Download Shh signalling and cell death in limb development

4811
Development 127, 4811-4823 (2000)
Printed in Great Britain © The Company of Biologists Limited 2000
DEV1593
Autoregulation of Shh expression and Shh induction of cell death suggest a
mechanism for modulating polarising activity during chick limb development
Juan Jose Sanz-Ezquerro*,‡ and Cheryll Tickle*
Department of Anatomy and Physiology, Wellcome Trust Biocentre, University of Dundee, Dow Street, Dundee DD1 5EH, UK
*This work was initiated at the authors’ previous address: Department of Anatomy and Developmental Biology, University College London, Gower Street,
London WC1 6BT, UK
‡Author for correspondence (e-mail: [email protected])
Accepted 17 August; published on WWW 24 October 2000
SUMMARY
The polarising region expresses the signalling molecule
sonic hedgehog (Shh), and is an embryonic signalling centre
essential for outgrowth and patterning of the vertebrate
limb. Previous work has suggested that there is a buffering
mechanism that regulates polarising activity. Little is
known about how the number of Shh-expressing cells is
controlled but, paradoxically, the polarising region appears
to overlap with the posterior necrotic zone, a region of
programmed cell death. We have investigated how Shh
expression and cell death respond when levels of polarising
activity are altered, and show an autoregulatory effect of
Shh on Shh expression and that Shh affects cell death in
the posterior necrotic zone. When we increased Shh
signalling, by grafting polarising region cells or applying
Shh protein beads, this led to a reduction in the endogenous
Shh domain and an increase in posterior cell death. In
contrast, cells in other necrotic regions of the limb bud,
including the interdigital areas, were rescued from death
by Shh protein. Application of Shh protein to late limb buds
also caused alterations in digit morphogenesis. When we
reduced the number of Shh-expressing cells in the
polarising region by surgery or drug-induced killing, this
led to an expansion of the Shh domain and a decrease in
the number of dead cells. Furthermore, direct prevention
of cell death using a retroviral vector expressing Bcl2 led
to an increase in Shh expression. Finally, we provide
evidence that the fate of some of the Shh-expressing cells in
the polarising region is to undergo apoptosis and contribute
to the posterior necrotic zone during normal limb
development. Taken together, these results show that there
is a buffering system that regulates the number of Shhexpressing cells and thus polarising activity during limb
development. They also suggest that cell death induced by
Shh could be the cellular mechanism involved. Such an
autoregulatory process based on cell death could represent
a general way for regulating patterning signals in embryos.
INTRODUCTION
retinoic acid (Tickle et al., 1985), or Shh protein (Yang et al.,
1997) applied: more cells or higher concentrations of retinoic
acid or Shh leading to specification of more posterior digit
identity. Rather curiously, however, when polarising region
cells (Tickle et al., 1975), retinoic acid (Tickle et al., 1985) or
Shh (Chang et al., 1994; Riddle et al., 1993; Yang et al., 1997)
are added to the posterior margin of chick limbs, normally
patterned digits are obtained. This points to a possible
buffering mechanism that compensates for an excess of
polarising signalling.
Very little is known about mechanisms controlling either
number of cells in the polarising region or the temporal extent
of its activity. It has been suggested that there is a positive
feedback loop between Shh expression in the polarising region
and Fgf4 expression in the apical ridge that links outgrowth
and patterning (Laufer et al., 1994; Niswander et al., 1994) and
in Shh knockout mice, limb-bud outgrowth is impaired, leading
to distal truncations and absence of digits (Chiang et al., 1996).
Wnt7a, which is expressed in dorsal ectoderm, also contributes
to maintenance of Shh expression. In chick limbs in which
During outgrowth of embryonic limb buds, a group of posterior
mesenchymal cells, known as the polarising region, produces
signal(s) that pattern the anteroposterior axis of the developing
limb. The polarising region was discovered by grafting small
pieces of tissue from the posterior margin of one chick limb
bud to the anterior margin of another limb bud (Saunders and
Gasseling, 1968). This operation leads to a mirror image
duplication of the digits. Retinoic acid was the first molecule
shown to reproduce this effect (Tickle et al., 1982) and now is
known to induce expression of sonic hedgehog (Shh) (Riddle
et al., 1993). Cells of the polarising region express Shh, which
is a secreted signalling molecule that can also produce
duplications (Riddle et al., 1993) and has been proposed to
mediate polarising region activity, probably via bone
morphogenetic proteins (BMPs) (Drossopoulou et al., 2000).
Polarising region signalling is dose dependent, since the
identity of duplicated digits depends on number of polarising
region cells transplanted (Tickle, 1981), or concentration of
Key words: limb, apoptosis, Shh, bcl-2, cell number, chick embryo
4812 J. J. Sanz-Ezquerro and C. Tickle
dorsal ectoderm has been removed (Yang and Niswander,
1995), and in limbs of mice in which Wnt7a is functionally
inactivated (Parr and McMahon, 1995), Shh expression is
reduced and this can lead, in both cases, to loss of posterior
structures.
The polarising region, paradoxically, is associated with a
major area of programmed cell death in developing limbs: the
posterior necrotic zone. Programmed cell death is a welldocumented feature of normal embryonic development
(Glücksmann, 1951) and the existence of an evolutionarily
conserved genetic programme that controls cell death is now
well accepted (Raff, 1998). This essential physiological cell
death is known to play several general roles during
development and morphogenesis, including the control of cell
number, particularly in the immune and nervous systems
(reviewed in Jacobson et al., 1997; Vaux and Korsmeyer,
1999. Some examples of the importance of cell death in
specific developmental processes have been reported, for
example, cavitation of the early embryo (Coucouvanis and
Martin, 1995), inner ear development (Fekete et al., 1997),
neural tube closure (Weil et al., 1997), tooth development
(Vaahtokari et al., 1996), and ductal morphogenesis and
lumen formation in the mammary gland (Humphreys et al.,
1996).
In limb development, the best known example of
programmed cell death occurs in the interdigital areas that are
involved in separation of the digits (Pautou, 1975; Saunders
and Fallon, 1967). Three other areas of well-defined massive
cell death have also been described in early chick limb buds
(Hinchliffe, 1982): one located in the anterior margin, the
anterior necrotic zone; another in central core mesenchyme, the
opaque patch; and a third in the posterior margin, the posterior
necrotic zone. The role of these areas of cell death is not clear.
It has been suggested that anterior and posterior necrotic zones
might control the number of mesenchymal cells available to
form digits and thus their prominence in chick limb buds would
be related to the decreased number of digits compared with
other vertebrates. However, the effects on the anterior and
posterior necrotic zones when parts of the limb are removed
are completely different (Hinchliffe and Gumpel-Pinot, 1981),
suggesting that each zone might have a specific function and
be differently regulated. Because the posterior necrotic zone is
associated with the polarising region, we tested the relationship
between cell death and Shh signalling from the polarising
region. Our results lead us to propose that apoptosis might play
a part in regulating the number of Shh-expressing cells during
chick limb development.
MATERIALS AND METHODS
Embryos and surgical manipulations
Fertilised White Leghorn chicken eggs were obtained from Needle
farm (UK). They were incubated at 38°C for three days, and then
windowed and embryos staged according to Hamburger and Hamilton
(1951). Limb buds were exposed and microsurgery was carried out
using fine watchmaker forceps and electrolytically sharpened tungsten
needles. For bead implantation, a small cut was made with a needle
at the desired position and a bead was introduced into the
mesenchyme with the aid of the needle and forceps. Polarising tissue
was removed by reference to maps of polarising activity (MacCabe et
al., 1973) and patterns of Shh expression (Riddle et al., 1993). A cut
was made in the flank just posterior to the base of the limb bud
(running anteriorly through the proximal part of the bud), a loop was
made in the posterior apical ectodermal ridge and another cut was
made in the middle of the limb. A piece of tissue was then taken away
with the aid of forceps, leaving the apical ridge intact. For polarising
region or control anterior grafts, wing buds were dissected from stage
21 embryos in growth medium (GM; MEM with Hepes plus 10%
foetal calf serum, 1% glutamine, 1% penicillin-streptomycin solution,
all from Gibco). After trypsinisation in 10× trypsin (Gibco) for 30
minutes at 4°C, the ectoderm was removed and the polarising region
or anterior tissue dissected. These blocks of tissue were transferred to
a host embryo with a Gilson pipette and grafted under a loop of apical
ridge lifted away from the posterior mesenchyme of the host wing
bud. Some grafts were labelled with DiI by placing them in a solution
of 100 µg/ml DiI in GM for 15 minutes at room temperature, before
grafting.
Beads
Shh (a gift from A. M. McMahon; Marti et al., 1995) was stored in
14 mg/ml aliquots at –70°C. Dilutions were made in storage buffer as
described (Yang et al., 1997). Fgf4 (R&D) was used at 0.75 mg/ml.
Staurosporin (Sigma) was used at 100 µM diluted in GM from a 10
mM stock in DMSO. Affi Gel-CM Blue beads (BioRad, 150-250 µm
diameter) were used for Shh and staurosporin, whereas heparin beads
(Sigma) were used for Fgf4. To soak the beads, 2 µl of the solutions
were placed in a bacterial Petri dish; beads were transferred with
forceps and soaked at room temperature for at least 1 hour. Soaked
beads were stored at 4°C and used within two weeks.
DiI labelling
DiI (Molecular Probes) was used at 3 mg/ml concentration in DMSO.
A Picospritzer (General Valve Corporation, N.J.) was used to inject
the solution in the posterior margin of wing buds using glass capillary
pipettes. Embryos were collected and analysed for fluorescent
labelling using a Leica MZ-FLIII microscope with a rhodamine
filter and subsequently stained for cell death with Nile Blue. After
washing in PBS, embryos were photographed and fixed in 4%
paraformaldehyde (PFA) for subsequent in situ hybridisation.
Nile Blue staining and TUNEL
For Nile Blue staining, embryos were dissected in PBS and incubated
in a 1/5000 solution of Nile Blue A (Sigma) in PBS for 15 minutes
at 37°C in a rolling incubator. Embryos were transferred to cold PBS
and washed for 15 minutes in PBS at 4°C, photographed and fixed in
4% PFA for subsequent in situ hybridisation. Some specimens that
were not analysed by in situ hybridisation were left to wash in PBS
overnight to improve staining and then photographed. In all cases the
operated limb was compared with the contralateral, which served as
a control, so that even small changes in cell death or Shh expression
could be detected. Quantitative analysis of the data was made by
counting the number of Nile Blue-positive cells in freshly stained
embryos or in pictures of stained embryos, or by measuring the areas
of the Shh-expression domains in pictures of in situ hybridisation
processed embryos using NIH Image software. Statistical analysis of
data was carried out using paired Student’s t-test comparing
experimental with contralateral limbs.
For TUNEL staining, embryos were fixed in 4% PFA, transferred
to a series of sucrose solutions in PBS (5%, 15% and 30%), embedded
in OCT compound and quick frozen in isopentane. 10 µm
cryosections of limb buds were cut and subjected to TUNEL staining
using the In Situ Fluorescein Cell Death Detection Kit from
Boehringer, following the manufacturer’s instructions. Fluorescein
labelling was observed using a Zeiss microscope.
In situ hybridisation and cartilage staining
In situ hybridisation was performed according to standard protocols
(Nieto et al., 1996). Probes used (Shh (Cohn et al., 1995), Gdf5
Shh signalling and cell death in limb development 4813
(Merino et al., 1999a) and RCAS p27gag, from C. Tabin (Goff and
Tabin, 1997) have been described elsewhere. Embryos were
photographed using a standard camera attached to a Zeiss microscope
or a digital camera attached to a Leica MZ-8 microscope and images
analysed using Photoshop software. Alcian Green was used to stain
cartilage in stage 36 embryos as described (Drossopoulou et al.,
2000).
Double labelling
For double-labelling experiments, embryos were subjected to wholemount in situ hybridisation to measure Shh expression and developed
with NBT/BCIP. Cryosections (10µm) of stained limbs were then
subjected to TUNEL staining as described above.
Retrovirus production and injection
Retroviruses were produced according to standard procedures
(Morgan and Fekete, 1996). Chicken embryo fibroblasts were grown
in DMEM supplemented with 10% FBS, 2% chicken serum and 1%
penicillin-streptomycin solution (all from Gibco). Cells were
transfected with RCAS(BP)B-hbcl2 plasmid (a gift from Prof. S.
Hughes), which contains the human BCL2 gene (Givol et al., 1994)
and has previously been reported to inhibit cell death in chick embryos
(Fekete et al., 1997), using Lipofectamine (Gibco). RCAS(BP)B
virus, with no insert, was used as control. Cells were passaged for a
week to amplify the virus. Supernatants from those cultures were
collected and concentrated by ultracentrifugation. Viruses were
injected using a Picospritzer at several spots in the prospective wing
regions (Morgan and Fekete, 1996) of stage 10-12 or stage 17 chick
embryos. Embryos were returned to the incubator and collected at
different times after injection for analysis of cell death and for in situ
hybridisation.
Fig. 1. Shh expression and cell death following grafts of
polarising region cells (A-H) or Shh beads (I-L) to the posterior
margin. (A) Graft of polarising region cells to posterior margin
of a stage 20 wing bud. Embryo collected immediately after the
operation and subjected to in situ hybridisation with a Shh
specific probe. Note Shh-expressing cells in graft (arrow), close
to endogenous Shh expression domain in host. (B) Embryo
showing Shh expression 24 hours after a polarising region graft.
Note reduction of the proximal part of the Shh expression
domain in right wing bud (arrow) and that the graft does not
express Shh. (C) Same embryo stained with Nile Blue for cell
death. Note position of graft containing cell death (arrowhead)
and an increased amount of dead cells in the posterior necrotic
zone of host (arrow) in the same area where reduction of Shh
expression was observed. (D) Another example of polarising
region graft showing persistence of cell death in the posterior
margin (arrow). (E) Ventral view of limbs showing Shh
expression 24 hours after grafting anterior cells to the posterior
margin. Note proximal expansion of Shh domain in operated
wing (arrow). (F) Nile Blue staining of same embryo. Note
reduction of cell death in the operated wing as compared with
the contralateral limb (arrow). (G,H) Same operated limb as in
D. This graft was labelled with DiI before implantation to follow
the position of grafted cells. As can be seen by comparing G
(DiI labelling) with H (cell death), Nile Blue-positive cells in the
posterior-most margin (arrows) are host cells. (I) Posterior Shh
bead. In situ hybridisation with a Shh-specific probe. Note
reduction in number of Shh-expressing cells. (J) Posterior
control bead. No change in Shh expression. (K) Embryo
showing Shh expression after implantation of a Shh bead.
(L) Same embryo stained with Nile Blue, showing increase in
cell death (arrow). Note that the area where extra cell death is
induced (arrow in L) corresponds to the region of reduced Shh
expression (arrow in K).
RESULTS
Effects of posterior polarising region grafts and Shh
beads on Shh expression and cell death
It has previously been described that increasing polarising
signalling in chick limb buds, by placing a polarising region
graft (Tickle et al., 1975) or extra Shh (Chang et al., 1994;
Riddle et al., 1993; Yang et al., 1997) at the posterior margin,
where the polarising region itself is located, does not have any
effect on digit patterning. This suggests that Shh expression
and/or signalling is regulative. To test this possibility, both
grafts of polarising region cells and Shh-soaked beads were
implanted in the posterior margin of stage 20 chick wing buds
and the effects on Shh expression analysed.
Additional polarising region cells from stage 21 embryos
were grafted. Some embryos (n=2) were fixed immediately and
in situ hybridisation confirmed that Shh-expressing cells had
been transplanted (Fig. 1A). 80% of embryos (n=15) fixed at
20-24 hours showed a marked reduction in endogenous Shh
expression (Fig. 1B, arrow) and no Shh expression could be
seen in the graft. As controls, anterior cells were grafted to the
posterior margin of limb buds. In most of these embryos, there
was no significant decrease in Shh expression (75%, n=16)
and, in some of them, Shh expression actually increased (Fig.
1E, arrow). To test the effects of Shh directly, beads soaked in
the N-terminal active peptide of Shh protein were implanted
posteriorly in stage 20 wing buds. 24 hours later, Shh
expression was reduced in 92% of embryos (n=26) (Fig. 1I).
4814 J. J. Sanz-Ezquerro and C. Tickle
Fewer cells expressed Shh and the most proximal part of the
domain was missing in the majority of manipulated limb buds.
Control beads had no effect on Shh expression (n=12) (Fig. 1J).
To investigate whether this effect was stage dependent we
implanted Shh beads posteriorly in later stage embryos (stage
22-23). A reduction in endogenous expression of Shh could
again be seen in 75% of treated embryos (n=4). These results
indicate that high levels of Shh can repress its own expression.
The posterior necrotic zone, an area of massive cell death
present during chick wing development, seems to co-localize
with the polarising region, and in other systems cell death has
been shown to control cell number. Therefore, we examined
cell death after the above manipulations, which we had found
repressed Shh expression. 20-24 hours after grafts of polarising
region cells to the posterior margin, the number of Nile Bluepositive dead cells in the posterior necrotic zone of the host
limb had clearly increased (40-300% more dead cells) in 50%
of the embryos (n=22) (Fig. 1C, arrow). Often this increase
reflected a distally expanded posterior necrotic zone (Fig. 1C
arrow) and/or cell death persisting for longer (Fig. 1D, arrow).
Furthermore, extensive cell death was often seen in the graft
(Fig. 1C, arrowhead). Evidence that cell death was induced in
the host was obtained by grafting DiI labelled cells (n=3). As
can be seen by comparing Fig. 1G with 1H, the cell death
present in Fig. 1H occurs in cells that are not labelled with DiI.
Moreover, as could be seen in some embryos which were first
stained for cell death with Nile Blue and subsequently fixed
and processed for Shh expression, the area where extra cell
death was induced corresponded to the area where a decrease
in Shh expression was observed (compare Fig. 1B and 1C,
arrows). In most control grafts of anterior cells there was no
increase in cell death (93% of cases, n=28) and in many cases
cell death was actually decreased (68% of embryos) (Fig. 1F).
When these latter limbs were analysed for Shh expression we
found that, in some of them, Shh expression was increased
proximally (19% of cases) (Fig. 1E, arrow).
Application of Shh beads to the posterior margin also led to
an increase in the number of Nile Blue-positive dead cells in
the posterior necrotic zone (Fig. 1L). In 15/17 of embryos, the
area of induced extra cell death coincided precisely with the
area in which Shh expression was reduced (compare Fig. 1L
with 1K, arrows). The reduction in Shh expression could be
detected 10-12 hours after bead implantation, the same time at
which a slight increase of cell death could be seen. When
control beads soaked in buffer were implanted, no change in
either cell death or Shh expression could be seen in most
embryos (8/10).
The observed reduction in endogenous Shh expression after
increasing Shh signalling is consistent with a buffering
mechanism that regulates the amount of Shh expression, and
the effects of Shh on apoptosis in posterior mesenchyme
suggest a possible cellular mechanism (see below).
Characterisation of the effects of Shh protein on
limb-bud programmed cell death
In most previous work, Shh has been reported to be a survival
factor rather than a cell death inducer, therefore, we carried out
another series of experiments to examine in detail the effects
of Shh protein on limb-bud cell death. We first implanted Shh
beads (14 mg/ml concentration) at the posterior margin of stage
20-22 chick wing buds and followed the effects on cell death
with time (Tables 1, 2). In 81% (n=16) of embryos examined
18-24 hours later, there was a substantial increase in the
number of Nile Blue-positive cells in the posterior necrotic
zone (Fig. 2A, arrow). More Nile Blue-positive cells were seen
in the normal region of cell death and/or the cell death domain
extended more dorsally and distally. In two cases, development
of the posterior necrotic zone was accelerated (Fig. 2E,
arrowhead). An increase in cell death in the posterior necrotic
zone was first seen in some embryos after 12 hours, peaked by
20 hours (Table 2) and persisted until 40 hours. By 48 hours,
the posterior necrotic zone appeared have returned to normal
(n=3). Shh beads also increased cell death in the posterior
necrotic zone when implanted into older limb buds (stages
24/25) (Table 2). We then examined the effects of different
doses of Shh. An increase in cell death in the posterior margin
Table 1. Effects on cell death when 14 mg/ml Shh beads are implanted to stage 20-22 chick limb buds at different
positions (Nile Blue-positive cells 18-24 hours after bead implantation)
Bead (limb)
Position
of bead
Shh (wing)
Posterior
Control (wing)
Posterior
Shh (wing)
Central
Shh (wing)
Anterior
Control (wing)
Anterior
Shh (leg)
Shh (leg)
Control (leg)
Posterior
Anterior
Anterior
Effects on cell death (percentage of embryos affected)
PNZ
n*
++ or +++ (81%‡)
= or – (19%)
+++ (0%)
= or – (100%)
+++ (33%)
= (67%)
+++ (25%)
= (75%)
= (100%)
16
– – – (100%)
5§
9
= (100%)
7§
++ (80%)
n.p.
n.p.
3§
8
ANZ
– – or – – – (100%¶)
– – – (100%)
2
= (100%)
5
– – or – – – (100%)
– – or – – – (100%)
= (100%)
n*
4
4§
2
4§
13
6
OP
– – or – – – 100%
= (100%)
– – or – – – (100%)
n*
16
9
3§
– – – (100%)
8
= (100%)
2
– – or – – – (100%)
– – or – – – (100%)
= (100%)
Key: Number of Nile Blue-positive cells. +++, marked (100-700%) increase; ++, moderate (50-100%) increase; +, slight (10-50%) increase; =, <10%
difference; –, slight (10-50%) reduction; – –, moderate (50-90%) reduction; – – –, absence.
*n=number of embryos.
‡Two cases: posterior necrotic zone appeared precociously.
§In rest of the embryos, cell death not yet present in that area.
¶Two of these beads were implanted in stage 23-24 embryos.
ANZ, anterior necrotic zone; OP opaque patch; PNZ, posterior necrotic zone; n.p., cell death not present at that time in that position.
5
9§
6
Shh signalling and cell death in limb development 4815
Table 2. Effects on cell death at different times after
application of 14 mg/ml Shh beads at various stages
Position
of bead
Stage
Posterior
20-22
24-25
Anterior
20-22
24-25
Time
after bead
implantation
(hours)
6-8
12
14
16
18
20
20-30
4
6
8
20-30
Effects on cell
death (percentage
of embryos affected)
PNZ
n*
= (100%)
+ (14%)
+ (50%)
+ (75%)
++ (43%)
+++ (100%)
+ (100%)
5
7
4
4
7
4
2
ANZ/OP
n*
= (100%)
– (82%)
– – or – – – (100%)
– – or – – – (100%)
3
11
12
9
Key: Number of Nile Blue-positive cells. +++, marked (100-700%) increase;
++, moderate (50-100%) increase; +, slight (10-50%) increase; =, <10%
difference; –, slight (10-50%) reduction; – –, moderate (50-90%) reduction;
– – –, absence.
*n=number of embryos.
ANZ, anterior necrotic zone; OP opaque patch; PNZ, posterior necrotic zone.
Fig. 2. Effects of Shh protein on cell death in
chick limb buds. (A-E) Nile Blue staining of
limb buds showing patterns of cell death 20-24
hours after Shh bead application to different
positions at stage 20. Left: contralateral limbs.
Right: operated limbs. (A) Posterior Shh bead in
wing bud. Note increase in cell death posterodistally (arrow). (B) Anterior Shh bead in leg
bud. Note absence of cell death in anterior
necrotic zone as compared to the anterior
necrotic zone in contralateral limb (arrow).
(C) Posterior control bead. Normal appearance
of the posterior necrotic zone. (D) Anterior
control bead. No change in cell death.
(E) Central Shh bead. Opaque patch clearly
present in the control limb (arrow) but absent in
operated limb bud. Note the precocious
appearance of an intense area of cell death
posteriorly in operated limb bud (arrowhead).
(F) Quantitative analysis of Shh or control beads
effects on cell death in limb buds. Graph shows
mean number±standard deviation of Nile Bluepositive cells of operated versus contralateral
limbs for the conditions described. *** P<0.001
in a paired Student’s t-test. (G-J) TUNEL
staining of cryosections from operated limb buds
(G,I control beads; H,J Shh beads) collected 24
hours after bead application. Dead cells show
fluorescent labelling. (G) Posterior control bead
in wing. Labelled cells represent the normal
posterior necrotic zone present at that time
(arrow). (H) Posterior Shh bead in wing. Note
marked increase in number of TUNEL-positive
cells postero-distally (arrow). (I) Anterior
control bead in leg. Note intense anterior
necrotic zone present at this time (arrow).
(J) Anterior Shh bead in leg. Arrow indicates the
area with a complete absence of cell death where
a anterior necrotic zone is normally located.
was also seen after application of beads soaked in 1mg/ml Shh
(17/19 embryos) but when beads soaked in 0.1 mg/ml Shh were
used, only one out of five embryos showed a slight increase in
Nile Blue-positive cells in the posterior necrotic zone. With
control beads, the number of dead cells in the posterior necrotic
zone was either normal (4/9) or slightly reduced (5/9) (Fig.
2C). Shh beads placed posteriorly also affected cell death in
the opaque patch and anterior necrotic zone (Table 1) (Fig. 2A).
In these regions, Nile Blue-positive cells were either
completely absent or their number much reduced in all cases.
Thus, Shh rescued cell death. This contrasts to the increase
seen in the posterior necrotic zone.
To investigate the rescue effects of Shh on cell death in the
opaque patch and anterior necrotic zone further, Shh beads
were applied anteriorly or centrally. At 18-24 hours, cell death
in the anterior necrotic zone and in the opaque patch was again
completely absent or much reduced (Table 1) (Fig. 2B compare
with control bead in Fig. 2D). In 3/11 cases, we could also
detect an increase in the size of the posterior necrotic zone
(Table 1). A slight decrease in cell death in the anterior necrotic
zone and the opaque patch could be detected as early as six
hours after bead implantation (Table 2) and substantial rescue
was maintained in all embryos up to 48 hours after bead
4816 J. J. Sanz-Ezquerro and C. Tickle
application. Shh also rescued cell death in older embryos (stage
24/25). Beads soaked in 1 mg/ml Shh could still prevent cell
death in these areas (11/11), but when 0.1 mg/ml Shh beads
were used, only a partial rescue was observed in the anterior
necrotic zone in 67% of embryos.
Similar bead implantations were carried out in chick leg
buds. Again, Shh increased cell death in the posterior margin
but rescued cell death in the anterior necrotic zone and the
opaque patch (Table 1).
A quantitative analysis of data obtained in experiments using
beads soaked in 14 mg/ml Shh is shown in Fig. 2F, where
number of Nile Blue-positive cells is compared between
contralateral and operated limbs after Shh bead application.
After posterior application of Shh beads, the number of Nile
Blue-positive cells in the posterior necrotic zone of the
operated wings significantly increased (117.0±40.7 in the
operated wings versus 71.4±29.7 in the contralateral wings,
P<0.001 as analysed by paired Student’s t-test). However,
application of control beads posteriorly did not have such an
effect (62.4±24.6 Nile Blue-positive cells in the operated limbs
versus 68.0±27.9 in the contralateral). Anterior application of
Shh-soaked beads led to a significant decrease in the number
of Nile Blue-positive cells in the anterior necrotic zone and
opaque patch of wings and legs (10.0±19.0 Nile Blue-positive
cells in the operated limb versus 76.2±46.7 in the contralateral,
P<0.001 according to a paired Student’s t-test). Control beads
applied anteriorly did not alter significantly the number of Nile
Blue-positive cells (76.1±25.0 in the operated limbs versus
78.1±31.0 in the contralateral).
Although Nile Blue staining has been shown to label
apoptotic cells specifically (Abrams et al., 1993) we confirmed
the apoptotic nature of the cell death regulated by Shh using
TUNEL labelling on sections of operated limbs. A marked
increase in the number of TUNEL-positive cells in the
posterior necrotic zone was seen in wing buds after
implantation of Shh beads posteriorly (Fig. 2H), while with a
control bead the posterior necrotic zone was normal (Fig. 2G).
A complete absence of TUNEL-positive cells in anterior
mesenchyme was also seen after anterior Shh bead
implantation in leg buds (Fig. 2J), while with a control bead
there was still extensive cell death in the anterior necrotic zone
(Fig. 2I).
Shh also rescued interdigital cell death when applied to later
limb buds. When Shh-soaked beads (14 mg/ml) were applied
to the third interdigital space of chick leg buds at stage 27-28
and interdigital cell death analysed by Nile Blue staining 48
hours later at stage 32, at a time when cell death is normally
well established, Nile Blue-positive cells were absent in the
operated third interdigital region and also much reduced in
number in the second interdigital region, when compared with
the contralateral limb (n=6) (Fig. 3A). The interdigital region
also appeared wider with no signs of membrane regression.
However, this rescue effect was transient, and by 66-72 hours
cell death started to be observed again distally (n=7) (Fig. 3B).
This later resumption of cell death, in the majority of cases,
did not restore the normal regression of the interdigital
membranes and led to soft tissue syndactyly with high
frequency (63% of cases, n=16) (Fig. 3C). In addition to this
syndactyly, we noticed that application of Shh at late stages
had remarkable effects on digit morphogenesis (see also a
recent report by Dahn and Fallon (2000), who independently
observed similar effects). When Shh-soaked beads (14 mg/ml)
were applied to the third interdigit at stage 27-28, in 87% of
cases (13/15) digit 2, anterior and far away from the bead, was
Fig. 3. Effects on cell death and digit morphology of application of Shh beads to late limbs. Shh-soaked beads (14 mg/ml) were applied to the
third interdigital space of stage 27-28 leg buds. (A,B) Nile Blue staining to show cell death. Note rescue of cell death at 48 hours after bead
implantation in A but reappearence of apoptosis at 72 hours in B (arrow). Note also widening of the operated interdigital space in A,B, and
noticeable lengthening of digits 2 and 4 in operated leg in B (asterisks). Numbers in control limb denote digit identity, from anterior 1 to
posterior 4. (C) Whole-mount legs showing syndactyly in the third interdigital area (arrow) four days after the operation. (D,E) Alcian Green
staining of leg buds four days after bead application. Note elongation of digit 2 in D and of digit 2 and 4 in E, with the formation of a new
phalanx (asterisks) and a new joint (arrows), and truncation of digit 3. Arrowheads mark the position of the bead. Numbers denote digit identity
as in B. (F) In situ hybridisation with the joint-specific marker Gdf5 to show the extra joints (arrows). A-C operated limbs to the left. D-F
operated limbs to the right.
Shh signalling and cell death in limb development 4817
longer (Fig. 3D,E), owing either to an increase in length of the
penultimate phalangeal element or to the formation of an extra
phalanx with a new joint, as confirmed by Gdf5 expression
(Fig. 3F), thus giving a digit with four phalanges instead of the
normal three. In most cases (80%), digit 3, close and anterior
to the beads, was shorter and truncated, with a
reduced number of phalangeal elements (Fig.
3D,E) while digit 4, close and posterior to the
beads was not affected in the majority of cases
(60%). In one case, digit 4 was elongated and had
an extra phalanx and joint (Fig. 3E).
The above results show that Shh has opposite
effects on apoptosis in the different regions of
programmed cell death in chick limb buds: Shh
rescues cells in the anterior necrotic zone,
opaque patch and interdigital areas, but increases
cell death in the posterior necrotic zone.
Relationship between cell death in the
posterior necrotic zone and Shh
expression in the polarising region after
experimentally manipulating the number
of Shh-expressing cells
The results presented above show that addition
of polarising region cells expressing Shh or
implantation of Shh beads to the posterior margin
leads to a decrease in endogenous Shh expression
and that this decrease is accompanied by an
increase in cell death. This suggests the
possibility that the buffering mechanism that
keeps Shh signalling constant is cell death. To
test this idea further, we carried out additional
experiments in which we either increased or
decreased the number of Shh-expressing cells at
the posterior margin of the limb and looked at the
effects on Shh expression and cell death.
To increase the number of Shh-expressing
cells, we applied Fgf4-soaked beads, an
operation that has been shown to increase the
extent of the Shh expression domain (Yang and
Niswander, 1995). Indeed, at 14 hours (1/1
cases), 18 hours (4/4 cases) or 24 hours (5/7
cases) after application of Fgf4-soaked beads to
the posterior margin of stage 20 wing buds, the
Shh expression domain expanded proximally
along the posterior margin (Fig. 4A, arrow).
However, by 28-40 hours, Shh expression was
now found to be restricted distally and resembled
the normal pattern of expression seen in the
contralateral limb bud (Fig. 4C). These changes
in Shh expression were accompanied by changes
in the extent of cell death. At 18-24 hours, the
number of Nile Blue-positive cells had decreased
(2/2 cases at 18 hours, 3/5 cases at 24 hours) (Fig.
4B) but at 28 or 40 hours after Fgf4 bead
application, cell death had increased in all
embryos (4/4 at 28 hours; 8/8 at 38-42 hours)
(Fig. 4D). These areas of extra cell death in the
proximal part of the bud corresponded to regions
where ectopic Shh expression had previously
been seen. Indeed, analysis in the same embryos
(2/2 at 28 hours; 8/8 at 38-42 hours) showed that areas of cell
death and of Shh expression were now almost mutually
exclusive, except for a small overlap between the distal-most
area of cell death and the proximal-most area of Shh expression
(compare Fig. 4C with 4D, arrows).
Fig. 4. Effects on Shh expression and cell death after increasing or reducing the
number of Shh-expressing cells in the polarising region. (A-D) implantation of Fgf4
beads. (A) Embryo showing Shh expression 18 hours after a Fgf4 bead was
implanted to the posterior margin of a stage 21 wing bud. Note proximal expansion
of Shh expression domain in operated limb bud (arrow). (B) Same embryo stained
with Nile Blue. Note reduction in cell death seen in the posterior necrotic zone as
compared with the contralateral wing (arrow). (C) Embryo processed for in situ
hybridisation to reveal Shh expression 40 hours after Fgf4 bead implantation. Shh
domain is now restricted distally in right limb bud and resembles normal expression
found in contralateral limb. (D) Same embryo stained with Nile Blue. Note marked
increase in cell death along posterior margin of right wing bud as compared with
normal posterior necrotic zone in contralateral limb. Note also that the area
undergoing cell death does not express Shh except for a small overlap (arrows in
C,D). (E-J) Effects of reducing number of polarising region cells. (E-G) Surgical
removal of Shh-expressing cells. (E) Embryo fixed immediately after the operation
and subjected to in situ hybridisation to reveal Shh expression. Note that right
experimental wing has a reduced number of Shh-expressing cells (arrow).
(F,G) Embryo collected 24 hours after surgery. F shows Shh expression and G shows
Nile Blue staining. Note that Shh expression is proximally expanded (arrow in F),
while cell death is absent in operated right limb as compared with the contralateral
left limb (arrow in G). (H-J) Staurosporin treatment. H shows embryo 4 hours after
a staurosporin bead was inserted in posterior margin of a stage 20 wing bud. In situ
hybridisation shows reduction in Shh expression (arrow). (I,J) Embryo collected 24
hours after treatment. I shows Shh expression and J shows cell death in the same
embryo. Note increase in Shh expression in operated limb with some cells
expressing Shh more proximally than in control (arrow in F) and reduction in cell
death in operated limb as compared with normal posterior necrotic zone present in
contralateral limb (arrow in E).
4818 J. J. Sanz-Ezquerro and C. Tickle
To reduce the number of Shh-expressing cells, a small area
of the posterior margin of stage 20-21 chick wing buds was
surgically removed, leaving the apical ectodermal ridge in
place. We confirmed that part of the Shh domain had been
excised by fixing some embryos just after the operation. As
shown in Fig. 4E, the number of Shh-expressing cells was
reduced after this operation (5/5 of embryos). However, 24
hours later, cells expressing Shh were present in the posterior
margin of the limb in all embryos and in many of these (50%,
n=24) there was a proximal expansion of the Shh expression
domain, when compared with the contralateral limbs (Fig. 4F,
arrow). In most other embryos (48%), the size and shape of the
Shh expression domain was similar to that in the contralateral
limb. In just one case was the number of Shh-expressing cells
reduced. Analysis of cell death in the same embryos showed a
variable degree of reduction in number of dead cells in the
posterior necrotic zone in 96% of embryos (n=24). Cell death
was completely absent in most cases (75%, n=24) (Fig. 4G).
This decrease in cell death coincided with the observed
expanded domain of Shh expression.
In another set of experiments, some cells expressing Shh at
stage 20 were killed by applying beads soaked in 100 µM
staurosporin (a protein kinase inhibitor known to induce
apoptosis (Jacobson et al., 1996). 4 hours after staurosporin
application, a reduction in number of Shh-expressing cells can
be detected (n=2) (Fig. 4H). However, at 22 hours, in all cases
the domain of Shh expression was restored, and in 2/3 cases
some Shh-expressing cells were found more proximally than
in the contralateral limb (Fig. 4I). Moreover, a clear reduction
in the number of Nile Blue-positive cells in the posterior
margin of the limb buds could be seen (3/3) (Fig. 4J).
All these results obtained after experimental manipulation of
the number of Shh-expressing cells indicate a regulatory ability
of the Shh expression domain and show that this is related to
cell death. There is a consistent inverse correlation between the
extent of Shh expression domain and cell death in the posterior
necrotic zone and altering the amount of Shh signalling can
affect both these parameters. When Shh signalling is increased,
i.e. by grafting polarising region cells or applying Shh beads,
or after Fgf-4 bead application, cell death is increased and Shh
expression reduced; when Shh signalling is reduced, i.e. by
surgical removal or chemical killing of polarising region cells,
cell death is reduced and Shh expression expanded. This
suggests a buffering model in which any imbalance in the
number of Shh-expressing cells will be sensed and adjusted
through appropriate regulation of cell death.
Effects of blocking cell death on the expression
domain of Shh
Our model predicts that blocking cell death will lead to an
expansion of the Shh expression domain. We tested this
hypothesis by directly preventing cell death.
We used the RCAS retroviral vector to overexpress
ectopically the proto-oncogene Bcl2, the product of which is a
protein known to be able to block cell death in the chicken
embryo (Fekete et al., 1997). RCAS-Bcl2 virus was injected
into the right side lateral plate mesoderm (prospective right
limb region) of chicken embryos at stages 9-12 or stage 17.
Analysis of some embryos at 48 hours (n=7), 72-80 hours
(n=7), 4 days (n=2) or 6 days (n=1) after injection by in situ
hybridisation with a viral-specific probe confirmed spread of
infection. A variable degree of infection was detected in the
targeted right limbs and an example of extensive infection of
the right wing is shown in Fig. 5A.
Infected embryos were analysed at stage 23-25 for both cell
death in the posterior necrotic zone and Shh expression. In 48%
of cases (n=25), the right wing had a 10% or more reduction
in the number of Nile Blue-positive cells in the posterior
necrotic zone (Fig. 5B). In one case, no dead cells could be
detected in the right wing bud, while in the contralateral left
wing bud a posterior necrotic zone was already established
(Fig. 5E). Most other embryos (11/25) showed less than a 10%
difference in the number of Nile Blue-positive cells.
Quantitation of these results showed that the decrease in the
Fig. 5. Effects of Bcl2
expression on cell death and Shh
expression. (A) In situ
hybridisation with a viral
message-specific probe. Embryo
fixed 80 hours after RCAS-Bcl2
virus injection to right lateral
plate mesoderm at stage 10. Note
extensive infection of right wing
bud (arrow). (B,D) Nile blue
staining (B) and Shh + Fgf8
expression (D) in same embryo
72 hours after RCAS-Bcl2
injection. Note reduced number
of Nile Blue-positive cells in
right wing bud (arrow in B) and
proximally expanded Shh
expression domain (arrow in D).
(C) Another example of
expansion in Shh expression
after RCAS-Bcl2 injection
(arrow). (E) Another example of decreased cell death after RCAS-Bcl2 injection. Note absence of dead cells in targeted right wing (arrow).
(F,G) Viral expression (F) and Nile Blue staining (G) of same wings, five days after injection with RCAS-Bcl2 virus of the nascent right wing
bud at stage 18-19. Note extensive infection of right wing, including interdigit (arrow in F). Cell death is absent in that area, as can be seen by
absence of Nile Blue-positive cells in the right wing interdigit (arrow in G) as compared to the left wing (arrowhead).
Shh signalling and cell death in limb development 4819
number of Nile Blue-positive cells in the infected versus
contralateral wings was significant (average number of cells
69.5±33.7 in the infected, 79.9±35.9 in the contralateral,
P<0.05 in a paired Student’s t-test). In most cases (9/12),
reduction in cell death was accompanied by an increase in
number of Shh-expressing cells and/or extension of the Shh
expression domain proximally (Fig. 5C,D). Quantitation of the
Shh expression domains showed a significant increase of the
Shh-expressing areas in infected wings versus control wings
(average percentage increase 116.5±11.8 of control limbs,
P<0.01 in a Student’s t-test). Thus, when cell death is blocked,
this leads to an expansion of Shh expression, which is
consistent with the model that cell death regulates the number
of Shh-expressing cells. Controls were carried out by infecting
stage 9-12 embryos in a similar way with a control virus
(RCAS with no insert). Infection of the right wing was
confirmed 72 hours after injection with an RCAS-specific
probe, showing spread of infection (n=6). When cell death and
Shh expression were analysed, no significant differences
between the infected wings and the contralateral ones were
observed either in the number of Nile Blue-positive cells
(79.1±40.6% in the right wings versus 76.2±38.0% in the
control) or the area of the Shh expression domains
(mean=99.9±0.9% of the control).
Functional activity of the delivered Bcl2 gene in infected
limbs was confirmed by the decreased number of Nile Bluepositive cells in the anterior necrotic zone in 7/14 cases and in
the interdigital region in 4/5 cases (compare viral message
expression in Fig. 5F with cell death in Fig. 5G in the same
embryo).
Some embryos (n=50) were left to develop until day 10
and several defects were observed; delay in regression of
interdigital membranes leading to partial soft tissue syndactyly
(n=4), blips of tissue in the distal part of digits/toes (n=3),
abnormal flexures at some joints leading to ‘straight’ limbs
(n=3) and, most remarkably, nodules of ectopic cartilage
associated with some long bones (radius/tibia) (n=2). All these
defects point to a possible role of cell death in other processes
of limb development such as joint formation and cartilage
morphogenesis.
Relationship between domain of Shh-expression
and the posterior necrotic zone in normal limb
development
The polarising region was discovered during investigations of
the properties of the posterior necrotic zone (Saunders and
Gasseling, 1968) and expression of Shh at the posterior margin
of the wing bud correlates with maps of polarising activity. Shh
expression is initiated at Hamburger and Hamilton (1951) stage
17-18 in posterior mesenchyme of chick wing buds,
maintained along the posterior bud margin as the bud grows
out and then becomes restricted distally at stage 24, where it
remains until stage 28 (Riddle et al., 1993). The posterior
necrotic zone, on the other hand, first appears as a discrete
patch of dead cells midway along the posterior margin of chick
wing buds at stage 23; by stage 24, a massive area of cell death
extends along the proximal two thirds of the posterior margin
and then cell death continues in the distal part of the limb until
stage 29-30 (Hinchliffe, 1981).
The results from our experimental manipulations pointed to
a direct link between Shh-expressing cells in the polarising
region and cell death in the posterior necrotic zone. To visualise
directly whether Shh expression and the posterior necrotic zone
overlap in normal limb development, we performed sequential
double labelling of stage 24 chick embryos. As can be seen in
Fig. 6A, the posterior necrotic zone, a group of cells labelled
with Nile Blue, extends along the proximal 2/3 of the posterior
margin of the wing bud. In the same wing bud (Fig. 6B), Shh
expression extends proximally from the distal tip, next to the
posterior boundary of the apical ectodermal ridge, up to almost
half way along the wing bud. Thus, the proximal part of the
Shh expression domain clearly overlaps with the distal part of
the posterior necrotic zone, as can be seen in Fig. 6C where a
superimposed image of Fig. 6A,B is shown.
To trace the cell lineage relationship between Shhexpressing cells and the posterior necrotic zone we injected DiI
into the posterior margin of stage 20 wing buds, at somite levels
19-20, in a subapical position (Vargesson et al., 1997; Fig. 6F).
Some embryos were fixed immediately and in situ
hybridisation confirmed that labelled cells were indeed in the
Shh-expressing domain (n=7) (Fig. 6G). Other embryos were
collected at 24 hours and, by this time, some DiI-labelled cells
had died and were present in the posterior necrotic zone as
confirmed by Nile Blue staining (n=11) (Fig. 6H,I, arrows);
Fig. 6J is a merged image showing double labelled DiI- and
Nile Blue-positive cells (arrows). Other labelled cells were
found proximal to the posterior necrotic zone (Fig. 6H,I,
arrowheads). When embryos were collected 48 hours after
labelling (n=7), Nile Blue-positive cells were still found in the
posterior necrotic zone. The same result was obtained when a
limb bud was labelled posterodistally at stage 22-23 and
analysed at stage 25-26.
Finally, to investigate at the single cell level whether Shhexpressing cells undergo apoptosis in the posterior necrotic
zone, we cut cryosections of limbs that had undergone wholemount in situ hybridisation to visualise Shh expression and
carried out TUNEL staining to reveal apoptosis. As can be seen
by comparing Fig. 6D and 6E (arrows), TUNEL-positive cells
are present in the Shh expression domain. Furthermore, we
observed some individual cells that expressed Shh (Fig. 6L,O)
and were also TUNEL positive (Fig. 6K,N, merged image in
Fig. 6M,P), thus providing direct evidence that Shh-expressing
cells die in the posterior necrotic zone.
DISCUSSION
Buffering model for regulating signalling in the limb
Our results suggest a model for the regulation of polarising
activity in chick limb development (Fig. 7). We have found that
Shh can repress its own expression and lead to increased cell
death, and both experimental manipulations and in vivo
evidence suggest that there is a link between the two. We
propose that cell death induced by Shh could act as a buffer to
regulate the number of Shh-expressing cells. In normal limb
development, there will be a balance between generation of
new cells expressing Shh, under the influence of Fgf4 and other
growth factors produced distally, and loss of cells expressing
Shh proximally. This will keep the Shh signal at the appropriate
level and restrict the Shh domain distally close to the apical
ridge. The model is compatible with previous results showing
that the removal of posterior apical ectodermal ridge, which
4820 J. J. Sanz-Ezquerro and C. Tickle
Fig. 6. Relationship between cell death in the posterior necrotic zone and Shh expression domain. (A) Stage 24 chick wing bud stained with
Nile Blue to reveal dead cells. Arrow indicates the distal part of the posterior necrotic zone. (B) Shh expression as revealed by in situ
hybridisation in the same limb. Note the distal restriction of expression and the overlap between the fainter proximal area of Shh expression
with the distal part of the cell death domain seen in A (arrow placed in the same position as in A). (C) Superimposed image of A and B showing
the overlap. (D,E) Cryosections of a stage 24 wing bud firstly stained for Shh expression by whole-mount in situ hybridisation (E) and
subsequently with TUNEL to reveal apoptosis (D). Note apoptotic cells (arrows) in the Shh expression domain. (F-J) Fate map of polarising
region cells. (F) Stage 20 wing bud injected with DiI (fluorescent labelling) in the posterior margin (arrow). (G) Same bud showing Shh
expression. Note that DiI labelled cells (arrow) are in the Shh expression domain. (H) Wing bud 24 hours after DiI injection. Note labelled cells
in medial part of posterior margin (arrow) and more proximally, close to base of limb bud (arrowhead). (I) Same limb stained with Nile Blue.
Note that medial labelled cells are in posterior necrotic zone (arrow) and that the more proximal labelled cells in H have passed through the
necrotic zone (arrowhead). (J) High resolution merged image of H and I showing DiI labelled cells that are Nile Blue-positive (arrows).
(K-P) High-magnification images of limb cryosections double stained for both Shh expression by whole-mount in situ hybridisation (L,O) and
apoptosis by TUNEL (K,N). Note double labelled cells in the polarising region (arrows, merged images in M,P). Posterior is at the bottom,
distal towards the right in all limbs.
leads to a decrease in Shh expression (Laufer et al., 1994;
Niswander et al., 1994), also results in reduction in cell death
in the posterior necrotic zone (Brewton and MacCabe, 1988).
A buffering system can account for several regulative
features of polarising region signalling. It can explain why
limbs with normal patterns develop after application of extra
Shh (polarising region cells (Tickle et al., 1975), Shhexpressing cells (Riddle et al., 1993) or Shh beads (Yang et al.,
1997) to the posterior margin of chick buds, and why normal
patterned limbs also develop after most, but not all, of the
polarising region is removed (Fallon and Crosby, 1975; Pagan
et al., 1996). It is important to note that this buffering
mechanism seems to operate only in posterior cells competent
to express Shh and/or that have been primed by posterior
polarising signals (supported by the finding that Shh expands
the normal posterior necrotic zone but it does not induce cell
death in other surrounding cells). This is why dose-dependent
effects of Shh signalling on digit pattern can be observed
anteriorly where no buffer exists and the effect of a given
number of polarising region cells can be analysed (Tickle,
1981).
Our model implies a positive-feedback loop between Shh
and Fgf4 and/or other Fgfs distally to maintain the population
of Shh-expressing cells. This is consistent with the finding that
Fgf4 application results in an initial increase in Shh expression.
The fact that this initial increase of Shh expression ultimately
leads to extra cell death and reduction of Shh fits well with an
autoregulatory buffering system. The model also implies a
negative feedback loop proximally, away from the influence of
the ridge, driven by Shh itself and involving apoptosis, to
eliminate Shh-expressing cells. It is not clear how this negativefeedback loop could operate. One possibility is that apoptosis
is induced in cells when the Shh signal in posterior
mesenchyme limb bud reaches a certain threshold. Polarising
region cells expressing Shh may also be particularly dependent
on the presence of survival ridge signals and/or be especially
sensitive to apoptotic stimuli, so that they initiate the cell death
programme once they have moved away from the apical
Shh signalling and cell death in limb development 4821
death in the developing limb can explain the absence of
necrotic zones in limbs of talpid3, a polydactylous chicken
mutant. In talpid3, a defect in the Shh signalling pathway,
involving inability to induce high levels of patched
expression, has been postulated to lead to a widespread
diffusion of Shh protein (Lewis et al., 1999). Thus, ectopic
Shh protein would be able to rescue cell death anteriorly and
in the interdigital spaces, while failure to localise high
concentrations of Shh protein posteriorly would also reduce
apoptosis in this area.
AER
apoptosis
growth
and
patterning
Shh
Fgfs
OUT
Shh
expressing
cells
IN
Fig. 7. Signal buffering model. Shh-expressing cells are added
distally under influence of Fgfs produced in ridge. It should be noted
that other growth factors produced distally are probably involved in
the input. Some cells are left behind as more cells are added distally
and are lost proximally through apoptosis, which is mediated by Shh
either directly or through an indirect mechanism. Fluctuations in the
number of Shh-expressing cells will be compensated by regulation of
cell death. AER, apical ectodermal ridge.
ectodermal ridge (AER). Alternatively, it can not be excluded
that cells of the polarising region first reduce Shh expression
as they become distant from the AER and only subsequently
initiate apoptosis. BMPs, which have been shown to act as
apoptotic signals, might mediate Shh-induced cell death and
expression of BMPs and/or BMPs inhibitors, which has been
shown to be regulated by Shh, could modulate the precise
amount of cell death. Indeed, application of gremlin, an
inhibitor of BMPs, to developing limb buds has been reported
to lead to an expansion of the Shh expression domain
(Capdevila et al., 1999; Merino et al., 1999b). This is
postulated to be due to an increase in Fgf4 signalling, but
apoptosis could be reduced and could also contribute to the
observed increase in Shh expression.
Different effects of Shh on cell death
Although Shh acts as a cell death signal in posterior limb bud
cells, Shh rescues anterior and interdigital limb cells from
death. Shh is better known as a survival factor and Shh has also
been shown to rescue cell death in other areas of the embryo
such as somites (Teillet et al., 1998) and neurones (Miao et al.,
1997). This highlights the fact that different cell populations
can respond in a different way to the same signal. Our time and
dose results suggest that the survival effect of Shh on anterior
cells may be direct.
The opposite effects of Shh on the different regions of cell
Roles of cell death in signalling regions
Cell death has been shown to play an important developmental
role in the control of cell number, which is essential for proper
organ size and tissue homeostasis (reviewed in Green (1998);
Jacobson et al. (1997); Vaux and Korsmeyer (1999)). Here, we
suggest that apoptosis may play a role in the regulation of the
number of signalling cells in the polarising region of the limb,
contributing to modulate its activity and extent. Regulation of
the number of Shh-expressing cells in limb development could
be important in the dose-dependent patterning properties of
polarising region signalling. It could also be important in
setting the signal level that gives the appropriate number of
digits. In chick wing development, the posterior necrotic zone
is a prominent feature, while in limbs with more digits (chick
leg buds and mouse limbs) the posterior necrotic zone is still
present but much reduced (Hinchliffe, 1981, 1982; Milaire and
Rooze, 1983). In chick legs, the smaller necrotic zone is
associated with a more-extensive Shh expression domain.
There could be different thresholds of Shh that induce
apoptosis, or the balance between growth and apoptotic signals
controlled by Shh could be different in the different limbs,
which would ensure the appropriate amount of Shh signalling
to achieve the characteristic final number of digits and limb
pattern. The ability to control polarising activity could also be
an important safety mechanism to avoid excessive signal
levels. Finally, apoptosis has been shown to mediate silencing
of the enamel knot, which acts as a signalling centre in tooth
development (Vaahtokari et al., 1996) and thus cell death might
be also involved in the silencing of Shh expression, when
outgrowth and patterning of the limb are completed. In this
context, our finding that Shh application to late limb buds,
when the endogenous signal has been switched off, can make
digits grow longer, sometimes forming new joints and thus
more phalangeal elements, could be very important in
understanding how the final steps of digit formation are
achieved. We have preliminary evidence that implicates both
Fgf and Bmp signalling in this process (J. J. S.-E.,
unpublished).
We suggest that autoregulation of signal production via cell
death might be a general developmental mechanism. Dosedependent Shh signalling has been demonstrated in the neural
tube (Ericson et al., 1996) and thus, according to our ideas,
apoptosis might regulate the number of Shh-expressing cells
here. Indeed, it has been reported recently that Shh induces cell
death in the floor plate, which is the region of the neural tube
that produces Shh (Oppenheim et al., 1999). It is also possible
that this mechanism could act in other regions to regulate the
levels of other signals, such as Fgfs. These are produced in the
AER, where cell death also occurs (Ferrari et al., 1998; Todt
and Fallon, 1984).
4822 J. J. Sanz-Ezquerro and C. Tickle
We thank A.M. McMahon for Shh protein and S.H. Hughes for the
RCAS-Bcl2 construct; Juan Hurle for the Gdf5 probe; and Litsa
Drossopoulou for her advice on bead implantation. This research was
supported by a long term EMBO postdoctoral fellowship to J. J. S.E. and a MRC programme grant to C. T.
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