Department of Craniofacial Development, King's College London, Floor 27, Guy's Tower, Guy's Hospital, London Bridge, London SE1 9RT, UK
* Author for correspondence (e-mail: susanne.dietrich{at}kcl.ac.uk)
Accepted 5 October 2005
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SUMMARY |
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Using the chick embryo as model, we investigated whether the neck, as the flank, has the competence to form a limb, and what mechanism may regulate its limblessness. We show that forelimb lateral mesoderm plus ectoderm grafted into the neck can continue limb development, suggesting that the neck does not actively inhibit this process. However, neck tissues themselves do not support or take part in limb formation. Hence, the neck is limb-incompetent. This is due to the dismantling of Fgf signalling at distinct points of the MAPK signalling cascade in the neck lateral mesoderm and ectoderm.
Key words: Limblessness, Limb development, Neck, Flank, Fgf signalling, Chick embryo
![]() |
Introduction |
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Interestingly, the neck as a limb-free area between head and forelimbs is a
recent acquisition of tetrapods, fully developed only in amniotes
(Clack, 2002). Initially, in
the ancestors of tetrapods (and still in extant fishes), the shoulder girdle
to which the forelimbs attach was an integral part of the breathing apparatus.
It provided the rear wall of the gill chamber and was held in place via a
series of bones that attached the shoulder girdle to the skull. These bones
were lost in stem-group tetrapods such as Acanthostega gunnari Jarvik
(Clack, 2002
). Next, the
function of the shoulder girdle in supporting the gills ceased when breathing
with lungs and through the skin became more prominent. An indicator for this
event is the absence of the postbranchial lamina on the cleithrum, as seen in
Pholiderpeton (Clack,
2002
). However, the shoulder girdle remained close to the head,
possibly because the branchiomeric muscles that insert here and previously
worked the gills were still required to facilitate breathing via air gulping.
This mode of breathing prevailed in modern amphibians
(Clack, 2002
). Amniotes then,
by developing a closed rib cage and intercostal and abdominal muscles to alter
its volume, found the means to more efficiently ventilate their lungs. At this
time, the shoulder girdle was `set free'. It relocated to a more posterior
position, yet maintaining the original muscular scaffold
(Clack, 2002
;
Matsuoka et al., 2005
). The
consequence of this evolution was the establishment of a neck as a limbless
area between shoulder girdle and head, which is of moderate length in mammals,
but considerably longer in birds (Clack,
2002
).
To unravel the events that may have established the limbless state of the
neck during amniote evolution, we have to investigate the control of limbness
versus limblessness, beginning with known amniote models. Possibly the best
understood model for amniote limb development is the chick embryo
(Capdevila and Izpisua-Belmonte,
2001). The prospective limb fields are specified in the lateral
mesoderm around stage HH10-11 (stages according to
Hamburger and Hamilton, 1992
).
Limb induction, i.e. the initiation of limb budding, occurs between stages
HH13 and 15 next to somites 15-20 (forelimb) and somites 25-30 (hindlimb). The
specification of the limb fields is known to involve the localized expression
of Tbx5 (forelimb) or Tbx4 (hindlimb) and the localized signalling of Wnt2b
(forelimb) (Ng et al., 2002
;
Takeuchi et al., 2003
) but is
otherwise poorly understood. Nevertheless, these factors establish Fgf10
expression in the lateral mesoderm of the limb fields, which signals to the
overlying surface ectoderm to induce the differentiation of the apical
ectodermal ridge (AER) at the interface between prospective dorsal and ventral
territories. Moreover, Fgf10, via Wnt3a as a mediator, triggers expression of
Fgf8 in the AER, which signals back to the mesoderm to maintain the expression
of Fgf10 (Kawakami et al.,
2001
; Kengaku et al.,
1998
; Ohuchi et al.,
1997
). Furthermore, the Fgf molecules promote cell proliferation
and hence outgrowth. Thus, the Fgf feedback loop is crucial to initiate limb
budding, simultaneously determining the proximodistal axis of the limb. Once
established, the AER participates in the installation of the zone of
polarizing activity (ZPA) at the posterior margin of the limb that controls
anteroposterior patterning and acts back onto the AER in a further feedback
loop. Finally, signalling from the ectodermal jacket to the underlying
mesoderm establishes the dorsoventral axis of the limb, crucial for the
correct patterning of bone, the development of extensor and flexor muscles and
their innervation.
As limb development depends on locally acting regulatory cascades, and
forelimbs were `moved' from the head to a more posterior position during
tetrapod evolution, it has been speculated that the set of regulatory cascades
for limb formation has simply been relocated
(Capdevila and Izpisua-Belmonte,
2001). However, while limbs were secondarily lost, for example in
snakes (Cohn and Tickle, 1999
),
in no species has the forelimb ever returned to its previous layout, with the
shoulder girdle joined to the head (Clack,
2002
). Moreover, the anteroposterior dimension of the limb is
closely regulated (Capdevila and
Izpisua-Belmonte, 2001
). Thus in addition to relocating the
positive signals for limb development, it is conceivable that a mechanism was
installed during evolution that both prevented the return of the forelimb to
the original position and that limited the anterior extent of the limb: there
may be a specific mechanism in the neck that ensures its limbless state.
Besides the neck, the flank is another prominent limbless area. However,
embryological studies in the chick have demonstrated that the flank supports
an ectopic limb grafted into this region
(Hamburger, 1938;
Capdevila and Izpisua-Belmonte,
2001
). Moreover, using exogenous Fgf10 to mimic the mesodermal
signalling or Fgf8 to substitute for the ectodermal signal, the Fgf feedback
loop can be kick-started, and the flank mesoderm plus ectoderm will generate a
limb (Yonnei-Tamura et al., 1999). Using this paradigm, we investigated the
state of limblessness in the neck.
First, grafting forelimb-derived lateral mesoderm plus ectoderm, we
investigated whether the neck environment may actively suppress limb
development. We found that the neck permits limb development. Second, as the
ectopic limb buds were poorly integrated into the neck, we investigated
whether neck lateral mesoderm and ectoderm may contribute to the ectopic limb
buds. However, this was not the case. Third, as the flank tissues participate
in limb development in response to Fgf signalling
(Crossley et al., 1996;
Yonnei-Tamura et al., 1999), we investigated the responsiveness of neck
ectoderm and lateral mesoderm to Fgf10 and Fgf8. We found that in the neck
neither the Fgf feedback loop nor bud outgrowth could be achieved. Fourth, we
investigated whether Fgf signalling was incapacitated in the neck due to the
loss of receptor. However, Fgfr2, the crucial receptor to transduce Fgf10 and
Fgf8 signalling in limb development
(Revest et al., 2001
;
Xu et al., 1998
), was
expressed. Finally, as Fgf signalling in limb development employs the MAPK
signal transduction system (Corson et al.,
2003
; Schlessinger et al., 2000), we investigated whether this
system was operational in the neck. We found that in the neck lateral
mesoderm, the MAPK signalling travels as far as the phosphorylation of the
kinases ERK1 and ERK2. However, this point is never reached in the ectoderm.
Thus, our study shows that limblessness in the neck is controlled by the
dismantling of the Fgf feedback loop through the interruption of MAPK
signalling at distinct points in the neck lateral mesoderm and ectoderm.
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Materials and methods |
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Protein-loaded beads
Heparin beads (Sigma) were washed in PBS and loaded overnight at 4°C
with Fgf8 or Fgf10 (R&D) at 500 µg/ml (limb induction assays) or at 1
mg/ml, 250 µg/ml or 50 µg/ml (assays for MAPK signalling) in PBS/1% BSA.
Before transplantation the beads were rinsed in PBS and kept on ice.
Tissue grafting
Host embryos were at HH10-14, as indicated in Table S1 in the supplementary
material. Using flame-sharpened tungsten needles
(Dietrich et al., 1997), a
slit was made in the occipital, neck or flank ectoderm plus lateral mesoderm,
next to the somites. Donor embryos were at HH14. They were pinned down in a
Sylgard (Dow Corning) dish dorsal side up, a fragment of forelimb lateral
plate mesoderm plus the covering ectoderm, or of forelimb lateral mesoderm
only (square of two somites length) was excised with tungsten needles,
aspirated with a serum-coated pipette, then released into the slit within the
host and manoeuvred into place with tungsten needles. The embryos were
incubated until they reached HH20-21.
Bead grafting
Host embryos were prepared as above, and protein-loaded beads were pipetted
into the slit and manoeuvred into place with tungsten needles.
In-ovo electroporation followed by tissue grafting
The plasmid pCaß-IRES-eGFP (Alvares
et al., 2003) was injected into the neck or the flank coelom of
HH12 or HH14-15 embryos, respectively, using a PV820 pneumatic picopump (WPI).
The lateral mesoderm corresponding to 2-3 somite-lengths was electroporated
with two 20 ms/18V rectangular pulses by an intracept TSS10 electroporator
(Intracell) with a 0.1 mm flame-sharpened tungsten (negative electrode) wire
placed under the embryo and a 0.5 mm platinum (positive electrode) wire placed
on top. The site of electroporation was recorded, using the position of the
neighbouring somites as reference. The electroporated embryos were then
incubated for 2-3 hours, and the tissue grafting was carried out into the
electroporated lateral plate mesoderm as described above.
In situ hybridization
Whole-mount in situ hybridization was carried out according to Mootoosamy
and Dietrich (Mootoosamy and Dietrich,
2002). Probes and their expression pattern are detailed in:
Bmp2 (Francis et al.,
1994
), En1 (Logan et
al., 1992
), Fgf4
(Streit and Stern, 1999
),
Fgf8 (Mahmood et al.,
1995
), Fgf10 (Ohuchi
et al., 1997
), Gremlin (Capdevilla et al., 1999),
Lmx1 (Riddle at al,
1995
), Myf5 (Saitoh
et al., 1993
), Shh
(Johnson et al., 1994
),
Tbx5 (Isaac et al.,
1998
), Wnt3a (unpublished PCR product), Wnt7a
(Dealy et al., 1993
),
Fgfr2 (Patstone et al.,
1993
).
Immunohistochemistry
RMO270 staining
Whole-mount tracing of the nervous system was carried out according to
Guthrie and Lumsden (Guthrie and Lumsden,
1992), using the RMO270 antibody (Zymed), which recognizes the 155
kDa intermediate neurofilament subunit. Primary antibodies were detected using
anti-mouse IgG conjugated with horseradish peroxidase (Dako). The detection
was made using diaminobenzidine (DAB) staining.
Detection of MAPK signalling
To detect activated MAPK signalling, rapid fixation in 4% PFA was required,
followed by dehydratation with MeOH and rehydratation. Embryos were incubated
serially with intervening washes in anti-diphosphorylated ERK2 mouse IgG
monoclonal antibody (1:250; # M8159, Sigma), Vectastain Biotinylated Goat
anti-mouse IgG secondary antibody (1:200; Vector labs), and Vectastain ABC
solution (Vectastain ABC Elite kit, Vector labs). The diphosphorylated ERK1
and 2 proteins were revealed by DAB staining.
|
Photomicroscopy
After completion of the staining reactions, embryos were cleared in 80%
glycerol/PBS and split midsagitally (except electroporated embryos). Embryos
and the vibratome sections were photographed on a Zeiss Axioskop, using
fluorescence or Normaski optics.
![]() |
Results |
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To establish if the ectopic limb buds obtained in the neck developed
properly, the expression of genes crucial for normal limb development was
investigated. Fgf10 is a key factor for limb induction, outgrowth and
proximodistal patterning (Ohuchi et al.,
1997; Yonei-Tamura et al.,
1999
). It induces the differentiation of the AER and the
expression of Fgf8 in the AER through the expression of
Wnt3a (Kawakami et al.,
2001
; Kengaku et al.,
1998
; Ohuchi et al.,
1997
). Fgf8, partially redundant with Fgf4
(Boulet et al., 2004
), then
signals back to the limb mesenchyme to maintain Fgf10
(Ohuchi et al., 1997
). A
mesodermal-ectodermal Fgf feedback loop is thus established. We found that
Fgf10 was expressed in the mesenchyme of the ectopic buds in the neck
(n=14/15, Fig.
1A,A'). An AER was differentiated at the distal tip of the
buds, expressing Wnt3a (n=17/17,
Fig. 1B,B'),
Fgf8 (n=6/6, Fig.
1C,C') and Fgf4 (n=8/9,
Fig. 1D,D').
Fgf4 expression in the AER is established upon Shh signalling from
the ZPA, which is situated in the posterior mesenchyme of the bud and
organizes the anteroposterior patterning of the limb
(Tickle et al., 1975). Fgf4
then acts back onto the ZPA to maintain Shh
(Laufer et al., 1994
;
Niswander et al., 1994
). The
establishment of this Shh-Fgf loop requires the expression of the Bmp
antagonists Gremlin and Noggin in the bud mesenchyme. These prevent Bmp2, 4
and 7 in the bud mesenchyme and AER from downregulating Fgf4 and
disorganizing the AER (Capdevilla et al., 1999;
Kawakami et al., 1996
;
Pizette and Niswander, 1999
;
Zuniga et al., 1999
). We found
that in the ectopic buds in the neck, Shh was expressed in the ZPA
(n=6/13, Fig.
1E,E'), and Bmp2 (n=20/24,
Fig. 1F,F') and
Gremlin (n=15/17, Fig.
1G,G') were expressed in the mesenchyme.
Tbx5 and Tbx4 expression distinguishes between forelimb
and the hindlimb fields, respectively
(Gibson-Brown et al., 1996;
Logan et al., 1998
), but both
genes have common roles in the initiation and maintenance of limb outgrowth
(Minguillon et al., 2005
).
Tbx5 was expressed in the ectopic buds (n=7/7,
Fig. 1H,H').
Dorsal and ventral limb territories are demarcated by the expression of
Wnt7a in the dorsal ectoderm, which induces Lmx1 in the
dorsal mesoderm and is antagonized by En1 in the ventral ectoderm
(Davis and Joyner, 1988;
Dealy et al., 1993
;
Loomis et al., 1996
;
Parr et al., 1993
;
Vogel et al., 1995
). In the
ectopic limb buds in the neck, Wnt7a (n=3/5,
Fig. 1I,I') and
Lmx1 (n=15/15, Fig.
1J,J') were correctly expressed. En1 was also
expressed, more weakly in the ventral ectoderm and strongly in the ventral
base of the ectopic buds (n=6/6,
Fig. 1K,K').
|
During the development of the chick embryo, motor axons extend from the
spinal cord towards the limb buds in order to innervate them
(Jacob et al., 2001). When
limb lateral mesoderm is grafted into the flank, the resulting ectopic limb is
properly innervated (Hamburger,
1939
). Using the RMO270 antibody to trace the developing nervous
system, we investigated whether the ectopic limb buds in the neck also
received innervation. We observed that whereas the forelimb and hindlimb buds,
as well as ectopic buds in the flank, were always properly innervated
(Fig. 2B,C), the ectopic limb
buds in the neck were not (Fig.
2A). However, in two cases, where the graft had been inserted at
the occipital-cervical interface, the hypoglossal nerve had formed a side
branch and innervated the limb (not shown).
The limb mesenchyme, i.e. the lateral mesoderm-derived tissue plus
immigrated myoblasts, produce chemoattractants for the growth cones of the
incoming axons (Jacob et al.,
2001). The fact that half of the ectopic limbs in the neck
recruited muscle precursors but only a few were innervated suggests that
failure of innervation was caused by the limb mesenchyme independent of the
presence of muscle. To confirm this, limb buds were generated in the
mid-cervical region, and double-stained for Myf5 and RMO270. We found
that, indeed, the ectopic limbs were not innervated, even in the presence of
Myf5 (n=2/2, Fig.
2D).
Neck mesoderm does not participate in limb bud formation
Ectopic buds in the flank are known to recruit host cells, which change
their fate to become limb cells and contribute to the bud
(Dhouailly and Kieny, 1972).
Thus, the bud is made from the grafted tissues but also from flank cells,
which allows it to be well integrated into the flank. Many of the ectopic limb
buds that develop in the neck had an abnormal pear-like shape
(Fig. 1H,J,L), and their base
was generally very thin and fragile in comparison with the wide base of a
forelimb bud (Fig.
1H',J',L', for instance), or the base of ectopic
limb buds in the flank (Fig.
4I). This suggests that in the neck the buds were not properly
integrated, possibly due to a failure of recruitment of host cells.
|
Fig. 3A,B shows the distribution of GFP in the flank and the neck tissues of control embryos that were electroporated but not grafted. The electroporated tissues developed normally. Notably, when the flank lateral mesoderm was electroporated and then received a graft, fluorescent cells were found inside the bud (n=6/6, Fig. 3C,E). The cells resided along the margins of the limb, indicating that they did not contribute to the limb via random cell mixing. Rather, they were incorporated into the limb in response to signals from the graft. By contrast, the ectopic buds that developed in the neck never contained fluorescent cells. The fluorescent mesodermal cells were all around the base of the bud but never inside. Moreover, they formed a sharp boundary with the mesoderm of the graft (n=6/6, Fig. 3D,F). Thus, the neck lateral mesoderm did not contribute to the ectopic buds. Consequently, the bud remained poorly integrated into the neck.
Neck ectoderm cannot differentiate into an AER
It has been shown that when the limb lateral plate mesoderm without its
covering ectoderm is grafted into the flank of a chick embryo, the host
ectoderm will cover the graft (Hamburger,
1938). Subsequently, the graft signals to the host ectoderm to
induce an AER from a subset of ectodermal cells, while the remaining ectoderm
will express the dorsal and ventral markers of the ectodermal jacket. Once the
AER is in place, this bud grows and differentiates normally.
To investigate whether the neck ectoderm, like the flank ectoderm, is able
to contribute to a limb bud, we grafted forelimb lateral mesoderm alone into
the neck. For this, forelimb lateral mesoderm from stage HH13-14 chick
embryos, i.e. already expressing Fgf10, was separated from the
ectoderm and inserted into the neck at the right side of HH10-14 embryos, and
in the flank of HH13-14 embryos as a control (see Table S2 in the
supplementary material). Embryos were harvested at HH20 as before. Whereas an
ectopic limb with a differentiated AER developed in the flank as previously
published (Fig. 4I)
(Hamburger, 1938), only a
small outgrowth was obtained in the neck
(Fig. 4A-H). No AER could be
seen in these outgrowths, and the lack of Wnt3a (n=10/10,
Fig. 4B,B') and
Fgf8 (n=5/5, Fig.
4C,C') expression confirmed that no AER was
differentiated.
Due to the failure of AER formation, we suspected that the AER-dependent signalling systems might be absent. Indeed, we found that Fgf10 expression was lost from the bud mesoderm (n=7/7, Fig. 4A,A'). Shh (n=8/8, Fig. 4D,D') and Bmp2 (n=10/10, Fig. 4E,E') were not expressed either. Moreover, Tbx5 was expressed in only two of five outgrowths, and the expression was barely detectable (Fig. 4F,F').
|
The neck ectoderm does not respond to Fgf10 signalling
We have shown that neck lateral mesoderm cannot be recruited into an
ectopic limb bud, and neck ectoderm cannot participate in limb development as
it does not form an AER. In normal limb development and during the development
of an ectopic limb in the flank, the recruitment of mesodermal and ectodermal
cells and the subsequent development of a bud depend on reciprocal Fgf
signalling between the lateral mesoderm and the overlying ectoderm
(Ohuchi et al., 1997). We thus
investigated whether the neck lateral mesoderm and ectoderm can respond to Fgf
signals, supplying exogenous sources of Fgf.
The first step in the establishment of the Fgf feedback loop is the
activation of Wnt3a and Fgf8 in the AER, in response to
Fgf10 from the lateral mesoderm (Kawakami
et al., 2001; Kengaku et al.,
1998
; Ohuchi et al.,
1997
). This is followed by reciprocal Fgf8 signalling to the
mesoderm, which stabilizes Fgf10 expression. To investigate if the
neck ectoderm is able to respond to Fgf10 and then signals back to the lateral
mesoderm, beads soaked in Fgf10 protein were grafted into the neck lateral
mesoderm at the right side of HH11-14 chick embryos at different positions
along the anteroposterior axis (see Table S3 in the supplementary material). As a positive control, Fgf10 beads were placed into the flank of HH13-15
embryos, as Fgf10 has been shown to trigger the development of an ectopic limb
(Yonei-Tamura et al., 1999
).
Indeed, Fgf10 beads induced an ectopic bud in the flank, the AER of which
expressed both Wnt3a (n=2/2,
Fig. 5D) and Fgf8
(n=2/2, Fig. 5G), and
consequently, the mesoderm of which expressed Fgf10 (n=2/2,
Fig. 5A). Thus, the beads
released a sufficient amount of Fgf10 protein to establish the Fgf10-Fgf8
regulatory loop and to induce the development of a limb in the flank. By
contrast, in the neck neither Wnt3a (n=17/17,
Fig. 5E) nor Fgf8
(n=6/6, Fig. 5H) were
expressed above the Fgf10 beads and, as a result, Fgf10 was not
expressed in the neighbouring neck mesenchyme (n=11/11,
Fig. 5B).
The neck mesoderm does not respond to Fgf signalling
The second step in the establishment of the Fgf feedback loop is the
maintenance of Fgf10 expression in the mesoderm by Fgf8, or when
ectopic limbs are induced in the flank, the de-novo activation of
Fgf10 expression in the mesoderm. Hence we tested if the neck
mesoderm was able to respond to Fgf8. Beads soaked in Fgf8 protein were
grafted into the neck lateral mesoderm at the right side of HH10-14 chick
embryos at different positions along the anteroposterior axis (see Table S4 in
the supplementary material). As a positive control, Fgf8 beads were inserted
into the flank of HH13-15 embryos. The beads grafted into the flank induced
ectopic limbs, which expressed Fgf10 (n=6/6,
Fig. 5J), Wnt3a
(n=4/4, Fig. 5M) and
Fgf8 (n=4/4, Fig.
5P). In the neck, however, Fgf10 (n=24/24,
Fig. 5K) was not expressed in
the mesoderm around the Fgf8 beads and, as a result, neither Wnt3a
(n=9/9, Fig. 5N) nor
Fgf8 (n=7/7, Fig.
5Q) was expressed in the neck ectoderm.
|
Expression of Fgf receptor 2 in the neck
The non-response of the neck tissues to Fgf8 and Fgf10 suggests that the
molecular network that transduces Fgf signals is missing or incomplete. We
thus investigated the presence of Fgf receptor 2 (Fgfr2), as its IIIb isoform
is exclusive to limb ectoderm and perceives the Fgf10 signal, while the IIIc
isoform is exclusive to lateral mesoderm and perceives Fgf8
(Miki et al., 1992;
Ornitz et al., 1996
;
Orr-Urtreger et al., 1993
;
Revest et al., 2001
;
Xu et al., 1998
). Using a
probe detecting the transcripts for all Fgfr2 isoforms, we simultaneously
assayed for the presence of Fgfr2IIIb in the neck ectoderm and Fgfr2IIIc in
the neck lateral mesoderm. We investigated the expression of this receptor at
stage HH14, i.e. at the stage of limb induction, when Fgf10 is specifically
expressed in the presumptive limb lateral plate mesoderm. We found that
Fgfr2 was expressed both in the neck mesoderm and ectoderm at this
stage (Fig. 6).
Dissociation of the Fgf signalling pathway in the neck
It has been shown that during limb development, Fgf receptors signal
through the MAPK pathway, which involves the diphosphorylation of the kinases
ERK1 and 2 (dpERK) (Corson et al.,
2003). To investigate if the MAPK pathway can be activated in the
neck in response to Fgf8 or Fgf10, we thus assayed for the presence of dpERK.
For this, beads soaked in Fgf10 protein or Fgf8 protein were grafted into the
neck lateral mesoderm of HH11-14 chick embryos and in the flank of HH13-15
embryos as a positive control. As we noticed that Fgf8 beads induced a bigger
ectopic limb in the flank than Fgf10 at a concentration of 500 µg/ml
(compare Fig. 5A,D,G and
J,M,P), we increased Fgf10 concentration to 1 mg/ml, and in some cases two
beads were grafted. Conversely, we decreased Fgf8 concentrations to 250 and 50
µg/ml. We then obtained ectopic buds in the flank of equivalent sizes with
both factors, which shows that they have equivalent properties at these
concentrations (Fig. 7, compare
A,C,E).
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
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The limbless neck is a recent acquisition of tetrapods, fully developed
only in amniotes (Clack, 2002).
It evolved as a result of the separation of the shoulder girdle from the
skull. Given its history, and given that limbs never returned to their
original position with the shoulder immediately posterior to the skull, we
wondered whether a specific mechanism was installed in the neck to ensure its
limbless state.
Our study shows that limb development is not actively suppressed in the neck. However, both neck lateral mesoderm and ectoderm are incompetent to participate in limb development, as they are unable to install the Fgf feedback loop. This is due to the dismantling of MAPK signal transduction cascades at distinct points in the lateral mesoderm and ectoderm. Our results are summarized in Table 1.
|
Neck tissues are unable to participate in limb development
When limb-derived lateral mesoderm plus ectoderm are grafted into the
flank, the grafted tissues provide the bulk of the ectopic bud
(Hamburger, 1938). However,
the ectopic buds also recruit surrounding cells to participate in limb
development (Dhouailly and Kieny,
1972
) (our results). This leads to the formation of a broad limb
base, ensures the integration of the limb into the host tissues and the
transmission of the limb-derived signals that organize innervation and
attraction of muscle progenitors
(Buckingham et al., 2003
;
Jacob et al., 2001
). In the
neck, by contrast, the ectopic buds soon showed deficiencies such as an
irregular morphology and a fragile base. Moreover, only half of the buds
attracted muscle precursors, and even fewer received innervation, indicating
that the molecular tools required to efficiently relay signals to the somites
and neural tube were absent or non-functional. This suggests that neck tissues
may not be able to actively participate in limb development.
To systematically address whether neck tissues may or may not contribute to limbs, we first labelled the lateral mesoderm with a fluorescent marker, followed by the insertion of forelimb mesoderm plus ectoderm. Our control experiments confirmed that flank lateral mesoderm becomes incorporated into the ectopic limb. However, this was never the case in the neck. Moreover, the host cells formed a sharp boundary with the mesoderm of the graft, indicating that they are unable to take an active part in limb development.
Next we performed an ectoderm recruitment assay. It has been shown that when lateral mesoderm of the forelimb is grafted into the flank alone, it recruits the host ectoderm to form the ectodermal jacket and AER of the limb, to participate in the Fgf and Shh-Fgf4 feedback loops, and to support the development of a normal limb. In the neck, by contrast, the grafted mesoderm formed a small outgrowth, possibly because in the donor it was loaded with Fgf10 protein. Markers for the establishment of the AER and the Fgf feedback loop failed, and consequently, the Shh-Fgf4 feedback loop also failed, leading to the absence of defined proximodistal and anteroposterior axes. Thus, the neck ectoderm was also not limb-competent.
Notably, the forelimb lateral mesoderm when grafted into the neck induced Wnt7a expression in the overlying host ectoderm. As a consequence, Lmx1 was strongly expressed in the grafted limb mesenchyme. This indicates that the neck ectoderm is not completely deaf to signals from the limb. However, both Wnt7a and Lmx1 showed aberrant expression patterns, indicating that the dorsoventral axis was also incorrect.
|
Presence of Fgf receptors
As the neck tissues were unable to install the Fgf feedback loop, it was
paramount to investigate at which point Fgf signalling was defective. It has
been established that in limb development Fgf signals are perceived through
the Fgf receptor 2, with the IIIb isoform binding Fgf10 and the IIIc isoform
taking up Fgf8 (Revest et al.,
2001; Xu et al.,
1998
). Moreover, expression studies showed that Fgfr2 IIIb is only
expressed in the limb ectoderm (Revest et
al., 2001
), while IIIc is exclusive to the lateral mesoderm
(Miki et al., 1992
;
Ornitz et al., 1996
;
Orr-Urtreger et al., 1993
).
Thus, using an Fgfr2 probe detecting all isoforms, we were able to
simultaneously assay for the presence of Fgfr2IIIc in the neck lateral
mesoderm and Fgfr2IIIb in the ectoderm. We found that they were correctly
expressed. Thus, if the transcripts are translated at sufficient levels, Fgf
perception in the neck is possible.
Fgf signalling is interrupted at specific points within the MAPK signalling pathway
In the neck lateral mesoderm, Fgf10 is expressed temporarily at the time
the future limb fields are established
(Ohuchi et al., 1997).
Moreover, our data showed persistent expression of at least Fgfr2
mRNA, suggesting that the system is geared up for functional Fgf signalling.
However, both Fgf10 signalling from the mesoderm to the ectoderm and Fgf8
signalling from the ectoderm to the mesoderm were unsuccessful. This suggests
that Fgf signalling is interrupted within the signal transduction cascade.
Fgf signalling predominantly operates through the MAPK pathway
(Corson et al., 2003;
Schlessinger et al., 2000). This pathway encompasses a series of protein
phosphorylation events and can be monitored using antibodies against the
diphosphorylated forms of the kinases ERK1 and ERK2
(Corson et al., 2003
).
Employing this approach, we show that in the neck Fgf8-loaded beads are able
to trigger phophorylation of ERK1 and ERK2. This confirms that functional
receptors are present in the neck to bind Fgf8. It furthermore indicates that
Fgf8 signal transduction is interrupted downstream of ERK1 and ERK2. By
contrast, Fgf10 did not trigger ERK phosphorylation, indicating that
signalling is interrupted upstream. Thus, in the neck lateral mesoderm and
ectoderm, Fgf signalling was dismantled separately and at distinct points of
the signalling cascades.
Model: the neck is limb-incompetent due to the dissociation of Fgf signalling cascades at distinct points in the lateral mesoderm and ectoderm
Limb induction, i.e. the initiation of limb budding, depends on reciprocal
Fgf signalling between lateral mesoderm and ectoderm
(Fig. 8A). At stage HH14 in the
chick, Fgf10 is expressed in the presumptive limb lateral plate mesoderm.
Fgf10 binds to the Fgfr2 IIIb present on the surface of the limb ectodermal
cells. The MAPK signalling pathway is activated in the ectodermal cells,
triggering expression of Wnt3a, which in turn induces expression of Fgf8 in
the AER. Fgf8 binds to Fgfr2IIIc present on the surface of the mesodermal
cells. MAPK signalling is activated and leads to the maintenance of the
expression of Fgf10. In the flank, this signalling cascade is silent until
exogenously applied Fgf10 or Fgf8 kick-starts the system. Thus, all components
of the molecular network involved in Fgf signalling are present in the flank -
the flank is a `limb in waiting'.
The neck, by contrast, is limb-incompetent. This is not due to the presence of specific inhibitors of limb development. Rather, key components of the signalling cascades positively regulating limb development have been lost (Fig. 8B). When exogenous Fgf10 is supplied to substitute for the signal emerging from the lateral mesoderm, MAPK signalling does not proceed to the point of ERK1/2 phosphorylation. Thus, signalling is interrupted upstream in the cascade, Wnt3a and consequently Fgf8 are not expressed in the ectoderm, and no AER develops. This prevents the activation of Fgf10 and the development of a limb. Our expression analysis suggests that the receptor to bind Fgf10 is present, although this needs to be confirmed at the protein level. If the receptor is functional, then either molecules that operate outside the cells to facilitate Fgf10 binding to the receptor (Fig. 8B-1), or factors that operate inside the cell, between the receptor and the phosphorylation of ERK1/2 (Fig. 8B-2), are missing.
When an exogenous source of Fgf8 is provided in the neck to substitute for the signals derived from the AER, then dpERK1/2 are produced in the lateral mesoderm. This indicates that Fgfr2IIIc is present and that signal transduction through the MAPK pathway has commenced. However, signal transduction downstream of ERK1/2 is not completed, as Fgf10 is not upregulated in the neck mesoderm. This indicates that the molecular pathway between ERK1/2 phosphorylation and FGF10 activation is defective (Fig. 8B-3).
Outlook
Our study shows that, in the neck, Fgf signalling was interrupted at
distinct points in the lateral mesoderm and ectoderm. This does not exclude
the possibility that further factors involved in limb development have also
been lost. For example, the regulators that act upstream of Fgf10, such as
Tbx5/4 and Wnt2b, may be required to install the components for successful
Fgf-MAPK signalling. These factors are absent from the neck. However, at least
at mRNA levels, there is some expression of these markers in the neck at the
time of limb field specification, as is the case for Fgf10
(Gibson-Brown et al., 1998;
Kawakami et al., 2001
;
Ohuchi et al., 1997
;
Ohuchi et al., 1998
). This
suggests that yet further factors may be involved. Candidates are Hox/HOM
genes, suspected to provide a `Hox-code' for fore- and hindlimbs in the
lateral mesoderm (Cohn et al.,
1997
), and amendment of their expression boundaries has been
suggested as a cause for the loss of forelimbs in snakes
(Cohn and Tickle, 1999
). By
contrast to the neck, the flank has all tools for limb development in store.
Thus, to investigate which signalling cascades have to be reconstituted in the
future to correct amelic conditions in humans, the neck is possibly the most
appropriate test system.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/132/24/5553/DC1
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ACKNOWLEDGMENTS |
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