Germany). PCR primers were custom-synthesized by BIOTEZ (Berlin, Germany). Preparation of Whole Murine Embryos and of Various Embryonic Tissues—All animal experiments were performed in strict adherence to the guidelines for experimentation with laboratory animals as set by the Chinese University of Hong Kong. Inbred Institute for Cancer Research pregnant mice were obtained from an animal house, and embryos in different developmental stages (E6.5 to postnatal day 5 (N5)) were prepared and kept in PBS (0.1% diethyl pyrocarbonate) for separation of extraembryonic tissues and the embryo proper. These preparations were carried out under a stereo microscope (Olympus, New York). At later developmental stages, embryonic brain and heart (from E10.5 to N5) were separately dissected. Different embryonic tissues from the same litter were pooled, and at least three dams were collected independently. Tissue samples were kept in RNAlater solution (Qiagen, Hilden, Germany) at 4 °C overnight and were then stored at -80 °C prior to RNA extraction. RNA Extraction and Reverse Transcription—Total RNA was extracted from the embryonic tissues using the RNeasy mini kit (Qiagen, Hilden, Germany). It was reverse transcribed into the corresponding cDNA using oligo(dT)15 primer and SuperScript III reverse transcriptase (Invitrogen) according to the vendor's instructions. Semiquantitative RT-PCR—The PCR samples (total volume of 25 μl) consisted of 40 mm Tricine buffer, pH 8.7, containing 0.5 μl of RT reaction, 15 mm potassium acetate, 3.5 mm magnesium acetate, 3.75 μg/ml bovine serum albumin, 0.005% Tween 20, 0.005% Nonidet P-40, 200 nm forward and reverse primers (Table 1), 200 μm dNTPs (final concentrations are given), and 0.5 μl of 50× BD Advantage 2 Polymerase Mix. The following PCR protocol was used to amplify the target gene products: preconditioning phase of 90 s at 95 °C; denaturing phase of 30 s at 95 °C followed by an annealing/extension phase of 60 s at 68 °C. After 35 cycles of amplification, a final extension phase (3 min at 68 °C) was run, and the samples were stored at 4 °C. For electrophoresis, usually 10 μl of the PCR was used, and the PCR products were separated in a 2% agarose gel. The DNA bands were routinely stained with ethidium bromide and quantified by densitometric evaluation of the gels in relation to a known mass ladder standard.TABLE 1Primers used for amplification of the different phGPx isoforms and for preparation of the labeled probes used for in situ hybridizationGene productDirectionPrimer SequenceSizebpRT-PCRGAPDHForward5′-CCA TCA CCA TCT TCC AGG AGC GA-3′447Reverse5′-GGA TGA CCT TGC CCA CAG CCT TG-3′m+c-GPx4Forward5′-CGC CTG GTC TGG CAG GCA CCA-3′467Reverse5′-ACG CAG CCG TTC TTA TCA ATG AGA A-3′m-GPx4Forward5′-GAG ATG AGC TGG GGC CGT CTG A-3′531Reverse5′-ACG CAG CCG TTC TTA TCA ATG AGA A-3′n-GPx4Forward5′-AGT TCC TGG GCT TGT GTG CAT CC-3′458Reverse5′-ACG CAG CCG TTC TTA TCA ATG AGA A-3′In situ hybridization probesm-GPx4 (exon 1A)Forward5′-CTC GGC CTC GCG CGT CCA TTG-3′146Reverse5′-TGG TGC CTG CCA GAC CAG GCG-3′n-GPx4 (exon 1B)Forward5′-AGC GGG GAC GCT GCA GAC AGC-3′230Reverse5′-CAA GCC CAG GAA CTC GGA GCT G-3′All isoforms (exons 2-3)Forward5′-GTG CAT CCC GCG ATG ATT GGC G-3′255Reverse5′-GAT TAC TTC CTG GCT CCT GCC TC-3′ Open table in a new tab Quantitative Real Time RT-PCR—Real time PCR was carried out with a Rotor Gene 3000 (Corbett Research, Mortlake, Australia) using the QuantiTect SYBR Green PCR Kit from Qiagen (Hilden, Germany). The primer combinations specified in Table 1 were used, and the following PCR protocol was applied: 15-min hot start at 95 °C, followed by 40 cycles of denaturation (30 s at 94 °C), annealing (30 s at 65 °C), and synthesis (30 s at 72 °C) in a total volume of 10 μl. Homogeneity of the amplified PCR products was tested, recording the melting curves. For this purpose, the temperature was elevated slowly from 60 to 99 °C. Data were acquired and analyzed with the Rotor-Gene Monitor software (version 4.6). The amplification kinetics were recorded in real time mode as sigmoid process curves, for which the fluorescence was plotted against the number of amplification cycles. To generate standard curves for exact quantification of gene expression levels, specific amplicons were used as external standards for each target gene and for GAPDH. The initial amplicon concentrations were set to values varying between 5 × 103 and 3 × 106 copy numbers. GAPDH mRNA was used as an internal standard to normalize expression of the target transcripts (m-GPx4, m+c-GPx4, and n-GPx4). Absolute ratios of the target mRNA species and the GAPDH mRNA, which exactly quantify the cellular expression levels of the target genes, were calculated using these standard curves. All RNA preparations were analyzed at least in triplicates, and means ± S.D. are given. Discrimination between Various GPx4 Isoforms in RT-PCR and in Situ Hybridization—Since GPx4-deficient mice die in utero at midgestation, we explored expression of different GPx4 isoforms during murine embryogenesis. Unfortunately, on the protein level, c-GPx4 and m-GPx4 cannot be distinguished, since the mitochondrial targeting sequence is cleaved off after mitochondrial import. However, such differentiation is possible on the mRNA level. We established isoform-specific real time PCR systems suitable for quantifying the expression kinetics of the different GPx4 mRNA species during the time course of murine embryogenesis. The reverse primer used for amplification of the three different GPx4 mRNAs was identical (Scheme 1B), but the forward primer for the m-GPx4 was placed inside the mitochondrial targeting sequence. In contrast, the forward primer for amplification of the n-GPx4 was placed in the alternative first exon (E1b), and both positions ensure selective amplification of the two isoforms. Selective quantification of the c-GPx4 mRNA was more complicated. To amplify this mRNA species, we placed the forward primer in exon E1a immediately after the transcriptional start site for the c-GPx4 (10Knopp E.A. Arndt T.L. Eng K.L. Caldwell M. LeBoeuf R.C. Deeb S.S. O'Brien K.D. Mamm. Genome. 1999; 10: 601-605Crossref PubMed Scopus (51) Google Scholar). Unfortunately, this primer combination did not allow separate quantification of the m-GPx4 and c-GPx4 mRNA; in fact, the sum of m-GPx4 and c-GPx4 mRNA species (m+c-GPx4) was amplified. However, we separately amplified m-GPx4 mRNA using the above mentioned m-GPx4-specific primer combination, and thus, we were able to calculate the c-GPx4 copy numbers using the following formula: [c-GPx4] = [m+c-GPx4] - [m-GPx4]. Immunohistochemistry—For immunohistochemical staining, murine brains were perfused with ice-cold 4% (w/v) paraformaldehyde solution and were immersion-fixed overnight. Sections were prepared on a McIllwain vibratome, quenched with NH4Cl for 15 min, and blocked with 10% goat serum plus 0.1% Saponin in PBS for 1 h at room temperature. Blocked sections were exposed to the primary monoclonal anti-GPx4 antibody (1:200 diluted) overnight at 4 °C. After washing in 0.1% Saponin/PBS, sections were incubated at +4 °C with a fluorescent anti-mouse IgG (diluted 1:500; Molecular Probes, Inc., Leiden, The Netherlands). Counterstaining of nuclei was carried out using the HOECHST dye (Roche Applied Science). Control staining was performed appropriately using a diluted preimmune serum. In Situ Hybridization—The expression of GPx4 isoforms at different developmental stages was studied by whole mount in situ hybridization. For this purpose, the whole embryos (E6.5 to E10.5) were fixed in 4% paraformaldehyde (in PBS) overnight, dehydrated in methanol, and stored at -20 °C prior to hybridization. The riboprobes were labeled with digoxigenin-11-UTP (Roche Applied Science) using the AmpliScribe kit (Epicenter Technologies). Whole mount in situ hybridization in the developing embryos was performed according to Wilkinson's methods (19Wilkinson D.G. In Situ Hybridisation. IRL Press, Oxford1992: 75-83Google Scholar) with minor modifications. In both, the prehybridization and hybridization solution SDS was used instead of CHAPS, and RNase treatment of the samples was omitted. For the posthybridization washes, formamide was not included in the washing solution. Both antisense and sense probes for each mRNA species were prepared from individual plasmids cloned into the PCR 2.1-TOPO vector (Invitrogen). For in situ hybridization, it was impossible to separately stain for c-GPx4 (Scheme 1C). In fact, the probe we used was placed in exon 2-3 and thus indicated expression of all three GPx4 isoforms (m+c+n-GPx4). However, the probes selected for m- and n-GPx4 were isoform-specific. Short Interfering RNA (siRNA) Experiments—m-GPx4 and n-GPx4 targeting siRNA probes were prepared from mouse cDNA plasmids. Antisense and sense RNA were separately amplified by T7 RNA polymerase using MEGAscript Kit (Ambion, Austin, TX). Annealed double-stranded RNA (15 μg) was digested with RNase III (15 units) for 1 h at 37°C using the Silencer siRNA mixture kit (Ambion). Purified and concentrated siRNAs (1.0 μg/μl) were first mixed with 0.01% Lipofectamine™ 2000 (Invitrogen) according to the manufacturer's instructions and then microinjected with an ASTP (application solution transgenic platform) micromanipulator (Leica, Wetzlar, Germany) into the amniotic cavity of the developing embryos at early headfold stage (E7.5). Sham-operated controls were injected with Lipofectamine alone without siRNA. Embryos were placed in a whole embryo culture roller incubator (BTC Engineering, Cambridge, UK) and allowed to develop for 72 h in 100% heat-inactivated rat serum with a continuous flow of gas mixtures (20Chan L.Y. Chiu P.Y. Siu N.S. Wang C.C. Lau T.K. Reprod. Toxicol. 2002; 16: 841-844Crossref PubMed Scopus (19) Google Scholar). Detection of Apoptotic Cells (TUNEL Assay)—After in situ hybridization, the siRNA-treated embryos and corresponding controls were fixed in 4% paraformaldehyde, washed at 4 °C in PBS, dehydrated, embedded in paraffin wax, and cut into 5-μm sections. Apoptotic cells were stained by the standard TUNEL technique. For this purpose, the tissue sections were incubated with the Tdt enzyme and conjugated with anti-digoxigenin peroxidase (Chemicon) for color development. Expression of GPx4 in Adult Murine Brain—Homozygous GPx4 knock-out mice die in utero during a time window that is important for early development of the central nervous and cardiovascular systems. To investigate whether or not retardations in brain development might contribute to embryonic lethality, we first tested expression of GPx4 in adult murine brain. Immunohistochemical staining with an anti-GPx4 antibody, which was raised against the pure full-length recombinant human enzyme (Sec-Cys mutant expressed in Escherichia coli) indicated that cortical neurons express GPx4 at high levels (Fig. 1). Furthermore, pyramidal neurons were highly immunopositive for GPx4. Interestingly, both the neuronal cell bodies and the corresponding dendrites were stained positive. Moreover, leptomeninges expressed GPx4, whereas white matter regions, glia cells, and astrocytes largely remained immune negative (Fig. 1). Since our antibody does not distinguish between the different GPx4 isoenzymes, isoform-specific differences could not be investigated. These data indicate for the first time expression of GPx4 protein in cortical neurons, suggesting a biological role of the enzyme for neuronal function and/or neuron development. Expression Kinetics of GPx4 Isoforms during Murine Embryogenesis—If GPx4 is important for neuronal function, systemic silencing of the enzyme during embryogenesis might contribute to premature lethality. To obtain evidence for GPx4 expression during the time course of murine embryogenesis, we first profiled expression kinetics of GPx4 mRNAs by semiquantitative RT-PCR (Fig. 2) and found that mRNA species encoding for all three GPx4 isoforms were continuously expressed. At late gestation (E14.5 and later), expression of m- and n-GPx4 mRNA was apparently somewhat reduced, and a similar decrease was observed for m+c-GPx4 (Fig. 2A). Exact quantification of the copy numbers of the different mRNA species indicated that during midgestation, c- and m-GPx4 were expressed at similar levels. From Fig. 2B, it can be seen that about 100-200 copies of the c-GPx4 mRNA and 100-200 copies of the m-GPx4 were found per 103 copies of GAPDH mRNA. In contrast, expression of n-GPx4 was significantly lower (30-60 mRNA copies per 103 copies of GAPDH mRNA). This expression kinetics indicates that at the peak of GPx4 expression during murine embryogenesis, almost 500 copies of GPx4 mRNA species were present per 1000 copies of GAPDH mRNA. Such values may be considered high level expression. Interestingly, expression of the n-GPx4 (Fig. 2B, inset) and m-GPx4 suddenly drops down at late gestation (between E14.5 and E17.5) and remains low until birth. In contrast, the expression levels of the c-GPx4 isoform remain unchanged. Taken together, these data indicate profound regulatory events in GPx4 expression during late embryogenesis. Localization of phGPx Isoforms in Murine Embryos—To investigate the intraembryonic distribution of GPx4 expression, we performed in situ hybridization using isoformspecific probes for the nuclear (n-GPx4) and the mitochondrial (m-GPx4) isoenzyme (Figs. 3 and 4). In addition, a probe (m+c+n-GPx4) was employed to test total GPx4 expression (Scheme 1C). At early embryonic development (Fig. 3; early bud gastrula E6.5 to late primitive streak embryos E7.5), a similar expression pattern of total GPx4 (m+c+n-GPx4) and m-GPx4 was observed (Fig. 3, A, C, D, and F). The most intense hybridization signals were seen in the ectoplacental cone, the extraembryonic ectoderm, the exocoelomic cavity, and the embryonic epiblast. In this developmental stage, it was impossible to decide whether the m- or the c-GPx4 were dominant at these sites. Using the n-GPx4-specific probe, the hybridization signals were somewhat less intense, suggesting low level expression of this isoform at early developmental stages. At E6.5, n-GPx4 (Fig. 3B) was mainly localized in the yolk sac endoderm, the exocoelomic cavity, and embryonic endoderm (amnion). At a later stage (E7.5), a similar expression pattern was observed, but the signals became even less intense (Fig. 3E). These data suggest that m-GPx4 and c-GPx4 (represented by the probe m+c+n-GPx4) may be of functional relevance during early gastrulation, mainly in ectoplacental and ectodermal tissues. In contrast, the nuclear isoform appears to be involved mainly in endodermal development. In addition, we found strong GPx4 expression (mitochondrial and/or cytosolic isoforms) in the primitive streak (arrows in Fig. 3, D and F) but not in embryonic mesoderm, which prompted us to conclude a function of m- and/or c-GPx4 in early embryonic lineage development.FIGURE 4Expression of GPx4 isoforms in somite stage murine embryos. In situ hybridization of embryo preparations was performed as described under “Materials and Methods.” In each panel, staining with a sense probe (left, negative control) and an antisense probe (right) is shown. The dark purple stains represent the positive in situ hybridization signals. A-C, E8.0 embryos with five or six pairs of somites; D-F, E8.5 embryos with 9-11 pairs of somites; G-I, E9.5 embryos with 20-25 pairs of somites; J-L, E10.0 embryos with 30-35 pairs of somites. Scale bar, 300 μm(A-F) and 800 μm(G-L). ec, exoplacental cone; xc, exocoelomic cavity; am, amnion; hf, headfold; rnt, rostral neural tube; cnt, caudal neural tube; fb, forebrain; mb, midbrain; hb, hindbrain; ov; optic vesicle.View Large Image Figure ViewerDownload Hi-res image Download (PPT) At late embryonic development (Fig. 4; early somite E8.0 to midsomite stages E10.0), total GPx4 expression (indicated by m-GPx4 and m+c+n-GPx4 probes) continues in extraembryonic ectoderm, but now we also detected hybridization signals in the developing embryos (Fig. 4, A, C, D, and F). Expression of the mitochondrial isoform (m-GPx4) was particularly intense in the head fold region of E8.0 embryos (Fig. 4A) but also throughout the rostral to caudal neural tube at E8.5 (Fig. 4D). A similar hybridization pattern was observed when the m+c+n-probe was used (Fig. 4, C and F). Because of the cross-hybridization properties of the latter probe, it was not possible to distinguish which of the two isoforms (c-GPx4 or m-GPx4) contributed more or less to the total signal intensity. The signals caused by the n-GPx4 probe in the endodermal layers were rather weak (Fig. 4, B and E). Expression of m- and/or c-GPx4 in the differentiating neuroepithelium of the rostral neural tube was extended to the forebrain, midbrain, and hindbrain at later developmental stages E9.5 and E10.0 (Fig. 4, G, I, J, and L). This stage-dependent expression pattern suggested a role of these isoforms in embryonic brain development. The intensity of the hybridization signals obtained with the probe for the n-GPx4 was rather weak in the developing head fold region and in the tail (Fig. 4, H and K). Expression of Different GPx4 Isoforms during Embryonic Development of Various Organ Systems—Abundant expression of GPx4 isoforms in the developing neuroepithelium (Fig. 4) prompted us to follow the kinetics of GPx4 expression during embryonic cerebral development in more detail. From Fig. 5, it can be seen that cerebral expression of the GPx4 isoforms is characterized by a unique profile (Fig. 5, top). The mRNA concentration for the cytosolic isoform does not undergo major alterations during perinatal brain development (E10.5-N3). In fact, it remains constant at a level of about 100 mRNA copied per 103 copies of GAPDH mRNA. In contrast, the m-GPx4 was expressed at higher levels until E13.5 but then dropped down to lower levels during E14.5-E17.5. Toward birth, expression levels recover, reaching copy numbers of up to 200 per 103 GAPDH copies. A similar transient drop was observed for n-GPx4 during these developmental stages. In contrast, such regulatory kinetics were not observed for other organs, such as the heart (Fig. 5, bottom). Here, we observed constant expression levels o
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