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Disruption of Cholesterol 7α-Hydroxylase Gene in Mice

1996; Elsevier BV; Volume: 271; Issue: 30 Linguagem: Inglês

10.1074/jbc.271.30.18017

1083-351X

Shun Ishibashi, Margrit Schwarz, Philip K. Frykman, Joachim Herz, David W. Russell,

Cancer, Lipids, and Metabolism

Mice deficient in cholesterol 7α-hydroxylase, the rate-limiting enzyme of bile acid biosynthesis, were constructed by targeted disruption of the Cyp7 gene. The introduced mutation removed exons 3-5 of the gene and gave rise to a null allele that encoded no immunoreactive or enzymatically active protein. Heterozygous carriers of the disrupted gene (Cyp7+/−) were phenotypically normal. Homozygous animals (Cyp7−/−) appeared normal at birth, but died within the first 18 days of life. Approximately 40% of the animals died between postnatal days 1 and 4 and 45% between days 11 and 18. The addition of vitamins to the water of nursing mothers prevented deaths in the early period, whereas the addition of cholic acid to chow prevented deaths in the later period. Newborn Cyp7−/− mice whose mothers were maintained on unsupplemented chow failed to gain weight at a normal rate and developed oily coats, hyperkeratosis, and apparent vision defects. These symptoms waned at 3 weeks of life, and their disappearance was accompanied by a marked increase in survival. In the accompanying study, the induction of an alternate pathway of bile acid biosynthesis is shown to underlie this unusual time course (Schwarz, M., Lund, E. G., Setchell, K. D. R., Kayden, H. J., Zerwekh, J. E., Björkhem, I., Herz, J., and Russell, D. W. (1996) J. Biol. Chem. 271, 18024-18031). We conclude that cholesterol 7α-hydroxylase is an essential enzyme for normal postnatal development. Mice deficient in cholesterol 7α-hydroxylase, the rate-limiting enzyme of bile acid biosynthesis, were constructed by targeted disruption of the Cyp7 gene. The introduced mutation removed exons 3-5 of the gene and gave rise to a null allele that encoded no immunoreactive or enzymatically active protein. Heterozygous carriers of the disrupted gene (Cyp7+/−) were phenotypically normal. Homozygous animals (Cyp7−/−) appeared normal at birth, but died within the first 18 days of life. Approximately 40% of the animals died between postnatal days 1 and 4 and 45% between days 11 and 18. The addition of vitamins to the water of nursing mothers prevented deaths in the early period, whereas the addition of cholic acid to chow prevented deaths in the later period. Newborn Cyp7−/− mice whose mothers were maintained on unsupplemented chow failed to gain weight at a normal rate and developed oily coats, hyperkeratosis, and apparent vision defects. These symptoms waned at 3 weeks of life, and their disappearance was accompanied by a marked increase in survival. In the accompanying study, the induction of an alternate pathway of bile acid biosynthesis is shown to underlie this unusual time course (Schwarz, M., Lund, E. G., Setchell, K. D. R., Kayden, H. J., Zerwekh, J. E., Björkhem, I., Herz, J., and Russell, D. W. (1996) J. Biol. Chem. 271, 18024-18031). We conclude that cholesterol 7α-hydroxylase is an essential enzyme for normal postnatal development. A 12-kilobase (kb) 1The abbreviations used are: kbkilobase(s)ESembryonic stem. fragment of the Mus musculus 7α-hydroxylase gene was enriched by sucrose gradient density ultracentrifugation of BamHI-digested genomic DNA isolated from the 129Sv strain. The resulting fraction was ligated into the bacteriophage λ DashII vector (Stratagene), and recombinant clones harboring the desired insert were identified by screening with a cDNA probe derived from the coding region of the mouse 7α-hydroxylase mRNA. The cDNA probe was originally amplified from mouse liver mRNA using oligonucleotide primers derived from the rat cDNA sequence (10Jelinek D.F. Andersson S. Slaughter C.A. Russell D.W. J. Biol. Chem. 1990; 265: 8190-8197Google Scholar). A replacement-type targeting vector was constructed as shown in Fig. 1A using the cloned genomic DNA fragment and a plasmid vector as templates. The short arm of the targeting vector (∼1.2 kb) represented a BamHI DNA fragment spanning intron 2 of the gene and was amplified from genomic DNA using two oligonucleotide primers (5′-primer = 5′-ATTTCATTACACTACTTCTGCGAAG-3′ and 3′-primer = 5′-TGGGTCTATGCTTCTGTGTCCAAAT-3′). The long arm of the targeting vector (∼12 kb) extended from a BamHI site in intron 5 to a BamHI site located 3′ of the 7α-hydroxylase gene. The Pol2sneobpA expression cassette (11Mansour S.L. Thomas K.R. Capecchi M.R. Nature. 1988; 336: 348-352Google Scholar) was positioned between the two murine DNA fragments (arms), and two copies of the herpes simplex virus thymidine kinase gene were located 5′ of the short arm of the vector (see Fig. 1A). kilobase(s) embryonic stem. Mouse AB-1 embryonic stem (ES) cells derived from the 129Sv strain were cultured on leukemia inhibitory factor-producing STO feeder cells as described (12Willnow T.E. Armstrong S.A. Hammer R.E. Herz J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4537-4541Google Scholar). Approximately 2 × 107 cells were electroporated (275 V, 330 millifarads) with 25 µg/ml targeting vector linearized with SalI using a device from Life Technologies, Inc. Electroporated cells were seeded onto irradiated (10,000 rads) feeder layers. After selection with 190 µg/ml G418 and 0.25 mM 1-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-5-iodouracil (Bristol Myers-Squibb), recombinant clones were identified by the polymerase chain reaction using a 5′-primer (5′-GAACCTCCTTTGGACAACGGGTTGA-3′) located in exon 2 of the 7α-hydroxylase gene and a 3′-primer (5′-AGGATTGGGAAGACAATAGCAGGCAT-3′) located within the bovine growth hormone polyadenylation sequence of the neo expression cassette. Two independent stem cell clones (clones 86 and 193) containing a disrupted 7α-hydroxylase allele were identified among 500 colonies screened. The homologous recombination events in these two clones were verified by Southern blot analysis of BglII-digested cellular DNA using an intron 1 hybridization probe (see Fig. 1B). Both ES cell clones were injected into C57Bl/6J blastocysts, yielding 18 chimeric males whose coat color (agouti) indicated a contribution from the stem cells ranging from 20 to 95%. Of these chimeric males, two (both derived from ES clone 193) transmitted the disrupted 7α-hydroxylase gene through the germ line. All experiments reported here were performed with mixed strain (C57Bl/6J-129Sv) descendants (F2 and subsequent generations) of these animals. Enzyme assays, immunoblotting, and RNA blotting were carried out as described previously (10Jelinek D.F. Andersson S. Slaughter C.A. Russell D.W. J. Biol. Chem. 1990; 265: 8190-8197Google Scholar). The normal diet was mouse/rat diet 7001 (Harlan Teklad, Madison, WI) and contained ≥4% (w/w) fat, ≥24% (w/w) protein, and ≤5% (w/w) fiber. Where indicated, this diet was supplemented with 1% (w/w) cholic acid (Sigma). A vitamin supplement (CritterVites, Mardel Labs, Glendale Heights, IL) containing both water-soluble vitamins (thiamine, 180 mg/kg; riboflavin, 300 mg/kg; pantothenic acid, 600 mg/kg; niacin, 1500 mg/kg; vitamin B12, 150 µg/kg; vitamin B6, 150 mg/kg; folic acid, 100 mg/kg; and ascorbic acid, 9000 mg/kg) and fat-soluble vitamins (vitamin A, 300,000 IU/kg; vitamin D3, 50,000 IU/kg; vitamin E, 750 IU/kg; and menadione, 250 mg/kg) was added to water bottles at the concentration (1 g/liter) recommended by the manufacturer. Vitamin supplements were replaced on a daily basis. The behavior of wild-type and 7α-hydroxylase-deficient mice in an open field was determined using a modification of a test described by Aiba et al. (13Aiba A. Kano M. Chen C. Stanton M.E. Fox G.D. Herrup K. Zwingman T.A. Tonegawa S. Cell. 1994; 79: 377-388Google Scholar). Disruption of the murine 7α-hydroxylase gene was carried out by standard methods in the 129Sv-derived ES cell line AB-1. A targeting vector was constructed as shown in Fig. 1A. Homologous recombination between this vector and a normal Cyp7 allele (14Tzung K. Ishimura-Ika K. Kihara S. Oka K. Chan L. Genomics. 1994; 21: 244-247Google Scholar) is expected to result in deletion of most of exon 3 and all of exons 4 and 5. Electroporation of the vector DNA followed by selection with neomycin and 1-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-5-iodouracil yielded two ES cell lines that harbored the desired recombination event. For unknown reasons, the frequency of such clones was very low (0.4%). Both ES cell lines were injected into blastocysts derived from C57BL/6J female mice and subsequently implanted in pseudopregnant females. Two of the resulting chimeric male animals transmitted the disrupted 7α-hydroxylase gene through the germ line as judged by Southern blot analysis of genomic DNA derived from tail segments. The data of Fig. 1B show that crossing male and female animals heterozygous for the mutant allele resulted in offspring of both sexes with all three of the expected genotypes at the Cyp7 locus. The 6.0-kb BglII DNA fragment characteristic of the disrupted gene and/or the 3.5-kb BglII DNA fragment characteristic of the normal gene was present in the expected Mendelian ratios in the offspring of this and other crosses. To confirm that deletion of exons 3-5 of the gene abolished 7α-hydroxylase enzyme activity, extracts of microsomal membranes prepared from the livers of animals with different genotypes were incubated with [14C]cholesterol in the presence of NADPH. As shown in Fig. 2A, wild-type mice express an enzyme activity capable of hydroxylating cholesterol at the 7α-position (lanes 5 and 6). Extracts prepared from animals heterozygous for the disrupted Cyp7 gene contain approximately one-half the amount of enzyme activity present in wild-type tissue (lanes 3 and 4), whereas extracts from Cyp7−/− animals contain little or no cholesterol 7α-hydroxylase activity (lanes 1 and 2). In control experiments, the production of 7α-hydroxycholesterol in the assay was dependent on the presence of both NADPH cofactor and microsomal protein (lanes 7 and 8). Immunoblotting of liver extracts prepared from mice homozygous for the disrupted gene also revealed the loss of a protein with a size and migration on SDS-polyacrylamide gels characteristic of 7α-hydroxylase (Fig. 2B). An antibody raised against a highly purified preparation of the rat enzyme (10Jelinek D.F. Andersson S. Slaughter C.A. Russell D.W. J. Biol. Chem. 1990; 265: 8190-8197Google Scholar) recognized a cluster of cytochrome P450 proteins in rat liver extracts that migrated with molecular weights in the 50,000 range (lanes 1 and 2). Within this group of proteins, the 7α-hydroxylase enzyme is known to have the slowest electrophoretic mobility (10Jelinek D.F. Andersson S. Slaughter C.A. Russell D.W. J. Biol. Chem. 1990; 265: 8190-8197Google Scholar). In liver extracts from wild-type mice, this antibody recognized two major proteins, one of which had a molecular weight identical to that of the rat 7α-hydroxylase enzyme (lanes 7 and 8). The intensity of this putative murine 7α-hydroxylase protein in liver extracts prepared from Cyp7+/− animals (lanes 5 and 6) was approximately one-half that detected in normal mice, while this protein was not detected in Cyp7−/− animals (lanes 3 and 4). The intensity of the other major cross-reacting protein, a presumed cytochrome P450 that has a lower molecular weight than the 7α-hydroxylase protein, also declined in the homozygous animals (lanes 3 and 4). This decline appeared to be due to an overall decrease in the content of cytochrome P450 enzymes present in the livers of Cyp7−/− mice (see below). RNA blotting of poly(A)+-enriched mRNA fractions from mice of different Cyp7 genotypes was performed next. As illustrated by the data of Fig. 3, wild-type mice contain several cholesterol 7α-hydroxylase mRNAs, with major species at ∼4.4 and ∼7.0 kb. Heterozygous animals appear to contain slightly more of these same mRNAs and, in addition, a new mRNA migrating with a size of ∼5.0 kb. Cyp7−/− animals contain the latter mRNA, but none of the mRNA species present in wild-type mice. RNA blotting, cDNA cloning, and DNA sequencing experiments revealed that the novel mRNA present in the Cyp7+/− and Cyp7−/− animals was a hybrid mRNA composed of sequences derived from the 5′- and 3′-ends of the mutated Cyp7 gene and the inserted neomycin resistance gene (data not shown). Control RNA blotting experiments (Fig. 3, lower panel) showed that the levels of sterol 27-hydroxylase mRNA and β-actin mRNA in the liver did not differ between wild-type and mutant Cyp7 mice. A phenotype associated with the absence of 7α-hydroxylase became apparent upon breeding heterozygous animals. Homozygous pups arising from these crosses failed to thrive and experienced a very high rate of perinatal mortality. We hypothesized that death was caused by a deficiency of bile acids and a subsequent failure to absorb fat-soluble vitamins from the diet. To test this hypothesis, nursing mothers were placed on diets supplemented with vitamins and cholic acid as described under “Experimental Procedures.” The survival rate of the Cyp7−/− animals increased dramatically upon dietary supplementation for a period of 30 days. Thereafter, the mutant animals could be switched to normal chow with no decrease in survival. Using this supplementation regime, the mutant mouse colony was expanded to produce sufficient animals to characterize in detail their phenotype and the effects of individual dietary supplements. We first determined the survival rate of animals born to Cyp7−/− mothers in the absence of supplementation. As shown in Fig. 4A, ∼85% of the pups arising from crosses of homozygous animals died within the first 18 days of postnatal life. Death occurred in two temporal waves, with ∼40% of the pups succumbing prior to day 4 and the remainder dying between days 11 and 17. The 10-15% of the homozygous animals that survived until day 18 showed a near normal survival rate over the following 12 days (Fig. 4A). In these experiments, all pups suckled regardless of genotype, and milk was visible in the stomachs and intestines of the animals. Thus, death could not be attributed to the absence of alimentation. Homozygous pups defecated clay-colored, fatty stools, indicative of fat malabsorption (steatorrhea). The effects of individual dietary supplements on animal survival were determined next. As shown in Fig. 4B, the presence of vitamins in the water decreased the numbers of deaths that occurred in the first few days of life, but did not prevent the second wave of death. Conversely, cholic acid supplementation of the mother's diet had no effect on the number of early deaths, but prevented the second temporal wave of death in the homozygous pups (Fig. 4B). The death curve of Cyp7−/− pups nursed by mothers given both vitamins and cholic acid is shown in Fig. 4C. When the supplements were begun on the day the pups were born, ∼60% of the pups died during postnatal days 1-14. All of the animals that survived to day 15 remained alive at day 30. In contrast, when the dual supplementation was begun on gestation day 12 (i.e. before the pups were born), the survival rate of homozygous pups (Fig. 4C) was indistinguishable from that of wild-type pups born to mothers fed normal chow (Fig. 4A). Taken together, the results of the feeding studies indicated that 7α-hydroxylase-deficient animals suffered from a vitamin shortage immediately after birth and from a bile acid shortage around the second week of life. Mutant mice born to Cyp7−/− mothers maintained on normal chow were noticeably smaller than age-matched heterozygous or wild-type pups. To quantify this difference, weight gain in newborn animals was measured as a function of age and genotype. The data of Fig. 5 indicate that wild-type pups gained weight at an essentially linear rate during postnatal days 1-30. In contrast, homozygous pups gained weight at a slower rate until day 5 and then experienced a period of weight stagnation until day 11 (Fig. 5). After day 11, surviving mutant pups sustained a progressive weight gain, but at a slower rate compared with wild-type animals. A comparison of the death curve shown in Fig. 4 and the weight gain curve of the homozygous animals shown in Fig. 5 indicates that weight stagnation in the postnatal day 5-11 period was not accompanied by death of the pups. Cyp7−/− pups born to mothers whose diets were supplemented with vitamins and cholic acid during and after gestation gained weight at a normal rate (data not shown). A second striking phenotype associated with disruption of the 7α-hydroxylase gene was an abnormality of the fur. As shown in Fig. 6 (upper panel), nursing wild-type mothers have a glossy smooth pelt. In contrast, nursing homozygous mothers and, more surprisingly, nursing heterozygous mothers developed an unusually oily coat (a “Brylcream phenotype”) between days 4 and 7 of the nursing period (center panel). Approximately 3-4 days after a mother developed this phenotype, her pups also developed an oily hide (lower panel). In mothers, this phenotype persisted for a period of at least 7-8 days and as long as 14 days. The duration and extent of the symptom appeared to be directly related to the number of pups nursed. Females nursing larger litters exhibited a more severe phenotype for longer periods of time compared with mothers with smaller litters. Pups remained oily for ∼7 days after the onset of symptoms, and most of them died within this period. In contrast, lactating females maintained on a 4% fat diet supplemented with 1% cholic acid did not develop the greasy phenotype, nor did the pups of these animals manifest the symptom. Vitamin supplementation alone had no effect on the development of the oily coat (data not shown). During the nursing period, pups and young animals deficient in 7α-hydroxylase also exhibited the phenotype shown in Fig. 7. The photograph shows the skin of a day 5 pup, which is characterized by a dry, scaly appearance. This feature of the deficient mice is easiest to visualize before the growth of fur (Fig. 7); however, the symptom persists throughout the nursing period. In addition, older Cyp7−/− animals showed eye abnormalities, including delayed opening and excess secretion emanating from the unopened oculi (data not shown). The eyelids of deficient animals opened ∼8-9 days later than those of control mice. The data of Fig. 8 show a histological analysis of the skin of wild-type and Cyp7−/− pups. The upper panel represents an abdominal skin section of a day 14 wild-type animal that exhibited a normal histology. In contrast, the skin of an age-matched Cyp7−/− pup that showed the scaly skin phenotype of Fig. 7 reveals a thickened and torn stratum corneum layer (hyperkeratosis) and a thickened granular layer (lower panel). The cells of the spinous and basal layers of the epidermis as well as the dermis of the homozygous animals appeared compressed relative to the wild-type specimen. Liver histology in the deficient animals was normal, with well formed lobules and alternating central vein and portal triad architecture (data not shown). Yellow crystalline deposits were observed in the bile canaliculi of young mice. The level of conjugated bilirubin was higher in the livers of Cyp7−/− mice (28.2 ± 2.8 µg/mg tissue, n = 3) than in age-matched wild-type controls (2.6 µg/mg ± 0.5, n = 3). Finally, spectrophotometric scans of liver membrane proteins revealed an ∼50% decrease in A450 absorbing material in the deficient versus wild-type animals (data not shown). These results, which indicate a decrease in liver cytochrome P450 content in the Cyp7−/− mice, further suggest a disruption of heme metabolism in this organ. Young Cyp7−/− animals, after they had opened their eyes, appeared to be less active than wild-type counterparts. To confirm this observation, the feet of animals of different genotypes were dipped in blue ink (wild-type) or red ink (Cyp7−/−), and the mice were placed in the upper right-hand corner of an enclosed sheet of filter paper (46 × 57 cm). The mice were then allowed to roam undisturbed for 1 min. This procedure was repeated 11 additional times for each mouse. The wild-type mouse actively explored the enclosure as indicated by the presence of blue footprints over much of the paper (Fig. 9). The majority of the movements of the wild-type mouse were confined to the perimeter of the enclosure, but the animal also crossed over the center of the paper. The concentration of blue footprints in each of the four corners reflects the animal's attempts to peer over and to climb out of the enclosure. In contrast to these results, a Cyp7−/− animal confined its movements to one side of the paper, persistently ambling up and down the wall adjacent to the corner in which it was placed (Fig. 9, red footprints). This behavior suggests that the Cyp7−/− mice have vision problems that render them dependent on tactile stimulation for location cues and subsequent movement. We provide evidence that 7α-hydroxylase activity is important in murine postnatal life. The majority of mice that lack this enzyme die within 18 days of birth. Death is preceded by abnormally slow weight gain, fatty stools, hyperkeratosis, an unusual appearance of the fur, and behavioral abnormalities. These symptoms are representative of those associated with fat malabsorption (wasting and steatorrhea) and fat-soluble vitamin malabsorption (skin, fur, and vision pathologies), and their occurrence in Cyp7−/− mice suggests that the bile acid pathway initiated in the endoplasmic reticulum by 7α-hydroxylase is crucial for nutritional homeostasis in the neonate. No decrease in fecundity (litter size) or effects on fertility were detected in Cyp7−/− females, suggesting that 7α-hydroxylase is not required for embryonic development or reproduction. At birth, 7α-hydroxylase-deficient mice appear phenotypically normal. However, shortly thereafter, many of the homozygous animals begin to succumb to the deficiency. Deaths cluster in two time periods, days 1-4 and days 12-17 (Fig. 4A). Animals that survive through day 4 remain alive until day 12, at which time another 40% expire during the second period of increased death. The animals that survive through day 17 (∼15% of the total) thereafter have lifespans that approximate those of wild-type mice. Dietary supplementation experiments provide a potential explanation for the observed biphasic death curve. Pups born to mothers supplemented with a combination of fat- and water-soluble vitamins survive the first wave of early postnatal demise, but succumb to the second (Fig. 4B). In these experiments, vitamins were added to the mothers' water supply, which the nursing pups cannot reach and thus do not have direct access to. Given this mode of delivery, the simplest interpretation of the rescue data is that the early deaths are a consequence of a vitamin deficiency due to a decrease in the vitamin content of the mother's milk. The metabolism of postpartum animals changes radically with the onset of lactation since vastly more nutritional resources must be committed to the production of milk. The stress imparted by these metabolic changes is presumably revealing a phenotype (avitaminosis) in the deficient mothers that is otherwise not detectable in non-nursing females. If this interpretation is correct, then the physiological stress imparted to the Cyp7−/− mothers by lactation and nursing overwhelms their capacity to solubilize sufficient vitamins to meet the needs of the pups. The resulting low levels of vitamins in the mother's milk, combined with the pups' deficiency in bile acids, leads to the failure of the pups to absorb sufficient vitamins to maintain survival. Supplementation of the mother provides high enough levels of vitamins to the pups such that a sufficient amount can be absorbed to stave off death. In the accompanying paper (15Schwarz M. Lund E.G. Setchell K.D.R. Kayden H.J. Zerwekh J.E. Björkhem I. Herz J. Russell D.W. J. Biol. Chem. 1996; 271: 18024-18031Google Scholar), we show that Cyp7−/− mice are deficient in vitamins D3 and E; thus, it seems likely that deficiency in one or more of the fat-soluble vitamins underlies the first wave of postnatal death. This deduction is in part based on the known role of bile acids in facilitating the uptake of fat-soluble vitamins (1Turley S.D. Dietschy J.M. Arias I. Popper H. Schachter D. Shafritz D.A. The Liver: Biology and Pathobiology. Raven Press, New York1982: 467Google Scholar) and the fact that mice lacking apolipoprotein B expression in the intestine, which are defective in fat-soluble vitamin uptake, also die at a high rate in early postnatal life (16Young S.G. Cham C.M. Pitas R.E. Burri B.J. Connolly A. Flynn L. Pappu A.S. Wong J.S. Hamilton R.L. Farese Jr., R.V. J. Clin. Invest. 1995; 96: 2932-2946Google Scholar). Alternatively, the phenotype of the 7α-hydroxylase-deficient mice may reflect a deficiency of both water- and fat-soluble classes of vitamins in that the malabsorption accompanying the defect could lead to a generalized loss of vitamins. The phenotype of the Cyp7−/− mice does not match the symptoms associated with a deficiency of any single water-soluble vitamin in the mouse (17Jones J.H. Foster C. Dorfman F. Hunter G.L. J. Nutr. 1945; 29: 127-136Google Scholar), except perhaps folic acid. This vitamin is moderately soluble in water (1.6 µg/ml at 25°C), and a deficiency in the mouse leads to weight loss and the appearance of an “untidy hair coat” (18Darby W.J. Jones E. Proc. Soc. Exp. Biol. Med. 1945; 60: 259-260Google Scholar), which resembles that of the 7α-hydroxylase-deficient animals (Fig. 6). We are carrying out supplementation studies in which individual vitamin classes or vitamins are given to the deficient animals in an attempt to identify the active substance in the current pan supplement. An unanswered question that arises from the feeding experiments is why supplementation with vitamins and bile acids beginning on postnatal day 1 did not prevent deaths in the initial period (Fig. 4C), whereas supplementation with vitamins alone did (Fig. 4B). A generalized toxicity of the combined supplements can be ruled out since their administration during gestation increased pup survival (Fig. 4C). These results suggest that the metabolism, maternal-fetal transfer, or uptake of bile acids or vitamins changes in some as yet undefined manner at term or shortly thereafter. Pups born to mothers fed a chow diet supplemented with 1% cholic acid die at the expected rate during the first lethal period, but survive the second wave of death (Fig. 4B). Death during the second period, but not the first, is preceded by the development of an oily coat in unsupplemented nursing mothers and their pups. In the vitamin supplementation experiments, we are certain that the pups did not gain direct access to the water bottles containing vitamins. However, in the case of the cholic acid supplementation experiments, the supplemented chow was strewn about the cage and adhered to the fur of both mothers and pups. Since the pups groom themselves, they may have consumed some of the cholic acid present in the chow. Thus, reversal of the second wave of death may have been due to alleviation of a maternal defect or an offspring defect associated with bile acid deficiency. A clue to understanding why the deficient mice die during postnatal days 11-17 may lie in determining the cause of the oily coat as cholic acid-supplemented animals did not die and did not develop this phenotype. We initially hypothesized that the second period of death might be due to the deficiency of an essential fatty acid. Diets lacking in these fats (linolenic acid and linoleic acid) give rise to hyperkeratosis and excess water loss in mice (19Holman R.T. Lipids. 1969; 9: 275-348Google Scholar), which is a phenotype that resembles the deficient mice of this study. However, as indicated in the accompanying paper (15Schwarz M. Lund E.G. Setchell K.D.R. Kayden H.J. Zerwekh J.E. Björkhem I. Herz J. Russell D.W. J. Biol. Chem. 1996; 271: 18024-18031Google Scholar), 7α-hydroxylase-deficient mice have near normal levels of essential fatty acids. Thus, the second period of death may result from the accumulation of a toxic intermediate in the bile acid pathway, a secondary sterol, or a lipid metabolite in the Cyp7−/− pups and their mothers. Alternatively, the animals may die of malnutrition caused by a failure to solubilize milk fats during the first 18 days of life. We thank Bob Hammer for advice regarding the construction of knockout mice; Ron Estabrook for help with spectrophotometric scans; Daphne Davis, Kristi Cala, and Kevin Anderson for excellent technical assistance; and Helen Hobbs, Joe Goldstein, and Mike Brown for critical reading of the manuscript.