Targeted Disruption of the Murine Dihydrolipoamide Dehydrogenase

Proc. Natl. Acad. Sci. USA Vol. 94, pp. 14512–14517, December 1997 Developmental Biology

Targeted disruption of the murine dihydrolipoamide dehydrogenase gene (Dld) results in perigastrulation lethality (gene targeting͞embryonic lethal mutation͞embryonic metabolism)

MARK T. JOHNSON*†,HSIN-SHENG YANG‡,TERRY MAGNUSON†, AND MULCHAND S. PATEL‡§

Departments of *Biochemistry and †Genetics, Case Western Reserve University School of Medicine, Cleveland, OH 44106; and ‡Biochemistry Department, School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, NY 14214

Edited by Shirley M. Tilghman, Princeton University, Princeton, NJ, and approved November 3, 1997 (received for review July 14, 1997)

ABSTRACT The Dld gene product, known as dihydroli- well as a source of one-carbon units for numerous metabolic poamide dehydrogenase or the E3 component, catalyzes the pathways. oxidation of dihydrolipoyl moieties of four mitochondrial The majority of studies investigating early murine embry- multienzyme complexes: pyruvate dehydrogenase, ␣- onic metabolism thus far have examined the metabolism in ketoglutarate dehydrogenase, branched-chain ␣-ketoacid de- vitro of exogenous substrates, particularly glucose. In mouse hydrogenase, and the glycine cleavage system. Deficiency of E3 embryos and other mammalian systems, it has been shown that activity in humans results in various degrees of neurological the fate of consumed glucose varies during development. In the dysfunction and organic acidosis caused by accumulation of preimplantation period, murine embryos shift from oxidative branched-chain amino acids and lactic acid. In this study, we metabolism to glycolytic metabolism of glucose (4). Embryos have introduced a null mutation into the murine Dld gene appear to continue to rely on glycolysis in the period imme- (Dldtm1mjp). The heterozygous animals are shown to have diately after implantation into the uterine wall with virtually all approximately half of wild-type activity levels for E3 and all glucose, greater than 95%, taken up in culture is converted to affected multienzyme complexes but are phenotypically nor- ؊ ؊ lactate (5). The importance of glucose metabolism during the mal. In contrast, the Dld / class dies prenatally with appar- early postimplantation period in vivo has been established by ent developmental delay at 7.5 days postcoitum followed by ؊ ؊ the observation that embryos lacking the glycolytic enzyme resorption by 9.5 days postcoitum. The Dld / embryos cease glucose-6-phosphate isomerase die within several days after to develop at a time shortly after implantation into the uterine implantation, coinciding with the disappearance of maternally wall when most of the embryos have begun to gastrulate. This derived glucose-6-phosphate isomerase (6, 7). In the following null phenotype provides in vivo evidence for the requirement days of postimplantation development, in vitro studies have of a mitochondrial oxidative pathway during the perigastru- demonstrated a gradual up-regulation of oxidative metabolism lation period. Furthermore, the early prenatal lethal condi- of glucose, although questions addressing the significance of tion of the complete deficiency state may explain the low these changes and their relevance to embryonic metabolism in incidence of detectable cases of E3 deficiency in humans. vivo remain unanswered (5, 8). Direct evidence for the metabolism of amino acids during murine embryogenesis has not The product of Dld assembles into a dimeric flavin-requiring been established, but studies have shown that the addition of oxidoreductase that catalyzes the oxidation of dihydrolipoyl amino acids to culture medium may improve growth and͞or moieties of several noncovalently associated proteins (1). The viability of preimplantation embryos (9). Dld gene product performs this catalytic function for three The generation of a complete deficiency state of E3 activity mitochondrial multienzyme ␣-ketoacid dehydrogenase com- by gene targeting should provide a means to assess the plexes: pyruvate dehydrogenase complex (PDC), ␣-ketogluta- importance of a combination of oxidative metabolic pathways rate dehydrogenase complex (KDC), and branched-chain ␣- during murine development. Although no mutations have ketoacid dehydrogenase complex (BCKDC) (2). Because this been identified that affect E3 activity or any of the E3- reaction is the third step in catalysis of the ␣-ketoacid dehy- dependent complexes in mice, there are several reports for drogenase complexes, this enzyme is often referred to as the each deficiency in humans. Patients with E3 deficiency suffer E3 component. There also is evidence indicating that the Dld from various degrees of neurologic impairment that are often gene product participates in the glycine cleavage system (GCS) lethal within the first few years of life. To date, 10 cases of E3 where it has been traditionally referred to as the L protein (3). deficiency, eight cases of KDC deficiency, and more than 100 The E3-dependent complexes catalyze key regulatory reac- cases of PDC deficiency have been reported in the literature tions in four different pathways of intermediary metabolism. (10–15). All of these cases have been diagnosed in the peri- PDC is essential for the oxidative metabolism of glucose and natal period or later, and no cases have been shown to select amino acids because it links the glycolytic and tricar- completely lack enzymatic activity, suggesting the importance boxylic acid pathways through the decarboxylation of pyruvate of PDC and KDC during prenatal development. In contrast, to produce acetyl-CoA. KDC catalyzes one of the enzymatic the limited biochemical͞molecular characterizations of cases steps of the tricarboxylic acid cycle, the central oxidative of BCKDC and GCS deficiencies suggest that complete defi- pathway for numerous metabolites, and a significant supplier ciency states of these complexes are compatible with survival of reducing equivalents to the electron transport chain. BCKDC catalyzes an irreversible step in the catabolism of This paper was submitted directly (Track II) to the Proceedings office. three essential amino acids, leucine, isoleucine, and valine. Abbreviations: PDC, pyruvate dehydrogenase complex; KDC, ␣- GCS is involved in the synthesis and degradation of glycine as ketoglutarate dehydrogenase complex; BCKDC, branched-chain ␣- ketoacid dehydrogenase complex; GCS, glycine cleavage system; dpc, The publication costs of this article were defrayed in part by page charge days postcoitum; ES, embryonic stem; RT-PCR, reverse transcription– coupled PCR; neo, neomycin phosphotransferase. payment. This article must therefore be hereby marked ‘‘advertisement’’ in §To whom reprint requests should be addressed at: Department of accordance with 18 U.S.C. §1734 solely to indicate this fact. Biochemistry, School of Medicine and Biomedical Sciences, State © 1997 by The National Academy of Sciences 0027-8424͞97͞9414512-6$2.00͞0 University of New York at Buffalo, 140 Farber Hall, 3435 Main PNAS is available online at http:͞͞www.pnas.org. Street, Buffalo, NY 14214. e-mail: [email protected].

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into the postnatal period (16, 17). This report describes the catalytic function (20). To select against random insertions of generation and phenotypic characterization of a null allele of the construct, a diphtheria toxin A-fragment cassette (19) was the murine Dld gene, Dldtm1mjp. inserted into the flanking region of homology. The construct was linearized by using a NotI site within the polylinker. Generation and Analysis of Dld؉/؊ Animals. The E14.1 MATERIALS AND METHODS embryonic stem (ES) cell line (21) was cultured under routine Construction of Targeting Vector. A 13-kb mouse genomic conditions similar to those described by Ramirez-Solis et al. clone was isolated from a 129SVJ genomic ␭ phage library (22). ES cells were electroporated with linearized targeting (Stratagene) by using two fragments from the murine Dld gene vector, selected with G418, and screened by Southern blot as probes that were previously isolated from a DBA͞2J analysis to identify recombinant clones (Fig. 1). Four inde- ϩ/Ϫ ͞ genomic library (18). A 7-kb XbaI subclone representing the 3Ј pendent Dld ES cell lines were injected into C57BL 6 half of the genomic ␭ clone was subcloned into pUC19 vector recipient blastocysts and transferred to the uteri of CH3B16 (Life Technologies) to serve as the region of homology in the pseudopregnant females. The chimeric animals were bred with targeting vector (Fig. 1A). The Dld gene was disrupted at an Black Swiss females to produce F1 progeny that were genotyped by Southern blot analysis or PCR using a trio of primers EcoRI site located at codon 301 within exon 10 by inserting a that simultaneously amplify regions of the wild-type and ␤-actin promoter-driven neomycin phosphotransferase gene targeted alleles (Fig. 2C): 5Ј common primer C1 (5Ј-GGTG- (neo) lacking a polyadenylylation signal (19). The cloning site GAATTGGAATTGACAT-3Ј), wild-type allele primer W1 is located upstream of key residues that have been shown in (5Ј-TTATTGACTGGAATTCTACCTTTGGGATCT-3Ј), studies of purified human E3 protein to be essential for and targeted allele primer T2 (5Ј-ACCCCCTCTCCCCTCC- TTTTG-3Ј). ϩ/Ϫ ϫ The Dld F1 progeny from the chimera Black Swiss matings were examined for any obvious phenotypic effect of carrying a mutant Dld allele by comparing behavior, gross anatomy, fertility, and growth curves with Dldϩ/ϩ littermates. Liver and kidney samples from the Dldϩ/ϩ and Dldϩ/Ϫ littermates were assayed for E3 activity as described by Patel et al. (23). The effects of the mutation on the E3-dependent multienzyme complexes were examined by assaying liver homog- enates of Dldϩ/ϩ and Dldϩ/Ϫ littermates for each of the four complexes. Citrate synthase activity was also measured to assess mitochondrial function (24–28). To determine the effect of the disruption mutation on gene expression, Dldϩ/ϩ and Dldϩ/Ϫ animals were subjected to Northern blot analyses using the murine Dld cDNA as a probe and glyceraldehyde-3- phosphate dehydrogenase cDNA as a loading control (18). Lower levels of Dldtm1mjp transcript were assessed by reverse transcription-coupled PCRs (RT-PCR) using the set of primers previously described or with an alternative targeted allele primer T1 (5Ј-CCTCCGCCCTTGTGGACACT-3Ј). Any ab- nornally spliced products that removed the neo sequence were detected by primer pairs that amplify across the EcoRI site: W2 (5Ј-CTCCCAGCACTCTATCTGTT-3Ј)orW3(5Ј-CTG- CAAATTCTTGGAACTGG-3Ј) (Fig. 2C). Identification and Analysis of the Dld؊/؊ Embryos. To ϩ/Ϫ assess the homozygous mutant phenotype, Dld F1 animals were intercrossed and F2 progeny were genotyped. The stage of prenatal death of DldϪ/Ϫ embryos was determined by dissecting out embryos at various stages of development, photographing, and genotyping them by PCR. Larger embryos at 7.5 days postcoitum (dpc) or later were genotyped by a single round of amplification with a trio of primers: a common intron 9 primer (5Ј-CACTAAGCTCCATCTTCAGCCATGAG-3Ј), a wild-type allele intron 10 primer (5Ј-GGTCTGTTTTTAT- CTTTAGAGAGAGCCAAAAA-3Ј), and a mutant allele ␤- FIG. 1. Targeting of the Dld gene. (A) Linearized targeting vector actin promoter primer (5Ј-CCTCCGCCCTTGTGGACACT- is composed of the pUC19 vector, the diptheria toxin fragment A Ј (Dt-A) negative marker, 7 kb of homology, and a neo cassette (neo). -3 ). The smaller preimplantation embryos were genotyped by (B) The wild-type Dld allele is represented by numbered boxes nested PCR in which an additional set of primers that were indicating exons with restriction enzyme sites listed above. The two external to the original set of primers were used for the first probes used for distinguishing the wild type from the targeted alleles round of amplification: C1, T2 (see above), and wild-type allele are designated by the solid boxes. The sizes of the various restriction primer (5Ј-CCTTTACAATATACCCGCCTCACCAT-3Ј). fragments and the enzymes used are shown below the restriction map. To assess the mutant phenotype in more detail, decidua at (C) The targeted allele after a recombination event is shown with the several time points around the established time of embryonic integrated neo cassette and the altered restriction map. (D) ES cell death were embedded in paraffin, serially sectioned, and colonies after electroporation and selection were initially screened by stained with hematoxylin͞eosin. using a SacI digest and probe A, which identified targeted alleles by a 2-kb reduction in size (lanes with asterisks). (E) The clones that appeared to be heterozygous by initial screen were subsequently RESULTS analyzed by additional digests by using the internal probe B with lane designations as follows: AϩK, ApaIϩKpnI; B, BstXI; EN, EcoNI; S, Disruption of the Dld Allele. A replacement targeting vector SacI. was designed in which the neo gene was inserted into the Downloaded by guest on October 1, 2021 14514 Developmental Biology: Johnson et al. Proc. Natl. Acad. Sci. USA 94 (1997)

Three of the four injected lines produced chimeras that showed germ-line transmission of the ES cell genome. In analyzing the ES cell-derived progeny, the presence of a 1:1 ratio of Dldϩ/ϩ:Dldϩ/Ϫ demonstrated that the introduced mutation had no dominant effects on viability. The Dldϩ/Ϫ animals were also similar to Dldϩ/ϩ littermates in gross appearance, behavior, and fertility. Liver samples were collected from the Dldϩ/ϩ and Dldϩ/Ϫ animals and analyzed by enzymatic assays to determine the effect of the mutation in vivo. The heterozygotes have approximately 50% of wild-type E3 activity levels compared with wild-type littermates, indicating that the disruption results in a loss of function (Fig. 2A). This half-fold change in E3 activity produces roughly similar reductions in each of the E3-dependent complexes in liver samples (Fig. 2A). As a control, the two genotypes were shown to have similar activity levels of the mitochondrial marker enzyme citrate synthase. Northern blot analysis showed that the Dldϩ/Ϫ animals have approximately 50% of wild-type levels of the 2.4-kb Dld mRNA with no additional bands being detected in liver and kidney samples (Fig. 2B). If the mutant transcript was processed by using the endogenous splice sites, then it would be expected to produce a transcript of 4.5 kb. To detect lower levels of mutant transcript, liver total RNA samples from Dldϩ/ϩ and Dldϩ/Ϫ animals were subjected to RT-PCR analysis using primers that amplify both wild-type and mutant alleles. No amplification product was detected with two different mutant-allele-specific primer pairs. The possibility of altered splicing patterns that remove all or part of the neo sequence was ruled out by demonstrating that primers amplifying across the neo insertion site detect only the wild-type amplification product (Fig. 2C). Phenotypic Characterization of Dld؊/؊ Animals. In the FIG. 2. Analysis of the effect of the Dldtm1mjp.(A) Liver homog- more than 200 progeny produced from intercrossing heterozy- ϩ ϩ ϩ Ϫ enates from Dld / and Dld / animals were assayed for E3 activity, gotes that originated from each of the three different clonal ES ϭ each of the E3-dependent complexes and citrate synthase (CS) (n cell lines, the litters at birth were found to be approximately 4 except for GCS where n ϭ 3). The graph indicates the percentage Ϯ Ϯ Ϯ Ϯ 25% smaller (7.6 1.1 mice vs. 9.8 1.6 mice) with the of activity relative to wild-type levels: E3, 21.0 3.0; PDC, 3.1 0.3; ϩ/ϩ ϩ/Ϫ Ϯ Ϯ Ϯ Ϯ expected Mendelian ratio of Dld to Dld (66:130) but KDC, 5.1 0.4; BCKDC, 0.79 0.05; GCS, 0.88 0.06; CS, 0.17 Ϫ/Ϫ Ϫ/Ϫ 0.01 (expressed as mean Ϯ SD in milliunits͞mg of total protein, except with no Dld class. The absence of Dld mutants indicates for GCS where milliunits͞mg of total mitochondrial protein was used). that a recessive prenatal lethal allele has been created. To (B) Northern blot analysis of total RNA from liver and kidney samples determine the time of death, embryos were collected at various of Dldϩ/ϩ and Dldϩ/Ϫ animals by using the full-length murine Dld stages of development and genotyped. At the blastocyst stage cDNA as a probe. For control of loading, the blots were simultaneously corresponding to 3.5 dpc, all embryos were similar in gross probed with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) appearance with the expected Mendelian distribution of ge- cDNA probe. A comparison of the normalized intensities with notypic classes: 16:35:14 for Dldϩ/ϩ͞Dldϩ/Ϫ͞DldϪ/Ϫ.At7.5 GAPDH is also presented. (C) RT-PCR analysis of total RNA from ϩ ϩ ϩ Ϫ dpc, corresponding to several days after implantation, two Dld / and Dld / liver samples. The locations of the primer annealing sites are depicted above the blot according to the wild-type and phenotypic classes of embryos were noted upon removal from targeted alleles: C1, common primer; W1, W2, and W3, wild-type the decidua (Fig. 3 A and B). Approximately 75% of the specific primers for exons 10, 11, and 14, respectively; T1 and T2, embryos (56 of 81) appeared to be of expected size and mutant specific primers. The amplifications were size-fractionated by morphology at pre- to early primitive streak stages. The gel electrophoresis, blotted, and probed with the murine Dld cDNA. remaining 25% of embryos were noticeably delayed in devel- The primers used for each amplification are listed below the lanes. The opment because they were much smaller and resembled nor- ϩ Ϫ templates used were as follows. Lanes: 1, Dld / genomic DNA; 2–5, mal 6.5 dpc egg cylinders. The expected Mendelian ratio of ϩ/ϩ ϩ/Ϫ Dld total RNA from liver; 6–9, Dld total RNA from liver. The genotypes at 7.5 dpc indicates that all DldϪ/Ϫ embryos survive amplification products of genomic DNA for the wild-type and mutant at least to this stage. As expected, the majority of phenotyp- alleles are 0.9 kb and 1.1 kb, respectively. The amplification products Ϫ/Ϫ ͞ ͞ ͞ ically delayed embryos were genotyped as Dld . The pres- from primer pairs C1 W1, C1 W2, and C1 W3 are 0.3, 0.6, and 0.85 Ϫ/Ϫ kb, respectively, for the wild-type allele cDNA. For the targeted allele, ence of two Dld embryos that were phenotypically normal the amplication products from primer pairs C1͞T1 and C1͞T2 are 0.4 demonstrates some variation in expressivity among the mutant and 0.5 kb, respectively. E, EcoRI. class. As would be expected, a small proportion of all embryos that contained a wild-type Dld allele were phenotypically abnormal due to some other cause of prenatal lethality. A coding region within exon 10 (Fig. 1 ). After electroporation, About one quarter of the embryos at 8.5 dpc (Fig. 3 C and approximately 30% of the G418-resistant clones had under- D) appeared to be similar in size to the mutant class at 7.5 dpc. gone the desired homologous recombination event, as indi- Surprisingly, only approximately 50% of these embryos were cated by Southern blot analysis (Fig. 1D). Additional digestion genotyped as DldϪ/Ϫ with the others being genotyped as Ј Ј ϩ Ϫ assays of 5 and 3 flanking restriction sites demonstrated that Dld / . The presence of the wild-type allele amplification the targeting vector had been inserted by homologous recom- product in these samples was attributed to maternal contam- bination into an endogenous gene. The absence of additional ination because these embryos were apparently being resorbed bands detected with an internal probe demonstrated that none as noted by the large amounts of extravasated blood surround- of the lines carried additional randomly inserted copies of the ing the embryos. The normal 8.5-dpc embryos, now at the targeting vector (Fig. 1E). head-fold stage of development, were genotyped as Dldϩ/ϩ or Downloaded by guest on October 1, 2021 Developmental Biology: Johnson et al. Proc. Natl. Acad. Sci. USA 94 (1997) 14515

Ϫ Ϫ ϩ Ϫ FIG. 3. Identification of Dld / embryos. Embryos from a Dld / ϫ Dldϩ/Ϫ mating were dissected from the decidua with the removal of Reichert’s membrane and ectoplacental cone tissue, photographed at ϫ15 magnification, and genotyped. The numbers below indicate the number of embryos identified with similar phenotypes and are then separated into genotypic classes.

Dldϩ/Ϫ with a ratio of roughly 1:2, which further supports the notion that none of the DldϪ/Ϫ class develop normally to 8.5 dpc. One day later at 9.5 dpc, 42 decidua were collected, of which 8 contained only resorption sites with insufficient embryonic material for genotypic analysis. These resorbed embryos were presumed to represent the DldϪ/Ϫ class based on the 34 normally staged embryos being genotyped as Dldϩ/ϩ or Dldϩ/Ϫ with a ratio of 11:23. The mutant phenotype was examined in more detail though histologic analysis of decidua at 6.5 through 8.5 dpc with the results summarized in Fig. 4. Of the 28 embryos examined at day 6.5, all of the embryos with the exception of one grossly malformed embryo appear to be normal egg cylinders, suggesting that the DldϪ/Ϫ class is not significantly delayed in ϩ Ϫ growth or differentiation at this stage (Fig. 4A). By 7.5 dpc, 7 FIG. 4. Histologic analysis of littermate embryos from Dld / ϫ embryos out of the 21 examined were phenotypically abnormal Dldϩ/Ϫ matings. The numbers in parentheses indicate the number of (Fig. 4C). These abnormal embryos resembled 6.5-dpc egg embryos with similar phenotypes (A–E, ϫ10). At 6.5 dpc, all embryos are cylinders with various degrees of disorganization. In 6 of the considered normal because there were no obvious phenotypic differences (A). At subsequent days of development, the littermate embryos could be 7 abnormal embryos, there was evidence of mesoderm for- divided into two phenotypic classes (B–E). A higher magnification of an mation, which signals the onset of gastrulation (Fig. 4F). One abnormal embryo at 7.5 dpc is included to allow for more detailed unusual feature of these abnormal embryos was the presence examination (F). Several important cell types are labeled and arrowheads of a thickened columnar layer of visceral endoderm. In normal indicate invaginations in the extraembryonic region. Downloaded by guest on October 1, 2021 14516 Developmental Biology: Johnson et al. Proc. Natl. Acad. Sci. USA 94 (1997)

7.5-dpc embryos, the visceral endoderm assumes a unicellular isomerase (7). The persistance of maternal E3 protein is flattened squamous morphology when in contact with the plausible considering both the long half-life of mitochondrial embryonic ectoderm. The presence of multiple folds in the proteins that was shown to be 43 h for E3 protein in 3T3-L1 extraembryonic region may be a result of either the initation preadipocytes (35) and the large number of mitochondria per of invaginations to form the three embryonic coela or contin- murine oocyte that has been estimated to be approximately 105 ued proliferation of the visceral endoderm in a spatially (36). Alternatively, metabolic studies in vitro provide evidence confined area. The normal process of formation of the three that the E3-dependent metabolic pathways may not be needed coela can be appreciated in the normally developing 7.5-dpc in the early postimplantation period based on the predomi- embryo (Fig. 3B). One day later at 8.5 dpc, 75% of the embryos nance of glycolysis (5, 8). (18 of 24) were at the expected head-fold stage having initiated The time of developmental cessation in the DldϪ/Ϫ embryos somitogenesis and organogenesis (Fig. 4D). The six abnormal coincides with the onset of gastrulation, a period of rapid embryos had not grown and were more disorganized relative growth and differentiation in which the three germ layers are to the abnormal embryos at 7.5 dpc. The hemorrhagic necrotic produced. Between 6.5 and 7.5 dpc, cell cycle times have been tissue surrounding these embryos indicates that these abnor- estimated to be as short as5hinembryonic tissue resulting in mal embryos were in the process of being resorbed by the a 20-fold increase in cell number and a 14-fold increase in maternal system (Fig. 4E). tissue volume (37). This large relative increase in biomass represents a significant energetic and biosynthetic demand on DISCUSSION the embryo. Presently, it is not possible to point definitively to which affected pathways contribute to this phenotype. When The murine Dld gene was disrupted by the insertion of a neo the critical roles of PDC and KDC are considered, it is gene into exon 10, which creates a loss of function mutation anticipated that deficiencies in these complexes would play a affecting gene expression predominantly at the mRNA level. significant role in the DldϪ/Ϫ phenotype. With the absence of The absence of detectable mRNA from the Dldtm1mjp allele in these two enzymatic activities, the DldϪ/Ϫ embryos would be liver and kidney tissues as determined by Northern blot and unable to metabolize glucose oxidatively. The restriction to RT-PCR analyses indicates that this mutation creates a null glycolysis would result in an ATP yield from glucose metab- allele. The most likely explanation for the lack of detectable olism that would be only about 5% of that if glucose was mutant transcript is mRNA instability. The phenomenon of metabolized oxidatively. Additionally, the loss of KDC activity mRNA instability caused by nonsense mutations has been would result in diminished concentrations of several of the documented in a number of different genes including ␤-globin tricarboxylic acid cycle intermediates that are required for the (29) and triosephosphate isomerase (30). The recessive nature biosynthesis of essential molecules such as porphyrins and of this mutation is in accordance with data showing that human pyrimidines. The roles of BCKDC and GCS deficiencies in the heterozygotes with approximately 50% of E3 activity levels are DldϪ/Ϫ phenotype are less clear. No data currently exists as to phenotypically normal (31). The half-fold reduction in E3 whether these complexes are expressed during early embryo- activity in Dld heterozygotes results in similar reductions in the genesis. One recent report has shown that a transcript of one activities of each of the E3-dependent complexes. The reduc- of the GCS component genes can be detected by RT-PCR in tion in GCS activity is particularly noteworthy in light of past porcine oocytes (38). The phenotypes of BCKDC and GCS studies demostrating that rat liver mitochondria have immu- deficiencies in humans result presumably from an accumula- nologically distinct dihydrolipoamide dehydrogenase compo- tion of the unmetabolizable substrates to neurotoxic levels. It nents for the GCS as compared with the ␣-ketoacid dehydro- is unclear how relevant such effects would be during the genase complexes (32). The observed reduction of GCS ac- perigastrulation period, a stage preceding neurogenesis, and tivity in Dldϩ/Ϫ mice suggests that one gene codes for the when the small size of the embryo should permit molecules to dihydrolipoamide dehydrogenase of all four multienzyme readily diffuse into the maternal system. complexes, which is also supported by studies of human Interestingly, it has been a long-held belief that early postim- heterozygotes (3). plantation development is essentially powered by anaerobic In the homozygous state, the disrupted Dld allele appears to metabolism. The basis for this view began with histologic be incompatible with early postimplantation viability. At sev- studies first performed by Krehbiel (39) that showed the eral days after implantation, DldϪ/Ϫ embryos form egg cylin- primary decidual zone surrounding the implanted embryo to ders but then become developmentally delayed. This finding be devoid of patent vessels. Further support has been provided was supported by histologic analyses of litters from heterozy- by the in vitro studies that have demonstrated virtually all gote matings in which an obvious abnormal phenotype was first glucose consumed by postimplantation embryos to be con- detected at 7.5 dpc. The aberrant 7.5-dpc embryos grossly verted to lactate. Comparisons have been drawn between the resemble 6.5-dpc egg cylinders in size and proportion, indi- rapidly developing embryonic tissue and other cell types such cating that virtually all cell types cease proliferation at the as transformed cells that rely on glycolysis presumably as a same time. One cell type, the visceral endoderm, appears to means of rapid ATP production. These studies have led to the proliferate relatively longer and forms a folded thickened cell hypothesis that oxidative metabolism does not play a signifi- layer. The prolonged survival of this cell type may be related cant role in postimplantation embryogenesis until after the to its ability to uptake nutrients because it is believed to act as formation of the chorioallantoic placenta when oxygen would a specialized transport mechanism for macromolecules prior to become more readily available. development of the chorioallantoic placenta (33). Interest- The Dld null phenotype provides direct evidence for the ingly, recent in situ hybridization studies of early postimplan- importance of oxidative metabolism during the early postim- tation mouse embryos have shown differences in the expres- plantation period. When the 18:1 differentiation ATP yield in sion of various glucose transporter isoforms in the visceral comparing aerobic to anaerobic glucose metabolism is con- endoderm compared with other embryonic cell types, which sidered, this result does not necessarily contradict the in vitro may reflect a differential ability to uptake this energy source studies. Even the small changes in relative utilization of these (34). The survival of the DldϪ/Ϫ embryos for almost 1 week two pathways recorded in vitro would translate to significant postconception may be attributed to either the persistence of differences in total energy yield. The assumption of an anaer- maternally encoded E3 protein or the lack of need for this obic postimplantation milieu has been challenged recently by enzyme before the perigastrulation period. There is a prece- several studies, demonstrating the high permeability of the dent for the persistence of a maternal protein into the postim- primary decidual zone (40). The permeable nature of this plantation period based on the studies of glucose-6-phosphate tissue may allow for adequate oxygenation merely by diffusion. Downloaded by guest on October 1, 2021 Developmental Biology: Johnson et al. Proc. Natl. Acad. Sci. USA 94 (1997) 14517

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