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Expression of the ETS Transcription Factor ER81 in theDeveloping Chick and Mouse HindbrainYAN ZHU AND SARAH GUTHRIE*MRC Centre for Developmental Neurobiology, King’s College, Guy’s Campus, London, UK

ABSTRACT ER81 is an ETS domain-con-taining transcription factor, which is expressedin various developing tissues and organs of theembryo and in pools of developing spinal motorneurons and proprioceptive sensory neurons.Analysis of mice lacking ER81 function showedthat this gene played an important role in theestablishment of sensory-motor circuitry in thespinal cord. Here, we investigate the expressionpattern of er81 in the hindbrain of both chick andmouse embryos. We find that er81 is expressed ina subpopulation of inferior olive neurons, whichsend their projections to the caudal cerebellum.© 2002 Wiley-Liss, Inc.

Key words: ER81; hindbrain; inferior olive; cere-bellum; olivocerebellar projection

INTRODUCTION

Transcription factor er81 belongs to the PEA3 groupof ETS family genes (reviewed by de Launoit et al.,1997). During chick and mouse development, er81 andother members of PEA3 group are expressed by severalembryonic tissues and display combinatorial expres-sion in specific regions of various organs (Chotteau-LeLievre et al., 1997, 2001). At later stages of develop-ment, er81 is expressed in cortical layer V neurons,motor neuron pools, and subsets of muscle sensoryneurons in the spinal cord (Lin et al., 1998; Xu et al.,2000; Arber et al., 2000). Er81 mutant mice displaysevere motor discoordination, which results at least inpart from the failure of Ia proprioceptive afferents toconnect with motor neurons in the ventral spinal cord(Arber et al., 2000). An implication from this work isthat er81 is involved in establishing the integrity ofmotor-sensory neuronal circuits in the spinal cord. Toelucidate whether er81 also plays a role in the devel-oping brain, we investigated the expression of er81 inthe hindbrains of both chicks and mice.

RESULTS AND DISCUSSION

Whole-mount in situ hybridisation on brains fromembryonic day (E) 3–E12 chick embryos showed thater81 was first expressed at E7 in two crescent-shapeddomains, one on either side of the ventral midline in

the caudal hindbrain (Fig. 1A). This expression patternpersisted until E12, which was the latest stage tested(Fig. 1B,C; data not shown). In situ hybridisation ondeveloping mouse hindbrains showed a closely similarexpression pattern, which was first detected at E13.5,and persisted until E17.5—the latest stage tested (Fig.1D,E; data not shown). Transverse sections of er81 insitu hybridised E9 chick hindbrains showed that er81-expressing cells were packed close to the pial surface ofthe neuroepithelium (Fig. 2A). Both the rostrocaudaland dorsoventral position of these er81-expressing cellscorrelated with the location of the inferior olive (IO)neurons at the same stages, suggesting that er81 wasexpressed in IO neurons (Chedotal et al., 1996). Toconfirm these findings, we used antibody to calbindin-D28K, a calcium-binding protein that was expressed inIO neurons at these embryonic stages (Chedotal et al.,1996). Immunohistochemistry, in both chick andmouse, by using anti-ER81 and anti-calbindin antibod-ies on adjacent sections of the caudal hindbrain showedthat the ER81-positive domain overlapped largely withthe calbindin-positive domain (Fig. 2B,C), confirmingthat er81 was indeed expressed in the IO nucleus.However, it must be noted that we cannot exclude thepossibility that some ER81-positive cells were calbin-din-negative.

The synaptic targets of the IO projection are thePurkinje cells of the cerebellum. We found that er81was expressed in the caudal region of the cerebellarprimordium in both the chick and the mouse (Fig. 3). Inthe chick, this expression was first detected at E7 inthe caudal margin of the cerebellar plate (Fig. 3A),where the expression domain corresponded with theventricular zone of the developing cerebellum (Fig. 3B).At E9 and E11, the er81 expression domain was locatedin the caudal lobule VIII and part of lobule VII as two

Grant sponsor: Wellcome Trust.*Correspondence to: Sarah Guthrie, MRC Centre for Developmen-

tal Neurobiology, 4th Floor New Hunt’s House, King’s College, Guy’sCampus, London SE1 1UL, United Kingdom.E-mail: sarah.guthrie@kcl.ac.uk

Received 19 November 2001; Accepted 26 August 2002DOI 10.1002/dvdy.10166Published online 11 October 2002 in Wiley InterScience (www.

interscience.wiley.com).

DEVELOPMENTAL DYNAMICS 225:365–368 (2002)

© 2002 WILEY-LISS, INC.

pairs of parasagittal stripes (Fig. 3C). Transverse sec-tion of this region showed that the er81 stripes span thePurkinje cell layer (Fig. 3D). In the E13.5 mouse cere-bellum, er81 was strongly expressed in the caudal re-gion (Fig. 3E), within differentiation zones, which aredestined to give rise to deep cerebellar neurons andPurkinje cells (Fig. 3F; Altman and Bayer, 1997).

We observed that the er81 expression domain inchick did not reflect the shape of the entire IO nucleus(Chedotal et al., 1997) and, therefore, sought to furtherdefine which IO neurons express er81. IO neurons sendcontralateral projections to the cerebellar primordiumat around E8.5 in chick and E15 in mouse (Chedotal etal., 1996; Bloch-Gallego et al., 1999). In chick, the early

Fig. 1. Whole-mount in situ hybridisation for er81 expression in thehindbrains of chick and mouse embryos at various developmental stages,viewed from the ventral (pial) side of the hindbrains. A–C: In situ hybridisa-tion with anti-sense chick er81 probe (including the entire coding sequence)on hindbrains of embryonic day (E) 7, E9, and E11 chick embryos. er81 isexpressed in the caudal hindbrain in two crescent-shaped domains close tothe midline (white arrows). D,E: In situ hybridisation with anti-sense mouseer81 probe (including the entire coding sequence) on hindbrains of E13.5and E17.5 mouse embryos. Er81 is expressed in the caudal hindbrain in twolongitudinal domains close to the midline (black arrows). This domain is widerin the middle and narrower at the ends. The orientation for all panels in this figureis rostral up, caudal down. HB, hindbrain. Scale bars � 1 mm in A–E.

Fig. 2. Er81 expression in inferior olive (IO) neurons. A: Transversesections of the caudal region of a chick embryonic day (E) 9 hindbrain thathas been in situ hybridised with er81 probe. Selected sections represent therostral (A1), middle (A2), and caudal (A3) level of the er81 expressiondomain. Er81 was expressed in cells that were located close to the ventral(pial) surface, on either side of the midline. B: ER81 (B1) and calbindin (B2)immunostaining on adjacent sections of the caudal region of a chick E9hindbrain, showing an overlap of the ER81-positive domain with the calbi-ndin-positive domain. The region of sections shown in (B1) and (B2) isapproximately similar to the boxed region in (A1) but slightly more caudal.C: ER81 (C1) and calbindin (C2) immunostaining on adjacent sections takenfrom the caudal region of a mouse E17.5 hindbrain also showed colocalisa-tion of these two proteins. The orientation for all panels in this figure is dorsalup, ventral down. Scale bars � 1 mm in A–C.

Fig. 3. In situ hybridisation for er81 in the cerebellar primordia of chickand mouse embryos. A,C,E: In situ hybridisation with anti-sense er81probe on whole-mount embryonic day (E) 7 and E11 chick cerebella andE13.5 mouse cerebellum, respectively. B,D,F: The transverse sectionsacross the er81-positive cerebellar region corresponding to A, C, E,respectively. In both chick and mouse, er81 was expressed in the caudalregion of the cerebella (indicated by black arrows). A: In chick, at E7, er81was expressed in a very thin horizontal stripe at the caudal edge of thecerebellar plate, and transverse sections showed this expression domainto be in the ventricular zone (black arrow in B). C: At E11, er81 wasexpressed in two pairs of parasagittal stripes on each side of the midline.The more medial pair of stripes is located in lobule VIII, and the morelateral pair extends into lobule VII. Transverse section of this regionshowed that the location of these stripes is typical of parasagittal Purkinjecell stripes (arrowhead and arrow in D). In mouse at E13.5, er81 wasstrongly expressed in the caudal cerebellum (E); in transverse sections,this expression was seen to correspond with the differentiation zones(arrow in F). The orientation for A, C, and E is rostral up, caudal down; forB, D, and F the orientation is dorsal up, ventral down. FP, floor plate; HB,hindbrain; CEP, cerebellar primordium; VZ, ventricular zone; DZ, differ-entiation zone. Scale bars � 1 mm in A–F.

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topography of this projection can be demonstrated byretrograde labelling of IO axons from the cerebellarplates of organotypically cultured hindbrains after 1–3days in vitro (Chedotal et al., 1997; Fig. 4A). Injectionof the fluorescent tracers DiI and DiO into the rostraland caudal cerebellum differentially labels two IO neu-ronal subpopulations (Fig. 4B–E). In the chick, a ros-tromedial and a caudolateral subpopulation of IO neu-rons project to the caudal and rostral cerebellar plate,respectively (Fig. 4B,C). In the mouse, there is aslightly different arrangement: the caudal and rostralcerebellar plates receive projections from the rostraland caudal half of the IO nucleus, respectively (Fig.4D,E). To investigate er81 expression in relation to thistopography, IO subnuclei were labelled retrogradelyand the same hindbrains were subsequently subjectedto er81 in situ hybridisation. The fluorescent image ofeach retrogradely labelled explant was aligned with

the image of er81 in situ hybridisation in the sameexplant, by using the caudal edge of the cerebellarplate as the reference point for rostrocaudal positioning(Fig. 5A–D). The rostrocaudal and mediolateral posi-tion of the er81 expression domain was then comparedwith the position of the retrogradely labelled IO sub-nuclei. This revealed that in the chick, er81 was ex-pressed in the rostromedial subpopulation of IO neu-rons (green label in Fig. 5A; compare with in situ

Fig. 5. Er81 is expressed in the subpopulation of inferior olive (IO)nucleus that projects to the caudal cerebellum. Organotypically culturedembryonic day (E) 7.5 chicken hindbrain (A) and E14.5 mouse hindbrain(C) were retrogradely labelled from the cerebellum. A: The tracers DiOand DiI were injected into the caudal and rostral regions of the leftcerebellar plate, resulting in contralateral labelling of the rostromedial(green) and the caudolateral (yellow) IO, respectively. C: DiI was injectedinto the rostral cerebellar plate on the left and the caudal cerebellar plateon the right, resulting in the labelling of the rostral IO on the left side (red)and the caudal IO on the ride side (red). B,D: After obtaining an image ofthe retrogradely labelled sample, the same explant in each case was thensubjected to in situ hybridisation with anti-sense er81 probe. The resultinglabelling patterns were compared side by side as shown in A vs. B forchick, and C vs. D for mice. The rostrocaudal coordinates of the twoimages were aligned by using caudal limit of the right cerebellar plate asa reference point (indicated by the top blue line). The bottom blue lineindicates the rostral limit of the retrogradely labelled rostromedial orrostral IO nucleus in each case. The er81-positive region correlates withthe rostromedial IO nucleus in chick and the rostral IO nucleus in mouse,although in the latter, there is weak expression of er81 the most medialportion of the caudal IO. E: A transverse section of a mouse hindbrainexplant that was retrogradely labelled from the caudal cerebellum on bothsides to label the rostral IO nucleus on both sides. F: A transverse sectionfrom the same axial level of a mouse hindbrain that has been in situhybridised with er81 anti-sense probe. Comparison of E and F shows thatthe dorsoventral position of er81 expression domain also correlates withthat of the rostral subpopulation of the IO nucleus. The white vertical barin A, C, E indicates the midline. The orientation for A–D is rostral up,caudal down. The orientation for E and F is dorsal up, ventral down. Scalebars � 1 mm in A–D, 0.5 mm in E,F.

Fig. 4. Retrograde labelling of subpopulations of inferior olive (IO)neurons from rostral and caudal cerebellum in chick and mouse.A: Schematics showing the procedures for the organotypic culture ofchick and mouse hindbrains and the DiI/DiO retrograde labelling from thecerebellar plates (adapted from Chedotal et al., 1997). DiI and DiOinjection sites are indicated by red and green dots, respectively. Embry-onic day (E) 7.5 chick (B,C) or E14.5 mouse (D,E) hindbrains wereorganotypically cultured for 3 days in vitro before DiI/DiO retrogradelabelling. C and E are higher magnification of the retrogradely labelled IOregion corresponding to B and D, respectively. An inverted topography ofolivocerebellar projection was already established at this stage of devel-opment. In chick, the rostromedial IO has been retrogradely labelled fromthe caudal cerebellar plate and the caudolateral IO from the rostralcerebellar plate (B,C). In mouse, the rostral IO has been retrogradelylabelled from the caudal cerebellar plate and the caudal IO from therostral cerebellar plate (D,E). Orientation of all panels is rostral up, caudaldown. FP, floor plate; CE, cerebellar plates; HB, hindbrain; SC, spinalcord; r, rostral; c, caudal. Scale bars � 1 mm in B–E.

367ER81 EXPRESSION IN THE DEVELOPING INFERIOR OLIVE

hybridisation in Fig. 5B, n � 6), whereas in the mouse,high er81 expression correlated with the rostral half ofthe IO nucleus (red label in the left side of the hind-brain in Fig. 5C; compare with in situ hybridisation inFig. 5D, n � 4). In general, therefore, in both chick andmouse, er81 is expressed in the subpopulation of IOneurons that project to the caudal cerebellum. Thater81 was expressed in the caudal region of the cerebel-lum at these embryonic stages (Fig. 3A–F), perhapssuggests the involvement of er81 in the establishmentand/or maintenance of connectivity between er81-posi-tive IO neurons and the Purkinje cells in the caudalcerebellum. It remains possible that er81-expressingIO neurons do not project exclusively to er81-express-ing synaptic targets in the cerebellum; for example thezones expressing er81 in the caudal cerebellum may bea subset of the target region of rostral/rostromedial IOneurons. This finding would parallel the situation inthe spinal cord, where of the motor neurons that aresynaptic targets of er81-expressing proprioceptive af-ferent neurons, only a subset express er81 (Lin et al.,1998).

We also analysed expression patterns of pea3, an-other ETS transcription factor, in the developing chickhindbrains at similar stages to those reported here.Pea3 together with er81 was shown to define subsets ofspinal motor pools and sensory afferent neurons duringdevelopment (Lin et al., 1998; Arber et al., 2000). How-ever, we did not detect any expression of pea3 in the IOat these stages, suggesting that it does not play acoordinate role with er81 in IO development or axonprojection formation (data not shown). It remains to beseen whether other ETS family genes might be ex-pressed in the developing IO in a manner complemen-tary to the er81 expression domain.

Our findings of er81 expression in both the IO neu-rons that project to the caudal cerebellar plate and inthe caudal cerebellum, in chick and mouse, suggeststhat er81 might play a role in the establishment and/orthe maintenance of the topography of the olivocerebel-lar projection. Our preliminary analysis of er81 mutantmice harbouring a tau-lacZ transgene in the er81 locus(Arber et al., 2000) showed that the initial axonal pro-jection from er81-expressing IO neurons to the cerebel-lum appeared normal (Y. Zhu, S. Arber, and S. Guthrie,data not shown). However, the precise topography andthe maintenance of this projection in these animalsawaits further investigation. Recently, it was shownthat er81 and pea3 might be involved in maintainingthe differential expression of members of type II cad-herins in the spinal motor pools (Price et al., 2002). Thesame study suggested that the expression of type IIcadherins in motor pools plays a role in motor poolsegregation. Therefore, it is possible that er81 in IOneurons control the expression of cadherins or otheradhesion molecules on the rostral or rostromedial IOneurons, which in turn regulates the topographic pro-jection of IO and/or the segregation of IO subpopula-tions.

EXPERIMENTAL PROCEDURESIn Situ Hybridisation

In situ hybridisation was performed on brains dis-sected from chick and mouse embryos as previouslydescribed (Varela-Echavarrıa et al., 1996).

Immunohistochemistry

Ten-micrometer adjacent cryosections were used forfluorescent immunohistochemistry as previously de-scribed (Varela-Echavarrıa et al., 1996). Monoclonalchick and mouse anti-ER81 antibody (kind gifts from S.Arber and T.M. Jessell) were used at 1:50 and 1:200dilution, respectively. Polyclonal anti-calbindin anti-body (Swant) was used at 1:1,000.

Organotypic Culture and Retrograde Labelling

The procedure for organotypic culture and retro-grade labelling was as described (Chedotal et al. 1997),except that, when culturing mouse hindbrains, we usedmedium composed of neurobasal medium supple-mented with 1% glutamax, 5% foetal calf serum, and1% B27 supplement (Gibco).

ACKNOWLEDGMENTSWe thank S. Arber and T.M. Jessell for kind gifts of

chick and mouse ER81 plasmids and antibodies.

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