distribution of neuregulin-1 (nrg1) and erbb4 transcripts in embryonic chick hindbrain

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MCNMolecular and Cellular Neuroscience 13, 237–258 (1999)

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istribution of Neuregulin-1 (nrg1) and erbB4ranscripts in Embryonic Chick Hindbrain

onica Dixon and Andrew Lumsdenepartment of Developmental Neurobiology, King’s College London, London, United Kingdom

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nterrhombomeric signalling has a role in development ofhe chick hindbrain. We have examined the transcriptistributions of neuregulin-1 (nrg1), a signalling moleculeith effects on nervous system development, and of onef its receptors, erbB4. Expression patterns of these twoolecules are, in contrast to the situation in mouse, not

onsistent with a role in interrhombomeric signalling, butre consistent with a role in signalling between dorsal andentral territories within individual rhombomeres. Otherreas of potential nrg1-erbB4 signalling are the cerebel-

um, the caudal rhombic lip, and dorsolateral mantle in theaudal hindbrain. nrg1 is additionally expressed in motoreurons and erbB4 in rostral rhombic lip plus a subset ofhombic lip derivatives. Both molecules are also ex-ressed in other parts of the nervous system and inonneuronal tissue. The expression data presented will

acilitate interpretation of future functional studies onrg1-erbB4 signalling during hindbrain development.

NTRODUCTION

We are interested in signalling within the embryonicindbrain and how this signalling regulates hindbrainevelopment. For instance, there is evidence for thexistence in chick of a signal produced by the even-umbered rhombomeres (r) r2, r4, r6 and received by

he odd-numbered rhombomeres r3, r5. This even todd signal controls neural crest apoptosis and genexpression in the receiving rhombomeres (Graham et al.,993, 1994; Graham and Lumsden, 1996). One family ofignalling molecules that has, along with its receptors inhe epidermal growth factor (EGF) receptor family, beenmplicated in a wide variety of functions in the cells ofhe developing nervous system by both in vivo and initro approaches comprises the many nrg1 isoformsreviewed by Lemke, 1996). These proteins and thencoding cDNAs were identified independently on the
asis of biological activity or of homology to previously
solated cDNAs and are thought to be encoded by aTl

044-7431/99 $30.00opyright r 1999 by Academic Pressll rights of reproduction in any form reserved.

ingle gene now referred to as nrg1. All the isoformsnclude an EGF-like domain, which can by itself activateyrosine phosphorylation of both receptors and theoreceptor (see below), but vary in which other domainsre present. There are soluble and cell-surface isoforms;he latter can be transmembrane or bound to thextracellular matrix via an immunoglobulin-like (Ig-ike) domain (Lemke, 1996; Meier et al., 1998; Rimer etl., 1998). Of the three types of nrg1 isoforms (Meyer etl., 1997), types I and II include Ig-like domains, whileype III does not; presumably the extracellular isoformsf type III nrg1 are diffusible. However, a membrane-ound isoform as well as a soluble one of type III nrg1xists at least in cultured cells (Schroering and Carey,998). Functions attributed to the various isoformsnclude survival and mitogenesis of Schwann cells andheir precursors, stimulation of acetylcholine receptorynthesis by subsynaptic muscle nuclei, the elongationf radial glia in the cerebellum and cortex that accompa-ies neuronal migration, enhancement of the rates ofigration of neurons on glia and of glia on neurons,

hoice of glial fate by neural crest stem cells, mitogen-sis of astrocytes, and effects on differentiation of oligo-endrocytes (Lemke, 1996; Anton et al., 1997; Nakao etl., 1997; Raabe et al., 1997; Rio et al., 1997). The effects ofrg1 are mediated by its two direct receptors, erbB3 andrbB4, and their common dimerization partner, erbB2.ignal transduction via erbB3 is thought to requireeceptor heterodimerization (often with erbB2) becauserbB3 has a defective tyrosine kinase domain, but erbB4an presumably act as a homodimer as well. The nrg1roteins are also expressed outside the nervous system,ften in the mesenchymal component of developingrgans such as the lung or mammary gland, where theyay be involved in the mesenchymal–epithelial interac-

ions necessary for the development of those organs.
he epithelial component of the mammary gland at
east expresses both of the nrg1 receptors (Yang et al., 1995).

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238 Dixon and Lumsden

Mouse knock-outs of nrg1, erbB2, erbB3, and erbB4Gassmann et al., 1995; Lee et al., 1995; Meyer andirchmeier, 1995; Kramer et al., 1996; Erickson et al.,997; Riethmacher et al., 1997; Britsch et al., 1998; Liu etl., 1998) have been reported, and their phenotypes givedditional information on the function of these sig-alling molecules during development. All four typesf mutant mouse display abnormalities of the nervousystem. nrg1, erbB2, and erbB3 homozygous mutantsll have undersized cranial ganglia and the connec-ion between the trigeminal (V) ganglion and the hind-rain is missing (nrg12/2, erbB22/2) or rudimentaryerbB32/2). nrg1 and erbB2 mutants additionally have a

uch reduced connection between the faciovestibulo-coustic (VII/VIII) ganglia and the hindbrain. Cells inhe cranial ganglia express these three molecules, andhey are thought to be required for survival of the neuralrest-derived component of these ganglia. The erbB4utant phenotype differs from the others in that cranial

anglia are apparently normal in size and all connectedo the hindbrain. However, each of the V and VII/VIIIanglia is connected both to r2 and r4 rather than the Vanglion only to r2 and the VII/VIII ganglia only to r4 as

n wild-type mice. For V and VII/VIII sensory neurons,s well as for V motor neurons, comparable numbers areorrectly and aberrantly connected, while VII motorxon connections are as in the wild-type. This pheno-ype implicates erbB4 in axonal pathfinding of trigemi-al and faciovestibuloacoustic neurons. Because erbB4 isxpressed in r3 and r5 of the embryonic hindbrain but ineither r2 and r4 nor in the misconnected ganglia, a

‘barrier’’ model was proposed to explain its mechanismf action (Gassmann et al., 1995; Lemke, 1996; Gassmannnd Lemke, 1997). This model states that erbB4 expres-ion is necessary for a barrier to axon growth to exist in3, r5, and the immediately adjacent paraxial mesen-hyme; when this barrier is absent, V and VII/VIIIanglionic axons are free to grow through the territorydjacent to r3 to use the entry point on the other side.ased on the fact that the erbB4 and nrg1 mutanthenotypes differ, it has been suggested that nrg1 mayot be the ligand for erbB4 in the hindbrain, even

hough nrg1 is expressed in r2, r4, and r6, and erbB4 inhe adjacent rhombomeres, r3 and r5. Several moleculesther than nrg1 have been shown to be capable ofctivating erbB4 in transfected cell lines, although embry-nic hindbrain expression at the relevant stage has noteen noted for any of them. They include nrg2 (Carr-way et al., 1997; Chang et al., 1997), also called diver-ent of neuregulin 1 (don1; Busfield et al., 1997), nrg3Zhang et al., 1997), betacellulin (btc; Riese et al., 1996a,
), heparin-binding-egf-like growth factor (hb-egf; Ele-ius et al., 1997b), epiregulin (Komurasaki et al., 1997)
da

nd neural-and-thymus-derived activator for ErbB ki-ases (NTAK; Higashiyama et al., 1997). All of theseolecules are, like nrg1, EGF family members. Another

xplanation for the difference in nrg1 and erbB4 knock-ut phenotypes has been put forward by Gassmann andemke (1997): it is possible that nrg1 is indeed the ligand

or erbB4 but that the nrg1 knock-out phenotype ofissing cranial connections to the hindbrain due to lack

f activation of erbB3-erbB2 masks the phenotype ofberrant connections due to lack of activation of erbB4.hile the mouse nrg1 and erbB mutants yield informa-

ion about neural roles for these molecules, they alsollustrate convincingly that they have nonneural roles as

ell. nrg1, erbB2, and erbB4 mutants die between embry-nic day (E) 10 and E11, apparently due to failure ofrabeculation in the ventricle of the heart, while mostrbB3 mutants die at E13.5, possibly due to abnormaleart valve formation and/or to reduced catecholamine

evels. Unfortunately, this means that only in erbB3utants, particularly those that survive to birth, has it

een possible to study later roles in neural developmentErickson et al., 1997; Rietmacher et al., 1997; Britsch etl., 1998).

Of the four genes, only nrg1 and erbB4 have beeneported to be expressed in the hindbrain prior to theime when the mutants die, and they must thus be the

ediators of any intrahindbrain signalling that occursia the nrg1/erbB system in the mouse at or before10–E11. Intriguingly, nrg1 in mouse is expressed in

hose rhombomeres found in the stage 10 chick to beignalling neural crest apoptosis in the hindbrain (r2, r4,6), while erbB4 in mouse is expressed in the rhombo-eres that receive the neural crest apoptosis signal in

hick (r3, r5). We thus considered it important toxamine whether the nrg1 and erbB4 expression patternsn chick were consistent with these molecules mediatinghe signalling previously demonstrated by us (Grahamt al., 1993, 1994; Graham and Lumsden, 1996). The worke present here shows that in chick, nrg1 and erbB4 are

ever expressed in a manner suggesting mediation ofven to odd rhombomeric signalling. However, the nrg1nd erbB4 expression patterns are consistent with an-ther type of signalling in the hindbrain, namely be-ween dorsal and ventral territories within individualhombomeres. There are additional sites of potentialrg1–erbB4 signalling during later chick hindbrain de-elopment. These are described in this paper, as are theultiple regions within the hindbrain where transcripts

re expressed. Extra-hindbrain expression domains areresented briefly. Particularly for erbB4, the work pre-ented here expands previously available expression
ata for the embryonic hindbrain and our expressionnalysis lays the groundwork for a functional analysis

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nrg1 and erbB4 in Embryonic Chick Hindbrain 239

f erbB4 receptor signalling during chick hindbrainevelopment. It will be interesting to see how theifferences in nrg1 and erbB4 expression patterns be-

ween mouse and chick relate to differences in functionuring development, and whether the differences inxpression may reflect heretofore unknown differencesn the way hindbrain development proceeds in the twopecies.

ESULTS

loning of Chick erbB4 cDNA

cDNA synthesized from an anchored oligo-dT primernd total RNA from stage 10 and 11 hindbrains wassed as template in a polymerase chain reaction (PCR)ith semi-nested primers kindly provided by Anthonyraham. The primers correspond to segments of the

yrosine kinase domain of the human erbB family ofeceptor tyrosine kinases. Sequencing the cloned PCRroduct revealed the presence of a single sequence (Fig.A). Its predicted amino acid sequence matches theuman erbB4 sequence (Plowman et al., 1993) at 69/69ositions and differs from human erbB1, erbB2 andrbB3 (Ullrich et al., 1984; Coussens et al., 1985; Kraus etl., 1989; Plowman et al., 1990) at 13, 8, and 13 positions,espectively, identifying the chick PCR product as erbB4.

The cloned chick erbB4 PCR product was used tocreen a stage 12–15 whole chick embryo cDNA libraryenerously provided by Angelo Nieto and David Wilkin-on. The sequence of the longest erbB4 cDNA obtained ishown in Fig. 1B. The predicted amino acid sequenceatches the human erbB4 sequence at 1064 of 1137 or

3% of the positions with only 18 gaps, as compared to8, 51, and 51% match to human erbB1, 2, and 3equences with 95, 68, and 98 gaps, respectively. ErbB4equences from other species found in the databases areot full length, but our chick sequence is 98% identicalver 146 amino acids and 85% over a distinct 68 aminocids of reported mouse erbB4, and 81% identical over a68 amino acid stretch of reported rat sequence. OurDNA starts with codon 153 of the human erbB4equence and includes 1.2 kb of 38 untranslated region.t encodes isoform JM-a of Elenius et al. (1997a), whichiffers from isoform JM-b in the juxtamembrane region.

rbB4 Transcript Localization during Earlyindbrain Development

Ventricular layer transcripts. erbB4 transcripts are
etected in chick hindbrain as early as stage 102 in r1
Fig. 2A, for stage 112). By stage 12, additional expres-ar

ion is visible in the isthmus, r3, r5, and caudal rhombo-eres, r7/8 (Fig. 2A, for stage 112; Fig. 2B and 2C, for

tage 13). By stage 13, r2 is also staining faintly (Fig. 2C)nd by stage 16, r4 and r6 expression has become visibles well (Fig. 2D). At stage 16, transcripts are expressedtrongly in the isthmus and rostral r1, moderately in r3nd r5, somewhat less in r2 and r7/8, and weakly in r4,6, and caudal r1. The transcripts in the isthmus andostral r1 cover the ventral two-thirds of the neural tubexcept the floorplate and cells just next to it (Fig. 2E).taining in the more caudal hindbrain regions consti-utes a band at a dorsoventral position that is closer tohe floorplate than to the roofplate, just dorsal of theslet-1/2 (isl-1/2) positive developing motor neuronsFigs. 2C, 2F, 4A, 4C, and 4G). By stage 26, the stainingntensity has become more uniform along the rostrocau-al axis, there being only a more intense component

rom r2 to r8 and a somewhat less intense componenthroughout r1 (data not shown). This epithelial domainersists until at least E7 but is no longer detectable on9. It does become complicated in the isthmus andostral r1 from stage 24, in that it gets progressively

ore dorsal at progressively more rostral levels until inhe isthmus it flanks the dorsal midline. Two additionalpithelial domains appear in r1 and the isthmus startingt stage 24 and E6. The former is ventral, at the levelhere motor neurons develop in more caudal parts of

he hindbrain; its rostrocaudal level is the region fromhe trochlear (IV) motor nucleus to the V motor nucleusFig. 2G). The latter is located dorsally, near, but not at,he midline, and gets progressively less intense atrogressively more caudal levels; it becomes undetect-ble somewhat caudally of the IV motor nucleus (dataot shown).Rhombic lip transcripts. A site of erbB4 expression

hat is more prominent but appears later than thepithelial domain is the rhombic lip, that portion of theeural tube adjacent to the hindbrain roofplate. We firstee transcripts in the rhombic lip at stage 16 from r4 to r6nclusive (Fig. 2D). These transcripts are localized at theial side of the neural tube (Figs. 2F, 2H, 4C, and 4K).xpression expands rostrally and caudally until thentire rhombic lip looks erbB4-positive in wholemountsy stage 21. The transcripts are most concentrated in r4o r6 at first, but their distribution becomes moreniform by stage 24 (Figs. 2I and 5G). Even then, the r1hombic lip appears fainter than more caudal rhombicip. Transverse sections through r1 at stage 24 reveal thatn fact r1 rhombic lip may not stain. Rather, it is nearbypithelium that stains very faintly and the surface layerf the neural tube that stains more strongly in a domain
lmost but not quite extending to the dorsal midline/oofplate (Fig. 2G). This domain is less intense and also

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roader than the more caudal rhombic lip domain.hus, it is not surprising that in wholemounts it appears

hat the rhombic lip in r1 and a swath of adjacent tissuere erbB4-positive, but less intensely so than the r2 to r8hombic lip.

Medullary rhombic lip. Migration of cells from thehombic lip of chick has been studied by Harkmark1954) for medullary levels and more recently by Rydernd Cepko (1994) for cerebellar levels; we will considerhe migrations from the two levels separately. Cells fromhe medullary rhombic lip accumulate in the adjacent

antle from about E4.5 and subsequently migrate toheir targets over a period lasting from E5 to at least9.5.The migration starts at the level of the vagal (X) nerve

xit point but later is found also in the rest of theedulla. Some of the cells migrate through the margin

r superficial mantle to contribute to the pontine, raphe,nd inferior olivary nuclei, while others migrate shorteristances through deeper mantle tissue to contribute toray matter of the dorsolateral mantle. Still othersifferentiate in situ to form the cochlear nuclei (Hark-ark, 1954).We observe erbB4 transcripts not just in the medullary

hombic lip but also in a subset of its derivatives,amely a subpopulation of cells migrating superficially,

he medial pontine nucleus, and possibly a cochlearucleus. We also see staining in patches of dorsolateralantle at caudal levels; these cells may or may not

riginate from the rhombic lip.Starting at E6 and continuing at E7, we see erbB4-

ositive cells in the superficial mantle and/or margin atostrocaudal levels from the abducens (VI) and VII tohe rostral hypoglossal (XII) motor nuclei. These areresumably migrating rhombic lip derived cells de-cribed as s-strand by Harkmark (1954). At E6, theseells are still quite close to the rhombic lip, extending nourther ventral than the VII/VIII exit point rostrally orhe ventrolateral X motor nucleus caudally (Fig. 2J).

owever, by E7 they occupy much more ventral regions

IG. 1. Sequence of chick erbB4. (A) Comparison of predicted aminomino acid sequences from the same region of the four known human et al. (1993). Dashes represent amino acids identical to the one in theosition of the first residue shown within our partial chick sequenceegree of identity between each human sequence and the chick seqequences of chick erbB4 cDNA. Numbering at right refers to positioosition, and italicized type for amino acid position. Position 1 in the cequence presented in Plowman et al. (1993); thus, approximately 456
verline marks the transmembrane domain and the tyrosine kinase domainere used for subcloning to generate templates for in situ probes. Arrowe
ragment of chick erbB4. The nucleotide sequence presented has been submi

f the margin, some even being located at the ventralidline (Fig. 2K). Cells at the midline occur in the region

etween the VI, VII and rostral accessory VI motoruclei rostrally and the glossopharyngeal/vagal (IX/X)nd rostral XII motor nuclei caudally. At E9, theserbB4-positive cells are still found ventrally but noonger at the midline (data not shown). Their caudalxtent has become reduced; they now reach only to theostral IX/X motor nuclei. Of the nuclei to whicharkmark’s (1954) s-strand cells contribute, we believe

he medial pontine nucleus but not the raphe or inferiorlivary nuclei to contain erbB4 transcripts. We observeranscripts appropriately positioned to correspond tohe medial pontine nucleus at E7 (Fig. 2L) and at E9.

At stage 21 (about E3.5), we begin to see erbB4ranscripts in the dorsolateral mantle of the caudalindbrain, initially in a longitudinal strip just medial to

he rhombic lip in the r6 to r8 area, and later (stages 24,6) in a few patches at rostrocaudal levels extendingrom the rostral VI motor nucleus to the rostral XII

otor nucleus (Figs. 2H and 2I). At E7, there are stillatches at similar rostrocaudal and dorsoventral posi-

ions. These patches are appropriately located to corre-pond to descending trigeminal and vestibular nuclei,hich are not regarded as rhombic lip derivatives (Fig.

M). However, it is not clear whether these erbB4-ositive cells are derived from those present earlier

stages 24, 26) or whether the earlier domains arise fromhe rhombic lip.

As for the final derivative of the medullary rhombicip, the cochlear nuclei, we detect erbB4 transcripts in

hat appears to be the lateral lemniscal nucleus at E7nd E9 (Fig. 2L). However, this assessment is basedolely on the position of the staining and this erbB4-ositive area could alternatively or additionally corre-pond to the principal sensory nucleus of the trigeminalystem.

Cerebellar rhombic lip. The cerebellar rhombic lipay not contain erbB4 transcripts we can detect, but

here is faint staining in the neuroepithelium close to the

equence of chick PCR product (top line in each group) with predictedamily members (lines 2 through 5 of each group) as given in Plowmank sequence. Numbers to the left of each sequence correspond to thethin each full-length human sequence. The table at the right lists thee over the region shown. (B) Nucleotide and predicted amino acidthin the partial cDNA presented, plain type numbers for nucleotideamino acid sequence corresponds to position 153 in the human erbB4eotides of chick coding sequence are predicted to be missing. Double

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hombic lip at stages 24 and 26 (Fig. 2G). It wouldppear from recent studies that the external germinalayer (EGL) is derived from epithelium near but notecessarily right at the caudal edge of the cerebellummediocaudal edge before cerebellar plate fusion at E8),.e., the rhombic lip (Ryder and Cepko, 1994). Cells fromhe ventricular zone (presumably the mediocaudal edgepecifically) appear in the EGL mainly in the periodrom E5 to E7 (Hanaway, 1967). These granule cellrecursors migrate from the caudal edge of the cerebel-

um in a rostral direction, starting in lateral regionsooner than in medial ones. By E7, the external granularells have started proliferating, and they continue to doo until E15. By E8 and lasting as late as E15, the EGLas a more superficial layer of mitotic cells and a deeper

ayer of postmitotic cells (Hanaway, 1967; Ryder andepko, 1994). After E15, the cells migrate inward in

arge numbers to form the internal granular layer (IGL;anaway, 1967).In addition to the faint neuroepithelial staining we see

or erbB4 transcripts at stages 24 and 26, there is morebvious staining in the dorsolateral mantle from stage0, extending ventrally of the ventricle by stage 22about E3.5; Fig. 2G), and at E6 an intense region oftaining appears on the surface of the cerebellum. Thisntense staining is restricted to caudolateral cerebellumt E6, but by E7 it covers much of the cerebellum (Fig.L) and by E9 the entire cerebellar surface is intenselyositive (Fig. 2N). At E7, the deeper mantle staining istill detected in the isthmus and rostral hindbraincaudal extent between IV and V motor nuclei) but notn caudal cerebellum. At E9, there are staining patches inhe deep mantle; presumably these are the deep nucleiFig. 2N). By E11, the intense superficial layer of stainingas moved slightly beneath the surface and in E15agittal sections, a more intense outer band separated bysmall gap from a less intense and parallel inner band isisible. The outer band is, as at E11, slightly below theurface (Fig. 2O).

Nonrhombic lip derivatives. Not all erbB4-positiveomains in hindbrains from embryos older than E5 are

ocated in a manner suggesting a rhombic lip origin.ased on their position with respect to morphological

andmarks or the isl-1/2-positive motor nuclei, weriefly outline possible identities of some of the do-ains present in E7 and E9 hindbrains below.erbB4-positive areas in the dorsolateral mantle at E7

nd E9 that are not thought to derive from the rhombicip include the previously mentioned area that is poten-ially the principal sensory trigeminal nucleus (and/or a
ateral lemniscal nucleus; Fig. 2L), the nucleus of theescending trigeminal tract, pars interpolaris (Fig. 2M) p
nd at E9 only, pars oralis (not shown). The caudalatches present at E7 and already mentioned includeestibular nuclei (Fig. 2M); at E9 nucleus tangentialisppears positive, as do the more rostral superior vestibu-ar nucleus and nucleus Deiters ventralis (not shown).

ore caudally located erbB4-positive areas present at E7nd E9 are nucleus solitarius and the external cuneateucleus (Figs. 2P, 2Q, and 2R). The latter is also promi-ent at E11 (not shown). At E7 there is mantle stainingetween the IX/X and XII motor nuclei that couldorrespond to the nucleus intermedius (Fig. 2R). At E9,e see erbB4 transcripts in nucleus interpeduncularis

Fig. 2N), nucleus semilunaris, and an area that isossibly the nucleus ventralis lemnisci thalami (nothown).

rg1 Transcript Localization during Early Hindbrainevelopment

We synthesized nrg1 in situ probes from two sub-lones we generated from a proacetylcholine-receptor-nducing-activity (proARIA, a nrg1 isoform) cDNAindly provided by Kenneth Rosen and Gerald Fisch-ach. Both probes include the EGF domain and wouldhus detect transcripts encoding all known nrg1 iso-orms.

Initial epithelial domain. We first detect nrg1 tran-cripts in chick hindbrain at stage 10, in r4 and r7. The r4omain soon expands rostrally across r3 and then r2,hile the r7 domain expands caudally into r8, with the

nlarged domains being present by stage 14 (Fig. 3A).he entirety of these domains persists until at least stage6 as bands in the neuroepithelium that are closer to theoofplate than to the floorplate (Figs. 3B, 3C, 4B, andH). By stage 19, the expression in r2 and r3 is gone,hile the r4 domain lasts until at least stage 23 and the

7/8 domain until about stage 22. Throughout theuration of this dorsal epithelial expression, it is r4 thattains most strongly.

isl-1/2-positive motor neurons. Besides the dorsalrg1 domains, by stage 15 a second site of expressionas appeared. The new nrg1 domain occurs in a bilateraltripe either side of the floorplate, extending from the1/r2 boundary all the way into caudal r8 (Fig. 3D for r2o r6 expression). At stage 18, a ventral patch either sidef the floorplate appears in rostral r1 as well. This is theV motor nucleus, judging from its position and itssl-1/2 immunoreactivity from stage 20 through to E9,

hen it is still expressing nrg1 transcripts (data nothown).

The ventral nrg1-positive stripe widens into an area ofatchy staining in r2 and by stage 16 also in rostral r3.

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his area is seen in transverse sections to coincide withsl-1/2-positive cells along the pial edge of the neuroepi-helium (Fig. 3E) and presumably marks newly differen-iated motor neurons migrating from their ventral birthlace to the position of the V motor nucleus. These cellsontinue to express nrg1 after their arrival and form aateral domain of nrg1 expression that has appeared inhe r2 mantle by stage 19 (Fig. 3F for stage 20). Thisateral domain represents the forming V motor nucleus,s judged by its position, time of appearance, andoincidence with isl-1/2 immunoreactivity (data nothown). The V motor nucleus still expresses nrg1 on E4stage 24), E5 (stage 26), E7, and E9, the latest stagexamined.

The narrower portion of the nrg1-positive stripe in r3o r8 at stage 15 and 16 also appears to be isl-1/2 positiven flatmounts but the nrg1 staining is too faint to see inections this early. Figure 3G shows a section through anlder embryo (stage 21) in which it is clear that the

sl-1/2 positive differentiating motor neurons in theantle are nrg1 positive. Note that, at least at this level

hrough the XII motor nucleus, the more ventrallyocated isl-1/2-positive cells still in the neuroepitheliumave no detectable nrg1 transcripts. At this stage, the VInd XII motor nuclei stain especially intensely for nrg1,resumably due at least in part to the relatively high
oncentration of cells at these positions (Fig. 3H). Inlder embryos (up to E9), cells in many hindbrain motor
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t staining in solitary nucleus and arrowheads point at staining in what mharynx; Vg, V ganglion. (A) represents 160 µm in (A), (G), (Q), 400 µm in (Bm in (I), 200 µm in (J), (K), (R), 700 µm in (O), and 300 µm in (P).

uclei express nrg1. Expression is relatively high in the7 lateral V, dorsal VII, accessory VI and especially XIIotor nuclei, and additionally in the IX/X motor

ucleus by E9 (Fig. 3I and data not shown). Thisxpression in developing hindbrain motor neurons andotor nuclei is in line with what has been reported inammals (Corfas et al., 1995; Ho et al., 1995).Later isl-1/2-negative domains. Other nrg1 expres-

ion domains are the caudal rhombic lip from stage 21,audal mantle patches from stage 22, dorsal isthmic anderebellar epithelium from stage 13, and cerebellarantle from stage 24. The mantle at the rhombic lip is

een to stain for nrg1 transcripts in the r5 to r7 regionrom stage 21 to at least stage 24 (Fig. 3H and 3J), butnlike erbB4 transcripts, nrg1 transcripts are neitheretected more rostrally in rhombic lip mantle norarticularly abundant in the lip. We do detect tran-cripts in the right position to be in the inferior olive, aaudal rhombic lip derivative (Harkmark, 1954; Ambro-iani et al., 1996), at E7 and E9, but not convincingly inells that could be migrating to the inferior olive fromhe lip at these or earlier stages (Fig. 3I). Some caudal

antle patches appearing about stage 22 occur in therea from just rostral of the otocysts to the IX exit pointFig. 3K). These are mainly in the alar plate, and like theimilarly located erbB4 patches, may or may not be
hombic lip derived.
We have detected nrg1 transcripts in epithelium at or

IG. 2. erbB4 transcript distribution in hindbrain. Wholemounts, flatmount, and sections of embryos subjected to in situ hybridization with anrbB4 probe. Reaction product is purple. Some embryos were also subjected to immunostaining with a-isl-1/2 antibody. This reaction product isrown and localized in motor neurons and in cranial ganglia. (A) Stage 112 wholemount. Arrowheads from left to right mark staining in r1, r3, and5. (B) Stage 13 wholemount. Arrowheads from left to right indicate staining in isthmus/r1, r3, and r5. (C) Stage 13 flatmount. Rhombomeredentities are shown across top of image and asterisk marks floorplate. (D) Stage 16 wholemount. Arrowheads indicate r3 (on left) and r5; arrowoints at staining in rhombic lip. (E) Transverse section through isthmus of a stage 16 embryo. (F) Transverse section through r5 of a stage 16mbryo. Arrowheads point at the pial rhombic lip staining. (G) Transverse section through r1 of a stage 24 embryo. Arrows indicate epithelialtaining near the roofplate; arrowheads point at ventralmost epithelial staining. (H) Section through otocysts and r6 of a stage 24 embryo.rrowheads mark dorsolateral mantle patches of staining. (I) Dorsal view into hindbrain of a stage 24 embryo. Arrowheads mark the rostrocaudal

imits of staining just medial to the rhombic lip staining. Arrows indicate the rostral and caudal limits of r1. (J) Transverse section through an E6indbrain at the level of the ventrolateral X motor nucleus. Arrowheads point at superficial staining probably corresponding to rhombic liperived cells migrating ventrally. (K) Transverse section through an E7 hindbrain at the level of the ventrolateral X motor nucleus (indicated by anrrow). Arrowhead points at staining in margin at midline. (L) Transverse section through E7 hindbrain just rostral of the V motor nucleus. Largerrowheads point at staining in medial pontine nucleus; small arrowheads indicate staining in what is either a lemniscal or the principal sensory Vucleus; arrows point at superficial cerebellar staining. Hindbrain had been flatmounted prior to sectioning, leading to irregular shape of lefterebellar plate. (M) Transverse section through E7 hindbrain at level of VI, ventral VII, and accessory VI (beside the asterisk) motor nuclei. Arrowsark the nucleus of the descending V tract, pars interpolaris, and arrowheads indicate staining in a vestibular nucleus. (N) Transverse section

hrough E9 hindbrain at level between IV and V motor nuclei. Arrow points at superficial cerebellar staining and asterisk is beside a putativeerebellar deep nucleus. Arrowhead points at nucleus interpeduncularis. (O) Sagittal section through an E15 cerebellum; anterior is to the left.rrow indicates the more superficial and arrowhead the deeper of the two bands of staining. (P) and (Q) Transverse sections through E9indbrain. (P) is at a more rostral level than (Q). Asterisks are beside the XII motor nucleus; more dorsal isl-1/2 positive cluster is the IX/X motorucleus; in (P) only, ventrolateral X motor nucleus is also present. Arrows point at staining in solitary nucleus; arrowheads point at staining inxternal cuneate nucleus. (R) Transverse section through E7 hindbrain at level of IX/X (marked with asterisk) and XII motor nuclei. Arrows point

ay be nucleus intermedius. mb, midbrain (tectum); ot, otocyst; ph,), (L), 80 µm in (C), (E), (F), 240 µm in (D), (M), (N), 100 µm in (H), 750

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IG. 3. nrg1 transcript distribution in hindbrain. Wholemount, flatmorobe. Reaction product is purple. Some embryos were also subjectednd localized in motor neurons and in cranial ganglia. (A) Dissected hdentities are indicated below the hindbrain. (B) Transverse section thtage 132 embryo. (D) Flatmount of stage 16 hindbrain. Asterisk marmage. (E) Transverse section through r2/r3 region of flatmount showtage 20 hindbrain at level of V motor neurons. Asterisk marks V ganglheir birth place; arrowheads indicate staining in V motor neurons thatransverse section through stage 21 hindbrain at level of XII motor nuco not contain detectable nrg1 transcripts. (H) Flatmounted stage 22taining in the V motor nucleus. Arrowhead indicates VI motor neuroaudal rhombic lip. (I) Transverse section through E9 hindbrain at levemotor nuclei. Arrowheads indicate staining in appropriate position t

t level of r7. Arrowheads point at staining in mantle of rhombic lip. (Kasterisk) and otocysts. Arrowheads point at patch in dorsolateral manucleus (asterisk). Arrow indicates epithelial staining at dorsal midlin

unts, and sections of embryos subjected to in situ hybridization with a nrg1to immunostaining with a-isl-1/2 antibody. This reaction product is brownindbrain from stage 121 embryo, lateral view. Dorsal is up. Rhombomere

rough r4 of a stage 132 embryo. (C) Transverse section through r7 of sameks floorplate and rhombomere identities are shown across the top of then in (D) after immunostaining for isl-1/2. (F) Transverse section through aion; arrows point at staining in V motor neurons still located medially nearhave migrated laterally to the position of the forming V motor nucleus. (G)leus (arrowheads). Arrows mark more ventrally located epithelial cells thathindbrain; light region down center is the floorplate. Asterisk is next to

ns. Short arrow points at XII motor neurons. Long arrow marks staining inl of very rostral XII motor nucleus (next to asterisk), IX/X, and ventrolateralo be in inferior olive. (J) Transverse section through flatmount shown in (H)) Transverse section through stage 24 hindbrain at level of VI motor nucleustle. (L) Transverse section through stage 24 hindbrain at level of IV motor

e and arrowheads point at staining in the mantle. Double asterisk is besidesl-1/2 positive cells of the mesencephalic V nucleus. (M) Transverse section through stage 26 hindbrain at level of V motor nucleus. Arrowheads
oint at staining in dorsal epithelium. Asterisk marks V ganglion. (N) Transverse section through E7 hindbrain at level between IV and V motoruclei. Arrowhead indicates medial epithelial staining in cerebellum; arrow marks staining in cerebellar mantle. *Scale bar in (A) represents 100m in (A), (L), 40 µm in (B), (C), 90 µm in (D), (E), 150 µm in (F), ( J), 60 µm in (G), 300 µm in (H), 200 µm in (I), (M), 120 µm in (K), and 240 µm in
N).

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ear the dorsal midline of the isthmus, r1, and cerebel-um from stage 13. Initially the staining is in the isthmusnd by stage 24 it is in r1, but at these times it is alwayst the midline, rostral to the roofplate (Fig. 3L). At stage6 and E7, the staining is in the cerebellar neuroepithe-ium, at stage 26 in more caudal cerebellum in a dorsalomain near but not quite at the roofplate (Fig. 3M), andt E7 in most of the cerebellar epithelium but moretrongly medially (Fig. 3N). Cerebellar mantle tran-cripts are detected from stage 24 on. The stainingattern changes from a relatively uniform distributiont stage 24 (Fig. 3L) to more complex by E7 (Fig. 3N),hen there are positive areas throughout the cerebellarantle, possibly including the deep nuclei.

omparison of erbB4 and nrg1 Domains in theeveloping Hindbrain

The difference in nrg1 and erbB4 mouse knock-outhenotypes suggested that nrg1 may not function as anrbB4 ligand in the hindbrain. By observation of em-ryos subjected either separately or simultaneously torbB4 and nrg1 in situs, we have found several regions inhe hindbrain where the nrg1 and erbB4 transcriptomains either overlap or abut and hence are in closenough proximity that they could act as ligand andeceptor, even if the nrg1 is not freely diffusible butemains bound on the surface of cells expressing it. A

ouse study (Meyer et al., 1997) has found that up to10, only type I nrg1 transcripts exist in the hindbrain.rom E12 to E18, type II and type III transcripts areresent but type I are not. If a similar situation obtains inhick, the early epithelial domains would express type Irg1 transcripts (presumably with nondiffusible proteinroducts), while the later domains would express type

I and III transcripts. The protein(s) encoded by theatter could be diffusible or in the plasma membranes ofhe expressing cells.

nrg1 versus epithelial erbB4. The earliest detectedrea of abuttal of nrg1 and erbB4 is in r3 at stages 12–13,here nrg1 in the ventral portion of the alar plate likely

buts erbB4 in the dorsal portion of the basal platecompare Figs. 4A and 4B). The same probably holds for2 from stage 13 on, and for r4 at stages 16 and 17 (Figs.C and 4D). Although both r4 domains persist untiltage 23 or beyond, by stage 18 it is already doubtfulrom double in situs that they abut anymore (data nothown). Comparison of the basal plate erbB4 domains in3 and r5 with the alar plate nrg1 domain in r4 viaouble-labeled flatmounts suggests that these domainso ‘‘touch corners’’ at stage 16 (Fig. 4E), but that from
tage 18 when the r4 nrg1 domain is shrinking, it islearly separated from the r3 and r5 erbB4 domains (Fig.
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F for stage 21). Finally, the basal erbB4 domain mustbut the alar nrg1 domain in the r7/8 region, as seen byomparing caudal hindbrain sections from stages 15–16mbryos (Figs. 4G and 4H).

Figure 4G also shows the ventral isl-1/2-positiveomain of motor neurons to be in close proximity to theasal erbB4 domain. As mentioned earlier, this isl-ositive domain is most probably nrg1 positive, al-

hough at this stage the nrg1 staining is too weak to beeen in sections, particularly over the isl staining. Atore rostral levels, this ventral nrg1 domain appears to

e separated by a gap from the basal erbB4 domain, aseen in stages 16 and 21 flatmounts (Figs. 4E and 4F).here are nevertheless two types of motor neurons atostral levels that do abut basal epithelial erbB4; this is aesult of their lateral migration through the mantle nexto the epithelium. One of these two types is the V motoreurons in r2 and r3 (Fig. 4I); they remain in closeroximity to the erbB4-positive epithelium from stage 15r 16 to stage 20 or 22. After this, the mantle has becomehicker and migrating V motor neurons are no longerdjacent to the epithelium. Some IV motor neurons arelso located close to epithelial erbB4 as seen in a sectionhrough this nucleus in very rostral r1 at stage 21 (Fig.J). In more rostral sections than this one, the IV motorucleus is more rounded in shape and separated from

he epithelial erbB4, while in more caudal sections, it isxtended laterally so that it lies adjacent to the erbB4omain. In the section shown, the IV motor nuclearhape is transitional between the two extremes.

nrg1 versus mantle erbB4. Both nrg1 and erbB4ranscripts have been detected in the rhombic lip. TherbB4 transcript staining appears earlier, is more exten-ive along the rostrocaudal axis, and is more intensehan the nrg1 transcript staining. Nevertheless, fromtage 21 to at least stage 24 in the r5 to r7 region, whererg1 transcripts are found, there is overlap of ligand-nd receptor-positive domains (e.g., Fig. 2H vs 3J). Thisverlap does not appear to carry over to rhombic liperivatives, with only nrg1 being detected in the inferiorlive and only erbB4 in migrating cells, medial pontineucleus, and the cerebellar surface. However, at E7 therg1-positive inferior olive surely abuts erbB4-positiveells migrating in the margin and superficial mantle (nothown).

Early in cerebellar development (stages 24, 26), theres extensive overlap between nrg1 and erbB4 expressingells in the dorsal and lateral cerebellar mantle (e.g., Fig.G vs 3L). Also, at stage 26 the small dorsal epithelialrbB4 domain may abut or overlap the epithelial nrg1omain in the caudal cerebellum (not shown). Later (E7
nd E9), the intense surface staining for erbB4 must abutt least in places the complex nrg1 pattern in the mantle

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e.g., Fig. 2L vs 3N). The erbB4 positive (at E9) putativeeep nuclei also likely come into contact with nrg1 in theeep mantle (not shown).The dorsolateral mantle patches expressing nrg1 and

rbB4 that are present at caudal hindbrain levels (caudalI/VII motor nuclei and caudad) at stages 24 and 26robably abut or overlap each other as well (Fig. 4Kersus 4L). At stage 26 this also seems to be the case forore rostral mantle domains of ligand and receptor (not

hown). A more medial caudal mantle patch expressingrbB4, possibly the nucleus intermedius, abuts the in-ensely nrg1 positive XII motor nucleus at E7 (Fig. 2R).

rbB4 Transcripts Outside the Hindbrain

erbB4 is not restricted to the hindbrain but is ex-ressed in all regions of the central nervous system

CNS). We have seen expression in the presumptiveorebrain as early as stage 9. A section through this areat stage 11 shows the transcripts are located in ventralresumptive diencephalon (Fig. 5A). The diencephalonontinues to express erbB4 relatively strongly until ateast stage 24, in the hypothalamus, including thenfundibulum and optic stalks until at least stage 20Fig. 5B). The medial portion of the lateral ventricles ofhe telencephalon contain erbB4 transcripts by stage 18nd these persist until at least stage 24. Sections showhe staining to be neuroepithelial and flanking theamina terminalis (Fig. 5C). Clear midbrain expressiontarts slightly later, with a bilateral spot in anterioridbrain floor at stage 21. A section through a stage 26idbrain (Fig. 5D) shows the spot to be in the mantle

mmediately adjacent to both the oculomotor (III) motorucleus and the neuroepithelium. Part of the tectumlso stains from stage 24 to at least E7 (data not shown).e detect expression in the neuroepithelium of the

pinal cord in a band at an intermediate dorsoventralosition from stage 16 to stage 20 (Figs. 5E and 5F). Attage 21/22, this domain is no longer detected but we doee transcripts in a dorsolateral patch in the spinal cordantle at this time (data not shown).We also find chick erbB4 transcripts in nonneuronal

issues, including the heart starting at about stage 10 to1, mesonephros from stage 16 or 17, pharynx by stage1, and lateral plate from stage 16 (Figs. 2D, 2F, 5E, andG). We additionally noted expression in the limb fieldsnd limb buds from stage 16 up to the latest stagexamined, stage 26. Initially the transcripts are in theesenchyme of both wing and leg fields, more concen-

rated laterally than medially (Figs. 5E and 5F). At stage7, they are in the lateral plate adjacent to the limb buds
nd in the distal ectoderm of the buds. By stage 24 therere two expression domains in each limb bud. One is
ms

esenchymal, concentrated in the anterodistal portionf the bud (Figs. 5G and 5H), and the other is in theistal ectoderm, in two stripes lying at or near the

unction of the apical ectodermal ridge (AER) with thedjacent ectoderm. The transcripts are excluded fromhe central portion of the AER (Fig. 5H). At stage 26,here is still an anterodistal patch in both leg and winguds.

rg1 Transcripts Outside the Hindbrain

Although its transcripts are detected first in theindbrain, nrg1 is, like erbB4, expressed in many places

n the embryo, including other parts of the CNS, theeripheral nervous system (PNS), and nonneuronal

issues. In the forebrain, transcripts are detected in theantle of the lateral ventricles of the telencephalon at

tages 24 and 26. This staining is mainly in the lateralortion of the ventricles (Fig. 5I). The diencephalon alsoxpresses nrg1, in a spot at or either side of the dorsalidline of the diencephalon from stage 20 to at least

tage 24 (Fig. 5J) and in a domain in the floor of theaudal diencephalon. This latter domain is present asarly as stage 15, and by stage 22, it is continuous withore intense staining in the III motor nucleus in theidbrain floor (Fig. 5K). The midbrain floor expression

s seen from stage 15 and the III motor nucleus is stilltaining strongly at E7 and E9, the latest stages exam-ned (Fig. 5K). An additional midbrain domain appearsy stage 15 and persists until at least stage 26 (Fig. 5J).his domain starts as a spot on the midline of the tectumear its caudal extreme where it joins the isthmus. Theingle spot splits into a pair of spots either side of theidline around stage 19. A section through this domain

t stage 20 shows that the expression is epithelial andanking the mesencephalic trigeminal nucleus (Fig. 5L).

n the spinal cord, nrg1 transcripts are detected in theentral, isl-1/2-positive motor neurons from stage 16 tot least stage 26 (Fig. 5M).In the PNS, the transcripts are detected in cranial

anglia as they form from stage 15 (Figs. 5J and 5N) andn dorsal root ganglia (DRG) by stage 20 (Fig. 5N).ranial ganglia in which expression was observed are

he two lobes of the trigeminal, the geniculate, theestibuloacoustic, the petrosal, and the nodose. The

ugular/superior ganglion is not seen by isl-1/2 stainingntil stage 24, at which point we do not observe nrg1

ranscripts in it.Outside the nervous system, we have noted nrg1

ranscripts in the heart from stage 13, at the ventral
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ISCUSSION

loning of Chick erbB4

IG. 4. Comparison of erbB4 and nrg1 transcript distributions inybridization with erbB4 and nrg1 probes either singly or doubly.

mmunostained with a-isl-1/2 antibody. Brown reaction product labeltage 13 embryo, erbB4 probe. (B) Transverse section through r3 of aisible only on the indicated side of the neural tube in this section. (Coint at the dorsal limit of the basal plate expression domain. (D) Tranrg1 probe. Arrows mark the ventral limit of the alar plate expressioloorplate is light strip at bottom; rhombomere identities are indicaloorplate is light strip at bottom, where rhombomere identities are alsindbrain, erbB4 probe. (H) Section through r7/8 of a previously flatotor neurons of a stage 16 embryo, erbB4 probe. Arrows mark the d

ection through IV motor nucleus (arrowheads) of a stage 21 embryootor nucleus, by asterisk, caudal extreme of VII/VIII ganglia, and ot

tocyst; ph, pharynx. *Scale bar in (A) represents 40 µm in (A), 35 µm im in ( J), (L), and 130 µm in (K).

We have cloned the chick homologue of erbB4 andhown it to be 93% identical to its human counterpart at

ds

he amino acid level. Our cDNA encodes the JM-asoform of the receptor. In humans, this isoform isotentially cleaved to release a soluble extracellularomain (Elenius et al., 1997a). In mouse and human,

ranscripts for both this isoform and the alternative,M-b, not thought to be cleaved, occur in the CNS,ncluding cerebellum and medulla oblongata, hindbrain

brain. Sections and flatmounts from embryos subjected to in situion product from both probes is purple. Some embryos were alsoor neurons and cranial ganglia. (A) Transverse section through r3 of a132 embryo, nrg1 probe. Arrowhead indicates the staining, which issverse section through r4 of a stage 16 embryo, erbB4 probe. Arrowse section through r4 of a stage 16 hindbrain, previously flatmounted,main. (E) Portion of a flatmounted stage 16 hindbrain, both probes.t top. (F) Portion of a flatmounted stage 21 hindbrain, both probes.icated. (G) Section through r7/8 of a previously flatmounted stage 16ted stage 15 hindbrain, nrg1 probe. (I) Transverse section through V

ventral extent of the isl-1/2 positive V motor neurons. (J) Transverseh probes. (K) and (L) Transverse sections through the same level (VIs) of a pair of stage 24 hindbrains. (K) erbB4 probe. (L) nrg1 probe. ot,80 µm in (C), 50 µm in (D), (E), (H), 60 µm in (F), (I), 70 µm in (G), 120

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erivatives (Elenius et al., 1997a). Since neither of our initu probes for erbB4 distinguishes between the iso-

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IG. 5. erbB4 and nrg1 transcript distributions outside hindbrain. Sections and wholemounts of embryos subjected to in situ hybridization withither erbB4 (A–H) or nrg1 (I–O) probes. Reaction product is purple. Some embryos were also immunostained for isl-1/2. Brown reaction productabels motor nuclei, the mesencephalic V nucleus, and DRG. (A) Transverse section through presumptive diencephalon of a stage 112 embryo.orsal is up. (B) Horizontal section through hypothalamus of a stage 20 embryo. Pharynx (ph) is up. Arrow indicates infundibulum and

rrowhead points at optic stalk. (C) Horizontal section through telencephalon of stage 24 embryo. Asterisk marks lamina terminalis. (D)orizontal section through midbrain (oblique through diencephalon, di) of stage 26 embryo. Asterisks are adjacent to the III motor nucleus

brown staining). (E) Transverse section through stage 16 embryo at level of wing field (W). Asterisk beside neural tube is at dorsoventral level ofpinal cord staining. Arrows point at staining in kidneys and arrowhead indicates staining in lateral plate. (F) Transverse section through stage 16mbryo at level of leg fields (L). Arrow points at spinal cord staining. (G) Stage 24 embryo. Asterisks mark wing and leg buds; arrow points attaining in kidneys; arrowhead points at staining in heart. (H) Sagittal section through wing bud of stage 24 embryo. Asterisk is beside the AER. D,orsal; V, ventral. (I) Horizontal section through telencephalon of stage 24 embryo. Arrows point at staining in telencephalic mantle. (J) Stage 16mbryo. Asterisk is beside staining in heart; arrowhead indicates staining in somites; small arrow points at staining at dorsal midline ofiencephalon; large arrow points at spot in caudal tectum. (K) Horizontal section through III motor nucleus (arrowheads) of stage 24 embryo.rrows point at diencephalic staining continuous with III motor nucleus staining. (L) Oblique (between horizontal and transverse) section

hrough tectum of a stage 20 embryo. Asterisk is at caudal midline and mesencephalic V nucleus. Arrowheads point at epithelial nrg1 staining. (M)ransverse section through spinal cord (wing level) of stage 20 embryo. Asterisk marks DRG. Arrowheads point at nrg1 staining in ventral motor
eurons. (N) Stage 22 embryo. Asterisks are beside staining in limb buds; arrow points at staining in DRG; arrowheads point at staining in cranialanglia. (O) Transverse section through r5 and otocysts of stage 13 embryo. Arrowhead indicates staining in ventral pharynx. Arrow points attaining in heart. I, II, III, first, second, third ventricles, respectively. *Scale bar in (A) represents 75 µm in (A), (M), (O), 120 µm in (B), (C), (D), (F),K), 100 µm in (E), 850 µm in (G), 30 µm in (H), 200 µm in (I), 650 µm in ( J), 170 µm in (L), and 700 µm in (N).
249

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orms, we cannot comment on which of them is presentn embryonic chick hindbrain, but the balance of iso-orms present could affect signalling function. For in-tance, a soluble ectodomain could conceivably act as aignal via nrg1, such that ‘‘ligand’’-positive cells ratherhan ‘‘receptor’’-positive ones respond to the molecularnteraction between them. That transmembrane iso-orms of nrg1 might transmit signals via their cytoplas-

ic domains has been postulated (Wen et al., 1992; Wangt al., 1998). A second possibility is that a solubleeceptor ectodomain can bind extracellular or transmem-rane isoforms of nrg1 so that nrg1 is no longervailable to activate membrane-bound, signal-transduc-ion-competent receptor. This would be a mechanismor limiting receptor activation that could operate inonjunction with, or instead of, other mechanisms suchs limiting expression or diffusion of nrg1.

arly Epithelial Expression of nrg1 and erbB4

There are two differences between our data on earlyrbB4 expression and those reported for mouse (Gass-ann et al., 1995; Gassmann and Lemke, 1997; Meyer et

l., 1997). First, in mouse, erbB4 transcripts are detectedn r3 and r5 (E9–9.5) prior to more rostral and moreaudal neural tube regions (E10), in contrast to ourbservation in chick that more rostral (isthmus/r1) andaudal (r7/8) expression precedes or coincides with r3nd r5 expression. Second, mouse transcripts in r3 and5 at E9–9.5 occur not only in the domain just dorsal toeveloping motor neurons that we observe in chick butlso in the roofplate (Jon Golding and Martin Gass-ann, personal communication). We do not detect this

oofplate domain in chick (Fig. 2C), nor do we ever seeny dorsal domain exclusively in r3 and r5.

The nrg1 expression pattern in chick also differs fromhat reported for mouse, where the earliest hindbrainxpression is at E8 in r2 and r4, followed by additionalxpression in r6 and dorsal spinal cord at E8.5 (Meyer etl., 1997). In E9.25 mouse embryos, it is still r2, r4, and r6hat express nrg1, while r3 and r5 apparently do notMeyer and Birchmeier, 1995). The earliest expression

e see in chick is in a different pair of rhombomeres, r4nd r7. We do subsequently also see expression in r2 butot without concomitant expression in r3. Because of

his expression in r3, and because nrg1 transcripts occurt best only caudally in r6 in chick, we never see the r2,4, r6 combination reported for mouse nrg1, althoughhe dorsoventral localization we see for caudal hind-rain nrg1 is similar to that observed in forelimb level
pinal cord of E9 mouse (Fig. 6E in Britsch et al., 1998).
These differences in the early patterns of nrg1 andre

rbB4 expression between chick and mouse cannot bexplained solely on the basis of differences in relativeiming of developmental events, and have implicationsor mechanisms by which these molecules may affectevelopment in the two species. In the mouse, nrg1 andrbB4 are expressed in alternating rhombomeres in the2 to r6 region of the hindbrain, allowing the possibilityhat they mediate interrhombomeric signalling. Suchignalling from even to odd rhombomeres has beenbserved in chick to regulate neural crest apoptosis in r3nd r5 (Graham et al., 1993) and gene expression in r3Graham et al., 1994; Graham and Lumsden, 1996).ndeed, it has been suggested that the misinnervationhenotype of erbB4 knock-out mice could arise if largerumbers of r3 and r5 neural crest survive in the mutantsnd contribute to the V and VII/VIII ganglia, leading toheir misspecification (Lemke, 1996; Gassmann andemke, 1997). This mechanism is unlikely to operate inhick because erbB4 is not expressed in the dorsalmosteural tube where neural crest cells arise.Furthermore, in chick there is no alternation of nrg1

nd erbB4 expression domains along the rostrocaudalxis. It seems more likely that in chick signallingediated by nrg1 and erbB4 would occur between

orsal and ventral neural tube within each of thehombomeres that expresses transcripts for both mol-cules. These are rhombomeres 2 to 4 and 7 to 8, startingt different stages according to when transcripts for botholecules have appeared. Curiously, the r5/r6 region

ever expresses the early dorsal epithelial nrg1 tran-cripts, nor does r1. (Isthmic and r1 nrg1 expression is athe dorsal midline, not in the ventral alar plate where it

ight abut the erbB4 domain.) Whether this indicates aack of erbB4 activation in these regions or activation bydifferent ligand is not known. Either way, r5 and r6 areefined as a single unit by other criteria, such asxpression of kreisler in the hindbrain, production of theI motor neurons, and position adjacent to the otocyst,hile r1 is set apart from the other rhombomeres in that

t gives rise to the cerebellum, expresses no hox genesnd produces motor neurons only at its rostral extrem-ty. Furthermore, r5/r6 and r1 share a functional prop-rty in that motor nuclei in both fields (VI, IV, respec-ively) innervate eye muscles. Any connection betweenhese distinguishing features of the r5/r6 and r1 territo-ies and the absence of the ventral alar nrg1 domainemains to be demonstrated.

In the rhombomeres where signalling between dorsalnd ventral territories via nrg1 and erbB4 is a possibility,his signalling could affect differentiation of interneu-
ons arising at intermediate dorsoventral positions,ither positively to allow or promote production of

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eurons or negatively to keep neuroepithelial cells in anndifferentiated, proliferative state. Alternatively, nrg1-rbB4 signalling could act to differentiate between thelar and basal plates. Differences in alar and basalharacteristics include types of neurons produced, tar-ets of exiting axons, entry sites of afferents, productionf neural crest, and rates of mitosis. Presumably anyquivalent roles in r1 and r5/r6 would be carried out byther mechanisms, possibly involving different erbB4

igands.Gassmann and colleagues (Gassmann et al., 1995;

emke, 1996; Gassmann and Lemke, 1997) proposed aarrier model to explain why, in the erbB4 knock-outouse, the sensory axons from the V and VII/VIII

anglia each enter at both r2 and r4. According to thisodel, erbB4 either is itself a barrier or is required for

he functional expression of a barrier, to axon growthcross rhombomeres expressing it and adjacent mesen-hyme. A prediction of the model would be that r2 and4, the rhombomeres with the entry points, should notxpress the putative inhibitory molecule, erbB4, beforehe sensory axons have entered r2 and r4. In chick, werst detect erbB4 transcripts in r2 at stage 13 and in r4 attage 16, coinciding with the time sensory axons enterhe hindbrain via these rhombomeres (Heaton and

oody, 1980; Vogel, 1992). Thus, the barrier modelould apply in chick with respect to sensory axonsespite the fact that in chick, unlike in mouse, erbB4

ranscripts are expressed in r2 and r4.

rg1 and erbB4 in Rhombic Lip and Its Derivatives

Although both nrg1 and erbB4 transcripts occur inaudal rhombic lip, it is unclear whether the same cellsr different cells express the two transcripts and whetherny signalling mediated by the two molecules is auto-rine or paracrine. In any event, because rhombic lipranscripts of erbB4 are concentrated in the mantle whileroliferation occurs in the ventricular layer, such signal-

ing probably does not affect proliferation of receptor-ositive cells. It may, however, be required for them to

nitiate their migration. Presumably it is no longerequired during the migration, at least not in a generalay by all cells leaving the rhombic lip, because we also

ee erbB4-negative cells in the margin. The cells in theaudal margin that do express erbB4 may depend uponts activation for their continued migration, but sincerg1 is not detected in these cells, it may not be thectivating ligand here. On the other hand, nrg1 in theentrally located inferior olive at E7 may be attracting
he erbB4-positive cells in the margin. It may addition-lly interact with these cells once they have reached the
it

entral level of the inferior olive, either to affect theirifferentiation or to determine whether they contribute

o the olive or migrate past it and contribute to othertructures. nrg1 has been shown in culture to be bothhemotactic and able to increase migration rates ofeuronal and glial cells (Mahanthappa, 1996; Anton etl., 1997).

There are additional possibilities for interaction be-ween nrg1-positive and erbB4-positive rhombic lip de-ivatives, besides those between the inferior olive andigrating rhombic lip-derived cells. For instance, if

xons from the inferior olive synapse onto the cerebellareep nuclei in chick as they do in rat (van der Want et al.,989), and if the inferior olivary axons express the nrg1rotein, they may activate erbB4 in the deep nuclei. The

ime we first detect erbB4 transcripts in putative deepuclei (E9) coincides with the time inferior olivary axonsnter the cerebellum in chick (just before E9; Chedotal etl., 1996). Similarly, if erbB4 protein is present on thexons from the erbB4-positive medial pontine nucleus,nd if nrg1 protein is expressed on the cerebellar cellsith which they synapse, it could be that cerebellar cells

ctivate the pontine erbB4 via nrg1 or that erbB4 onontine axons activates cerebellar nrg1, i.e., that cerebel-

ar nrg1 functions as a receptor. It should be notedhough that nrg1 transcripts are present in the medialontine nucleus of adult rat (Chen et al., 1994; Corfas etl., 1995). Hence, it is conceivable that the embryonichick nucleus also contains nrg1 transcripts but at levelselow our threshold of detection, and that pontinerbB4 is activated by pontine nrg1.

erbB4-positive and nrg1-positive patches appear in theorsolateral mantle of the caudal hindbrain at about3.5, before Harkmark (1954) noted an accumulation ofells at the rhombic lip (E4.5) or cells migrating intoorsolateral mantle (E5). Nevertheless, it is conceivable

hat the erbB4-positive and/or nrg1-positive cells arehombic lip derived and start to express these genesefore being numerous enough to detect by the histologi-al staining methods used by Harkmark. Indeed, theay in which erbB4 staining seems to be ‘‘breaking

way’’ from the rhombic lip (Fig. 4K) makes it reason-ble to speculate that the erbB4-positive cells originate athe rhombic lip.

Regardless of their origin, the dorsolateral mantleatches of nrg1-positive and erbB4-positive cells are inlose enough proximity that they may signal each other,erhaps being instrumental in the differentiation of oner both. The nrg1-positive patches could respond to theignalling either by nrg1 acting as a receptor or by
nvolvement in reciprocal interactions. Examples ofhese have been reported to involve secretion of survival

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erebellar Expression of nrg1 and erbB4

erbB4 transcripts appear in r1 mantle at about E3.5stage 20), which is before E5 (stage 26) when EGLormation begins according to Hanaway (1967), butther aspects of erbB4 expression parallel EGL develop-ent. Thus, the appearance of erbB4 transcripts on the

erebellar surface by E6, and their spread over theurface from caudal to rostral, lead us to believe that it ishe granule cell precursors of the EGL that express erbB4ranscripts. The appearance of the second, deeper bandf erbB4-positive cells reminiscent of the IGL at E15hen granule cells start their radial migration supports

his notion. Preliminary measurements suggest that thehickness of the staining layer dorsal and lateral of theentricle does not change between stage 22 and E9. This

s consistent with, but by no means proof of, theorsolateral mantle cells that express erbB4 at an interme-iate level at stages 20 to 26 being the same cells as aren the cerebellar surface expressing erbB4 at a high levelrom E6 onwards. If this is true, it may be that therbB4-positive cells in r1 dorsolateral mantle at stages 20o 26 are granule cell precursors and that EGL formationegins sooner than previously believed. The faint neuro-pithelial staining we see near the rhombic lip at stages4 and 26 may be in granule cell precursors before theynter the EGL.

At stages 24 and 26, nrg1 transcripts are also found inorsal and lateral cerebellar mantle, in a domain overlap-ing extensively with the erbB4 domain. Whether erbB4

n this region is active before we detect nrg1 there isnclear, nor do we know whether any activation by nrg1

s autocrine or paracrine. Possible roles of nrg1 signal-ing in this area would be effects on survival or differen-iation of these cells, such as the putative upregulationf erbB4 transcripts at E6.By E7 and continuing at E9, the overlap between nrg1

nd erbB4 domains is reduced, with intensely erbB4-ositive cells on the cerebellar surface apparently notxpressing nrg1, and many areas in the mantle where weetect nrg1 not expressing erbB4. However, these do-ains do at least contact each other and possibly

verlap along the deep edge of the superficial erbB4omain. Conceivably, nrg1 in the mantle triggers theroliferation in the EGL that has started by E7 (Han-way, 1967; Ryder and Cepko, 1994). The increase in
rbB4 transcripts in the EGL at E6 may be in preparationor this proliferative phase. However, maintenance of
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roliferation beyond E9 is not likely to involve erbB4irectly, as its transcripts are not detected in the superfi-ial mitotic layer of the EGL at E11 or E15.

At stage 26, there may be abutting or overlap betweenrg1- and erbB4-expressing cells in the dorsal epitheliumf the caudal cerebellum. If the erbB4-positive cells doive rise to the EGL as suggested, the nrg1-positive cellsay signal them to leave the epithelium and migrate

ver the surface. This would be consistent with theiming of EGL formation according to Hanaway (1967),ut as mentioned above, EGL formation may commenceooner than this (stage 20). However, it may be that only

later migrating population of epithelial cells makesse of nrg1–erbB4 signalling and that a different mecha-ism operates in cells migrating between stage 20 and5. The continued expression of erbB4 in the deeperand in the E15 cerebellum suggests that the granuleells in the IGL are still or also erbB4-positive. erbB4 mayhus also be involved in the radial migration of cellsetween EGL and IGL, as has been demonstrated in ratRio et al., 1997), although in this organism, erbB4 isxpressed in glial cells rather than granule cells duringhe radial migration.

Brainstem and cerebellar expression of erbB4 in embry-nic chick (Francoeur et al., 1995), as well as pontine andedullary expression in embryonic mouse (Meyer et al.,

997), have already been reported and these previouseports are consistent with ours. The mouse study alsoound E18 expression in both cortex and medulla of theerebellum, in line with our findings in chick of tran-cripts both on the surface of the cerebellum and in theeep mantle at E9. A report of erbB4 protein in both EGLnd IGL of fetal human (Gilbertson et al., 1998) broadlygrees with our data, except that these authors foundrbB4 protein throughout the EGL rather than only inhe deeper, postmitotic layer.

erbB4 expression in developing rat cerebellum is, byontrast, potentially different from what we observe inhick. Although Ozaki et al. (1997) report erbB4 RNA inhe EGL and protein in granule cells of P14 rat near thend of the radial granule cell migration from external tonternal granular layers, Rio et al. (1997) have noted that,uring this migration of granule cells, radial glia but notranule neurons express erbB4 protein. This expression

s down-regulated after the migration, and in the adulthe bulk of the expression is in the mature granule cells.he latter study potentially contrasts with our finding

hat at E15 in chick when the EGL and IGL are bothresent, they both express erbB4. Because the spread ofhick erbB4 staining from caudolateral cerebellum at E6
o cover the whole surface by E9 is reminiscent of thepread of granule cells to form the EGL in both pattern

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nd timing, we favor the idea that chick erbB4 tran-cripts occur in the granule cells and their precursors,ut we cannot rule out that the transcripts are in radiallial cell bodies and appear in granule neurons onlyfter their radial migration into the IGL.

Cerebellar expression of nrg1 has also been noted inhick previously, albeit much later (E19) than we lookedFalls et al., 1993). Studies in mouse suggest there is nrg1xpression in r1 (Gassmann and Lemke, 1997) and inmbryonic cerebellum, the latter being in the medullaut not the cortex at E18 (Meyer et al., 1997; Meyer andirchmeier, 1994). These data are consistent with ourndings in chick. In fetal human (15 weeks and older),rg1 protein has been detected in the outer layer of theGL as well as in deeper cerebellum (Gilbertson et al.,998). While we noted only deeper nrg1 transcripts inhick, we did not examine comparably advanced devel-pmental stages and, therefore, cannot say whether therg1 expression in human EGL contrasts with the chickrg1 pattern.

xtra-Hindbrain Expression of nrg1 and erbB4

Our data on expression of erbB4 outside the hindbrainre largely consistent with previous studies in chick andther organisms. Specifically, our data on CNS erbB4xpression agree with a study of protein localization inlder chick embryos (Francoeur et al., 1995), as well asith transcript distribution in similarly aged mouse

mbryos (Gassmann et al., 1995; Meyer et al., 1997),xcept that the latter authors do not find erbB4 tran-cripts in the ventricular layer of the telencephalon untiluch later than we do. This could simply reflect

ifferences in the relative rates of development ofarious brain regions in the two species. NonneuronalrbB4 expression in chick is also similar to that describedn previous reports. Transcripts have been noted inmbryonic heart for mouse (Gassmann et al., 1995;eyer and Birchmeier, 1995; Meyer et al., 1997) and

uman (Srinivasan et al., 1998), and in fetal humanidney and gut (Srinivasan et al., 1998). We know of norevious reports of transcripts in lateral plate or limbs.Our data on nrg1 transcript distribution also generally

gree with previous reports though there are someifferences. nrg1 transcripts in spinal cord motor neu-ons have been documented for chick (Falls et al., 1993;oodearl et al., 1995) and mammals (Marchionni et al.,

993; Orr-Urtreger et al., 1993; Meyer and Birchmeier,994; Corfas et al., 1995; Ho et al., 1995; Meyer et al.,997), but the transient dorsal band of nrg1 transcripts
bserved in E10 mouse spinal cord (Meyer et al., 1997)as not detected in our chick in situ hybridization
tn

xperiments. The expression we observe in developingelencephalon, diencephalon, and midbrain is consis-ent with previous reports of expression in embryonic

ouse and rat at comparably young stages (Marchionnit al., 1993; Orr-Urtreger et al., 1993; Meyer and Birch-eier, 1994; Corfas et al., 1995; Meyer et al., 1997). Of the

eports of nrg1 transcripts in embryonic mammalianranial ganglia (Marchionni et al., 1993; Meyer andirchmeier, 1994; Ho et al., 1995; Meyer et al., 1997),eyer et al. (1997) is the most detailed. They find nrg1

ranscripts in trigeminal, geniculate, glossopharyngeal,nd vagal ganglia of E9.5 mice. The latter two gangliaust be the petrosal and nodose, as the jugular and

uperior have not yet formed. Thus, these findings inouse are in keeping with our results in chick, except

hat the mouse report did not mention nrg1 transcriptsn the vestibuloacoustic ganglion. The DRG expressionas been observed previously in embryonic chick (Good-arl et al., 1995), as well as in mammalian embryosMarchionni et al., 1993; Orr-Urtreger et al., 1993; Meyernd Birchmeier, 1994; Shah et al., 1994; Corfas et al., 1995;o et al., 1995; Meyer et al., 1997). Expression in

mbryonic heart has been noted previously in chickGoodearl et al., 1995) and mammals (Meyer and Birch-

eier, 1994; Corfas et al., 1995; Meyer and Birchmeier,995; Meyer et al., 1997), as has somite expression in E5hick (Goodearl et al., 1995) and embryonic frog (Yang etl., 1998). Transcripts in pharynx have not been de-cribed, but intestinal expression in mammalian em-ryos has (Holmes et al., 1992; Meyer and Birchmeier,994). To our knowledge, there are no previous reportsf limb bud expression.

ummary

Both nrg1 and erbB4 are expressed at multiple sitesnside and outside the nervous system and over a largeevelopmental time window. The breadth of their loca-

ion and time of expression as observed by us in chicknd by others in various species is matched by theiversity of functions attributed to them by others; these

nclude effects on the survival, proliferation, migration,nd differentiation of target cells (reviewed by Lemke,996). Our study focussing on expression of nrg1 andrbB4 in the embryonic chick hindbrain shows that evenithin this single region of the CNS, both molecules

ave multiple expression domains, some of them abut-ing or overlapping each other. Our data lay a founda-
ion for interpreting phenotypes found in this part of theervous system during our planned functional studies.

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XPERIMENTAL METHODS

nimals

Fertile hen eggs from a mixed flock were obtainedrom Poyndon Farm (Enfield, UK) and incubated in aumidified 38°C room to the desired stage. The embryosere removed from the eggs, rinsed in Howard’singer, and the vitelline and any extra-embryonic mem-ranes removed. Embryos were staged according to theystem of Hamburger and Hamilton (1951) and wereurther processed for RNA extraction or in situ hybridiza-ion as detailed below.

solation of Chick erbB4 PCR Fragment and cDNA

Stages 10 and 11 embryos were incubated in 1 mg/mlispase with 35 µg/ml DNaseI in L15 culture medium

GibcoBRL) 10–15 min, until mesenchyme started tooosen from the neural tube. Hindbrains from thesthmus to somewhat caudal of the r6/r7 border wereissected free of surrounding tissue and collected inoward’s Ringer. The Ringer was replaced with solu-

ion D, 20 µg glycogen carrier added, and total RNAxtracted essentially by the method of Chomczynskind Sacchi (1987), except that the total RNA pellet fromine hindbrains was directly resuspended in 100 µl ofater rather than being resuspended in solution D andrecipitated a second time. First-strand cDNA wasynthesized in a total volume of 20 µl from 4 µl (40%) ofhe total RNA and an anchored oligo-dT primer, usingn Amersham cDNA synthesis kit. Reverse transcrip-ase was heat inactivated after the reaction.

The single-stranded hindbrain cDNA was used asemplate for nested PCR with primers designed againsthe tyrosine kinase domains of the human erbB familyHER1-4) and provided by Anthony Graham. Primerrb1 sequence is CGGAATTCCAA/G ATT/C GCNAA/G GGN ATG where the first block includes ancoRI site to simplify cloning the PCR product and the

riplets encode residues 826 to 831 of HER4 (Plowman etl., 1993). Primer erb2 is CGGGAATTC G/ATN TTNTA/C/T AAA/G TGG ATG, including a 58 EcoRI sitend triplets for residues 882 to 887 of HER4. Primer erb3s GCTCTAGA A/GTC NGC A/GTC A/G/TAT CATCA with a 58 XbaI site to simplify cloning and the

omplement of triplets encoding residues 957 to 962 ofER4. PCR conditions were as follows. Round 1 wasone in a 50-µl reaction with 2 µl (1/10) cDNA template,µM each erb1 and erb3 primers, 200 µM each dNTP, 2
M MgCl2, and 1 unit Taq DNA polymerase. Cycling
arameters were 94° 28, 303(94° 309, 43° 28, 72° 28), 72°mb

08. Round 2 was done in a 50-µl reaction with 2 µl1/25) product from round 1 (used directly withouturification), 1 µM each erb2 and erb3 primers, 200 µMach dNTP, 1 mM MgCl2, and 1 unit Taq DNA polymer-se. Cycling parameters were as for round 1 except thathe annealing temperature was dropped to 40°C.

The major product of the second PCR was a band ofhe predicted size of about 255 base pairs (bp). AftercoRI and XbaI digestion of this product, the major bandas purified from an agarose gel and cloned intoluescript SK1 (Stratagene). Eighteen independentlones were sequenced. Predicted amino acid sequencesf the region between erb2 and erb3 primers matchedER4 perfectly in 17/18 clones. The remaining cloneiffered only in that it encoded asparagine rather thanerine at the position corresponding to position 91 ofER4 (Plowman et al., 1993). Since asparagine does not

ccur at this position in any human erbB sequence, andince the change was due to a single nucleotide differ-nce, this likely represents a PCR artefact. A stage 12–15hole chick embryo cDNA library in lzapII provided

y Angela Nieto and David Wilkinson was screenedith one of the 17 clones of PCR product that matchedER4 perfectly. Prehybridization and hybridization were

oth at 55°C in 53 SSPE, 5% SDS (Shain et al., 1995). Of.4 3 105 library clones screened, 4 independent onesooked positive after three rounds of hybridization.xcision of the Bluescript plasmid with cDNA insert

rom each of these with R408 helper phage was followedy Southern blotting of restriction-enzyme-digested plas-ids to nylon membrane. Membrane was hybridizedith the same probe under the same conditions as used

or the library screen. Three of the four plasmids carriednserts that hybridized with the erbB4 probe. These werealled clones 2, 4, and 6. Restriction mapping and partialequencing revealed that clone 6 corresponds to the 38nd of clone 2 and that these are both erbB4 cDNAs.lone 4 is a different erbB and will not be discussed

urther. The clone 2 cDNA was sequenced in its entiretyn both strands using standard subcloning and internalrimer strategies.

n Situ Hybridization

Embryos up to E5 were treated to avoid trapping ofrobe and antibody in body cavities by cutting holes orlits into the neural tube, eyes, otic vesicles, and heart.or embryos at E6 to E15, hindbrains with a little caudalidbrain and rostral spinal cord attached were dis-

ected out and all tissue external to the basement
embrane pulled off with forceps. Embryos or hind-
rains were fixed in 4% paraformaldehyde in phosphate-

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uffered saline (PBS) at least a few hours at roomemperature for younger embryos but generally over-ight or longer at 4°C. E15 hindbrains were rinsed inBS, embedded in 20% gelatin in PBS, refixed at leastvernight at 4°C, and sectioned at 200 µm on a vibra-ome. The sections were collected in fixative, one sectioner well in a 24-well plate, in preparation for in situybridization. All younger embryos or hindbrains wereubjected to in situ hybridization in wholemount.

For all nrg1 and nrg1 erbB4 double in situs, and forrbB4 in situs on E5 or older tissue, the in situ hybridiza-ion protocol was closely based on an unpublishedrotocol of Timothy Sanders and Clifton Ragsdale. ForrbB4 in situs on E4 or younger embryos, the protocolsed was either the one based on Sanders and Ragsdale

unpublished) or one based on Henrique et al. (1995),ith some modifications from Wilkinson (1992).

n Situ Hybridization Probes

erbB4. The probe generally used was synthesizedrom the 670-bp BamHI restriction fragment at the 58 endf the chick erbB4 cDNA (58 BamHI site in linker; Fig.B). Occasionally the 1-kb BamHI fragment nearer the 38nd of the cDNA was used instead. In some earlyxperiments a sense probe generated from the formeremplate was used; no signal was observed with thisegative control probe.nrg1. A proARIA-1 cDNA obtained from Kenneth

osen and Gerald Fischbach was used to make tem-lates for in situ probes. The 538-bp Asp700 fragment

nucleotide positions 468 to 1006; Falls et al., 1993) andhe 835-bp EcoRI–PvuII fragment (positions 15 to 842;coRI site in linker) were subcloned into Bluescript SK1nd used to generate antisense probes. Both probesnclude all or most of the sequence encoding theGF-like domain and should thus detect mRNAs encod-

ng all known nrg1 isoforms. The 835 nucleotide probelso includes the Ig domain. While this probe sometimesave a stronger signal than the 538-nucleotide probe, itlso sometimes produced higher background in olderindbrains, and for this reason the 538-nucleotide probeas the more often used one. An initial experiment

ncluded the sense version of the 835-nucleotide probes a negative control; no signal was observed with thisrobe.Probe preparation. About 625 ng of linearized plas-id was used as template for in vitro transcription usingPromega kit. Probes were resuspended in 100 µl in situybridization buffer after precipitation from 300 mM
iCl and 2.3 vol ethanol and were typically diluted00-fold in hybridization buffer before use. w
sl-1/2 Immunostaining

Some embryos or hindbrains were subjected to immu-ostaining with a-isl-1/2 antibody (Thor et al., 1991),indly provided by Thomas Edlund, after in situ hybrid-

zation. This antibody recognizes isl1 and isl2 transcrip-ion factors and labels motor nuclei in the chick brain-tem (Varela-Echavarrıa et al., 1996). It thus allowed uso use the motor nuclei in the hindbrain as landmarks tossist in identification of regions labeled with our in situybridization probes. Immunostaining was performedfter the in situ hybridization color reaction had beentopped by washing with 20 mM EDTA in PBS. Speci-ens were refixed in 4% paraformaldehyde in PBS a

ew hours at room temperature or overnight at 4°C. Thisas followed by blocking with 0.03–0.06% hydrogeneroxide, 1% Triton X-100, and 5% goat serum in PBSvernight at 4°C. Incubation in a-isl-1/2 primary anti-ody was at 4°C for 3 to 6 days at a dilution of 1 in 500 inBS with 10% goat serum, 1% Triton X-100, and 0.02%odium azide. Secondary antibody was horse radisheroxidase (HRP)-conjugated donkey a-rabbit Ig frommersham used at 1 in 100 in PBS with 1% goat serum

nd 1% Triton X-100 at 4°C overnight. Color substrateas diaminobenzidine (DAB) at 0.5 mg/ml in PBS at

°C for 2 to 3 h followed by 0.5 mg/ml DAB, 0.009%ydrogen peroxide in PBS at room temperature.

latmounting and Sectioning

Some specimens were flatmounted or sectioned aftern situ hybridization and/or immunostaining. For flat-

ounts, hindbrains were dissected free of surroundingissue, cut open along the dorsal midline, and flattenedetween a glass slide and coverslip in 80–90% glycerol

n PBS.For sections, specimens were embedded in 27% albu-in, 18% sucrose, 0.45% gelatin in PBS, to which 5–10%

ol of 25% glutaraldehyde was added in the embeddingould. Blocks were sectioned on a vibratome at a

hickness of 50 µm. Sections were collected on gelatin-oated glass slides and covered with 80–90% glycerol inBS under a glass coverslip. A minority of specimensas embedded in 20% gelatin in PBS. These blocks were

llowed to harden at 4°C for a minimum of an hourefore trimming, then fixed at least overnight at 4°C in% paraformaldehyde in PBS. Sectioning and mountingas as for albumin/sucrose/gelatin-embedded tissue.

hotography and Image Processing

Photography was under a dissecting microscope forhole embryos or under a Zeiss Axiophot for flat-

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ounted or sectioned material. Either a conventionalamera loaded with Fujichrome 64T film or a SPOTamera and digital image collection system was used forhotography on either microscope. Color slide imagesere converted to digital images using a Nikon Cool-

can slide scanner. All digital images were processednd Figs. 2 to 5 assembled in Adobe Photoshop.

CKNOWLEDGMENTS

We are grateful to Anthony Graham for PCR primers, to Angelaieto and David Wilkinson for the chick cDNA library, to Kennethosen and Gerald Fischbach for the proARIA cDNA, to Thomasdlund for the a-isl-1/2 antibody, and to Timothy Sanders and Cliftonagsdale for their in situ hybridization protocol. We thank Luis Puelles

or invaluable help in identification of erbB4-positive nuclei in E7 and9 hindbrains, and Jon Golding and Martin Gassmann for communi-ating unpublished data. This work was supported by the Wellcomerust.

EFERENCES

mbrosiani, J., Armengol, J. A., Martinez, S., and Puelles, L. (1996).The avian inferior olive derives from the alar neuroepithelium of therhombomeres 7 and 8: An analysis by using chick-quail chimericembryos. Neuroreport 7: 1285–1288.nton, E. S., Marchionni, M. A., Lee, K.-F., and Rakic, P. (1997). Role ofGGF/neuregulin signaling in interactions between migrating neu-rons and radial glia in the developing cerebral cortex. Development124: 3501–3510.

ritsch, S., Li, L., Kirchhoff, S., Theuring, F., Brinkmann, V., Birchmeier,C., and Riethmacher, D. (1998). The erbB2 and erbB3 receptors andtheir ligand, neuregulin-1, are essential for development of thesympathetic nervous system. Genes Dev. 12: 1825–1836.

usfield, S. J., Michinick, D. A., Chickering, T. W., Revett, T. L., Ma, J.,Woolf, E. A., Comrack, C. A., Dussault, B. J., Woolf, J., Goodearl,A. D. J., and Gearing, D. P. (1997). Characterization of a neuregulin-related gene, don-1, that is highly expressed in restricted regions ofthe cerebellum and hippocampus. Mol. Cell. Biol. 17: 4007–4014.

arraway III, K. L., Weber, J. L., Unger, M. J., Ledesma, J., Uy, N.,Gassmann, M., and Lai, C. (1997). Neuregulin-2, a new ligand oferbB3/erbB4-receptor tyrosine kinases. Nature 387: 512–516.

hang, H., Riese II, D. J., Gilbert, W., Stern, D. F., and McMahan, U. J.(1997). Ligands for erbB-family receptors encoded by a neuregulin-like gene. Nature 387: 509–511.

hedotal, A., Pourquie, O., Ezan, F., San Clemente, H., and Sotelo, C.(1996). BEN as presumptive target recognition molecule during thedevelopment of the olivocerebellar system. J. Neurosci. 16: 3296–3310.

hen, M. S., Bermingham-McDonogh, O., Danehy Jr., F. T., Nolan, C.,Schere, S. S., Lucas, J., Gwynne, D., and Marchionni, M. A. (1994).Expression of multiple neuregulin transcripts in postnatal ratbrains. J. Comp. Neurol. 349: 389–400.

homczynski, P., and Sacchi, N. (1987). Single-step method of RNA
isolation by acid guanidinium thiocyanate-phenol-chloroform extrac-tion. Anal. Biochem. 162: 156–159.
orfas, G., Rosen, K. M., Aratake, H., Krauss, R., and Fischbach, G. D. H

(1995). Differential expression of ARIA isoforms in the rat brain.Neuron 14: 103–115.

oussens, L., Yang-Feng, T. L., Liao, Y.-C., Chen, E., Gray, A., McGrath,J., Seebrug, P. H., Libermann, T. A., Schlessinger, J., Francke, U.,Levinson, A., and Ullrich, A. (1985). Tyrosine kinase receptor withextensive homology to EGF receptor shares chromosomal locationwith neu oncogene. Science 230: 1132–1139.

lenius, K., Corfas, G., Paul, S., Choi, C. J., Rio, C., Plowman, G. D.,and Klagsbrun, M. (1997a). A novel juxtamembrane domain isoformof HER4/erbB4. J. Biol. Chem. 272: 26761–26768.

lenius, K., Paul, S., Allison, G., Sun, J., and Klagsbrun, M. (1997b).Activation of HER4 by heparin-binding EGF-like growth factorstimulates chemotaxis but not proliferation. EMBO J. 16: 1268–1278.

rickson, S. L., O’Shea, K. S., Ghaboosi, N., Loverro, L., Frantz, G.,Bauer, M., Lu, L. H., and Moore, M. W. (1997). ErbB3 is required fornormal cerebellar and cardiac development: a comparison witherbB2- and heregulin-deficient mice. Development 124: 4999–5011.

alls, D. G., Rosen, K. M., Corfas, G., Lane, W. S., and Fischbach, G. D.(1993). ARIA, a protein that stimulates acetylcholine receptorsynthesis, is a member of the neu ligand family. Cell 72: 801–815.

rancoeur, J. R., Richardson, P. M., Dunn, R. J., and Carbonetto, S.(1995). Distribution of erb-B2, erb-B3, and erb-B4 in the developingavian nervous system. J. Neurosci. Res. 41: 836–845.assmann, M., Casagranda, F., Oriolo, D., Simon, H., Lai, C., Klein, R.,and Lemke, G. (1995). Aberrant neural and cardiac development inmice lacking the erbB4 neuregulin receptor. Nature 378: 390–394.assmann, M., and Lemke, G. (1997). Neuregulins and neuregulinreceptors in neural development. Curr. Op. Neurobiol. 7: 87–92.ilbertson, R. J., Clifford, S. C., MacMeekin, W., Wright, C., Perry,R. H., Kelly, P., Pearson, A. D. J., and Lunec, J. (1998). Expression ofthe ErbB-Neuregulin signaling network during human cerebellardevelopment: Implications for the biology of medulloblastoma.Cancer Res. 58: 3932–3941.oodearl, A. D. J., Yee, A. G., Sandrock Jr., A. W., Corfas, G., andFischbach, G. D. (1995). ARIA is concentrated in the synaptic basallamina of the developing chick neuromuscular junction. J. Cell Biol.130: 1423–1434.raham, A., Heyman, I., and Lumsden, A. (1993). Even-numberedrhombomeres control the apoptotic elimination of neural crest cellsfrom odd-numbered rhombomeres in the chick hindbrain. Dev. 119:233–245.raham, A., Francis-West, P., Brickell, P., and Lumsden, A. (1994). Thesignalling molecule BMP4 mediates apoptosis in the rhombence-phalic neural crest. Nature 372: 684–686.raham, A., and Lumsden, A. (1996). Interactions between rhombo-meres modulate Krox-20 and follistatin expression in the chickembryo hindbrain. Dev. 122: 473–480.amburger, V., and Hamilton, H. L. (1951). A series of normal stages inthe development of the chick embryo. J. Morphol. 88: 49–92.anaway, J. (1967). Formation and differentiation of the externalgranular layer of the chick cerebellum. J. Comp. Neurol. 131: 1–14.arkmark, W. (1954). Cell migrations from the rhombic lip to theinferior olive, the nucleus raphe and the pons. A morphological andexperimental investigation on chick embryos. J. Comp. Neurol. 100:115–209.eaton, M. B., and Moody, S. A. (1980). Early development andmigration of the trigeminal motor nucleus in the chick embryo. J.Comp. Neurol. 189: 61–99.enrique, D., Adam, J., Myat, A., Chitnis, A., Lewis, J., and Ish-
Horowicz, D. (1995). Expression of a Delta homologue in prospec-tive neurons in the chick. Nature 375: 787–790.igashiyama, S., Horikawa, M., Yamada, K., Ichino, N., Nakano, N.,

H

H

K

K

K

L

L

L

M

M

M

M

M

M

N

O

O

P

P

R

R

R

R

R

R

R

S

S

S

S

T

U


Nakagawa, T., Miyagawa, J., Matsushita, N., Nagatsu, T., Taniguchi,N., and Ishiguro, H. (1997). A novel brain-derived member of theepidermal growth factor family that interacts with ErbB3 andErbB4. J. Biochem. 122: 675–680.o, W.-H., Armanini, M. P., Nuijens, A., Phillips, H. S., and Osheroff,P. L. (1995). Sensory and motor neuron-derived factor. J. Biol. Chem.270: 14523–14532.olmes, W. E., Sliwkowski, M. X., Akita, R. W., Henzel, W. J., Lee, J.,Park, J. W., Yansura, D., Abadi, N., Raab, H., Lewis, G. D., Shepard,H. M., Kuang, W.-J., Wood, W. I., Goeddel, D. V., and Vandlen, R. L.(1992). Identification of heregulin, a specific activator of p185erbB2.Science 256: 1205–1210.

omurasaki, T., Toyoda, H., Uchida, D., and Morimoto, S. (1997).Epiregulin binds to epidermal growth factor receptor and ErbB-4and induces tyrosine phosphorylation of epidermal growth factorreceptor, ErbB-2, ErbB-3, and ErbB-4. Oncogene 15: 2841–2848.

ramer, R., Bucay, N., Kane, D. J., Martin, L. E., Tarpley, J. E., andTheill, L. E. (1996). Neuregulins with an Ig-like domain are essentialfor mouse myocardial and neuronal development. Proc. Natl. Acad.Sci. USA 93: 4833–4838.

raus, M. H., Issing, W., Miki, T., Popescu, N. C., and Aaronson, S. A.(1989). Isolation and characterization of ERBB3, a third member ofthe ERBB/epidermal growth factor receptor family: Evidence foroverexpression in a subset of human mammary tumors. Proc. Natl.Acad. Sci. USA 86: 9193–9197.

ee, K.-F., Simon, H., Chen, H., Bates, B., Hung, M.-C., and Hauser, C.(1995). Requirement for neuregulin receptor erbB2 in neural andcardiac development. Nature 378: 394–398.

emke, G. (1996). Neuregulins in development. Mol. Cell. Neurosci. 7:247–262.

iu, X., Hwang, H., Cao, L., Buckland, M., Cunningham, A., Chen, J.,Chien, K. R., Graham, R. M., and Zhou, M. (1998). Domain-specificgene disruption reveals critical regulation of neuregulin signalingby its cytoplasmic tail. Proc. Natl. Acad. Sci. USA 95: 13024–13029.ahanthappa, N. K., Anton, E. S., and Matthew, W. D. (1996). Glialgrowth factor 2, a soluble neuregulin, directly increases Schwanncell motility and indirectly promotes neurite outgrowth. J. Neurosci.16: 4673–4683.archionni, M. A., Goodearl, A. D. J., Chen, M. S., Bermingham-McDonogh, O., Kirk, C., Hendricks, M., Danehy, F., Misum, D.,Sudhalter, J., Kobayashi, K., Wroblewski, D., Lynch, C., Baldassare,M., Hiles, I., Davis, J. B., Hsuan, J. J., Totty, N. F., Otsu, M., McBurney,R. N., Waterfield, M. D., Stroobant, P., and Gwynne, D. (1993). Glialgrowth factors are alternatively spliced erbB2 ligands expressed inthe nervous system. Nature 362: 312–318.eier, T., Masciulli, F., Moore, C., Schoumacher, F., Eppenberger, U.,Denzer, A. J., Jones, G., and Brenner, H. R. (1998). Agrin can mediateacetylcholine receptor gene expression in muscle by aggregation ofmuscle-derived neuregulins. J. Cell Biol. 141: 715–726.eyer, D., and Birchmeier, C. (1994). Distinct isoforms of neuregulinare expressed in mesenchymal and neuronal cells during mousedevelopment. Proc. Natl. Acad. Sci. USA 91: 1064–1068.eyer, D., and Birchmeier, C. (1995). Multiple essential functions ofneuregulin in development. Nature 378: 386–390.eyer, D., Yamaai, T., Garratt, A., Riethmacher-Sonnenberg, E., Kane,D., Theill, L. E., and Birchmeier, C. (1997). Isoform-specific expres-sion and function of neuregulin. Development 124: 3575–3586.akao, J., Shinoda, J., Nakai, Y., Murase, S., and Uyemura, K. (1997).Apoptosis regulates the number of Schwann cells at the premyelin-
ating stage. J. Neurochem. 168: 1853–1862.rr-Urtreger, A., Trakhtenbrot, L., Ben-Levy, R., Wen, D., Rechavi, G.,Lonai, P., and Yarden, Y. (1993). Neural expression and chromo-
somal mapping of Neu differentiation factor to 8p12-p21. Proc. Natl.Acad. Sci. USA 90: 1867–1871.zaki, M., Sasner, M., Yano, R., Lu, H. S., and Buonanno, A. (1997).Neuregulin-b induces expression of an NMDA-receptor subunit.Nature 390: 691–694.

lowman, G. D., Whitney, G. S., Neubauer, M. G., Green, J. M.,McDonald, V. L., Todaro, G. J., and Shoyab, M. (1990). Molecularcloning and expression of an additional epidermal growth factorreceptor-related gene. Proc. Natl. Acad. Sci. USA 87: 4905–4909.

lowman, G. D., Culouscou, J.-M., Whitney, G. S., Green, J. M.,Carlton, G. W., Foy, L., Neubauer, M. G., and Shoyab, M. (1993).Ligand-specific activation of HER4/p180erbB4, a fourth member ofthe epidermal growth factor receptor family. Proc. Natl. Acad. Sci.USA 90: 1746–1750.

aabe, T. D., Suy, S., Welcher, A., and DeVries, G. H. (1997). Effect ofneu differentiation factor isoforms on neonatal oligodendrocytefunction. J. Neurosci. Res. 50: 755–768.

iese II, D. J., Bermingham, Y., van Raaij, T. M., Buckley, S., Plowman,G. D., and Stern, D. F. (1996a). Betacellulin activates the epidermalgrowth factor receptor and erbB-4, and induces cellular responsepatterns distinct from those stimulated by epidermal growth factoror neuregulin-b. Oncogene 12: 345–353.

iese II, D. J., Kim, E. D., Elenius, K., Buckley, S., Klagsbrun, M.,Plowman, G. D., and Stern, D. F. (1996b). The epidermal growthfactor receptor couples transforming growth factor-a, heparin-binding epidermal growth factor-like factor, and amphiregulin toneu, erbB-3 and erbB-4. J. Biol. Chem. 271: 20047–20052.

ietmacher, D., Sonnenberg-Riethmacher, E., Brinkmann, V., Yamaai,T., Lewin, G. R., and Birchmeier, C. (1997). Severe neuropathies inmice with targeted mutations in the erbB3 receptor. Nature 389:725–730.

imer, M., Cohen, I., Lomo, T., Burden, S. J., and McMahan, U. J.(1998). Neuregulins and erbB receptors at neuromuscular junctionsand at agrin-induced postsynaptic-like apparatus in skeletal muscle.Mol. Cell. Neurosci. 12: 1–15.

io, C., Rieff, H. I., Qi, P., and Corfas, G. (1997). Neuregulin and erbBreceptors play a critical role in neuronal migration. Neuron 19: 39–50.

yder, E. F., and Cepko, C. L. (1994). Migration patterns of clonallyrelated granule cells and their progenitors in the developing chickcerebellum. Neuron 12: 1011–1029.

chroering, A., and Carey, D. J. (1998). Sensory and motor neuron-derived factor is a transmembrane heregulin that is expressed on theplasma membrane with the active domain exposed to the extracellu-lar environment. J. Biol. Chem. 273: 30643–30650.

hah, N. M., Marchionni, M. A., Isaacs, I., Stroobant, P., and Anderson,D. J. (1994). Glial growth factor restricts mammalian neural creststem cells to a glial fate. Cell 77: 349–360.

hain, D. H., Neuman, T., and Zuber, M. X. (1995). A novel initiatorregulates expression of the non-tissue-specific helix-loop-helix geneME1. Nucleic Acids. Res. 23: 1696–1703.

rinivasan, R., Poulsom, R., Hurst, H. C., and Gullick, W. J. (1998).Expression of the c-erbB-4/HER4 protein and mRNA in normalhuman fetal and adult tissues and in a survey of nine solid tumourtypes. J. Pathol. 185: 236–245.

hor, S., Ericson, J., Brannstrom, T., and Edlund, T. (1991). Thehomeodomain LIM protein Isl-1 is expressed in subsets of neuronsand endocrine cells in the adult rat. Neuron 7: 881–889.llrich, A., Coussens, L., Hayflick, J. S., Dull, T. J., Gray, A., Tam, A. W.,
Lee, J., Yarden, Y., Libermann, T. A., Schlessinger, J., Downward, J.,Mayes, E. L. V., Whittle, N., Waterfield, M. D., and Seeburg, P. H.(1984). Human epidermal growth factor receptor cDNA sequence

V

V

V

V

W

W

W

Y

Y

Z


and aberrant expression of the amplified gene in A431 epidermoidcarcinoma cells. Nature 309: 418–425.

an der Want, J. J. L., Wiklund, L., Guegan, M., Ruigrok, T., and Voogd,J. (1989). Anterograde tracing of the rat olivocerebellar system withphaseolus vulgaris leucoagglutinin (PHA-L). Demonstration ofclimbing fiber collateral innervation of the cerebellar nuclei. J. Comp.Neurol. 288: 1–18.

arela-Echavarrıa, A., Pfaff, S. L., and Guthrie, S. (1996). Differentialexpression of LIM homeobox genes among motor neuron subpopu-lations in the developing chick brain stem. Mol. Cell. Neurosci. 8:242–257.

erdi, J. M., Groves, A. K., Farinas, I., Jones, K., Marchionni, M. A.,Reichardt, L. F., and Anderson, D. J. (1996). A reciprocal cell-cellinteraction mediated by NT-3 and neuregulins controls the earlysurvival and development of sympathetic neuroblasts. Neuron 16:515–527.

ogel, K. S. (1992). Origins and early development of vertebratecranial sensory neurons. In Sensory Neurons: Diversity, Development,and Plasticity (S. A. Scott, Ed.), pp. 171–193. Oxford Univ. Press,
Oxford, UK.ang, J. Y., Frenzel, K. E., Wen, D., and Falls, D. L. (1998). Transmem-
kinase implicated in development of visuospatial cognition. J. Biol.Chem. 273: 20525–20534.en, D., Peles, E., Cupples, R., Suggs, S. V., Bacus, S. S., Luo, Y., Trail,G., Hu, S., Silbiger, S. M., Levy, R. B., Koski, R. A., Lu, H. S., andYarden, Y. (1992). Neu differentiation factor: a transmembraneglycoprotein containing an EGF domain and an immunoglobulinhomology unit. Cell 69: 559–572.ilkinson, D. G. (1992). Whole-mount in situ hybridisation of verte-brate embryos. In In Situ Hybridisation, a Practical Approach (D. G.Wilkinson, Ed.), pp. 1–83. I.R.L., Oxford, UK.

ang, J. F., Zhou, H., Pun, S., Ip, N. Y., Peng, H. B., and Tsim, K. W. K.(1998). Cloning of cDNAs encoding Xenopus neuregulin: expressionin myotomal muscle during embryo development. Mol. Brain Res.58: 59–73.

ang, Y., Spitzer, E., Meyer, D., Sachs, M., Niemann, C., Harmann, G.,Weidner, K. M., Birchmeier, C., and Birchmeier, W. (1995). Sequen-tial requirement of hepatocyte growth factor and neuregulin in themorphogenesis and differentiation of the mammary gland. J. CellBiol. 131: 215–226.

hang, D., Sliwkowski, M. X., Mark, M., Frantz, G., Akita, R., Sun, Y.,
Hillan, K., Crowley, C., Brush, J., and Godowski, P. J. (1997).Neuregulin-3 (NRG-3): A novel neural tissue-enriched protein that
brane neuregulins interact with LIM kinase 1, a cytoplasmic protein binds and activates erbB4. Proc. Natl. Acad. Sci. USA 94: 9562–9567.

Received January 25, 1999Accepted March 3, 1999

distribution of neuregulin-1 (nrg1) and erbb4 transcripts in embryonic chick hindbrain

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