Human Molecular Genetics, 2000, Vol. 9, No. 8 1219-1226
© 2000 Oxford University Press
Inactivation of the Friedreich ataxia mouse gene leads to early embryonic lethality without iron accumulation
nig§Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), CNRS/INSERM/Université Louis Pasteur, Hôpitaux Universitaires de Strasbourg, 1 rue Laurent Fries BP 163, 67404 Illkirch, France
Received 21 January 2000; Revised and Accepted 7 March 2000.
DDBJ/EMBL/GenBank accession nos AF223568 and AF223569.
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ABSTRACT |
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Friedreich ataxia (FRDA), the most common autosomal recessive ataxia, is caused in almost all cases by homozygous intronic expansions resulting in the loss of frataxin, a mitochondrial protein conserved through evolution, and involved in mitochondrial iron homeostasis. Yeast knockout models, and histological and biochemical data from patient heart biopsies or autopsies indicate that the frataxin defect causes a specific iron–sulfur protein deficiency and mitochondrial iron accumulation leading to the pathological changes. Affected human tissues are rarely available to further examine this hypothesis. To study the mechanism of the disease, we generated a mouse model by deletion of exon 4 leading to inactivation of the Frda gene product. We show that homozygous deletions cause embryonic lethality a few days after implantation, demonstrating an important role for frataxin during early development. These results suggest that the milder phenotype in humans is due to residual frataxin expression associated with the expansion mutations. Surprisingly, in the frataxin knockout mouse, no iron accumulation was observed during embryonic resorption, suggesting that cell death could be due to a mechanism independent of iron accumulation.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Friedreich ataxia (FRDA), the most common hereditary ataxia, is characterized by progressive ataxia of the limbs, loss of deep tendon reflexes and of vibration sense in the lower limbs, cerebellar dysarthria and pyramidal signs (1). Most patients have hypertrophic cardiomyopathy, while skeletal deformities (scoliosis and pes cavus) are found in two-thirds of cases, and diabetes or carbohydrate intolerance in a third of patients (2–5). The first pathological changes are progressive degeneration of large sensory neurons in the dorsal root ganglia, followed by deterioration of the posterior columns, spinocerebellar and pyramidal tracts of the spinal cord, and atrophy of the large sensory fibers in peripheral nerves (6). Mild degenerative changes are also observed in the dentate nucleus of the cerebellum. Neuronal degeneration is considered to be the cause of most of the symptoms, including the skeletal deformities. However, the cardiomyopathy and diabetes are most likely due to independent sites of primary degeneration.
FRDA is due to a deficiency of frataxin, a 210 amino acid nuclear-encoded mitochondrial protein highly conserved through evolution (from 
-purple bacteria to human) (7–9). Human and mouse studies have shown that frataxin is expressed in heart, dorsal root ganglia and pancreas, in keeping with the pattern of degeneration observed in the disease, as well as in liver and skeletal muscle. Tissue-specific expression of frataxin was reported during mouse development after embryonic day (E) 10.5 (9,10).
FRDA is most commonly caused by a large GAA triplet repeat expansion within the first intron of the gene encoding frataxin (7). Ninety-six percent of patients are homozygous for GAA repeat expansions, while the remaining cases are compound heterozygotes for a GAA expansion and a point mutation altering or truncating the coding region of the gene. Western blot analysis of protein extracts from patients has shown a reduction in frataxin levels, indicating, in combination with occurrence of truncating point mutations, that the disease is due to a loss of function of the FRDA gene (11). Analyses of frataxin mRNA from patients homozygous for the GAA expansion have demonstrated that the reduction of expression is due to an inhibition of the transcriptional machinery along the expanded sequence, rather than to a splicing defect (12,13).
Frataxin is associated with mitochondrial membranes and is involved in mitochondrial iron homeostasis. Deletion of the frataxin yeast homologue (YFH1) results in mutant strains that show a growth defect on fermentable carbon sources, accumulate mitochondrial iron and exhibit a high sensitivity to oxidative stress induced by oxidant agents such as hydrogen peroxide (H2O2) or iron, as well as a reduction in oxidative phosphorylation (OXPHOS) (9,14–16). Such a mitochondrial iron accumulation is consistent with the observation of iron accumulation in some myocardial fibres in post-mortem examination of hearts from 15 FRDA patients (17). Furthermore, biochemical studies of endomyocardial biopsied tissue from two unrelated FRDA patients revealed deficient activities of the iron–sulfur (Fe-S) cluster-containing subunits of mitochondrial respiratory complexes I, II and III, as well as a deficiency of both mitochondrial and cytosolic aconitases, the latter being an Fe-S protein involved in iron homeostasis (18). These Fe-S proteins are extremely sensitive to oxidative stress. The most commonly accepted hypothesis is that the primary effect of the absence of frataxin is mitochondrial iron accumulation; iron being a potent oxidizing agent through the Fenton reaction, its accumulation generates superoxide (O·)-free radicals that destabilize complexes I, II and III of the respiratory chain, leading to oxidative stress and cell death. A recent study in yeast has demonstrated that the presence of iron chelator in the culture media restores normal mitochondrial iron levels and normal oxidative respiration in 
YFH1 yeast strain, but that the activity of aconitase remains low (19). This suggests that the reduction in the activity of the respiratory chain complexes is a consequence of mitochondrial iron accumulation, while the reduced aconitase activity is directly linked to frataxin deficiency. The mitochondrial defect may explain why degeneration is restricted to some neurons and to cardiomyocytes, for which energy production is exclusively dependent on the OXPHOS system.
The homologous mouse gene (Frda) is predicted to encode a 207-amino acid protein showing 73% identity to the human frataxin, with very high identity in exons 3–5 and less conservation in the first two exons (9). In order to investigate the function of frataxin and the mechanism of the disease, and to develop a system for testing antioxidant and iron chelator therapies, we generated a mouse model by inactivation of the Frda gene via homologous recombination. We found that homologous inactivation of Frda leads to early embryonic lethality, demonstrating an important role for frataxin during mouse development. The embryonic cell death occurs both by apoptosis and necrosis. Surprisingly, no iron deposits were observed in degenerating embryos, suggesting that cell death is not the consequence of abnormal iron accumulation.
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RESULTS |
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Cloning and targeted disruption of the mouse frataxin gene
A phage genomic library from the 129/sv-ter mouse strain was screened with the human cDNA, and several clones corresponding to two highly homologous pseudogenes were obtained. Major differences between the mouse cDNA (9) and the pseudogenes (EMBL/GenBank/DDBJ accession nos AF223568 and AF223569) were found over the 3'-untranslated region (UTR) and included several substitutions and a 35 bp deletion (data not shown). A specific probe of the 3'-UTR of the mouse cDNA was therefore used for an additional screen of the same genomic library. Two overlapping phage clones containing exons 4 and 5, and exon 5 only, respectively, were obtained. A detailed restriction map was made (Fig. 1A) and intron–exon boundaries were sequenced.
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We chose to delete frataxin exon 4 by homologous recombination (Frdadel4) because the corresponding part of the protein is probably functionally important, as this sequence is highly conserved through evolution and contains various missense mutations in compound heterozygous patients. Furthermore, deletion of the 98 bp exon 4 results in a frameshift of exon 5 coding sequence, leading to a severe truncation of frataxin.
We constructed a targeting vector by replacing a 3.7 kb genomic fragment containing exon 4 and its flanking intronic sequences by a PGKneo(polyA) cassette (Fig. 1A). After electroporation of the vector in embryonic stem (ES) cells, four out of 271 G418-resistant cell clones were correctly targeted without 3' rearrangement or additional vector integration.
Three ES cell clones were injected into host blastocysts of the C57Bl/6J strain and male chimeras were obtained for two of them (MI95 and MI97). Two independent lines of heterozygous mice were generated (Fig. 1b) in both pure 129/sv-ter background and mixed 25% 129/sv–75% C57Bl/6J background obtained after two backcrosses with C57Bl/6J mice. In addition, Cre-mediated excision of the neoR cassette was obtained for one line of heterozygous mice. In both genetic backgrounds, the heterozygous mice were indistinguishable from their wild-type littermates in physical and behavioural phenotype.
Early embryonic lethality in nullizygous frataxin mice
Intercrosses of heterozygotes from the three lines in both genetic backgrounds failed to produce homozygous frataxin –/– progeny. In 24 pregnancies (nine from lines with the neoR cassette and 15 without the neoR cassette), the genotypes of surviving offspring showed that 53 were wild type (+/+) and 83 were heterozygous for the deleted (Frdadel4) allele (+/–) (Table 1). The complete absence of nullizygous mutants, the wild type and heterozygous progeny approximating a 1:2 ratio, and the small litter sizes (mean, 5.7) suggested that homozygosity for the frataxin null mutation resulted in embryonic lethality.
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To determine the stage of embryonic death, pregnant females from matings between heterozygous (+/–) mice were sacrified between embryonic days (E) 7.5 and E9.5, and the embryos were dissected and genotyped using a multiplex PCR assay (Fig. 2). All morphologically normal embryos were wild type (+/+) or heterozygous (+/–) for the deleted Frdadel4 allele (Table 1). Approximately a third of the embryos (29/89) showed gross morphological abnormalities. At E7.5, the abnormal embryos were much smaller than normal littermates and consisted of a small spherical mass, surrounded by adherent haemorrhagic tissue (Fig. 2). At E8.5 and E9.5, the abnormal embryos were reduced to small masses of haemorrhagic tissue, indicating that they were being resorbed (data not shown). PCR genotyping confirmed the presence of homozygous (–/–) mutants for the Frdadel4 allele among the abnormal E7.5 and E8.5 embryos (Table 1), although some of these were scored as heterozygotes, probably as a result of maternal tissue contamination due to the difficulty in separating the haemorrhagic envelope from the abnormal embryo. Maternal infiltration can also explain the heterozygous (+/–) genotyping of the E9.5 resorbed embryos (Table 1).
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Early post-implantation degeneration of frataxin null embryos
To further analyse the Frda null phenotype, we examined histological sections of embryos from heterozygote intercrosses, collected in utero from E4.5 to E8.5. No abnormality was detected in E4.5 and E5.5 embryos (n = 8 and n = 10, respectively), with the exception of one empty decidua at E5.5 (not shown). At E6.5, all 22 embryos examined from two pregnancies exhibited normal egg-cylinder morphology and differentiation of the ectodermal and endodermal cell layers. However, two of these embryos were growth-retarded, especially at the level of the embryonic portion of the egg-cylinder (data not shown).
At E6.75, analysis of 15 embryos from two pregnancies revealed one empty decidua, three slightly abnormal and growth-retarded embryos (not shown), and one highly abnormal embryo which had started to degenerate, although its embryonic, extra-embryonic and ectoplacental regions could still be identified (compare Fig. 3A and B). Its parietal endoderm had detached from the trophoblast, loose cells (likely corresponding to degrading visceral endoderm) spread in the collapsed yolk cavity, and no proamniotic cavity was visible. The extra-embryonic portion was particularly narrow and contained abnormal cells with pycnotic nuclei (Fig. 3B).
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Thirty embryos from four pregnancies were analysed at E7.5. Decidual vacuity was observed in four cases. Five embryos were abnormal, with different degrees of severity. Four of these were much smaller than their littermates and appeared to have stopped their development at the egg-cylinder or early gastrulation stage (compare Fig. 3C and D). The embryonic germ layers (ectoderm, mesoderm and endoderm) could be histologically identified. However, the extra-embryonic region was particularly reduced and lacked distinct exocoelomic and chorionic cavities. Massive blood infiltration was seen between the parietal endoderm and trophoblast cells (Fig. 3D). Cells with pycnotic nuclei were seen throughout the embryo, but were most abundant in the extra-embryonic portion. One embryo was reduced to a small mass of abnormal cells, most of them with pycnotic nuclei (compare Fig. 3E and F).
At E8.5, three out of 19 embryos examined were almost or entirely resorbed, such that the embryonic cavity was filled with exudate containing maternal lymphocytes and red blood cells (compare Fig. 3G and H).
Apoptotic and necrotic cell death in frataxin null embryos
The presence of pycnotic nuclei with condensed chromatin in the abnormal E6.75 and E7.5 embryos suggested that abnormal cell death occurs by apoptosis in the Frda null embryos. To confirm this hypothesis, TUNEL (TdT-mediated dUTP-biotin nick-end labelling) assays were performed to detect the fragmented DNA characteristic of apoptotic cells. At E6.75, a number of TUNEL-positive cells were seen in the extra-embryonic region of the abnormal embryos, whereas labelled cells were rare or undetectable in normal embryos (data not shown). Furthermore, TUNEL-positive cells were abundant in the extra-embryonic region and appeared as clusters in the embryonic portion of the E7.5 abnormal embryos (data not shown).
The presence of apoptotic cells was further confirmed by electron microscopy analysis of E6.75 embryos. Analysis of presumptive Frda null embryos (n = 4), identified by their abnormal appearance on preparative semi-thin sections, showed cells with chromatin condensation characteristic of apoptosis (Fig. 4A). However, most cells showed nuclear and cytoplasmic features of necrosis (Fig. 4A).
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Absence of mitochondrial iron accumulation
To test for the presence of iron deposits, as previously found in cardiac muscle from FRDA patients and in yeast mutants of the frataxin homologue, we performed Perls staining of abnormal (presumptive Frda null) embryos. No iron-specific staining was observed in one E6.5 embryo, two E6.75 embryos, four E7.5 embryos and one E8.5 embryo analysed. Futhermore, no evidence of dense granules, indicative of iron deposits, was seen in the four resorbed E6.75 embryos analysed by electron microscopy, either in mitochondria, or in the cytosol (Fig. 4B).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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We have generated a mouse model for Friedreich ataxia by targeted disruption of the frataxin gene (Frda). Our construct is likely to result in a complete loss of frataxin function, since deletion of exon 4 causes a frameshift of exon 5, leading to a truncated product lacking more than half of the frataxin protein sequence. Moreover, this missing half contains a 27-amino acid segment, which is the most conserved domain of frataxin (with 22 of 27 amino acids identical between human and yeast sequences) (7).
The early embryonic lethality of homozygous knockout mice indicates that frataxin is an essential protein in mammals. This is in contrast to the phenotype of the FRDA patients, where frataxin mutations are compatible with normal development and survival into adulthood. Unlike mice, humans may have compensatory protection against the lack of frataxin, such as additional antioxidant defences, since oxidative stress has been implicated in the pathogenesis of the disease (14,16). However, a striking difference between the FRDA patients and our mouse mutants is the presence in FRDA patients of at least one copy of the intronic GAA triplet repeat expansion as the disease-causing mutation. Small expansions are known to correlate with a milder and later disease (2–5) and FRDA expansions allow the synthesis of residual levels of frataxin in a repeat length dependent manner (11). In lymphoblastoid cell lines, large expansions allow at least 3–4% of normal frataxin expression levels (11). It is therefore plausible that even very low residual frataxin levels allow the viability of the FRDA patients and the late, slowly progressive nature of the human disease. The absence of known FRDA diagnosed patients with two truncating point mutations might also be explained by a more severe presentation or by early lethality with null mutation (complete frataxin loss). However, such patients are expected to be very rare [less than four in 10 000 patients (20,21)] and may not have been ascertained yet. A similar discrepancy between a human disease and a knockout mouse model has been reported for spinal muscular atrophy, where homozygous mouse mutants are embryonic lethal before implantation (22). In this case, survival of the patients until after birth is explained by residual levels of the survival of motorneuron (SMN) protein expressed from a duplicated gene, which is absent from the mouse genome.
At E7.5, the Frdadel4 (–/–) mutant embryos were found to be grossly abnormal. The early resorption of the Frdadel4 (–/–) mutant embryos makes them difficult to analyse and, in particular, hampers the determination of the genotype in parallel with morphological analyses. As a consequence, study of the (–/–) embryos was not possible prior to the appearence of morphological alterations, at E6.75. At this stage, however, the proportion of abnormal embryos was ~25%, as expected for an autosomal recessive phenotype. At E6.75, embryos enter gastrulation, with the formation of the primitive streak at one edge of the embryonic ectoderm that marks the posterior end of the future embryo. Gastrulation is characterized by a high rate of cell division and migration allowing the differentiation of three layers: ectoderm, mesoderm and endoderm. In our model, histological analysis of the null mutants suggest that developmental arrest occurs 1–2 days after blastocyst implantation (E4.5), at the egg-cylinder stage, followed by rapid embryonic resorption during the gastrulation period. During resorption, the ectodermal, mesodermal and endodermal cell layers can still be identified, suggesting that the frataxin inactivation does not affect a particular cell type. However, the alterations seem more severe in the extra-embryonic region of the conceptus, where size reduction and signs of cell death first occur, before extending to the embryonic portion. This region, where the embryo interacts with maternal tissues, is characterized by high metabolic activity (23) and appears to be particularly sensitive to frataxin deficiency. It is not known whether normal development until E6.25 is related to an absence of need for frataxin, or to the presence of residual maternal frataxin. However, our results suggest that frataxin expression even at very low levels is essential for embryonic development as early as E6.5.
The present embryonic lethality is reminiscent of observations of embryonic resorption in female rats fed a vitamin E-deficient diet for several months (24). Vitamin E is a well known antioxidant which protects biological membranes against lipid peroxidation, and patients with isolated ataxia with vitamin E deficiency (AVED) exhibit a clinical phenotype highly similar to FRDA, suggesting that the two diseases share pathological pathways. Here again, the severe fetal resorption in female rats under low vitamin E diet could be explained by an almost complete absence of vitamin E, while the comparatively mild course of the disease in AVED could be due to residual vitamin E obtained from nutrition.
In the light of the rapid and severe cell death of Frdadel4 (–/–) embryos, the absence of clear iron accumulation in degenerating cells is somewhat unexpected, since it was hypothesized to be the primary cause of FRDA pathology (14,16). The recent results of Isaya et al. (25), indicating that frataxin plays a ferritin-like role within the mitochondria, might reconcile our results with frataxin function. Cell death in our model might be the consequence of lack of mitochondrial iron stores rather than the consequence of mitochondrial iron accumulation. Iron–frataxin particles might serve as a reservoir for Fe-S cluster biogenesis, which could be impaired in the absence of frataxin. This would explain the specific Fe-S protein deficiency seen in FRDA but not in other mitochondrial diseases (18), the absence of the restoration of aconitase activity in iron chelator-treated YFH1 mutants (19) and the specific mitochondrial iron accumulation seen in other yeast mutants of the Fe-S cluster biogenesis (Atm1, Nfs1, Ssq1) (26,27)
In order to circumvent the early embryonic lethality, we are constructing a conditional knockout model, which should allow us to induce nullisomy for frataxin in a tissue controlled or inducible manner and to establish a cell model for further analysis of the mechanism of the disease.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Construction of the targeting vector
A phage genomic library from the 129/sv-ter mouse strain was screened using the mouse frataxin 3' untranslated sequences (9) as a probe. The targeting vector (Fig. 1A), constructed in pBluescript SK+ from subcloned mouse genomic fragments, contained a 1.9 kb XbaI–BamHI genomic fragment 5' to exon 4, followed by a 2.0 kb phosphoglycerate kinase promoter-neomycin resistance gene (PGK-neo)–poly(A) cassette flanked by loxP sites in the same orientation as the frataxin gene, and a 6.3 XhoI–XhoI genomic fragment downstream from exon 4. A positive control vector for PCR analysis of recombinant clones was made by cloning into pBluescript SK+ a 2.4 kb NotI (in phage polylinker)–BamHI fragment and the neomycin cassette, therefore containing 500 additional base pairs 5' to the targeting construct, which contains the P1 primer sequence (see below).
Gene targeting in ES cells and generation of chimeras
Following linearization at the unique NotI site, 10 µg of targeting vector were electroporated into 129/Sv-ter ES cells grown on feeder layers (28). After G418 selection (150 µg/ml), DNA of resistant clones was analysed by PCR using the frataxin primer P1: 5'-TTACAATGTGGGGGTATTTCTGA-3' and the neomycin primer P2: 5'-CGCCTCCCCTACCCGGTAGAATTC-3' generating a 2.0 kb fragment for the correctly targeted ES cells. The positive control vector was integrated randomly into mouse cells which were used for PCR analysis, demonstrating the presence of the 2.0 kb fragment. To generate chimeras, targeted ES cells were injected into C57BL/6J host blastocysts that were transferred into pseudo-pregnant females. Male chimeras were selected by coat colour and crossed with C57BL/6J and 129/Sv-ter females to obtain germline transmission of the targeted Frdadel4 allele.
Genotype analysis
Genomic DNA was prepared from tail tips of mice, yolk sac of E9.5 embryos or whole E7.5 and E8.5 embryos. Multiplex PCR for mouse genotyping (Fig. 1A) included a common forward primer (P3: 5'-CTGTTTACCATGGCTGAGATCTC-3') and two reverse primers specific for the wild type (P4: 5'-CCAAGGATATAACAGACACCATT-3') and mutant (P2: neo) Frda alleles. The PCR products were 520 bp for the wild type allele and 245 bp for the knockout allele. PCR amplification was performed in 25 µl reactions containing 0.2 µg of genomic DNA from tails or 2.5 µl of PCR lysis buffer containing embryos, 20 pmol (0.8 µM) of primer P3, 10 pmol of primer P4, 2.5 pmol of primer P2, 10 mM Tris–HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 200 µM of each dNTP, 5% DMSO and 1 U of Ampli-Taq DNA polymerase (Sigma, St Louis, MO). Cycling conditions were 30 cycles with the following steps: 94°C for 20 s, 54°C for 20 s, 72°C for 20 s in a PTC-100 (MJ Research Inc., Watertown, MA) machine. PCR fragments were separated by electrophoresis on a 2% agarose gel. PCRs derived from embryos were subsequently transferred to Nytran N+ membrane (Schleicher & Schuell, Keene, NH) and hybridized with an internal [-32P]ATP end-labelled oligoprobe (P5: 5'-ATGAGCTTCAGGCATTCATC-3') in 10% formamide at 42°C, washed in 6x SSC, 0.1% SDS at 50°C, and autoradiographed.
Genomic DNA Southern-blot was performed by standard procedure (29). The 5' internal probe (Fig. 1A) was the 2.4 kb NotI–BamHI fragment in intron 3 (targeting vector left arm). The 2.3 kb 5' external probe was generated by PCR of genomic mouse DNA using a primer in frataxin exon 3 (P6: 5'-TGGCCGAGTTCTTTGAAGACCTCGCAGAC-3') and the reverse primer in the 500 bp fragment upstream from the targeting construct (P7: 5'-ACCACACCAGGGACAGCTC- TCCTAACCACT-3') (Fig. 1B).
Histological analyses
The deciduae from gravid uteri were dissected, fixed overnight at 4°C in 4% paraformaldehyde, 0.1 M phosphate buffered saline pH 7.3 (PBS), washed three times (5 min each) in PBS–0.1% Tween 20, dehydrated in graded ethanol series, cleared and embedded in paraffin wax. The paraffin blocks were cut at 7 µm and placed successively on three series of slides per embryo. The first series of slides was stained with haematoxylin and eosin, while the second and the third were used for TUNEL analysis and Perls staining, respectively.
TUNEL analysis. After deparaffinization and rehydration, the slides were treated for 10 min with 20 µg/ml proteinase K in 0.5 M Tris pH7.5–50 mM EDTA pH 8, and bleached in 2% H2O2 to block endogenous peroxidase activity. TUNEL reactions were performed with the APOTag In Situ Apoptosis Detection Kit peroxidase (Appligene-Oncor, Illkirch, France) according to the manufacturer’s protocol, and revealed using diaminobenzidine as substrate (DAB substrate kit for peroxidase, Roche Molecular Biochemicals, Mannheim, Germany). The slides were counterstained with 0.01% safranine. Negative control reactions consisted in the omission of the TdT enzyme in corresponding slides.
Perls Fe3+ staining. After deparaffinization and rehydration, the slides were incubated for 30 min in 1% potassium ferrocyanide, 0.12 N HCl, and then counterstained with 0.01% safranine.
Electron microscopy
E6.75 deciduae were fixed by immersion in 2.5% glutaraldehyde in PBS for 48 h at 4°C, rinsed in PBS, post-fixed in 1% osmium tetroxide in the same buffer for 2 h at 4°C, dehydrated and embedded in Epon. Embryos were localized and characterized with the light microscope on 2 µm sections stained with toluidine blue. Ultrathin sections from selected areas were stained with uranyl acetate and lead citrate, and examined with a Philips 208 electron microscope, operating at 80 kV.
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ACKNOWLEDGEMENTS |
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We wish to thank J.-L. Mandel for fruitful discussions and comments, P. Kastner for the PGK-Neo cassette, L. Reutenauer, T. Ding, M. Digelmann, M. Gendron and E. Blondelle for technical support. This work was supported by funds from the Association Française contre les Myopathies (AFM), the Muscular Dystrophy Association of America (MDA), the CNRS (grant no. 96084), and the Human Frontier Science Program (HFSP) (to M.K.) and by the INSERM and the Hôpitaux Universitaires de Strasbourg. M.C. is supported by the INSERM, H.P. by the Association de Recherche sur le Cancer and H.K. by the AFM and the Association Française de l’Ataxie de Friedreich.
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FOOTNOTES |
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+ Present address: Neurogenetics Branch, NINDS, NIH, Bethesda, MD, USA
§ To whom correspondence should be addressed. Tel: +33 3 88 65 33 99; Fax: +33 3 88 65 32 46; Email: mkoenig@igbmc.u-strasbg.fr
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REFERENCES |
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