Embryonic Expression of the Soma-Restricted Products
of the Myelin Proteolipid Gene in Motor Neurons
Erin C. Jacobs,1,4Ernesto R. Bongarzone,1,3Celia W. Campagnoni,1
and Anthony T. Campagnoni1,2
(Accepted September 15, 2003)
In addition to classic proteolipid protein (PLP) and DM20, the mouse myelin proteolipid gene
produces the sr-PLP and sr-DM20 proteins. The sr-isoforms are localized to the cell bodies of
both oligodendrocytes and neurons. However, they are expressed to a greater extent in neurons
than they are in glia. In this study, we examined expression of the sr-proteolipids in the mouse
embryo using immunohistochemistry with an sr-PLP/DM20 specific antibody. Widespread expres-
sion of the sr-proteins was found in many nonmyelinating cell types. In particular, strong
immunoreactivity was detected in motor neurons of both the autonomic and somatic nervous sys-
tems as well as in striated muscle. This pattern of expression persisted throughout the embryonic
period studied. Thus, the sr-proteolipids are expressed prior to the onset of myelination and in a
much broader array of cell types than their classic counterparts. These results support the con-
clusion that the sr-isoforms of the PLP gene have a biological role independent of myelination.
KEY WORDS: Development; motor neurons; muscle; PLP gene expression; sr-DM20; sr-PLP.
The proteolipid protein (PLP) gene is alternatively
spliced to produce two transmembrane proteins: PLP and
DM20. These proteolipids comprise nearly 50% of the
proteins in myelin and are believed to serve an integral
role in stabilizing the compaction of the myelin sheath (1).
PLP and DM20 were originally thought to be expressed
solely by interfascicular oligodendrocytes. It is now
known that the gene is also expressed in the peripheral
nervous system (PNS), nonmyelinating cells in the embry-
onic central nervous system (CNS), as well as in the heart
and the immune system (2–6).
A new exon was recently identified in the mouse
PLP/DM20 gene and mapped to the genomic segment
between exons 1 and 2 (7). Within this exon is a cryptic
translation initiation start site that when used produces
full-length PLP and DM20 proteins with an additional 12-
amino-acid leader sequence linked to their N-terminals.
This leader sequence appears to target these isoforms to
the cell body away from the myelin sheath and thus have
been termed soma-restricted (sr)-PLP and sr-DM20 to
denote this distinction (7,8).
Though sr-PLP and sr-DM20 are expressed in
oligodendrocytes, the highest levels of immunore-
activity occurs in neurons. We have previously shown
0364-3190/04/0500–0997/0 © 2004 Plenum Publishing Corporation
Neurochemical Research, Vol. 29, No. 5, May 2004 (© 2004), pp. 997–1002
*Special issue dedicated to Dr. Nicole Baumann.
1Developmental Biology Group, Mental Retardation Research Cen-
ter, UCLA School of Medicine, Los Angeles, California 90024.
2Brain Research Institute, UCLA School of Medicine, Los Angeles,
3Present address: Laboratory for Gene Therapy of Neurodegenerative
Disorders, San Raffaele Telethon Institute for Gene Therapy, Milan,
4Address reprint requests to: Dr. Erin C. Jacobs, Neuropsychiatric
Research Institute, Room 47-444, UCLA School of Medicine, 760
Westwood Plaza, Los Angeles, California 90024. Tel: (310)825-0459;
Fax: (310)206-5050; E-mail: firstname.lastname@example.org
998 Jacobs, Bongarzone, Campagnoni, and Campagnoni
Fig. 1. Diagram of the mouse PLP/DM20 gene including the new
exon 1.1 and the products generated from that exon. Exon 1.1
(shaded) is spliced in frame to produce sr-PLP and sr-DM20
mRNAs. The translation initiation start site for the sr-proteolipids
is contained in exon 1.1. Use of this site leads to the synthesis
of PLP and DM20 proteins with 12 additional amino acids at their
sr-PLP/DM20 immunoreactivity in many neuronal
populations along the neuraxis including cells in the
olfactory bulb, neocortex, hippocampus, thalamus, and
cerebellum (9). The expression in these cell types is
developmentally regulated with the strongest levels of
staining occurring in late embryonic and early postna-
tal development. One exception to this observation is
the robust expression of the sr-proteins in spinal cord
and dorsal root ganglia during early embryogenesis. In
this report, we describe the expression of sr-PLP and
sr-DM20 in the embryonic mouse spinal cord, cranial
nuclei, and muscle.
Tissue Preparation. Timed-pregnant females (E0 ? day of insemi-
nation) were anesthetized and sacrificed by cervical dislocation. Embryos
were removed, rinsed briefly in PBS, and immersion-fixed in 4% buffered
paraformaldehyde for 4–6 h. All tissues were cryoprotected in sucrose
and frozen in OCT (optimal cutting temperature) embedding compound
(Tissuetek, Miles, Elkhart, IN, USA). Frozen cryostat sections (20-?m
thick) were cut on a sagittal plane, collected onto Superfrost Plus micro-
scope slides (Fisher, Pittsburgh, PA, USA) and stored at ?20°C until
Immunohistochemistry. Recombinant peptide corresponding to
the first 16 amino acids of the srPLP and srDM20 specific sequence
was used to generate polyclonal antisera (7). In this study, we used
an affinity purification of the rabbit polyclonal antiserum at a dilution
Frozen sections were dried at 37°C for 30 min and rinsed in PBS
prior to immunohistochemistry. Endogenous peroxidase activity was
quenched by incubating the slides in a solution containing 0.3% H2O2
and 10% methanol in PBS for 10 min. After rinsing in PBS, nonspe-
cific labeling was blocked by preincubation in 0.1% Triton-X, 0.1%
casein PBS, and 3% normal goat serum for 1 h at room temperature.
Sections were then incubated with the primary antibody diluted in
0.1% Triton-X, 0.1% casein PBS, and 1.5% normal goat serum
overnight at 4°C. The primary antibody was visualized by the
immunoperoxidase method using avidin–biotin–horseradish peroxi-
dase (HRP) complex (ABC kit; Vector Labs, Burlingame, CA, USA)
with 3,3?-diaminobenzidine (DAB, Roche, Indianapolis, IN, USA) as
the chromagen per manufacturer’s instructions. Images were obtained
using a Leica DM RXA microscope (Nussloch, Germany).
The organization of the PLP/DM20 gene is dia-
grammed in Fig. 1. The sr-PLP/DM20 specific exon
(1.1; shaded) is located between exons 1 and 2 and con-
tains a translation initiation start site separate from the
site in exon 1 used to produce the “classic” PLP and
DM20 proteins. Use of the site in exon 1.1 results in
the production of full-length PLP and DM20 proteins
with an additional 12-amino-acid peptide linked to their
We previously identified sr-PLP/DM20 immunore-
activity in the mouse CNS with the highest levels of
staining occurring between embryonic day 17 (E17) and
postnatal day 21 (P21) (7,9). However, as early as E13,
robust staining was detected in the spinal cord and dor-
sal root ganglia. To examine this early embryonic
expression in more detail, we have taken tissue from
E12 to E17 mice and used immunohistochemistry with
an affinity-purified form of the antibody specific to the
sr-PLP/DM20-unique peptide. The characteristics of this
antibody have been reported (7).
As shown in Fig. 2, the embryonic spinal cord
stained intensely for the antibody for sr-PLP/DM20. At
E12, staining for the sr-proteolipids was detected in the
cell bodies within each layer of the developing spinal
cord [i.e., the dorsal gray (DG), intermediate gray (IG),
and ventral gray (VG) layers] (Fig. 2A). At later ages,
there was a moderate decline in staining intensity
throughout the spinal cord. However, populations of
large motor neurons in the medial and lateral motor
columns (MMC and LMC, respectively) remained
intensely labeled (Figs. 2B–2D). These nuclei were par-
ticularly evident at the level of the lumbar (Fig. 2B) and
cervical (Figs. 2C and 2D) regions of the spinal cord
where their axons exit the ventral horn to innervate the
skeletal muscles of the limbs. The efferents from the
ventral spinal column, however, remained unlabeled.
This lack of staining in the processes of the motor neu-
rons is consistent with our previous findings that the
subcellular localization of the sr-proteins is restricted
largely to the cell body.
Expression of PLP Isoforms in Embryonic Motor Neurons and Muscle 999
Fig. 2. Robust expression of sr-PLP/DM20 protein in the embryonic spinal cord. (A) At E12, sr-PLP/DM20 staining was evident in all layers
of developing spinal column including the dorsal gray (DG), the intermediate gray (IG), and the ventral gray (VG). Sagittal section with dorsal
(D) and caudal (C) orientation markers indicated. At older ages, staining of the sr-proteins persisted and was particularly evident in the large
motor neurons of the (B) lumbar and (C, D) cervical enlargements. Within these regions, label was particularly evident in the cell bodies of the
motor neurons in (B) the medial motor column (MMCn) at E15 and the lateral motor column (LMCn) at (C) E14 and (D) E16. DH ? dorsal
horn. Scale bar: 100 ?m (A, C); 120 ?m (B, D).
In addition to neurons in the spinal cord, strong
immunoreactivity for the sr-proteolipids was detected in
many cranial nerve nuclei and ganglia (Fig. 3). Expres-
sion was particularly evident in the cranial motor nucleus
of the facial nerve (VII, Fig. 3A), the somatomotor
nucleus of the hypoglossal nerve (XII, Fig. 3B), as well
as the oculomotor nucleus and the motor nucleus of the
trigeminal nerve (data not shown). Interestingly, neurons
within both the migratory population (VIInm) and the
nucleus (VIIn) of the facial nerve were labeled at E12
(Fig. 3A). Low levels of staining were also detected in
the pre- and postganglionic motor neurons of the autono-
mic nervous system (ANS; data not shown). In addition
to motor neuron populations, immunoreactivity for the
sr-proteolipids was detected in the sensory pseudounipo-
lar neurons of the trigeminal (V, Fig. 3C) and the glos-
sopharyngeal (IX, Fig. 3D) ganglia.
Though the sr-proteins were expressed in a vari-
ety of neuronal subtypes, many of the labeled nuclei
in the brain stem and spinal cord share a common
characteristic in their innervation of striated skeletal
muscle. Throughout the developmental period stud-
ied, strong immunoreactivity for sr-PLP/DM20 was
detected in the target musculature of many of these
nuclei. For example, the intrinsic and extrinsic muscles
of the tongue stained intensely for the sr-PLP/DM20
1000 Jacobs, Bongarzone, Campagnoni, and Campagnoni
Fig. 3. Staining of sr-PLP/DM20 proteins in cranial nuclei and ganglia. (A) Labeled motor neurons in the nucleus of the seventh nerve (VIIn,
facial). At E13, immunostaining of the motor neurons migrating to the facial nucleus (VIInm) were also evident. (B) Immunoreactive neurons
in the nucleus of the twelfth nerve (XII, hypoglossal) in an E14 animal. Sensory pseudounipolar neurons in (C) the trigeminal (Vg) and in both
(D) the inferior (IXgi) and superior (IXgs) glossopharyngeal ganglia stained with the sr-PLP/DM20 antibody at E13. V4 ? fourth ventricle.
Scale bar: 60 ?m (A–D).
antibody (Fig. 4A). The axial skeletal muscle groups
were also labeled. These muscle groups include mus-
cles along the spine and rib cage (Fig. 4B) as well
as in the fore and hind limbs (data not shown). Strong
immunoreactivity to the sr-proteins was also detected
in the axial muscles that form the abdominal wall
(Fig. 4C). In addition to striated muscle groups,
myocardial fibers in the heart were intensely labeled
(Fig. 4D) and at low levels in smooth muscle along
the gut (data not shown). Low immunoreactivity was
also detected in a number of organs including the
large and small intestine, stomach, liver, brown adi-
pose tissue, lung, kidney, and thymus. Label in some
of these organs may be due in part to sr-PLP/DM20
staining of smooth muscle and/or in cell bodies of
It has become increasingly evident that the prod-
ucts of the PLP gene are expressed in cell types other
than oligodendrocytes and that they have functions
beyond the compaction of the myelin sheath (for a
review see Ref. 10). In this study, immunohistochem-
ical analyses indicated that the sr-proteolipids are pre-
sent in motor neurons of the spinal cord and ANS,
various cranial ganglia of the PNS, and in muscle.
Expression of PLP Isoforms in Embryonic Motor Neurons and Muscle 1001
Fig. 4. The sr-proteins are present in muscle fibers throughout the mouse embryo. (A) As early as E13, sr-PLP/DM20 immunoreactivity was
evident in both the intrinsic (T: transversus) and extrinsic (G: genioglossus, S: styloglossus) muscles of the tongue. (B) Similarly, at E13 the
axial-skeletal muscles were immunoreactive for the sr-proteins including the muscles along the spine such as the semispinalis cervicis (SC) and
between the ribs [internal intercostals (IIC) and the external intercostals (EIC)]. (C) Immunostaining was also evident in the muscles that form
the body wall: transversus abdominis (TA), internal oblique (IO) and external oblique (EO) in an E15 animal. (D) Intense staining for sr-
PLP/DM20 in cardiac muscle (C) at E13. Scale bar: 160 ?m (A), 100 ?m (B, D), 50 ?m (C).
Moreover, the expression of the sr-PLPs persisted in
these cell types throughout the developmental period
In previous studies, sr-PLP and sr-DM20 proteins
were detected in the cell bodies of both oligodendro-
cytes and neurons with the highest levels of immunore-
activity occurring in neurons during the early, postnatal
period (7–9). However, during early embryonic devel-
opment, staining for the sr-proteins was much greater in
the spinal cord than in the rest of the CNS. In this report,
robust immunostaining for the sr-PLPs was present in
the spinal cord as early as E12 and more moderate stain-
ing persisted in the embryonic spinal cord, particularly
in large motor neurons.
Neuronal expression of the sr-proteolipids was not
limited to motor neurons in the embryonic CNS. Anti-
body for sr-PLP/DM20 also labeled pre- and postgan-
glionic motor neurons of the ANS, interneurons in the
spinal cord, as well as sensory neurons in the spinal and
cranial ganglia of the PNS. Interestingly, the nonclassic
products of the golli-mbp gene are also expressed
primarily in neurons and have been detected in many
neuronal populations in the CNS and PNS during devel-
opment (11–14). Combined, these data suggest a general
role for the nonclassic isoforms of the myelin protein
genes during neuronal development.
It is important to note that the antibody used in this
study does not distinguish between the two sr-isoforms.
However, like classic PLP and DM20 mRNAs, sr-DM20
mRNA expression precedes that of sr-PLP mRNA dur-
ing development (7). Thus, it is likely that the predom-
inant form of the sr-proteins being expressed during
embryonic development is sr-DM20.
One of the most striking areas of sr-PLP/DM20
expression was in the striated muscle fibers labeled
throughout the embryo. To our knowledge, there are no
reports of immunostaining for PLP/DM20 in muscle tis-
sue. DM20 transcripts have been detected in myocardial
1002 Jacobs, Bongarzone, Campagnoni, and Campagnoni Download full-text
cells by Northern blot, reverse transcription-polymerase
chain reaction (RT-PCR), and in situ hybridization (3,
15). PCR fragments containing PLP/DM20 exons 3 to 4
have also been identified in a variety of mouse tissues
including liver, intestine, adipose tissue, lung, muscle,
kidney, and thymus (15). In this report, immunostain-
ing for sr-PLP/DM20 was detected in many of these
same tissues. Currently, it is not clear whether the mRNA
products detected in these studies reflect DM20 or
sr-DM20 transcripts. However, the primers used in the
RT-PCR experiments by Nadon et al. (15) would recog-
nize and amplify an equivalent size product from either
In summary, the sr-products of the PLP gene are
expressed throughout the embryo in a variety of cell
types. The widespread pattern of expression during mul-
tiple stages of development supports the notion that
sr-PLP and sr-DM20 serve a function unrelated to mye-
lination. In nearly all cell types that express sr-PLP/
DM20, the proteins have been localized in the cell body.
In oligodendrocytes, sr-PLP/DM20 co-localize with traf-
ficking molecules clathrin and syntaxin 6 suggesting a
possible role of the sr-PLPs in vesicular transport (8).
Whether the sr-PLPs serve a similar function in other
cell types remains to be determined.
The authors wish to thank Dr. Ellen Carpenter and Dr. Robin Fisher
for their helpful discussions during the preparation of this manuscript.
This work was supported by grants from the National Institutes of Health
(NS23022 and NS33091) and from the National Multiple Sclerosis Soci-
ety (RG2693). E. C. J. was supported in part from an National Research
Service Award grant (MH199250304b).
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