Sonic hedgehog signaling in forebraindevelopment and its interactions withpathways that modify its effectsNicolas Bertrand1 and Nadia Dahmane2

1 IBDML, UMR 6216, Campus de Luminy, Universite de la Mediterranee, 13288 Marseille cedex 09, France2 Wistar Institute, 3601 Spruce Street, Philadelphia, PA19103, USA

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During the development of the nervous system andother organs in the embryo, a limited set of mastersignaling pathways are used repeatedly for induction,patterning and growth. Among these, the Sonic hedge-hog (Shh) pathway is crucial for the development ofmany structures in the brain. How the context-specificinterplay between these various signaling pathwaysproduces distinct temporal and spatial outcomes isnot clear. Resolving this problem is a major goal inthe study of cell and organ development. Here, we focuson signaling events during dorso-ventral patterning ofthe embryonic forebrain in vertebrates. In particular, wediscuss the role of the Shh pathway in this process andon its interactions with the FGF, retinoic acid and Nodalpathways and other information cascades that modifyits effects.

IntroductionThe formation of the body plan and the construction of thebrain are central areas of research in developmental biol-ogy. The mechanistic study of these events has focused oncell differentiation, migration and, more recently, on theaction of specific signaling pathways, triggered by pattern-ing molecules that activate specific intracellular signaltransduction cascades. These activate specific sets of tran-scription factors that then dictate the fate of respondingcells by orchestrating the genomic response to extracellu-lar information. Mechanistic information on pathwayactivity is key to understanding how the brain, and in thisreview, how the forebrain, develops. However, these ‘whatand how’ issues need to be accompanied by a preciseknowledge of when particular events take place. Althoughsuch a statement might seem obvious in the context of arapidly changing embryo, the changing context is likely tohave impact on how signaling pathways interact andintegrate. Indeed, this is the main challenge that wenow face: understanding the temporal and spatial controland codes of information integration that lead to distinctand precise outcomes during development.

Here, we focus on the role of theHedgehog (Hh) pathwayduring early forebrain development and its interactionswith information cascades such as the FGF, retinoic acid(RA) and Nodal signaling pathways that appear to modify

Corresponding author: Dahmane, N. (ndahmane@wistar.org).Available online 9 October 2006.

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its effects. We summarize recent advances in zebrafish,mouse and chick models and speculate on future directionsof research. We suggest that the context-dependent role ofHh signaling results from such modifying interactions intime and space. Understanding these interactions holds akey to unraveling how the brain develops and how, ingeneral, this essential signaling pathway is redeployedmultiple times in the life of an organism with a myriadof specific effects. Such knowledge will also shed light onseveral human pathologies, including holoprosencephalyand many types of cancer (Box 1).

Forebrain development starts in the early embryo. Itsinduction in the early ectoderm is a result of planar andvertical signals from the adjacent axial mesoderm. Theaxial mesoderm occupies a midline position under theventral central nervous system (CNS) and gives rise tothe notochord, which underlies most of the CNS except forthe anterior forebrain, and the prechordal plate, whichunderlies the anterior forebrain [1–4] (Figure 1). Followinginduction, the forebrain rapidly grows and divides into twosecondary vesicles, the telencephalon at the anterior (ros-tral) end and the diencephalon just posterior (caudal) to it(Figure 1). The cerebral cortex, the striatum, the hypotha-lamus and the thalamus of the adult brain are derived fromthese two vesicles. In the developing telencephalon, majorsubdivisions include the cerebral cortex (on themost dorsalside) and the ganglionic eminences (on the ventral side;Figure 1).

The Hh signaling pathway and its essential role in theforebrainOne important step in the elaboration of the adult fore-brain is its correct dorso-ventral (DV) patterning, which iscontrolled by a set of secreted morphogens, including Sonichedgehog (Shh) ventrally and bonemorphogenetic proteins(BMPs, which are members of the TGFb superfamily)dorsally (Box 2).

In the developing forebrain, as in the spinal cord, Hhsignaling has a crucial role in the DV patterning of cell typedifferentiation [1,5], for example, the formation of motorneurons ventrally and the differentiation of commissuralinterneurons dorsally (Box 3). Mice lacking Shh do notdevelop ventral CNS structures (e.g. motor neurons) andare cyclopic (having only one eye) [6]. Moreover, mis-expression studies, both in vitro and in vivo, indicate that

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Box 1. The main human pathologies associated with

deficient or enhanced Hh–Gli signaling

Loss of Shh signaling leads to deficient ventral induction of the CNS

and causes holoprosencephaly (HPE) and cyclopia. HPE is one of the

most frequent brain defects in humans: it occurs in 1 in 250 embryos

and in 1 in 10 000–20 000 live births (reviewed in Refs [69,70]). This

birth defect is characterized by a failure in the development of the

forebrain, which can lead to a variety of clinical features, from

severe to mild. In the most extreme cases, called alobar HPE, the

forebrain is not divided into two lateral ventricles. As a result, there

is only one single brain lobe and severe craniofacial anomalies,

including cyclopia.

In humans, several factors are associated with the emergence of

HPE, among them early exposure to alcohol or retinoic acid, and

hypocholesterolemia. The HPE phenotype has also been described

in sporadic and familial cases. Twelve genetic loci (HPE1 to HPE12)

linked to HPE have so far been identified in the human genome

(reviewed in Ref. [70]) and are thought to alter Shh and BMP

signaling. These include mutations in the SIX3, ZIC2, TGIF and SHH

genes [70]. Moreover, mutations in PTC1 [71] and GLI2 [72] genes

have also been recently identified. Mutations in SHH are the most

frequent identified mutations in HPE, although they represent a

minority of cases [69]. HPE is also associated with Smith-Lemli-Opitz

syndrome, Pallister-Hall syndrome and Rubenstein-Taybi syndrome

(reviewed in Ref. [69]). It is interesting to note that the gene mutated

in Pallister-Hall syndrome is GLI3, encoding a member of the Shh

signaling pathway [73]. In animal models, HPE can be induced in

zebrafish by loss of the axial mesoderm, the tissue that expresses

Shh ventrally [4,74], in chick embryos by overexpressing BMP5 [75],

and in mice by deletion of the Shh gene [6].

In contrast to HPE, enhanced Hh–Gli signaling has been found to

be involved in several types of sporadic human cancers, such as

those of the prostate, brain, pancreas, muscle and skin (reviewed in

Refs [76,77]). Moreover, inappropriate Shh–Gli signaling induces

tumor formation in experimental animals [76,77].

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Shh expression is sufficient to induce ventral forebrainmarkers ectopically in the dorsal forebrain [2,7].

In both zebrafish and mice mutant for the single Smogene (which encodes critical components of the Hh recep-tion complex), there is loss of Hh signaling, resulting instrong DV patterning defects [8–11]. These results,together with the finding that other signaling pathways(such as BMP signaling) that contribute to the elaborationof ventral identity in mice and zebrafish are also active inother species (reviewed in Refs [2,7,12]), support the ideathat the molecular mechanisms involved in DV forebrainpatterning are conserved in vertebrates.

The roles of the different Gli genes in DV forebraindevelopmentMice that are mutant for Gli1 or Gli2, which encodetranscription factors that are mediators of Hh signaling,do not seem to have ventral defects in the forebrain [13]. Bycontrast, Gli3 mutants (Gli3Xt) show alterations in DVpatterning and there is a reduction or loss of dorsalmarkers [14–16]. Double mutant mice for Gli1 and Gli2,however, show diminished expression of the ventral mar-ker Nkx2.1 in the diencephalon [13], indicating deficientventral differentiation. A role for Gli3 in diencephalicventral specification has been also suggested followingepistasis analysis using mice mutant for Ptc (whichencodes an Hh receptor), Gli2 and Gli3 [17]. Gli3 thusseems to have at least two roles: one as a repressor ofventral differentiation dorsally and another one as a pro-moter of intermediate values more ventrally. The first role

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is thought to be associated with the C-terminally trun-cated, repressing version of Gli3 that is produced post-transcriptionally, whereas the second is likely to be asso-ciated with the activity of a full-length, activating form.

Other results have shown that the balance betweenGli3and Shh activities is crucial for DV patterning in the spinalcord and forebrain: in mice mutant for both Gli3 and Shhthere is a partial rescue of ventral cell differentiationcompared with Shh mutant mice [18,19]. This result isinterpreted to mean that the repressor function of Gli3,normally dorsally restricted, is inhibited ventrally by Shhduring normal development. When Shh is absent, therepressive role of Gli3 (which is dominant over the positiveactivities of Gli1 andGli2) is unleashed ventrally to repressventral differentiation. Dorsally, Gli3 might be induced bypathways that antagonize Shh signaling, such as the BMPor Wnt pathways (see later).

This role of Gli3 as a repressor that needs to be inhibitedby Shh might not, however, exist in zebrafish [20]. In thetelencephalon of this species, Gli3 does not seem to act indorsalization and, instead,Gli2 is involved in repression ofgenes expressed in the ventral telencephalon [20,21].Furthermore, it seems that Gli3 possesses weak activatorfunction that, together with the activator role of Gli1,induces expression of Shh targets in the ventral forebrain[20]. Thus, species differences need to be taken intoaccount when considering the function of Gli3 in dorsalforebrain specification. In addition, the combinatorial andcooperative action of Gli proteins [22–24] makes solvingthese issues a complex task. Together, these results raisethe possibility that Shh is dispensable for telencephalic DVpatterning as long as the repressor function of Gli3 isinhibited and there is persistent ventral positive activityof Gli1 and/or Gli2. Normally, the repressor activity of Gli3is inhibited by Hh signaling, which is known to antagonizeformation of the repressor forms of Gli3 and of its flyhomolog Cubitus interruptus (reviewed in [24,25]).

The Nodal and Hh pathwaysZebrafish

The phenotypic analysis of zebrafish mutant in a ligand ofthe Nodal family of TGFb molecules, such as Cyclops (Cyc),has uncovered a role for Nodal signaling in the DV pattern-ing of the forebrain (reviewed in [26]). It has been shownthat embryos lacking Cyclops, or those lacking One-eyedpinhead (Oep, a membrane protein of the EGF–CFC familyessential for Nodal signaling [27]), have defects in theventral forebrain, including loss of expression of Nk2.1b(one zebrafish ortholog of mouse Nkx2.1) in the ventraltelencephalon [28]. Nodal signaling seems to act upstreamof the Shh pathway}, because Shh expression is almostcompletely abolished in Cyc and Oep mutants and Shhoverexpression can rescue ventral telencephalic specifica-tion in Cyc or Oep mutant embryos [28]. How Nodalsignaling regulates Shh signaling, however, is not comple-tely clear.

In the ventral diencephalon, the development of thehypothalamus requires both Nodal and Hh activities.However, these secreted factors do not have the same role.Nodal signaling is first required for hypothalamic specifi-cation, because in Cyc mutant embryos or maternal and

Figure 1. Schematic diagrams of a vertebrate brain during (a) early (�E9.5–E10) and (b) late (�E13.5) CNS development in the embryo, and localization of Shh, Gli1 and Gli3

expression during early mouse development. (a) The brain vesicles expand soon after neural tube closure, and the brain is subdivided along the anteroposterior

(rostrocaudal) axis. At this stage, most Wnt and BMP expression is dorsal, whereas Shh is expressed ventrally, first in the underlying mesoderm of the notochord (green)

and prechordal plate (red) and then in the floor plate at the ventral midline of the neural tube. It is also expressed in the zona limitans intrathalamica (ZLI, blue). (b) Following

the formation of the cephalic flexures (bends) and the disproportionate growth of the forebrain, the brain is clearly subdivided. The cerebellum begins to show its

characteristic growth and other regions also expand, including the tectum and the hypothalamus. (c) Telencephalon morphology and localization of Shh, Gli1 and Gli3

expression during early mouse development. E12.5 coronal mouse forebrain sections were hybridized with probes for Shh, Gli1 and Gli3; dashed lines indicate the midline,

separating the two hemispheres. Note the localized expression of Shh in the differentiated zone of the medial ganglionic eminence (MGE) and the response to its action by

cells expressing Gli1 in the boundary between the MGE and the lateral ganglionic eminence (LGE). As they are located close to the ventricle, these cells are precursors of

differentiated cells, including neurons. The domain of Gli3 expression extends just to the MGE–LGE boundary, probably negating the effects of Shh in these regions in a

dose-dependent manner, as its expression is stronger close to the dorsal midline than close to the region adjacent to that expressing Gli1. DVB, dorso-ventral boundary; CP,

choroid plexus; Hip, hippocampus; Ctx, neocortex; VZ, ventricular zone.

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zygotic Oep mutant embryos, the expression of severalhypothalamic markers is lost [28,29]. Importantly, over-expression of Shh in these embryos does not rescue thisphenotype, pointing to different requirements of the Hhpathway downstream to Nodal signaling in ventral fore-brain induction [28]. Nodal is also involved in patterningthe hypothalamic tissue: Nodal signaling is required cell-autonomously in the cells that will form the posterior-ventral (PV) hypothalamus, whereas it controls the devel-opment of the anterior-dorsal (AD) hypothalamic compart-ment in a non-cell-autonomousmanner (the signal is activein different cells from the cells being specified), and it

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probably does so through its regulation of the Hh pathway[29]. During hypothalamic patterning the two signalingpathways interact. In Smu mutant embryos (Smu is thezebrafish homolog of Smoothened) or in embryos treatedwith cyclopamine (a specific inhibitor of Smoothened andthus of Hh signaling [30]), the PV hypothalamic compart-ment is enlarged at the expense of the AD compartment.Conversely, Shh overexpression in wild-type embryosdiminishes or suppresses development of the PV hypotha-lamic compartment [29]. This mechanism is likely to con-tribute to the precise definition of the two hypothalamiccompartments, possibly by acting on progenitors that then

Box 2. Signaling pathways involved in forebrain development

Hedgehog

Sonic Hedgehog (Shh) encodes a secreted glycoprotein that is

implicated in many developmental processes, including the regula-

tion of cell identity, proliferation and survival (reviewed in Refs

[25,78]). The Shh gene is one of several vertebrate Hh genes; mice and

humans have three but zebrafish and other species have more. In

mice and humans, Shh is expressed in specific cell groups in many

organs such as brain and lungs, whereas Indian hedgehog (Ihh) is

expressed prominently in the bone and Desert hedgehog (Dhh) in the

gonads. Hh signals are conveyed intracellularly by the membrane

proteins Patched1 (Ptc1) and Smoothened (Smo). The reception of the

Shh signal in the responding cell enables Smo to signal and results in

the activation of target genes through the transcription factors of the

Gli family.

In vertebrates, three Gli genes have been identified, encoding

proteins with partially divergent functions (reviewed in Ref. [24]).

Their single homolog in Drosophila is Cubitus interruptus, also acting

downstream of Hh signaling. In general, Gli1 acts primarily as an

activator, Gli2 as both an activator and repressor and Gli3 mostly as a

repressor. It is thought that they regulate the same sets of genes, but

it cannot be ruled out that they have differential roles that are target-

specific. Moreover, their function, is context-dependent (e.g.

[22,79,80]), that is, they regulate targets differently at different times

and in different cells [22,79]. Indeed, they show partial redundancy in

different tissues (e.g. [13,80]). How this occurs is not known and is a

central aspect of signaling that needs to be understood if we are to

provide a mechanistic explanation of forebrain development.

Nodal

Nodal proteins are secreted factors of the TGFb superfamily, and,

based on the fact that they predominantly bind to activin receptors,

they are classified in the activin family [27].

Bone morphogenetic proteins

BMP ligands belong to the TGFb family and, like Nodal ligands, act on

transmembrane serine/threonine kinase receptors to modulate the

activity of Smad proteins – transcription factors that regulate genome

activity [81] – by phosphorylation and association with diverse

partners (Figure I).

Fibroblast growth factors

FGF signaling is active in many cell types and organs in vertebrates.

To date 22 FGF ligands have been described, which activate

transmembrane receptor tyrosine kinases at the cell surface [82]

(Figure I). This signaling activates the small GTPase Ras and several

cascades, most notably the mitogen activated protein (MAP) kinase

cascade (through Ras, the serine/threonine kinase Raf and the MAP/

ERK kinase MEK), which can lead to the specific phosphorylation and

activation of key transcription factors.

Retinoids

Retinoids, including all-trans retinoic acid (RA) and 13-cis retinoic

acid, are active signaling molecules that can act non-cell-autono-

mously on nuclear receptors of the RAR and RXR families [83] (Figure

I). Ligand binding drives the activity of these receptors as specific

transcription factors that regulate target gene expression.

Wnts

Wnt ligands, which are secreted glycoproteins, act on Frizzled

receptors to induce a complex intracellular signaling cascade that

leads to the regulation of target gene expression through the

activation of complexes between b-catenin and lymphoid enhancer-

binding factor/T-cell factor (TCF; Figure I). There are 19 Wnt ligands

and some act through non-canonical signaling routes that have

different effects (http://www.stanford.edu/�rnusse/wntwindow.html).

Figure I. Diagrams of selected essential components of five signaling pathways involved in forebrain patterning. The pathways are complex and for simplicity, in most

case only the ligands, receptor modules and final transcription factors regulating the genomic response are depicted. See the references cited in the text for details. The

overall strategy taken by the Hh and Wnt pathways is similar; the FGF pathway (and other similar peptide growth factor pathways that activate receptor tyrosine kinases

and MAP kinase cascades) has a true cascade of intracellular effectors; and the RA pathway is exceedingly simple, with the ligand penetrating the cell and acting in the

nucleus to activate transcription after binding to its nuclear receptor. In all cases, signaling leads to the activation of regulators that are already made, but inactive.

Abbreviations: Act-TFs, activated transcription factors; FGFR, fibroblast growth factor receptor; Frz, Frizzled; I-Smad, inhibitory Smad; LRP, low-density lipoprotein

receptor-related protein; RAR and RXR, retinoic acid receptors; R-Smad, receptor-activated Smad.

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Box 3. DV patterning of the mouse and chick spinal cord

Figure Ia shows a diagrammatic cross section of the neural tube,

showing the sources of patterning signals, while Figure Ib shows the

neural progenitors that arise from different DV regions of the neural

tube in response to these patterning signals. The types of post-mitotic

neuron that are formed at each position within the spinal cord at later

stages of development are shown in Figure Ic (colors correspond to

those in Figure Ib). (In Figure I: dI, early born dorsal interneurons; dIL,

late born dorsal interneurons; dp, dorsal progenitor domain; p,

ventral progenitor domain.)

The neural tube, which forms the spinal cord at caudal levels of the

embryo, is patterned along the DV axis by signals coming from

signaling centers located at the midline, the floor plate and the

notochord ventrally and the roof plate dorsally, but also from the

somites (Figure Ia; S; formed from the paraxial mesoderm). The floor

plate is the source of the Shh signal implicated in patterning the ventral

neural tube [from which motor neurons and V0 to V3 interneurons will

arise (Figure Ic)]; the roof plate is the source of TGFb (such as BMPs,

Gdf7 and activin) and Wnt molecules that are involved in patterning the

most dorsal part of the neural tube (from which dI1 to dI3 interneurons

will differentiate (Figure Ic); the somites contribute via the production

of retinoic acid (RA) in specifying part of the intermediate compartment

of the neural tube [from which V0 and V1 interneurons develop (Figure

Ic)] and, together with Shh, in inducing motor neurons (MN). The

interneurons dI4–dI6, dILA and dILB differentiate independently from

roof plate inductive signals; the role of RA, which has been shown to

act on the dorsal compartment, in their specification remains to be

addressed. The implication of RA in general dorsal neural tube

patterning (Figure Ia) also needs to be clarified.

The interpretation and integration of all of these signals lead to the

establishment of several domains in the neuroepithelium (n) along

the DV axis (Figure Ib). In each of those domains, neuronal

progenitors are fated to give rise to one particular population of

neurons composing the spinal cord (Figure Ic). Shh is known to act in

a concentration-dependent manner, thus keeping control of the

ventral progenitor domains. A combination of qualitative and

quantitative cues probably acts in the dorsal neural tube. Indeed, if

data are in agreement with a role for a graded BMP signaling in

specification of interneurons dI1–dI3, it seems that some of these

interneurons could rely on specific roof plate factors for their

determination. For more detailed reviews, see Refs [1,67,68].

Figure I.

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expand to give rise to the appropriate number of cells thatmake up each compartment.

The study of the interactions between the Nodal and Hhpathways in ventral telencephalon patterning suggest thatNodal signaling acts to upregulate Shh expression (e.g.[28]). The mechanisms that could underlie this regulationhave been studied, and the results argue for a directtranscriptional control of Shh expression in the neuraltube [31]. The question of how the Hh pathway interactswith Nodal signaling in hypothalamus patterning remainsto be addressed directly.

Mouse

Inmice, loss of Nodal function leads to early developmentalarrest at gastrulation [32] and thus the function of Nodal inlate forebrain patterning cannot be examined with thismutant allele. Conditional alleles of Nodal, which makeexamination of its function after gastrulation possible,have been generated [33,34], but so far the analysis hasbeen focused on patterning of the anterior embryo as awhole rather than the forebrain. These studies have shown

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that Nodal is required for the establishment of theproximo–distal axis into the anterior–posterior axis andin maintenance of anterior patterning in the early embryo.The defects that have been noted in the mutant embryosare likely to be due to defective function of the anteriorvisceral endoderm (an early tissue involved in forebrainpatterning) and absence of the prechordal plate [33,34],tissues that are both crucial for anterior and ventraldevelopment [35]. Thus, those studies do not yet clarifythe possible interactions between Nodal and Shh in DVforebrain patterning in the mouse. Moreover, precise andextensive cell lineage and fate analyses are lacking. Never-theless, it is still possible that a molecular mechanismintegrating Nodal and Shh signaling, in common withzebrafish and other vertebrates, could be at work.

The FGF and Hh pathwaysZebrafish

Several results support the involvement of FGF signalingin forebrain patterning in zebrafish [3,36,37]. The expres-sion of Fgf3 and Fgf8 changes quickly in the developing

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forebrain (i.e. in the anterior neural border, the dorsaltelencephalon and the diencephalon) and they have spe-cific and redundant functions in patterning this structure[38,39]. In particular, they are implicated in telencephalicand diencephalic ventral patterning, and their loss offunction leads to defects in neuronal differentiation. Inter-estingly, the expression of two Hh molecules, Shh andTiggywinkle hedgehog (Twhh), seems to be dependenton signaling by FGF3 and FGF8 in the hypothalamus,the ventral thalamus and the zona limitans intrathalamica(ZLI) [38]. Moreover, the expression of Fgf3 and Fgf8 is, atleast in part, under the control of Hh signaling in theforebrain [39]. This suggests that some of the effects ofboth these FGF ligands could be due to their ability toregulate elements of the Hh pathway and that FGF8 andFGF3 could be involved in a positive feedback loop in someregions of the forebrain. As discussed later, FGF and Hhsignaling have an intimate relationship and a positivefeedback loop could also exist in other situations.

Recently, the zebrafish Fgf19 gene (an ortholog of thehuman FGF19 and mouse Fgf15 genes) has been identi-fied; it is expressed in the telencephalon and diencephalon[39]. Fgf19 is involved in the general growth of the fore-brain, midbrain and hindbrain and this function is due toits role in cell proliferation and survival [39]. Moreover, theexamination of patterning defects in embryos injected withmorpholino oligonucleotides to knock down the expressionof Fgf19, and the analysis of forebrain marker expression,demonstrated that Fgf19 is also involved in specification ofthe ventral telencephalon and anterior-ventral thalamus.Consequently, neuronal differentiation is impaired in theaffected regions. Fgf19 expression depends on a functionalHh pathway, suggesting that, similarly to Fgf3 and Fgf8,Fgf19 acts downstream of the Hh pathway. Moreover,aspects of the phenotype caused by inhibition of Hh signal-ing, such as loss of telencephalic and diencephalic GABAer-gic interneurons (generated ventrally) can be rescued byFgf19 overexpression [39].

Consequently, in zebrafish, FGF ligands are requiredfor correct forebrain patterning, and interactions betweenthe FGF andHh pathways are essential. How this occurs isnot known. It is also not clear whether or how the Gliproteins are involved in pathway integration or coordina-tion. In frog embryos, however, it has been shown thatGli2and Gli3 are involved in a positive feedback loop with FGFsignaling during mesoderm induction and patterning [40],suggesting that a mode of interaction between the Hh andFGF pathways might be applicable to other situations.Interestingly, recent data suggest regulation of Gli func-tion by extracellular signal regulated kinases (ERKs) [41],possibly providing a molecular explanation for phenotypicinteractions.

Chick

Data obtained in the chick embryo has also led to theconclusion that FGF signaling is an important player inforebrain patterning and more specifically in telencepha-lon patterning [42,43]. Indeed, Fgf8, which is secreted fromthe anterior neural ridge [2] located at the most anterioredge of the early neural plate (at around embryonicday E8 inmouse), is believed to be involved inmaintenance

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of the ventral telencephalic character at the time theintermediate region of the telencephalon is specified (seelater) [43].

FGF signaling, however, is also involved in the devel-opment of the dorsal telencephalon in chick embryos [42].This dual function of the FGF pathway in telencephalonpatterning, even if occurring approximately at the samestages of development, is thought to imply two differentsources of FGF ligands: Fgf8 is first expressed anteriorlyand influences cells that will contribute to the ventraltelencephalon, but then its expression is upregulated inthe dorsal rostral midline, so that it influences dorsaldevelopment [42]. Moreover, interactions with two differ-ent signaling pathways take place in the ventral and dorsaldeveloping telencephalon: FGF contributes to ventraliza-tion, at least in part, by counteracting the action of theretinoid pathway (see below) [43] whereas it contributes todorsalization acting together with the Wnt pathway (seebelow) [42]. Interestingly, Fgf3 and Fgf8 are also expressedin the dorsal telencephalon in zebrafish [38] and the ques-tion of whether FGFs are also implicated in some aspects ofdorsal telencephalon development in this model systemremains to be addressed. In this regard, it has been shownthat FGFR1 is present in the dorsal telencephalon inzebrafish [44]. Like in chick and mouse, the action ofFGF and Hh signaling in zebrafish is likely to be complex.

Mouse

In mouse, Fgf8 has been shown to ventralize dorsal tele-ncephalic tissue in explants, independently of Shh signal-ing, and mice mutant for Fgf8 show defects in aspects ofventral telencephalic specification, with loss of Shh andNkx2.1 expression (e.g. [45]). This suggests that, in thisinstance, FGF signaling is upstream of Shh, although thiscould reflect the requirement for FGF signaling in thespecification and/or proliferation of cells that will thenexpress Shh. By contrast, Shh and FGF8 cooperate toinduce dopaminergic neurons in the ventral forebrainand midbrain [46], although here again there could be adistinct temporal order of action of each signaling pathwayand not a direct integration of action at the same time.

It has also been proposed that FGF8 takes part in theventralization of the telencephalon in Gli3�/� mouseembryos [47]. The expression of Fgf8 in the rostral tele-ncephalon depends on Hh signaling, as Gli3 is a repressorofFgf8 expression [48,49] and Shh counteracts the action ofGli3. The recent specific knockdown of several FGF recep-tors in the mouse telencephalon has clearly demonstratedthat the FGF pathway is implicated in ventral forebrainspecification, and genetic data indicate that it acts down-stream of the Shh–Gli3 antagonism [50].

The results obtained in chick, mouse and zebrafish thushighlight the roles of the Hh and FGF pathways and across-regulation between the two pathways at distinct andprecise times in telencephalon ventralization (Figure 2).

Retinoic acid and Hh signalingIn mice, the role of RA in forebrain development andpatterning has recently been evaluated using embryoslacking a retinal dehydrogenase enzyme (Raldh2) respon-sible for the production of retinoic acid [51]. The production

Figure 2. Selected interactions between signaling pathways at early (left; �E8.5 to E9) and later (right) forebrain development. The localization and ranges of signaling

molecules change with time and are affected by tissue growth. Arrows represent positive influences and T bars negative ones. These influences can be at many levels,

including transcriptional, post-transcriptional and effects on cells that then express other signaling molecules. See text for details.

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of RA in the early developing forebrain and surroundingmesenchyme is dependent on Raldh2 [51]. Raldh2-deficient embryos present strong defects in the developingforebrain: they have a single hypoplastic vesicle, with nodistinction between telencephalon and diencephalon.These abnormalities have been ascribed to a combinedeffect resulting from increased apoptosis, decreased pro-liferation and patterning defects [51]. Indeed, the expres-sion of Foxg1 (encoding a winged helix/forkheadtranscription factor required for telencephalic develop-ment), of the dorsal marker Emx2 and of the ventralmarkerNkx2.1 are all reduced in expression. RA deficiencyaffects dorsal and ventral patterning, acting on FGF andShh signaling, as described earlier, which might explainnot only these patterning defaults, but also the reducedsurvival and proliferation potential of forebrain progenitorcells [51].

These results are similar to that obtained in chickembryos in which RA signaling was inhibited via theuse of antagonists of the RAR and RXR retinoic acidreceptors [52]. Treatment of chick embryos with theseantagonists early during embryogenesis (Hamburger-Hamilton stage 10) leads to the absence of telencephalonand to a hypoplastic diencephalon. Shh and Fgf8 expres-sion is highly reduced in embryos with reduced RA signal-ing, suggesting that these two signaling pathways areinvolved in the mediation of RA signaling in the formationof a normal forebrain in the chick embryo [52].

Another role for RA signaling in the forebrain has alsobeen reported. The developing mouse telencephalon iscomposed of three compartments along the DV axis: thedorsal compartment, the ventral compartment, andbetween them an intermediate compartment that spansboth dorsal structures and ventral structures (includingthe lateral ganglionic eminence; reviewed in [12]). Theexistence of the intermediate compartment is revealed,for example, in Shh-null mice, in which ventro-lateralregions of the telencephalon are not affected (reviewedin [12]). This suggests that this intermediate domain doesnot depend solely on Shh function, but also on RA signal-ing. Recent work carried out in the chick embryo has led to

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the conclusion that the RA signaling pathway is necessaryand sufficient to specify intermediate character in telence-phalic tissue [43] (Figure 2). The source of RA is thought tobe the head ectoderm, which expresses Raldh3, an enzymeinvolved in RA production. The presumptive telencephalicterritory is first exposed to the ventralizing action of Shhduring gastrulation [53]. Yet the role of RA in inducingtelencephalic cells to adopt an intermediate fate might beindependent of Shh signaling, because the intermediatecompartment is present in Shh-deficient mice [18]. More-over, induction of the intermediate telencephalic domaindoes not seem to imply inhibition of the Shh pathway,because when RA signaling is blocked in vitro or in vivo, itdoes not lead to complete ventral differentiation [43]. It ispossible that the range of Shh activity, in combination withthe growth of the telencephalic vesicle, leads to the gen-eration of a presumptive intermediate region that is notsubject to the ventralizing effect of Shh. However, a morelikely explanation can be deduced from the fact that dor-salizing signals (see later), which can reverse ventralizingeffects [42], act before RA and therefore create an area inwhich an intermediate identity can be imprinted. Consis-tent with this second hypothesis, we note that RA inducesintermediate cells in early dorsal telencephalic explantsmuch more efficiently than in early ventral telencephalicexplants [43].

BMP and Wnt signalingIn mammals, BMPs and Wnts, which are present in thedorsal midline and adjacent cells, have been shown to beinvolved in dorsal telencephalon patterning (reviewed inRef. [12]), but their role and their potential interactionswith Shh signaling and Gli proteins are still not wellunderstood. In chick, Wnt factors have a prominent rolein specification of the dorsal telencephalon in combinationwith FGF ligands, whereas BMPs do not seem to have anydorsalizing activities [42].

The role of b-catenin, a mediator of the canonical Wntsignaling pathway (one of the two ways in which Wntssignal), in forebrain patterning was addressed inmice withgain and loss of function of b-catenin [54]. Analysis of these

604 Review TRENDS in Cell Biology Vol.16 No.11

transgenic mice show that early inhibition of b-cateninleads to downregulation of dorsal marker gene expressionand upregulation of ventral markers, such as the tran-scription factor-encoding genes Gsh2 and Dlx2, suggestingthat canonical Wnt signaling has a role in patterning theearly mouse forebrain. However, another study using micein which b-catenin had been inactivated using a floxedcatenin allele and Foxg1-Cre mice [55] also found strongdefects in the forebrain formation but attributed them to arole of b-catenin in cell adhesion within the early forebrainneuroepithelium rather than to a role including canonicalWnt signaling [56].

Wnt signaling might generally act downstream of Glifunction. Gli3 is required for dorsal telencephalic develop-ment in mouse [16], and analysis of Gli3�/� embryos hasshown that BMPs andWnts seem to lie downstream of Gli3function [47,57] (Figure 2). However, the mechanismsregulating these interactions have not yet been investi-gated in these anterior structures. In the frog embryo, Gliproteins regulate a battery of Wnt genes [58]. Neverthe-less, Wnt, BMP and Hh signaling show positive feedbackloops in different directions in different tissues. Forinstance, in the hair follicles, the Wnt pathway activityis thought to act upstream of Hh signaling [59]. Moreover,in mice, BMP signaling has been implicated in dorsalmidline specification [60,61], and cooperation betweenShh and BMP (BMP7) signaling has also been shown toinduce diencephalic ventral midline markers (reviewed in[7]).

ConclusionsThe data summarized here point to important effect ofcombinations of TGFb (BMP or Nodal), Wnt, FGF, RA andHh signaling pathways in regulating cellular responsesand patterning in the forebrain and elsewhere in the CNSand other organs. How they act together to cause so manydifferent outcomes is unclear. The possibility that theinformation encoded in pathway signaling is integratedat the level of transcription factor function is intriguing,and future research needs to address this issue by eluci-dating the molecular mechanisms regulating final path-way mediators, such as Gli and Smad proteins (which aredownstream of BMPs). In some instances, the differentpathways can affect the same cell populations simulta-neously or at different times [62], or they can even affectdistinct yet adjacent subpopulations [63]. Interestingly,the Hh–Gli signaling pathway seems to have been rede-ployed often during evolution and could be a key target ofevolutionary change, underlying, for example, the rapidmodification of brain shape during evolution (e.g. [64,65];for a review see Ref. [66]). Future research might unravelnot only the kinds of molecular interactions underlyingsignaling integration but also the strength of signalingrequired and the precise timing of events.

AcknowledgementsWe apologize to our colleagues whose work was not cited due to spacelimitations. Work in N.D.’s laboratory was supported by a CNRS ATIPEgrant, l’Association pour la Recherche contre le Cancer, la Fondation pourla Recherche Medicale while at the Developmental Biology Institute ofMarseille, France, and by grants from the A. Taxin Brain Tumor Center, VFoundation and The WW Smith Charitable Trust at the Wistar Institute.

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