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The dorsal telencephalon gives rise to discrete regions of the adultbrain including the cerebral cortex, the most highly evolved region ofthe vertebrate central nervous system. Differences in morphology,gene expression and connectivity permit a subdivision of the devel-oping dorsal telencephalon into domains from which the differentadult structures originate1,2. Genetic studies have provided evidencethat cross-regulatory interactions between transcription factors con-tribute to the regionalization of the dorsal telencephalon3,4. Less isknown, however, about the secreted signals that induce the initialdorsal character of telencephalic cells before the patterning of thedorsal telencephalon.

In chick and zebrafish embryos, the exclusion of Wnt signaling fromthe anterior neural plate is required to establish the telencephalic char-acter of the neural plate5–7. Anterior neural plate cells in chickembryos are exposed to Shh activity at gastrula stages and initiallybecome specified as prospective ventral telencephalic cells8. Thesefindings indicate that telencephalic cells of early dorsal character maybe generated from cells that have evaded Wnt signaling and have beenexposed to Shh activity at earlier developmental stages.

At caudal levels of the neural tube, bone morphogenetic proteins(BMPs) are required for the generation of dorsal neural cell types9,10.Several genes that encode BMPs are expressed in dorsal midline cellsand in the epidermal ectoderm surrounding the prospective telen-cephalon11,12, and exposure of the developing telencephalon to BMPsperturbs the generation of ventral telencephalic cells13,14. Activation ofthe gene encoding BMP receptor 1A (Bmpr1a) is required for the for-mation of the dorsal midline derivative, the choroid plexus, but is notrequired for the specification of dorsolateral cells that give rise to thecerebral cortex15.

At the neural plate stage, Wnt signaling is required for the specifica-tion of caudal neural cells6. By neural tube stages, Wnt signals arerequired for the proliferation of neural progenitor cells16,17 and alsofor the differentiation of interneurons in the dorsal spinal cord18. Atthe neural fold stage, ectodermal cells that abut prospective dorsal cellsof the telencephalon express the genes encoding Wnt proteins. After

neural tube closure, Wnt genes are expressed in cells that give rise tothe dorsal region of the telencephalon19,20, and genetic studies haveshown that Wnts are required for the proliferation of hippocampalprecursor cells20 and for the maintenance of Emx2 expression in thedorsal telencephalon21. Nevertheless, it is unclear whether Wnt signal-ing has a role in the initial specification of dorsal telencephalic cells.

At neural fold stages, Fgf8 is expressed in the anterior margin ofthe neural plate, termed the anterior neural ridge. As the anteriorneural tube closes, the expression of Fgf8 extends caudally along theboundary of the neural and non-neural regions, which will latercontribute to the prosencephalic roof plate22–24. Later, Fgf15, Fgf17and Fgf18 are expressed in overlapping domains of the dorsal fore-brain25. Reduced Fgf8 expression leads to an impairment in thegrowth of the telencephalon and to an apparent mis-specification ofdorsal midline cells23,26. Moreover, ectopic FGF8 seems to induce astructure that resembles a rostral midline24,27. These results indicatethat FGF8 may be required for the generation of dorsal midline cellsin the telencephalon, but again, a role for FGF signaling in the initialspecification of dorsal telencephalic cells of cortical character hasyet to be demonstrated.

To identify the extrinsic signals that induce the initial dorsal charac-ter of telencephalic cells, and to define when and how these signals act,we used explant and whole-embryo assays of neural differentiation inthe chick embryo. These assays showed that Wnt signals suppress ven-tral cell fates and that sequential Wnt and FGF signaling induces dorsaltelencephalic character.

RESULTSTo define the positional character of neural cells in chick neural tissueexplants, we analyzed transcription factors that are restricted to differ-ent dorsoventral regions of the developing telencephalon. In Hamiltonand Hamburger (HH)28 stage-15 chick embryos (Fig. 1, top), Nkx2.1is expressed in ventral cells8,29 and Pax6 is expressed in dorsal cells30,whereas very few Ngn2+ and no Meis2+ or Emx1+ cells were detectedin the telencephalon (Fig. 1). In stage-22, or embryonic day (E) 3.5,

1Umeå Center for Molecular Medicine, Building 6M, 4th floor, Umeå University, S-901 87 Umeå, Sweden. 2Department of Biochemistry and Cell Biology, StateUniversity of New York, Stony Brook, New York 11794, USA. 3Howard Hughes Medical Institute, Department of Biochemistry and Molecular Biophysics, ColumbiaUniversity, New York, New York 10032, USA. Correspondence should be addressed to T.E. (thomas.edlund@molbiol.umu.se).

Specification of dorsal telencephalic character bysequential Wnt and FGF signalingLena Gunhaga1, Matthew Marklund1, My Sjödal1, Jen-Chih Hsieh2, Thomas M Jessell3 & Thomas Edlund1

Dorsoventral patterning of the telencephalon is established early in forebrain development and underlies many of the regionalsubdivisions that are critical to the later organization of neural circuits in the cerebral cortex and basal ganglia. Sonic hedgehog(Shh) is involved in the generation of the ventral-most telencephalic cells, but the identity of the extrinsic signal(s) that inducedorsal character in telencephalic cells is not known. Here we show in chick embryos that sequential Wnt and fibroblast growthfactor (FGF) signaling specifies cells of dorsal telencephalic character.

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embryos (Fig. 1, bottom), Nkx2.1 is still expressed in ventral cells,Meis2 is highly expressed in cells in the intermediate region31 and dor-sal cells co-express Pax6, Ngn2 and Emx1 (ref. 32). Thus, from stages15 to 22, cells in the prospective telencephalon progress from an earlydorsal Pax6+/Ngn2+ expression state to a later definitive dorsalPax6+/Ngn2+/Emx1+ expression state.

By HH stages 8 and 10, fate maps have shown that cells fated to gen-erate ventral and dorsal regions of the telencephalon are located at dif-ferent positions along the rostrocaudal axis of the anteriorprosencephalon (Fig. 2; refs. 33–35). Guided by these fate maps, weisolated explants of the prospective ventral and dorsal regions of thetelencephalon and monitored the positional character of cells thatemerged after 45–56 h in culture (Fig. 2). Ventral explants isolatedfrom stage-8 and stage-10 embryos generated Nkx2.1+ cells but notPax6+, Ngn2+, Meis2+ or Emx1+ cells (Fig. 2a; data not shown and

ref. 8), a profile indicative of the ventral telencephalon. By stage 8, dor-sal explants generated Pax6+ and Ngn2+ cells but not Emx1+, Meis2+

or Nkx2.1+ cells (Fig. 2b), a profile indicative of an early dorsal charac-ter. In stage-10 dorsal explants, ∼ 65% of the cells expressed Pax6,∼ 35% of the cells expressed Emx1 and cells expressing Ngn2 were alsopresent (Fig. 2c), a profile indicative of a later dorsal character. Bystage 15, when the dorsoventral axis of the telencephalon is estab-lished, most cells in dorsal explants expressed Pax6, Emx1 and Ngn2(Fig. 2d), a profile indicative of the definitive dorsal telencephalon.Thus, cells fated to generate the dorsal telencephalon seem to acquiretheir dorsal character gradually, between stages 8 and 15.

Epidermal ectoderm induces dorsal telencephalic cellsAt caudal levels of the neural axis, the epidermal ectoderm flanking theneural plate is a source of signals that induce cells of dorsal neuralcharacter9,10. To examine whether the epidermal ectoderm promotesthe generation of dorsal telencephalic cells, we isolated stage-8 dorsalexplants that alone do not generate Emx1+ cells, together with theflanking epidermal ectoderm, the neural fold region (dorsal neuralfold explants; Fig. 2). These explants generated Pax6+, Emx1+ andNgn2+ cells, whereas no Nkx2.1+ or Meis2+ cells were present (Fig. 2e),a profile indicative of the dorsal telencephalon. To test whether dorsalepidermal ectoderm could induce dorsal character in prospective ven-tral telencephalic cells, we cultured chick stage-8 dorsal epidermalectoderm with quail stage-8 ventral explants (Fig. 3a). In co-culturedexplants, the generation of neural Nkx2.1+ cells was blocked, andPax6+, Ngn2+ and Emx1+ cells were generated (Fig. 3b). These resultsindicate that signals derived from the epidermal ectoderm may inducedorsal telencephalic character.

BMPs do not induce early dorsal telencephalic cellsIn prospective forebrain regions at stage 8, Bmp4 is expressed in thedorsal epidermal ectoderm, and by stage 10, Bmp4 expression isdetected in the epidermal ectoderm surrounding the entire prospec-tive telencephalon. These expression patterns indicate that prospectivedorsal telencephalic cells may be exposed to BMP activity (seeSupplementary Fig. 1 online; refs. 11,12). The generation of thechoroid plexus from the most dorsal midline cells requires BMP sig-naling15; however, these midline cells do not express Pax6, Ngn2 orEmx1 (Fig. 1).

Figure 1 Domains of expression of transcription factors define ventral,intermediate and dorsal subdivisions of the developing telencephalon. Left,drawing of HH stage-15 (top) and stage-22 (E3.5, bottom) chick embryos.The dorsal (D) to ventral (V) line indicates the level of the transverse sectionsshown in the corresponding panels. In the telencephalon of a stage-15 chickembryo (top panels), Nkx2.1 was expressed in the ventral region and Pax6was expressed in the dorsal region of the telencephalon. Although neitherMeis2 nor Emx1 was expressed in the telencephalon at this stage, very few cells expressed Ngn2 in the dorsal region. In the telencephalon of astage-22 chick embryo (bottom panels), Nkx2.1 was expressed in the ventralregion, Meis2 (red) was expressed in the intermediate region and Pax6(green) was expressed in the dorsal region and in the most dorsal part of theintermediate region of the telencephalon. Double labeling (yellow) showscells that express Pax6 and Meis2. Emx1 and Ngn2 were expressed in thedorsal region of the telencephalon.

Figure 2 Telencephalic cells gradually acquire dorsal character. On the leftare schematic drawings of a dorsal view of stage-8 and stage-10 chickembryos (rostral is up) and a side view of a stage-15 embryo (rostral is tothe right). Ventral (V; light gray) and dorsal (D; dark gray) neuroectodermexplant regions are indicated in embryos at stages 8, 10 and 15. Theexplant region of dorsal neuroectoderm together with adjacent epidermalectoderm (dorsal neural fold; D-nf) is indicated in a stage-8 embryo.Explants were analyzed for expression after 30–56 h in culture. (a) Stage-8(st8) ventral explants (n = 20) generated Nkx2.1+ cells (> 90%), but notMeis2+, Pax6+, Emx1+ or Ngn2+ cells. (b) Stage-8 dorsal explants (n = 15)generated Pax6+ cells (58 ± 5%) and Ngn2+ cells, but not Nkx2.1+, Meis2+

or Emx1+ cells. (c) Stage-10 dorsal explants (n = 20) generated Pax6+ cells(64 ± 5%) and some Emx1+ (35 ± 4%) and Ngn2+ cells, but not Nkx2.1+ orMeis2+ cells. (d) Stage-15 dorsal explants (n = 15) generated Pax6+ cells(73 ± 3%), Emx1+ cells (> 90%) and Ngn2+ cells, but not Nkx2.1+ orMeis2+ cells. (e) Stage-8 dorsal neural fold explants (n = 15) generatedPax6+ cells (72 ± 4%), Emx1+ cells (> 90%) and Ngn2+ cells, but notNkx2.1+ or Meis2+ cells. Scale bar, 30 µm.

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We next examined whether BMP activity is required for the genera-tion of dorsal cells. In the presence of Noggin and/or soluble dominant-negative BMP receptor 1A and 1B derivatives, stage-8 dor-sal neural fold explants and stage-10 dorsal explants still generatedEmx1+, Pax6+ and Ngn2+ cells, but no Meis2+ or Nkx2.1+ cells weredetected (Supplementary Fig. 1; data not shown). Furthermore, BMP4(0.05–10 nM) did not induce Emx1+ cells in stage-8 dorsal or ventralexplants (data not shown). Thus, BMP activity seems to be neitherrequired nor sufficient for the generation of dorsal telencephalic cellsin vitro, a finding consistent with genetic evidence in the mouse15.

Wnts induce dorsal character in ventral cellsWnt genes are expressed either adjacent to or in prospective dorsal telen-cephalic cells at stages during which they acquire dorsal character. Atstage 8, Wnt1 and Wnt4 are expressed in the epidermal ectoderm adja-cent to the prospective dorsal telencephalon (Fig. 4a; data not shownand ref. 19). By stage 10, these genes are no longer expressed in the epi-dermal ectoderm surrounding the telencephalon, and instead, Wnt8b isexpressed in the prospective dorsal telencephalon (Fig. 4a; data notshown and ref. 19). To test whether Wnt activity induces dorsal cells, weexposed stage-8 ventral explants to different concentrations of solubleWnts. Both XWnt8 and Wnt3A signal through the β-catenin path-way20,36. Soluble Wnt3A and XWnt8-myc show similar activities inassays of neural induction and of rostrocaudal neural patterning inchick5,6, but Wnt3A had a higher and more reproducible activity thanXWnt8-myc. We therefore used Wnt3A in most of our experiments butconfirmed key findings with XWnt8-myc. In the presence of Wnt3A (1 ×), ∼ 70% of the cells expressed Nkx2.1 and ∼ 30% expressed Pax6 and

Ngn2, but no cells expressed Emx1 in stage-8 ventral explants (Fig. 4b).In contrast, in the presence of Wnt3A (2.5 ×), ∼ 50% of the cellsexpressed Nkx2.1, ∼ 50% expressed Pax6 and Ngn2 and < 5% of the cellsexpressed Emx1 (Fig. 4b). In the presence of Wnt3A (7.5 ×), the genera-tion of Nkx2.1+ cells was blocked and most cells expressed Pax6, Ngn2and Emx1 (Fig. 4b). Explants exposed to Wnt3A (7.5 ×) were ∼ 30%larger than control explants (data not shown), supporting the idea thatWnt signaling promotes the proliferation of telencephalic progenitorcells20. Thus, graded Wnt activity blocks the generation of ventral telen-cephalic cells and induces dorsal telencephalic cells in vitro.

We next assayed Wnt activity using New culture methods37, inwhich chick embryos are isolated from the yolk and cultured on theirvitelline membranes. Beads soaked in conditioned medium contain-ing Wnt3A or in control medium were implanted in the ventral side ofthe anterior prosencephalon of stage 9–10 embryos, and the embryoswere allowed to develop to stage 20–22. All embryos grafted with con-trol (n = 5) or Wnt3A (n = 5) beads showed a normal morphology(Fig. 4c). Control embryos showed normal dorsoventral patterning of

Figure 3 Signals derived from the dorsal epidermal ectoderm could inducedorsal telencephalic character in prospective ventral telencephalic cells. (a) Chick stage-8 dorsal epidermal ectoderm flanking the prospective dorsaltelencephalon was cultured with quail stage-8 ventral explant for 52–56 h.(b) Expression of transcription factors after culturing. Quail stage-8 ventralexplants (n = 10) generated Pax6+ cells (61 ± 5%), Emx1+ (red) cells (47 ± 7%) and Ngn2+ cells, but not Nkx2.1+ cells. Quail neural ectodermwas identified by the QCPN marker (green). Scale bar, 30 µm.

Figure 4 Wnt3A induces dorsal character in prospective ventraltelencephalic cells in a concentration-dependent manner. (a) At stage 8(upper panel), Wnt1 was expressed in the neural folds flanking theprospective dorsal telencephalon, and by stage 10 (lower panel), Wnt8bwas expressed in the presumptive dorsal telencephalon. (b) Expression of transcription factors in stage-8 ventral explants (n = 15 for eachconcentration) cultured for 52–56 h in the presence of differentconcentrations of conditioned medium containing Wnt3A. Explants exposedto Wnt3A (1 ×) generated Nkx2.1+ cells (72 ± 3%), Pax6+ cells (26 ± 4%)and Ngn2+ cells, but not Emx1+ cells. Explants exposed toWnt3A (2.5 ×)generated Nkx2.1+ cells (43 ± 3%), Pax6+ cells (53 ± 4%), Ngn2+ cells anda few Emx1+ cells (< 5%). Explants exposed to Wnt3A (7.5 ×) generatedPax6+ cells (83 ± 4%), Emx1+ cells (87 ± 3%) and Ngn2+ cells, but notNkx2.1+ cells. In the presence of control conditioned medium, explantsgenerated the same combination of cells as did explants that were grownalone. Scale bar, 30 µm. (c) Lateral views of E3 to E3.5 chick embryosgenerated in New culture from stage-9 embryos grafted with control (upperpanel) or Wnt3A (lower panel) beads; both treatments produced embryoswith normal morphology. Te, telencephalon. (d) Transverse sections of Newculture embryos showing the expression of transcription factors in thetelencephalon. Embryos (n = 5) grafted with control beads in the prospectivetelencephalon (upper panels) had Nkx2.1+ cells in the ventral region andPax6+ and Emx1+ cells in the dorsal region of the telencephalon. Embryos (n = 5) grafted with Wnt3A beads in the prospective ventral telencephalon(lower panels) had no Nkx2.1+ cells. In two of five embryos, Pax6+ andEmx1+ cells were located in the ventral region of the telencephalon(compare arrowheads). The insets show nuclear staining of Pax6 and Emx1,respectively.

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the telencephalon, whereas embryos grafted with Wnt3A beads failedto express Nkx2.1 and, in some cases, their ventral cells ectopicallyexpressed Pax6 (Fig. 4d). Moreover, Emx1+ cells were found in a moreventral position than in control embryos (Fig. 4d). Thus, Wnt activityblocked ventral and induced dorsal character in prospective ventraltelencephalic cells in intact chick embryos.

Wnt is required to generate dorsal telencephalic cellsTo examine whether Wnt signaling is required for the generation oftelencephalic cells of early dorsal character, we exposed chick explantsto a soluble fragment of the mouse frizzled receptor 8 protein(mFrz8CRD-IgG), an antagonist of Wnt signaling5,6,36. In the pres-ence of mFrz8CRD-IgG, the generation of Emx1+, Pax6+ and Ngn2+

cells was blocked in stage-8 dorsal neural fold explants and stage-10dorsal explants; in addition, Nkx2.1+ cells, but not Meis2+ cells, weregenerated (Fig. 5a; data not shown), a profile indicative of the ventraltelencephalon. Stage-8 dorsal neural fold explants exposed tomFrz8CRD-IgG were ∼ 40% smaller than explants cultivated undercontrol conditions, consistent with evidence that Wnt signaling isrequired for the proliferation of progenitor cells in the dorsal telen-cephalon20. Thus, our in vitro results provide evidence that Wnt sig-naling is involved in the specification and proliferation of dorsaltelencephalic cells.

We also used New culture methods37 to examine whether Wnt sig-naling is required for the generation of dorsal telencephalic cells inintact embryos. Beads soaked in control medium or in conditionedmedium containing mFrz8CRD-IgG were implanted in the dorsal sideof the anterior region of the prosencephalon of stage-9 embryos.Embryos grafted with control beads (n = 5) showed normal morphol-ogy and dorsoventral patterning of the telencephalon (data not

shown). Embryos implanted with mFrz8CRD-IgG beads (n = 5) hadtruncated telencephalic vesicles (Fig. 5b), supporting the idea that Wntactivity promotes the proliferation of telencephalic progenitor cells.Moreover, in all embryos, Pax6+ and Emx1+ cells were absent or theirnumbers were severely reduced (Fig. 5c; data not shown), providingevidence that in intact embryos, Wnt signaling is involved in the gen-eration of telencephalic cells of early dorsal character. In theseembryos, Nkx2.1+ cells were still present in the ventral region of thetelencephalon, and in some cases, the domain of Nkx2.1+ cellsexpanded dorsally (Fig. 5c ; data not shown).

Sequential Wnt and FGF signalingWe next examined whether Wnt signaling induces dorsal character inprospective dorsal telencephalic cells by testing whether Wnt3Ainduces Emx1+ cells in stage-8 dorsal explants. Wnt3A (7.5 ×)increased the number of Pax6+ cells but did not induce Emx1+ cells(Fig. 6a; data not shown), suggesting that an additional signal isrequired to generate Emx1+ cells in these explants. Because Wnt3A caninduce Pax6+, Ngn2+ and Emx1+ cells in stage-8 ventral explants (Fig. 4b), prospective ventral but not prospective dorsal cells may beexposed to a signal that acts in concert with Wnts to induce dorsalEmx1+ telencephalic cells.

The pattern of expression of Fgf8 in the developing forebrain led usto consider whether FGF8 serves as such a signal. At stages 7 and 8,Fgf8 is expressed in the anterior neural ridge that flanks prospectiveventral telencephalic cells (Fig. 6b; ref. 24) but not in the dorsal epider-mal ectoderm (Fig. 6b). By stages 9 and 10, the expression of Fgf8 nowincludes the dorsal epidermis and neural ectoderm of the rostral mid-line that abuts the prospective dorsal telencephalic cells (Fig. 6b;ref. 24). These data indicate that prospective ventral telencephalic cellsmay be exposed to FGF8 as early as stage 7, whereas prospective dorsalcells become exposed to FGF8 by stage 9.

To test whether Wnt activity requires FGF signaling to induce theexpression of Pax6, Ngn2 or Emx1 in prospective ventral cells, weexposed stage-8 ventral explants to Wnt3A (7.5 ×) and SU5402

Figure 5 Wnt and FGF activity are required to induce dorsal character inprospective dorsal telencephalic cells. (a) Expression of transcription factorsin stage-8 dorsal neural fold explants cultured alone or with mFrz8CRD,SU5402 or dnFGFR4 (n = 15 for each) for 52–56 h. Explants grown aloneor in control conditioned medium generated Pax6+ cells (72 ± 4%), Emx1+

cells (> 90%) and Ngn2+ cells, but not Nkx2.1+ or Meis2+ cells. In thepresence of mFrz8CRD, the generation of Pax6+, Emx1+ and Ngn2+ cellswas blocked, and Nkx2.1+ cells (> 90%), but not Meis2+ cells, weredetected. In the presence of SU5402, the generation of Emx1+ cells wasblocked, and Pax6+ cells (72 ± 3%) and Ngn2+ cells, but not Nkx2.1+ orMeis2+ cells, were detected. In the presence of dnFGFR4, the generation ofEmx1+ cells was blocked, and Pax6+ cells (65 ± 5%) and Ngn2+ cells, butnot Nkx2.1+ or Meis2+ cells, were detected. Scale bar, 30 µm. (b) Lateralviews of E3 to E3.5 chick embryos generated in New culture from stage-9embryos grafted with mFrz8CRD (upper panel) or SU5402 (lower panel)beads. mFrz8CRD produced embryos with smaller telencephalic vesiclesthan control embryos; SU5402 did not affect morphology. Te,telencephalon. (c) Transverse sections of New culture embryos showing theexpression of transcription factors in the telencephalon. Embryos (n = 5)grafted with mFrz8CRD beads in the prospective dorsal telencephalon(upper panels) lacked or showed reduced number of Pax6+ and Emx1+ cells.Nkx2.1+ cells were still present in the ventral region of the telencephalon,and in one of five embryos the domain of Nkx2.1+ cells had expandeddorsally. Embryos (n = 5) grafted with SU5402 beads in the prospectivedorsal telencephalon (lower panels) lacked (2 of 5) or showed a severelyreduced number of (3 of 5) Emx1+ cells, whereas the number and pattern ofPax6+ and Nkx2.1+cells was normal in all embryos.

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(5 µM), an inhibitor of FGF receptor signaling38. In the presence ofSU5402, Wnt3A blocked the generation of Nkx2.1+ cells and inducedPax6+ cells, but Emx1+ and Meis2+ cells were not generated (Fig. 6a;data not shown). In the presence of SU5402 alone, however, explantsstill generated Nkx2.1+ cells, and no Pax6+ cells were detected (datanot shown). Thus, FGF signaling seems to be required for Wnt activityto induce the expression of Emx1 in prospective ventral telencephaliccells, whereas Wnt activity induces dorsal Pax6+ and Ngn2+ cells inde-pendently of FGF signaling.

To test whether a combination of Wnt and FGF signals inducesdefinitive dorsal character in prospective dorsal telencephalic cells, weadded FGF8 (1–5 nM) alone or in combination with Wnt3A (7.5 ×) orXWnt8-myc to stage-8 dorsal explants. FGF8 did not induce Emx1+

cells, whereas Pax6+ and Ngn2+ cells (but not Nkx2.1 or Meis2+ cells)were detected (Fig. 6a; data not shown). In contrast, Wnt3A (7.5 ×) or XWnt8-myc in combination with FGF8 induced Emx1+ cells instage-8 dorsal explants. (Fig. 6a; data not shown). Thus, Wnt activityinduces Pax6+ and Ngn2+ cells, whereas the induction of Emx1+ cellsin the telencephalon seems to require both Wnt and FGF signaling.

To examine whether FGF signaling is required for the specificationof dorsal telencephalic cells, we used SU5402. In the presence of 5 µMSU5402, the generation of Emx1+ cells was blocked in stage-8 dorsalneural fold explants and in stage-10 dorsal explants, whereas Pax6+

and Ngn2+ cells were still generated and no Meis2+ or Nkx2.1+ cellswere detected (Fig. 5a; data not shown). FGF8 binds FGFR4 andFGFRIIIc with high affinity39, and both soluble FGFR4 and FGFRIIIcmimicked the effect of SU5402 (Fig. 5a; data not shown). These resultsindicate that FGF signaling in vitro may be required for the inductionof Emx1+ cells but not for the induction of Pax6+ or Ngn2+ cells.

To test the requirement of FGF signaling for the generation of telen-cephalic Emx1+ cells in intact embryos, we implanted beads soaked inSU5402 in the dorsal side of the anterior region of the prosencephalonof stage-9 embryos. Embryos implanted with SU5402 beads showednormal morphology (n = 5) and a normal pattern of Pax6+ andNkx2.1+ cells (Fig. 5b,c). In the dorsal telencephalon, however, Emx1+

cells either were absent or were present in reduced numbers (Fig. 5c;data not shown). These results support the view that FGF signaling isrequired to generate telencephalic Emx1+ cells, whereas Pax6+ cells aregenerated independently of FGF signaling.

DISCUSSIONEarly prospective telencephalic cells seem to become specified as cellsof ventral character in response to Shh signals at gastrula stages8, sug-gesting that dorsal telencephalic cells are generated through therespecification of cells of an intrinsic ventral character. The signalsthat induce the initial dorsal character in telencephalic cells have, how-ever, remained elusive. In this study, we provide evidence that Wnt sig-nals block ventral and induce early dorsal character in telencephaliccells. At the neural plate stage, the generation of the anterior forebrainregion depends on the exclusion of Wnt signaling. At this stage, Wntsignals seem to specify cells of caudal neural character6. Thus, the earlydevelopment of the forebrain depends on a shift in the response ofprospective telencephalic cells to Wnt activity. Wnt signaling also pro-motes the proliferation of dorsal telencephalic progenitor cells20, andour results are consistent with this observation. Several Wnt genes areexpressed in the dorsal region of the developing forebrain19, butgenetic studies have not resolved whether Wnt signaling is required forthe specification of early cell identity in the telencephalon20, possiblyreflecting redundancy in Wnt signals. Thus, we cannot assign theobserved patterning activity to a specific Wnt ligand.

The pattern of expression of Fgf genes in the anterior region of theembryo indicates that cells along the entire anteroposterior (futuredorsoventral) axis of the prospective telencephalon may be exposed toFGF activity. We find that FGF signaling is not required for the genera-tion of dorsal Pax6+ and Ngn2+ telencephalic cells but, in combinationwith Wnt activity, is required to induce definitive dorsal Emx1+ telen-cephalic cells. At later stages of development, Fgf15, Fgf17 and Fgf18are expressed in the dorsal midline region of the forebrain25, and cellsin this region do not express Emx genes. At these stages, FGF8 seems tocontribute to the specification of dorsal midline cells23,26, in part bysuppressing the expression of Emx2 (refs. 24,27). Consistent with thesefindings, FGF8 suppressed the generation of Emx1+ cells in stage-10and stage-12 dorsal explants (data not shown). The expression of

Figure 6 The temporal expression of Fgf8 in the developing forebraincontrols the generation of telencephalic cells of definitive dorsal character.(a) Expression of transcription factors in stage 8 dorsal explants cultured for52–56 h with conditioned medium containing Wnt3A or FGF8, either aloneor in combination, or with conditioned medium containing XWnt8-myc andFGF8 and in stage-8 ventral explants cultured for 52–56 h with both Wnt3A(7.5 ×) and SU5402 (n = 15 for each). In the presence of Wnt3A, dorsalexplants generated Pax6+ cells (83 ± 4%), but not Nkx2.1+ or Emx1+ cells.In the presence of FGF8, dorsal explants generated Pax6+ cells (25 ± 3%),but not Nkx2.1+ or Emx1+ cells. In the presence of both Wnt3A and FGF8,dorsal explants generated Pax6+ cells (80 ± 3%) and Emx1+ cells (> 90%),but not Nkx2.1+ cells. In the presence of both XWnt8-myc and FGF8, dorsalexplants generated Pax6+ cells (60 ± 5%) and Emx1+ cells (41 ± 4%), butnot Nkx2.1+ cells. Ventral explants exposed to both Wnt3A (7.5 ×) andSU5402 generated Pax6+ cells (74 ± 4%), but not Nkx2.1+ or Emx1+ cells.In the presence of control conditioned medium, explants generated the samecombination of cells as explants that were grown alone. Scale bar, 30 µm.(b) Expression of Fgf8 in stage-8 prospective ventral and dorsal and stage-10 prospective dorsal telencephalon (tel.). At stage 8, Fgf8 wasexpressed in the anterior neural ridge flanking the prospective ventraltelencephalon, but no Fgf8 expression was detected in the region of theprospective dorsal telencephalon. Upper and middle panels represent ventraland dorsal regions of the telencephalon located at different positions alongthe rostrocaudal axis shown in Figure 2. By stage 9–10, Fgf8 was expressed in the dorsal midline cells abutting the presumptive dorsal telencephalon. In the bottom panel, the section level represents the dorsal region of thetelencephalon shown in Figure 2.

a b

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several Fgf genes in the developing dorsal forebrain again makes it dif-ficult to assign precise roles for individual FGFs in these processes.

The stage at which different regions of the chick anterior neuralplate are specified to express Pax6 and Emx1 has been examined bygrafting neural tissue from this region to the midbrain region of theneural tube30. These results indicated that Emx1 expression isautonomous in the anterior neural plate from as early as HH stage 8.Because the midbrain region is a source of both Wnt and FGF signals,the grafted forebrain tissue may be exposed to the signals that inducePax6 and Emx1, raising the possibility that this grafting assay may nottest for specification of dorsal telencephalic cells.

Taken together, our findings suggest a working model of progressivesteps in the early specification of cells in the dorsal region of the telen-cephalon (Supplementary Fig. 2). At gastrula stages, most or allprospective telencephalic cells become specified as cells of ventralcharacter in response to node-derived Shh signals8. At the early neuralfold stage, prospective dorsal telencephalic cells seem to be exposed toWnt and BMP signals from the adjacent epidermal ectoderm. But atthis stage, only prospective ventral telencephalic cells seem to beexposed to FGF signals derived from the anterior neural ridge. Inresponse to Wnt activity, ventral telencephalic character is blocked,and prospective dorsal telencephalic cells become specified as earlydorsal (Pax6+ and Ngn2+) cells. By the early neural tube stage, Wnt8b isexpressed in prospective dorsal telencephalic cells, and the domain ofFgf8 expands dorsally and now also includes the dorsal midline regionthat abuts cells of the prospective dorsal telencephalon. The coinci-dence of Wnt and FGF signaling seems to be sufficient to induce defin-itive dorsal telencephalic Emx1+ cells. Subsequently, BMP and FGFsignaling seems to be required to specify dorsal midline cells15,23,26.Our findings indicate, therefore, that the restriction of Wnt expressionto the prospective dorsal telencephalon, in combination with the dor-sal spread of FGF signaling in the forebrain, may be a critical step inspecifying the fate and time of generation of dorsal telencephalic cells.

METHODSEmbryos. Fertilized white leghorn chicken eggs were obtained from AgriseraAB, Umeå, Sweden. Chick embryos were staged according to the protocol ofHamburger and Hamilton28. The use of chick embryos in this study wasapproved by the ethical committee at Umeå University.

Whole-embryo culture. The New culture method was essentially carried out aspreviously described37. Heparin acrylic beads (Sigma) were soaked in SU5402or PBS (control) or in conditioned medium containing mFzCRD-IgG, Wnt3Aor control conditioned medium. The beads were inserted in the prospectiveprosencephalon of HH stage 9–10 chick embryos and were placed in contactwith the neural ectoderm. The embryos were maintained in New culture for46–52 h.

Isolation of explants. Explants of the prospective ventral and dorsal telen-cephalon were dissected from HH stage-8 (three or four somites) chickembryos34,35 and cultured for 50–56 h as described40,41. Explants from theprospective dorsal telencephalon were dissected from HH stage 10 (9 or 10 somites) and HH stage 15–16 (25–27 somites) chick embryos8,33 and cul-tured for 30–48 h.

Culture of explants. Explants were cultured as previously described8. XenopusNoggin (provided by R. Harland) was used at 75 nM and BMPR-1A/Fc andBMPR-1B/Fc (R&D Systems) were used at 50 nM each. FGF8 (R&D Systems)was used at 0.5–5 nM. Mouse recombinant soluble dominant-negativeFGFRIIIc and FGFR4 (R&D Systems) were used at 60–180 nM together with0.5 µg/ml heparin (Sigma). SU5402 (Calbiochem) was used at 5–10 µM.Soluble Wnt3A and control conditioned medium were obtained from stablytransfected mouse L cells, and Wnt3A was used at an estimated concentrationof 4–30 ng/ml (1–7.5 ×) in explant assays6. Soluble XWnt8-myc and control

conditioned medium were obtained from stably transfected Drosophila S2 cellsand XWnt8-myc was used at an estimated concentration of 3 ug/ml in explantassays36. Soluble Frizzled 8 and control conditioned medium were obtainedfrom HEK-293 cells transfected with mFrz8CRD or LacZ reporter construct36.

In situ hybridization. In situ RNA hybridization histochemistry using chickdigoxigenin-labeled Ngn2 (ref. 42), Bmp4 (ref. 43), Wnt1, Wnt8b19 and Fgf8were carried out essentially as described44.

Immunohistochemistry. The anti-Emx1 rabbit antibody45 was used on chickembryos and explants fixed in MeOH:DMSO (4:1) at –20 °C for 80 min(explants) and at –80 °C for 5 d (E3.5 embryos) before freezing in embeddingmedium. For staining with the anti-Nkx2.1 rabbit antibody (BIOPATImmunotechnologies), anti-Meis2 rabbit antibody31 and monoclonal antibodyPax6 (ref. 46), embryos and explants were fixed as described8. Quail tissue wasdetected with monoclonal antibody QCPN (B. Carlson, Developmental StudiesHybridoma Bank). To quantify the percentage of cells that expressed the anti-gen, each explant was serially sectioned at 8 µm and stained, and the cells werecounted; the total number of cells was determined by counting the number ofnuclei as visualized with DAPI (Boehringer Mannheim).

Note: Supplementary information is available on the Nature Neuroscience website.

ACKNOWLEDGMENTSWe thank D. Anderson, P. Brickell, G. Corte, K. Campbell, J. Nathans and A. Streitfor kindly providing us antibodies, plasmids and cell lines. We are grateful tomembers of the Edlund lab for discussions. T.E. is supported by the SwedishMedical Research Council, the Foundation for Strategic Research and theEuropean Union research programs. T.M.J. is supported by grants from theNational Institute of Neurological Disorders and Stroke and is an Investigator ofthe Howard Hughes Medical Institute.

COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.

Received 28 February; accepted 29 April 2003Published online 25 May 2003; doi:10.1038/nn1068

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