control of tectum territory by shh - development

INTRODUCTION

Midbrain consists of two major components along dorsoventral(DV) axis, the tectum and the tegmentum. It has been shownin chick-quail chimera that the optic tectum derives exclusivelyfrom the midbrain alar plate (Senut and Alvarado-Mallart,1987). After day 3 of incubation (E3), the alar plate undergoesrapid cell proliferation to form an enlarged tectum. In contrast,the basal plate gives rise to the midbrain tegmentum. Insteadof massive cell proliferation, neural differentiation takes placein this ventral area to produce many nuclei related to motorfunction. Although these two structures undergo quite differentdevelopmental programs, it is largely unknown how thedifferences are molecularly controlled along the DV axis.

Many transcription factors and growth factors are involvedin midbrain pattern formation along anteroposterior (AP) axis(reviewed by Lumsden and Krumlauf, 1996). The factors sofar reported are: En-1/2 (Wurst et al., 1994; Millen et al., 1994),Pax-2/5 (Krauss et al., 1992; Urbánek et al., 1994; Brand et al.,1996; Funahashi et al., 1999; Okafuji et al., 1999), Fgf8(Crossely et al., 1996) and Otx-2 (Acampora et al., 1995;Matsuo et al., 1995; Ang et al., 1996). Misexpression of oneof these factors can induce ectopic midbrain structure in the

diencephalon or in the hindbrain (Araki and Nakamura, 1999;Okafuji et al., 1999; Funahashi et al., 1999; Crossely et al.,1996; Shamin et al., 1999; Broccoli et al., 1999; Katahira etal., 2000). In such cases, ectopic tectum emerges exclusivelyfrom the alar plate, i.e., DV polarity of the induced structure isconserved. This suggests that a distinct mechanism exists fordetermining regional identity of midbrain structure along DVaxis that is independent of that of the AP axis.

Sonic hedgehog (Shh) is produced in the notochord and floorplate, and is implicated in inducing ventral cell types in CNS(Tanabe and Jessell, 1996). In neural explants, aminoterminuscleavage product of Shh protein (Shh-N) can induce both floorplate and motor neurons depending on its concentration(Roelink et al., 1995). It is now accepted that the amino-terminus cleavage product of Hedgehog family protein (Hh-N)has all the signaling activity (Porter et al., 1995; Ekker et al.,1995; Fan et al., 1995; Martí et al., 1995; Roelink et al., 1995).In vertebrates, Shh-N acts as long-range signaling for somitepatterning (Fan and Tessier-Lavigne, 1994; Fan et al., 1995).Shh-N can regulate cells in neural plate explants to differentiateinto floor plate cells, motor neurons and interneurons accordingto its concentration (Ericson et al., 1996). In vivomisexpression experiments of Shh revealed that ectopic Shh

1131Development 127, 1131-1140 (2000)Printed in Great Britain © The Company of Biologists Limited 2000DEV1486

Chick midbrain comprises two major components alongthe dorsoventral axis, the tectum and the tegmentum. Thealar plate differentiates into the optic tectum, while thebasal plate gives rise to the tegmentum. It is largelyunknown how the differences between these two structuresare molecularly controlled during the midbraindevelopment. The secreted protein Sonic hedgehog (Shh)produced in the notochord and floor plate inducesdifferentiation of ventral cell types of the central nervoussystem. To evaluate the role of Shh in the establishment ofdorsoventral polarity in the developing midbrain, we haveectopically expressed Shh unilaterally in the brain vesiclesincluding whole midbrain of E1.5 chick embryos in ovo.Ectopic Shh repressed normal growth of the tectum,producing dorsally enlarged tegmentum region. Inaddition, the expression of several genes crucial for tectumformation was strongly suppressed in the midbrain andisthmus. Markers for midbrain roof plate were inhibited,indicating that the roof plate was not fully generated. After

E5, the tectum territory of Shh-transfected side wassignificantly reduced and was fused with that ofuntransfected side. Moreover, ectopic Shh induced aconsiderable number of SC1-positive motor neurons,overlapping markers such as HNF-3β (floor plate), Isl-1(postmitotic motor neuron) and Lim1/2. Dopaminergic andserotonergic neurons were also generated in the dorsallyextended region. These changes indicate that ectopic Shhchanged the fate of the mesencephalic alar plate to that ofthe basal plate, suppressing the massive cell proliferationthat normally occurs in the developing tectum. Takentogether our results suggest that Shh signaling restricts thetectum territory by controlling the molecular cascade fortectum formation along dorsoventral axis and byregulating neuronal cell diversity in the ventral midbrain.

Key words: Midbrain, Tectum, Tegmentum, Dorsoventral, Sonichedgehog, Chick

SUMMARY

Control of chick tectum territory along dorsoventral axis by Sonic hedgehog

Yuji Watanabe* and Harukazu Nakamura

Department of Molecular Neurobiology, Institute of Development, Aging and Cancer, Tohoku University, Aoba-ku,Sendai 980-8575, Japan*Author for correspondence (e-mail: [email protected])

Accepted 1 December 1999; published on WWW 8 February 2000

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can induce floor plate cells in dorsal neuraltube (Echelard et al., 1993; Roelink et al.,1994). However, it has not been elucidatedhow the ventral cell types other than floorplate cells are regulated in vivo along DVaxis by Shh signal.

In this study, we focus on the role of Shhin DV pattern formation of the midbrain byShh misexpression in the embryonic brain.We examined the expression of the tectum-related genes in Shh-transfected midbrainand followed tectal development. Drasticchanges in morphology along DV axis led toan investigation of cell proliferation andneural differentiation, which normally occur

Y. Watanabe and H. Nakamura

Fig. 1. In ovo electroporation into chick embryos.pEGFP-N1 alone (C,E,G) or pEGFP-N1 pluspMiwII-Shh (D,F,H) was transfected. (A) Dorsalview of brain vesicle. Plasmid DNA (red) wasinjected into the central canal at stage 10. (B) DNAwas electroporated by pulses charged between a pairof electrodes placed besides of the head.(C,D) Efficiency of transfection was monitored inovo by GFP expression 1 day after electroporation.Lateral view of the right side of the head is shown.Rostral is to the right. Introduced GFP wasabundantly observed in diencephalon, midbrain andhindbrain. There is no significant difference in GFPlocalization between C and D. (E,F) GFPlocalization 2 days after electroporation. (E, right)Dorsal view shows GFP is exclusively localized inunilateral half of the brain vesicle. (E, left) NoteGFP is dispersed in whole midbrain. (F) GFP is notobserved in dorsal midbrain. Dorsal limit of GFP-positive region is indicated with an arrow.(G,H) Expression of Shh mRNA in the brain vesiclesisolated from the embryos shown in E and F,respectively. Lateral views (left) and anterior views(right). (G) Endogenous Shh expression is detectedin the normally developed brain vesicle. (H) Ectopicexpression of Shh is introduced in lateral region.Note that the tectum is not properly developed inShh-transfected side. Dorsal limit of GFP-positiveregion shown in F is colocalized with that ofintroduced Shh expression indicated with an arrow.Abbreviations: tel, telencephalon; di, diencephalon;mid, midbrain; hind, hindbrain. Bars, 500 µm.

Fig. 2. Expression of the tectum-related genes inShh-transfected brains at E3.5. Localization ofShh mRNA is detected in red except in D.Expression of Pax-7 is shown in black (F) andother gene expression is stained dark blue (B-E,G-Q). The level of the isthmus is indicated withan arrow in A. Expressions of Shh-transfectedside (right) were compared with untransfectedside (left) after bisecting the brain at the midline(B-I). Lateral (left) and dorsal (right) views areshown in J-M. Expression of normal (J,L,N,P)and Shh-transfected embryos (K,M,O,Q) areshown. Abbreviations: tel, telencephalon; di,diencephalon; mid, midbrain; hind, hindbrain.Bar, 500 µm.

1133Control of tectum territory by Shh

in the developing tectum and tegmentum, respectively.Moreover the localization of the ventral cell types wasdetermined to analyze the effects of Shh in cell typespecification. The results of these studies indicated that Shh isinvolved in the patterning of the tectum territory by controllingthe molecular cascade for tectum formation along DV axis andby regulating the neuronal cell diversity in ventral midbrain.

MATERIALS AND METHODS

In ovo electroporationIn ovo electroporation method (Muramatsu et al., 1997) was modifiedto obtain efficient transfection into brain vesicles (Funahashi et al.,1999; Nakamura and Funahashi, 2000).

Fertile chick eggs were incubated at 38°C in a humidifiedatmosphere for 1.5 days. Embryos were staged according toHamburger and Hamilton (1951). Chick Shh-coding region (Riddle etal., 1993) was inserted into pMiwII expression vector derived frompMiwSV, which carries chick β-actin promoter and RSV enhancer(Wakamatsu et al., 1997). This Shh construct (pMiwII-Shh) wasmixed with GFP expression vector (pEGFP-N1; Clonetech) or lacZexpression vector (pMiwZ; Suemori et al., 1990) in a finalconcentration at 1 µg/µl. pEGFP-N1 or pMiwZ alone was used forthe control experiment. For the experiment shown in Fig. 7, Shh-coding region was inserted into another expression vector (pIRES2-EGFP; Clonetech) containing the internal ribosomal entry site of theencephalomyocarditis virus, which permits Shh and GFP genes to betranslated from a single bicistronic mRNA.

DNA solution of 0.1-0.2 µl in Tris-EDTA buffer was injected intothe central canal with a micropitette at stage 10 as described in Fig.1A. A pair of electrodes (0.5 mm diameter, 1.0 mm length and 4 mmdistance between the electrodes) were put beside brain vesicles on thevitelline membrane as shown in Fig. 1B. A rectangular pulse of 25 V,50 mseconds was charged 4 times in 1 second intervals by theelectroporator (CUY21, Tokiwa Science, Fukuoka, Japan; T820 andOPTIMIZERTM500, BTX, San Diego, CA, USA). Efficiency ofelectroporation was monitored by GFP expression under afluorescence dissection microscope (MZ FLIII, Leica).

Expression of the transfected gene can be detected 2-3 hours afterelectroporation and strong expression was observed during thefollowing 24-48 hours (Funahashi et al., 1999; Fig. 1). Transfectionof GFP or lacZ construct alone did not cause any morphologicalabnormalities. DNA was transfected into the right side unless detailedotherwise. Details of the procedure are also described elsewhere(Nakamura and Funahashi, 2000).

In situ hybridization of whole mounts and sections For whole-mount staining, embryos were fixed in 4% formaldehydein PBS at 4°C for 2 hours to overnight and the brains were dissectedout. They were rinsed twice with PBT (PBS-0.1% Tween 20) anddehydrated in a graded series of methanol in PBS to store in 100%

Fig. 3. Development of normal (A,C,F) and Shh-transfected brain(B,D,E,G) at E5.5 (A,B), E12.5 (C-E) and E18.5 (F,G). Shh wastransfected into right (B,D,G) and left (E) brain hemisphere.(A,B) Three views of E5.5 brain from dorsal (upper), lateral (left)and anterior (right) are shown. The level of the dorsal midline isindicated by arrows. The tectum territory of Shh-transfected side issignificantly reduced and the dorsal midline dividing right-lefttectum is unclear (B). (C-E) Dorsal views of E12.5 brain. HRP wasinjected into the right eye ball to detect retinal fibers. The fusedtectum is mainly composed of the untransfected hemisphere (D,E).(F,G) Dorsal views of E18.5 brain 2-3 days before hatching. Onefused optic lobe was generated in the dorsal position. Abbreviations:Tel, telencephalon; Di, diencephalon; Tc, tectum; Cer, cerebrum;Cel, cerebellum. Bar, 2 mm.

Fig. 4. BrdU incorporation in lacZ-transfected (A) and lacZ- plusShh-transfected embryo (B,C) in E5.5 whole brain (A,B) and thetransversal section of E3.5 midbrain (C). Dorsal (upper) and lateral(lower) views are shown (A, B). β-gal stains blue and BrdUlocalization stains red. Ventral limit of BrdU localization wasindicated with an arrow in B. While lacZ is detected in wholeunilateral hemisphere including the developing tectum in the controlbrain (A), lacZ is restricted in the tegmentum in Shh-transfectedbrain (B). In E3.5 midbrain (C), Hu-positive cells (brown) arelocalized only at the level of oculomotor nucleus in untransfectedside (small arrowhead), but extend dorsally in transfected side(between two arrows). Massive cell proliferation is observed on theuntransfected side, but is repressed on the transfected side.Presumptive dorsal boundary between both sides is indicated (largearrowhead). Bars, 1 mm (B); 200 µm (C).

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methanol at −20°C. For cryostat sections, fixed embryos wereimmersed in 20% sucrose in PBS overnight, and embedded in Tissue-Tek OCT compound. Cryosections of 10-15 µm were transferred toVector Bond (Vector)-coated slides.

In situ hybridization on whole-mount and cryostat sections wereperformed as described by Stern (1998) and Henrique (1997),respectively. Production of RNA probes of Shh (Riddle et al., 1993),HNF-3β (Ruiz i Altaba et al., 1995), Pax-6 (Araki and Nakamura,1999), Otx-2 (Katahira et al., 2000), En-2 (Itasaki and Nakamura,1996), Pax-2 (Okafuji et al., 1999), Pax-5 (Funahashi et al., 1999),Fgf8, Wnt-1 (Sugiyama et al., 1998) and Msx-1 (Suzuki et al., 1991)was described elsewhere. Digoxigenin- or FITC-labeled RNA probeswere synthesized according to the manufacturer’s protocol (Promega).For double in situ hybridization on whole-mount specimens, alkalinephosphatase (ALP)-conjugated anti-FITC (Boeringer Manheim) wasused for the first detection of Shh mRNA with FAST RedTR/Naphthol AS/MX (Sigma FASTTM; Sigma Chemical Co., StLouis, MO, USA). ALP-conjugated anti-DIG (Boeringer) was usedfor the second detection with 4-nitroblue tetrazorium chloride (NBT)and 5-bromo-4-chloro-3-indolyl-phosphate (BCIP). The ALP for thefirst staining was inactivated by incubating in 0.1 M glycine-HCl pH2.2 for 15 minutes at room temperature. The untransfected side wasregarded as a good control after bisecting the embryo in the midline(Fig. 2B-I).

Whole-mount immunostaining and immunohistochemistryEn2 and PAX7 proteins on whole-mount specimens were detectedwith 4D9 (Patel et al., 1989) and anti-Pax7 monoclonal antibody(Kawakami et al., 1997; Developmental Studies Hybridoma Bank,Iowa University, IA, USA), respectively. After in situ hybridization,embryos were postfixed in 4% paraformaldehyde in PBS, washed inPGT (PBS-0.2% gelatine-0.25% Triton X-100) and incubatedovernight at 4°C in half-diluted hybridoma supernatant with PGTcontaining 5% heat-inactivated goat serum. The embryos werewashed with PGT several times for 1 hour, reacted with HRP-conjugated anti-mouse IgG (Jackson, West Groove, PA, USA) andcolored with 3,3′-diaminpbenzidine (DAB) substrate containing0.03% CoCl2. Nerve fibers were detected with anti-neurofilamentantibody 3A10 (DSHB, 1/2 supernatant) after treatment of fixedembryos with 0.25% trypsin in PBS.

For immunohistochemistry, cryosections were washed severaltimes in PBS. For E7.5 and E5.5 embryos, endogenous peroxidasewas inactivated in 0.3% H2O2-0.1% NaN3 in PBS for 10 minutes.Monoclonal antibodies against SC1 (a gift from Dr Tanaka, 1/2supernatant), Shh-N (5E1, DSHB, 1/2 supernatant), Isl-1 (40.2D6,DSHB, 1/4 supernatant), Lim1/2 (4F2, DSHB, 1/100) and tyrosinehydroxylase (Chemicon, 1/100) or antiserum against GFP (MolecularProbes, 1/200) and 5-HT (Incstar, 1/500) were used, followed byHRP- or Cy3-conjugated goat anti-mouse and HRP- or Alexa488-conjugated goat anti-rabbit secondary antibodies.

BrdU incorporation BrdU was injected into the central canal of the midbrain of E3.5 orE5.5 transfected embryos. The embryos were fixed in 4%paraformaldehyde in PBS at 2 hours after BrdU injection.Incorporation of BrdU was subsequently detected using in situ cellproliferation kit, AP (Boehringer Mannheim) with Fast Red staining.For sections, immunohistochemistry with anti-Hu antibody (16A11,a gift from Dr Wakamatsu, 1/500 dilution) was performed oncryosections, followed by staining for β-gal and BrdU detection.

Labeling of retinal fibers with HRPPBS solution containing 30% HRP (horseradish peroxidase) wasinjected into the right eye ball of E11.5 chick embryos with a glassmicropitette. The next day embryos were killed and the brain wasisolated in PBS. The HRP-positive retinal fibers were stained usingp-cresol-diaminobenzisine method (Streit and Reubi, 1977). The

reaction was stopped in 4% paraformaldehyde in PBS and the wholebrain was stored in the same fixative.

RESULTS

Transfection of ectopic Shh by in ovoelectroporationMisexpression of Shh was achieved by in ovo electroporationperformed on chick embryos at stage 10, with GFP as anindicator for efficiency of transfection. (Fig. 1A,B; see Materialsand Methods).

In the control experiment, GFP-expression vector alone wasintroduced and GFP was detected abundantly in the unilateralside of the diencephalon, midbrain, isthmus and rostral hindbrain1 day after electroporation (Fig. 1C). This expression wassustained on the 2nd day exclusively in the unilateral half of thebrain (Fig. 1E). As the dorsal midbrain grows rapidly from E3onward, the whole enlarging tectum of the transfected side waslabeled with GFP.

When pMiwII-Shh was electroporated together with pEGFP-N1, no obvious difference in GFP expression from the controlwas discerned until E2.5 (1 day after electroporation; compareFig. 1C,D). The difference became apparent in subsequent 24hours; GFP was positive in the midbrain but did not cover thedorsal side of the midbrain (compare Fig. 1E,F). Since GFP waslocalized in all of the lateral half of the tectum in the controlembryo, the result suggests that the GFP-positive domain of Shh-transfected embryo did not contribute to the tectum formation.

After isolation of the brain vesicle, whole-mount in situhybridization with Shh probe was carried out. Introduction ofGFP alone did not affect endogenous Shh expression or tectaldevelopment (Fig. 1G). In contrast, in Shh-transfected embryos,ectopic expression of Shh was detected in lateral region whereGFP was co-localized (Fig. 1F,H). Moreover, tectal developmentwas significantly repressed, resulting in a flat, undevelopedmidbrain on the transfected side (Fig. 1H). No significantdifference in the number of cell deaths was observed betweenthe control and the experimental side after staining with NileBlue Sulfate (data not shown).

Shh suppresses the expression of the tectum-relatedgenesAs ectopic Shh affected tectal development, we havesubsequently analyzed the expressions of genes related to thetectum formation in Shh-transfected embryos at E3.5.

The transcription factor HNF-3β required for thedifferentiation of floor plate cells is expressed in the midline ofthe neural plate after stage 5 (Ruiz i Altaba et al., 1995). At E3.5HNF-3β expression is observed in the zona limitansintrathalamica (ZLI) and the floor plate posterior to thediencephalon (Fig. 2B left). On the transfected side, ectopicexpression of HNF-3β was exclusively induced in the introducedShh-positive region of diencephalon, midbrain and hindbrain(Fig. 2A,B right).

In diencephalon Pax-6 is expressed at the dorsal sidecomplementary to Shh expression at ventral side (Fig. 2C left).Pax-6 expression totally disappeared in the ectopic Shh-positiveregion, making a distinct boundary between Shh- and Pax-6-positive domains as in ZLI (Fig. 2C right).

Otx-2 transcripts are restricted anterior to the isthmus in an



early phase of embryogenesis (Fig. 2D left; Millet et al., 1996).Genetic alterations of Otx genes in mice suggested that they arerequired to establish midbrain-hindbrain boundary along APaxis (Matsuo et al., 1995; Acampora et al., 1997). Misexpressionof Shh in our experiment did not affect the Otx-2 expressiondomain and its posterior limit was conserved (Fig. 2D right).

En-2 is also involved in midbrain and hindbrain developmenttogether with En-1 (Hanks et al., 1995). Gradient of En-2expression is a determinant of AP polarity in the midbrain(Itasaki and Nakamura, 1996; Logan et al., 1996; Shigetani etal., 1997). AP gradient of En-2 expression in the midbrain andisthmus was diminished in Shh-transfected area (Fig. 2E). Asimilar pattern was observed after 4D9 antibody staining todetect En-2 protein localization (data not shown).

PAX-7 localization is detected in the pretectum, tectum anddorsal hindbrain (Fig. 2F left; Nomura et al., 1998). It was shownthat PAX-7 expression in the dorsal midbrain is suppressed bythe heterotopic grafting of the midbrain floor plate, producingtegmentum-like structure instead of the tectum. (Nomura et al.,1998). Ectopic Shh extinguished expression of PAX-7 in dorsalmidbrain (Fig. 2F right).

Pax-2 and Pax-5 are involved in the organization of the tectum(Okafuji et al., 1999; Funahashi et al, 1999). Expression of bothgenes in the isthmus was inhibited by Shh transfection (Fig.2G,H). Another gene of isthmic organizer, Fgf8, is localized inthe narrow band at the midbrain-hindbrain boundary (Fig. 2I left;Crossley et al., 1996). Interestingly, in the transfected embryo,the band of Fgf8 expression shifted to the rhombic lip while itwas almost extinguished in the isthmus (Fig. 2I right).

We have further examined the expression of Wnt-1 and Msx-1, whose transcripts are localized on the dorsal midlinefrom posterior diencephalon to midbrain (Fig. 2J,L; Holliday etal., 1995). In the Shh-transfected embryos, caudal expression ofboth genes in the midbrain was totally absent with diminishedexpression rostral to it (Fig. 2K,M).

These changes caused by Shh transfection were alreadydetected at E2.5 (data not shown), indicating that Shh cansuppress the expression of genes involved in early tectaldevelopment. Moreover, the loss of dorsal midline markerssuggests that the roof plate might not have been fully generatedwhen the ectopic Shh was introduced in the lateral hemisphere.

We have finally examined the expression of Shh-target genesPtc and Gli-1. Both genes are expressed in the neural tubeadjacent to Shh-positive domain, which reflects the Shh signalreception (Marigo and Tabin, 1996; Goodrich et al., 1996). Atmidbrain level, Ptc is expressed in a longitudinal linecomplementary to Shh expression (Fig. 2N). In Shh-transfectedembryos, Ptc expression was induced in the domain where Shhwas introduced. Moreover, another line of ectopic Ptc expressionappeared at the dorsal side complementary to the introduced Shhexpression (Fig. 2O). In contrast, the Gli-1 transcripts that areobserved in the ventral half of the midbrain in the normalembryos (Fig. 2P; Platt et al., 1997) were absent with ectopicShh expression (Fig. 2Q).

Shh inhibits the tectum formation and itssubdivision into optic lobes To investigate the long-term effects of Shh transfection in thetectal development, the embryos were kept alive until E5.5.From E3 onward, dorsal midbrain continues rapid expansionto form the optic tectum. In the E5.5 embryo, the tectum has

just started to subdivide into right and left hemispheres at thedorsal midline (Fig. 3A). In Shh-transfected embryos,reduction of the tectum size was apparent (Fig. 3B).Subdivision of the tectum into two hemispheres was notobserved at the dorsal midline. Most importantly, thetransfected tectum was significantly reduced in size, whileventral tegmentum region was dorsally enlarged.

Thereafter the tectum becomes completely separated into theright and the left hemispheres to form optic lobes, which laterrotate between E7 and E12 so that the rostrocaudal axis of thetectum accords with the ventrodorsal axis of the body(Goldberg, 1974; Itasaki and Nakamura, 1992). At E12.5, therotation is almost complete and the two optic lobes are situateddorsolaterally to the tegmentum (Fig. 3C). In contrast, thetectum of Shh-transfected embryos was not separated no matteron which side Shh was introduced. Rotation of the tectum wasalso incomplete; the fused tectum was located at the dorsal sideof the tegmentum (Fig. 3D,E). Histologically, the fused tectumstill had the laminar structure of the tectum (data not shown).At E18.5, total brain size was reduced to some extent intransfected embryos and the fused tectum was in a dorsalposition (Fig. 3F,G).

To determine to what extent each compartment of the tectalprimodium has contributed to form the fused tectum, retinalfibers were traced with HRP. In normal E12.5 embryos, retinalfibers are distributed throughout the contralateral tectum in ananteroposterior direction (Fig. 3C). When HRP was injectedinto the eye ipsilateral to the transfected side, most of the fusedtectum was stained, although the number of fibers was reduced(Fig. 3D). In contrast when HRP was injected into the eyecontralateral to the transfected side, a limited number of theretinal fibers were detected on the lateral portion of the fusedtectum in the transfected side (Fig. 3E). These staining patternsconfirm that the fused tectum derives mostly from theuntransfected side, while the transfected tectal anlagen hasonly poorly enlarged.

Shh controls cell proliferation-differentiation in themidbrainChanges in gene expression and morphogenesis of Shh-introduced embryos suggest that ectopic Shh extended thetegmentum territory and thus reduced the tectum territory. Itremains unclear how these changes take place at the cellularlevel. Since the transfected tectum is significantly reduced, itis possible that Shh-positive cells did not proliferate enough toform the tectal hemisphere. To test this idea, cell proliferationwas examined by BrdU incorporation. This time, lacZexpression vector was co-transfected with Shh expressionvector. BrdU was injected into the midbrain ventricule of atE3.5 or E5.5 embryos, which were subsequently fixed after 2 hours to detect β-galactosidase activity and BrdUincorporation.

In the control embryo in which lacZ alone was introduced,β-gal staining covered whole tectum and tegmentum at E5.5(Fig. 4A), showing that the alar and basal plates where lacZwas transfected have differentiated into the tectum and thetegmentum, respectively. This is consistent with the result thatGFP was detected throughout the unilateral midbrain at E3.5when GFP alone was introduced (Fig. 1E). In contrast, β-galstaining was restricted to the tegmentum region of Shh-transfected embryos even though lacZ must also have been

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introduced into the alar plate (Fig. 4B). While the tectumexpands as a result of extensive cell proliferation revealed byactive BrdU incorporation in tectal region, much lessincorporation was detected in the lacZ-positive tegmentumarea, making a clear boundary of the signal between them (Fig.4B). It indicates that, after Shh transfection, not only the basalplate but also the alar plate ceased active cell proliferation anddeveloped into the tegmentum without contributing to thetectum formation.

The assumption that Shh represses cell proliferation andenhances neural differentiation in the midbrain is confirmedhistologically in E3.5 embryos. Double staining with anti-BrdU and anti-Hu antibody was carried out (Fig. 4C). It hasbeen shown that anti-Hu antibody 16A11 recognizes HuC andHuD proteins, which are detected in postmitotic cells in thespinal cord (Marusich et al., 1994; Wakamatsu and Weston1997). At the untransfected side, many BrdU-positive cellswere found near the pial surface and Hu protein was localizedat the level of oculomotor nucleus (Fig. 4C). On the transfectedside where β-gal staining was positive, there were only a smallnumber of BrdU-positive cells, but many Hu-positive cellswere found.

Shh regulates cell type differentiation along DV axisDifferentiation of the tectal region and enhanced neuraldifferentiation in Shh-transfected midbrain prompted us toexamine the neuronal cell types along DV axis.

First, nerve fibers were stained with anti-neurofilamentantibody 3A10 and observed on whole mounts. On the controlside, fibers in subcutaneous positions such as trigeminal nervesare visible (Fig. 5A). On the experimental side, many ectopicnerves emerged from dorsolateral midbrain (Fig. 5B). Since itwas suggested that tegmental region extended dorsally, wewondered if these fibers were motor fibers or not. Cryosectionswere stained with SC1 antibody, which specifically stainsmotor neurons and their fibers (Tanaka and Obata, 1984). Onthe control side, SC1-positive cells were located just lateral tothe floor plate in the oculomotor nucleus (Fig. 5D). On the

experimental side, the SC1-positive zone extended to the alarplate where Shh was introduced and ectopic nerves were alsoSC1 positive (Fig. 5C,D).

As ectopic Shh induced additional motor neurons in dorsalregion, we next examined ventral cell markers in the normaland Shh-transfected midbrain. In the normal embryo, ShhmRNA is present in the floor plate colocalized with HNF-3βexpression (Fig. 5E; data not shown). Shh protein detected with5E1 antibody against Shh-N diffuses from Shh mRNA-positivedomain to form a weak gradient laterally (Fig. 5F). In the


Fig. 5. Neuronal cell types along DVaxis in E3.5 midbrain (A,B) Nervesin subcutaneous position weredetected with anti-neurofilament3A10 antibody in Shh-transfectedembryo. Ectopic nerves emerged inthe transfected side (B) which didnot exist in untransfected side (A).(C,D) Transversal sections of Shh-transfected embryo at the level ofoculomotor nucleus. Motor neuronmarker SC1 was positive in ectopicnerves and in the lateral hemisphere(D) where Shh was introduced (C).(E-L) Serial transversal sections ofnormal (E-H) and Shh-transfectedembryos (I-L), showing thelocalization of Shh mRNA (E,I),Shh-N protein (F,J), Isl-1 (G,K) and Lim1/2 (H,L). Ectopic Shh induced both Isl-1- and Lim1/2-positive cells in overlapping region, whichnormally appear in distinct domains along DV axis. Bars, 500 µm (B); 200 µm (D,L).

Fig. 6. Distribution of dopaminergic (A,B) and serotonergic (C,D)neurons in the E7.5 midbrain and E5.5 hindbrain. Normal (A,C) andShh-transfected brain (B,D) was stained with antibodies against TH(A,B) or 5HT (C,D). In normal embryos, TH-positive cells arelocalized in substatia nigra and 5HT-positive cells are adjacent to thefloor plate. They are induced ectopically in the dorsal region on theShh-transfected side. Abbreviations: Tc, tectum; Tg, tegmentum.Bars, 500 µm (A,B); 200 µm (C,D).


mantle layer laterally to the Shh-positive domain, oculomotor nucleiare stained with anti-Isl-1 antibody,which labels postmitotic motorneurons (Fig. 5G; Ericson et al.,1992). More laterally Lim1/2-positiveneurons are localized (Fig. 5H). In theShh-transfected midbrain, Shh mRNAwas ectopically expressed in theventricular layer and Shh-N wasdispersed to the mantle layer of thecorresponding region (Fig. 5I,J).Interestingly, both Isl-1- and Lim1/2-positive neurons appeared ectopicallyin a much more lateral domain thantheir normal localization (Fig. 5K,L).Both types of neurons werecolocalized with Shh-N in a dorsallyextended position. Thus, in spite of the normal cell diversityordered along DV axis, ectopic Shh induced these ventralmarkers in overlapping regions.

We also examined the neuronal cell types in the later stagesof development. It has been reported that Shh is required fordifferentiation of midbrain dopaminergic and hindbrainserotonergic neurons in rat neural explants (Ye et al., 1998). Shhcan also induce dopaminergic neurons in rat and chick midbrainexplants in vitro (Hynes et al., 1995; Wang et al., 1995).Localization of dopaminergic and serotonergic neurons wasdetected in normal and Shh-transfected midbrain-hindbrainusing antibodies against tyrosine hydroxylase (TH) andserotonin (5-HT), respectively. In normal E7.5 midbrain,abundant TH immunoreactivity is detected in the areacorresponding to substatia nigra (Fig. 6A). In Shh-transfectedmidbrain, the distribution of TH-positive cells was extendeddorsally compared with the control side (Fig. 6B). In E5.5hindbrain, 5-HT neurons were present at the ventral midlineadjacent to the floor plate, especially in the hindbrain rostral tothe pontine flexure (Fig. 6C). In Shh-transfected hindbrain, theregion of 5-HT-positive cells was dorsally extended (Fig. 6D).

Enlarged distribution of both early and late neuronal cellmarkers suggests that Shh regulates neuronal cell diversity alongDV axis in the midbrain.

Fig. 7. Distribution of Shh protein (A,D),Isl-1- (B,E) and Lim1/2- (C,F)- positivecells (red) in compared to the GFPpositive cells (green) in the section ofShh-transfected midbrain. The regionsbetween two arrowheads in A-C areshown in D-F at higher magnification. AsIRES sequence was inserted between Shhand GFP gene in a single expressionvector, transfected Shh must be expressedin GFP-positive cells. Many of Isl-1- orLim1/2-positive cells do not coexpressGFP (E,F). Bars, 200 µm (C); 50 µm (F).

Fig. 8. Results of cell type determination in Shh-transfected midbrain(A) and the role of Shh in normal midbrain development (B) aresummarized schematically. In E3.5 midbrain, both Isl-1- and Lim1/2-positive cells are induced in the dorsal region where ectopic Shh isintroduced. The ventral limit of each cell types has not changed (A).While differentiation is promoted in the basal plate where Shhregulates the ventral cell fate, the alar plate cells continues activeproliferation to form enlarging tectum (B).

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Shh promotes cell differentiation non-cellautonomouslyAs Shh promotes cell differentiation in ectopic sites, it ispossible that differentiation proceeded cell autonomously in thecells into which Shh expression vector was transfected. We havetested this possibility by transfection with another expressionvector containing IRES sequence between Shh and GFP gene,which enables both genes to be expressed in the same cells (seeMaterials and Methods).

GFP was detected in the cells of unilateral midbrainhemisphere (Fig. 7A-C). GFP-positive cells were found at alllevels between the ventricule and the outer surface of themidbrain, indicating that both mitotic and postmitotic cells areincluded (Fig. 7D-F). While Shh-transfected cells correspond tothe GFP-positive cells, Shh protein was localized ubiquitouslyin the transfected domain (Fig. 7A,D). Isl-1- and Lim1/2-positive cells were found in the same domain (Fig. 7B,C).Although some of Isl-1- and Lim1/2-positive cells coexpressGFP, many of them were GFP negative, suggesting that theywere not the cells into which Shh-expression vector wastransfected (Fig. 7E,F). These results demonstrate that thedifferentiation into ventral cell types by transfected Shhproceeded non-cell autonomously. Since ubiquitous expressionof Shh mRNA is detected in the transfected domain (Figs 1H,5C,I), it may be induced by Shh signal from the transfected cellsto promote cell differentiation in surrounding cells.

DISCUSSION

In the developing midbrain, the alar plate and basal plate undergodistinct developmental processes to form the tectum and thetegmentum, respectively. From E3, midbrain alar plate cellsproliferate rapidly, resulting in considerable expansion of thetectal wall with little change in its thickness. On the contrary,many nuclei related to motor function form in the tegmentum.To evaluate the role of Shh in the control of the differences alongDV axis in the midbrain development, we have generatedembryos in which Shh was transfected unilaterally in the brainvesicles. In this study, we have demonstrated that, in Shh-transfected midbrain: (1) the expression of the genes crucial forthe tectum formation was repressed and the roof plate markerswere inhibited, (2) the tectum territory was significantly reducedand replaced by the dorsally extended tegmentum, generating afused tectum, (3) neural differentiation was promoted and cellproliferation was suppressed, and (4) ventral cell types wereectopically induced in the dorsal domain through cell non-autonomous process. All these results suggest that ectopic Shhinhibited the molecular cascade for tectum formation and thuschanged the fate of the mesencephalic alar plate for the basalplate derivatives. Taken together, we conclude that Shhnegatively regulates tectal development along the DV axis byrepressing tectal gene expression, which results in the promotionof neural differentiation to restrict the tectum territory fromventral side.

It is noteworthy that many of the tectum-related genes suchas En-2, Pax-7, Pax-2, Pax-5 and Fgf8 were inhibited by ectopicShh, whereas the Otx-2-positive domain was not affected. It issuggested that Otx-2 is involved in the early determination ofmidbrain region along AP axis (Acampora et al., 1995; Matsuoet al., 1995; Ang et al., 1996). Moreover, Otx-2 interacts with

Gbx-2 at the midbrain-hindbrain boundary to determine theposterior limit of the tectum territory (Broccoli et al., 1999;Katahira et al., 2000). Our observation suggests that Otx-2 iscontrolled independently of DV polarity directed by Shh. In fact,as the boundary of dopamine neurons and serotonin neuronsalong AP axis was not changed in Shh-transfected embryos (datanot shown), Shh may not disturb the AP patterning of cell typesin the tegmentum area.

It has previously been reported that the induction ofdopaminergic neurons in the midbrain is dependent on Shh andFgf8, and serotonergic neurons appear posterior to the site ofFgf8 expression (Ye et al., 1998). In our experiments, however,although the expression of Fgf8 was repressed in the isthmusand shifted to the rhombic lip at E3.5, both dopaminergic andserotonin neurons were well induced at the ectopic sites (Fig.6B,D). We found that, in the early stages of Shh-transfectedembryos, the shift of Fgf8 expression is detected at 24 hours buthas not commenced at 12 hours after electroporation (data notshown). This suggests that early exposure to Fgf8 signal iscrucial for the induction of dopaminergic neurons. Consistentwith this, early blocking of FGF8 by the application of FGFR3-IgG in the rat midbrain explant can completely inhibit thedevelopment of dopaminergic neurons, while it partiallysucceeds or fails with later application (Ye et al., 1998).

At E5.5, Shh-transfected embryos had reduced tectumterritory and showed dorsal extension of the tegmentum (Fig.3B). This suggests that the alar plate changed its fate to developinto the tegmentum by Shh-misexpression. Therefore, it ispossible that Shh-transfected alar plate transformed to basalplate and contacted the contralateral alar plate in the dorsalregion. We suspect that this is a major reason why the roof platestructure was not formed, leading to fusion of the tectum at thedorsal side of the tegmentum (Fig. 3D,E,G). This idea issupported by the result of the surgical ablation experiment.When unilateral midbrain alar plate was removed at stage 9-11, the roof plate region was absent in the regenerated tectum,producing a fused tectum similar to the Shh-transfected embryoin our experiment (Cowan and Finger, 1982). Direct contact ofthe alar and basal plates may disturb the formation of the roofplate.

Dorsal extension of ventral cell types by Shh-misexpression is summarized in Fig. 8A. On the control side,as in the normal embryo, Shh mRNA is expressed in the floorplate region. At the level of midbrain, Shh-positive domain isbroader than at the trunk level. It is suggested that Shh-Ndiffuses from floor plate to form a ventrodorsal gradient(Tanabe and Jessell, 1996), although it is not clear how farShh-N can diffuse in the neural tissue. Isl-1-positive cells atthe oculomotor nucleus are located at the lateral limit of ShhmRNA-positive region. Lim1/2-positive cells are localizedmore laterally to Isl-1 domain but they do not overlap. On thecontrary, on the Shh-transfected side, misexpressed ShhmRNA is positive in the ventricular layer and Shh-N diffusedto the mantle layer in the correspondent neural tissue. Isl-1-and Lim1/2-positive domains extend dorsally and coexist inthe same area in the mantle layer, although the ventral limitof each cell type is maintained.

This is inconsistent with the results of culture of trunk-levelneural plate. Lim1/2-positive cells can be generated from naiveneural plate without exposure to Shh while high concentrationsof Shh induced exclusively Isl-1/2-positive cells (Ericson et al.,



1996). In our experiment, introduced Shh was so high that HNF-3β- and Isl-1-positive cells were generated. Coexistence of Isl-1- and Lim1/2-positive domain in Shh-transfected embryossuggests that the mode of determination of these cell types atrostral level may not be the same as that at the trunk level.Consistent with this idea, in the diencephalon, Isl-1-positiveneurons are generated in Shh-positive domain, and theycoexpress Lim-1 (Ericson et al., 1995). In addition, MNR2, atranscription factor that directs Shh-mediated differentiation ofsomatic motor neurons, is not expressed in oculomotor andtrochlear motor neurons (Tanabe et al., 1998). Therefore,although Shh acts as a common ventralizing signal, midbrain hasdistinct properties for ventral cell type determination from thetrunk neural tube.

Before the Shh-transfection at stage 10, cell types may not befixed in the basal plate, as both Isl-1- and Lim1/2-positive cellswere induced by ectopic Shh. Thus Shh signaling after stage 10may be critical for these ventral cell type determination. On thecontrary, Lim1/2- and Isl-1-positive cells were never induced inoculomotor nucleus and floor plate, respectively. In theseregions, the cell fate of motor neuron or floor plate progenitorwas already fixed before the transfection. These observationssupport the idea that the ventral cell type determinations occursequentially from ventral to dorsal direction as in the spinal cord(Erickson et al., 1996). In contrast, both TH- and 5-HT-positivecells, normally located at the most ventral position, were induceddorsally by ectopic Shh. Since the initial expressions of thesecell markers are relatively late, after E3, it is possible both celltypes should normally be determined much later.

Whereas anti-Shh antibody stains limited ventral region ofCNS, the following suggest that Shh can diffuse over largedistances. Functional blocking of Shh with antibody can preventthe induction of ventral cell types in neural explant (Ericson etal., 1996). The Shh target genes Ptc and Gli-1 are expressed inthe neural tube adjacent to Shh-positive domain, which mayreflect the Shh signal reception (Marigo and Tabin, 1996;Goodrich et al., 1996). In fact, at the midbrain level, Gli-1transcripts are observed in the ventral half of the midbrain andPtc is expressed in ventral longitudinal line complementary toShh expression (Fig. 2N,P), suggesting Shh can act as a long-range signal within the basal plate.

Finally, Fig. 8B proposes a possible role for Shh in midbraindevelopment. In the basal plate, Shh regulates ventral cell fate,which results in the promotion of neural differentiation to formthe tegmentum. On the contrary, the alar plate does not receiveShh signaling and so continues massive cell proliferation to formthe enlarging tectum. There may be some factors or genenetwork to support this proliferative property in the developingtectum.

We are indebted to Drs. K. Kitamura, S. Noji, I. Araki, M. Wassefand C. Tabin for providing us the probes for Otx-2, Fgf8, Pax-6, Wnt-1, Ptc and Gli-1. We are also grateful to Drs H. Tanaka and Y.Wakamatsu for the gifts of SC1 and 16A11 antibodies. The hybridomasand monoclonal antibodies were also obtained from the DevelopmentalStudies Hybridoma Bank developed under the auspices of NICHD andmaintained by The University of Iowa, Department of BiologicalSciences, Iowa City, IA52242. We thank Drs Christiane Ayer-Le Lièvre,Olivier Pourquié and Hajime Fujisawa for helpful discussions. Thiswork was supported by the Ministry of Education, Culture and Sports,Japan, New Energy and Industrial Technology DevelopmentOrganization, and the Agency of Science and Technology, Japan.

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