gpr126 is essential for peripheral nerve development and ... · axons in the vertebrate nervous...

2673RESEARCH ARTICLE

INTRODUCTIONMyelin is a multilayered membrane formed by glial cells aroundaxons in the vertebrate nervous system. In the peripheral nervoussystem (PNS), Schwann cells form the myelin sheath by wrappingtheir membrane around an axonal segment many times. Schwanncell precursors arise from neural crest progenitor cells, andprevious work has defined several steps in the developmentalprogression leading to mature Schwann cells (Jessen and Mirsky,2005). During embryogenesis, Schwann cell precursors co-migratewith axons in the developing PNS and differentiate into immatureSchwann cells that remain associated with many axons. ImmatureSchwann cells may become either non-myelinating or myelinatingSchwann cells. Non-myelinating Schwann cells remain associatedwith multiple small-caliber axons. These axons are ensheathed bynon-myelinating Schwann cells in Remak bundles, but are notmyelinated. Other immature Schwann cells begin to sort large-caliber axons and develop into promyelinating Schwann cells thatassociate with a single axonal segment. Promyelinating Schwanncells differentiate into myelinating Schwann cells, which repeatedlywrap their membrane around an axonal segment to eventually forma compact myelin sheath.

Investigations in mammals have defined a number of essentialregulators of Schwann cell development, including the axonal signalneuregulin 1 and the Schwann cell transcription factors Pou3f1 (alsoknown as Oct-6), Pou3f2 (also known as Brn-2) and Egr2 (alsoknown as Krox-20) (Nave and Salzer, 2006; Svaren and Meijer,2008). Neuregulin 1 (Nrg1) signaling through ErbB receptors iscrucial for many steps in Schwann cell development, including

proliferation, ensheathment and myelination (Garratt et al., 2000;Michailov et al., 2004; Taveggia et al., 2005). The transcriptionfactors Pou3f1, Pou3f2 and Egr2 are required for the development ofmature myelinating Schwann cells. Pou3f1 and Pou3f2 are requiredfor the timely differentiation of myelinating Schwann cells; Schwanncells in Pou3f1-null mice transiently arrest at the promyelinating stageand 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 thepromyelinating stage and fail to wrap an axonal segment more than1.5 turns (Topilko et al., 1994; Zorick et al., 1999). Less is knownabout the genes controlling the development of non-myelinatingSchwann cells, but these cells express many of the same markers asimmature Schwann cells (Jessen and Mirsky, 2002).

We recently reported that the orphan G-protein-coupled receptorGpr126 is required autonomously in Schwann cells for theexpression of pou3f1 and egr2, and that Gpr126 is essential forpromyelinating Schwann cells to initiate myelination in zebrafish(Monk et al., 2009). In the present study, we show that this functionof Gpr126 is conserved in mammals through the analysis of Gpr126knockout mice. As in zebrafish, Schwann cells in Gpr126–/– mutantmice are arrested at the promyelinating stage. Mutant mice have limbposture abnormalities, are smaller than littermates and die beforeweaning. In mutant sciatic nerves, we also observe delayed sortingof axons by Schwann cells, aberrant fasciculation throughout theendoneurium by perineurial fibroblasts, progressive loss of axons inthe postnatal period and an absence of Remak bundles. Our findingsshow that Gpr126 is essential for several aspects of peripheral nervedevelopment in mammals.

MATERIALS AND METHODSMiceGpr126 knockout mice were generated by Lexicon Pharmaceuticals (TheWoodlands, TX, USA) and heterozygous animals were obtained fromTaconic (Hudson, NY, USA; catalog number TF0909) on a mixedbackground (129/SvEv-C57BL/6). These mice and their offspring werecrossed to generate the animals analyzed in this study. We used thefollowing PCR primers to determine the genotype of the mutant mice:

Development 138, 2673-2680 (2011) doi:10.1242/dev.062224© 2011. Published by The Company of Biologists Ltd

1Department 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 UniversitySchool of Medicine, St Louis, MO 63110, USA†Author for correspondence ([email protected])

Accepted 6 April 2011

SUMMARYIn 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 areincompletely understood. In this study, we show that Gpr126 is essential for myelination and other aspects of peripheral nervedevelopment in mammals. A mutation in Gpr126 causes a severe congenital hypomyelinating peripheral neuropathy in mice, andexpression 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-myelinatingSchwann cells associated with small caliber axons) are not observed, and Schwann cells are ultimately arrested at the promyelinatingstage. 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 axonalsorting, formation of mature non-myelinating Schwann cells and organization of the perineurium.

KEY WORDS: Schwann cell, Myelin, Gpr126, Mouse

Gpr126 is essential for peripheral nerve development andmyelination in mammalsKelly R. Monk1,*, Kazuo Oshima2, Simone Jörs2, Stefan Heller2 and William S. Talbot1,†

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Primer A, 5�-GTAGGATTGTTGCTTTATTATAC-3�; Primer B, 5�-CCTGATCCTGACCATTCGC-3�; and Primer C, 5�-CCCTAGGA -ATGCTCGTCAAGA-3�. All animal procedures were approved byStanford University’s Administrative Panel on Laboratory Animal Care orby Washington University’s Institutional Animal Care and Use Committee.

RT-PCR and quantitative real-time PCRFor standard RT-PCR, we isolated RNA from Gpr126–/– and heterozygotesibling sciatic nerves at postnatal day (P)4 using the RNeasy Micro Kit(Qiagen) according to the manufacturer’s instructions. We reversetranscribed the RNA using the SuperScript First-Strand synthesis systemfor RT-PCR (Invitrogen) with Superscript II reverse transcriptase andrandom hexamers per manufacturer’s instructions. Samples lacking reversetranscriptase were used to control for genomic DNA contamination. Weamplified 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)]. Weused the following primers to amplify Gpr126: 5�-CCAAAGT -TGGCAATGAAGGT-3� and 5�-GCTGGATCAG GTAGGAACCA-3�. Forquantitative real-time PCR (qPCR), the same heterozygote P4 sciatic nervecDNA and primers were used as described above. Mouse dorsal rootganglia (DRGs) were collected from embryonic day (E)13.5 and neuronswere purified as described (Sasaki et al., 2009). RNA was extracted fromDRG neurons using Ribozol (Amresco) and cDNA was synthesized usingQScript cDNA Supermix (VWR) according to the manufacturer’sinstructions. qPCR was performed in an ABI 7500 using SYBR GreenMaster Mix (ABI) according to standard protocols.

Transmission electron microscopySciatic nerves and optic nerves were removed from mice of the indicatedages 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% osmiumtetraoxide in 0.1M sodium cacodylate, pH 7.4, dehydrated, and embedded inepoxy resin (EMBed-812, Electron Microscopy Sciences). We stained semi-thin sections (1 m) with Toluidine Blue for light microscope examination(Zeiss Axioplan2). Ultrathin sections (70 nm) were stained and imaged asdescribed (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, threeGpr126+/–) and three mutants at P8; five siblings (one WT, four Gpr126+/–)and four mutants at P12.

Morphometric analysisFor P1 animals (n2 pairs of WT and Gpr126–/– siblings), images of thesciatic nerve were taken in an overlapping series and montaged in Photoshop.We then counted the total number of axons in P1 sciatic nerve. For WTanimals, the tibial branch of the nerve was analyzed; Gpr126 mutant nerveswere difficult to dissect and we did not observe branches in the nerve in theregions examined. For P8 WT and Gpr126+/– siblings (n3: one WT andtwo Gpr126+/–), we imaged Toluidine Blue-stained semi-thin sections (seeabove) on a Zeiss Axioplan2 and digitally imaged the entire nerve with aZeiss AxioCam. We then counted the total number of myelinated axons inthe tibial branch of P8 sciatic nerve. To estimate the total number of axonsin WT nerve, we imaged ten randomly selected regions of the same nervesusing ultrathin sections at 8,000� magnification. We then counted the totalnumber of unmyelinated axons and myelinated axons in these regions andobtained an average ratio of unmyelinated:myelinated axons (576:168). Weused this ratio and the counted number of myelinated axons to estimate thetotal number of axons in WT sciatic nerve. For Gpr126–/– mutants (n3),we imaged ultrathin sections (see above) at 4000� magnification. Images ofthe entire nerve were taken in an overlapping series and montaged inPhotoshop. We then counted the total number of axons in P8 sciatic nerve.

ImmunohistochemistryCochlear dissections, sample preparation, cryosectioning andimmunostaining were performed as described (Oshima et al., 2009). Weused 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), mouse-anti-myelin protein zero (1:1000) (Vargas et al., 2010) and rabbit-anti-neurofilament-M (1:200, Chemicon). Samples were incubated withappropriate fluorescently labeled secondary antibodies (1:1000, MolecularProbes; 1:200, Jackson ImmunoResearch), visualized by epi-fluorescencemicroscopy (Zeiss Axioplan2 for sciatic nerves and Zeiss Axioimager forcochleae) and digitally photographed (Zeiss AxioCam). We examined threeGpr126+/– littermates and two Gpr126–/– mutants at P8 for sciatic nervestains, and we examined five littermates (two wild type, three Gpr126+/–)and three Gpr126–/– mutants at P14 for cochlear stains.

Statistical analysisFor axon quantification, statistical comparisons were made using a two-tailed t-test. Differences were considered to be significant at P<0.05.

RESULTSNerve and limb defects in Gpr126–/– miceStarting with a genetic screen for mutants with abnormaldevelopment of myelinated axons, we previously described anessential role for Gpr126 in Schwann cell myelination in zebrafish(Monk et al., 2009). To determine whether the function of Gpr126 isconserved in mammalian myelination, we obtained and analyzedGpr126 knockout mice from Taconic/Lexicon. In the mutant allele,exons 2 and 3 are replaced by a selection cassette (Fig. 1A,B). ByRT-PCR using primers that span exons 3 and 4, we detected Gpr126mRNA in Gpr126+/– sciatic nerves, but not in wild-type neuronspurified from E13.5 dorsal root ganglia (DRG; Fig. 1C,D). Becausemost mRNAs in the sciatic nerve are transcribed by Schwann cells,this result suggests that, as in zebrafish, mammalian Schwann cellsexpress Gpr126. As expected for a deletion allele lacking exons 2and 3, mutant sciatic nerves did not express detectable Gpr126mRNA using primers that span exons 3 and 4 (Fig. 1C).

Crosses of Gpr126+/– heterozygotes yielded <25% mutant pupsat birth (26 homozygous mutants out of 334 pups; 7.8%),indicating that the majority of Gpr126 mutants did not survive tobirth. At birth, mutant pups were generally the same size as theirwild type (WT) and heterozygous siblings, but they failed to growat equal rates and were substantially smaller than littermates withina week of birth (Fig. 1E; see Movie 1 in the supplementarymaterial). Gpr126–/– mice died before weaning, usually betweenpostnatal day (P)8 and P16. Gpr126–/– mice that survived to P12were generally immotile; when movement was occasionallyattempted, the mutants flailed their limbs and locomotion wasuncoordinated (see Movie 1 in the supplementary material).

Mutants were distinguishable from their WT and heterozygouslittermates by abnormal joint contractures of the forelimbs andhindlimbs (Fig. 1E; see Movie 1 in the supplementary material).This phenotype was sometimes observed as early as birth, and wasreadily observable by P4. This limb phenotype is reminiscent ofhuman patients with arthrogryposis multiplex congenita (AMC)(Bamshad et al., 2009) and has also been reported in mouseAdam22 mutants (Nishino et al., 2010) and mouse claw pawmutants (Henry et al., 1991; Darbas et al., 2004), which have amutation in the Lgi4 gene (Bermingham et al., 2006). In additionto 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, weexamined several markers of Schwann cell development by RT-PCR using cDNA template prepared from P4 sciatic nerve.Gpr126–/– sciatic nerves expressed Sox10 (Fig. 1C), a transcriptionfactor crucial for early Schwann cell development (Kuhlbrodt et al.,1998; Britsch et al., 2001). This result suggests that Schwann cells

RESEARCH ARTICLE Development 138 (13)

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are present and expressing Sox10 in the mutant nerves, similar toour previous studies in zebrafish (Monk et al., 2009). Expressionof other Schwann cell markers, including Dhh and Egr2, was notdetectable in Gpr126–/– nerve (Fig. 1C). Additionally, Pou3f1 andmyelin basic protein (Mbp) expression were not detected inGpr126–/– sciatic nerve (Fig. 2B,D). This analysis providesevidence that Schwann cells are present in Gpr126–/– peripheralnerve, but that these cells do not develop normally.

Multiple ultrastructural abnormalities areobserved in Gpr126–/– nervesTo determine whether Gpr126 is required for the differentiation ofmyelinating Schwann cells in mammals, we examined myelinationand nerve ultrastructure at several developmental time points inWT, Gpr126+/– and Gpr126–/– mice (see Materials and methods).We did not observe any abnormalities in heterozygous animals, andthe development of myelinated axons was similar in WT andGpr126+/– sciatic nerves (see Fig. S1 in the supplementarymaterial; Fig. 3). Additionally, myelination of optic nerve axons byoligodendrocytes, the myelinating glial cells of the central nervoussystem (CNS), appeared normal in Gpr126–/– mutants at P12 (n4;see Fig. S2 in the supplementary material), suggesting that Gpr126is not essential for myelination in the CNS.

The ultrastructural studies revealed striking abnormalities inGpr126–/– 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 thepromyelinating stage, and a few thinly myelinated axons wereobserved (Fig. 3A). By contrast, Schwann cell development wasmarkedly delayed in Gpr126–/– nerves. In the mutant sciatic nerveat P1, most Schwann cells were at early sorting stages located atthe periphery of axon bundles and had sorted very few axons awayfrom the bundles (Fig. 3E). From P4 to P12 in WT and Gpr126+/–animals, large caliber axons were progressively myelinated, andRemak bundles developed as non-myelinating Schwann cellsensheathed small caliber axons (Fig. 3B-D; Fig. 4A). From P4 toP12 in Gpr126–/– nerves, Schwann cells eventually sorted axonsand formed a basal lamina, but all Schwann cells that wereassociated with axons in a 1:1 relationship were arrested at thepromyelinating stage and myelin was never observed (Fig. 3F-H;Fig. 4B,E). Additionally, we did not observe developing Remakbundles in Gpr126–/– sciatic nerve (Fig. 3G,H; Fig. 4B).

To determine whether the loss of Remak bundles might reflect theloss of small caliber axons that are normally ensheathed by non-myelinating Schwann cells, we analyzed the axons in Gpr126–/–sciatic nerves. At P1, the total number of axons in Gpr126–/– sciaticnerve (5853±2109 s.d.) was somewhat reduced compared with thetotal number of axons in WT nerve (9808±2720 s.d.; P0.07),although the difference in axon number was not significant. Thereduction 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

2675RESEARCH ARTICLEGpr126 is essential for PNS myelination

Fig. 1. Gpr126–/– mice have limb and nerve abnormalities. (A)Diagrams of Gpr126 gene (top) and Gpr126 protein (bottom). In the Gpr126gene, 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-transmembranedomain (7TM) (Stehlik et al., 2004). (B)Diagram depicting the targeting strategy employed to generate Gpr126–/– mice. (C)RT-PCR analysis ofexpression of Schwann cell developmental markers in cDNA derived from P4 Gpr126+/– (+/–) or Gpr126–/– (–/–) sciatic nerve. Sox10 is expressed inboth +/– and –/– nerve, but expression of Gpr126, Dhh and Egr2 is not detectable in mutant nerve. Gapdh is included as a loading control, andsamples lacking reverse transcriptase (RT–) in the cDNA reaction mixture show no amplification. (D)Table showing average cycle threshold ofexpression for Gapdh and Gpr126 in neurons purified from E12.5 dorsal root ganglia (DRG) and in P4 sciatic nerve (sciatic), as determined byquantitative real-time PCR (qPCR). We did not detect Gpr126 in DRG neurons by qPCR, although with extended cycling under conditions of highsensitivity 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 andhindlimbs (arrowheads). (F,G)Sciatic nerves from P12 littermates (arrows). (F)WT sibling nerves are white and opaque; a Gpr126+/– (+/–) animal isshown. (G)By contrast, Gpr126–/– nerves are thinner than wild-type nerves and are translucent, indicative of hypomyelination.

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axons in WT nerve (1855±275 s.d.), and much less than theestimated number of total axons in WT nerve (extrapolatedvalue8216±1220, P0.006). To determine whether small caliber C-fiber axons were absent in the mutants, we examined the expressionof peripherin, a marker of C-fibers (Goldstein et al., 1991).Gpr126–/– sciatic nerves expressed peripherin (Fig. 2D), indicatingthat the loss of Remak bundles is not simply due to absence of small

caliber C-fiber axons. This analysis shows that Gpr126 is requiredfor the development of myelinating Schwann cells in mammals andsuggests that Gpr126 also has a role in the development of non-myelinating Schwann cells. In addition, axon number was markedlyreduced in Gpr126–/– nerves at P8, indicating that Gpr126 functionis required for the maintenance of axons.

A fasciculation defect was evident in Gpr126–/– nerves as earlyas P4. Throughout the endoneurium in mutant nerves, flattened cellsencircled 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, asthey contained numerous caveolae and possessed a patchy basallamina (Fig. 4C) (Gamble and Eames, 1964; Parmantier et al., 1999).A similar phenotype has been reported in Dhh–/– mice and has alsobeen described in a human patient with a DHH mutation. In thesecases, minifascicles are present throughout the endoneurium, muchlike Gpr126–/– nerves. The Dhh and Gpr126 mutant phenotypesdiffer, however, in that Dhh mutant Schwann cells are able tomyelinate axons (Parmantier et al., 1999; Umehara et al., 2000). Wedid not observe Dhh mRNA expression in Gpr126–/– nerves (Fig.1C), supporting the possibility that Gpr126 is involved in theregulation of Dhh expression in Schwann cells, together with othergenes that drive the myelination program. Together, ourultrastructural analyses show that Gpr126 is essential for several keyaspects of peripheral nerve development in mammals, includingprogression of Schwann cells beyond the promyelinating stage,axonal sorting, maintenance of axons, formation of Remak bundlesand normal perineurium formation.

Abnormalities in the auditory nerve in Gpr126–/–mutantsThe auditory nerve carries information from the sensory hair cellsof the cochlea to the cochlear nucleus in the brainstem. The afferentauditory neuron cell bodies are located in the spiral ganglion in thecentral stalk of the cochlea. Peripheral myelination of the afferentfibers is mediated by Schwann cells and reaches from the habenula


Fig. 2. Gpr126–/– peripheral nerve lacks late Schwann cellmarkers. (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�) andPeripherin (C�) are expressed in Gpr126+/– sciatic nerve at P8. (D)Mbpis not detected in Gpr126–/– sciatic nerve at P8 (D,D�,D�). Peripherin isexpressed in Gpr126–/– sciatic nerve at P8 (D�). Boxed areas in C� andD� are enlarged in C� and D�, respectively.

Fig. 3. Numerous ultrastructural abnormalities are observed in Gpr126–/– sciatic nerves. (A-H)Transmission electron micrographs showingultrastructure of sciatic nerve in wild-type (+/+) and Gpr126+/– (+/–; B-D) or Gpr126–/– (–/–; E-H) animals at the indicated stages. (A)In +/+ sciaticnerve 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 aremyelinated (m) in +/– sciatic nerve at P4. (F)In –/– sciatic nerve at P4, axonal sorting remains delayed and minifascicles (arrow) including axons andSchwann 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 andminifascicles including axons and Schwann cells are readily observed (arrow in G). Scale bars: 2m in all panels. D

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perforata, which demarks the entry point of the afferent fibers intothe organ of Corti. Schwann cell myelination continues until thecentral-peripheral transitional zone, also known as the gliallimitans, which is located towards the base of the cochlearmodiolus (Fraher and Delanty, 1987). Central myelination,mediated by oligodendrocytes, stretches from the glial limitans tothe cochlear nucleus, the first central echelon of the auditorypathway. We previously reported that gpr126 mutant zebrafishlarvae have visibly swollen ears but did not pursue furtherphenotypic analyses (Monk et al., 2009).

To investigate the possibility that Gpr126 is required fordevelopment of the auditory system in mammals, we examinedinner ear morphology and various markers in mid-modiolarcochlear sections at P14. No obvious defects were observed in earor cochlea morphology upon gross examination, and hair cellmorphology and innervation appeared normal as assessed byneurofilament-M immunostaining in Gpr126–/– animals comparedwith WT siblings (Fig. 5A; data not shown).

We also examined myelin protein expression in the central-peripheral transitional zone of the auditory nerve in mid-modiolarcochlear sections at P14. In WT mice, myelin basic protein (MBP)was evident in both the CNS and the PNS regions of the nerve, asassessed by immunostaining (Fig. 5). In the peripheral portion ofthe auditory nerve of WT and Gpr126+/– mice, MBP wascolocalized with myelin protein zero (P0), a major structuralcomponent of Schwann cell, but not oligodendrocyte, myelin(Lemke et al., 1988). MBP was strongly expressed in CNS regionsof Gpr126–/– cochlear nerve (Fig. 5), providing further evidencethat oligodendrocytes can form myelin in the absence of Gpr126function. In the PNS region of the nerve, however, no P0 or MBPexpression was detected in Gpr126–/– mutants, specifically in thespiral ganglia and in peripheral neurites between the spiral ganglionand the habenula perforata, where the fibers enter the organ ofCorti (Fig. 5B,C). Schwann cell numbers in these regions did notappear to be disrupted, as shown by DAPI staining (Fig. 5C).Interestingly, the part of the auditory nerve known as the centraltissue projection (Fraher and Delanty, 1987), which is myelinatedby oligodendrocytes and strongly labeled with MBP antibody, was

consistently expanded deeper into the modiolus of Gpr126–/–nerves (Fig. 5B,D). At the glial limitans, where the transitionbetween central and peripheral myelin occurs (Hurley et al., 2007),we observed many central MBP-positive cells projecting fartherinto the peripheral region of Gpr126–/– nerves than in WT nerves(Fig. 5A,B,D). Similar expansions of CNS myelin into the PNShave recently been reported in the absence of peripheral glia inzebrafish (Kucenas et al., 2009) and in mammals when Schwanncells or Egr2 function are absent (Coulpier et al., 2010). Together,this analysis shows that Schwann cells do not express MBP or P0in Gpr126–/– nerves, and that Gpr126–/– oligodendrocytes expressMBP and project aberrantly into PNS regions of the cochlear nervein the absence of peripheral myelin but in the presence ofGpr126–/– Schwann cells.

DISCUSSIONBeginning with a forward genetic screen for mutations disruptingthe development of myelinated axons, we recently described anessential role for Gpr126 in Schwann cell myelination in zebrafish(Monk et al., 2009). Through analysis of Gpr126–/– mice, weshow that the function of Gpr126 in myelination is conserved inmammalian peripheral nerve: Schwann cells are arrested at thepromyelinating stage in Gpr126–/– mice, as in zebrafish. Thismyelination defect is specific to the PNS, as CNS myelinationappears to be normal in Gpr126–/– mice (Fig. 5; see Fig. S2 in thesupplementary material). In addition, this study defines newfunctions of Gpr126 by analyzing other defects in Gpr126–/– micethat were not evident in gpr126 zebrafish mutants.

The analysis of peripheral nerves in Gpr126 mutant mice definesa previously unknown function for Gpr126 in the organization ofthe perineurium. By P4 in Gpr126–/– mutants, the ectopiclocalization of apparent perineurial fibroblasts is observedthroughout the endoneurium (Fig. 3F). This phenotype worsenswith age and, by P12, the endoneurium of Gpr126–/– mutants ispartitioned into numerous distinct minifascicles by the fibroblasts(Fig. 3G, Fig. 4B). This defect is likely to be due to loss of Gpr126in Schwann cells and not in fibroblasts: loss of desert hedgehog(Dhh) in Schwann cells also leads to ectopic fascicle formation by


Fig. 4. Perineurial fibroblasts are found throughoutGpr126–/– sciatic nerve and endoneurial collagenfibrils are disrupted. (A)Myelinated axons (blackasterisks) and developing Remak bundles (white asterisks)are observed in wild-type and Gpr126+/–(+/–) nerve atP12. Fibroblasts are never observed to bundle axons andSchwann cells into minifascicles. (B)Minifascicles (outlinedin white) are observed throughout the endoneurium of–/– sciatic nerve. (C)The minifascicles are formed byperineurial fibroblasts, identified by numerous caveolae(arrowheads) and a patchy basal lamina (arrow).(D)Schwann cells in wild-type and +/– nerve aresurrounded by a basal lamina (arrowheads), and collagenfibrils are well organized (arrow). (E)Collagen fibrils areoften poorly organized in –/– sciatic nerve (arrow),although Gpr126–/– Schwann cells are surrounded by abasal lamina (arrowheads). (A,B)Scale bars: 5m in A,B;0.5m in C-E.

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perineurial fibroblasts (Parmantier et al., 1999). These fibroblastsexpress the hedgehog receptor patched homolog 1 (Ptch1), andrequire Dhh from Schwann cells during nerve development forproper perineurium formation (Parmantier et al., 1999). Dhh is notexpressed in Gpr126–/– sciatic nerve (Fig. 1C), suggesting that theminifascicle pathology found throughout the endoneurium ofGpr126–/– nerves is due to loss of Dhh in Schwann cells ratherthan a direct role for Gpr126 in fibroblast development.

Gpr126–/– nerve pathology is also reminiscent of defectsobserved in Adam22 and Lgi4 mutants (Henry et al., 1991; Darbaset al., 2004; Sagane et al., 2005; Ozkaynak et al., 2010; Nishino etal., 2010). Adam22 is a receptor for Lgi4; Adam22 is expressedneuronally, and Lgi4 is secreted principally by Schwann cells inperipheral nerve (Sagane et al., 2008; Ozkaynak et al., 2010). Inaddition to reduced Schwann cell myelination, Adam22 and Lgi4mutant peripheral nerves also possess minifascicle pathologiesthroughout the endoneurium (Henry et al., 1991; Darbas et al., 2004;Sagane et al., 2005; Ozkaynak et al., 2010). Axonal sorting is alsodelayed in Lgi4 mutants (Darbas et al., 2004). Although thesephenotypic similarities raise the possibility that Gpr126 activationrequires Adam22 and/or Lgi4, it is important to note that Adam22and Lgi4 mutants express Pou3f1 (Ozkaynak et al., 2010; Darbas etal., 2004; Nishino et al., 2010), whereas Gpr126 mutants do not (Fig.2B) (Monk et al., 2009). Future studies are required to examine thepossible interaction between Gpr126 and Adam22 and Lgi4.

Impaired radial sorting in Gpr126–/– nervesDuring embryonic nerve development, in a process known as radialsorting, Schwann cells associated with multiple axons interdigitatecytoplasmic extensions throughout the bundle to separate largecaliber axons and eventually myelinate them. At P1 in Gpr126–/–nerve, Schwann cells are found mainly at the periphery of axonbundles, and very few axons have been sorted (Fig. 3E). Similar

defects in radial sorting are found in peripheral nerves of mice withSchwann cell-specific mutations affecting laminin isoforms (Chenand Strickland, 2003; Yu et al., 2005), the laminin receptor b1-integrin (Feltri et al., 2002; Nodari et al., 2007), the tyrosine kinaseFAK, integrin-linked kinase and downstream signaling componentsin Schwann cells (Benninger et al., 2006; Grove et al., 2007; Nodariet al., 2007; Pereira et al., 2009). Although Schwann cells inGpr126–/– mutant mice eventually recover and sort axons, theseearly phenotypic similarities suggest a possible connection betweenGpr126 and signaling between laminins and their integrin receptors.It is also possible that activation of Gpr126 in Schwann cells leadsto similar downstream signaling cascades as integrin activation.

Reduced axon number in Gpr126–/– nervesThe total number of axons in Gpr126–/– sciatic nerve wassubstantially reduced compared with the estimated total number ofaxons in WT sciatic nerve at P8. However, at P1, this reductionwas less severe. Additionally, when we viewed the entire sciaticnerve by electron microscopy montage at P1, we observed regionsof mutant nerve with dying axons (data not shown). In ErbB3mutant mice, which lack Schwann cells associated with developingsensory and motor neurons, loss of trophic support from Schwanncells 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 Gpr126cannot confer vital trophic support to associated axons, resulting inthe loss of axons we observe at P8.

Absence of Remak bundles in Gpr126–/– nervesWe observed no developing Remak bundles in Gpr126–/– sciaticnerves as late as P12. Axonal sorting was initially delayed, andSchwann cells eventually sorted axons into a 1:1 relationship.Bundles of unsorted axons were occasionally observed at P12 (data


Fig. 5. Cochlear histology appears grossly normal in Gpr126–/– mice, but Schwann cells do not express P0 or MBP, and CNS myelin isexpanded into peripheral zones. (A)Mid-modiolar cross sections showing the middle turn of wild-type (+/+) and Gpr126–/– (–/–) mice, stainedwith antibodies to myelin basic protein (MBP; red) and neurofilament-M (NF-M; green). (B) Adjacent sections to those shown in A, labeled withantibodies to MBP (red) and peripheral myelin protein zero (P0; green). The brackets indicate the region between the habenula perforata and thespiral ganglion; the arrow indicates the spiral ganglion. (C)Higher magnification of the peripheral section of the auditory nerve fibers. The habenulaperforata (hp) and the spiral ganglion (sg) are indicated. (D)Higher magnification of the glial limitans, where the transition between central andperipheral 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: 200m in A,B; 100m in C,D.

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not shown), but these axons were not surrounded by non-myelinating Schwann cell cytoplasm; rather, Schwann cellsremained at the periphery of axon bundles, reminiscent of thesorting defect we observed at earlier stages (Fig. 3E,F). Althoughthe total number of axons is reduced in Gpr126–/– nerves, smallcaliber C-fibers are present in mutants (Fig. 2D). This resultsuggests Gpr126 plays a role in the development of non-myelinating Schwann cells, leading to the loss of Remak bundlesin Gpr126–/– mutants. Previous results suggest that Gpr126functions to elevate cAMP levels in promyelinating Schwann cellsprior to myelination (Monk et al., 2009). cAMP elevation inpostmitotic Schwann cells is thought to downregulatepremyelinating signals and upregulate myelinating signals (Morganet al., 1991; Monuki et al., 1989; Scherer et al., 1994). It is possiblethat non-myelinating Schwann cells also require elevation ofcAMP levels in order to differentiate. Indeed, when Schwann cellsare proliferative, elevation of cAMP does not initiate myelin geneexpression but instead induces expression of the cell surface lipidantigen O4, an early Schwann cell differentiation marker (Mirskyet al., 1990; Morgan et al., 1991; Jessen et al., 1991). The levels ofNrg1TypeIII on the surface of an axon are thought to instructSchwann cells to become myelinating or non-myelinating(Taveggia et al., 2005). It was recently shown that levels of cAMPdetermine how Schwann cells respond to Nrg1 in vitro (Arthur-Farraj et al., 2011). Future studies are required to determine the roleof cAMP in non-myelinating Schwann cell development and toascertain how Gpr126 and Nrg signaling interact in vivo.

It is also possible that non-myelinating Schwann cells require asignal from promyelinating or myelinating Schwann cells that isaltered in the absence of Gpr126; one such candidate signalingmolecule is Dhh. Non-myelinating Schwann cells express thehedgehog receptor patched homolog 2 (Ptch2) in adult sciatic nerveand after injury (Bajestan et al., 2006). Although Dhh-Ptch2signaling has not been directly shown to be required for thedevelopment of non-myelinating Schwann cells, Dhh mutantnerves possess significantly more axons sorted in a 1:1 relationshipand fewer axons in Remak bundles (Sharghi-Namini et al., 2006),a pathology that is reminiscent of the defects observed inGpr126–/– peripheral nerve. Future studies utilizing cell-typespecific ablation of Gpr126 will be useful to determine the role ofGpr126 in non-myelinating Schwann cell development.

Other roles for Gpr126 and future directionsZebrafish mutants for gpr126 are viable and fertile, whereasGpr126–/– mice are not born in Mendelian ratios and no mutantsurvived past P16. In a recent study, loss of Gpr126 in mouseresulted in mid-gestation embryonic lethality (Waller-Evans et al.,2010). These embryos died of cardiovascular defects beforeanalysis of peripheral myelination could take place. It is thereforelikely that cardiovascular defects also account for some of theembryonic lethality we observe in our studies. Strain differences orthe region of Gpr126 targeted might explain why we obtainedsome live mutants at birth and why Waller-Evans et al. observedfully penetrant embryonic lethality. Waller-Evans et al. deleted theseven-transmembrane region of Gpr126 in their mutant allele,whereas functional domains in the N-terminus were targeted in theGpr126 mutation that we analyzed. Gpr126 is also highlyexpressed in mouse and rat lungs (Moriguchi et al., 2004; Haitinaet al., 2008) so it is possible that an abnormality in the lungcontributes to the mortality of Gpr126 mutants. Furthermore, threesingle-nucleotide polymorphisms (SNPs) in GPR126 were recentlyassociated with lung function in humans (Hancock et al., 2010).

Bergmann glial cells of the mouse cerebellum also express highlevels of Gpr126 (Koirala and Corfas, 2010), as does the collectingduct of the mouse kidney (Pradervand et al., 2010). Future studiesare required to determine the function of Gpr126 in these tissuesand 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 signalingbetween these cells is required for the proper development,organization and function of peripheral nerves. This work shows thatGpr126 is essential for myelination in the mammalian PNS. We alsoshow that Gpr126 is required for timely Schwann cell differentiationand peripheral nerve organization in mice. Many key drug targets areG protein-coupled receptors (Lundstrom, 2005); as a crucialregulator of myelination in mammals, Gpr126 might represent anattractive therapeutic target for re-myelination therapy in humans.

AcknowledgementsThis work was supported by grants to W.S.T. from the Muscular DystrophyAssociation (92233) and the National Institutes of Health (NIH; R01NS050223). K.R.M. was supported by a National Multiple Sclerosis Societypostdoctoral award (FG 1719-A-1) and by the Stanford Genome TrainingProgram (NHI/NGHRI). S.H. was supported by NIH R01 DC004563 and P30DC010363. We thank Ben Emery for assistance with optic nerve dissections,John Perrino for electron microscopy advice, the Barres lab for the gift of theP0 antibody, and members of the Talbot and Kingsley laboratories for helpfuldiscussions. We are also grateful to Yo Sasaki and Jeffrey Milbrandt forproviding DRG neuron cDNA samples, and to Valeria Cavalli, John Heuser,David Ornitz, Naren Ramanan and Lila Solnica-Krezel for sharing laboratoryspace and reagents. Deposited in PMC for release after 12 months.

Competing interests statementThe authors declare no competing financial interests.

Supplementary materialSupplementary material for this article is available athttp://dev.biologists.org/lookup/suppl/doi:10.1242/dev.062224/-/DC1

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