Laboratory of Developmental Neurobiology, The Rockefeller University, New York, New York 10021
Zellweger syndrome is a peroxisomal biogenesis disorder that results in abnormal neuronal migration in the central nervous system and severe neurologic dysfunction. The pathogenesis of the multiple severe anomalies associated with the disorders of peroxisome biogenesis remains unknown. To study the relationship between lack of peroxisomal function and organ dysfunction, the PEX2 peroxisome assembly gene (formerly peroxisome assembly factor-1) was disrupted by gene targeting.
Homozygous PEX2-deficient mice survive in utero but die several hours after birth. The mutant animals do not feed and are hypoactive and markedly hypotonic. The PEX2-deficient mice lack normal peroxisomes but do assemble empty peroxisome membrane ghosts. They display abnormal peroxisomal biochemical parameters, including accumulations of very long chain fatty acids in plasma and deficient erythrocyte plasmalogens. Abnormal lipid storage is evident in the adrenal cortex, with characteristic lamellar-lipid inclusions. In the central nervous system of newborn mutant mice there is disordered lamination in the cerebral cortex and an increased cell density in the underlying white matter, indicating an abnormality of neuronal migration. These findings demonstrate that mice with a PEX2 gene deletion have a peroxisomal disorder and provide an important model to study the role of peroxisomal function in the pathogenesis of this human disease.
NEURONAL migration disorders are a diverse group
of human brain malformations that primarily affect development of the cerebral cortex. Over 25 syndromes with abnormal neuronal migrations have been
described and many have been proven to be genetic in origin (Dobyns and Truwit, 1995 Zellweger syndrome is a severe, autosomal recessive human neuronal migration disorder. It is a prototype for peroxisome biogenesis disorders (PBDs)1 in which the organelle is not correctly assembled, leading to multiple defects
in peroxisome function (Lazarow and Moser, 1994 In the Zellweger central nervous system, there is disordered neuronal migration leading to characteristic cytoarchitectonic abnormalities involving the cerebral hemispheres,
the cerebellum, and inferior olivary complex (Volpe and Adams, 1972 In the PBDs, there is a block in the posttranslational import of peroxisomal matrix proteins into the organelle,
whereas peroxisomal membrane proteins are assembled in
the cell into "membrane ghost" structures that appear to
be devoid of content (Santos et al., 1987 PBDs are genetically heterogeneous disorders that may
arise from defects in at least 11 different genes (Moser et al.,
1995 Zellweger syndrome is one of the few human neuronal
migration disorders for which genetic defects have been
defined. Elucidation of the causal link between absence of
peroxisome function and the neuronal migration defect
will certainly require the use of animal models, yet no naturally occuring models exist. We have generated a null
mutation of the murine PEX2 gene locus and demonstrated that homozygous PEX2-deficient mice show abnormal brain development with a prominent defect in the
formation of the cerebral cortex. These mice provide a
model system for analyzing the role of peroxisomal function in early phases of development, when cell migration is establishing the form of different brain regions.
Cloning of the Mouse PEX2 Gene
A 658-bp fragment of the mouse PEX2 gene was isolated by PCR using
genomic DNA isolated from C57Bl/6 mouse liver and primers based on
the rat PEX2 sequence (Tsukamoto et al., 1991 The PCR fragment was used to probe a 129SVJ mouse genomic
Gene Targeting and Generation of PEX2-deficient Mice
A targeting construct (Fig. 1 A) was prepared by ligation of a 5.05-kb
BsteII (blunted)-XbaI PEX2 genomic fragment into a pKS-NT vector
(Wurst et al., 1994 Chimeric mice were generated by injection of four ES cell clones into
blastocysts of C57BL/6 donor mice as described (Papaioannou and
Johnson, 1993 Southern Blot Analysis
Genomic DNA of ES cell clones was isolated as described (Wurst and
Joyner, 1993 PCR Analysis
PCR of genomic tail DNA of wild-type, heterozygous, and homozygous
mice was carried out with a set of three primers: primer a (5 Northern Blot Analysis
Total RNA was isolated by the single-step guanidinium thiocyanate-phenol method (Chomczynski and Sacchi, 1987 Biochemical Analyses
VLCFA and Plasmalogens.
Blood (~50-100 µl) was collected from newborn mice after decapitation and 10 µl of 7.5% K3EDTA added to prevent clotting. The sample was centrifuged for 10 min at 1,000 g and plasma
separated from the pelleted erythrocytes. Samples were stored at Liver Fractionation and Catalase Assay.
The liver from newborn mice
was collected, weighed, and placed in ice-cold 0.25 M sucrose, 1 mM
EDTA, 0.1% ethanol, pH 7.4 (SVE) and processed essentially as described (Lazarow et al., 1991 Immunohistochemistry
Fibroblasts were isolated from skin of newborn mice. The skin was finely
minced, digested in trypsin, and plated in DME, 10% heat inactivated fetal calf serum, 100 mM L-glutamine, and 1× antibiotics. Cells were plated
on printed microscopic slides (Cel-Line, Newfield, NJ) with 12-mm diam
wells that had previously been coated with 0.01 mg/ml poly-L-lysine and
fixed 16-20 h after plating with 4% paraformaldehyde for 20 min at room
temperature. Antibodies against bovine catalase and rat liver PxIMPs were
a gift from P.B. Lazarow (Mount Sinai Hospital, New York, NY); antibody against peroxisomal 3-ketoacyl-CoA thiloase was a gift from T. Hashimoto (Shinshu University School of Medicine, Matsumoto, Nagano, Japan). The procedure for indirect immunofluorescence was essentially as
described (Santos et al., 1988 Mice at P0 and embryos at E15 were deeply anesthetized and perfused
intracardially with 4% paraformaldehyde in PBS, pH 7.4. The brain was
removed and postfixed at 4°C for 24 h in the same fixative, rinsed in PBS,
and 100-µm sections prepared on a vibratome (coronal plane for forebrain; saggital plane for cerebellum). Sections were permeabilized and
blocked in 1% Tween, 20% normal goat serum in PBS for 90 min. Antibodies were diluted in the above buffer and incubated with the sections
overnight at 4°C, including: rabbit anti-GFAP (Dako Corporation, Carpinteria, CA) at 1:200, rabbit anti-BLBP (a gift from N. Heintz, Rockefeller
University) at 1:1,000, mouse monoclonal antibody RC2 (Misson et al.,
1988 Cells and sections were photographed with a microscope (Axiovert;
Zeiss, Inc., Thornwood, NY) fitted with fluorescent illumination plan-neofluor 2.5, 5, 10, 20, 40, and 100× objectives, 1.6× zoom capability, and
an Axiophot camera module. For RC2, BLBP, and calbindin D-28k staining, sections were imaged with a confocal scan head (MRC600; Bio Rad,
Hercules, CA) and host computer system (PC-AT; IBM, Danbury, CT).
Histology
Brain, spinal cord, and kidney from newborn mice were removed and
fixed by immersion in Bouin's fixative for 4 to 20 h at room temperature,
followed by rinsing in 70% ethanol. Liver, adrenal, and patella were fixed
in 10% formalin. Tissues were embedded in paraffin following standard
procedures, sectioned at 7 to 10 µm, and stained with hematoxylin-eosin.
Carefully matched coronal sections of brains were photographed with a
microscope (Axiovert; Zeiss, Inc.) and print pictures compared to analyze
histologic abnormalities in mutant versus control animals.
Whole mount alizarin red/alcian blue staining of newborn mouse skeletons was performed as previously described (Lufkin et al., 1992 Electron Microscopy
For morphologic studies, liver and adrenal gland from newborn mice were
diced in 2.5% glutaraldehyde in 100 mM cacodylate, pH 7.4, and fixed for
3 to 4 h on ice. The tissues were then postfixed in 1% osmium tetroxide in
the same buffer, treated with uranyl acetate, dehydrated in ethanol, propylene oxide, and embedded in Epon.
For DAB catalase cytochemistry, liver pieces were fixed in 2% paraformaldehyde containing 2.5% glutaraldehyde in 100 mM cacodylate, pH
7.4, for 3 h. The alkaline 3,3 Grids were viewed in an electron microscope (100 CX; Jeol Ltd., Tokyo, Japan) operated at 80 kV.
Generation of PEX2-deficient Mice
A fragment of the mouse PEX2 gene was isolated by PCR
using genomic mouse liver DNA and primers based on the
rat PEX2 sequence (see Materials and Methods). This fragment was used as a probe to isolate genomic and cDNA
clones for PEX2, and the genomic organization was determined (Fig. 1 A). To disrupt the PEX2 locus, a targeting vector was constructed in which exon 5, containing the
translation initiation site and entire coding sequence for
the gene, was replaced by the neomycin phosphotransferase gene (Fig. 1 A). The targeting construct was electroporated into R1 ES cells (Nagy et al., 1993 No homozygous mutant mice were found in 175 surviving offspring at 3 to 4 wk of age. A normal Mendelian ratio
of wild-type/heterozygous mice was observed at this age
(60 +/+:112 +/
Biochemical Abnormalities in PEX2-deficient Mice
Plasma and erythrocyte samples were collected from newborn mice to assay for VLCFA and erythrocyte plasmalogens. Tables I and II show plasma VLCFA and erythrocyte
plasmalogen levels, respectively, in control and homozygous-mutant mice along with comparative data from patients
with peroxisomal biogenesis disorders. In the PEX2-deficient mice there is a marked increase in plasma VLCFA,
both saturated and monounsaturated, with an ~8-12-fold
increase in the c26/22 ratio and 9.5-fold increase in the c26:0
concentration versus control mice (Table I). These elevations are similar to that seen in human peroxisomal biogenesis disorders. The erythrocyte plasmalogen levels
(Table II) are severely deficient in the mutant mice, with a
50-60-fold decrease relative to control mice. This plasmalogen deficiency is most similar to that seen in the disorder RCDP, where the plasmalogen deficiency is most severe. Mice heterozygous for the PEX2 gene deletion show
no significant difference from control mice in plasma
VLCFA and erythrocyte plasmalogens.
Table I.
Plasma Total Very Long Chain Fatty Acids
Table II.
Eythrocyte Plasmalogens
). Classical studies on the
formation of cortical architecture in human brain have revealed four basic steps, including neuronal precursor proliferation in germinal zones, directed migration from germinal zones, assembly of postmigratory cells into discrete
layers, and the formation of synaptic connections. The migration of immature cortical neurons on a scaffold of radial glial cells during mid to late gestation is a remarkable
feature of cortical formation (Rakic, 1972
; Hatten, 1990
),
providing mechanisms for the disposition of different classes
of neurons into specific neuronal layers. While attention has
focused on neuron-glia interactions during neuronal locomotion on glial fibers (Zheng et al., 1996
), cell organelles
such as the peroxisome, which function in cellular metabolism, are also critical to this process.
). These
infants are readily recognized in the early postnatal period by their characteristic dysmorphic facial features, profound generalized hypotonia, psychomotor delay, and seizures. There is progressive dysfunction of the liver and
central nervous system, culminating in death within the
first year of life. Morphologic changes are present in multiple organ systems including central nervous system malformations, renal cysts, hepatic fibrosis, joint calcifications, striated adrenocortical cells (Goldfischer et al., 1973
), and ocular abnormalities. The clinical spectrum of PBDs also
includes the milder disorders neonatal adrenoleukodystrophy (NALD) and infantile Refsum's disease as well as
classic rhizomelic chondrodysplasia punctata (RCDP). In
these latter disorders, brain malformations are absent or
much less prominent than in Zellweger syndrome.
; Evrard et al., 1978
). The malformation of the cerebral cortex is most severe and reproducibly results in
gyral abnormalities centered around the Sylvian fissure
with a stereotypic medial pachygyria and lateral polymicrogyria. These gyral abnormalities reflect a reduced neuronal population in the cortex and large numbers of subcortical heterotopic neurons. These architectonic features,
encountered postnatally as well as in pathologic studies on
Zellweger fetuses (Powers et al., 1985
, 1989
), indicate that
this malformation results from a developmental disturbance in the migration of neuroblasts to form the cerebral
cortical plate throughout much or all of the cytogenetic
epoch. In the cerebellum, one finds heterotopic Purkinje cells (PCs) in the white matter, subjacent to intact Purkinje and granule cell laminae, or combinations of abnormally arranged PCs and granule cells (heterotaxias). Dysplastic changes of the principal olivary nucleus and dentate
nucleus are seen with a simplification in the normal serpiginous course, laminar discontinuities and condensation of
neurons around the periphery of the nuclear islands. In addition, abnormalities develop postnatally in white matter
including decreased myelination, reactive astrocytosis, and
lipid accumulations in astrocytes.
, 1988
). The import of matrix proteins bearing the COOH-terminal PTS1
and/or the NH2-terminal PTS2 peroxisomal topogenic targeting signal (Purdue and Lazarow, 1994
; Rachubinski and
Subramani, 1995
) may be differentially affected in these
patients (Motley et al., 1994
; Slawecki et al., 1995
). Biochemical studies have shown a reduced activity of multiple
peroxisomal matrix enzymes in the PBDs. In Zellweger
syndrome, the peroxisomal dysfunction is characterized by
accumulation of very long chain fatty acids (VLCFA), deficient plasmalogen synthesis, and accumulation of pipecolic acid, phytanic acid, and bile acid intermediates (Lazarow and Moser, 1994
).
). Complementation group 10 (group F in Japan) was
the first complementation group for which a genetic defect
was defined (Shimozawa et al., 1992
) and demonstrated to
be a mutation in the peroxisome assembly factor-1 gene
(now called PEX2; Distel et al., 1996
). This zinc-finger- containing, 35-kD peroxisomal integral membrane protein
was originally described in a Chinese hamster ovary cell
line selected for absence of peroxisomes (Tsukamoto et al.,
1990
, 1991
) and subsequently found to be mutated in another patient with severe Zellweger syndrome (Shimozawa et al., 1993
) and two additional Chinese hamster
ovary cell lines (Thieringer and Raetz, 1993
). The majority
of mutations are nonsense mutations leading to premature
termination of this protein. The role of Pex2p in peroxisome assembly is not yet understood.
Materials and Methods
). Primers were: forward,
5
-CAGTGCATGAATTTTGTGGTTGGA-3
extending from position 567; and reverse 5
-CCAGTGATTAGAACTGAGTTGTGC-3
extending from position 1,225 of the rat mRNA sequence. (PCR cycle parameters are for a thermocycler [480; Perkin Elmer, Norwalk, CT]). After 4 min of denaturation at 94°C and 2 min of renaturation at 60°C, 30 cycles
were run with 1.5 min at 72°C, 1 min at 94°C, 1 min at 60°C, followed by a
final extension for 10 min at 72°C. A single appropriately sized fragment
was obtained, subcloned into pCRII plasmid vector (Invitrogen Corp.,
San Diego, CA), and sequenced to confirm the identity as a PEX2 gene
(data not shown).
FIX
II phage library (946309; Stratagene, La Jolla, CA) and a C57BL/6 P6 cerebellum cDNA library in
ZAP (kindly provided by G. Dietz, Rockefeller
University, New York, NY). These sequence data are available from Genbank/EMBL/DDBJ under accession number AF031128. The deduced genomic structure of the mouse PEX2 gene is shown in Fig. 1 A.
Fig. 1.
Generation of PEX2-deficient mice. (A) Schematic
representation of the PEX2 genomic locus (top), targeting vector
(middle), and targeted allele (bottom). Exon sequences are indicated by the black boxes. Selectable neomycin resistance (NEO)
and thymidine kinase (TK) cassettes, under control of the PGK
promoter (shaded box), are shown. Dashed lines indicate the region of homology between locus and targeting vector. The origins
of the 5 and 3
probes (X and Y) and the size of the resulting restriction fragments (long arrows) used to identify the targeted ES
cell lines are shown. In the targeted locus, exon 5, containing the translation initiation site and entire coding sequence, is replaced by the NEO gene, resulting in a null allele. Bg, BglII; Bx, BstXI; E, EcoRI; H, HindIII; X, XbaI. (B) Southern blots of genomic
DNA isolated from targeted ES cell lines. (Lanes 1-3) EcoRI digest gives a wild-type 6-kb fragment and targeted 9-kb fragment.
(Lanes 4-6) BglII digest gives a wild-type 13.8-kb fragment and
targeted 9.6-kb fragment. (C) PCR analysis of tail DNA of wild-type (+/+), heterozygous (+/
), and homozygous (
/
) mice.
Primers used for the PCR (a-c) are indicated. M, BRL 1-kb ladder marker. (D) Northern blot hybridization analysis of RNA
from liver (lanes 1-3), brain (lanes 4-6), and kidney (lanes 7-9).
20 µg of total RNA from +/+ (lanes 1, 4, and 7), +/
(lanes 2, 5,
and 8), and
/
(lanes 3, 6, and 9) newborn mice were hybridized
with an exon 5 PEX2 genomic probe or GAPDH probe. Arrowheads indicate the three PEX2 transcripts identified. Size marker,
BRL RNA ladder.
[View Larger Version of this Image (43K GIF file)]
) digested with EcoRI (blunted) and XbaI to yield pNT-BX , followed by ligation of a blunt 2.5-kb HindIII-BstXI PEX2 genomic
fragment into the pNT-BX vector digested with SalI (blunted). The targeting vector (50 µg) was linearized with XhoI and electroporated into R1
embryonic stem (ES) cells (5.6 × 106) as previously described (Wurst and
Joyner, 1993
). Transfected cells were plated onto neor, mitomycin-C-inactivated primary embryonic fibroblasts and double selection (250 µg/ml
G418; 0.2 µm gancyclovir) was started 24 h after the electroporation. Resistant ES clones were selected 7 to 9 d after the transfection. Targeted
clones were identified by Southern blot hybridization analysis of genomic
DNA (Fig. 1 B) using probes external to the targeting vector (see below). The frequency of homologous recombination was 10 and 20% of double drug-resistant colonies in two separate electroporations.
). Highly chimeric males from three of the four ES cell lines
were intercrossed with C57BL/6 mice, and agouti offspring were tested for
germline transmission by Southern blot analysis. Homozygous PEX2-deficient mice were obtained by interbreeding F1 heterozygotes; F2 offspring
were genotyped by PCR analysis (see below).
) and was restriction digested with EcoRI or BglII for the analysis of homologous recombinants. DNA was transferred to Gene-screen
Plus (New England Nuclear, Boston, MA) and hybridized according to
the manufacturer's protocol. Southern blots were probed with a 0.7-kb
BsteII-ClaI PEX2 genomic fragment (5
-probe, probe X) for EcoRI digests
or a 0.8-kb BstXI PEX2 genomic fragment (3
-probe, probe Y); both of
these probes flank the regions of homology in the targeting vector (Fig. 1 A).
-GGGATAGGGTCAAGATA-TAAAGG-3
), primer b (5
-TAGATGGTGAACCTCCACAGGAAA-3
), and primer c (from neo gene; 5
-ATGCCTGCTTGCCGAATATCATG-3
). A standard 20-µl reaction contains:
~0.25 µg DNA and 16 pmol of each primer in reaction buffer containing
10 mM Tris-HCl, pH 8.8, 75 mM KCl, and 1.5 mM MgCl2. After denaturation for 3 min at 94°C and renaturation for 2 min at 60°C, 30 cycles were
run with 1.5 min at 72°C, 1 min at 94°C, 1 min at 60°C followed by a final extension for 10 min at 72°C. PCR products were analyzed on 2% agarose
gels and yielded the wild-type allele of 400 bp and targeted allele of 800 bp
(Fig. 1 C).
). RNA (20 µg) from brain,
liver, and kidney of newborn mice (P0) was heat denatured and fractionated by electrophoresis through a 1.2% formaldehyde-agarose gel and
transferred onto a nylon membrane (Gene-screen; New England Nuclear)
by capillary blotting. The filter was hybridized according to the manufacturer's instructions with a randomly 32P-labeled 1.25-kb EcoRI PEX2 genomic fragment that contains exon 5 (Fig. 1 A). The final high stringency
wash was done in 0.2× SSC, 1% SDS at 65°C. The filter was then stripped
of radioactivity and reprobed with a 1.2-kb PstI fragment of GAPDH DNA.
70°C
before analysis. Analyses of VLCFA in plasma and erythrocyte plasmalogens were kindly performed for us by Dr. H. Moser and A. Moser
(Kennedy Krieger Institute, Baltimore, MD) as described (Bjorkhem et
al., 1986
; Moser and Moser, 1991
).
). Briefly, each liver was homogenized in 0.75 ml
of SVE (10-15 vol) containing 5 µg/ml of leupeptin, pepstatin, and antipain using a motor-driven Potter-Elvehjem homogenizer and a postnuclear supernatant (PNS) prepared. Two thirds of the PNS was centrifuged at 17,000 g and separated into supernatant and pellet. Catalase (peroxisomes) and N-acetyl-
-glucosaminidase (lysosomes; as an internal control
that homogenization was not excessive) were assayed in the postnuclear
supernatant, 17,000 g supernatant and pellet as previously described (Lazarow et al., 1988
). Protein was measured by BCA reagent (Pierce, Rockford, IL).
) and used 1% NP40 to permeabilize the
cells.
) at 1:1, and rabbit anti-calbindin D-28k (SWant, Bellinzona, Switzerland) at 1:1,000. Sections were washed three times for 30 min in PBS. Secondary antibody conjugated to fluorescein (Cappel Research Products,
Cochranville, PA) was used at 1:200.
).
-diaminobenzidine (DAB) reaction was carried out as previously described (Shio and Lazarow, 1981
). After reaction,
the slices were washed, postfixed in osmium tetroxide, and processed as
above for morphology.
Results
) and targeted clones identified by Southern blot hybridization (Fig. 1 B). Highly chimeric males from three ES cell lines
were intercrossed with C57BL/6 mice, and all of them transmitted the mutation through the germline. F1 heterozygous offspring from these three lines were interbred to
produce homozygous PEX2-deficient offspring. The phenotype of the homozygous PEX2 mutant mice was the
same in all three lines. F2 offspring were genotyped by PCR analysis (Fig. 1 C). Northern blot analysis of total RNA of
liver, brain, and kidney of newborn mice revealed an absence of PEX2 transcripts in homozygous mutant mice
when an exon 5 genomic fragment was used as a probe
(Fig. 1 D). Note that in addition to the major mRNA transcript of ~1.8 kb, two other less abundant transcripts are
detected (3.9 and 3.25 kb), and all are missing in the homozygous mutant mice. A less abundant transcript of 2.5 kb
has been described for human PEX2 (Shimozawa et al.,
1992
).
), indicating normal survival of heterozygotes. However, among 331 newborn pups (P0), there
were 87 wild-type (+/+), 162 heterozygous (+/
), and 82 homozygous (
/
) mice, the ratio being close to 1:2:1.
The majority of PEX2-deficient mice were observed to die
shortly after birth (usually <12 h), do not feed, and display
a variable in utero growth retardation (mean 29% weight
reduction versus littermate controls, range 12-42%; see
Fig. 7 A). Although the mutant mice do not feed, one can
elicit the normal rhythmic suckling movement of the jaw
by mechanical stimulation of the lip (Kutsuwada et al.,
1996
). Approximately 2% of newborn mutant mice do show
small amounts of milk in their stomachs. Rarely, mutant
mice have survived up to 1 or 2 d after birth (n = 3). There
is no obvious facial or limb dysmorphism (see Fig. 7 A),
and seizures have not been observed. The animals respond
normally to noxious stimuli. The PEX2-deficient mice
move their limbs well but are hypoactive and tend to maintain a "C-shaped," contracted posture. When placed on their
paws, the mutants lift their heads poorly and often fall over rapidly. This apparent hypotonia of the mutant mice
was evident even in litters of mice obtained very soon after
birth and in which none of the animals had fed. In addition, several runted, nonmutant mice have been observed
and shown not to display this degree of hypotonia.
Fig. 7.
External appearance and cranial morphology of PEX2
mutant mice. (A) Litter of newborn mice with three mutant animals (top row) and four control animals (bottom row). Note the
absence of milk in the stomach and variable size in the mutant
animals. Abnormal facies are not evident in the mutant mice.
(B-H) Alcian blue (cartilage) and alizarin red (bone) staining of
newborn mouse calvarium. The weight of each mouse is shown.
(B) Typical appearance of the calvarium in control animals (70%).
f, Frontal bone; p, parietal bone; ip, interparietal bone. (C) Control animal with slightly open "anterior fontanelle" but relatively
normal bone density (19%). (D) Runted, control animal showing
slight enlargement of fontanelle space and reduced bone density
in medial frontal and interparietal bone. (E-H) Homozygous mutant mice, demonstrating the correlation of increasing severity in
reduction of bone density in frontal and interparietal bones and
enlargement of fontanelle spaces with decreasing size of the animal. Bars, 1 cm.
[View Larger Version of this Image (95K GIF file)]
Absence of functional peroxisomes may also be inferred
by the lack of sedimentable catalase in cell homogenates.
To directly demonstrate the abnormal cellular localization
of catalase, a postnuclear supernatant was prepared from
newborn mouse livers and separated into a high speed pellet, containing the cell organelles, and a supernatant. As
shown in Table III, catalase was 80% sedimentable in
wild-type and heterozygous mice but was 97% soluble in
the PEX2-deficient mice. The distribution of the lysosomal enzyme marker N-acetyl--glucosaminidase was not
affected by the PEX2 gene mutation (Table III).
Table III. Subcellular Distribution of Organelle Enzymes in Liver |
PEX2-deficient Mice Have a Peroxisome Assembly Defect
To determine the cellular localization of peroxisomal matrix and membrane proteins, fibroblast cultures were derived from skin of newborn mice and stained by immunofluorescence using antibodies against catalase, peroxisomal
3-ketoacyl-CoA thiolase, and rat liver peroxisomal integral membrane proteins (PxIMPs). In control fibroblasts,
the matrix markers catalase and thiolase show a punctate pattern of immunofluorescence typical of peroxisomes
(Fig. 2, A and C). Fibroblasts derived from PEX2-deficient
mice show only diffuse cytoplasmic staining with these antibodies (Fig. 2, B and D). Thus, both peroxisomal targeting
signal (PTS) pathways for import of peroxisomal matrix
proteins, as defined here by catalase (PTS1) and thiolase
(PTS2), are disrupted in the mutant fibroblasts. Assembly
of peroxisomal membrane proteins into "membrane ghosts"
(Santos et al., 1988) can be identified in PEX2-deficient fibroblasts with the anti-PxIMP antibody (Fig. 2 F). As has
been described for Zellweger fibroblasts, the membrane
ghosts are generally larger in size and fewer in number
than normal peroxisomes (compare with Fig. 2 C) and are
often found in small clusters. Thus, integral membrane
proteins of the peroxisome are assembled in fibroblasts of
PEX2-deficient mice, but matrix proteins are not imported into the organelle.
Morphologic Consequences of a Lack of Peroxisomes
Abnormal Lamination in the Cerebral Cortex.There were no
external abnormalities evident in the brain of homozygous
PEX2-deficient mice at PO; the size and proportion were
similar to those of the wild-type and heterozygous mice. Carefully matched coronal sections of the cerebrum were
examined by hematoxylin and eosin (H&E) staining and revealed a major abnormality in the developing cerebral cortex of homozygous mutant mice. Low power examination
revealed an altered distribution of cells within the developing cortical plate of mutant mice, with a lack of normal layering as well as an increased cellular density in the underlying white matter (intermediate zone) where neurons are
still migrating to form the cortex (Fig. 3). The subplate
neurons, easily visible in the wild-type brains (Fig. 3, A
and C, black arrows), are obscured by the increased density of cells in the lower cortical plate and intermediate
zone in mutant mice. These changes are seen over a large
expanse of neocortex from medial (Fig. 3 B) and lateral (Fig. 3 D) regions. In addition, there is a gradient of severity in the morphologic change from medial to lateral cortical regions. In more lateral cortical regions (Fig. 3, C and
D), the layer VI/V boundary (indicated by white arrows) is
easier to discern in the mutant mouse but becomes increasingly blurred as one proceeds medially (third white
arrow is absent in Fig. 3 D, as boundary is not evident).
This boundary is not evident in medial cortical regions
(Fig. 3 B).
At high power, the abnormal distribution of cells in the
cortical plate can be further appreciated (Fig. 4). Note that
only layers VI, V, and I (marginal zone) are completely
formed in newborn mice and layer IV cells are just arriving in the cortex. In the mutant mice, the marginal zone
and subplate are normally formed, and the thickness of the
cortical plate is not consistently different from control mice.
In medial cortical regions (Fig. 4, A and B), there is severe
effacement of the lamination in the remaining cortical layers seen as a loss of distinction between the layer boundaries (indicated by white dotted lines in Fig. 4 A) and an altered cell distribution in layers V and VI. This change is
most prominent in the region corresponding to layer V;
cells that appear to be pyramidal neurons are difficult to
identify and are obscured by a large number of cells with
rounder, more immature-appearing nuclei, as well as cells
with dark-staining, elongated nuclei. These latter cells may
represent migrating neuroblasts. In addition, there is a reduction in the amount of neuropil separating cells (normal
neuropil shown in white circle, Fig. 4 A) in the mutants, leading to an apparent alteration in cell packing density. In ventral-lateral cortical regions, the cortex in mutant mice
has a more normal lamination with milder abnormalities in
cell distribution (Fig. 4, compare C and D). In this region, it
is possible to more clearly visualize the layer boundaries,
and the reduction in intervening neuropil is not as severe.
One can still visualize an increased number of cells that appear to be migrating neuroblasts in in the mutant cortex.
Rostrocaudal differences in the cortical abnormality were
also evident. While the medial cortex was generally abnormal throughout the brain, anterior and posterior cortical
regions showed a lesser degree of abnormality throughout
a greater extent of lateral cortex (similar to that shown in
Fig. 4 D; data not shown). Other forebrain regions, including hippocampus, basal ganglia, thalamus, and olfactory
bulb, showed no obvious abnormality in the mutant mice.
Cortical lamination was normal in heterozygous mice. A
total of 16 brains from P0 mutant mice were examined, and all were recognizably abnormal to blinded observers.
There was significant variation in the cortical malformation between mutant mice with different parents. In some animals the defect was much less severe (similar to that in lateral cortex, Fig. 4 D), with fewer cells in the underlying white matter and a more limited rostrocaudal extent, being apparent only in central regions of the brain (data not shown). The presence of the cortical malformation and variation within was not attributable to the size of the animal since (a) mutant mice of similar size showed significant differences, and (b) control, runted animals did not show similar cortical changes (data not shown).
To examine the radial glial scaffold, which serves as the
guidance substrate for cortical neuronal migration, brains
from E15 mice were stained with antibodies against RC2
and brain lipid binding protein (BLBP), both markers for
radial glia (Misson et al., 1988; Feng et al., 1994
). RC2 and
BLBP immunostains showed no obvious abnormality in
the radial glial scaffold in brains from homozygous PEX2-deficient mice (Fig. 5 A; BLBP not shown).
Brains of P0 mutant mice stained with antibodies against the astrocyte marker GFAP and neuronal marker MAP2 revealed a normal distribution of labeled cells (data not shown).
Inferior Olivary Nucleus, Cerebellum and Spinal Cord
Coronal sections of the hindbrain of P0 mice were examined by serial sections throughout the rostrocaudal extent of the inferior olivary nucleus. In rodents, the principal olivary nucleus is a simple u-shaped structure that lacks the convolutions seen in humans. In the homozygous mutant mice, the component olivary nuclei were normal in position, and there was no evidence of discontinuities in the principal olivary nucleus (data not shown). Preliminary histologic analysis of the principal olivary nucleus in the homozygous PEX2 mutant mice suggests that there may be subtle abnormalities in the shape as well as neuropil of this nucleus; however, these findings remain to be substantiated.
Histologic sections of the cerebellum in P0 mutant mice
revealed that the deep cerebellar nuclei and the external
granular cell layer were normally formed and folial development was appropriate for the age of the animal. Examination of the P0 cerebellum with antibodies against calbindin-D28k, a PC marker, demonstrated that the vast majority
of PCs in mutant mice had reached a normal position within a multilayer beneath the cerebellar surface (Fig. 5 B). These cells had a normal morphology for an early postmigratory PC with numerous thin perisomatic processes
(Baptista et al., 1994). In one of three mutant mice examined, a small cluster of PCs was seen slightly beneath the
PC multilayer (Fig. 5 B, arrow). These cells had a bipolar
morphology with a few long processes, consistent with an
earlier embryonic migratory phase (Fig. 5 C), indicating a
slight delay in the migration and differentiation of these
few cells relative to the majority of PCs. Similar cells were
not identified in wild-type mice at P0. Due to the early death of the mutant animals and the immature status of
the mouse cerebellum at birth, it is not possible to exclude
abnormal positioning of PCs and/or granule cells once the
internal granule cell layer has formed (complete by ~P14).
The absence of major morphologic changes in the inferior olive or cerebellum is not due to regional differences in expression of the PEX2 gene product. RNA in situ hybridization studies showed a diffuse distribution of the PEX2 transcript in normal mouse brain (data not shown).
Histologic sections of the spinal cord in P0 mutant mice
were examined from cervical, thoracic, lumbar, and sacral
regions and revealed no obvious abnormality. Lipidosis of
neurons, as described by Powers et al. (1987) in Zellweger
patients, was not seen.
Liver, Adrenal Gland, Kidney, and Eye
The liver of newborn mutant mice was normal in size and
showed no light microscopic abnormalities (data not shown).
In electron microscopic analysis of control livers, peroxisomes stain with the cytochemical procedure for catalase
(Fig. 6 A) and appear as small round or oval forms with an
electron-dense urate oxidase core (6 A, inset), characteristic of rodent peroxisomes. Catalase-reactive particles were
not identified in livers from PEX2-deficient mice, and there
was no obvious morphologic abnormality in the mitochondria (Fig. 6 B), which reportedly show distortion in shape
and matrix condensation in some Zellweger patients (Goldfischer et al., 1973). Immunofluorescence staining of frozen livers from newborn mice with antibodies against catalase also showed diffuse cytoplasmic staining in all of the
hepatocytes in the mutant mice, consistent with the cytosolic localization of catalase (data not shown).
Light microscopic examination of the adrenal gland in
newborn PEX2-deficient mice revealed adrenocortical cells
containing clear clefts (Fig. 6 C). These inclusions tended
to be located in adrenocortical cells within deeper regions
of the adrenal cortex. Similar structures were not observed
in wild-type or heterozygote littermate controls. Electron
microscopic examination confirmed the presence of gently
curved to curvilinear clefts associated with electron dense
leaflets (Fig. 6, D-F). The clefts ranged in size from 40 to
435 nm. These structures are consistent with the lamellar
lipid profiles seen in the adrenal cortex of Zellweger and
adrenoleukodystrophy patients (Powers and Schaumberg,
1973; Goldfischer et al., 1983
). Complex, multilamellate
inclusions were not identified.
Light microscopic analysis of the kidneys from newborn mice showed only a slight tubular ectasia in PEX2-deficient mice versus littermate controls (data not shown). There was no evidence of tubular or glomerular cysts in mutant mice. Light microscopic examination of the eyes of newborn mutant mice showed no obvious abnormality (data not shown).
Skeleton
In the Zellweger syndrome, there are characteristic dysmorphic facial features that occur in essentially all patients
and stippled calcifications of patellae, femora, and humeri
(occur in ~69% of patients; Moser et al., 1995). Characteristic
facial features include high forehead, hypertelorism, epicanthal folds, hypoplastic supraorbital ridge, and depressed
bridge of nose. Many patients also have wide cranial sutures and large fontanelles. In the newborn PEX2-deficient
mice, there is no obvious external facial dysmorphism (Fig.
7 A). Examination of the entire mouse skeleton by alcian
blue (cartilage) and alizarin red (bone) staining and by radiographic examination showed a normal axial and extremity skeleton and no evidence of calcific stippling in the
patella or epiphyses of long bones (data not shown). The
bones of the calvarium in the homozygous mutant mice
did show a delay in membranous ossification characterized
by (a) a reduced amount of bone with a mottled appearance in the medial frontal bones and the interparietal bone
(Fig. 7, E-H), and (b) an expanded, oval shape at the medial apposition of the frontal bones (analogous to the anterior fontanelle region in humans) and enlargement of the
"posterior fontanelle region" formed by the juncture of the
parietal and interparietal bones (see especially Fig. 7, G
and H). The majority (70%) of calvaria in control animals
are more extensively mineralized and have closely opposed frontal bones (Fig. 7 B). In 19% of control mice,
there was a slight opening between the medial frontal
bones (Fig. 7 C) but no significant reduction in bone density. However, the calvarial phenotype of the mutant mice
correlated with the size of the animal rather than the genotype, as (a) runted wild-type or heterozygous animals
showed a similar defect (11% of control animals; weight
<1.12 gm; Fig. 7, compare D and F), and (b) larger mutant
mice showed a lesser degree of abnormality (Fig. 7, E and F). The remaining facial bones and base of skull bones
were normal in appearance and proportionate in size in
the mutant mice.
Zellweger syndrome is a unique peroxisomal disorder that results in characteristic central nervous system malformations. We have demonstrated that mice with a complete absence of the PEX2 gene product have a peroxisomal biogenesis disorder with deficiencies in peroxisomal biochemical functions and morphologic changes in a number of organ systems, including the presence of a prominent defect in the development of the cerebral cortex. These findings confirm the genetic linkage of the PEX2 defect in humans and the prime importance of absent peroxisomal function in causing the multiple anomalies associated with the Zellweger syndrome.
In Zellweger syndrome, multiple peroxisomal matrix
enzymes are deficient due to abnormal assembly of the
peroxisomal organelle. Homozygous PEX2-deficient mice
have a marked increase in plasma VLCFA and deficient
synthesis of plasmalogens in erythrocytes. Fractionation of
liver demonstrated that the matrix enzyme catalase was
present in the cytosol in mutant mice rather than in its normal particulate location. Morphologic studies on fibroblasts and liver from mutant animals confirmed the absence of normal peroxisomes in these tissues. Hepatocytes
in the mutant mice did not contain any normal peroxisomes, indicating absence of mosaicism in the liver, as has
been reported in some Zellweger patients as well as in the
recently described peroxisomal fatty acyl-coenzyme A oxidase-deficient mice (Fan et al., 1996). In fibroblasts, both the PTS-1 and -2 targeting pathways for import of peroxisomal matrix proteins were disrupted in mutant animals,
and peroxisomal membrane proteins were assembled into
structures consistent with the "membrane ghosts" described
in Zellweger patients (Santos et al., 1988
). Motley et al.
(1994)
and Slawecki et al. (1995)
have described heterogeneity in the disruption of the PTS-1 and -2 import pathways amongst fibroblasts from PBD patients in different complementation groups, such that one or both pathways
are disrupted. Patients with a PEX2 defect (complementation group 10) were found to have complete disruption of
both pathways (Slawecki et al., 1995
), as also seen in
PEX2 mutant mice.
The central nervous system malformations are the most
consistent feature of the Zellweger syndrome with the
characteristic array of cerebral cortical, inferior olivary,
and cerebellar changes. In PEX2-deficient mice, there is
disordered lamination of the cerebral cortex and an increased cell density in the underlying white matter, indicating an abnormality of neuronal migration. Similar to
the human condition, the cortex was not uniformly affected; both medial-lateral and anterior-posterior gradients in severity were observed in the mutant mice brains.
While the less severe involvement of the ventral-lateral
cortex might reflect the greater degree of maturation normally found in the earlier-formed lateral neocortex, the
observed anterior-posterior changes do not follow the
normal anterior to posterior gradient of cortical maturation (Bayer and Altman, 1991), suggesting an independent
mechanism. In the human Zellweger brain, the cortical plate
is significantly thinner than in normal brains. However, this
was not consistently observed in the PEX2-deficient mice,
suggesting that the malformation is less severe in the mouse
than in humans. This may reflect (a) the much smaller size
of the mouse neocortex, which contains ~1,000-fold fewer
neurons than the human neocortex (Rakic, 1995
), and
consequently the much smaller distance that migrating
neurons must traverse to reach the cortical plate; (b) a major difference in the duration of the migratory epoch in humans (months) as opposed to mice (days), with cumulative
damage accruing over time; and (c) differences in genetic
background, with other as yet undefined genes affecting the phenotypic expression of the peroxisomal defect. Immunohistochemical examination of the radial glial scaffold, which serves as the guidance substrate for cortical
neuronal migration, did not reveal any obvious structural
abnormalities in the mutant mice. However, this does not
exclude the presence of ultrastructural or functional abnormalities in the radial glial cells induced by the lack of peroxisomal function.
The inferior olive and cerebellum also show characteristic changes in the Zellweger syndrome. In the PEX2-deficient mice, major morphologic changes were not observed
in the inferior olive or dentate nucleus. These nuclei are
much simpler, nonlaminated structures in the rodent, reflecting the much smaller size of the lateral cerebellar cortex in rodents versus humans (Altman and Bayer, 1997).
Preliminary histologic analysis of the principal olivary nucleus in the PEX2 mutant mice suggests that there may be
subtle abnormalities in the shape as well as neuropil of this nucleus; however, these findings remain to be substantiated with more detailed studies of olivary morphology and
olivocerebellar connectivity. In the cerebellum of mutant
mice, subcortical PC heterotopias were not detected. Although the vast majority of PCs reached the multilayer beneath the cerebellar surface, a small cluster of PCs with a
slight delay in migration was detected in one mutant
mouse. Thus there may be limited and subtle abnormalities in PC migration in the PEX2-deficient mice. Due to
the early death of the mutant animals and the immature
status of the mouse cerebellum at birth, it is not possible to
evaluate many aspects of cerebellar development. Therefore, we cannot exclude that during the inward migration
of external granule cells from the surface of the cerebellum to form the internal granule cell layer that PC heterotopias might result subjacent to the internal granule cell
layer as well as the abnormal arrangements of granule cells
and PCs that characterize heterotaxias seen in the Zellweger syndrome. In the neurologic mutant mouse reeler,
there are extensive subcortical PC heterotopias (more severe than seen in Zellweger syndrome) and shape changes in the principal olivary nucleus (Goffinet et al., 1984
), suggesting a relationship between the migratory abnormalities in these cell populations, as has also been suggested in
Zellweger syndrome (Evrard et al., 1978
). As olivocerebellar topography is established as early as E15 in the mouse
(Paradies and Eisenman, 1993
), the absence of major olivary changes or subcortical PC heterotopias suggests that
malformations that might occur in the PEX2-deficient mice,
were they to survive longer, are likely to be mild.
Light and electron microscopy analysis of the adrenal
gland in mutant mice demonstrated structures consistent
with the lamellar-lipid inclusions described in Zellweger syndrome (Goldfischer et al., 1983). The chemical composition of these inclusions is unknown. They are postulated to
be phospholipids containing saturated VLCFA.
Some morphologic features of the Zellweger syndrome
were not modeled by the PEX2-deficient mice. As structural changes in the liver, eye, and brain white matter occur during the postnatal period in Zellweger syndrome,
one would not expect to see changes in a newborn animal.
Renal cysts, seen in ~93% of Zellweger patients (Moser
et al., 1995) and evident in the fetal period (Powers et al.,
1985
), were not found in kidneys of newborn mutant mice. The slight tubular ectasia seen in the PEX2 mutant mouse
kidneys may reflect early damage and a precursor lesion.
Stippled joint calcifications, seen in ~69% of Zellweger
patients, were also not identified in PEX2-deficient mice.
The facial dysmorphism of this syndrome is a central diagnostic feature. However, abnormal facies were not evident
in the mutant mice. Zellweger patients are also reported to
have enlarged cranial fontanelles. Examination of the mouse
skeleton initially suggested the presence of delayed cranial
bone mineralization and a larger fontanelle space in the mutant mice. However, this was found to correlate with
growth retardation of the animal rather than the mutant
genotype. It remains possible that the enlarged fontanelles
found in Zellweger patients are also an effect of growth retardation.
The exact cause of death in the PEX2-deficient animals has not been established. Shortly after birth, the mutant mice are normal in color and respiratory pattern, and the heart and lungs are normal in size, suggesting absence of a primary respiratory or circulatory defect. While the inability of the mice to feed certainly contributes to their early demise, the mutant animals die sooner than control animals that have not fed. This suggests toxic accumulation of a metabolic product(s) due to the lack of peroxisomal function, which may be exacerbated by dehydration in these animals.
Establishment of normal cerebral cortical lamination involves several defined steps, including proliferation and
early specification of neurons within the ventricular zone,
attachment to and migration along radial glial guides, followed by detachment from radial glia and assembly into
cortical layers. Examination of migrating neurons, both in
vivo (Rakic, 1972) and in vitro (Gregory et al., 1988
; Hatten,
1990
; Rivas and Hatten, 1995
), reveals a characteristic morphology with a thick leading process that wraps around the radial glial fiber, a posterior positioning of the nucleus,
and a thin trailing process. Neuronal-glial interactions mediated by the molecule astrotactin (Fishell and Hatten,
1991
; Zheng et al., 1996
), as well as cell-matrix adhesions
(Jessell, 1988
; Fishman and Hatten, 1993
; Sheppard et al.,
1995
) and cytoskeletal elements (Rivas and Hatten, 1995
),
are all believed to be important in the migratory process.
It remains to be established how the absence of peroxisomal function in Zellweger syndrome may disrupt any or all
of these processes. It has been postulated that elevations of VLCFA and bile acid intermediates may play a toxic
role in the Zellweger neuronal migration defect (Powers
et al., 1989
; Kaufmann et al., 1996
). Accumulations of lipid
products in migrating neurons and radial glia have been
documented in Zellweger fetuses (Powers et al., 1989
). In
addition, specific cellular processes may be disrupted. Recently, mutations in homologues for the PEX2 gene (Berteaux-Lecellier at al., 1995) and the LIS-1 gene (Xiang et al.,
1995
), which is abnormal in the Miller-Dieker lissencephaly syndrome (Reiner et al., 1993
; Hattori et al., 1994
),
have been identified in filamentous fungi, and both cause
abnormalities in processes involving nuclear migrations
within the cell. While Miller-Dieker lissencephaly has a more
severe neuronal migration defect than seen in Zellweger
syndrome, it shares a common spectrum of affected brain
regions, including cerebral cortical gyral abnormalities (pachygyria or agyria), inferior olive dysplasia and heterotopias, and PC heterotopias (Norman et al., 1995
). Thus,
these diverse gene mutations may affect cellular mechanisms common to nuclear migrations and the translocation
of the neuronal cell soma along the radial glial fiber.
The PEX2-deficient mice provide an important animal model for Zellweger syndrome and a major step toward deciphering the cellular mechanisms of this neuronal migration disorder. Zellweger syndrome displays abnormalities in cortical foliation pattern (pachgyria, polymicrogyria) as well as heterotopias that are commonly seen in many human neuronal migration disorders. As cell migration and the formation of neuronal layers occurs prenatally in the mouse, this system can be used to study the role of peroxisomes in these key steps of brain development. Despite the many differences between human and mouse brain development, the presence of a cortical defect in the PEX2-deficient mice indicates a conservation in the mechanisms for neuronal migration. Thus, this mouse model can serve as a system to elucidate the role of peroxisomal function in neuronal migration as well as to unveil general mechanisms for cortical neuronal migration and the establishment of lamination.
Received for publication 28 July 1997 and in revised form 16 September 1997.
Address all correspondence to Phyllis Faust, Department of Pathology, Columbia University, PH Stem 15-124, 630 West 168th Street, New York, NY 10032. Tel.: (212) 305-7339. Fax: (212) 305-4548. E-mail: plf3{at}columbia.eduWe would like to thank Dr. H. Moser and A. Moser for performing the VLCFA and plasmalogen analyses; J.M. Powers for electron microscopy on the adrenal glands and help with the histologic analysis; A. Nagy for providing the R1 ES cells; K. Millen for assistance in making the chimeric mice; H. Shio for technical assistance with the electron microscope; and F. Vito and R. Alcarez for technical assistance with paraffin sections.
This work was supported by National Institutes of Health grants NS-15429 (M.E. Hatten) and the Howard Hughes Medical Institute Physician Postdoctoral Fellowship Program (P.L. Faust).
While this manuscript was in review, Baes et al.
published another mouse model for Zellweger syndrome by targeted deletion of the Pxr1 gene (PEX5) that encodes the PTS-1 receptor (Nat. Genet. 1997. 17:49-57). The phenotype of the Pxr1 /
mouse is very similar to the PEX2
/
mouse.
BLBP, brain lipid binding protein; ES, embryonic stem; PBD, peroxisome biogenesis disorder; PC, Purkinje cell; PTS, peroxisomal targeting signal; VLCFA, very long chain fatty acids.
1. | Altman, J., and S.A. Bayer. 1997. Comparative anatomy of the cerebellum: an evolutionary perspective. In Development of The Cerebellar System. CRC Press Inc., New York. 2-25. |
2. | Baptista, C.A., M.E. Hatten, R. Blazeski, and C.A. Mason. 1994. Cell-cell interactions influence survival and differentiation of purified purkinje cells in vitro. Neuron. 12: 243-260 |
3. | Bayer, S.A., and J. Altman. 1991. Overview of global neurogenetic gradients in the neocortex and limbic cortex. In Neocortical Development. Raven Press, New York. 30-45. |
4. | Berteaux-Lecellier, V., M. Picard, C. Thompson-Coffe, D. Zickler, A. Panvier-Adoutte, and J.-M. Simonet. 1995. A nonmammalian homolog of the PAF1 gene (Zellweger syndrome) discovered as a gene involved in caryogamy in the fungus Podospora anserina. Cell. 81: 1043-1051 |
5. | Bjorkhem, I., L. Sisfontes, B. Bostrom, B.F. Kase, and R. Blomstrand. 1986. Simple diagnosis of the Zellweger syndrome by gas-liquid chromatography of dimethylacetals. J. Lipid Res. 27: 786-791 [Abstract]. |
6. | Chomczynski, P., and N. Sacchi. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159 |
7. | Distel, B., R. Erdmann, S.J. Gould, G. Blobel, D.I. Crane, J.M. Cregg, G. Dodt, Y. Fujiki, J.M. Goodman, W.W. Just, et al . 1996. A unified nomenclature for peroxisome biogenesis factors. J. Cell Biol. 135: 1-3 |
8. | Dobyns, W.B., and C.L. Truwit. 1995. Lissencephaly and other malformations of cortical development: 1995 update. Neuropediatrics. 26: 132-147 |
9. | Evrard, P., V.S. Caviness Jr., J. Prats-Vinas, and G. Lyon. 1978. The mechansim of arrest of neuronal migration in the Zellweger malformation: an hypothesis based on cytoarchitectonic analysis. Acta Neuropathol. (Berl.). 41: 109-117 |
10. |
Fan, C.-Y.,
J. Pan,
R. Chu,
D. Lee,
K.D. Kluckman,
N. Usuda,
I. Singh,
A.V. Yeldandi,
M.S. Rao,
N. Maeda, et al
.
1996.
Hepatocellular and hepatic peroxisomal alterations in mice with a disrupted peroxisomal fatty acyl-coenzyme A oxidase gene.
J. Biol. Chem.
271:
24698-24710
|
11. | Feng, L., M.E. Hatten, and N. Heintz. 1994. Brain lipid-binding protein (BLBP): a novel signaling system in the developing mammalian CNS. Neuron. 12: 895-908 |
12. | Fishell, G., and M.E. Hatten. 1991. Astrotactin provides a receptor system for CNS neuronal migration. Development. 113: 755-756 [Abstract]. |
13. | Fishman, R.B., and M.E. Hatten. 1993. Multiple receptor systems promote CNS neural migration. J. Neurosci. 13: 3485-3495 [Abstract]. |
14. | Goffinet, A.M., K.-F. So, M. Yamamoto, M. Edwards, and V.S. Caviness Jr.. 1984. Architectonic and hodological organization of the cerebellum in reeler mutant mice. Dev. Brain Res. 16: 263-276 . |
15. | Goldfischer, S., C.L. Moore, A.B. Johnson, A.J. Spiro, M.P. Valsamis, H.K. Wisniewski, R.H. Ritch, W.T. Norton, I. Rapin, and L.M. Gartner. 1973. Peroxisomal and mitochondrial defects in the cerebro-hepato-renal syndrome. Science. 182: 62-64 |
16. | Goldfischer, S., J.M. Powers, A.B. Johnson, S. Axe, F.R. Brown, and H.W. Moser. 1983. Striated adrenocortical cells in cerebro-hepato-renal (Zellweger) syndrome. Virchows Arch. A Pathol. Anat. Histopathol. 401: 355-361 |
17. | Gregory, W.A., J.C. Edmondson, M.E. Hatten, and C.A. Mason. 1988. Cytology and neuron-glial apposition of migrating cerebellar granule cells in vitro. J. Neurosci. 8: 1728-1738 [Abstract]. |
18. | Hatten, M.E.. 1990. Riding the glial monorail: a common mechanism for glial-guided neuronal migration in different regions of the developing mammalian brain. Trends Neurosci. 13: 179-184 |
19. | Hattori, M., H. Adachi, M. Tsujimoto, H. Arai, and K. Inoue. 1994. Miller-Dieker lissencephaly gene encodes a subunit of brain platelet-activating factor acetyl-hydrolase. Nature. 370: 216-218 |
20. | Jessell, T.M.. 1988. Adhesion molecules and the hierarchy of neural development. Neuron. 1: 3-13 |
21. | Kaufmann, W.E., C. Theda, S. Naidu, P.A. Watkins, A.B. Moser, and H.W. Moser. 1996. Neuronal migration abnormality in peroxisomal bifunctional enzyme defect. Ann. Neurol. 39: 268-271 |
22. | Kutsuwada, T., S. Kenji, T. Manabe, C. Takayama, N. Katakura, E. Kushiya, R. Natsume, M. Watanabe, Y. Inoue, T. Yagi, et al . 1996. Impairment of suckling response, trigeminal neuronal pattern formation, and hippocampal LTD in NMDA receptor e2 subunit mutant mice. Neuron. 16: 333-344 |
23. | Lazarow, P.B., and H.W. Moser. 1994. Disorders of peroxisome biogenesis. In The Metabolic and Molecular Basis of Inherited Disease. C.R. Scriver, A.L. Beaudet, W.S. Sly, and D. Valle, editors. McGraw Hill, New York. 2287- 2324. |
24. | Lazarow, P.B., G.M. Small, M. Santos, H. Shio, A. Moser, H. Moser, A. Esterman, V. Black, and J. Dancis. 1988. Zellweger syndrome amniocytes: morphological appearance and a simple sedimentation method for prenatal diagnosis. Pediatr. Res. 24: 63-67 [Abstract]. |
25. | Lazarow, P.B., R. Thieringer, G. Cohen, T. Imanaka, and G. Small. 1991. Protein import into peroxisomes in vitro. In Methods in Cell Biology, Vol. 34. A.M. Tartakoff, editor. Academic Press, San Diego. 303-326. |
26. | Lufkin, T., M. Mark, C.P. Hart, P. Dolle, M. LeMeur, and P. Chambon. 1992. Homeotic transformation of the occipital bones of the skull by ectopic expression of a homeobox gene. Nature. 359: 835-841 |
27. | Misson, J.P., M.A. Edwards, M. Yammamoto, and V.V. Caviness Jr.. 1988. Identification of radial glial cells within the developing murine central nervous system: studies based upon a new immunohistochemical marker. Dev. Brain Res. 44: 95-108 |
28. | Moser, H.W., and A.B. Moser. 1991. Measurements of saturated very long chain fatty acids in plasma. In Techniques in Diagnostic Human Biochemical Genetics. F.E. Hommes, editor. Wiley-Liss, New York. 177-191. |
29. | Moser, A.B., M. Rasmussen, S. Naidu, P.A. Watkins, M. McGuiness, A.K. Hajra, G. Chen, G. Raymond, A. Liu, D. Gordon, et al . 1995. Phenotype of patients with peroxisomal disorders subdivided into sixteen complementation groups. J. Pediatr. 127: 13-22 |
30. | Motley, A., E. Hettema, B. Distel, and H. Tabak. 1994. Differential protein import deficiencies in human peroxisome assembly disorders. J. Cell Biol. 125: 755-767 [Abstract]. |
31. |
Nagy, A.,
J. Rossant,
R. Nagy,
W. Abramow-Newerly, and
J. Roder.
1993.
Derivation of completely cell culture-derived mice from early passage embryonic stem cells.
Proc. Natl. Acad. Sci. USA.
90:
8424-8428
|
32. | Norman, M.G., B.C. McGillivray, D.K. Kalousek, A. Hill, and K.J. Poskitt. 1995. Neuronal migration disorders and cortical dysplasias. I. Migration disorders. In Congenital Malformations of the Brain: Pathologic, Embryologic, Clinical, Radiologic and Genetic Aspects. Oxford University Press, New York. 223-277. |
33. | Papaioannou, V., and R. Johnson. 1993. Production of chimeras and genetically defined offspring from targeted ES cells. In Gene Targeting: A Practical Approach. A.L. Joyner, editor. Oxford University Press, Oxford. 107-146. |
34. | Paradies, M.A., and L.M. Eisenman. 1993. Evidence of early topographic organization in the embryonic olivocerebellar projection: a model system for the study of pattern formation processes in the central nervous system. Dev. Dyn. 197: 125-145 |
35. | Powers, J.M., and H.H. Schaumberg. 1973. The adrenal cortex of adrenoleukodystrophy. Arch. Pathol. 96: 305-310 |
36. | Powers, J.M., H.W. Moser, A.B. Moser, J.K. Upshur, B.F. Bradford, S.G. Pai, P.H. Kohn, J. Frais, and C. Tiffany. 1985. Fetal cerebrohepatorenal (Zellweger) syndrome: dysmorphic, radiologic, biochemical and pathologic findings in four affected fetuses. Hum. Pathol. 16: 610-620 |
37. | Powers, J.M., R.C. Tummons, A.B. Moser, H.W. Moser, D.S. Huff, and R.I. Kelley. 1987. Neuronal lipidosis and neuroaxonal dystrophy in cerebro-hepato-renal (Zellweger) syndrome. Acta Neuropathol. (Berl.). 73: 333-343 |
38. | Powers, J.M., R.C. Tummons, V.C. Caviness Jr., A.B. Moser, and H.W. Moser. 1989. Structural and chemical alterations in the cerebral maldevelopment of fetal cerebro-hepato-renal (Zellweger) syndrome. J. Neuropathol. Exp. Neurol. 48: 270-289 |
39. |
Purdue, P.E., and
P.B. Lazarow.
1994.
Peroxisomal biogenesis: multiple pathways of protein import.
J. Biol. Chem.
269:
30065-30068
|
40. | Rachubinski, R.A., and S. Subramani. 1995. How proteins penetrate peroxisomes. Cell. 83: 525-528 |
41. | Rakic, P.. 1972. Mode of cell migration to the superficial layers of fetal monkey neocortex. J. Comp. Neurol. 145: 61-84 |
42. | Rakic, P.. 1995. A small step for the cell, a giant leap for mankind: a hypothesis of neocortical expansion during evolution. Trends Neurosci. 18: 383-388 |
43. |
Reiner, O.R.,
R. Carrozzo,
Y. Shen,
M. Wehner,
F. Faustinella,
W.B. Dobyns,
C.T. Caskey, and
D.H. Ledbetter.
1993.
Isolation of Miller-Dieker lissencephaly gene containing G-protein ![]() |
44. | Rivas, R.J., and M.E. Hatten. 1995. Motility and cytoskeletal organization of migrating cerebellar granule neurons. J. Neurosci. 15: 981-989 [Abstract]. |
45. |
Santos, M.J.,
T. Imanaka,
H. Shio, and
P.B. Lazarow.
1987.
Peroxisomal integral membrane proteins in control and Zellweger fibroblasts.
J. Biol. Chem.
263:
10502-10509
|
46. | Santos, M.J., T. Imanaka, H. Shio, G.M. Small, and P.B. Lazarow. 1988. Peroxisomal membrane ghosts in Zellweger syndrome: aberrant organelle assembly. Science. 239: 1536-1538 |
47. | Sheppard, A.M., J.E. Brunstrom, T.N. Thornton, R.W. Gerfen, T.J. Broekelmann, J.A. McDonald, and A.L. Pearlman. 1995. Neuronal production of fibronectin in the cerebral cortex during migration and layer formation is unique to specific cortical domains. Dev. Biol. 172: 504-518 |
48. | Shio, H., and P.B. Lazarow. 1981. Relationship between peroxisomes and endoplasmic reticulum investigated by combined catalase and glucose-6-phosphatase cytochemistry. J. Histochem. Cytochem. 29: 1263-1272 [Abstract]. |
49. | Shimozawa, N., T. Tsukamoto, Y. Suzuki, T. Orii, Y. Shirayoshi, T. Mori, and Y. Fujiki. 1992. A human gene responsible for Zellweger syndrome that affects peroxisome assembly. Science. 255: 1132-1134 |
50. | Shimozawa, N., Y. Suzuki, T. Orii, A.B. Moser, H.W. Moser, and R.J.A. Wanders. 1993. Standardization of complementation grouping of peroxisome- deficient disorders and the second Zellweger patient with peroxisome assembly factor-1 (PAF-1) defect. Am. J. Hum. Genet. 52: 843-844 |
51. |
Slawecki, M.L.,
G. Dodt,
S. Steinberg,
A.B. Moser,
H.W. Moser, and
S.J. Gould.
1995.
Identification of three distinct peroxisomal protein import defects in patients with peroxisome biogenesis disorders.
J. Cell Sci.
108:
1817-1829
|
52. |
Thieringer, R., and
C.R.H. Raetz.
1993.
Peroxisome-deficient Chinese hamster
ovary cells with point mutations in peroxisome assembly factor-1.
J. Biol.
Chem.
268:
12631-12636
|
53. | Tsukamoto, T., S. Yokota, and Y. Fujiki. 1990. Isolation and characterization of chinese hamster ovary cell mutants defective in assembly of peroxisomes. J. Cell Biol. 110: 651-660 [Abstract]. |
54. | Tsukamoto, T., S. Miura, and Y. Fujiki. 1991. Restoration by a 35K membrane protein of peroxisome assembly in a peroxisome-deficient mammalian cell mutant. Nature. 350: 77-81 |
55. | Volpe, J.J., and R.D. Adams. 1972. Cerebro-hepato-renal syndrome of Zellweger: an inherited disorder of neuronal migration. Acta Neuropathol. (Berl.). 20: 175-198 |
56. | Wurst, W., and A.L. Joyner. 1993. Production of targeted embryonic stem cells. In Gene Targeting: A Practical Approach. A.L. Joyner, editor. Oxford University Press, New York. 33-61. |
57. |
Wurst, W.,
A.B. Auerbach, and
A.L. Joyner.
1994.
Multiple developmental defects in Engrailed-1 mutant mice: an early mid-hindbrain deletion and patterning defects in forelimbs and sternum.
Development.
120:
2065-2075
|
58. | Xiang, X., A.H. Osmani, S.A. Osmani, M. Xin, and N.R. Morris. 1995. NudF, a nuclear migration gene in Aspergillus nidulans, is similar to the human LIS-1 gene required for neuronal migration. Mol. Biol. Cell. 6: 297-310 [Abstract]. |
59. | Zheng, C., H. Heintz, and M.E. Hatten. 1996. CNS gene encoding astrotactin, which supports neuronal migration along glial fibers. Science. 272: 417-419 [Abstract]. |