Making a vertebrate limb: newplayers enter from the wingsGail Martin

SummaryWhat initiates vertebrate limb development and induceslimbs to form where they do? For several years theanswer to this intriguing question has been framed interms of a working model that limb induction depends ona dialogue between two members of the FibroblastGrowth Factor (FGF) family of intercellular signalingmolecules, FGF8 and FGF10. Now, a recent paper haswritten roles for signals encoded by WNT genes, thevertebrate relatives of the Drosophila wingless gene, intothe script.(1) BioEssays 23:865±868, 2001.ß 2001 John Wiley & Sons, Inc.

Observing limb developmentÐin front of the

curtain and behind the scenes

Much of what we know about vertebrate limb development has

been learned by watching the chicken embryo develop in ovo

through a window in the egg's shell. The first sign that limbs are

developing is the appearance of a small protrusion on each

side of the body, near the anterior end of the torso. Several

hours after these forelimb buds become visible, the hindlimb

buds begin to protrude near the posterior end. Each limb bud is

composed of lateral plate mesoderm (LPM) and the overlying

surface ectoderm. The mesoderm contains the progenitors of

skeletal elements, tendons, and other connective tissue of the

mature limb. The surface ectoderm gives rise to skin and

cutaneous appendages such as feathers on chick wings or

hairs on mouse legs.

A few hours after the limb buds first become visible,

changes in cell shape and position within the surface ectoderm

result in the appearance of a ridge that runs along the distal

margin of each limb bud. Once this apical ectodermal

ridge (AER) has formed, the limb bud elongates along its

proximodistal axis (e.g., shoulder to fingers), flattens along

its dorsoventral axis (e.g., back of hand to palm), and becomes

asymmetric along its anteroposterior axis (e.g., thumb to

little finger). AER formation is a critical event, because the

ridge produces signals that are essential for limb development.

If the AER fails to form, the nascent limb bud regresses and no

limb develops.(2) If the AER is removed, the limb that develops

is truncated, i.e., it contains proximal skeletal elements but

lacks distal ones.(3)

To gain insight into what causes limb buds to develop at

specific positions along the body axis, tissue grafting and other

experimental manipulations have been performed in ovo.(4) A

key finding was that the LPM along most of the length of the

torso is competent to respond to a limb-inducing signal. This

implies that under normal circumstances, the induction of limb

development at specific positions involves some mechanism

for restricting the availability of the limb inducer. A second

important finding was that the signal(s) for limb induction is

produced by tissue medial to the LPM (Fig. 1). But what is the

limb-inducing signal? Studies by Cohn et al.(5) turned the

spotlight on FGFs.

Outlining the scriptÐFGFs have a starring role

FGFs are secreted proteins that signal by binding to high-

affinity tyrosine kinase receptors on the cell surface (FGF

receptors; FGFRs), thereby activating intracellular signaling

pathways, including the RAS-MAP kinase pathway.(6,7) FGFs

are known to play major roles in multiple aspects of embryonic

development,(8) including outgrowth of the limb bud.(4) But

their function as signals that initiate limb development was not

appreciated until a bead soaked in FGF protein was inserted

into the prospective interlimb region.(5) This induced cells that

would not normally participate in limb development to form an

ectopic limb (Fig. 1A). The type of limb that forms (wing or leg)

depends on whether the FGF source was placed closer to the

prospective forelimb or hindlimb territory, respectively.

These results suggested that the endogenous limb inducer

might be an FGF. However, several different members of the

FGF family (of which there are now 22 in mouse and human

Ref. 9) proved capable of inducing ectopic limbs. Studies of

FGF gene expression patterns at early stages of normal and

ectopic chick limb development identified FGF8 and FGF10 as

potential regulators of limb development.(10±12) The model

that was proposed to explain their function (Fig. 1B) posits that

FGF10 produced in the LPM is the signal that initiates limb

development. At early stages of embryogenesis, however,

Fgf10 is expressed throughout the LPM, so the key to inducing

limbs at specific locations is to restrict Fgf10 expression to the

appropriate territories at the stage when limb induction occurs.

FGF8 produced in medial tissues (intermediate mesoderm for

the forelimb and primitive streak for the hindlimb) was

proposed to be the signal that maintains Fgf10 expression in

BioEssays 23:865±868, ß 2001 John Wiley & Sons, Inc. BioEssays 23.10 865

Department of Anatomy, Box 0452, University of California, Rm.

S-1457, 513 Parnassus Ave, San Francisco, CA 94143-0452.

E-mail: gmartin@itsa.ucsf.edu

What the papers say

the prospective limb territories while it ceases in the interlimb

region. The FGF10 that is thus produced was proposed to

stimulate limb bud outgrowth and induce Fgf8 expression in

prospective limb bud surface ectoderm. Finally, it was

hypothesized that FGF8 produced in the AER maintains

expression of Fgf10 in the underlying mesenchyme, and

FGF10 produced in the mesenchyme maintains Fgf8 expres-

sion in the AER.

Figure 1. A: Protocol for inducing an ectopic

limb. (Left panel) A schematic diagram represent-

ing a dorsal view of the right side of a chick

embryo at the stage when normal limb inductionis thought to occur (Hamburger-Hamilton stage

14). The prospective forelimb (FL) and hindlimb

(HL) territories in the lateral plate mesoderm(LPM) and surface ectoderm (SE) are indicated in

yellow. Medial to the LPM is the intermediate

mesoderm (IM), the somites (SO) / presomitic

mesoderm (PSM), and the neural tube (NT) atthe midline of the body. A bead loaded with a

limb-inducing signal such as FGF8 is inserted

into the LPM in the prospective interlimb region,

which lies at approximately the level of somites21±25. At this stage, prospective somites 24±29

are still within the PSM. (Middle panel) After �2

days of incubation, an ectopic limb bud hasformed in the interlimb region (arrow). The

arrowhead points to Fgf8 RNA detected in the

AER of the normal hindlimb bud. (Right panel)

After longer incubation, the ectopic limb buddevelops into a relatively complete limb (arrow).

A$P, anteroposterior axis. B: A working model

for FGF signaling events responsible for limb

induction and outgrowth of the limb bud along theproximodistal (P$D) axis. (Left panel) FGF8

produced in the IM in the prospective forelimb

territory functions to maintain expression of Fgf10in the adjacent LPM. The proposed role for FGF8

in hindlimb induction is not illustrated. (Middle

panel) FGF10 produced in the prospective limb

mesoderm then induces Fgf8 expression in thesurface ectoderm (SE). (Right panel) Once the

limb bud has developed, FGF8 produced by cells

in the AER functions to maintain Fgf10 expres-

sion in the underlying distal limb bud mesench-yme. In turn, FGF10 produced in the

mesenchyme functions to maintain Fgf8 expres-

sion in the AER. The dotted arrows indicate

proposed steps in the signaling cascade that arenot yet supported by genetic evidence. C: A

revised model, illustrating how WNT signaling via

b-catenin might fit into the limb-induction path-way. WNT signaling, via WNT2B in the forelimb

and WNT8C in the hindlimb, function to maintain

Fgf10 expression in the LPM of the prospective

limb-forming territories. FGF10 signaling to thesurface ectoderm (via FGFR2-isoform IIIb) is

required for AER formation, but the target genes

that are necessary for the development of the

AER are not yet known. One possibility is thatFGF8 and WNT/b-catenin signaling (via WNT3A

in the chick, and its ortholog in the mouse) both

contribute to AER formation.

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866 BioEssays 23.10

To validate this model, which is based primarily on data

from ``gain-of-function'' experiments (i.e. inserting a source of

a specific FGF in the interlimb region and determining what

genes are subsequently expressed), some form of genetic test

needs to be performed. If one assumes that the signals for limb

induction have been evolutionarily conserved, then an

analysis of the limb phenotypes in mutant mice can provide a

critical test of the model, as well as offer new insights into the

specific functions of individual molecules in the proposed

signaling cascade. Importantly, the central tenet of the working

model, namely the concept that an FGF10 signal is essential

for limb development, has been confirmed by loss-of-function

studies in the mouse. The data show that, although limb bud

outgrowth is initiated, the AER does not form and limb

development therefore fails in mutant mouse embryos that

cannot produce either FGF10(13,14) or its receptor, an isoform

of FGFR2 (FGFR2-IIIb).(15,16) Furthermore, as predicted by

the working model, Fgf8 is not expressed in the surface

ectoderm of Fgf10ÿ /ÿor Fgfr2ÿ /ÿ limb buds.(13,14,16) There

are, however, some conflicting data on this point, since Fgf8

RNA has been detected in the limb bud rudiments of embryos

that cannot produce FGFR2-IIIb, the specific isoform of

FGFR2 that transduces the FGF10 signal in the limb bud.(15)

On the other hand, the genetic data clearly contradict the

assumption implicit in the working model that induction of Fgf8

expression by FGF10 is vital for limb development. When Fgf8

is inactivated specifically in prospective limb ectoderm, the

AER forms and a limb develops, although it is not completely

normal.(17,18) Therefore, the failure of AER formation in

Fgf10ÿ /ÿ or Fgfr2ÿ /ÿ limb buds cannot be attributed solely

to a lack of FGF8, and other targets of FGF10 signaling must

be required for the formation of an AER.

Questions about other features of the working model

remain unresolved. For example, does FGF8 produced in

medial tissue act upstream of Fgf10? This hypothesis has

not yet been tested genetically because complete loss of

Fgf8 function in the embryo causes early lethality,(19) and

the mice necessary for conditionally inactivating Fgf8 in the

intermediate mesoderm or primitive streak at the appropriate

stages of development are not yet available. Furthermore, it

has been difficult to test the proposal that there is an FGF8/

FGF10-positive regulatory loop in the established limb bud.

Embryos that lack Fgf8 function in the AER continue to

express Fgf10, suggesting that signaling via FGF8 is not

essential to maintain Fgf10 expression.(17,18) This result is not

so easily interpreted, however, because, as the AER devel-

ops, other FGF family members in addition to FGF8 begin to be

produced by AER cells, and these other FGFs might maintain

Fgf10 expression in the absence of FGF8. The question of

whether FGF10 signaling is required to maintain Fgf8

expression in the AER cannot be answered by analyzing

mutant mouse embryos that cannot signal via FGF10,

because they never develop an AER. In principle, however,

this question could be addressed by inactivating Fgf10 in the

limb bud mesenchyme or its receptor in the AER, after the AER

has been established.

In summary, we know that FGF10 produced in the

mesoderm is necessary for AER formation. But we do not

yet know for certain what signals are responsible for ensuring

that Fgf10 is expressed appropriately in prospective limb

territories or what targets of FGF10 signaling are responsible

for forming the AER (Fig. 1B). The recent paper by Kawakami

et al.(1) turns the spotlight on members of another family of

signaling molecules, the WNTs.

Introducing new playersÐWNTs take center

stage

Like FGFs, members of the WNT superfamily are secreted

proteins that play major roles in multiple aspects of embryonic

development.(20) WNTs act as ligands for transmembrane

receptors belonging to the Frizzled family. Some WNTs signal

via an intracellular pathway that involves the stabilization of

cytoplasmic b-catenin, which then translocates to the nucleus

and becomes a transcriptional co-activator of WNT target

genes.(21) One member of the WNT gene family, Wnt7a, which

is expressed in the dorsal ectoderm of the limb bud, is essential

for normal dorsoventral patterning of the mouse limb.(4,22) But

until now it had not been shown that WNT genes play a role at

the earliest stages of limb development (Fig. 1C).

Kawakami et al.(1) performed both gain- and loss-of-

function experiments to show that signaling via b-catenin

functions upstream of FGF10 in the limb-induction pathway.

First, they used a retroviral vector to focally express an

activated form of b-catenin in the chick interlimb region. This

resulted in ectopic Fgf10 expression and the formation of an

ectopic limb. Second, they used an adenoviral vector to

express Axin, a negative regulator of b-catenin, in the normal

limb-forming territory several hours before limb induction is

thought to occur. This completely blocked limb formation.

Together these data provide strong support for the hypothesis

that signaling via b-catenin functions to ensure that Fgf10

expression is maintained in the limb-forming territories,

which in turn is required for AER formation in the nascent

limb bud.

The question of which specific WNT family members might

be responsible for stabilizing b-catenin and thus affecting

Fgf10 expression was addressed by studying the normal

patterns of WNT family gene expression in the early chick

embryo. Interestingly, two different WNT genes were identified

as candidate limb inducers. WNT2B was proposed to be

involved in forelimb and WNT8C in hindlimb induction. In

support of the hypothesis that these genes function upstream

of Fgf10 during limb development, the authors showed that

ectopic expression of either Wnt2b or Wnt8c in the chick

interlimb region can lead to Fgf10 expression in cells

near the source of the WNT signal and, subsequently, to the

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BioEssays 23.10 867

induction of an ectopic limb bud. Furthermore, they provided

evidence that both of these WNT signals act by stabilizing

b-catenin.

In addition to its role upstream of FGF10 signaling,

Kawakami et al. propose that WNT signaling via b-catenin

also functions downstream of FGF10. This hypothesis is

based on the finding that Fgf8 expression is severely reduced

and the AER is disrupted in the normal limb when Axin is

expressed throughout the limb territory after Fgf10 expression

has become normally restricted to the LPM of the limb-forming

region. They suggest that the WNT family member that effects

this b-catenin-dependent function is Wnt3a, which is normally

expressed in prospective chick limb bud ectoderm, and

subsequently in the AER.(23) In support of the concept that

activation of Fgf8 expression and AER formation by FGF10 is

mediated by WNT3A (Fig. 1C), they showed that implanta-

tion of a pellet of FGF10-producing cells in the interlimb region

induces first Wnt3a expression and then Fgf8 expression in

the overlying surface ectoderm.

An open casting call

From its inception, the model that FGF signaling is responsible

for inducing limb development was regarded as a framework

upon which to build a more complete description of the

molecular mechanism of limb induction. The evidence that

WNT family genes play a role in this process adds a new

dimension to the model. If the hypothesis that a medial FGF8

signal maintains Fgf10 expression in the limb-forming regions

is incorrect, then WNT/b-catenin signaling (via WNT2B in the

forelimb and WNT8C in the hindlimb) may prove to be the

earliest known regulator of Fgf10 expression in the prospec-

tive limb territories.

Perhaps the most interesting question raised by the data

accumulated to date is what are the target genes of FGF10

signaling to the surface ectoderm that are required for AER

formation? As noted above, genetic studies have shown that

Fgf8, which is the only FGF family member known to be

expressed in prospective limb ectoderm prior to AER forma-

tion, is not necessary for AER formation.(17,18) Kawakami et al.

propose that WNT3A plays a critical role in AER formation.

However, when b-catenin signaling is blocked by Axin

expression after Fgf10 expression has become restricted to

the limb-forming territory, a truncated limb develops, in which

proximal elements are present and distal elements are

missing. This phenotype is more similar to what is observed

when the AER is removed at a relatively early stage in the

outgrowth of an established limb bud than to what is seen when

the AER fails to form. One intriguing possibility is that FGF and

WNT signals contribute to AER formation, and that the failure

to form an AER in mouse embryos that cannot signal via

FGF10 is due to a lack of both FGF8 and the WNT3A ortholog

in mice. As research into the question of what induces limb

development continues, it will be exciting to learn how the

known players interact and what other molecules have a place

at the center of the stage.

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