www.elsevier.com/locate/brainresrev

Brain Research Reviews

Review

Modulation of Fgf8 activity during vertebrate brain development

Diego Echevarriaa,*, Jose Antonio Belob,c, Salvador Martineza,*

aInstitute of Neuroscience, University Miguel Hernandez (UMH-CSIC), Carretera de Valencia (N332), San Juan, Alicante 03550, SpainbCentro de Biomedicina Molecular e Estrutural (CBME), Universidade do Algarve. Campus de Gambelas, 8005-139 Faro, Portugal

cInstituto Gulbenkian de Ciencia, Rua da Quinta Grande, 6. Apartado 14, 2781-901 Oeiras, Portugal

Accepted 16 December 2004

Available online 16 February 2005

Abstract

In recent years much emphasis has been placed on investigation of the precise control of FGF signaling during brain development. Such

control is achieved in part by regulatory elements that determine the domains and levels of expression of genes coding for the diverse FGF

ligands via specific molecular signaling pathways. There is new knowledge on the operation of such mechanisms in regions of the neural tube

involved in the correct patterning of adjacent territories (known as secondary organizers of neural tube pattern). In the present minireview we

intend to summarize recent evidence and emerging conclusions on potent modulators that govern the activity of Fgf8 signals in the

developing vertebrate brain, focusing our attention on the best known secondary organizer, the isthmic organizer.

D 2005 Elsevier B.V. All rights reserved.

Theme: Development and regeneration

Topic: Pattern formation, compartments, and boundaries

Keywords: Isthmic organizer; Mouse; Brain; MAPK phosphatase; ERK1/2; PI3K; Fgf8; Mkp3; Sprouty; Sef

Contents

1. The isthmic organizer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150

2. FGF8 signal and the isthmus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151

3. FGF intracellular pathways. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152

4. FGF modulators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152

4.1. Sprouty genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152

4.2. Sef gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152

4.3. Mkp3 gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153

5. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155

0165-0173/$ - see front matter D 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.brainresrev.2004.12.035

* Corresponding authors. Fax: +34 965919555.

E-mail addresses: diegoaza@umh.es (D. Echevarria)8

smartinez@umh.es (S. Martinez).

1. The isthmic organizer

The regionally differential specification state of the brain

(molecular regionalization) evolves and diversifies over

time. Preexistent cellular specification states may either

change into new ones or become increasingly fixed and

49 (2005) 150–157

D. Echevarria et al. / Brain Research Reviews 49 (2005) 150–157 151

irreversible (fate determination), under the control of

various cell-autonomous and microenvironmental effects.

Several classes of developmental genes are influential in this

process, and the lack of their functions can lead to profound

alterations in the development of specific brain regions. At

each developmental stage and locus there occurs both

intrinsic genetic regulation (genes up-, downregulated, or

maintained) and epigenetic intercellular signaling. These

dynamic phenomena are all encompassed in the concept of

bpatterningQ (Fig. 1A; [52]). Thus, the process of ordered

embryogenesis requires cell-to-cell communication media-

ted by secreted factors and/or cell contacts and further

involves complex interactions of multiple intracytoplasmic

signaling pathways. The specification or patterning of the

entire brain (as a part of the body), which involves setting

up the D/V and A/P axes, is dependent upon signals

produced by discrete embryonic regions – the primary

organizers – at gastrulation and early neural plate stages.

These are represented by the anterior visceral endoderm or

AVE and the node and its derivatives (reviewed in [1,58]).

The term bsecondary organizerQ is applied at early neural

tube stages to neuroepithelial subregions experimentally

shown to have polarizing and inductive properties. These

secondary organizers usually develop within the previously

broadly regionalized neuroectoderm at given genetic boun-

Fig. 1. (A) Schematic representation of an E9.5 mouse neural tube in which the ne

zones by shadowed areas (following the prosomeric model [52,54]). The position

activity along the neural tube (arrows) is also shown. (B) This represents at higher m

some of the gene expression patterns at the isthmic organizer (IsO). The expression pa

this minireview but extended information elsewhere [15]. (C) Drawing that represen

data from recent literature cited in the text, inclusive of the negative modulators of

dary loci and their subsequent activity refines local neural

identities along the A/P or D/V axes [15,17,23,41,54,65].

The best studied secondary organizer is the isthmic

organizer (IsO), which is located at the junction between

the mesencephalic and rhombencephalic (mes/rhomb) terri-

tories, and is marked by the isthmic constriction [9,10,37]

(Figs. 1A and B).

2. FGF8 signal and the isthmus

Among the molecules with known morphogenetic

activity, there is now extensive evidence that FGF8 signal-

ing is essential for the development of the vertebrate

midbrain and cerebellum (reviewed in Refs. [15,31,37,44,

68]). Fgf8 is expressed in the isthmic organizer from early

neural plate stages (~embryonic day [E] 8.25) to mid-

gestation (~E12.5) stages (Fig. 1B). The murine Fgf8 gene

contains six exons and its alternate splicing has been shown

to produce isoforms with different efficiencies [34]. Eight

Fgf8 isoforms were found so far [9] and it was reported that

Fgf8b has stronger transforming activity than Fgf8a [35]. In

fact, the Fgf8b isoform can induce a rhombencephalic

phenotype in the diencephalon or in the mesencephalon

[32,38] but not so the Fgf8a isoform [57].

uromeric boundaries are delineated by transversal lines and the longitudinal

of known secondary organizers and their postulated morphogenetic field of

agnification the area enclosed by a dashed rectangle in panel A to illustrate

ttern ofFgf15, a possible downstreamof FGF8 signal, will not be discussed in

ts schematically the intracellular Fgf8 signals occurring at the IsO, collecting

FGF8 found acting at different levels of different intracellular pathways.

D. Echevarria et al. / Brain Research Reviews 49 (2005) 150–157152

Mutational analysis in mice indicated that FGF signaling

has distinct functions at different stages of mid/hindbrain

development. At early stages, FGF8 is required to maintain

the expression of genes that play a role in patterning the

neural tube [15]. Moreover, it is essential for cell survival, as

evidenced by the finding that inactivation of Fgf8 in the early

neural plate causes extensive cell death throughout the

mesencephalon and rostral hindbrain between the 7 and 30

somite stages (E8.5 and E10, respectively), resulting in full

deletion of the midbrain and cerebellum. Interestingly, when

Fgf8 expression is modestly reduced, rather than eliminated,

the rostral-most portion of the midbrain is spared and appears

normal, whereas the remaining dorsal midbrain, the isthmus,

and cerebellum are absent [7]. This suggests that there are

regional differences in sensitivity to FGF signaling within

the mesencephalon and/or rhombencephalon. Thus, FGFs

activate signal transduction pathways required for multiple

developmental processes including cell fates, determination

of axial polarity, and promotion of cell survival.

3. FGF intracellular pathways

Substantial progress has been made towards under-

standing the intracellular response and transductional

mechanism of the FGF8 signaling pathway. FGF signaling

is mediated via tyrosine–kinase receptors (RTKs). The

transmembrane FGF receptors (FGFRs) activate several

signaling cascades including the phospholipase C gamma

(PLC-gamma), phosphatidylinositol-3 kinase (PI3K), and

Ras–ERK pathways, the latter being a subclass of mitogen-

activated protein kinase pathway (Fig. 1C; MAPK;

reviewed in Refs. [36,45,61]; the PLC-gamma pathway

will not be discussed further in this review). The mentioned

pathways ultimately regulate gene transcription. While the

activation of the MAPK cascade promotes neural prolifer-

ation, differentiation, and apoptosis, activation of the PI3K

pathway promotes cell survival [6,46,50]. In the mouse

neural tube at E9.5, Fgfr1 is widely expressed throughout

the entire developing brain, while Fgfr2 appears restricted

to the diencephalon, hindbrain, and spinal cord but is clearly

absent in the isthmic organizer itself [60]. Interestingly, it

was found by the latter authors that after establishment of

the midbrain and hindbrain regions, Fgfr1 is required for

the normal response to the signals coming from the isthmic

organizer. This study further suggests that FGF signaling

through FGFR1 is directly involved in the regulation of

subsequent gene expression in both the mid- and hindbrain.

The diversity of signaling pathways triggered by FGFs and

the importance of this signal for many developmental

processes entrail a tight positional regulation of its signal

intensity and duration (Fig. 1C). Here we intend to

summarize recent evidence on control mechanisms that

govern the Fgf8 signal activity in the neural tube,

emphasizing available information on the best known

morphogenetic brain area, the isthmic organizer.

4. FGF modulators

In developmental embryology and particularly in develop-

mental molecular biology, the term bsynexpression-groupgenesQ is given to those genes in a complex hierarchy that

share a distinct expression pattern and accordingly may be

involved in the same biological processes [48]. Recent

investigations demonstrated that FGF signaling is negatively

modulated by complex intracellular regulatory systems. It

was previously shown that there exist other genes that are

co-expressed with Fgf8 in vertebrate embryos [61]. Among

these are included cytosolic elements such as the sprouty

proteins (Spry1, 2, 4) and MAP-kinase-phosphatase-3

(Mkp3), as well as the transmembrane protein Sef (Fig. 2)

[18,21,27,29,62,64,70]. Spry1, 2, 4, Mkp3, and Sef genes

represent a synexpression group with Fgf8.

4.1. Sprouty genes

Sprouty (Spry) was the first member of the synexpression

group found. The Drosophila Spry gene was identified by its

property of regulating tracheal branching morphogenesis via

intracellular antagonism of the FGF signaling pathway [21].

Four Spry were later identified in vertebrates [11] and their

respective expression patterns were studied [42]. Only

Spry1/2 are expressed at the isthmus [5,30,33,39], where

their RNA signal overlaps topographically with Fgf8

expression, they function as negative regulators of the

Ras–MAPK signaling pathway [4,28,33]. Spry proteins

seem to specifically inhibit the Ras/Raf/Erk pathway,

leaving the phosphatidylinositol 3-kinase (PI3K) and other

MAP kinase pathways intact [71]. Spry1/2 are thought to be

largely redundant functionally. This inhibitory activity has

been localized downstream of the RTKs and upstream of

Ras activation (Fig. 1C). Although some interactions are

known to exist between Spry proteins and components of

the Ras/Raf/Erk pathway, such as Grb2 [20] and c-Cbl [67],

the precise molecular mechanism by which the Fgf8 signal

is blocked remains unknown. Vertebrate sprouty genes can

cause chondrodysplasia when overexpressed [42]. In the

context of the isthmic organizer and other brain regiona-

lization phenomena observed in vertebrates, it would be

of great interest to perform gain-of-function and loss-

of-function experiments, i.e., by generating conditional

mouse mutant models, in order to advance in our under-

standing of Fgf8 signal regulation by the sprouty genes.

4.2. Sef gene

Another member of the FGF synexpression group is

called Sef (Similar expression to Fgfs); it codes for a

transmembrane protein which is highly conserved across

anamniotes and amniotes (zebrafish, Xenopus, mouse, and

human) but does not exist in invertebrates [30]. As its name

implies, and as in the case of Sprouty, Sef is expressed in the

same neuroepithelial places where Fgf8 is expressed

Fig. 2. Expression pattern of Fgf8 and Fgf8-modulator genes. (A) Expression of Fgf8 in an E10 mouse embryo. (B) Expression pattern of Mkp3, the new

negative modulator of Fgf8 signal. Note that the expression pattern is almost identical, besides small differences (see text). The white dashed line indicates the

plane of the section shown in panel C. (C) Transversal section of an E10 mouse embryo in which the isthmic Mkp3 expression pattern can be examined.

Interestingly, Mkp3 is expressed also in the mesenchyme close to the expression in the neuroepithelium; the arrowhead indicates the roof plate. (D) Sagittal

section of a littermate in which theMkp3 expression can be observed at the isthmic organizer, both in the alar and basal plates, and including the floor plate. (E)

Mkp3 expression pattern in the wnt1�/� mutant mouse. The arrowhead indicates the lack of Mkp3 as well as of Fgf8 expression in the IsO suggesting a

correlation between the two genes. (F) A cartoon sketched by S.Martinez that summarizes the experimental methodology used for explant-culturing a whole

mouse neural tube as a key for the following images. Briefly, the roof plate is cut along the entire neural tube and the lateral walls are flattened down as an open

book upon membrane rafts floating over a small amount of culture medium (see for details Ref. [15]). (G–J) Neural tube culture explants of an E9.5 mouse

neural tube after 12 h in vitro with different synexpressed gene patterns. We have used only one half of the explant to illustrate the identical position and

maintenance of gene expressions and some characteristic brain structures in vitro as compared to the normal case. The dashed red line indicates the ventral

midline of the neural tube (median floor plate). The outer profile of the cultured explant corresponds to the roof plate. ANR, anterior neural ridge; BI, branchial

arch one; BII, branchial arch two; cf, cephalic flexure; D, diencephalon; fp, floor plate-; flb, forelimb bud; hlb, hindlimb bud; IsO, isthmic organizer; M,

mesencephalon; os, optic stalk; ov, otic vesicle; s, somites; Tel, telencephalon; zli, zona limitans intrathalamica.

D. Echevarria et al. / Brain Research Reviews 49 (2005) 150–157 153

normally, though in a broader fashion than Fgf8 expression.

In fact, Sef is also an antagonist of Fgf8 signaling in

zebrafish [18] and Xenopus [62] and is recently held to have

this function in mammals as well [51]. Ectopic induction of

Sef by injection into Xenopus blastomeres blocks FRS2

(FGF receptor subtrate-2), the key mediator of the Ras–

MAPK and PI3K pathways [62]. Furthermore, injection of

antisense morpholino oligos mimics the effects produced by

ectopic Fgf8 expression. Such functional results were

adduced to claim that the action of Sef is to modulate the

dorsalizing and mitogenic effects of FGF8 signals in

zebrafish and Xenopus brain development, thereby limiting

the intensity and regional spread of positive upregulation of

Fgf signaling in diverse parts of the brain [18,62].

The conventional mechanism by which Sef functions is

by inhibiting tyrosine phosphorylation of FGFR1 [27] and

FGFR2, but not of FGFR3 [69]. As a consequence, there

occurs a reduction in the phosphorylation state of Raf-1.

This indicates that Sef must act at the level of FGFR1/2

themselves or between the FGFRs and Ras (probably by

inactivating FRS2 [62]). This would lead to a downstream

reduction in the activation of components of the Ras–

MAPK and PI3K/AKT pathways [47] (Fig. 1C). However,

this scenario may have changed since two alternatively

spliced isoforms of human Sef were identified recently,

which show functional heterogeneity. One ubiquitous iso-

form (called Sef-a) is a transmembrane element and

subserves the functional profile sketched above, acting

upstream of Ras. The second isoform, called Sef-b, lacks

transmembrane character and is not ubiquitous like Sef-a,

being localized instead to the cellular cytosol at neuro-

epithelial sites characteristic of the Fgf8 synexpression

group (i.e., the isthmic organizer). This form is able to

suppress FGF signaling downstream of MEK and does not

block the PI3K pathway [19,51]. These recent investigations

thus uncover multiple interactions of Sef at different levels

of the Ras–MAPK pathway, which suggests different

functional effects depending on the biological context [59].

4.3. Mkp3 gene

As we mentioned above, one of the major pathways

activated by FGF signals is the conserved protein cascade

referred to as mitogen-activated-protein (MAP) kinase

D. Echevarria et al. / Brain Research Reviews 49 (2005) 150–157154

module (Ras N Raf N MEK N ERK or Ras–MAPK

pathway). Activation of ERK is dually phosphorylated by

MEK and MEK is dually phosphorylated by Raf. Thus, it is

considered that the amplification of this cascade is actually

dependent on 5% of Ras molecules for fully activation of

ERK [22]. Activation of the MAPK cascade promotes

neural proliferation, differentiation, and apoptosis [3,50,55].

Upon activation, ERK and MEK detach from the surround-

ing proteins and translocate into the nucleus. Thereafter,

MEK is exported again to the cytoplasm, whereas ERK is

retained in the nucleus, increasing synthesis and trans-

cription of nucleotides [66] (Fig. 1C). ERK has two

isoforms (1 and 2), of which ERK2 is of great interest

due to the lethal phenotype produced at E6.5 when the gene

is knocked down [56].

Negative regulation of ERK1/2 activation is performed

by a family of MAP dual-specificity phosphatases (MKPs).

There are up to nine MKPs. Several of these are under tight

transcriptional control and display distinct cellular and

subcellular expression patterns [49]. Among them Mkp3

appears exceptional in that it specifically inactivates ERK2

[3,43,72]. In fact, monomeric Mkp3 selectively binds the

monomeric phosphorylated form of ERK2 and the conse-

quent activation of the catalytic subunit of Mkp3 leads to a

z4000-fold rate enhancement for the dephosphorylation of

ERK2 [25].

The zebrafish, chick, mouse, and human Mkp3 genes

were cloned recently [12,13,24,26,63]. The expression

pattern of mouse Mkp3 is of interest because it also belongs

to the synexpression group of Sef, Sprouty, and Fgf8. In

E8.5 to E10.5 mouse embryos (Figs. 2A and B) the

expression patterns of Mkp3 and Fgf8 are maintained

roughly co-localized, although the expression domain of

Fgf8 gradually becomes more restricted than that of Mkp3.

From E9.5 onwards Mkp3 expression was observed in some

non-neural tissues (limb bud, branchial arches, tail bud, and

mesenchyme in the neighborhood of Mkp3-positive neuro-

epithelial cells; Figs. 2B–D) [12,13,24,26,63]. Interestingly,

whole mount in situ hybridization for Mkp3 on E9.5 neural

tube explant cultures [53], or on thick sections of embryonic

brain, detected throughout the neural tube a longitudinal line

of expression along the floor plate/paramedian plate (lateral

part of the floor plate according to Ref. [14]) (Figs. 2C and

3A). Thus, during the early development of the vertebrate

brain both Mkp3 and Fgf8 are expressed in correlative

domains in the neuroepithelium, particularly in association

to secondary organizers, such as the forebrain ANR and the

isthmic organizer. Interestingly the strongest domains of

ERK signaling also coincide with areas where a morpho-

genetic role is postulated for Fgf8 signaling [8]. A further

correlation between Fgf8 and Mkp3 role was noted in E9.5

Wnt1�/� mutant mice; these lack Fgf8 expression in the

isthmic organizer, where the expression domain of Mkp3

was also absent (Fig. 2E; [40]).

To further investigate the effect of Fgf8 morphogen on

Mkp3 induction, FGF8-soaked beads were implanted in

ectopic locations of mouse neural tube explants [19]. The

results showed that there was an induction of Mkp3

expression around the bead, suggesting an immediate

dependency on Fgf8 signaling (Figs. 3A and B). Moreover,

to investigate which is the signaling pathway responsible for

Mkp3 induction, specific inhibitors for the classical MAPK

(MEK1/2 inhibitor) and PI3K kinase (PI3K inhibitor)

pathways were used. The results showed that induction/

maintenance by FGF8 of the endogenous Mkp3 expression

in the isthmic organizer occurs mainly through the PI3K

signaling cascade (a survival/apoptosis pathway [6,46,50]),

rather than the MAPK pathway (cell proliferation). This

induction/maintenance seems important for mesencephalic

and rhombencephalic development since it results in a

severe reduction of genes activated downstream of Fgf8,

such as En2 [37,68]. Loss-of-function experiments using

siRNA to silence endogenous Mkp3 expression through

focal microelectroporated gene transfer in the mouse

isthmus (Figs. 3C–D) produced complete abolishment of

the endogenous Mkp3 expression as well as of Fgf8

endogenous expression within 24 h in vitro (Fig. 3E). A

hypothetical explanation for these unexpected observations

might be that initially the increasing levels of ERK1/2 due

to Mkp3 inactivation by siRNA may result from an

upregulation of the pathway leading to Sef expression

[18,62]. This switch of pathways would explain the

disappearance of Fgf8 expression from 7 to 24 h, since

the presence of Sef would inhibit ERK1/2 activation and

therefore its pathway [16].

5. Concluding remarks

Extensive information was obtained during the last 20

years regarding the relevance in developmental biology of

secondary organizers. Among them, the isthmic organizer

was studied most intensively, and its morphogenetic/

inductive activity during brain regionalization (leading to

differential and polarized tissue specification) was well

described and variously tested experimentally, even if that

chapter may not yet be finished. An exciting breakthrough

was made when specific molecular determinants were

identified and shown to act in combination, representing

the cause and consequence of the previously recorded

morphogenetic activity. This phase led to focusing of the

research on Fgf8 signaling, in parallel to complementary

ongoing progress on molecular fate specification in the

midbrain–isthmus–hindbrain continuum, increasingly sup-

ported by molecular loss-of-function and gain-of-function

approaches. Recent synthesis of the accumulated knowledge

highlights a scenario where the cellular mechanisms

triggered by Fgf8 signals bring together in a rather

satisfactory way the manifold data coming from expe-

rimental embryology, molecular biology, and cell biology.

The present minireview aimed to summarize this scenario

with emphasis in an important emergent issue, represented

Fig. 3. Functional relationship between FGF8 and Mkp3. (A) Ectopic induction of Mkp3 after implantation of an FGF8-soaked bead in the mesencephalon.

Although this image shows the induced expression after 24 h, Mkp3 usually was detectable already after 7 h of FGF8 bead implantation. (B) Same type of

experiment, but the fluorochromes for both genes are interchanged and the FGF8 bead was placed in the diencephalon. Note that in mammals the FGF8 protein

seems unable to induce its own expression. (C) Schematic representation of the microelectroporation technique used for Mkp3 siRNA experiments at the IsO.

(D and F) Controls of the electroporation assay, showing the green-fluorescent-protein [GFP]-electroporated region of the isthmus after 36 h. (E) An example of

the results obtained 24 h after microelectroporating Mkp3 siRNA at the isthmus (see [19]). Note that the endogenous Mkp3 expression is abolished at the left

side, in contrast to the control right side.

D. Echevarria et al. / Brain Research Reviews 49 (2005) 150–157 155

by the study of modifiers/attenuators of Fgf8 signaling

activity in the context of the vertebrate isthmic organizer.

Given the present abundance of biochemical protagonists

and the fact that two new negative modulators of Fgf8 have

been discovered within the last 2 years, it is plausible that

additional key molecular elements exist which must be also

investigated before we fully understand the control of brain

patterning at this single site during early development. The

results reported so far increasingly reveal considerable

complexity of the mechanisms underlying the intracellular

FGF signal. The question might be raised now whether there

exist molecules important for promoting or making more

selective Fgf signaling. The answer apparently is yes and

new data are coming in that address this issue [2].

Acknowledgments

The work presented by the authors has been supported by

the following EuropeanUnion Grants: U.E. QLG2-CT-1999-

00793; UE QLRT-1999-31556; UE QLRT-1999-31625;

QLRT-2000-02310; Spanish Grants: DIGESIC-MEC

PM98-0056; FEDER-1FD97-2090; BFI2002-02979; the

Spanish Multiple Sclerosis Foundation and Generalitat

Valenciana CTIDIA/2002/91, GV04B/673, and Ramon y

Cajal contract 2004 to Dr. Echevarria. Portuguese Grants:

POCTI/NSE/46420/2002 and IGC/Fundacao Calouste

Gulbenkian.

References

[1] R.S. Beddington, E.J. Robertson, Axis development and early

asymmetry in mammals, Cell 96 (1999) 195–209.

[2] R.T. Bottcher, N. Pollet, H. Delius, C. Niehrs, The transmembrane

protein XFLRT3 forms a complex with FGF receptors and promotes

FGF signalling, Nat. Cell Biol. 6 (2004) 38–44.

[3] M. Camps, A. Nichols, C. Gillieron, B. Antonsson, M. Muda, C.

Chabert, U. Boschert, S. Arkinstall, Catalytic activation of the

phosphatase MKP-3 by ERK2 mitogen-activated protein kinase,

Science 280 (1998) 1262–1265.

[4] T. Casci, J. Vinos, M. Freeman, Sprouty, an intracellular inhibitor of

Ras signaling, Cell 96 (1999) 655–665.

[5] D. Chambers, A.D. Medhurst, F.S. Walsh, J. Price, I. Mason,

Differential display of genes expressed at the midbrain–hindbrain

junction identifies sprouty2: an FGF8-inducible member of a family

of intracellular FGF antagonists, Mol. Cell. Neurosci. 15 (2000)

22–35.

[6] Y. Chen, X. Li, V.P. Eswarakumar, R. Seger, P. Lonai, Fibroblast

D. Echevarria et al. / Brain Research Reviews 49 (2005) 150–157156

growth factor (FGF) signaling through PI 3-kinase and Akt/PKB is

required for embryoid body differentiation, Oncogene 19 (2000)

3750–3756.

[7] C.L. Chi, S. Martinez, W. Wurst, G.R. Martin, The isthmic organizer

signal FGF8 is required for cell survival in the prospective midbrain

and cerebellum, Development 130 (2003) 2633–2644.

[8] L.B. Corson, Y. Yamanaka, K.M. Lai, J. Rossant, Spatial and temporal

patterns of ERK signaling during mouse embryogenesis, Develop-

ment 130 (2003) 4527–4537.

[9] P.H. Crossley, G.R. Martin, The mouse Fgf8 gene encodes a family

of polypeptides and is expressed in regions that direct outgrowth

and patterning in the developing embryo, Development 121 (1995)

439–451.

[10] P.H. Crossley, S. Martinez, G.R. Martin, Midbrain development

induced by FGF8 in the chick embryo, Nature 380 (1996) 66–68.

[11] A.A. de Maximy, Y. Nakatake, S. Moncada, N. Itoh, J.P. Thiery,

S. Bellusci, Cloning and expression pattern of a mouse homologue

of Drosophila sprouty in the mouse embryo, Mech. Dev. 81 (1999)

213–216.

[12] R.J. Dickinson, M.C. Eblaghie, S.M. Keyse, G.M. Morriss-Kay,

Expression of the ERK specific MAP kinase phosphatase PYST1/

MKP3 in mouse embryos during morphogenesis and early organo-

genesis, Mech. Dev. 113 (2002) 193–196.

[13] M.C. Eblaghie, J.S. Lunn, R.J. Dickinson, A.E. Munsterberg, J.J.

Sanz-Ezquerro, E.R. Farrell, J. Mathers, S.M. Keyse, K. Storey, C.

Tickle, Negative feedback regulation of FGF signaling levels by

Pyst1/MKP3 in chick embryos, Curr. Biol. 13 (2003) 1009–1018.

[14] D. Echevarria, C. Vieira, S. Martinez, Mammalian neural tube grafting

experiments: an in vitro system for mouse experimental embryology,

Int. J. Dev. Biol. 45 (2001) 895–902.

[15] D. Echevarria, C. Vieira, L. Gimeno, S. Martinez, Neuroepithelial

secondary organizers and cell fate specification in the developing

brain, Brain Res. Rev. 43 (2003) 179–191.

[16] D. Echevarria, S. Martinez, S. Marques, V. Lucas-Teixeira, J.A. Belo,

Mkp3 is a negative feedback modulator of Fgf8 signaling in the

mammalian Isthmic organizer, Dev. Biol. 277 (2004) 114–128.

[17] M.C. Figdor, C.D. Stern, Segmental organization of embryonic

diencephalon, Nature 363 (1993) 630–634.

[18] M. Furthauer, W. Lin, S.L. Ang, B. Thisse, C. Thisse, Sef is a

feedback-induced antagonist of Ras/MAPK-mediated FGF signalling,

Nat. Cell Biol. 4 (2002) 170–174.

[19] A.L. Garda, D. Echevarria, S. Martinez, Neuroepithelial coexpression

of Gbx2 and Otx2 precedes Fgf8 expression in the isthmic organizer,

Mech. Dev. 101 (2001) 111–118.

[20] I. Gross, B. Bassit, M. Benezra, J.D. Licht, Mammalian sprouty

proteins inhibit cell growth and differentiation by preventing ras

activation, J. Biol. Chem. 276 (2001) 46460–46468.

[21] N. Hacohen, S. Kramer, D. Sutherland, Y. Hiromi, M.A. Krasnow,

Sprouty encodes a novel antagonist of FGF signaling that patterns

apical branching of the Drosophila airways, Cell 92 (1998) 253–263.

[22] B. Hallberg, S.I. Rayter, J. Downward, Interaction of Ras and Raf

intact mammalian cells upon extracellular stimulation, J. Biol. Chem.

269 (1994) 3913–3916.

[23] A.L. Joyner, A. Liu, S. Millet, Otx2, Gbx2 and Fgf8 interact to

position and maintain a mid-hindbrain organizer, Curr. Opin. Cell

Biol. 12 (2000) 736–741.

[24] Y. Kawakami, J. Rodriguez-Leon, C.M. Koth, D. Buscher, T. Itoh,

A. Raya, J.K. Ng, C.R. Esteban, S. Takahashi, D. Henrique, M.F.

Schwarz, H. Asahara, J.C. Izpisua Belmonte, MKP3 mediates the

cellular response to FGF8 signalling in the vertebrate limb, Nat. Cell

Biol. 5 (2003) 513–519.

[25] Y. Kim, A.E. Rice, J.M. Denu, Intramolecular dephosphorylation of

ERK by MKP3, Biochemistry 42 (2003) 15197–15207.

[26] A. Klock, B.G. Herrmann, Cloning and expression of the mouse dual-

specificity mitogen activated protein (MAP) kinase phosphatase Mkp3

during mouse embryogenesis, Mech. Dev. 116 (2002) 243–247.

[27] D. Kovalenko, X. Yang, R.J. Nadeau, L.K. Harkins, R. Friesel, Sef

inhibits fibroblast growth factor signaling by inhibiting FGFR1

tyrosine phosphorylation and subsequent ERK activation, J. Biol.

Chem. 278 (2003) 14087–14091.

[28] S. Kramer, M. Okabe, N. Hacohen, M.A. Krasnow, Y. Hiromi,

Sprouty: a common antagonist of FGF and EGF signaling pathways in

Drosophila, Development 126 (1999) 2515–2525.

[29] I. Lax, A. Wong, B. Lamothe, A. Lee, A. Frost, J. Hawes, J.

Schlessinger, The docking protein FRS2alpha controls a MAP kinase

mediated negative feedback mechanism for signaling by FGF

receptors, Mol. Cell 10 (2002) 709–719.

[30] W. Lin, M. Furthauer, B. Thisse, C. Thisse, N. Jing, S.L. Ang,

Cloning of the mouse Sef gene and comparative analysis of its

expression with Fgf8 and Spry2 during embryogenesis, Mech. Dev.

113 (2002) 163–168.

[31] A. Liu, A.L. Joyner, Early anterior/posterior patterning of the

midbrain and cerebellum, Annu. Rev. Neurosci. 24 (2001) 869–896.

[32] A. Liu, K. Losos, A.L. Joyner, FGF8 can activate Gbx2 and transform

regions of the rostral mouse brain into a hindbrain fate, Development

126 (1999) 4827–4838.

[33] A. Liu, J.Y. Li, C. Bromleigh, Z. Lao, L.A. Niswander, A.L. Joyner,

FGF17b and FGF18 have different midbrain regulatory properties

from FGF8b or activated FGF receptors, Development 130 (2003)

6175–6185.

[34] C.A. MacArthur, A. Lawshe, D.B. Shankar, M. Heikinheimo, G.M.

Shackleford, FGF-8 isoforms differ in NIH3T3 cell transforming

potential, Cell Growth Differ. 6 (1995) 817–825.

[35] C.A. MacArthur, D.B. Shankar, G.M. Shackleford, Fgf-8, activated by

proviral insertion, cooperates with the Wnt-1 transgene in murine

mammary tumorigenesis, J. Virol. 69 (1995) 2501–2507.

[36] G.R. Martin, The roles of FGFs in the early development of vertebrate

limbs, Genes Dev. 12 (1998) 1571–1586.

[37] S. Martinez, The isthmic organizer and brain regionalization, Int. J.

Dev. Biol. 45 (2001) 367–371.

[38] S. Martinez, P.H. Crossley, I. Cobos, J.L. Rubenstein, G.R. Martin,

FGF8 induces formation of an ectopic isthmic organizer and

isthmocerebellar development via a repressive effect on Otx2

expression, Development 126 (1999) 1189–1200.

[39] J.M. Mason, D.J. Morrison, B. Bassit, M. Dimri, H. Band, J.D. Licht,

I. Gross, Tyrosine phosphorylation of Sprouty proteins regulates their

ability to inhibit growth factor signaling: a dual feedback loop, Mol.

Biol. Cell 15 (2004) 2176–2188.

[40] A.P. McMahon, A. Bradley, The Wnt-1 (int-1) proto-oncogene is

required for development of a large region of the mouse brain, Cell 62

(1990) 1073–1085.

[41] H. Meinhardt, Cell determination boundaries as organizing regions for

secondary embryonic fields, Dev. Biol. 96 (1983) 375–385.

[42] G. Minowada, L.A. Jarvis, C.L. Chi, A. Neubuser, X. Sun, N.

Hacohen, M.A. Krasnow, G.R. Martin, Vertebrate Sprouty genes are

induced by FGF signaling and can cause chondrodysplasia when

overexpressed, Development 126 (1999) 4465–4475.

[43] M. Muda, A. Theodosiou, N. Rodrigues, U. Boschert, M. Camps,

C. Gillieron, K. Davies, A. Ashworth, S. Arkinstall, The dual

specificity phosphatases M3/6 and MKP-3 are highly selective for

inactivation of distinct mitogen-activated protein kinases, J. Biol.

Chem. 271 (1996) 27205–27208.

[44] H. Nakamura, Regionalisation and acquisition of polarity in the optic

tectum, Prog. Neurobiol. 65 (2001) 473–488.

[45] C. Niehrs, H. Meinhardt, Modular feedback, Nature 417 (2002)

35–36.

[46] S.H. Ong, Y.R. Hadari, N. Gotoh, G.R. Guy, J. Schlessinger, I.

Lax, Stimulation of phosphatidylinositol 3-kinase by fibroblast

growth factor receptors is mediated by coordinated recruitment of

multiple docking proteins, Proc. Natl. Acad. Sci. U. S. A. 98 (2001)

6074–6079.

[47] K. Ozaki, R. Kadomoto, K. Asato, S. Tanimura, N. Itoh, M. Kohno,

ERK pathway positively regulates the expression of Sprouty genes,

Biochem. Biophys. Res. Commun. 285 (2001) 1084–1088.

D. Echevarria et al. / Brain Research Reviews 49 (2005) 150–157 157

[48] E.M. Pera, J.I. Kim, S.L. Martinez, M. Brechner, S.Y. Li, O. Wessely,

E.M. De Robertis, Isthmin is a novel secreted protein expressed as part

of the Fgf-8 synexpression group in the Xenopus midbrain-hindbrain

organizer, Mech. Dev. 116 (2002) 169–172.

[49] J. Pouyssegur, P. Lenormand, Fidelity and spatio-temporal control

in MAP kinase (ERKs) signalling, Eur. J. Biochem. 270 (2003)

3291–3299.

[50] C.J. Powers, S.W. McLeskey, A. Wellstein, Fibroblast growth

factors, their receptors and signaling, Endocr. Relat. Cancer 7 (2000)

165–197.

[51] E. Preger, I. Ziv, A. Shabtay, I. Sher, M. Tsang, I.B. Dawid, Y. Altuvia,

D. Ron, Alternative splicing generates an isoform of the human Sef

gene with altered subcellular localization and specificity, Proc. Natl.

Acad. Sci. U. S. A. 101 (2004) 1229–1234.

[52] L. Puelles, J.L. Rubenstein, Forebrain gene expression domains and

the evolving prosomeric model, Trends Neurosci. 26 (2003) 469–476.

[53] L. Puelles, G. Domenech-Ratto, M. Martinez-de-la-Torre, Location of

the rostral end of the longitudinal brain axis: review of an old topic in

the light of marking experiments on the closing rostral neuropore,

J. Morphol. 194 (1987) 163–171.

[54] J.L. Rubenstein, K. Shimamura, S. Martinez, L. Puelles, Region-

alization of the prosencephalic neural plate, Annu. Rev. Neurosci. 21

(1998) 445–477.

[55] H. Rubinfeld, R. Seger, The ERK cascade as a prototype of MAPK

signaling pathways, Methods Mol. Biol. 250 (2004) 1–28.

[56] M.K. Saba-El-Leil, F.D. Vella, B. Vernay, L. Voisin, L. Chen, N.

Labrecque, S.L. Ang, S. Meloche, An essential function of the

mitogen-activated protein kinase Erk2 in mouse trophoblast develop-

ment, EMBO Rep. 4 (2003) 964–968.

[57] T. Sato, I. Araki, H. Nakamura, Inductive signal and tissue

responsiveness defining the tectum and the cerebellum, Development

128 (2001) 2461–2469.

[58] C.D. Stern, Initial patterning of the central nervous system: how many

organizers? Nat. Rev., Neurosci. 2 (2001) 92–98.

[59] S. Torii, M. Kusakabe, T. Yamamoto, M. Maekawa, E. Nishida, Sef is

a spatial regulator for Ras/MAP kinase signalling, Dev. Cell 7 (2004)

33–44.

[60] R. Trokovic, N. Trokovic, S. Hernesniemi, U. Pirvola, D.M. Vogt

Weisenhorn, J. Rossant, A.P. McMahon, W. Wurst, J. Partanen,

FGFR1 is independently required in both developing mid- and

hindbrain for sustained response to isthmic signals, EMBO J. 22

(2003) 1811–1823.

[61] M. Tsang, I.B. Dawid, Promotion and attenuation of FGF signaling

through the Ras–MAPK pathway, Sci. STKE 228 (2004) pe17.

[62] M. Tsang, R. Friesel, T. Kudoh, I.B. Dawid, Identification of Sef, a

novel modulator of FGF signaling, Nat. Cell Biol. 4 (2002) 165–169.

[63] M. Tsang, S. Maegawa, A. Kiang, R. Habas, E. Weinberg, I.B. Dawid,

Role for MKP3 in axial patterning of the zebrafish embryo,

Development 131 (2004) 2769–2779.

[64] T. Wakioka, A. Sasaki, R. Kato, T. Shouda, A. Matsumoto, K.

Miyoshi, M. Tsuneoka, S. Komiya, R. Baron, A. Yoshimura, Spred is

a Sprouty-related suppressor of Ras signalling, Nature 412 (2001)

647–651.

[65] M. Wassef, A.L. Joyner, Early mesencephalon/metencephalon pat-

terning and development of the cerebellum, Perspect. Dev. Neurobiol.

5 (1997) 3–16.

[66] A.J. Whitmarsh, R.J. Davis, A central control for cell growth, Nature

403 (2000) 255–256.

[67] E.S. Wong, J. Lim, B.C. Low, Q. Chen, G.R. Guy, Evidence for direct

interaction between Sprouty and Cbl, J. Biol. Chem. 276 (2001)

5866–5875.

[68] W. Wurst, L. Bally-Cuif, Neural plate patterning: upstream and

downstream of the isthmic organizer, Nat. Rev., Neurosci. 2 (2001)

99–108.

[69] S. Xiong, Q. Zhao, Z. Rong, G. Huang, Y. Huang, P. Chen, S. Zhang,

L. Liu, Z. Chang, hSef inhibits PC-12 cell differentiation by

interfering with Ras mitogen-activated protein kinase MAPK signal-

ing, J. Biol. Chem. 278 (2003) 50273–50282.

[70] W. Ye, M. Bouchard, D. Stone, X. Liu, F. Vella, J. Lee, H. Nakamura,

S.L. Ang, M. Busslinger, A. Rosenthal, Distinct regulators control the

expression of the mid-hindbrain organizer signal FGF8, Nat. Neurosci.

4 (2001) 1175–1181.

[71] P. Yusoff, D.H. Lao, S.H. Ong, E.S. Wong, J. Lim, T.L. Lo, H.F.

Leong, C.W. Fong, G.R. Guy, Sprouty2 inhibits the Ras/MAP kinase

pathway by inhibiting the activation of Raf, J. Biol. Chem. 277 (2002)

3195–3201.

[72] B. Zhou, L. Wu, K. Shen, J. Zhang, D.S. Lawrence, Z.Y. Zhang,

Multiple regions of MAP kinase phosphatase 3 are involved in its

recognition and activation by ERK2, J. Biol. Chem. 276 (2001)

6506–6515.