THE JOURNAL OF BIOLOGICAL CHEMISTRY© 1999 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 274, No. 43, Issue of October 22, pp. 30843–30848, 1999
Printed in U.S.A.

Embryonic Lethality and Defective Neural Tube Closure in Mice
Lacking Squalene Synthase*(Received for publication, March 30, 1999, and in revised form, June 22, 1999)

Ryu-ichi Tozawa‡, Shun Ishibashi‡§, Jun-ichi Osuga‡, Hiroaki Yagyu‡, Teruaki Oka¶,
Zhong Chen‡, Ken Ohashi‡, Stéphane Perrey‡, Futoshi Shionoiri‡, Naoya Yahagi‡,
Kenji Harada‡, Takanari Gotoda‡, Yoshio Yazakii, and Nobuhiro Yamada‡From the ‡Department of Metabolic Diseases, the iDepartment of Cardiology, and the ¶Department of Pathology,
Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan

Squalene synthase (SS) catalyzes the reductive headto-head condensation of two molecules of farnesyl
diphosphate to form squalene, the first specific intermediate in the cholesterol biosynthetic pathway. We usedgene targeting to knock out the mouse SS gene. The miceheterozygous for the mutation (SS1/2) were apparently
normal. SS1/2 mice showed 60% reduction in the hepatic mRNA levels of SS compared with SS1/1 mice.
Consistently, the SS enzymatic activities were reduced
by 50% in the liver and testis. Nevertheless, the hepaticcholesterol synthesis was not different between SS1/2
and SS1/1 mice, and plasma lipoprotein profiles were
not different irrespective of the presence of the low
density lipoprotein receptor, indicating that SS is not a
rate-limiting enzyme in the cholesterol biosynthetic
pathway. The mice homozygous for the disrupted SS
gene (SS2/2) were embryonic lethal around midgestation. E9.5–10.5 SS2/2 embryos exhibited severe growth
retardation and defective neural tube closure. The lethal phenotype was not rescued by supplementing thedams either with dietary squalene or cholesterol. We
speculate that cholesterol is required for the development, particularly of the nervous system, and that thechorioallantoic circulatory system is not mature enoughto supply the rapidly growing embryos with maternalcholesterol at this developmental stage.

Squalene synthase (SS)1 (farnesyl-diphosphate:farnesyldiphosphate farnesyltransferase, EC 2.5.1.21) catalyzes the reductive head-to-head condensation of two molecules of farnesyl
diphosphate to form squalene, the first specific intermediate in
the cholesterol biosynthetic pathway (1). This enzyme is an
attractive target for cholesterol-lowering therapy, because theinhibition of this step theoretically may not perturb the nonsterol pathway, which is a potential problem in the use of theinhibitors of cholesterol synthesis (2). Indeed, several potent SS
inhibitors, such as squalestatins or zaragozic acids (3), 1,1bisphosphonates (4), and quinuclidine derivatives (5), successfully lower plasma cholesterol levels without adverse effects invivo.
Accumulating evidence supports the notion that cholesterol
metabolism plays an essential role in development, particularly of a like nervous system (see Ref. 6 for review). All of thenaturally occurring inborn errors in cholesterol metabolism,
such as Smith-Lemli-Opitz (SLO) syndrome (7, 8), desmosterolosis (9), and mevalonate kinase deficiency (10), are associatedwith severe developmental abnormalities. Moreover, mice lacking apoB (11, 12), microsomal triglyceride transfer protein (13),and megalin/gp330 (14) had anomalies in the nervous system.
Recently, the Hedgehog family of proteins, which are crucial forthe pattern formation in the vertebrate embryogenesis (see Ref.
15 for review), was shown to undergo post-translational modification by covalent attachment of a cholesterol molecule to the
biologically active amino-terminal fragment of these peptides(16). Furthermore, Patched, a putative cognate receptor for the
Hedgehog protein, shares homology with cholesterol-binding
proteins (17, 18). Together, it is tempting to hypothesize thatthe derangement of cholesterol synthesis affects the development of the nervous system through distorting Hedgehog
signaling.
To test the hypothesis that cholesterol itself is essential fornormal embryogenesis and whether the reduction of SS activities is associated with cholesterol-lowering effects, we havegenerated SS knockout mice. The SS2/2 mice were lethal
between E9.5 and E12.5 and exhibited severe retardation ofdevelopment. The SS1/2 mice expressed only 50% SS activities in the liver and testes compared with SS1/1 mice,
whereas their plasma lipoprotein profiles and responses to the
dietary challenge were unaffected.
EXPERIMENTAL PROCEDURES* This work was supported by Grant-in-aid for Scientific Research
10557104 from the Ministry of Education, Science, and Culture, thePromotion of Fundamental Studies in Health Science of the Organization for Pharmaceutical Safety and Research (OPSR), and Health Sciences Research Grants (Research on Human Genome and Gene Therapy) from the Ministry of Health and Welfare. The costs of publicationof this article were defrayed in part by the payment of page charges.
This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. Tel.: 181-3-38155411 (ext. 33113); Telefax: 181-3-5802-2955; E-mail: ishibash-tky@umin.ac.jp.
1
The abbreviations used are: SS, squalene synthase; SLO, SmithLemli-Opitz; apo, apolipoprotein; kb, kilobase(s); LDL, low density lipoprotein; VLDL, very low density lipoprotein; HDL, high density lipoprotein; HMG-CoA, 3-hydroxy-3-methyglutaryl-coenzyme A; HPLC,
high performance liquid chromatography; TC, total cholesterol; TG,
triglycerides; LDLR, low density lipoprotein receptor; E, embryonic day.
This paper is available on line at http://www.jbc.org

Generation of SS Knockout Mice—A fragment (528 base pairs) ofmouse SS cDNA was amplified by using primers that were designed
based on reported sequences of mouse SS cDNA (sense primer, 59GTCGCAAGGATGGAGTTCGT-39, and antisense primer, 59-GTGGCAGTACTTGTCCCAGT-39) (19). This polymerase chain reaction
product was used as a probe to clone a genomic DNA from the 129/Sv
mouse genomic library as described previously (20). A replacement-typetargeting vector was constructed; the short arm containing a 1.2-kb
PstI/BglII fragment in intron 3 and the long arm containing a 8.5-kb
EcoRV fragment spanning exons 6 –7 were inserted into the XhoI and
NotI sites, respectively, of the vector pPolIIshort-neobpA-HSVTK as
described previously (21). Thus, exons 4 –5, which contained a putative
catalytic site (22), were replaced by a neomycin-resistant cassette.
After linearization by digestion with SalI, the vector was electroporated into JH1 embryonic stem cells. Targeted clones, which had been
selected in the presence of G418 and 1-(2-deoxy, 2-fluoro-b-D-arabinofuranosyl)-5-iodouracil, were identified by polymerase chain reaction

30843

30844

Neural Tube Defects in Squalene Synthase Knockout

FIG. 1. Targeted disruption of the SS gene. A, a map of the SS locus, together with the sequence replacement gene-targeting vector and thetargeted SS allele. Exons (closed boxes) and introns (thick lines) are indicated. The gene targeting event replaced exon 4 –5 coding sequences andintron 4 with a cassette containing a neomycin resistance gene (Neor) driven by the RNA polymerase II promoter. Two copies of herpes simplex
virus thymidine kinase (HSV-TK) expression cassettes were used as a negative selection marker. Analysis of gene-targeting events and mouse
genotyping was performed by Southern blot analysis of BamHI/EcoRI-digested genomic DNA using a 0.5-kb probe (closed bar) in intron 3. P1,
primer 1; P2, primer 2; TGA, stop codon; Kbp, kilobase pairs. B, Southern blot of BamHI/EcoRI fragment in the targeted allele is 4.0 kb versus 5.0
kb in the wild-type allele. DNA was isolated from whole E10.5 embryos.
using the following primers: primer 1, 59-ATACAGGGGAGTGTGCCTTTCTTGTG-39 and primer 2, 59-GATTGGGAAGACAATAGCAGGCATGC-39 (Fig. 1). Homologous recombination was verified by Southern blot analysis after BamHI/EcoRI double digestion using a 0.5kilobase pair PstI/XhoI fragment as a probe (Fig. 1). Targeted
embryonic stem clones were injected into the C57BL/6 blastocysts yielding five lines of chimeric mice, which transmitted the disrupted allele
through the germ line. All experiments reported here were performed
with 129/Sv-C57BL6 hybrid descendants (F1 and subsequent generations) of these animals.
Breeding Experiments—SS1/2 mice were cross-bred to the LDL
receptor knockout mice (LDLR2/2) (21) to produce mice that were
heterozygous for the disrupted alleles of both SS and the LDL receptor
loci. An intercross of these animals was performed.
Diets—Three diets were used: (i) a normal chow (MF, Oriental
Yeast), (ii) a 1.25% cholesterol diet, which contains 1.25% (w/w) cholesterol, 5% (w/w) cocoa butter, and 0.5% (w/w) cholic acid (23), and (iii) a
2% squalene diet, which contains 2% (w/w) squalene (WAKO) (24).
Timed mating was performed and dietary supplementation was initiated 2 weeks before the mating.
SS Activity Assay—Liver and testes were homogenized in a buffer
containing 15 mM nicotinamide, 2 mM MgCl2, and 100 mM potassium
phosphate, pH 7.4, and centrifuged at 10,000 3 g for 20 min at 4 °C. The
supernatants were centrifuged at 105,000 3 g for 1 h at 4 °C, and theresultant pellets, a microsome fraction, were washed, resuspended in
the same buffer, and stored in aliquots at 280 °C. SS activities were
measured according to a modified method of Cohen et al. (25). In brief,
the microsome fractions (;20 mg) were incubated in 50 ml of a buffer
containing 20 mM [1-3H] farnesyl pyrophosphate (25 mCi/mmol), 1 mM
NADPH, 5 mM MgCl2, 6 mM glutathione, and 100 mM potassium phosphate, pH 7.4, at 37 °C for 15 min. Reaction was terminated by theaddition of 150 ml of chloroform/methanol (1:2, v/v) containing 0.2%
unlabeled squalene. After 50 ml of chloroform and 50 ml of 3 M NaOH

were added, the reaction mixtures were vortexed and centrifuged. The
infranatant organic phase was used for the determination of the radioactivities in the squalene produced.
Measurement of Hepatic Cholesterol Synthesis—Cholesterol synthesis in the liver was estimated by a modified method of Eisele et al. (26).
In brief, 6 SS1/1 and 6 SS1/2 mice (9 –10-week-old females) were
given food and water ad libitum and injected intraperitoneally with
[2-14C]acetate (37 kBq/kg body weight). After 30 min, animals wereeuthanized, and two pieces of the liver (0.3 g/each) were removed.
[1,2-3H]cholesterol (;10,000 dpm) was added as an internal standard,
and tissue samples were saponified in 2 ml of 15% KOH, 90% ethanol at
70 °C overnight. After the addition of 2 ml of water, nonsaponified lipids
were extracted with n-hexane, evaporated, and dissolved in ethanol/
acetone (1:1, v/v). Sterols were precipitated with digitonin. After washing the precipitates with 50% acetone, the radioactivities were determined. The results were expressed as 14C dpm/g wet weight of liver/30
min.
Northern Blot Analyses—Poly(A1) RNA was isolated and pooled fromthe livers of six animals. 1.2 mg were subjected to 1% agarose electrophoresis in the presence of formalin. The fractionated RNA was transferred to Hybond N (Amersham Pharmacia Biotech). The filters were
hybridized to 32P-labeled cDNA probes: the LDL receptor, 3-hydroxy-3methylglutaryl-coenzyme A (HMG-CoA) reductase, and cholesterol
7a-hydroxylase.
Plasma Lipoprotein Analyses—After a 12-h fast, blood was collected
in tubes containing EDTA. Plasma levels of total cholesterol (TC) andtriglycerides (TG) were determined enzymatically using kits (Determiner TC555 and Determiner TG555, Kyowa Medex). Lipoproteins
were fractionated by high performance liquid chromatography (HPLC)
as described (23), and the cholesterol contents in each lipoprotein fraction were determined.
Morphological Studies—Embryonic tissues were examined by standard histological techniques.

Neural Tube Defects in Squalene Synthase Knockout

30845

TABLE I
Genotypes of offspring from intercrosses of SS1/2 mice
Genotypes were confirmed by Southern blot analysis.
Age

SS1/1

SS1/2

SS2/2

Resorbeda

Total

E9.5
E10.5
E9.5–10.5b
E12.5
E13.5
Postnatal

12
19
4
6
12
50

18
42
14
7
25
99

2
8
3
0
0
0

9
14
12
4
21
NAc

41
83
33
17
58
149

a
Significant number of embryos exhibited appearance of resorption. Genotyping of these embryos were ambiguous because of small DNA
contents.
b
E9.5 and E10.5 embryos were analyzed together.
cNA, not applicable.

Statistics—Data are represented as mean 6 S.D. The Student’s t test
was used to compare the mean values between two groups.
RESULTSSS Knockout Mice—Two genomic clones were isolated froma library with a cDNA probe. The restriction mapping revealed that one genomic clone (Fig. 1A) consisted of 8 exons
spanning more than 26 kb. A replacement-type vector, whichallowed the deletion of exon 4 –5, was constructed and used togenerate heterozygous SS knockout mice (SS1/2). SS1/2
mice, which were viable and fertile, were intercrossed toobtain homozygous SS knockout mice (SS2/2). No viable
SS2/2 mice were identified among 149 weaned offspring,
although 99 were heterozygotes (Table I). To determine thedevelopmental stage where the embryos were lethal, the embryos were genotyped at four different embryonic stages: 9.5,
10.5, 12.5, and 13.5 days postcoitum (Table I, Fig. 1B). At
E9.5–10.5, SS2/2 was identified at the frequency significantly lower than the expected mendelian frequency (x2 58.35, p , 0.05). All SS2/2 embryos were significantly
smaller than SS1/1 embryos with comparable developmental age. SS2/2 embryos exhibit a wide variety of morphology. Among 13 SS2/2 embryos, only 1 at E9.5 showed anearly normal shape with a small forebrain (Fig. 2C), 6 had ashape that is similar to E8.5 embryos, early somite-stageembryos, with an open head fold (Fig. 2D). Their microscopic
examination showed necrotic neuronal cells with condensation of nuclei, i.e. apoptotic cells (Fig. 2, F and G). Four weremuch smaller and ambiguous and did not have somites (Fig.
2B). At E12.5–13.5, however, no SS2/2 were identified(Table I).
Reduction in mRNA Levels and Activities of SS in Heterozygotes of SS Knockout Mice—The SS mRNA levels in theSS1/2 mice were reduced by 60% in the liver (Fig. 3A).
Consistently, their SS activities were significantly reduced
by 50% both in the liver and testes (Fig. 3B). In the liver, themRNA levels of the LDL receptor appeared to be increased by
20% in the SS1/2 mice (Fig. 3A). On the other hand, themRNA of cholesterol 7a-hydroxylase appeared to be decreased by 20%. There was no significant change in themRNA levels of HMG-CoA reductase between the SS1/1
and SS1/2 mice.
Cholesterol Synthesis in the Liver—If SS is the rate-limiting
step for TC synthesis, cholesterol synthesis is expected to be
reduced in the liver. However, the amounts of [14C]acetate
incorporated into cholesterol in the liver were not different
between the wild-type and SS1/2 mice (10,173 6 6444 versus
13,992 6 10,276 dpm/g liver/30 min, p . 0.05, n 5 6).
Plasma Lipoprotein Analyses—To determine whether the
reduction in the SS activities in the liver is associated with thechanges in plasma lipoprotein profile, we determined theplasma lipid levels. Neither plasma TC nor TG levels weredifferent between the wild-type and SS1/2 mice, which were

FIG. 2. Phenotype of SS2/2 embryos. A, an E9.5 SS1/1 embryowith normal developmental morphology; B, an E9.5 SS2/2 embryo thatlacked somite structure; C, an E9.5 SS2/2 embryo that was muchsmaller than wild-type embryos but apparently almost normal except
for an undeveloped forebrain; D, E10.5 SS2/2 embryos that had open
heads (indicated by white arrows). Bar, 1 mm. E, a sagittal section of an
E9.5 SS1/1 embryo as a normal control; F, a sagittal section of an
E10.5 SS2/2 embryo that is shown in D. The unclosed head is indicatedby an arrow. G, microscopic examination of a part of the head that isshown in F. Some of the cells with condensed nuclei are indicated bywhite arrows. Hematoxylin eosin staining was used.

fed a normal chow (Table II). There was no significant difference in the amounts of VLDL, LDL, or HDL cholesterol as
measured by HPLC (Table III). To evaluate the effects of the
suppression of SS activities in the liver on the plasma lipoprotein profile in the setting of LDL deficiency, we further crossbred these animals with LDL receptor knockout mice to generate SS1/2;LDLR2/2 mice. Neither plasma TC nor TG
levels were different between the SS1/1;LDLR2/2 andSS1/2;LDLR2/2 mice (Table II).
Attempt to Rescue the Homozygotes by Feeding with Squaleneor Cholesterol—If the embryonic lethality of the homozygotes
results from the cholesterol deficiency, it is theoretically possible that dietary supplementation of squalene or cholesterol
would reverse the phenotype. To test this hypothesis, we fed
the pregnant SS1/2 mice with the diets supplemented witheither 2% squalene or 1.25% cholesterol. No viable SS2/2
mice were identified among 21 offspring for squalene rescue (430846

Neural Tube Defects in Squalene Synthase Knockout

FIG. 3. SS expression and SS activities in SS1/2 mice. A, Northern blot for
SS, LDL receptor, HMG-CoA reductase,cholesterol 7a-hydroxylase, and b-actin
as a loading control using liver poly(A1)RNA. W, wild-type; H, SS1/2. B, SS activities in the liver from SS1/1 andSS1/2 mice. SS activities in the microsome prepared from the liver (n 5 6) andtestis (n 5 3) were measured as describedunder “Experimental Procedures.”

TABLE II
Plasma lipid levels
Mice aged 10 –11 weeks were maintained on normal chow. After a
12-h fast, blood was collected for determination of TC and TG. Data areexpressed as mean 6 S.D. No significant difference was noted between
SS1/1 and SS1/2 for either plasma TC or TG levels, irrespective of thepresence of the LDLR mutation.
LDLR locus

SS locus

n

TC

TG

1/1
1/1
2/2
2/2

1/1
1/2
1/1
1/2

7
18
11
7

96.4 6 18.6
93.8 6 25.0
306.4 6 21.4
294.4 6 24.2

132.3 6 54.5
119.1 6 38.7
167.5 6 30.9
119.1 6 38.7

TABLE III
HPLC lipoprotein profiles
Five animals aged 8 –10 weeks were maintained on normal chow.
After a 12-h fast, blood was collected in tubes containing EDTA. Plasma
was subjected to HPLC lipoprotein analyses. Total cholesterol contents
in three lipoprotein fractions (VLDL, LDL, and HDL) were calculated
and expressed as mean 6 S.D. No significant differences were noted
between SS1/1 and SS1/2 for each lipoprotein.

SS1/1
SS1/2

VLDL

LDL

HDL

6.3 6 4.4
4.7 6 2.0

5.5 6 2.4
7.7 6 3.4

61.8 6 14.6
65.9 6 7.5

1/1 versus 17 1/2 versus 0 2/2, x2 5 6.9, p , 0.05,) and 40
offspring for cholesterol rescue (10 1/1 versus 30 1/2 versus 0
2/2, x2 5 6.7, p , 0.05).
DISCUSSION

In the present study, we demonstrate that SS is essential forthe normal development of the embryo. 50% reduction in the
SS activities in the liver did not result in alterations in theplasma lipoprotein profiles. Moreover, the lethal phenotypes ofSS2/2 embryos were not reversed by oral administration ofeither cholesterol or squalene to the dams.
During the early development of animals, particularly during fetal stage, there is a marked demand for new sterol.
Cholesterol is required for new membrane synthesis, for maintenance of existing membranes, and for the synthesis of hormone and bile acids. As in the adult, this cholesterol is suppliedby either de novo synthesis within the fetal compartment or bytransfer from the maternal compartment to the fetus throughuptake of cholesterol carried in lipoproteins. Previous studies
have shown that sterol synthesis is markedly increased in thedeveloping fetus including a preimplantation embryo (27–29).
In addition, transport of maternal LDL and HDL to the fetal

yolk sac is drastically increased (30). Evidence suggests thatseveral lipoprotein receptor systems mediate the transfer ofmaternal lipoprotein: the LDL receptor, megalin/gp330, VLDL
receptor, and low density lipoprotein receptor-related protein
for the uptake of lipoproteins containing either apoB-100 or
apoE (reviewed in Refs. 6 and 31), and scavenger receptor B-1
for the binding of apoA-I containing lipoproteins (32). Because
mice lacking the LDL receptor (21) or VLDL receptor (33) are
fertile, there may be some redundancy in the receptor function.
On the other hand, mice lacking low density lipoprotein receptor-related protein (34) or megalin/gp330 (14) are embryoniclethal, suggesting the pivotal role of these receptor inembryogenesis.
In this concern, it is noteworthy that SS2/2 embryos develop to the stage around E9.5. Results indicate that cholesterol required for growth is exclusively supplied from the dams
in SS2/2 mice. Indeed, it has not been reported that pharmacological doses of HMG-CoA reductase inhibitors or SS inhibitors cause preimplantation lethality (35). It is known that
nonpolar squalene, an immediate product of squalene synthase, is transported in association with VLDL in the plasma(36) and subsequently by a squalene and sterol carrier protein(37). Therefore, embryos may utilize squalene that is diffusedfrom extra embryonic tissues for cholesterol synthesis. Furthermore, in the rodents, the nutritional supply of the embryo
during the immediate postimplantation period is dependent on
the resorptive and synthetic capacity of the yolk sac (38). Presumably, cholesterol transported across this maternal fetal
barrier is utilized for development.
The embryonic lethality of the SS2/2 mice may result fromeither cholesterol deficiency, toxic precursor buildup, or both.
The cholesterol-supplying system in the early gestational period may no longer be effective beyond midgestation when afunctional chorioallantoic circulatory system is established(38). It is conceivable that the placental system is too immature
to fully compensate for the complete deficiency of the de novocholesterol synthesis around this developmental stage.
Belknap and Dietschy (27) have shown that the de novo cholesterol synthesis remains highly active during the whole midgestational period and that the contents of newly synthesizedsterols are maximal at E17 in the rat fetus. Similar failure tocatch up with the increasing demand may underlie the lethality of mice lacking apoB (11, 12, 39) or microsomal triglyceridetransfer protein (13), because both mutants die at the samedevelopmental stage. Alternatively, precursor buildup may
have lethal effects on the embryos as postulated for SLO syndrome (7, 40). Inhibition of SS by compounds resulted in mas-

Neural Tube Defects in Squalene Synthase Knockout
sive urinary excretion of farnesol-derived dicarboxylic acids(41), indicating the elevation of the plasma concentration of
farnesol and its metabolites. Farnesol has recently been foundto be a biologically active substance; it inhibits arterial vasoconstriction through blocking L-type Ca21 channels in vascularsmooth muscle cells (42, 43). Other studies have reported theexistence of a farnesol-specific, orphan nuclear receptor in vertebrate cells, the farnesoid X-activated receptor, but its precise
functions remain unknown (44). Therefore, it is possible thatfarnesol or its metabolites may have toxic effects on the embryos. This toxic precursor buildup hypothesis may be moreplausible, because supplementation of the dams with dietarysqualene failed to rescue the lethal phenotype.
As mentioned above, neural developmental anomalies arecommon in mice with the induced mutations in cholesteroltransport systems (11–14), humans with genetic defects inthe enzymes of the cholesterol biosynthetic pathway (7–9),
and fetuses born to animals treated with the inhibitors ofthese enzymes (36, 40). These observations lead us to the
speculation that SS2/2 mice develop anomalies in the nervous system. In this regard, it is noteworthy that SS2/2 hadan open neural plate (Fig. 2D). Because the general growth ofSS2/2 was severely impaired, the defective neural tube
closure may simply reflect general growth retardation. Interestingly, the phenotypes of SS2/2 mice are different fromthose of infants with SLO syndrome, despite the fact that the
responsible enzymes are involved in the proximal part ofcholesterol synthesis (8). In the SLO syndrome, D7-reductase
deficiency may be partially compensated by D24-reductase, analternative enzyme involved in the terminal step of cholesterol biosynthesis, thereby leading to a milder phenotype (9).
The differences may also arise from either species differences
or build up of different precursors.
Recently, a link has emerged between cellular cholesterol
metabolism and signaling molecules that are crucial for early
development, the Hedgehog proteins (16 –18). Drosophila
Hedgehog and murine sonic Hedgehog proteins undergo posttranslational modification in which a cholesterol moiety is attached to its amino terminus (16). Patched, the putative receptor for Hedgehog protein (17), shares homology with the
Nieman-Pick C1 protein (18), HMG-CoA reductase (45), andsterol regulatory element-binding protein cleavage-activating
protein (46). This shared domain may function as a cholesterolbinding domain that is crucial for their functions. A certain
part of the developmental anomalies of SLO syndrome may beascribed to the defective function of the sonic Hedgehog signaling pathway; indeed, sonic Hedgehog knockout mice develop aholoprosencephalic phenotype similar to that in some SLO
syndrome cases and in megalin-deficient mice (47). The phenotypes of SS2/2 are rather similar to Patched-deficient micethat have open and overgrown neural tubes (48). Further studies will be needed to elucidate the similarity in the phenotypesof these two mutants.
SS1/2 mice are apparently normal. Both mRNA levels andenzymatic activities for SS were reduced by 50% in the liver,
but the overall hepatic cholesterol synthesis was not reduced,
confirming that SS is not the rate-limiting enzyme in cholesterol biosynthesis (Fig. 3). The apparent changes in the LDL
receptor and cholesterol 7a-hydroxylase mRNA levels may reflect hepatic cholesterol homeostasis. In this concern, it is noteworthy that zaragozic acid, an SS inhibitor, caused a compensatory increase in the hepatic mRNA levels of the LDL
receptor, HMG-CoA reductase, and HMG-CoA synthase as well
as SS itself along with marked suppression of cholesterol synthesis (49). Our results, however, strongly indicate that inhibition of SS activities by 50% is not sufficient to reduce the30847

plasma LDL cholesterol levels with or without the functional
LDL receptor at least in mice (Table II).
Acknowledgments—We thank Mitsuyo Okazaki (Tokyo Medical Dental University) for the HPLC lipoprotein analyses; Kimiko Saito,
Megumi Herai, and Mihoko Kusubae for animal maintenance; and Yoko
Iizuka, Yuan Xunmei, Yoshiaki Tamura, Tetsuya Kitamine, andHitoshi Shimano for helpful discussion.
Note Added in Proof—Bile acids have been found to be natural
ligands for the FXR (50 –52).
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