DEVELOPMENTALDYNAMICS222:40–51(2001)

InEarlyDevelopmentoftheRatmRNAfortheMajor

MyelinProteinP0 IsExpressedinNonsensoryAreasof theEmbryonicInnerEar,,EntericNervous System,andOlfactoryEnsheathingCells

MENG-JENLEE,1 ESTERCALLE,1 ANGELABRENNAN,1 SABRINAAHMED,1 ELENASVIDERSKAYA,2 1 1 KRISTJANR.JESSEN, ANDRHONAMIRSKY * 1DepartmentofAnatomyandDevelopmentalBiology,UniversityCollegeLondon,London,UnitedKingdom 2DepartmentofAnatomy,StGeorge’sHospitalMedicalSchool,London,UnitedKingdom

ABSTRACT ThemyelinproteinP0 hasama- INTRODUCTION jorstructuralroleinSchwanncellmyelin,andthe TheP0 geneandthemechanismsthatcontrolithave expressionofP0 proteinandmRNAinthe beenofgreatinteresteversinceitwasrealizedthatP0 Schwanncelllineagehasbeenextensivelydocu- proteinwasbyfarthemostabundantproteinofmyelin mented.Weshowhere,usinginsituhybridization, inthePNS,andthatitsexpressionwassubjectto thattheP0 geneisalsoactivatedinanumberof dramaticregulationbysignalsfromneurons(Gieseet othertissuesduringembryonicdevelopment.P0 al.,1992;LemkeandAxel,1985;SommerandSuter, mRNAisfirstdetectablein10-day-oldembryos 1998).Inmammals,P0 appearedinitiallytobean (E10)andisatthistimeseenonlyincellsinthe exampleparexcellenceofacelltypespecificgene, cephalicneuralcrestandintheoticplacode/pit.P0 beingrestrictedtothoseSchwanncellsinducedtoform expressioncontinuesintheoticvesicleandatE12 myelinsheaths(Colmanetal.,2001).Ithasnowbe-

P0 expressioninthisstructurelargelyoverlaps comeclearthattheP0 geneisactivated,albeitatrel- withexpressionofanothermyelingene,proteo- ativelylowlevels,priortomyelinationnotonlyinall lipidprotein.InthedevelopingearatE14,P0 ex- immatureSchwanncellsbutalsoinSchwanncellpre- pressioniscomplementarytoexpressionofserrate cursors(Burronietal.,1988;ChengandMudge,1996; andc-retmRNAs,whichlaterareexpressedinsen- Leeetal.,1997;MirskyandJessen,2001;Zhangetal., soryareasoftheinnerear,whileexpressionof 1995).P0 isalsoseenevenearlierinasubpopulationof bonemorphogeneticprotein(BMP)-4andP0, cellsinthetrunkneuralcrest,thetissuethatgivesrise thoughlargelycomplementary,showssmallareas toSchwanncellprecursors,whereitssignificancehas ofoverlap.P0 mRNAandproteinaredetectablein becomecontroversial(Bhattacharyyaetal.,1991; thenotochordfromE10toatleastE13.Inaddition Hagedornetal.,1999;Leeetal.,1997).Furthermore, thereissomeevidencefromimmunochemicalandim- toP0 expressioninasubpopulationoftrunkcrest cellsatE11/E12andinSchwanncellprecursors munohistochemicalstudiesthatP0 proteinisseenin populationsofneuronsintheCNS(Satoetal.,1999; thereafter,P0 mRNAisalsopresenttransiently inasubpopulationofcellsmigratingintheen- SatoandEndo,2000),andtransgenicmicegenerated tericneuralcrestpathway,butisdown-regu- withareportergenedrivenbya1.1-kbstretchofreg- ulatorysequencefromtheP0 geneshowtransgene latedinthesecellsatE14andthereafter.P0 is alsodetectedintheplacode-derivedolfactoryen- expressioninnumerousplacesincludingsomeperiph- sheathingcellsfromE13andismaintainedinthe eralneurons(Yamauchietal.,1999).Thus,ourpicture adult.Nosignalisseenincellsinthemelanocyte ofP0 geneexpressionduringmammalianembryonic developmentischanging.Itwillcertainlyturnoutto migrationpathwayorinTUJ1positiveneuronal bemorecomplexthanoriginallyenvisagedandmuchis cellsintissuesections.TheactivationoftheP 0 stillunclearabouttissuesandtimingofgeneactiva- geneinspecifictissuesoutsidethenervoussys- tion,letaloneaboutwhatmightbethefunctionofP temwasunexpected.Itremainstobedetermined 0 outsidethemyelinsheath.Withtheaimofprovidinga whetherthisisfunctionallysignificant,or whetheritisanevolutionaryrelic,perhapsre-

flectingancestraluseofP0 asanadhesionmole- Grantsponsor:WellcomeTrust;Grantnumber:042257/Z/94/Z; cule. ©2001Wiley-Liss,Inc. Grantsponsor:EuropeanCommission;Grantnumber:ERBFM- BICT98-3106. *Correspondenceto:R.Mirsky,DepartmentofAnatomyandDevel- Keywords:neuralcrest;Schwanncells;oticpit; opmentalBiology,UniversityCollegeLondon,GowerStreet,London WC1E6BT,UK.E-mail:[email protected] oticvesicle;glia;serrate;BMP-4;c- Received26March2001;Accepted14May2001 ret Publishedonline20July2001;DOI10.1002/dvdy.1165

©2001WILEY-LISS,INC. P0 IN RAT PERIPHERAL GLIAL LINEAGE 41

more comprehensive view of P0 expression during and the vagal crest migrating into the gut to form the mammalian embryonic development, we have, in the enteric . In enteric cells, P0 mRNA ex- present work, monitored P0 mRNA expression during pression was strikingly transient, resembling, on a embryonic rat development using whole embryos, tis- shorter time scale, the transient expression of P0 sue sections, and dissected organs. mRNA in the development of nonmyelin-forming

P0 comprises approximately 50% of the total protein Schwann cells (Lee et al., 1997). Moreover, in the in Schwann cell myelin in peripheral nerves where it oesophageal part of the gut P0 mRNA expression was plays a major stabilizing role, acting via homophilic first detected a day after the first appearance of the interactions (Colman et al., 2001). The levels of mRNA early neuronal marker ␤3-tubulin, a finding with clear expressed in Schwann cell precursors and immature implications for questions concerning the lineage sta-

Schwann cells prior to myelination represent constitu- tus of P0-positive cells in the . P0 was not tive basal expression levels of P0 that are many times detected in the dorsolateral crest melanocyte migration lower than the peak levels of P0 mRNA seen in those pathway, nor was it found in crest cells in the jaw Schwann cells that are induced to myelinate large di- region. These findings indicate that P0 expression is ameter axons (Lee et al., 1997; Trapp et al., 1988). low or absent from non-neural cells of the crest lineage. Conversely, in those cells that develop to form mature Lastly, in contrast to the general finding in the rat that non-myelinating Schwann cells, the basal P0 levels are basal P0 expression is down-regulated in mature non- down-regulated. This divergent regulation of basal P0 myelin-forming glia (Lee et al., 1997), we found that levels is reversible: if Schwann cells lose contact with olfactory ensheathing cells, which start expressing P0 axons, P0 levels in myelinating and nonmyelinating at E13, continue to do so in the adult. This is of interest cells fall and rise, respectively, as the cells de-differen- because P0 protein promotes axonal growth in vitro and tiate. Thus, denervated adult Schwann cells express olfactory ensheathing cells uniquely associate with ax- basal P0 mRNA levels comparable to those seen in ons that continue to regrow throughout life (Schneider- immature Schwann cells (Lee et al., 1997). Basal P0 Schaulies et al., 1990). mRNA levels in developing satellite cells in rat dorsal root ganglia (DRG) are also suppressed as these cells RESULTS mature (Lamperth et al., 1989; Lee et al., 1997). P0 at To determine the onset of P0 mRNA expression in the cell surface can take part in homophilic and het- embryogenesis, in situ hybridization using digoxygenin erophilic interactions with P0 and other adhesion mol- (DIG)-labelled mRNA probes was performed on whole ecules on the surface of neighbouring cells outside the mount preparations of rat embryos at E9–12. context of the myelin sheath, and in this way mediate cell–cell adhesion (Doyle et al., 1995; D’Urso et al., P0 Is First Detectable Within the 1990; Filbin et al., 1990; Schneider-Schaulies et al., and Developing Inner

1990). While it is possible that basal P0 expression acts No signal was detectable at E9. At E10 (10 somite to promote cell adhesion, there is as yet no direct evi- stage), some cells in the /pit were strongly dence for this, and the significance of nonmyelin re- P0 positive, and by E11 intense expression of P0 was lated P0 expression in developing Schwann cells re- seen in most areas of the otic vesicle (Fig. 1A,B). By mains uncertain. E12 it was still widely expressed in the vesicle but Perhaps because of the highly restricted distribution showed a more variable distribution, being low in some of P0 to myelinated nerves in the adult, previous stud- areas, remaining extremely high in others, including ies did not pay much attention to whether P0 expres- the developing endolymphatic duct and the adjacent sion during development was limited to the peripheral dorsomedial area (Figs. 1C and 2A). To test whether nervous system (PNS). Even within the developing other myelin related genes might also be expressed in

PNS, although it has been noted that P0 mRNA is the developing ear, we compared the expression of P0 expressed in a subpopulation of with that of PLP/DM20, another myelin gene that has cells, P0 expression has not been examined in other been detected in glial cells early in development divisions of the rat neural crest and little is known (Spassky et al., 1998; Timsit et al., 1992). Using in situ about the relationship between P0 and other markers hybridization, PLP/DM20 labelling was clearly seen in of early crest differentiation. the otic vesicle at E11, and was, like P0 mRNA, located Since it would be useful to have a comprehensive mainly in the dorsal part of the vesicle (Fig. 2B). We picture of the expression pattern and developmental also examined the expression of a lacZ reporter con- regulation of this important gene, we have used in situ struct expressed under the control of PLP regulatory hybridization to detect P0 mRNA in the rat embryo sequences in a transgenic mouse line where labelling between E9 and birth. P0 gene activation was seen in has been reported in the otic vesicle at E10 (equivalent two unanticipated locations, i.e., in the notochord at to E12 rat) (Spassky et al., 1998). Again, the PLP/ E10–E13 and in the developing ear, starting in the otic DM20 transgene was expressed within the otic vesicle, placode/pit at E10 and selectively in nonsensory areas in a broadly similar pattern to that of P0 (Fig. 2C). of the developing inner ear at E14. In the crest, we The distribution of P0 was next compared with found that P0 expression extends to the cephalic crest several markers known to be present in restricted 42 LEE ET AL.

Fig. 1. A: At E10, P0 mRNA, revealed by DIG-labelled in situ hybrid- anterior somites now extends to the tail region and labelling in the ization, is seen in the developing otic placode/pit (arrow) and also in notochord is clearly seen (arrowhead in C); E,F: P0 labelling in sections migrating cephalic crest cells (arrowheads). B: At E11 the otic vesicle through the hindlimb regions at E12. E: Anterior part of somite, located by

(ov) is strongly P0 positive and a positive signal is seen clearly in the labelling alternate sections for erbB3 and P0. Note that P0 positive cells ophthalmic and submandibular branches of the developing trigeminal are restricted to the ventral part of the condensing DRG/neural crest and nerves and within the trigeminal ganglion (V), and also in the position of that P0 is essentially absent in the most dorsal region of the ventromedial the developing acoustic/facial (VII/VIII) nerves (big arrow). Wedge- pathway of crest migration/dorsal roots (arrows in E; compare E to G). shaped streams of positive neural crest cells can be seen in the trunk Cells in the dorsolateral region of the embryo, where the melanoblasts region down to the level of the 18th–19th somite. From the first to the migrate, are also P0 negative. Cells in the ventral root exit zone and the 10th–12th somite position, strong P0 expression appearing as a slightly notochord (nc) are strongly P0 positive. The (nt) is negative. interrupted line along the neural tube represents glial cells associated F: Posterior part of somite. P0 labelling is confined to the ventral root exit with the emerging motor axons (arrows). Ventral to this, weaker P0 signal zone (arrows) and notochord. G,H: erbB3 mRNA expression in the could be seen representing cells forming the sympathetic chain; this hindlimb region at E12 reveals the location of the neural crest. G: Anterior location is not in focus in micrograph B. C,D: E12 (C) whole mount view part of somite. Cells expressing erbB3 (arrows) have a more widespread of P0 mRNA and (D) TUJ1 labelling to reveal neurons. Ophthalmic (op), distribution and are seen more dorsally than cells expressing P0 (com- maxillary (mx), and submandibular (md) branches of the trigeminal pare G with E). In particular, note erbB3-positive cells in the position of nerves are shown and other cranial nerves are indicated by their roman the most dorsal migrating pathways between the and neural numbers. At E12, the otic vesicle (ov) and trigeminal ganglion (T) are tube in the position of the forming dorsal roots. P0 positive cells are never strongly P0 positive, and the acoustic/facial ganglia and nerves are also seen at this stage in this position. H: Posterior part of somite. Note erbB3 positive (arrow in C and D). TUJ1 labelling is clearly seen within the in an aggregated group of cells above the sclerotome between the neural trigeminal ganglion (V) and nerves and the acoustic/facial ganglia and tube and dermamyotome; P0 is never seen in this location (compare H nerves. P0 positive cells can be seen in the developing glossopharyngeal with F). I: E13 P0 protein expression in the notochord. Immunoperoxidase (IX), vagus (X), and accessory (XI) cranial nerves. P0 labelling in the labelling with antibodies to P0 protein.

territories or cell types in the developing inner ear. non-overlapping. As c-ret marks out cells in the fu- At E14, in a series of sections through the endolym- ture sensory area in other species, this suggested phatic duct, the semicircular canals and part of the that P0 might be selectively expressed in cells in the utricle, P0 was seen in cells of the endolymphatic nonsensory component of the inner ear (Robertson duct and part of the semicircular canals (not shown). and Mason, 1995; Torres and Giraldez, 1998). This More anteriorly in a section through the cochlea and idea was confirmed when expression of BMP-4 and part of the utricle, P0 was restricted to approxi- serrate was compared with P0 (Fig. 4). Serrate ex- mately half the cochlea with a sharp boundary be- pression is restricted to the sensory epithelium in the tween positive and negative territories (Fig. 3A). In developing chick ear and BMP-4 is also highly ex- adjacent parasaggital sections in the vestibule region pressed in future sensory patches although it is also hybridized with P0 and c-ret, respectively, c-ret expressed outside the sensory area (Myat et al., marked a small area of the utricle, the putative 1996; Torres and Giraldez, 1998; Whitfield et al., sensory area, while P0 expression was more wide- 1997; Wu and Oh, 1996). Again the patterns of ex- spread (Fig. 3B,C). The patterns of labelling were pression of both BMP-4 and serrate were distinctly P0 IN RAT PERIPHERAL GLIAL LINEAGE 43

Fig. 2. P0 and PLP/DM20 in the otic vesicle. A: At E12, expression of P0 mRNA within the rat otic vesicle is not uniform. Labelling is more evident in dorsomedial and ventrolateral areas and in the developing endolymphatic duct (arrow). B: At E11, PLP/DM20 mRNA is visible in the dorsal area of the otic vesicle. C: At E10 in mouse otic vesicle (equivalent Fig. 4. Comparison of P0 labelling (A,C) with that of BMP-4 (B) and to E12 rat), lacZ driven by the PLP/DM20 promoter in a transgenic mouse serrate-1 (D) using in situ hybridization of consecutive sections through is seen in dorsomedial and ventrolateral areas in agreement with P0 the cochlea. The distribution of P0 positive cells is essentially complimen- mRNA expression in the rat. In all sections, the hindbrain lies to the left tary to that of cells expressing BMP-4 or serrate-1. of the section, and dorsal regions are at the top of the figure.

P0 Is Expressed in Glial Cells Within the Head Region From E10 Onwards

At E10, P0 was also detectable in migrating crest cells in the head (Fig. 1A). By E11, P0 signal was clearly seen in the ophthalmic and submandibular branches of the developing trigeminal nerves and within the ganglion (Fig. 1B). These nascent nerves and ganglia can be identified using antibodies to ␤3- tubulin (TUJ1), which specifically labels neurons (Mayer-Proschel et al., 1997; Moody et al., 1987) (not shown). Since at this stage the only cells within the nerves are glial cells, we can conclude that at E11 the

P0-positive cells within the nerves are almost certainly Schwann cell precursors (Jessen et al., 1994). At E12 when all the cranial ganglia and nerves except the

olfactory nerves have been formed (but see below), P0 positive cells were seen in all nerves except the 4th and 6th cranial nerves, which were difficult to see, and the optic nerve, cells of which are derived from the central

nervous system (CNS) (Fig. 1C). No P0-positive cells Fig. 3. P0 expression in the developing ear at E14. A: In a horizontal were seen in the region of the developing jaw at E10, section through the hindbrain area, P labelling is evident in the acoustic 0 11, or 12, indicating that neural crest cells destined to ganglion (g) and nerve. Restricted labelling is seen within the cochlea (c) differentiate into non-neural lineages were not P0 pos- and vestibule (v). B,C: P0 and c-ret labelling of consecutive sections through the vestibule (v) and cochlea (c). P0 labelling is widespread but itive. non-uniform while c-ret labelling is highly restricted to small areas in the saccule and cochlea. The P0 and c-ret labelling is largely nonoverlapping. P0 Is Expressed in Cells of the Notochord

Strong P0 mRNA hybridization signal was seen in the notochord from E10 onwards, until at least E13 different from and broadly complementary to the (Fig. 1C,E,F). P0 protein could also be visualized in the expression of P0 in the cochlea although some overlap notochord at E13 (Fig. 1I), although the protein could of BMP-4 and P0 territories may be present (Fig. 4). not be detected immunocytochemically in the migrat- Thus, in the E14 cochlea P0 is expressed within ing crest and only weakly in the otic vesicle in sections sharply demarcated boundaries and probably re- under labelling conditions where protein expression stricted to the nonsensory portion of the tissue. No was visible in E14 nerves in sections and was strongly

P0-positive cells were seen in areas outside the co- expressed in neural crest cell cultures (not shown). chlea where migrating melanocytes are normally seen, in agreement with the lack of labelling of cells P0 Is Expressed in the Trunk Region From E11 in the dorsolateral melanocyte migration pathway in Onwards the trunk neural crest (see below) (Nakayama et al., In the trunk, P0 mRNA expression was first seen in 1998; Steel et al., 1992). migrating neural crest cells at E11, as reported previ- 44 LEE ET AL. ously (Lee et al., 1997) (Fig. 1B). It was seen down to erbB3 is expressed in a group of aggregated cells sitting the level of the 18th–19th somites and was confined to in a wedge between the neural tube and the dermamyo- known migratory pathways for neurons and glia. It tome, but above the sclerotome (Fig. 1H). This is con- was not seen in the dorsolateral melanocyte migration sistent with the position of neural crest cells migrating pathway. P0-positive cells were distributed in triangu- at the most dorsal part of the ventromedial pathway of lar streams emanating from the dorsal aspect of the crest migration. No P0-positive cells are seen in this neural tube, although there were no P0-positive cells in position (Fig. 1F). These observations show that P0- the most dorsal region of crest migration between the positive cells are not present at the initial stages of neural tube, ectoderm, and the tip of the dermamyo- crest migration. Rather, the P0 gene is activated in a tome. As expected, P0-labelled cells avoid the posterior subpopulation of crest cells at the time they have mi- part of the somites. Strong P0 expression was also seen grated about halfway, or further, along the tube in a running in a longitudinal line, punctuated by small ventral direction. negative areas, down the length of the embryo from the first somite to the 10th–12th somites. This line corre- P0 Is Expressed in a Subpopulation of Cells in sponds to cells situated at the exit points of emerging the Enteric Neural Crest Pathway and in motor axons (Fig. 1B). Between the 3rd–6th somites Migrating Cells Within the Developing Gut, and Is Later Down-Regulated in These Cells P0-positive cells are also seen in the area of the con- densing sympathetic ganglia. Towards the tail of the When E11 embryos were double labelled with a DIG- embryo, little expression is seen. labelled P0 probe and TUJ1 antibodies and then sec- At E12, P0-positive cells are again seen in a repeti- tioned, TUJ1 labelled neurons could be seen in the tive pattern corresponding to expression in cells mi- region of the developing oesophageal wall while no grating in the anterior half of each somite (Fig. 1C). In P0-positive cells were seen (Fig. 5A). In enteric cells, serial cross-sections of the embryo at the hindlimb c-ret is considered to be a late marker for crest cells and level, P0-positive cells migrate in the ventromedial a marker for early neurons (Natarajan et al., 1999). pathway, in locations immediately adjacent to the neu- When c-ret was used as a probe for migrating enteric ral tube (Fig. 1E). Again, no positive cells are seen in cells, ret-positive cells were also present in this region the dorsolateral pathway where melanoblasts migrate at E11 in the absence of P0 expression (not shown). (Fig. 1E,F). P0 cells are at first concentrated in the most However, at E12, P0 expression is evident in many cells ventral areas of each condensing ganglion, then appear in the crest migratory region of the gut wall (Fig. 5B). around the edges and at later time points are more This suggests, firstly, that in the gut, significant P0 evenly distributed within the ganglion and in the de- expression starts at E12 and, secondly, that in this veloping spinal nerves (not shown). P0 is not seen in the environment P0 positive cells are first seen after the developing dorsal roots at this stage. Later, at E13 and appearance of ret positive cells and TUJ1 positive cells.

E14, expression of P0 within the DRGs drops to lower When P0 expression at E12 was examined in whole levels, while strongly positive cells are concentrated at mounts of the gut, P0-positive cells, which presumably the poles of the ganglion and in the dorsal and ventral can give rise to enteric glial cells, were clearly seen in roots (Lee et al., 1997). the region of the migration pathways of neural crest cells that will populate the developing gut. High levels P0 Is Detectable in a Subpopulation of Neural of P expression were present in the nodose ganglia, Crest Cells: Comparison With erbB3 0 with streams of P0-positive cells following the course of The tyrosine kinase receptor erbB3, to which neu- the vagus nerve towards and along the exterior of the regulins bind with high affinity, is expressed on many oesophagus (Fig. 5C). P0-positive cells were also neural crest cells, including early migrating crest cells present throughout the small intestine, reaching as far (Lemke, 1996; Meyer and Birchmeier, 1995; Meyer et as, but not into, the caecum and the large intestine. al., 1997). To compare P0 labelling with that of erbB3, C-ret-positive cells follow similar pathways, as re- sections taken from the hindlimb region of E12 em- ported previously (Durbec et al., 1996; Robertson and bryos labelled with an erbB3 probe (Fig. 1G,H) were Mason, 1995). Although c-ret cells are more numerous, examined together with those labelled with a P0 probe they appear to reach the same level of the gut as the (Fig. 1E,F). It is clear from this comparison that in the P0-positive cells at this stage of development. There- ventromedial pathway of crest migration, erbB3 ex- fore, P0-positive cells form a subpopulation of the total pression extended more dorsally than P0. In agreement migrating cell population. with the distribution reported in mouse, at the level of At E13, the highest level of P0 expression was seen at anterior somites, erbB3-positive cells are present in an the junction of the oesophagus and stomach (Fig. 5D). area that is nearly continuous with the dorsal edge of The signal is associated with the distal ends of the the tube and that extends ventrally about two thirds of vagus nerves as they enter the stomach, becoming the way down the tube; some positive cells are also seen more diffuse in the body of the stomach. P0-positive within the myotome and sclerotome (Britsch et al., cells were clearly seen in the small intestine although 1998; Meyer and Birchmeier, 1995; Meyer et al., 1997) none were present in the caecum or lower parts of the (Fig. 1G). In the posterior half of the same somite, gut, in agreement with the observations at E12 (above) P0 IN RAT PERIPHERAL GLIAL LINEAGE 45

Fig. 6. A: Portion of whole mount of olfactory region at E13. P0 labelling is seen in the developing olfactory nerve (arrow), as it projects from the olfactory epithelium (e) to the olfactory bulb (ob); B: In a

horizontal section taken at E14 through the olfactory epithelium, P0 labelling is not seen within the epithelium (e) but is seen in developing nerves (arrows) just outside it. C: Adult olfactory nerve, teased nerve preparation. Heavy P labelling of all Schwann cells is evident. D: P Fig. 5. P0 expression in the enteric nervous system. A: TUJ1 positive 0 0 /early neurons (arrows) are seen in the foregut region of an labelling in a teased preparation of the adult sympathetic trunk. Non- myelinating Schwann cells are unlabelled, as previously reported, while E11 embryo double labelled with P0 and TUJ1. No P0 positive cells are P labelling is seen in occasional myelinating Schwann cells. seen. B: By E12, P0-positive cells are seen in the region of the oesoph- 0 agus (arrows). C: In a whole mount preparation at E12, streams of

P0-positive cells can be seen emanating from the direction of the nodose ganglion (arrow) and migrating along the course of the vagus nerves (big positive cells appeared similar (Fig. 5D-G). The large arrowhead). P0-positive cells are seen in the stomach (small arrowhead). D: At E13, the highest level of P0 expression is seen at the junction of the intestine remained P0 negative. The level of expression oesophagus and stomach (arrow). E: At E13, P0 signal is also seen in the of c-ret was similar at E13 and E14, and it was ex- small intestine (arrow) but is not seen in the caecum (arrowhead) or pressed in a similar number of cells (Fig. 5E, inset). beyond. At this stage c-ret labelling is evident beyond the caecum in the We, therefore, conclude that by E14, down-regulation colon (inset). F,G: At E14, P0 expression is down-regulated. F: Very little labelling is now evident in the stomach (arrow in F and G). G: In the small of P0 mRNA has occurred in all the P0-positive cell intestine (arrowheads), individual cells show much weaker signal than at population. E13 (compare to E), although their distribution is similar, i.e., no positive In adult gut, no P0-positive cells could be detected in cells are seen in the caecum or beyond. This suggests that factors within the enteric glia, visualized with antibodies to GFAP, the gut environment down-regulate P mRNA expression. H,I: In the 0 within the enteric plexuses in any area of the tract (Fig. enteric plexuses of adult gut, P0-positive cells are never seen. H: GFAP labelling to show enteric glia within the enteric plexus of the adult small 5H,I) (Jessen and Mirsky, 1983). Therefore, the down- intestine. I: No P0 labelling is seen with P0 in situ hybridization of the same regulation of P0 that begins at about E14 persists into section. adult life.

P0 Is Expressed in Olfactory Nerve Cells (Fig. 5E). In contrast, in E13 embryos c-ret-positive Throughout Life cells are seen in the colon (Fig. 5E, inset). P0 was not expressed in the olfactory placode at E11 A comparison of P0 signal in the developing gut at or in the olfactory pit at E12. It was, however, detected E13 and E14 shows that the signal is lower in individ- in the developing olfactory nerves at E13 (Fig. 6A). In ual cells at E14 than at E13, although the number of sections of E14 olfactory epithelium, p75NGF recep- 46 LEE ET AL. tors, which mark the olfactory nerves at this stage, cells within the ear. Intriguingly, a majority of patients were detected immunohistochemically in the connec- with a clinically distinct late-onset Charcot-Marie- tive tissue just outside the epithelium (not shown), in Tooth neuropathy associated with a missense mutation locations where P0 signal was present (Fig. 6B). When (Thr124Met) in the P0 gene are also deaf (De Jonghe et adult teased olfactory nerves were hybridized for P0 in al., 1999). Whether this is due solely to neuropathy of the same experiment as teased adult cervical sympa- the vestibulocochlear ganglia and nerves, or also due to thetic trunk, the olfactory ensheathing cells in the other defects in the inner ear, is unknown. olfactory nerve remained P0-positive, whereas the non- It may be significant that another myelin gene that is myelinating Schwann cells in the trunk were P0-nega- expressed early in development in both the CNS and tive as previously reported (Lee et al., 1997). This sug- PNS, PLP/DM20, is also expressed in the otic vesicle gests that the regulation of basal P0 levels is different (see also Spassky et al., 1998). Likewise, the tyrosine in olfactory ensheathing cells from regulation in other kinase receptor erbB3, which is strongly expressed by nonmyelinating PNS glia such as nonmyelinating neural crest cells, Schwann cell precursors, and Schwann cells or enteric glial cells of the gut. Schwann cells, is also expressed in the otic vesicle and inner ear (Meyer et al., 1997). These molecules shared DISCUSSION in common with the neural crest might argue for a

The picture of P0 gene expression in embryonic rat common evolutionary origin for the otic placode and development is considerably more complex than antic- neural crest (Baker and Bronner-Fraser, 1997). ipated in the early studies that established P0 as a key, In general, P0 is highly expressed in areas of the and apparently specific, protein of Schwann cell mye- inner ear in a broadly reciprocal fashion to that of lin. This study raises in particular the question of what markers of the future sensory areas such as Delta-1, the function of P0 outside the myelin sheath might be. serrate, c-ret, and BMP-4 (reviewed in Torres and Gi- A more careful analysis of mice in which the P0 gene raldez, 1998). In particular, serrate and P0, and c-ret has been inactivated (Giese et al., 1992) and mice com- and P0 expression appear to be mutually exclusive bining P0 inactivation with inactivation of other poten- (Figs. 3 and 4). Furthermore, at E12 in rat, P0 expres- tial adhesion molecules with similar distribution may sion is widespread within nonsensory ear structures be informative in this regard (Carenini et al., 1999). such as the endolymphatic duct and the cochlear duct Another issue raised by the present work concerns the (Figs. 2–4) (Bissonnette and Fekete, 1996; Martin and mechanisms that regulate P0 gene expression. The ex- Swanson, 1993; Whitfield et al., 1997; Wu and Oh, pression of P0 mRNA on distinct schedules in diverse 1996). Unfortunately, no markers for specific develop- tissues suggests that a relatively complex set of cis- ing nonsensory structures within the cochlea, such as and trans-acting elements direct P0 expression and this the stria vascularis or Reissner’s membrane, have been will be revealed in further work. described for this early stage of inner ear development.

The expression of P0 mRNA in the otic placode/pit We, therefore, cannot at present ascertain whether P0 and subsequently in the otic vesicle and developing is restricted to particular structures or includes most inner ear was intriguing. Many of the studies on otic nonsensory areas. vesicle markers that identify future territories in the The significance of the finding that P0 is expressed by developing ear or associated ganglia have been carried cells within the notochord is at present unknown, al- out in other species, in particular in Xenopus, fish or though it may be relevant that PLP/DM20 transcripts chick (Fekete, 1996). It is, therefore, often difficult to can also be detected there (Lee, unpublished data), and make specific stage comparisons, particularly when the that activation of a PLP/DM20-lacZ transgene can be final ear structures vary quite significantly between detected in mouse notochord (Spassky et al., 1998). different species. Nevertheless, if we take somite stage Coexpression of P0 and PLP proteins has a long evolu- as a reasonable reference point, bearing in mind that tionary history, both being found together in myelin of somite generation occurs later in the rat than the chick the earliest vertebrates. The disappearance of P0 from (Brown, 1990), some valid comparisons can be made. CNS myelin occurred relatively late in evolution, so The earliest cells to delaminate from the otic vesicle are perhaps the presence of both of these proteins in the the future sensory neurons of the VIIIth ganglion (re- inner ear and notochord should not surprise us (Yosh- viewed in Legan and Richardson, 1997; Torres and ida and Colman, 1996).

Giraldez, 1998). It is possible that the cells that de- Our results on the expression of P0 mRNA in migrat- laminate from the otic vesicle could contain committed ing neural crest cells in rat extend our previous obser- glial precursors although all available evidence sug- vations and comparable results of others in migrating gests that, with the exception of the olfactory placode, crest in chick, where both mRNA and protein were glial cells associated with placode-derived neurons are detectable (Bhattacharyya et al., 1991; Lee et al., 1997; derived from the neural crest (Chuah and Au, 1991; Zhang et al., 1995). A comprehensive evaluation of P0 D’Amico and Noden, 1983). Furthermore, at later mRNA expression at several different developmental stages, the persistence of P0-positive cells within the ages, including both the head and gut regions, revealed otic vesicle and developing inner ear subsequent to this that P0 mRNA was first detectable at E10, with an delamination argues against a glial fate for P0-positive extremely restricted expression: it was seen only in P0 IN RAT PERIPHERAL GLIAL LINEAGE 47 some cells of the cephalic crest and, as discussed above, crest migration taken by developing melanocytes, nor some cells in the otic placode/pit. In migrating crest was P0 protein seen in melanocyte cultures (not shown) cells in the trunk region, P0 was seen in the ventrome- and P0 mRNA was not detected in areas of the devel- dial pathway only in cells that had migrated about oping jaw that contain neural crest cells that give rise halfway down the dorsoventral axis of the neural tube. to non-neural lineage cells. Importantly, TUJ1-positive In cells in the most dorsal area of this pathway, erbB3 neuroblasts were detectable prior to the appearance of expression was seen, but P0-positive cells were not P0-positive cells in tissue sections taken from the re- seen. This is consistent with the reported expression of gion of the oesophagus (Fig. 5). This is hard to reconcile erbB3 in the mouse (Meyer et al., 1997). In the present with the idea that TUJ1-positive neurons develop from experiments, glial cells associated with the ventral root P0-positive precursors. In view of the general finding in exit zone were strongly positive for P0 mRNA. These the CNS and PNS that neuronal differentiation pre- cells may well be of mixed neural crest and spinal cord cedes glial differentiation (Mayer-Proschel et al., 1997; origin (Grim et al., 1992; Lunn et al., 1987), and strong Moody et al., 1987), this sequence of in vivo events, i.e.,

P0 expression in the ventral root exit zone is consistent TUJ1 expression followed by the appearance of P0- with the report that dorsal root entry zone cells express positive cells, is however consistent with the notion

P0 protein at E14 (Golding and Cohen, 1997). P0 was that P0 is an early indicator of glial specification. It is also expressed by a subpopulation of migrating crest important to note that although initial entry of a cell to cells in the enteric nervous system at E12 and E13. In a lineage is indicative of the normal fate of that cell, it these cells, P0 was first seen a day after the appearance is not indicative of developmental commitment. On the of c-ret, which is a late crest cell marker and present on contrary, early steps in a lineage often remain revers- early neurons (Natarajan et al., 1999). In the oesoph- ible for some time, the cells retaining other develop- agus, P0 appeared a day after the early neuronal mental options that can be revealed if the cells are marker TUJ1 was detected. While P0 has not previ- challenged in appropriate ways (Edlund and Jessell, ously been reported in migrating enteric crest cells, 1999; Henion and Weston, 1997; Morrison et al., 1999; this pattern of expression is consistent with the view Sherman et al., 1993). The finding that some P0-posi- that P0 is activated as crest cells enter the glial lineage tive crest-derived cells can be induced to generate neu- of cells destined to become enteric glial cells. The signal rons by exposure to BMP-2 is consistent with this view is strongly down-regulated by E14 and no P0 was found (Hagedorn et al., 1999; Morrison et al., 1999; 2000). in the enteric glial cells of adult enteric ganglia, in Although we have argued that our results suggest that agreement with previous comments (Bhattacharyya et the P0 gene is activated in cells that are about to enter al., 1991; Zhang et al., 1995). Comparable down-regu- or have entered the glial lineage, the results of Yam- lation of basal P0 levels takes place in adult nonmyeli- auchi et al. (1999) might suggest an earlier activation nating Schwann cells and in satellite cells in the DRG of P0 in the neural crest. In this study, mice containing (Lamperth et al., 1989; Lee et al., 1997). In contrast, it a transgene where Cre recombinase expression was is interesting to note the persistence of basal P0 mRNA driven by a 1.1-kb fragment of the P0 promoter were expression in adult olfactory nerve ensheathing cells, crossed with indicator transgenic mice containing a cells that support continuous axonal regeneration floxed lacZ reporter gene, driven by the chicken actin throughout life and promote CNS axonal regeneration promoter. Expression of lacZ was associated with a (Gudino-Cabrera et al., 2000; Li et al., 1997; Ramon- larger subset of neural crest cells and their derivatives, Cueto et al., 1998). Another protein, Schwann cell my- including neurons, and ventral craniofacial mesen- elin protein, shows a rather similar pattern of regula- chyme in addition to the expected expression in periph- tion in chick although it is first expressed later than P0 eral glial cells and notochord. The question arises of (Cameron-Curry et al., 1993; Dulac and Le Douarin, how to reconcile the clearly different distribution of the 1991). lacZ reporter gene in the transgenic mice with that of

Most studies on glial development from the rat neu- the endogenous gene. The P0 sequences used to drive ral crest have used S-100 or glial fibrillary acidic pro- the transgene comprise a relatively small region of the tein (GFAP) immunoreactivity to monitor glial differ- P0 gene regulatory sequences and are unlikely to in- entiation (Morrison et al., 2000; Shah et al., 1994; clude all the regulatory sequences involved in control-

Smith-Thomas and Fawcett, 1989). S-100 marks a rel- ling the cell type specific expression of the P0 gene. atively late stage in embryonic glial development, i.e., Numerous examples exist of mismatch of expression the generation of Schwann cells from Schwann cell patterns between reporter genes driven by promoter precursors and GFAP appears at a similar time (Jessen constructs consisting of portions of the total gene reg- et al., 1994). These proteins, therefore, cannot be used ulatory sequences and reporter genes driven by more to mark early determination events in this lineage. A extensive segments of the relevant regulatory se- number of observations in the present paper indicates quences. In general, the constructs driven by shorter that during normal development of the nervous sys- sequences show less faithful cell type specific expres- tem, the P0 gene is activated in cells that are about to sion than constructs driven by longer sequences (e.g., enter, or have entered, the glial lineage. Thus, P0 for myelin basic protein, Farhadi et al., 1999; Landry et mRNA was not seen in the dorsolateral pathway of al., 1998). We, therefore, suggest that the expression 48 LEE ET AL. pattern described by Yamauchi et al. (1999) may reflect sion is, at least in some cases, accompanied by expres- the use of a relatively short portion of the P0 regulatory sion of protein, albeit at relatively low levels. sequences. Additionally, the use of the strong chicken actin promoter to drive the lacZ gene in the reporter EXPERIMENTAL PROCEDURES Materials mouse may result in detection of lacZ in these mice at extremely low levels of activation of the 1.1 P0 pro- Sources of materials used for in situ hybridization moter region. These levels, which are not detectable by and immunocytochemistry have been detailed in pre- in situ hybridization of the native gene, are unlikely to vious papers (Dong et al., 1995; Jessen et al., 1994; Lee be biologically relevant. et al., 1997; Morgan et al., 1994; Stewart et al., 1996). The P polyclonal antibody prepared in this laboratory Expression of P0 in olfactory ensheathing cells is in 0 agreement with previous results in the chick (Norgren was used as previously described (Lee et al., 1997) and et al., 1992). These cells are the only nonmyelinating the TUJ1 antibody recognising the ␤3 isoform of tubu- lin was a gift from Dr. A. Frankfurter (Moody et al., glia that continue to express basal P0 mRNA levels throughout life. Uniquely, these cells associate with 1987). axons that continuously regrow in the normal intact Probes Used for In Situ Hybridization nerve. This, together with the upregulation of P0 ex- pression in nonmyelinating Schwann cells in regener- These include: cDNA pmcret 7 encoding a 2.8-kb ating nerves, suggests the possibility that basal non- fragment of the c-ret gene subcloned into Bluescript (SK) (Schuchardt et al., 1994); cDNA encoding a 1.8-kb myelin related P0 protein expression has a role in regulating axonal growth (Lee et al., 1997). In support sequence of Serrate-1 (Jagged-1) subcloned into Rvin of this is the observation that in culture P expression pKs (Mitsiadis et al., 1997); cDNA encoding human 0 Ј on the cell surface, and outside the context of the my- erbB3 bp1250-3 UTR of the erbB3 gene subcloned into elin sheath, can promote axonal elongation (Schneider- Bluescript (Meyer et al., 1997); cDNA encoding 750-bp PstI-ApaI fragment of the Krox-20 gene subcloned into Schaulies et al., 1990). P0, however, does not appear to be essential for early development of either the PNS, Bluescript (KS) (Chavrier et al., 1990; Irving et al., 1996; Wilkinson et al., 1989); cDNA (SN63c) encoding notochord, or inner ear, since knockout of P does not 0 the P coding sequence subcloned into pGEM4 (Grif- result in development that is obviously aberrant (Calle 0 fiths et al., 1989; Lemke and Axel, 1985); cDNA encod- and Martini, unpublished observations). ing part of the PLP/DM20 coding sequence (Timsit et Expression of P protein in vivo is a precondition for 0 al., 1992); cDNA encoding bp 707–1,275 in the BMP-4 any function for nonmyelin related P expression 0 sequence, prepared by RT-PCR from a mouse E12.5 whether in migrating crest cells, in the Schwann cell heart and lung library and subcloned into PCR2 plas- lineage prior to myelination, in nonmyelinating mid using Original TA cloning kit and the manufactur- Schwann cells in regenerating nerves, or in ear devel- er’s instructions by Dr. E. Parmantier. opment. Although basal P0 levels can been detected in vitro (see below), in vivo, these cells have not been In Situ Hybridization found previously to bind P0 antibodies using conven- In most cases, this was performed on whole embryos tional methods. It is possible that this was due to the at E10–12 (plug day is E0) using a modification of the absence of protein and that P0 is only expressed at the method of Rex and Scotting (1994) using DIG-labelled mRNA level in these cells. Alternatively, the absence of riboprobes incubated overnight at 65°C, alkaline phos- P0 immunoreactivity could be due to technical reasons, phatase coupled antibodies to DIG and X-phosphate/ perhaps related to masking of antigenic epitopes or NBT to visualize the alkaline phosphatase. In experi- other factors. This possibility is supported by a number ments where in situ hybridization was combined with of observations. In E18 rat sciatic nerves, P0 protein is immunocytochemistry, after in situ hybridization em- present in Western blots although it is not seen in bryos were blocked in phosphate buffered saline con- tissue sections (Lee et al., 1997); P0 protein appears taining 0.1% Tween 20 (PBT) containing 10% foetal during maturation of neural crest cells in vitro (Hage- calf serum (FCS) for 1 hr and then incubated in TUJ1 dorn et al., 1999); Schwann cell precursors from E14 antibody (10 ␮g/ml) for 5 days, washed extensively nerves express membrane-associated P0 immunoreac- prior to overnight incubation at 4°C in goat anti-mouse tivity when examined 3 hr after dissociation from em- Ig peroxidase (1:100 in PBT/FCS) and visualization as bryonic nerves, even in the presence of cycloheximide previously described (Morgan et al., 1994). In all exper- to inhibit new protein synthesis (Lee et al., 1997); in iments, embryos were washed and post-fixed in 4% tissue sections P0 protein is detectable in chick neural paraformaldehyde for 20 min and either cleared in 90% crest cells and chick Schwann cells prior to myelina- glycerol/PBS or processed for cryosectioning in 15% tion; similarly it is seen at low levels in embryonic sucrose containing 7.5% gelatin. Ten to 20 ␮M serial mouse nerves prior to myelination (Jaegle and Meijer, sections were mounted on gelatin-coated glass slides.

1998) and it is seen in E12 mouse nerves by antibody To study P0 mRNA expression in the inner ear at E14, labelling of tissue sections (Lange et al., unpublished the head of an E14 embryo was horizontally sectioned data). All of this suggests that early P0 mRNA expres- after fixation in 4% paraformaldehyde for 4 hr, followed P0 IN RAT PERIPHERAL GLIAL LINEAGE 49 by incubation for several hours in 30% sucrose in PBS Brown NA. 1990. Routine assessment of morphology and growth: to cryoprotect the tissue. This was then mounted in scoring systems and measurements of size. In: Copp AJ, Cockcroft OCT compound, then 10–14 ␮M cryosections were cut, DL, editors. Postimplantation Mammalian Embryos. Oxford: IRL Press at OUP. followed by in situ hybridization, essentially as de- Burroni D, White FV, Ceccarini C, Matthieu JM, Costantino-Cecca- scribed above. For guts from E12-14 embryos, whole rini E. 1988. Expression of myelin components in mouse Schwann embryos were fixed in 4% paraformaldehyde for 4 hr, cells in culture. J Neurochem 50:331–336. dehydrated and rehydrated as for whole mount in situ. Cameron-Curry P, Dulac C, Le Douarin NM. 1993. Negative regula- Then the oesophagus, stomach, intestine, and colon tion of Schwann cell myelin protein gene expression by the dorsal were dissected out as a single structure, washed in root ganglionic microenvironment. Eur J Neurosci 5:594–604. Carenini S, Montag D, Schachner M, Martini R. 1999. Subtle roles of PBS, refixed in 4% paraformaldehyde for 4 hr, and neural cell adhesion molecule and myelin-associated glycoprotein subjected to whole mount in situ hybridization as de- during Schwann cell spiralling in P0 deficient mice. Glia 27:203– scribed. For adult gut, samples from various regions of 212. the gastrointestinal tract were dissected, cut open, and Chavrier P, Vesque C, Galliot B, Vigneron M, Dolle P, Duboule D, flat mounted prior to fixation with 4% paraformalde- Charnay P. 1990. The segment-specific gene Krox-20 encodes a transcription factor with binding sites in the promoter region of the hyde for 6 hr, followed by embedding in polyester wax, Hox-1.4 gene. EMBO J 9:1209–1218. sectioning, and in situ hybridization followed by immu- Cheng L, Mudge AW. 1996. Cultured Schwann cells constitutively nolabelling for GFAP or S-100. express the myelin protein P0. Neuron 16:309–319. DIG-labelled probes were transcribed using the Chuah MI, Au C. 1991. Olfactory Schwann cells are derived from Boehringer SP6/T7 transcription kit using the manu- precursor cells in the olfactory epithelium. J Neurosci Res 29:172– facturer’s instructions. P transcripts were hydrolyzed 180. 0 Colman DR, Pedraza L, Yoshida M. 2001. Concepts in myelin sheath to give an average probe length of 150 bases and the evolution. In: Jessen KR, Richardson WD, editors. Glial cell devel- probe was used at a dilution of 1:200. Other probes opment: basic principles and clinical relevance. 2nd ed. London: were used unhydrolyzed at a dilution of 1:200. Colour Oxford University Press. p 161–176. development of hybridized probes was carried out over D’Amico A, Noden DM. 1983. Contributions of placodal and neural crest cells to avian cranial ganglia. Am J Anat 166:445–468. a period of 1–4 days. Immunolabelling of P0 protein was performed as described previously for high sensi- De Jonghe P, Timmerman V, Cueterick C, Nelis E, De Vriendt E, Lofgren A, Vercruyssen A, Verellen C, Van Maldergem L, Martin tivity detection using either FITC-conjugated or ABC J-J, Van Broeckhoven C. 1999. The Thr124Met mutation in the Vectastain antibodies to visualize the signal (Lee et al., peripheral myelin protein zero (MPZ) gene is associated with a 1997). Immunolabelling for GFAP and S-100 was car- clinically distinct Charcot-Marie-Tooth phenotype. Brain 122:281– ried out as described previously (Brennan et al., 2000; 290. Jessen et al., 1994; Morgan et al., 1994). Dong Z, Brennan A, Liu N, Yarden Y, Lefkowitz G, Mirsky R, Jessen KR. 1995. Neu differentiation factor is a neuron-glia signal and regulates survival, proliferation and maturation of rat Schwann cell ACKNOWLEDGMENTS precursors. Neuron 15:585–596. We thank Dr. Bernard Zalc and Dr. Cecile Goujet- Doyle JP, Stempak JG, Cowin P, Colman DR, D’Urso D. 1995. Protein Zalc for the gift of the PLP/DM20 lacZ embryos and the zero, a nervous system adhesion molecule, triggers epithelia rever- PLP/DM20 probe. We thank Dr. V. Pachnis, Dr. D. sion in host carcinoma cells. J Cell Biol 131:465–482. Dulac C, Le Douarin NM. 1991. 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