Download Gpr126 is essential for peripheral nerve development and

RESEARCH ARTICLE 2673
Development 138, 2673-2680 (2011) doi:10.1242/dev.062224
© 2011. Published by The Company of Biologists Ltd
Gpr126 is essential for peripheral nerve development and
myelination in mammals
Kelly R. Monk1,*, Kazuo Oshima2, Simone Jörs2, Stefan Heller2 and William S. Talbot1,†
SUMMARY
In peripheral nerves, Schwann cells form the myelin sheath that insulates axons and allows rapid propagation of action potentials.
Although a number of regulators of Schwann cell development are known, the signaling pathways that control myelination are
incompletely understood. In this study, we show that Gpr126 is essential for myelination and other aspects of peripheral nerve
development in mammals. A mutation in Gpr126 causes a severe congenital hypomyelinating peripheral neuropathy in mice, and
expression of differentiated Schwann cell markers, including Pou3f1, Egr2, myelin protein zero and myelin basic protein, is reduced.
Ultrastructural studies of Gpr126–/– mice showed that axonal sorting by Schwann cells is delayed, Remak bundles (non-myelinating
Schwann cells associated with small caliber axons) are not observed, and Schwann cells are ultimately arrested at the promyelinating
stage. Additionally, ectopic perineurial fibroblasts form aberrant fascicles throughout the endoneurium of the mutant sciatic nerve.
This analysis shows that Gpr126 is required for Schwann cell myelination in mammals, and defines new roles for Gpr126 in axonal
sorting, formation of mature non-myelinating Schwann cells and organization of the perineurium.
INTRODUCTION
Myelin is a multilayered membrane formed by glial cells around
axons in the vertebrate nervous system. In the peripheral nervous
system (PNS), Schwann cells form the myelin sheath by wrapping
their membrane around an axonal segment many times. Schwann
cell precursors arise from neural crest progenitor cells, and
previous work has defined several steps in the developmental
progression leading to mature Schwann cells (Jessen and Mirsky,
2005). During embryogenesis, Schwann cell precursors co-migrate
with axons in the developing PNS and differentiate into immature
Schwann cells that remain associated with many axons. Immature
Schwann cells may become either non-myelinating or myelinating
Schwann cells. Non-myelinating Schwann cells remain associated
with multiple small-caliber axons. These axons are ensheathed by
non-myelinating Schwann cells in Remak bundles, but are not
myelinated. Other immature Schwann cells begin to sort largecaliber axons and develop into promyelinating Schwann cells that
associate with a single axonal segment. Promyelinating Schwann
cells differentiate into myelinating Schwann cells, which repeatedly
wrap their membrane around an axonal segment to eventually form
a compact myelin sheath.
Investigations in mammals have defined a number of essential
regulators of Schwann cell development, including the axonal signal
neuregulin 1 and the Schwann cell transcription factors Pou3f1 (also
known as Oct-6), Pou3f2 (also known as Brn-2) and Egr2 (also
known as Krox-20) (Nave and Salzer, 2006; Svaren and Meijer,
2008). Neuregulin 1 (Nrg1) signaling through ErbB receptors is
crucial for many steps in Schwann cell development, including
1
Department of Developmental Biology, Stanford University School of Medicine,
Stanford, CA 94305, USA. 2Department of Otolaryngology-Head and Neck Surgery,
Stanford University School of Medicine, Stanford, CA 94305, USA.
*Present address: Department of Developmental Biology, Washington University
School of Medicine, St Louis, MO 63110, USA
†
Author for correspondence ([email protected])
Accepted 6 April 2011
proliferation, ensheathment and myelination (Garratt et al., 2000;
Michailov et al., 2004; Taveggia et al., 2005). The transcription
factors Pou3f1, Pou3f2 and Egr2 are required for the development of
mature myelinating Schwann cells. Pou3f1 and Pou3f2 are required
for the timely differentiation of myelinating Schwann cells; Schwann
cells in Pou3f1-null mice transiently arrest at the promyelinating stage
and deletion of both Pou3f1 and Pou3f2 exacerbates this phenotype
(Bermingham et al., 1996; Jaegle et al., 1996; Jaegle et al., 2003).
Schwann cells in Egr2-null mice remain arrested at the
promyelinating stage and fail to wrap an axonal segment more than
1.5 turns (Topilko et al., 1994; Zorick et al., 1999). Less is known
about the genes controlling the development of non-myelinating
Schwann cells, but these cells express many of the same markers as
immature Schwann cells (Jessen and Mirsky, 2002).
We recently reported that the orphan G-protein-coupled receptor
Gpr126 is required autonomously in Schwann cells for the
expression of pou3f1 and egr2, and that Gpr126 is essential for
promyelinating Schwann cells to initiate myelination in zebrafish
(Monk et al., 2009). In the present study, we show that this function
of Gpr126 is conserved in mammals through the analysis of Gpr126
knockout mice. As in zebrafish, Schwann cells in Gpr126–/– mutant
mice are arrested at the promyelinating stage. Mutant mice have limb
posture abnormalities, are smaller than littermates and die before
weaning. In mutant sciatic nerves, we also observe delayed sorting
of axons by Schwann cells, aberrant fasciculation throughout the
endoneurium by perineurial fibroblasts, progressive loss of axons in
the postnatal period and an absence of Remak bundles. Our findings
show that Gpr126 is essential for several aspects of peripheral nerve
development in mammals.
MATERIALS AND METHODS
Mice
Gpr126 knockout mice were generated by Lexicon Pharmaceuticals (The
Woodlands, TX, USA) and heterozygous animals were obtained from
Taconic (Hudson, NY, USA; catalog number TF0909) on a mixed
background (129/SvEv-C57BL/6). These mice and their offspring were
crossed to generate the animals analyzed in this study. We used the
following PCR primers to determine the genotype of the mutant mice:
DEVELOPMENT
KEY WORDS: Schwann cell, Myelin, Gpr126, Mouse
2674 RESEARCH ARTICLE
RT-PCR and quantitative real-time PCR
For standard RT-PCR, we isolated RNA from Gpr126–/– and heterozygote
sibling sciatic nerves at postnatal day (P)4 using the RNeasy Micro Kit
(Qiagen) according to the manufacturer’s instructions. We reverse
transcribed the RNA using the SuperScript First-Strand synthesis system
for RT-PCR (Invitrogen) with Superscript II reverse transcriptase and
random hexamers per manufacturer’s instructions. Samples lacking reverse
transcriptase were used to control for genomic DNA contamination. We
amplified Gapdh as a control for each sample using published primers
(Peirano et al., 2000). Sox10, Egr2 and Dhh were amplified as described
[Sox10 and Egr2 (Peirano et al., 2000); Dhh (Parmantier et al., 1999)]. We
used the following primers to amplify Gpr126: 5⬘-CCAAAGTTGGCAATGAAGGT-3⬘ and 5⬘-GCTGGATCAGGTAGGAACCA-3⬘. For
quantitative real-time PCR (qPCR), the same heterozygote P4 sciatic nerve
cDNA and primers were used as described above. Mouse dorsal root
ganglia (DRGs) were collected from embryonic day (E)13.5 and neurons
were purified as described (Sasaki et al., 2009). RNA was extracted from
DRG neurons using Ribozol (Amresco) and cDNA was synthesized using
QScript cDNA Supermix (VWR) according to the manufacturer’s
instructions. qPCR was performed in an ABI 7500 using SYBR Green
Master Mix (ABI) according to standard protocols.
Transmission electron microscopy
Sciatic nerves and optic nerves were removed from mice of the indicated
ages and genotypes and immediately immersed in fresh fixative (4%
paraformaldehyde, 2% glutaraldehyde in 0.1M sodium cacodylate, pH 7.4)
for three hours. Nerves were contrasted by postfixation in 2% osmium
tetraoxide in 0.1M sodium cacodylate, pH 7.4, dehydrated, and embedded in
epoxy resin (EMBed-812, Electron Microscopy Sciences). We stained semithin sections (1 m) with Toluidine Blue for light microscope examination
(Zeiss Axioplan2). Ultrathin sections (70 nm) were stained and imaged as
described (Pogoda et al., 2006). We examined six siblings [three wild type
(WT), three Gpr126+/–] and three mutants at P1; five siblings (two WT,
three Gpr126+/–) and three mutants at P4; five siblings (two WT, three
Gpr126+/–) and three mutants at P8; five siblings (one WT, four Gpr126+/–)
and four mutants at P12.
Morphometric analysis
For P1 animals (n2 pairs of WT and Gpr126–/– siblings), images of the
sciatic nerve were taken in an overlapping series and montaged in Photoshop.
We then counted the total number of axons in P1 sciatic nerve. For WT
animals, the tibial branch of the nerve was analyzed; Gpr126 mutant nerves
were difficult to dissect and we did not observe branches in the nerve in the
regions examined. For P8 WT and Gpr126+/– siblings (n3: one WT and
two Gpr126+/–), we imaged Toluidine Blue-stained semi-thin sections (see
above) on a Zeiss Axioplan2 and digitally imaged the entire nerve with a
Zeiss AxioCam. We then counted the total number of myelinated axons in
the tibial branch of P8 sciatic nerve. To estimate the total number of axons
in WT nerve, we imaged ten randomly selected regions of the same nerves
using ultrathin sections at 8,000⫻ magnification. We then counted the total
number of unmyelinated axons and myelinated axons in these regions and
obtained an average ratio of unmyelinated:myelinated axons (576:168). We
used this ratio and the counted number of myelinated axons to estimate the
total number of axons in WT sciatic nerve. For Gpr126–/– mutants (n3),
we imaged ultrathin sections (see above) at 4000⫻ magnification. Images of
the entire nerve were taken in an overlapping series and montaged in
Photoshop. We then counted the total number of axons in P8 sciatic nerve.
Immunohistochemistry
Cochlear dissections, sample preparation, cryosectioning and
immunostaining were performed as described (Oshima et al., 2009). We
used the following primary antibodies: rat-anti-myelin basic protein (1:10,
AbD Serotec), rabbit-anti-peripherin (1:100, Millipore Bioscience Research
Reagents), goat-anti-Pou3f1 (1:100, Santa Cruz Biotechnology), mouseanti-myelin protein zero (1:1000) (Vargas et al., 2010) and rabbit-antineurofilament-M (1:200, Chemicon). Samples were incubated with
appropriate fluorescently labeled secondary antibodies (1:1000, Molecular
Probes; 1:200, Jackson ImmunoResearch), visualized by epi-fluorescence
microscopy (Zeiss Axioplan2 for sciatic nerves and Zeiss Axioimager for
cochleae) and digitally photographed (Zeiss AxioCam). We examined three
Gpr126+/– littermates and two Gpr126–/– mutants at P8 for sciatic nerve
stains, and we examined five littermates (two wild type, three Gpr126+/–)
and three Gpr126–/– mutants at P14 for cochlear stains.
Statistical analysis
For axon quantification, statistical comparisons were made using a twotailed t-test. Differences were considered to be significant at P<0.05.
RESULTS
Nerve and limb defects in Gpr126–/– mice
Starting with a genetic screen for mutants with abnormal
development of myelinated axons, we previously described an
essential role for Gpr126 in Schwann cell myelination in zebrafish
(Monk et al., 2009). To determine whether the function of Gpr126 is
conserved in mammalian myelination, we obtained and analyzed
Gpr126 knockout mice from Taconic/Lexicon. In the mutant allele,
exons 2 and 3 are replaced by a selection cassette (Fig. 1A,B). By
RT-PCR using primers that span exons 3 and 4, we detected Gpr126
mRNA in Gpr126+/– sciatic nerves, but not in wild-type neurons
purified from E13.5 dorsal root ganglia (DRG; Fig. 1C,D). Because
most mRNAs in the sciatic nerve are transcribed by Schwann cells,
this result suggests that, as in zebrafish, mammalian Schwann cells
express Gpr126. As expected for a deletion allele lacking exons 2
and 3, mutant sciatic nerves did not express detectable Gpr126
mRNA using primers that span exons 3 and 4 (Fig. 1C).
Crosses of Gpr126+/– heterozygotes yielded <25% mutant pups
at birth (26 homozygous mutants out of 334 pups; 7.8%),
indicating that the majority of Gpr126 mutants did not survive to
birth. At birth, mutant pups were generally the same size as their
wild type (WT) and heterozygous siblings, but they failed to grow
at equal rates and were substantially smaller than littermates within
a week of birth (Fig. 1E; see Movie 1 in the supplementary
material). Gpr126–/– mice died before weaning, usually between
postnatal day (P)8 and P16. Gpr126–/– mice that survived to P12
were generally immotile; when movement was occasionally
attempted, the mutants flailed their limbs and locomotion was
uncoordinated (see Movie 1 in the supplementary material).
Mutants were distinguishable from their WT and heterozygous
littermates by abnormal joint contractures of the forelimbs and
hindlimbs (Fig. 1E; see Movie 1 in the supplementary material).
This phenotype was sometimes observed as early as birth, and was
readily observable by P4. This limb phenotype is reminiscent of
human patients with arthrogryposis multiplex congenita (AMC)
(Bamshad et al., 2009) and has also been reported in mouse
Adam22 mutants (Nishino et al., 2010) and mouse claw paw
mutants (Henry et al., 1991; Darbas et al., 2004), which have a
mutation in the Lgi4 gene (Bermingham et al., 2006). In addition
to these limb posture abnormalities, sciatic nerves in Gpr126–/–
mice were severely reduced in size with a hypomyelinated (i.e.
translucent) appearance (Fig. 1F,G).
To learn more about the defects in Gpr126–/– sciatic nerves, we
examined several markers of Schwann cell development by RTPCR using cDNA template prepared from P4 sciatic nerve.
Gpr126–/– sciatic nerves expressed Sox10 (Fig. 1C), a transcription
factor crucial for early Schwann cell development (Kuhlbrodt et al.,
1998; Britsch et al., 2001). This result suggests that Schwann cells
DEVELOPMENT
Primer A, 5⬘-GTAGGATTGTTGCTTTATTATAC-3⬘; Primer B, 5⬘CCTGATCCTGACCATTCGC-3⬘; and Primer C, 5⬘-CCCTAGGAATGCTCGTCAAGA-3⬘. All animal procedures were approved by
Stanford University’s Administrative Panel on Laboratory Animal Care or
by Washington University’s Institutional Animal Care and Use Committee.
Development 138 (13)
Gpr126 is essential for PNS myelination
RESEARCH ARTICLE 2675
are present and expressing Sox10 in the mutant nerves, similar to
our previous studies in zebrafish (Monk et al., 2009). Expression
of other Schwann cell markers, including Dhh and Egr2, was not
detectable in Gpr126–/– nerve (Fig. 1C). Additionally, Pou3f1 and
myelin basic protein (Mbp) expression were not detected in
Gpr126–/– sciatic nerve (Fig. 2B,D). This analysis provides
evidence that Schwann cells are present in Gpr126–/– peripheral
nerve, but that these cells do not develop normally.
Multiple ultrastructural abnormalities are
observed in Gpr126–/– nerves
To determine whether Gpr126 is required for the differentiation of
myelinating Schwann cells in mammals, we examined myelination
and nerve ultrastructure at several developmental time points in
WT, Gpr126+/– and Gpr126–/– mice (see Materials and methods).
We did not observe any abnormalities in heterozygous animals, and
the development of myelinated axons was similar in WT and
Gpr126+/– sciatic nerves (see Fig. S1 in the supplementary
material; Fig. 3). Additionally, myelination of optic nerve axons by
oligodendrocytes, the myelinating glial cells of the central nervous
system (CNS), appeared normal in Gpr126–/– mutants at P12 (n4;
see Fig. S2 in the supplementary material), suggesting that Gpr126
is not essential for myelination in the CNS.
The ultrastructural studies revealed striking abnormalities in
Gpr126–/– peripheral nerve at P1, the earliest time point examined.
In P1 WT and Gpr126+/– animals, axonal sorting was well
underway in sciatic nerve; many Schwann cells had reached the
promyelinating stage, and a few thinly myelinated axons were
observed (Fig. 3A). By contrast, Schwann cell development was
markedly delayed in Gpr126–/– nerves. In the mutant sciatic nerve
at P1, most Schwann cells were at early sorting stages located at
the periphery of axon bundles and had sorted very few axons away
from the bundles (Fig. 3E). From P4 to P12 in WT and Gpr126+/–
animals, large caliber axons were progressively myelinated, and
Remak bundles developed as non-myelinating Schwann cells
ensheathed small caliber axons (Fig. 3B-D; Fig. 4A). From P4 to
P12 in Gpr126–/– nerves, Schwann cells eventually sorted axons
and formed a basal lamina, but all Schwann cells that were
associated with axons in a 1:1 relationship were arrested at the
promyelinating stage and myelin was never observed (Fig. 3F-H;
Fig. 4B,E). Additionally, we did not observe developing Remak
bundles in Gpr126–/– sciatic nerve (Fig. 3G,H; Fig. 4B).
To determine whether the loss of Remak bundles might reflect the
loss of small caliber axons that are normally ensheathed by nonmyelinating Schwann cells, we analyzed the axons in Gpr126–/–
sciatic nerves. At P1, the total number of axons in Gpr126–/– sciatic
nerve (5853±2109 s.d.) was somewhat reduced compared with the
total number of axons in WT nerve (9808±2720 s.d.; P0.07),
although the difference in axon number was not significant. The
reduction in axon number was far more pronounced at later stages.
At P8, the total number of axons in Gpr126–/– sciatic nerve
(1314±383 s.d.) was somewhat less than the number of myelinated
DEVELOPMENT
Fig. 1. Gpr126–/– mice have limb and nerve abnormalities. (A)Diagrams of Gpr126 gene (top) and Gpr126 protein (bottom). In the Gpr126
gene, exons 1 and 23 are denoted, as is the targeted locus (red arrows). The protein diagram depicts functional domains in Gpr126: CUB domain
(Complement, Uegf, Bmp1; CUB), a Pentraxin domain (PTX), a G protein-coupled receptor proteolytic site (GPS) and a type II seven-transmembrane
domain (7TM) (Stehlik et al., 2004). (B)Diagram depicting the targeting strategy employed to generate Gpr126–/– mice. (C)RT-PCR analysis of
expression of Schwann cell developmental markers in cDNA derived from P4 Gpr126+/– (+/–) or Gpr126–/– (–/–) sciatic nerve. Sox10 is expressed in
both +/– and –/– nerve, but expression of Gpr126, Dhh and Egr2 is not detectable in mutant nerve. Gapdh is included as a loading control, and
samples lacking reverse transcriptase (RT–) in the cDNA reaction mixture show no amplification. (D)Table showing average cycle threshold of
expression for Gapdh and Gpr126 in neurons purified from E12.5 dorsal root ganglia (DRG) and in P4 sciatic nerve (sciatic), as determined by
quantitative real-time PCR (qPCR). We did not detect Gpr126 in DRG neurons by qPCR, although with extended cycling under conditions of high
sensitivity using traditional RT-PCR, weak Gpr126 expression was observed in the same DRG neuron sample (data not shown). (E)Wild-type (WT)
and Gpr126–/– littermates at P8. Gpr126–/– mutants are smaller than their siblings and display abnormal joint contractures in the forelimbs and
hindlimbs (arrowheads). (F,G)Sciatic nerves from P12 littermates (arrows). (F)WT sibling nerves are white and opaque; a Gpr126+/– (+/–) animal is
shown. (G)By contrast, Gpr126–/– nerves are thinner than wild-type nerves and are translucent, indicative of hypomyelination.
Fig. 2. Gpr126–/– peripheral nerve lacks late Schwann cell
markers. (A)Pou3f1 is expressed in nuclei of Gpr126+/– nerve at P8
(A⬘,A⬙, arrowheads). (B)Pou3f1 is not detected in nuclei of Gpr126–/–
nerve at P8 (B⬘,B⬙). (C)Myelin basic protein (Mbp; C,C⬙,C⵮) and
Peripherin (C⬘) are expressed in Gpr126+/– sciatic nerve at P8. (D)Mbp
is not detected in Gpr126–/– sciatic nerve at P8 (D,D⬙,D⵮). Peripherin is
expressed in Gpr126–/– sciatic nerve at P8 (D⬙). Boxed areas in C⬙ and
D⬙ are enlarged in C⵮ and D⵮, respectively.
axons in WT nerve (1855±275 s.d.), and much less than the
estimated number of total axons in WT nerve (extrapolated
value8216±1220, P0.006). To determine whether small caliber Cfiber axons were absent in the mutants, we examined the expression
of peripherin, a marker of C-fibers (Goldstein et al., 1991).
Gpr126–/– sciatic nerves expressed peripherin (Fig. 2D), indicating
that the loss of Remak bundles is not simply due to absence of small
Development 138 (13)
caliber C-fiber axons. This analysis shows that Gpr126 is required
for the development of myelinating Schwann cells in mammals and
suggests that Gpr126 also has a role in the development of nonmyelinating Schwann cells. In addition, axon number was markedly
reduced in Gpr126–/– nerves at P8, indicating that Gpr126 function
is required for the maintenance of axons.
A fasciculation defect was evident in Gpr126–/– nerves as early
as P4. Throughout the endoneurium in mutant nerves, flattened cells
encircled groups of axons and Schwann cells, forming minifascicles
(see Fig. S1 in the supplementary material; Fig. 3F,G; Fig. 4B).
These flattened cells are most likely to be perineurial fibroblasts, as
they contained numerous caveolae and possessed a patchy basal
lamina (Fig. 4C) (Gamble and Eames, 1964; Parmantier et al., 1999).
A similar phenotype has been reported in Dhh–/– mice and has also
been described in a human patient with a DHH mutation. In these
cases, minifascicles are present throughout the endoneurium, much
like Gpr126–/– nerves. The Dhh and Gpr126 mutant phenotypes
differ, however, in that Dhh mutant Schwann cells are able to
myelinate axons (Parmantier et al., 1999; Umehara et al., 2000). We
did not observe Dhh mRNA expression in Gpr126–/– nerves (Fig.
1C), supporting the possibility that Gpr126 is involved in the
regulation of Dhh expression in Schwann cells, together with other
genes that drive the myelination program. Together, our
ultrastructural analyses show that Gpr126 is essential for several key
aspects of peripheral nerve development in mammals, including
progression of Schwann cells beyond the promyelinating stage,
axonal sorting, maintenance of axons, formation of Remak bundles
and normal perineurium formation.
Abnormalities in the auditory nerve in Gpr126–/–
mutants
The auditory nerve carries information from the sensory hair cells
of the cochlea to the cochlear nucleus in the brainstem. The afferent
auditory neuron cell bodies are located in the spiral ganglion in the
central stalk of the cochlea. Peripheral myelination of the afferent
fibers is mediated by Schwann cells and reaches from the habenula
Fig. 3. Numerous ultrastructural abnormalities are observed in Gpr126–/– sciatic nerves. (A-H)Transmission electron micrographs showing
ultrastructure of sciatic nerve in wild-type (+/+) and Gpr126+/– (+/–; B-D) or Gpr126–/– (–/–; E-H) animals at the indicated stages. (A)In +/+ sciatic
nerve at P1, many axons are ensheathed by promyelinating Schwann cells (p) and a few axons are surrounded by a thin myelin sheath (m). (E)In –/–
sciatic nerve at P1, axonal sorting is delayed and Schwann cells (s) are predominately found on the edge of axon bundles (a). (B)Several axons are
myelinated (m) in +/– sciatic nerve at P4. (F)In –/– sciatic nerve at P4, axonal sorting remains delayed and minifascicles (arrow) including axons and
Schwann cells (s) are observed. (C,D)Many axons are myelinated (m) in +/– sciatic nerve at P12 and myelin sheath thickness increases. Additionally,
developing Remak bundles (R) are observed. (G,H)Axons (a) are sorted by Schwann cells (s) in –/– sciatic nerve at P12, no myelin is observed and
minifascicles including axons and Schwann cells are readily observed (arrow in G). Scale bars: 2m in all panels.
DEVELOPMENT
2676 RESEARCH ARTICLE
Gpr126 is essential for PNS myelination
RESEARCH ARTICLE 2677
Fig. 4. Perineurial fibroblasts are found throughout
Gpr126–/– sciatic nerve and endoneurial collagen
fibrils are disrupted. (A)Myelinated axons (black
asterisks) and developing Remak bundles (white asterisks)
are observed in wild-type and Gpr126+/–(+/–) nerve at
P12. Fibroblasts are never observed to bundle axons and
Schwann cells into minifascicles. (B)Minifascicles (outlined
in white) are observed throughout the endoneurium of
–/– sciatic nerve. (C)The minifascicles are formed by
perineurial fibroblasts, identified by numerous caveolae
(arrowheads) and a patchy basal lamina (arrow).
(D)Schwann cells in wild-type and +/– nerve are
surrounded by a basal lamina (arrowheads), and collagen
fibrils are well organized (arrow). (E)Collagen fibrils are
often poorly organized in –/– sciatic nerve (arrow),
although Gpr126–/– Schwann cells are surrounded by a
basal lamina (arrowheads). (A,B)Scale bars: 5m in A,B;
0.5m in C-E.
consistently expanded deeper into the modiolus of Gpr126–/–
nerves (Fig. 5B,D). At the glial limitans, where the transition
between central and peripheral myelin occurs (Hurley et al., 2007),
we observed many central MBP-positive cells projecting farther
into the peripheral region of Gpr126–/– nerves than in WT nerves
(Fig. 5A,B,D). Similar expansions of CNS myelin into the PNS
have recently been reported in the absence of peripheral glia in
zebrafish (Kucenas et al., 2009) and in mammals when Schwann
cells or Egr2 function are absent (Coulpier et al., 2010). Together,
this analysis shows that Schwann cells do not express MBP or P0
in Gpr126–/– nerves, and that Gpr126–/– oligodendrocytes express
MBP and project aberrantly into PNS regions of the cochlear nerve
in the absence of peripheral myelin but in the presence of
Gpr126–/– Schwann cells.
DISCUSSION
Beginning with a forward genetic screen for mutations disrupting
the development of myelinated axons, we recently described an
essential role for Gpr126 in Schwann cell myelination in zebrafish
(Monk et al., 2009). Through analysis of Gpr126–/– mice, we
show that the function of Gpr126 in myelination is conserved in
mammalian peripheral nerve: Schwann cells are arrested at the
promyelinating stage in Gpr126–/– mice, as in zebrafish. This
myelination defect is specific to the PNS, as CNS myelination
appears to be normal in Gpr126–/– mice (Fig. 5; see Fig. S2 in the
supplementary material). In addition, this study defines new
functions of Gpr126 by analyzing other defects in Gpr126–/– mice
that were not evident in gpr126 zebrafish mutants.
The analysis of peripheral nerves in Gpr126 mutant mice defines
a previously unknown function for Gpr126 in the organization of
the perineurium. By P4 in Gpr126–/– mutants, the ectopic
localization of apparent perineurial fibroblasts is observed
throughout the endoneurium (Fig. 3F). This phenotype worsens
with age and, by P12, the endoneurium of Gpr126–/– mutants is
partitioned into numerous distinct minifascicles by the fibroblasts
(Fig. 3G, Fig. 4B). This defect is likely to be due to loss of Gpr126
in Schwann cells and not in fibroblasts: loss of desert hedgehog
(Dhh) in Schwann cells also leads to ectopic fascicle formation by
DEVELOPMENT
perforata, which demarks the entry point of the afferent fibers into
the organ of Corti. Schwann cell myelination continues until the
central-peripheral transitional zone, also known as the glial
limitans, which is located towards the base of the cochlear
modiolus (Fraher and Delanty, 1987). Central myelination,
mediated by oligodendrocytes, stretches from the glial limitans to
the cochlear nucleus, the first central echelon of the auditory
pathway. We previously reported that gpr126 mutant zebrafish
larvae have visibly swollen ears but did not pursue further
phenotypic analyses (Monk et al., 2009).
To investigate the possibility that Gpr126 is required for
development of the auditory system in mammals, we examined
inner ear morphology and various markers in mid-modiolar
cochlear sections at P14. No obvious defects were observed in ear
or cochlea morphology upon gross examination, and hair cell
morphology and innervation appeared normal as assessed by
neurofilament-M immunostaining in Gpr126–/– animals compared
with WT siblings (Fig. 5A; data not shown).
We also examined myelin protein expression in the centralperipheral transitional zone of the auditory nerve in mid-modiolar
cochlear sections at P14. In WT mice, myelin basic protein (MBP)
was evident in both the CNS and the PNS regions of the nerve, as
assessed by immunostaining (Fig. 5). In the peripheral portion of
the auditory nerve of WT and Gpr126+/– mice, MBP was
colocalized with myelin protein zero (P0), a major structural
component of Schwann cell, but not oligodendrocyte, myelin
(Lemke et al., 1988). MBP was strongly expressed in CNS regions
of Gpr126–/– cochlear nerve (Fig. 5), providing further evidence
that oligodendrocytes can form myelin in the absence of Gpr126
function. In the PNS region of the nerve, however, no P0 or MBP
expression was detected in Gpr126–/– mutants, specifically in the
spiral ganglia and in peripheral neurites between the spiral ganglion
and the habenula perforata, where the fibers enter the organ of
Corti (Fig. 5B,C). Schwann cell numbers in these regions did not
appear to be disrupted, as shown by DAPI staining (Fig. 5C).
Interestingly, the part of the auditory nerve known as the central
tissue projection (Fraher and Delanty, 1987), which is myelinated
by oligodendrocytes and strongly labeled with MBP antibody, was
2678 RESEARCH ARTICLE
Development 138 (13)
Fig. 5. Cochlear histology appears grossly normal in Gpr126–/– mice, but Schwann cells do not express P0 or MBP, and CNS myelin is
expanded into peripheral zones. (A)Mid-modiolar cross sections showing the middle turn of wild-type (+/+) and Gpr126–/– (–/–) mice, stained
with antibodies to myelin basic protein (MBP; red) and neurofilament-M (NF-M; green). (B) Adjacent sections to those shown in A, labeled with
antibodies to MBP (red) and peripheral myelin protein zero (P0; green). The brackets indicate the region between the habenula perforata and the
spiral ganglion; the arrow indicates the spiral ganglion. (C)Higher magnification of the peripheral section of the auditory nerve fibers. The habenula
perforata (hp) and the spiral ganglion (sg) are indicated. (D)Higher magnification of the glial limitans, where the transition between central and
peripheral myelin occurs. Arrowhead indicates projections of MBP-positive CNS myelin into the peripheral region in –/– nerves. In A,B and D,
asterisks indicate CNS myelin. Scale bars: 200m in A,B; 100m in C,D.
Impaired radial sorting in Gpr126–/– nerves
During embryonic nerve development, in a process known as radial
sorting, Schwann cells associated with multiple axons interdigitate
cytoplasmic extensions throughout the bundle to separate large
caliber axons and eventually myelinate them. At P1 in Gpr126–/–
nerve, Schwann cells are found mainly at the periphery of axon
bundles, and very few axons have been sorted (Fig. 3E). Similar
defects in radial sorting are found in peripheral nerves of mice with
Schwann cell-specific mutations affecting laminin isoforms (Chen
and Strickland, 2003; Yu et al., 2005), the laminin receptor b1integrin (Feltri et al., 2002; Nodari et al., 2007), the tyrosine kinase
FAK, integrin-linked kinase and downstream signaling components
in Schwann cells (Benninger et al., 2006; Grove et al., 2007; Nodari
et al., 2007; Pereira et al., 2009). Although Schwann cells in
Gpr126–/– mutant mice eventually recover and sort axons, these
early phenotypic similarities suggest a possible connection between
Gpr126 and signaling between laminins and their integrin receptors.
It is also possible that activation of Gpr126 in Schwann cells leads
to similar downstream signaling cascades as integrin activation.
Reduced axon number in Gpr126–/– nerves
The total number of axons in Gpr126–/– sciatic nerve was
substantially reduced compared with the estimated total number of
axons in WT sciatic nerve at P8. However, at P1, this reduction
was less severe. Additionally, when we viewed the entire sciatic
nerve by electron microscopy montage at P1, we observed regions
of mutant nerve with dying axons (data not shown). In ErbB3
mutant mice, which lack Schwann cells associated with developing
sensory and motor neurons, loss of trophic support from Schwann
cells results in increased neuronal death in the embryo
(Riethmacher et al., 1997). Schwann cells are present in Gpr126–/–
mutants, but our results suggest that Schwann cells lacking Gpr126
cannot confer vital trophic support to associated axons, resulting in
the loss of axons we observe at P8.
Absence of Remak bundles in Gpr126–/– nerves
We observed no developing Remak bundles in Gpr126–/– sciatic
nerves as late as P12. Axonal sorting was initially delayed, and
Schwann cells eventually sorted axons into a 1:1 relationship.
Bundles of unsorted axons were occasionally observed at P12 (data
DEVELOPMENT
perineurial fibroblasts (Parmantier et al., 1999). These fibroblasts
express the hedgehog receptor patched homolog 1 (Ptch1), and
require Dhh from Schwann cells during nerve development for
proper perineurium formation (Parmantier et al., 1999). Dhh is not
expressed in Gpr126–/– sciatic nerve (Fig. 1C), suggesting that the
minifascicle pathology found throughout the endoneurium of
Gpr126–/– nerves is due to loss of Dhh in Schwann cells rather
than a direct role for Gpr126 in fibroblast development.
Gpr126–/– nerve pathology is also reminiscent of defects
observed in Adam22 and Lgi4 mutants (Henry et al., 1991; Darbas
et al., 2004; Sagane et al., 2005; Ozkaynak et al., 2010; Nishino et
al., 2010). Adam22 is a receptor for Lgi4; Adam22 is expressed
neuronally, and Lgi4 is secreted principally by Schwann cells in
peripheral nerve (Sagane et al., 2008; Ozkaynak et al., 2010). In
addition to reduced Schwann cell myelination, Adam22 and Lgi4
mutant peripheral nerves also possess minifascicle pathologies
throughout the endoneurium (Henry et al., 1991; Darbas et al., 2004;
Sagane et al., 2005; Ozkaynak et al., 2010). Axonal sorting is also
delayed in Lgi4 mutants (Darbas et al., 2004). Although these
phenotypic similarities raise the possibility that Gpr126 activation
requires Adam22 and/or Lgi4, it is important to note that Adam22
and Lgi4 mutants express Pou3f1 (Ozkaynak et al., 2010; Darbas et
al., 2004; Nishino et al., 2010), whereas Gpr126 mutants do not (Fig.
2B) (Monk et al., 2009). Future studies are required to examine the
possible interaction between Gpr126 and Adam22 and Lgi4.
not shown), but these axons were not surrounded by nonmyelinating Schwann cell cytoplasm; rather, Schwann cells
remained at the periphery of axon bundles, reminiscent of the
sorting defect we observed at earlier stages (Fig. 3E,F). Although
the total number of axons is reduced in Gpr126–/– nerves, small
caliber C-fibers are present in mutants (Fig. 2D). This result
suggests Gpr126 plays a role in the development of nonmyelinating Schwann cells, leading to the loss of Remak bundles
in Gpr126–/– mutants. Previous results suggest that Gpr126
functions to elevate cAMP levels in promyelinating Schwann cells
prior to myelination (Monk et al., 2009). cAMP elevation in
postmitotic Schwann cells is thought to downregulate
premyelinating signals and upregulate myelinating signals (Morgan
et al., 1991; Monuki et al., 1989; Scherer et al., 1994). It is possible
that non-myelinating Schwann cells also require elevation of
cAMP levels in order to differentiate. Indeed, when Schwann cells
are proliferative, elevation of cAMP does not initiate myelin gene
expression but instead induces expression of the cell surface lipid
antigen O4, an early Schwann cell differentiation marker (Mirsky
et al., 1990; Morgan et al., 1991; Jessen et al., 1991). The levels of
Nrg1TypeIII on the surface of an axon are thought to instruct
Schwann cells to become myelinating or non-myelinating
(Taveggia et al., 2005). It was recently shown that levels of cAMP
determine how Schwann cells respond to Nrg1 in vitro (ArthurFarraj et al., 2011). Future studies are required to determine the role
of cAMP in non-myelinating Schwann cell development and to
ascertain how Gpr126 and Nrg signaling interact in vivo.
It is also possible that non-myelinating Schwann cells require a
signal from promyelinating or myelinating Schwann cells that is
altered in the absence of Gpr126; one such candidate signaling
molecule is Dhh. Non-myelinating Schwann cells express the
hedgehog receptor patched homolog 2 (Ptch2) in adult sciatic nerve
and after injury (Bajestan et al., 2006). Although Dhh-Ptch2
signaling has not been directly shown to be required for the
development of non-myelinating Schwann cells, Dhh mutant
nerves possess significantly more axons sorted in a 1:1 relationship
and fewer axons in Remak bundles (Sharghi-Namini et al., 2006),
a pathology that is reminiscent of the defects observed in
Gpr126–/– peripheral nerve. Future studies utilizing cell-type
specific ablation of Gpr126 will be useful to determine the role of
Gpr126 in non-myelinating Schwann cell development.
Other roles for Gpr126 and future directions
Zebrafish mutants for gpr126 are viable and fertile, whereas
Gpr126–/– mice are not born in Mendelian ratios and no mutant
survived past P16. In a recent study, loss of Gpr126 in mouse
resulted in mid-gestation embryonic lethality (Waller-Evans et al.,
2010). These embryos died of cardiovascular defects before
analysis of peripheral myelination could take place. It is therefore
likely that cardiovascular defects also account for some of the
embryonic lethality we observe in our studies. Strain differences or
the region of Gpr126 targeted might explain why we obtained
some live mutants at birth and why Waller-Evans et al. observed
fully penetrant embryonic lethality. Waller-Evans et al. deleted the
seven-transmembrane region of Gpr126 in their mutant allele,
whereas functional domains in the N-terminus were targeted in the
Gpr126 mutation that we analyzed. Gpr126 is also highly
expressed in mouse and rat lungs (Moriguchi et al., 2004; Haitina
et al., 2008) so it is possible that an abnormality in the lung
contributes to the mortality of Gpr126 mutants. Furthermore, three
single-nucleotide polymorphisms (SNPs) in GPR126 were recently
associated with lung function in humans (Hancock et al., 2010).
RESEARCH ARTICLE 2679
Bergmann glial cells of the mouse cerebellum also express high
levels of Gpr126 (Koirala and Corfas, 2010), as does the collecting
duct of the mouse kidney (Pradervand et al., 2010). Future studies
are required to determine the function of Gpr126 in these tissues
and define the cause of postnatal lethality in the mutant mice.
Peripheral nerves are complex structures containing axons,
Schwann cells, fibroblasts and other cell types. Precise signaling
between these cells is required for the proper development,
organization and function of peripheral nerves. This work shows that
Gpr126 is essential for myelination in the mammalian PNS. We also
show that Gpr126 is required for timely Schwann cell differentiation
and peripheral nerve organization in mice. Many key drug targets are
G protein-coupled receptors (Lundstrom, 2005); as a crucial
regulator of myelination in mammals, Gpr126 might represent an
attractive therapeutic target for re-myelination therapy in humans.
Acknowledgements
This work was supported by grants to W.S.T. from the Muscular Dystrophy
Association (92233) and the National Institutes of Health (NIH; R01
NS050223). K.R.M. was supported by a National Multiple Sclerosis Society
postdoctoral award (FG 1719-A-1) and by the Stanford Genome Training
Program (NHI/NGHRI). S.H. was supported by NIH R01 DC004563 and P30
DC010363. We thank Ben Emery for assistance with optic nerve dissections,
John Perrino for electron microscopy advice, the Barres lab for the gift of the
P0 antibody, and members of the Talbot and Kingsley laboratories for helpful
discussions. We are also grateful to Yo Sasaki and Jeffrey Milbrandt for
providing DRG neuron cDNA samples, and to Valeria Cavalli, John Heuser,
David Ornitz, Naren Ramanan and Lila Solnica-Krezel for sharing laboratory
space and reagents. Deposited in PMC for release after 12 months.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.062224/-/DC1
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