Local regulation of fat metabolism
in peripheral nerves
Mark H.G. Verheijen,1Roman Chrast,1Patrick Burrola, and Greg Lemke2
Molecular Neurobiology Laboratory, The Salk Institute, La Jolla, California 92037, USA
We comprehensively analyzed gene expression during peripheral nerve development by performing microarray
analyses of premyelinating, myelinating, and postmyelinating mouse sciatic nerves, and we generated a
database of candidate genes to be tested in mapped peripheral neuropathies. Unexpectedly, we identified a
large cluster of genes that are (1) maximally expressed only in the mature nerve, after myelination is
complete, and (2) tied to the metabolism of storage (energy) lipids. Many of these late-onset genes are
expressed by adipocytes, which we find constitute the bulk of the epineurial compartment of the adult nerve.
However, several such genes, including SREBP-1, SREBP-2, and Lpin1, are also expressed in the endoneurium.
We find that Lpin1 null mutations lead to lipoatrophy of the epineurium, and to the dysregulation of a battery
of genes required for the regulation of storage lipid metabolism in both the endoneurium and peri/epineurium.
Together with the observation that these mutations also result in peripheral neuropathy, our findings
demonstrate a crucial role for local storage lipid metabolism in mature peripheral nerve function, and have
important implications for the understanding and treatment of peripheral neuropathies that are commonly
associated with metabolic diseases such as lipodystrophy and diabetes.
[Keywords: Schwann cells; peripheral nerve development; fat metabolism; Charcot-Marie-Tooth disease;
peripheral neuropathy; microarray analysis]
Supplemental material is available at http://www.genesdev.org.
Received May 27, 2003; revised version accepted August 4, 2003.
Among the most complex cellular interactions in verte-
brate neural development is the interplay of neurons,
glia, and mesenchymal cells that leads to the formation
of peripheral nerves. A subset of these interactions oc-
curs between neurons and Schwann cells, and results in
the elaboration of myelin. This Schwann cell organelle
consists of an extended sheet of specialized glial mem-
brane that is repeatedly wrapped and then tightly com-
pacted around axons (Webster 1971; Low 1976; Garbay et
al. 2000). It provides the insulation upon which the
rapid, saltatory conduction of neuronal action potentials
is entirely dependent, and the loss of myelin integrity in
demyelinating diseases is invariably debilitating (Keegan
and Noseworthy 2002).
Peripheral nerves are composed of three distinct tissue
compartments: the epineurium, perineurium, and endo-
neurium. The outermost epineurium surrounds a thin
lamellated perineurium, which in turn surrounds the
fascicle of axons, Schwann cells, and fibroblasts that
make up the endoneurium. Schwann cells, the most
abundant cells of the endoneurium, are derived from the
neural crest via a multistep differentiation process in-
volving the formation of a Schwann cell precursor that is
present in mouse nerves beginning at embryonic day 12
to 13 (E12–E13), and subsequently, of an immature
Schwann cell, present from E15 to birth (Jessen and Mir-
sky 1999). During the first postnatal week, a subset of
immature Schwann cells exit the cell cycle, lose suscep-
tibility to apoptosis, synthesize a basement membrane,
and begin to myelinate their associated axons (Zorick
and Lemke 1996). Myelination of peripheral nerves
peaks in the second postnatal week in the mouse, after
which the process substantially slows; only steady-state
maintenance levels of myelin protein synthesis are ob-
served in adult nerves (Garbay et al. 1998). In addition to
adopting a myelinating phenotype, immature Schwann
cells may alternatively differentiate into nonmyelinat-
ing Schwann cells, which loosely ensheath, without
multilayered wrapping or compaction, multiple small-
caliber axons. These mature nonmyelinating Schwann
cells first differentiate in the second postnatal week, and
continue to express many of the genes characteristic of
immature cells (Zorick and Lemke 1996).
The choice between a myelinating versus a nonmy-
elinating fate is highly significant with regard to
Schwann cell metabolism and gene expression. Two
Schwann cell transcription factors have been shown to
play a pivotal role in myelinative differentiation: the
1These authors contributed equally to this work.
E-MAIL firstname.lastname@example.org; FAX (858) 455-6138.
Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/
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POU protein Scip (Oct-6/Tst-1; Monuki et al. 1990; Ber-
mingham et al. 1996; Jaegle et al. 1996; Zorick et al.
1996) and the zinc finger protein Krox-20 (Egr2; Swiatek
and Gridley 1993; Topilko et al. 1994; Zorick et al. 1999).
In humans, the failure to assemble or properly main-
tain peripheral nervous system (PNS) myelin results in a
heterogeneous group of disabling peripheral neuropa-
thies. Most of these neuropathies are metabolic and ac-
quired, notably as sequelae of diabetes. Diabetic periph-
eral neuropathy, whose molecular basis remains ob-
scure, represents a substantial medical problem in
humans, and accounts for the bulk of the 8% of the aged
human population in which peripheral nerve damage is
thought to occur (Martyn and Hughes 1997). In addition
to these acquired conditions, a set of inherited peripheral
neuropathies, collectively referred to as Charcot-Marie-
Tooth disease (CMT) or hereditary motor and sensory
neuropathy (HMSN), have also been described. Together,
these familial disorders constitute a relatively common
set of genetic diseases, with an estimated 1 in 2500 per-
sons affected (Skre 1974).
The molecular mechanisms underlying many of the
events of peripheral nerve development are poorly un-
derstood, and a systematic analysis of gene expression
during these events has yet to be described. We therefore
performed Affymetrix-based GeneChip analyses on
mRNA populations isolated from sciatic nerves of the
mouse, from late embryogenesis to adult. Unexpectedly,
we identified a large cluster of genes that are most highly
expressed only after myelination is complete. This clus-
ter contains an abundance of genes related to storage
lipid metabolism, many of which are expressed in the
adipocytes that populate the epineurium of the mature
peripheral nerve. Most interestingly, we found that sev-
eral of these genes, including SREBP-1, SREBP-2, and
Lpin1 are also expressed in the endoneurium. We dem-
onstrate that null mutation of Lpin1, which results in a
peripheral neuropathy, also leads to lipoatrophy of the
epineurium and to dysregulation of storage lipid metabo-
lism genes and their products in the mature nerve.
Developmental expression profiling of mouse
samples isolated from mouse sciatic nerves dissected at
E17 and at postnatal days 0, 2, 4, 10, and 56 (P0–P56; Fig.
1). Schwann cells account for the clear majority of the
cells present in the sciatic nerve during the first 3 post-
natal weeks (Low 1976; Brockes et al. 1979; Nakao et al.
1997), and the levels of neuronal mRNAs present in the
sensory and motor axons of the nerve are thought to be
very low (Mohr 1999). The time points selected therefore
allow for the sampling of Schwann cell mRNA popula-
tions from premyelinating (E17), promyelinating (P0,
P2), and myelinating nerves (P4, P10). (For raw array
data, see Supplementary Table S1.)
The differentiation of Schwann cells in the sciatic
analyseswere performedwith mRNA
nerve progresses in a proximal-to-distal wave (where
proximal is close to the spinal cord), such that Schwann
cells in a distal segment of the nerve are at approxi-
mately the same developmental stage as more proximal
cells were 1–2 d earlier (Zorick et al. 1996). Because we
dissected a relatively large segment of the nerve, from
distal of the sciatic notch to proximal of the knee, we
selected for the analysis those mRNA hits that were
scored as “present” (see Materials and Methods) in du-
plicate samples from at least two consecutive time
points between E17 and P56. The E17 and P56 samples
lack a consecutive time point, and we therefore included
those mRNAs scored as “present” in duplicate samples
of these single time points. This filter yielded an initial
group of 5376 mRNAs expressed in the nerve from E17
to P56 (Fig. 1). Of this group of 5376 genes, 2808 were
found to systematically vary (increase or decrease) in ex-
pression level as a function of developmental time, and
were classified as differentially expressed during sciatic
nerve development (Fig. 1). For 712 genes of this group,
expression across the different time points showed sig-
nificant differences with a P-value cutoff of 0.01 in
ANOVA. Analysis using the NCBI Blast, Unigene, and
SOURCE databases revealed that 27 of the 712 genes
were represented by two probe sets. Of these 27 dupli-
cated sets, 26 yielded essentially identical expression
data for both probe sets (see Supplementary Fig. S2). This
correspondence is indicative of a reproducible and semi-
quantitative assay. Thus, 685 unique genes/ESTs were
selected as displaying the most significant expression
changes during sciatic nerve development (Fig. 1).
Systematic identification of peripheral neuropathy
For about half of the 35 described forms of hereditary
peripheral neuropathy, the causal genes remain to be
development. For a detailed description of software-based selec-
tion, see Materials and Methods.
Scheme of GeneChip analysis of mouse sciatic nerve
Energy mobilization in peripheral nerves
GENES & DEVELOPMENT2451
identified, although they have been mapped to more or
less well-delimited regions in the genome (Bennett and
Chance 2001). To facilitate the identification of these
genes, and of those mapped in the future, we used our
GeneChip data to establish biological selection criteria.
For all of the 685 genes exhibiting the most pronounced
developmental regulation, we determined the human
homolog and its chromosomal location, whenever avail-
able, using the public databases SOURCE and Locuslink
(Supplementary Table S3A). We thereby identified 22
candidate genes for nine distinct CMT loci (Table 1,
Supplementary Table S3B). For example, the intermedi-
ate disease I-CMT2 has been mapped to a locus contain-
ing 133 genes (10q24.1–q25.1, D10S1709–D10S1795), of
which three were selected by our screen. The fact that
these three genes exhibit significant changes in expres-
sion during peripheral nerve development makes them
attractive candidate genes for this disease.
To establish proof-of-principle for this approach, we
performed a post hoc retrospective analysis of our list of
candidate genes (Table 2). We asked whether previously
cloned peripheral neuropathy disease genes, such as
those encoding PMP22, CX32, EGR2, and MPZ (Bennett
and Chance 2001) would have been selected as candidate
genes by our screen. We found that the majority of genes
whose mutant alleles have previously been tied to de-
myelinating forms of CMT were indeed identified by the
screen, with the exception of PRX and LITAF (which
were detected as being expressed, but not differentially
so, throughout peripheral nerve development) and
MTMR13 [which was detected as being differentially ex-
pressed, but did not make the significance threshold
(p < 0.01) for selection]. Ganglioside-induced differentia-
tion-associated protein-1 (GDAP1), which causes both
axonal and demyelinating forms of CMT4A (Baxter et al.
2002; Cuesta et al. 2002), could not be detected. This is
in agreement with published observations that GDAP1
mRNA expression is high in dorsal root ganglia but very
low in sciatic nerve (Cuesta et al. 2002). In addition, we
have observed that the gene encoding glycyl tRNA syn-
thetase (GARS), previously described as being mutated
in an axonal form of CMT2D, was also identified by our
screen. This, together with previous observations that
mutations in a gene expressed in Schwann cells (MPZ)
can lead to both demyelinating and axonal phenotypes
(Vance 2000), extends the validity of our candidates to
both demyelinating and axonal forms of CMT.
Primary expression profiles during peripheral
To identify distinct temporal expression profiles and
thereby segregate the 685 highly differentially expressed
genes, K-means clustering was performed. This yielded
five clusters (Fig. 2A), which we validated with a set of
marker genes whose expression levels during the course
of Schwann cell differentiation have already been well
described. We also independently validated the Ge-
neChip data for each of these marker genes with quan-
titative polymerase chain reaction (Q-PCR; Fig. 2B).
Cluster 1 consisted of 247 genes that were highly ex-
pressed at E17, and then down-regulated during the
course of subsequent peripheral nerve development. Sev-
eral genes with this expression profile have been de-
scribed, among which is Ncam, a marker for nonmyelin-
ating and immature promyelinating Schwann cells that
is markedly down-regulated upon differentiation into
myelinating cells (Jessen et al. 1987). Ncam was present
on our Affymetrix GeneChips, was indeed included in
Candidate disease genes for different types of peripheral neuropathy
Total genes in locusb
Selected genes in locusc
I-CMT2606483 Intermediate 10q24.1–q25.1
I-CMT1 606482 Intermediate
dHMN VII158580 Axonal
HMN II 158590Axonal
SMAL 600175 Axonal
aLocus of peripheral neuropathy based on OMIM database information.
bTotal amount of genes in locus based on search of NCBI Map Viewer.
cDifferentially expressed with p < 0.01 during Schwann cell differentiation based on GeneChip analysis.
Verheijen et al.
2452 GENES & DEVELOPMENT
cluster 1 by our clustering paradigm, and served as a
validating marker for this cluster (Fig. 2B).
Cluster 2 contained a small group of 42 genes exhib-
iting down-regulation after E17 and then modest up-
regulation in adult (P56) sciatic nerves. This “U-like”
pattern has not been previously described for peripheral
nerve. Comparison of GeneChip data and Q-PCR for this
cluster was carried out with lactate dehydrogenase (Ldh;
Fig. 2B), an enzyme demonstrated to be highly expressed
in cultured Schwann cells (Rust et al. 1991).
Cluster 3 consisted of 49 genes up-regulated early in
postnatal development, with an expression maximum at
P0–P2, and then down-regulated as peripheral nerve de-
velopment proceeds (P10 and P56). This expression pat-
tern, with a transient peak corresponding to the presence
of promyelinating Schwann cells, has also been previ-
ously documented, notably for the transcription factor
Scip (Zorick et al. 1996). As predicted, Scip was included
in cluster 3, and served as a reliable validating marker for
this set of “transitional” genes (Fig. 2B).
The 61 genes in cluster 4 exhibited an expression pro-
file similar to those of cluster 3, but with peak expres-
sion later in development (around P10), and an extended
period of elevation. Cluster 4 contained all of the well
known markers for myelinating Schwann cells that were
present on our chips, for example, Krox-20 and the my-
elin structural protein genes Mpz, Pmp22, and Mbp. Plp,
a gene required for both PNS and CNS myelination (Gar-
bern et al. 1997), served as a validation marker for this
major cluster (Fig. 2B).
Prior to undertaking our analysis, we anticipated that
cluster 4 would represent the last significant cluster of
genes to be identified in the course of sciatic nerve de-
velopment, because Schwann cells account for the ma-
jority of the cells present in the sciatic nerve (Low 1976;
Brockes et al. 1979; Nakao et al. 1997) and have been
thought to exist in the mature nerve in a relatively qui-
escent “maintenance” state (Peters et al. 1991). We were
therefore surprised to identify a large cluster of 286 genes
(the largest cluster) that were expressed at low levels in
perinatal sciatic nerve and were up-regulated only in
adult (P56) mouse nerves, after the bulk of peripheral
myelination is complete. This expression profile is re-
markable but not unprecedented, in that it has been de-
scribed for at least one gene—Itgb4 (?4-integrin; Einhe-
ber et al. 1993; Feltri et al. 1994). Again, this gene was
binned into cluster 5 by our clustering paradigm, and
served as a validating marker for this set of late-onset
genes (Fig. 2B). Comparison of GeneChip with Q-PCR
data for the validating markers of each of the clusters
revealed strong agreement, both in qualitative expres-
sion profiles, and in most cases, in quantitative (abso-
lute) measurements. This validates our clustering pa-
rameters, and supports the reproducibility and semi-
quantitative aspects of the GeneChip assay.
Expression profiles and biological functions
We next organized the genes of clusters 1–5 as a function
of known or hypothesized biological activity (see Supple-
mentary Table S4). Broad annotations (nodes) revealed
characteristic spectra of biological functions for each of
the five clusters (Fig. 3), and detailed annotations (sub-
nodes) were used to plot the expression profiles for more
than 100 gene groups with similar biological functions
(see Supplementary Fig. S5).
Cluster 1 was notable for the prominence of genes in-
volved in the regulation of the cell cycle, in nucleic acid
metabolism, and in apoptosis (Fig. 3). Among the latter
were Acinus, Apaf1, Pdd5, and Peg3, which are known
positive mediators of apoptosis. The maximal expression
of these pro-apoptotic genes in dividing premyelinating
Retrospective analysis of cloned peripheral neuropathy disease genes
No linkage data (10q22)
aLocus of peripheral neuropathy as described prior to identification of disease gene.
b,cSee Table 1 for definitions.
dDefines if mutated gene is among selected genes (p < 0.01) in locus.
eDetected as being differentially expressed (but not reaching p < 0.01 significance).
Energy mobilization in peripheral nerves
GENES & DEVELOPMENT 2453
(A) K-means clustering of 685 highly differentially expressed genes resulted in five distinct temporal expression patterns. mRNA expression is depicted as fold
increase over the mean expression during depicted developmental stages. (B) Validation of GeneChip data and clustering method by comparing expression data obtained with
GeneChip (dotted lines) vs. Q-PCR (solid lines) for genes with a known expression profile representative for their cluster. Data are expressed as the level of change compared
to the minimal expression measured for the given gene. Standard deviations were calculated from triplicate experiments. (R) Correlation coefficient for GeneChip vs. Q-PCR
Verheijen et al.
2454 GENES & DEVELOPMENT
cells contrasts with the presence of anti-apoptotic genes
in later clusters associated with myelination (Mapk8ip
in cluster 4 and Bcl2l2, Serpinf1, and Tax1bp1 in cluster
5). Cluster 1 also included 46 genes associated with the
positive regulation of the cell cycle, including key regu-
lators such as Cdk4 and Cdc2, and cyclin D1, cyclin D2,
cyclin A2, cyclin B1, and cyclin B2, and 13 genes in-
volved in DNA metabolism, for example, the histones
H2afx, H2afy, and H2bfs, and Top2 (DNA topoisomerase
II). The maximal expression of these proliferation genes
in premyelinating cells was in contrast to the presence of
cell-cycle arrest genes in later stages of development (in-
cluding Pcbp4 in cluster 3, p21Cip1 in cluster 4, and
Ndrg1 in cluster 5). Also prominent in cluster 1 were
genes involved in RNA metabolism, protein synthesis,
protein folding, and nucleocytoplasmic transport.
An interesting feature of cluster 2 was the relatively
high number of genes (9 out of 42) involved in energy
pathways, including glucose metabolism as well as cy-
tochrome and NADH dehydrogenase subunits. These
were expressed in premyelinating nerves, down-regu-
lated during early postnatal development, and subse-
quently up-regulated in adults.
Cluster 3 genes are characterized by an expression pro-
file that peaks in the first few days immediately after
birth. In addition to the transcription factor Scip (see
above), this cluster contained a large number of other
transcription factors, such as Sox4, Nab2, and Id2.
As expected, cluster 4—mRNAs whose expression was
maximal at P10—contained all of the structural genes
associated with the deposition and compaction of the
myelin sheath, for example, Mbp, Pmp22, and Mpz. In
addition, this cluster contained a large number of genes
(12) involved in the synthesis, transport, and metabolism
of structural lipids. The latter observation is consistent
with the fact that myelinating cells must synthesize and
extend an extraordinary volume of specialized mem-
brane per day (Webster 1971).
As noted above, both the existence of a cluster 5
(mRNAs highly expressed only in adult nerves) and its
large size (286 genes) were unexpected findings. Included
in this cluster were genes involved in cell adhesion, ion
transport, and responses to external stimuli (RES), which
includes antigen presentation and complement activa-
tion. In addition, this cluster exhibited a provocative
composition with regard to lipid metabolism.
Differential regulation of lipid metabolism
Nearly all genes annotated as relevant to “lipid metabo-
lism” were maximally expressed at either P10 (myelina-
tion cluster 4) or P56 (mature nerve cluster 5). More de-
tailed annotation, however, revealed that the segregation
into either cluster 4 or cluster 5 was tightly correlated
with the extent to which individual genes were tied to
the production of structural (myelin membrane) lipids
versus the metabolism of storage (energy) lipids (Fig. 4A).
Included in the genes of cluster 4, for example, are 10
genes involved in the biosynthesis of cholesterol and ga-
lactocerebroside, two major components of the myelin
membrane (see Supplementary Fig. S5). Among the 15
genes encoding cholesterol biosynthetic enzymes that
were present on the chip, 12 followed the cluster 4 ex-
pression profile, and the remaining three genes did not
display significant changes in expression during develop-
ment (Fig. 4B,C). Similar expression was observed for
seven genes involved in biosynthesis or metabolism of
plasma membrane glyco- and phospholipids (Fig. 4B).
for each cluster. Significant classes for particular clusters are highlighted. For methods of annotation, see Materials and Methods.
Association of clusters with biological function. The number of genes per class is depicted as a fraction of the total genes
Energy mobilization in peripheral nerves
GENES & DEVELOPMENT2455
Most interestingly, two genes involved in the biosynthe-
sis of oleate, Scd2 and Masr (also called Lce), appeared in
cluster 4 rather than cluster 5, and unusually, this spe-
cific fatty acid serves primarily as a structural compo-
nent of PNS myelin rather than as an energy source (Gar-
bay et al. 1998; Nagarajan et al. 2002).
In marked contrast, the “lipid metabolism” genes of
cluster 5 encoded proteins involved in the metabolism of
Expression profile of highly differentially expressed genes regulating the metabolism of myelin membrane lipids (green) and storage
lipids (black). mRNA expression is presented as fold increase over the mean expression during depicted developmental stages. (B)
Expression profile of selected genes involved in biosynthesis of cholesterol and membrane lipids. All genes are expressed at some point
during myelination, with the differentially expressed genes marked as *, and the highly differentially expressed genes (p < 0.01) marked
as **. (C) Scheme of the cholesterol biosynthesis pathway in which the site of action is shown for the genes presented in B. Genes in
white boxes were not present on the chip. (D) Expression profile during peripheral nerve development of selected genes known to be
involved in distinct aspects of storage lipid metabolism in adipocytes and hepatocytes.
Different temporal expression of genes involved in myelin membrane lipid biosynthesis and storage lipid metabolism. (A)
Verheijen et al.
2456 GENES & DEVELOPMENT
fatty acids and storage lipids, and in the catabolism of
phospholipids. Given this unexpected result, we further
searched for differentially expressed genes known to be
involved in storage lipid metabolism in other cells, most
notably adipocytes and hepatocytes (Fig. 4D). Nearly all
such genes that we assayed are also strongly up-regulated
in adult nerves. The adult-onset genes we detected are
involved in multiple aspects of fatty acid and triacylglyc-
erol (TAG) metabolism. In addition, several transcrip-
tion factors known to regulate the expression of lipid
metabolism genes in adipocytes and hepatocytes, includ-
ing C/EBP?, C/EBP?, and SREBP-1, also showed strong
up-regulation in adult nerves. A similar adult-onset ex-
pression profile was found for two secreted factors of
adipocytes, Acrp30 and Resistin, as well as for lipid
transport genes, such as Apoc1, Apod, Apoe, Pltp, Lpl,
and Abca2, which are all involved in extracellular lipid
transport and all appeared in cluster 5. Q-PCR assays
were performed for Scd1, SREBP-1, Lpl, and Acrp30,
which confirmed their peak expression in adult sciatic
nerves (data not shown). Thus, genes tied to the struc-
tural lipids of myelin (e.g., cholesterol, phospholipids,
and galactolipids) or to storage lipids (e.g., fatty acids and
TAGs) segregated alternatively into cluster 4 and cluster
Although Schwann cells account for the vast majority
of cells in the peripheral nerve (Low 1976; Brockes et al.
1979; Nakao et al. 1997), it is important to establish
whether genes binned into each of the above clusters are
or are not expressed by these cells. First, hematoxylin
and eosin stainings were performed on dissected sciatic
nerves to determine differences in cellular composition
of P10 versus adult sciatic nerves (Fig. 5). An unantici-
pated large amount of adipose-like tissue was observed
in the epineurial compartment of adult sciatic nerves, in
marked contrast to P10 nerves. Interestingly, the
amount of adipose-like tissue in the adult nerve in-
creased from proximal to distal, where proximal is close
to the spinal cord and distal is close to the knee joint
In situ hybridization on the distal region of the adult
sciatic nerve was used to determine the site of expres-
sion of many of the storage lipid metabolism genes in the
peripheral nerve. Figure 6 shows that expression patterns
for this group of genes can be roughly divided into three
classes. One class consisted of genes only expressed in
the epineurium; these included Acrp30, Resistin, and
Lpl, consistent with the observed preponderance of adi-
pocytes in this sheath. A second class contained genes
expressed in both the endoneurium and peri/epineu-
rium, such as SREBP-1, SREBP-2, Scd1, Apoe, and Fasn.
Itgb4 (?4-integrin) was previously shown to be expressed
in both the endoneurium and peri/epineurium (Feltri et
al. 1994), and was therefore used as a control. A third
class (two genes) showed expression only in the endo-
neurium: Scd2 and Apod. Importantly, for all of the en-
doneurial mRNAs in Figure 6, comparison with the
mRNA localization of myelin P0(Mpz) strongly suggests
that Schwann cells are the main site of their expression.
Indeed, immunostaining staining of SREBP-1 in the
adult sciatic nerve demonstrated that SREBP-1 protein is
expressed by myelinating Schwann cells in the endoneu-
rium as well as by adipocytes in the epineurium, where
it is localized both within and close to the nuclei of both
cell types (Fig. 7).
For selected genes, the contribution in mRNA expres-
sion made by the endoneurium versus the peri/epineu-
rium of the adult sciatic nerve was also determined by
Q-PCR, after physical separation of these compartments
(Fig. 8A). That this separation was successful is demon-
strated by the fact that the peri/epineurial fraction con-
tains almost no Schwann cell-specific mRNAs (e.g.,
Mpz, Plp, and Krox-20), whereas the endoneurial fraction
contains almost no adipocyte-specific mRNAs (e.g., Lpl
and Acrp30). These data show that for genes expressed
only in the peri/epineurium compartments (Thrsp,
C/EBP?, Resistin, Lpl, and Acrp30), the observed in-
crease in mRNA expression in adults (cluster 5) is en-
tirely due to the appearance of adipocytes in the mature
epineurium. For cluster 5 genes that are expressed in
both the endoneurium and the peri/epineurium (Apoe,
?4-integrin, C/EBP?, SREBP-1, Fasn, Lpin1, and Scd1),
the onset of expression in adults is mainly, although not
entirely, derived from the appearance of adipocytes in
ing of P10 (longitudinal section) and adult
(P56) sciatic nerve (both cross- and longi-
tudinal sections). Adipocytes are abundant
in distal regions of the adult nerve. The
sciatic nerves shown are representative for
at least five nerves studied per stage. Bar,
Hematoxylin and eosin stain-
Energy mobilization in peripheral nerves
GENES & DEVELOPMENT2457
adult nerves. Apod is the only gene we analyzed for
which the increase in adults is mainly derived from ex-
pression in the endoneurium. Figure 8B shows that for
genes specific to the endoneurium (Mpz), peri/epineu-
rium (Thrsp and Acrp30), or both compartments (Fasn
and Apoe), the expression of protein correlates very well
with mRNA expression.
Lpin1 is expressed in the peripheral nerve and is
required for its function
Lipin1 is the product of the Lpin1 gene, which is mu-
tated in fatty liver dystrophy (fld) mice (Peterfy et al.
2001). These mice are characterized by a neonatal fatty
liver that resolves at weaning, lipoatrophy, and a progres-
sive demyelinating neuropathy affecting peripheral
nerves (Langner et al. 1989, 1991; Reue et al. 2000). Ob-
servations on the sciatic nerve in these mice show a
variety of abnormalities, including poorly compacted
myelin sheaths, active myelin breakdown, and hypertro-
phic Schwann cells (Langner et al. 1991). We have found
that the lipodystrophy in adult Lpin1 mutants extends to
the epineurium of the sciatic nerve. Staining of storage
lipids in the epineurium with Oil Red O highlights the
presence of large fat deposits in the epineurium of wild-
type sciatic nerves, and these fat deposits are severely
reduced in the Lpin1 mutants (Fig. 9A). Detailed obser-
vations on the mid-region of the nerve revealed that
(red) and neurofilament (green) or DAPI
staining (blue) shows that SREBP-1 protein
is expressed in both Schwann cells and adi-
pocytes (Ad) of adult mouse sciatic nerve.
(B) SREBP-1 is expressed in and close to nu-
clei (N) of myelinating Schwann cells (SC)
surrounding an axon (Ax). Result shown is
representative of at least three immunos-
tainings. Bars, 10 µm.
(A) Immunolabeling of SREBP-1
(P56) mouse sciatic nerve for depicted genes illustrates
their expression in endoneurium only (Mpz, Scd2, and
Apod), in both endoneurium and peri/epineurium
(SREBP-2, Scd1, Apoe, SREBP-1, Fasn, and ?4-integrin),
or only in peri/epineurium (Acrp30, Resistin, and Lpl).
Results shown are representative for at least a duplicate
experiment. Bar, 50 µm.
33P-RNA in situ hybridization signal on adult
Verheijen et al.
2458GENES & DEVELOPMENT
lipid-filled adipocytes are uniformly shaped and linearly
arranged in wild-type mice, mainly between branches of
nerves. In marked contrast, in the Lpin1 mutants lipid-
filled adipocytes are strongly reduced in number and ir-
regularly shaped (Fig. 9B). Cell counts of lipid-filled adi-
pocytes in the mid-region of the nerve revealed a reduc-
tion of ∼70%, which is comparable to the reduction in
adipose mass in Lpin1 mutant mice documented for
adult inguinal and epididymal fat pads (Reue et al. 2000).
Lpin1 mRNA has been reported to be undetectable in
the mouse sciatic nerve at P21 (Peterfy et al. 2001), lead-
ing to the conclusion that the neuropathy in Lpin1 mu-
tant mice is a secondary effect of Lipin1 deficiency in
other tissues. However, our GeneChip and Q-PCR data
demonstrate that Lpin1 mRNA is indeed expressed in
the adult (P56) sciatic nerve (Figs. 4D, 8A). We therefore
examined the expression of this mRNA during sciatic
nerve development using Q-PCR, and included a P28
time point in our analysis. Lpin1 mRNA is unambigu-
ously expressed during the first 4 wk of peripheral nerve
development, albeit at low levels (Fig. 9C). In agreement
with our GeneChip data, we find that this expression is
strongly up-regulated thereafter, such that Lpin1 segre-
gates as a “cluster 5” gene; its late-onset profile is due
largely to expression in the peri/epineurium. Finally,
Western blotting confirmed expression of Lipin1 protein
(MW ∼140 kD) in both the endoneurium and the peri/
epineurium fractions of wild-type but not mutant adult
sciatic nerves (Fig. 9D).
Interestingly, the epineurium of Lpin1 mutants dis-
plays reduced Fasn protein levels. This is in agreement
with our observations that mutant epineurium has re-
duced storage lipid content. Importantly, the endoneu-
rium of Lpin1 mutants also displays lower Fasn protein
levels, as well as reduced Mpz levels, whereas Apoe lev-
els are strongly increased (Fig. 9D). Together, these ob-
servations suggest that the neuropathy and lipoatrophy
of the peripheral nerve are direct consequences of the
absence of Lipin1—and the attendant dysregulation of
storage lipid metabolism—within the nerve itself.
Regulation of gene expression during peripheral
Earlier studies have indicated that premyelinating
Schwann cells exit the cell cycle and lose susceptibility
to apoptosis at the onset of myelination (Stewart et al.
1993; Syroid et al. 1996; Nakao et al. 1997; Zorick et al.
1999). Our expression data reinforce this view, because
genes associated with proliferation and apoptosis are
highly expressed in late embryonic nerves and are down-
regulated during subsequent development, in contrast to
cell-cycle arrest and anti-apoptotic genes, which are up-
regulated during myelinative differentiation. Our data
further demonstrate that the transition to myelination is
associated with decreased expression of genes involved
in RNA metabolism, protein biosynthesis, protein folding,
nucleocytoplasmic transport, and energy metabolism.
The onset of active myelination is associated with the
up-regulation of a large battery of genes required for the
synthesis of the myelin membrane (cluster 4). Our ob-
servation of strict coexpression of myelin protein genes
with genes involved in the biosynthesis of cholesterol
and galactolipids in developing Schwann cells, together
with similar observations recently reported for tran-
sected and crushed nerves (Nagarajan et al. 2002), are of
particular interest given the demonstration that myelin
lipid metabolism genes in different com-
partments of the adult sciatic nerve. (A) Q-
PCR on endoneurium and peri/epineurium
fractions of adult sciatic nerve (P56) for de-
picted genes. Numbers next to bars depict
the cluster associated with that mRNA.
(NOC) Not on chip. Data are averages of
triplicate experiments. (B) Western blots on
total lysates (40 µg) of P10 and adult (P56),
or endoneurium (24 µg) or peri/epineurium
(16 µg) fractions of adult sciatic nerve, for
depicted proteins. Note that the protein
amount of adult endoneurium and peri/epi-
neurium fractions loaded are relative to
their contribution to the protein amount of
total adult nerve. Protein samples were de-
rived from at least eight sciatic nerves.
Quantitation of expression of
Energy mobilization in peripheral nerves
GENES & DEVELOPMENT2459
structural proteins are delivered to the myelin sheath via
membrane rafts enriched in cholesterol and galactolipids
(Simons et al. 2000). The finding that genes involved in
the biosynthesis of distinct components of the myelin
sheath follow the same timecourse of expression during
Schwann cell development may indicate coordinate
transcriptional control by the same factor(s). We have
identified SREBP-2 as a candidate transcriptional regula-
tor of myelin lipid metabolism in the peripheral nerve. A
large number of studies have demonstrated that SREBP-2
regulates lipid and cholesterol homeostasis in fat and
liver (for review, see Horton et al. 2002). The fact that
SREBP-2 expression in liver is induced by cholesterol
depletion is in keeping with its expression in myelinat-
ing Schwann cells, whose synthesis of the cholesterol-
rich myelin membrane may lead to transient cytosolic
cholesterol depletion (Fu et al. 1998), especially because
nearly all accumulated cholesterol in the developing sci-
atic nerve is synthesized within the nerve (Jurevics and
Lipid metabolism in the mature peripheral nerve
Our data demonstrate that adult Schwann cells express
multiple genes involved in fatty acid metabolism (e.g.,
SREBP-1, Fasn, Scd1) and extracellular lipid transport
(e.g., Apod and Apoe). SREBP-1 is known to be an im-
portant transcriptional regulator in liver for genes in-
volved in fatty acid metabolism, including Fasn and
Scd1 (Shimano et al. 1997; Horton et al. 1998), and is
likely to have a similar function in the peripheral nerve.
Apod and Apoe, which are required for the formation and
transport of lipids via lipoproteins, probably function in
lipid transport in the mouse peripheral nerve, as has been
shown for Apoa-I in the chick (LeBlanc et al. 1989). Im-
portantly, inactivation of the mouse Apoe gene affects
the morphology of nonmyelinating Schwann cells and
results in sensory nerve defects (Fullerton et al. 1998),
which ties lipoprotein-mediated lipid transport in/from
Schwann cells to the functioning of the mature nerve.
Direct evidence of the importance of local storage lipid
metabolism to the maintenance of mature peripheral
nerve functions comes from our analysis of Lpin1 mu-
tant mice. In contrast to earlier reports (Peterfy et al.
2001), we demonstrate that Lpin1 mRNA and protein are
expressed in both the endoneurium and peri/epineurium
of the peripheral nerve, where they follow a “cluster 5”
developmental expression profile. Mutation of Lpin1 af-
fects both of these nerve compartments: It leads to a
strong reduction in the adipose mass in the epineurium,
it is required for storage lipid metabolism. (A) Staining
of storage lipids with Oil Red O in adult wild-type and
Lpin1 mutant (fld/fld) sciatic nerve. Note the strong
reduction in size of fat deposits (dark red; arrowheads)
in the Lpin1 mutant nerve. Data are representative of
at least three animals studied per genotype. (B) Higher
magnification of the central section of the nerves pre-
sented in A. Arrowheads point to fat-filled adipocytes.
Note the lines of uniformly sized adipocytes in the
wild-type nerve, in contrast to the few irregularly
shaped adipocytes in the Lpin1 mutant. (C) Q-PCR
measurements on Lpin1 mRNA at depicted develop-
mental stages of total sciatic nerve. Extra measure-
ments at P56 illustrate mRNA expression in endoneu-
rium (?) or peri/epineurium (?). Standard deviations
were calculated from triplicate experiments. (D) West-
ern blots of endoneurium (24 µg) or peri/epineurium
(16 µg) fractions of wild-type (wt) or Lpin1 mutant (fld/
fld) adult sciatic nerve, for depicted proteins. Note that
the difference in detection of Apoe in wild-type
samples compared with Figure 8B is due to shorter ex-
posure time. Protein samples are derived from at least
three animals per genotype. Bars: A,B, 700 µm.
Lpin1 is expressed in the sciatic nerve where
Verheijen et al.
2460 GENES & DEVELOPMENT
and to the altered expression of proteins—including Fasn
and Apoe—required for storage lipid metabolism in the
endoneurium. This strongly suggests that the pro-
nounced peripheral neuropathy evident in the mutants is
a direct consequence of the absence of Lipin1, and the
attendant dysregulation of storage lipid metabolism, in
the nerve itself, and not a secondary effect from neonatal
hypertriglyceridemia and fatty liver during the first 4
postnatal weeks (Langner et al. 1989, 1991).
Although adipose tissue in adult peripheral nerves has
been noted previously (Sunderland 1945, 1965), its pres-
ence has for the most part been neglected (Thomas and
Olsson 1984; Peters et al. 1991; Stolinski 1995). Adipo-
cytes have been classically viewed as a storage depot for
lipids, and as such may serve as a source of fatty acids for
the endoneurium. It should be noted that fat also func-
tions as an endocrine organ, through the production and
secretion of adipokines that act on muscle, brain, and fat
to regulate energy homeostasis and insulin action (Sal-
tiel and Kahn 2001). Although the perineurium forms a
diffusion barrier between the epineurium and the endo-
neurium, these two compartments are nonetheless con-
nected by the vascular network of the peripheral nerve
(Rechthand and Rapoport 1987). Our finding that two
adipokines—Acrp30 and Resistin—are highly expressed
in the epineurium suggests that this compartment may
regulate lipid metabolism in Schwann cells and/or axons
via supply of both fat metabolites and adipokines. Meta-
bolic studies have demonstrated that both fatty acids and
TAGs have a high turnover rate in the endoneurial com-
partment of the adult sciatic nerve, in contrast to cho-
lesterol and galactolipids (Yao 1985). These fatty acids
and storage lipids, delivered to the endoneurium through
adipokine-mediated release from epineurial adipocytes,
Schwann cells and/or axons.
Implications for storage lipid metabolic disorders
associated with peripheral neuropathy
Our study may provide insight into human diseases pro-
ducing demyelinating peripheral neuropathies associ-
ated with altered lipid metabolism. The Lpin1 mutant
mouse described in this study is a model for human li-
podystrophy (Langner et al. 1989, 1991; Reue et al. 2000;
Peterfy et al. 2001), for which numerous cases with pe-
ripheral neuropathy have been reported (Fessel 1971;
Tuck et al. 1983). Also, Refsum’s disease is caused by
mutation in phytanoyl-CoA hydroxylase (Pahx), which
encodes for a peroxisomal enzyme regulating branched
chain fatty acid oxidation (Jansen et al. 1997; Mihalik et
al. 1997). We observe that Pahx is also expressed in the
sciatic nerve, where it follows a “cluster 5” expression
profile (see Supplemenatry Table S1). Furthermore, on-
going storage lipid metabolism in both the adipose epi-
neurium and in Schwann cells may be related to the high
prevalence (∼30%) of peripheral neuropathies in both in-
sulin-dependent (type I) and independent (type II) diabe-
tes mellitus (Tesfaye et al. 1996). Diabetic peripheral
neuropathy (DPN) results in reduced conduction veloc-
ity and demyelination of the peripheral nerve, but there
is no general agreement as to whether the demyelination
is the primary effect or whether it is a secondary effect of
axonal damage. Several competing hypotheses have been
advanced to explain the pathogenesis of DPN, including
perturbed lipid metabolism, but its biological basis re-
mains obscure (for review, see Sugimoto et al. 2000). Li-
poprotein lipase (Lpl) activity in the sciatic nerve is re-
duced in diabetic rats and corrected by treatment with
insulin, which links Lpl and fatty acids to the patho-
physiology of diabetic neuropathy (Ferreira et al. 2002).
Our demonstration that epineurial adipocytes are the
main contributors of Lpl expression in the mature sciatic
nerve suggests that production of Lpl by these cells pro-
motes the hydrolysis of triglyceride-rich lipoproteins in
the bloodstream, and thereby facilitates uptake of fatty
acids by Schwann cells.
It should be noted that both Lpin1 mutation as well as
diabetes cause demyelinating neuropathy in the PNS,
whereas myelin in the CNS is generally not affected
(Brownlee et al. 1986; Langner et al. 1991; Sugimoto et al.
2000). This is striking given the presence of adipocytes
specifically in the peripheral nerve—these cells have not
been detected apposed to axon tracts in the CNS. Indeed,
the peripheral epineurium ends at the point at which
peripheral nerves enter the CNS, where it becomes con-
tiguous with the dura mater (Peters et al. 1991). We ex-
amined one “nerve-like” CNS axon tract in the mouse—
the optic nerve that connects the eyes to the brain—and
observed that it does not contain an adipose epineurium
(M.H.G. Verheijen, R. Chrast, P. Burrola, and G. Lemke,
In conclusion, our transcriptional analysis of develop-
ing sciatic nerve provides a newly comprehensive de-
scription of biological processes underlying Schwann
cell maturation. Together with the identification of hu-
man homologs and their mapping positions for the
highly differentially expressed genes, these data will con-
tribute to the identification of new peripheral neuropa-
thy disease genes. Most importantly, our findings pro-
vide important new functional insights into the regula-
tion and role of lipid metabolism in the endoneurium
and peri/epineurium of peripheral nerve, and may have
important implications for the understanding and treat-
ment of peripheral neuropathies associated with lipid
metabolic disorders, including lipodystrophy and diabetes.
Materials and methods
ICR mice were obtained from Harlan Sprague Dawley. Female
Lpin1fld/fldmice were obtained from The Jackson Laboratory.
Throughout the text, mice of the Lpin1fld/fldphenotype are re-
ferred to as “Lpin1 mutant”, and mice of both Lpin1fld/+and
Lpin1+/+are referred to as “wild-type”.
Total RNA was isolated from sciatic nerves of at least 100 ICR
mice for each developmental stage (E17, P0, P2, P4, P10, and
Energy mobilization in peripheral nerves
GENES & DEVELOPMENT2461
P56) using TRIzol (Life Technologies), and subsequently puri-
fied by RNeasy kit (QIAGEN). Five micrograms of total RNA
was labeled and hybridized to MG-U74Av2 chips (Affymetrix),
as specified by the manufacturer. All labelings and hybridiza-
tions were done in duplicate.
Microarray data were analyzed using the Affymetrix Microarray
Suite 4.0; per chip normalization was performed by scaling the
Average Intensity of every chip to a Target Intensity of 2500.
Subsequently, those genes that were called “expressed” and
“differentially expressed” according to Affymetrix Microarray
Suite 4.0 were selected using the software Bullfrog (Zapala et al.
2002). Next, data were imported into GeneSpring (Silicon Ge-
netics), and per gene normalization was performed by using the
median for each gene’s expression values over all six time points
(if expression was at least 0.01, negative values were set to 0).
Subsequently, the genes showing high differential expression
(p < 0.01 in ANOVA) were selected, and K-means cluster analy-
ses were performed using Pearson correlation with GeneSpring.
For each of the highly differentially expressed genes, the se-
quence was updated via BLAST search (http://www.ncbi.nlm.
nih.gov/BLAST) when required, and biological function anno-
tation was determined using the SOURCE database (http://
When required, biological function annotation was created
based on literature and according to the definitions of the Gene
Ontology Consortium using the AmiGO database (http://www.
godatabase.org/cgi-bin/go.cgi). Subsequently, genes were classi-
fied based on their biological function, and expression profiles of
the different groups were analyzed using GeneSpring. Annota-
tions for groups with putative outliers (with clearly distinct
expression profile compared to other members of the same
group) were re-examined. Human homologs and their map po-
sitions were determined by BLAST search (when required), and
searching the SOURCE database. Critical regions for each pe-
ripheral neuropathy were obtained via the Inherited Peripheral
Neuropathies Mutation Database (http://molgen-www.uia.ac.
be/CMTMutations) and the Online Mendelian Inheritance in
Man database (http://www.ncbi.nlm.nih.gov/entrez/query.
fcgi?db=OMIM).To determine whether a gene maps to the criti-
cal regions for a peripheral neuropathy, we used a human map
cgi?). Because annotation of the human genome is a dynamic
process, all data reflect the status of the genome as of July 2003.
Microdissection of sciatic nerve
Sciatic nerves from adult (P56) mice were placed in ice-cold
phosphate-buffered saline (pH 7.4). The perineurium, with the
epineurium attached to it, was gently dissected away from the
endoneurium along the whole length of the nerve, with the aid
of fine forceps and a binocular dissecting microscope. In those
cases where the nerve was heavily fasciculated and proper mi-
crodissection was therefore not possible, that part of the nerve
Quantitative (real-time) PCR
cDNA was prepared using the SuperScript system (Life Tech-
nologies) starting with 250 ng of total RNA from the following
samples: (1) E17, P0, P2, P4, P10, P28, and P56 unfractioned
sciatic nerves; (2) dissected endoneurium form P56 sciatic
nerves; and (3) dissected peri/epineurial part of P56 sciatic
nerves. RNA was isolated from an independent pool of at least
10 animals, and was not used for array experiments. Relative
quantification of expression of selected genes was performed
using an ABI PRISM 7700 Sequence Detection System with
SYBR green chemistry (Applied Biosystems) as described
Dissociation curve analysis was performed using Dissociation
Curve 1.0 software (ABI) for each PCR reaction to detect and
eliminate possible primer-dimer artifacts. Oligonucleotides (se-
quences available upon request) were selected to amplify a frag-
ment containing sequences from two adjacent exons in order to
avoid contaminating genomic DNA amplification. To standard-
ize the amount of cDNA in each reaction, we measured the
amount of the ubiquitin-conjugating enzyme Ube2l3 (GB#:
NM_009456), which showed no variation in expression in the
sciatic nerve between E17 and P56 as detected by array experi-
ments, and as was confirmed by independent Q-PCR measure-
ments (data not shown).
In situ hybridization
Expression patterns of selected genes were analyzed by in situ
clones obtained from ResGen (Invitrogen; see Supplementary
Table S6), as described (Zhadanov et al. 1995). Dissected sciatic
nerves were immersion-fixed in 4% paraformaldehyde/PBS
overnight at 4°C, followed by overnight infiltration with 20%
sucrose/PBS. Cryoprotected tissues were then embedded in
OCT medium (Miles). Hybridization was performed with 6-µm
longitudinal frozen sections of adult (P56) sciatic nerves. Con-
trol hybridizations with sense strand-labeled riboprobes gave no
significant signals for all selected genes (data not shown).
33P-radiolabeled riboprobes using EST
Total and/or microdissected fractions of sciatic nerves were
taken up in liquid nitrogen and thawed using boiling lysis buffer
(BLB; 10 mM Tris-HCl at pH 7.4, 1% SDS, 1 mM sodium vana-
date, and Complete protease inhibitor cocktail; Roche). After
thawing, extra protease inhibitors were added and the nerves
were homogenized using a dounce and a polytron. Subse-
quently, lysates were centrifuged and the protein concentration
of the supernatant was measured using a Pierce BCA protein
assay kit. After SDS-PAGE, proteins were blotted to Hybond-P
PVDF membrane and incubated with appropriate primary anti-
bodies: rabbit anti-Lipin [provided by Dr. J.C. Lawrence Jr. (Uni-
versity of Virginia School of Medicine, Charlottesville, VA)] was
used at 2 µg/mL, mouse anti-Mpz monoclonal antibody P07
[provided by Dr. J.J. Archelos (Karl-Franzens-Universitat, Graz,
Austria)] was used at a dilution of 1:10,000, rabbit anti-Acrp30
[provided by Dr. H. Lodish (Whitehead Institute for Biomedical
Research, Cambridge, MA)] was used at 1:250, mouse mono-
clonals against Fasn and Thrsp (BD Biosciences) were used at
1:250, rabbit-anti Apoe (Calbiochem) was used at 1:2000, and
mouse monoclonal against actin (Oncogene) was used at 1:5000.
Subsequently, the blots were incubated with peroxidase-labeled
secondary antibody, followed by ECL.
Immunohistochemistry and histology
To analyze the expression of SREBP-1, we blocked 6-µm cross-
sections of immersion-fixed (4% paraformaldehyde/PBS over-
night at 4°C) adult (P56) sciatic nerves in 15% NDS/0.1% Tri-
tonX in PBS for 1 h before incubation with rabbit anti-mouse
SREBP-1 [provided by Dr. H. Shimano, (University of Tsukuba,
Tsukuba, Ibaraki, Japan)] at a dilution of 1 to 50 together with
Verheijen et al.
2462 GENES & DEVELOPMENT
mouse anti-pig Neurofilament 160 (Sigma) at a dilution of 1 to
200. Slides were subsequently incubated with Cy3-conjugated
donkey anti-rabbit IgG (Jackson Immunoresearch Labs) together
with Alexa Fluor 488-conjugated goat anti-mouse IgG (Molecu-
lar Probes), and staining was completed using a DAPI-Vecta-
shield mounting medium (Vector Laboratories).
Immersion-fixed (4% paraformaldehyde/PBS overnight at
4°C) adult (P56) sciatic nerves were stained with Mayer’s He-
matoxylin (on 6-µm frozen sections) for 5 min or Oil Red O (on
intact nerves) for 10 min, followed by washing with tapwater for
5 min. Hematoxylin-stained nerves sections were subsequently
incubated in Eosin Y (Alcoholic) for 1 min, washed with 100%
ETOH for 1 min, followed by a 5-min incubation with Histo-
clear (National Diagnostics). All incubations were done at room
temperature. Sections were mounted using Permount (Fisher).
We thank Dan Syroid for advice and help with tissue isolations,
and Joe Hash for technical assistance. We thank J.C. Lawrence
Jr., H. Shimano, J.J. Archelos, and H. Lodish for generous supply
of antibodies. This work was supported by a stipend of the Ter
Meulen Fund, Royal Netherlands Academy of Arts and Sci-
ences, and a long-term fellowship of the Human Frontier Sci-
ence Program (to M.H.G.V); by a postdoctoral fellowship from
the Swiss National Science Foundation (to R.C., grant 823A-
064660); and by grants from the NIH (to G.L.).
The publication costs of this article were defrayed in part by
payment of page charges. This article must therefore be hereby
marked “advertisement” in accordance with 18 USC section
1734 solely to indicate this fact.
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