1 Departamento de Anatomía y Biología Celular, Universidad de
Cantabria, 39011 Santander, Spain
2 Department of Anatomy, University of Wisconsin-Madison, 1300 University
Avenue, Madison, WI 53706, USA
3 Department of Animal Sciences, University of Wisconsin-Madison, Madison, WI
53706, USA
* Authors for correspondence (e-mail: jffallon{at}facstaff.wisc.edu and rosm{at}unican.es)
Accepted 31 October 2002
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SUMMARY |
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Key words: Chick mutant, ZPA, Shh, Shh pathway, Limb development, Pattern formation
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INTRODUCTION |
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Evidence from a variety of sources points to an interdependence of the limb
bud signaling centers for continued synthesis of effector molecules and
signaling function. The AER is necessary for the induction
(Crossley et al., 1996;
Grieshammer et al., 1996
;
Noramly et al., 1996
;
Ros et al., 1996
) and
maintenance of Shh expression
(Riddle et al., 1993
) by ZPA
cells. AER induction and maintenance has long been known to be dependent on
the limb bud mesoderm (e.g. Saunders,
1977
). Recent data suggest the Bone morphogenetic protein (BMP)
inhibitor gremlin (Gre) is downstream of Shh and required for AER maintenance
(Capdevila et al., 1999
;
Merino et al., 1999
;
Zúñiga et al.,
1999
). Thus, the molecular framework of a possible
AER-to-ZPA-to-AER feedback loop is emerging. The possibility that FGF10 is the
effector growth factor of AER induction has been suggested
(Ohuchi et al., 1997
). The
complexity of limb signaling center interaction is further demonstrated by the
observation that Wnt7a-/- mice showed reduced Shh
expression and posterior limb deficiencies
(Parr and McMahon, 1995
).
The mechanisms that precisely define the location and subsequent
maintenance of the limb bud signaling centers are poorly understood. This is
especially true of the ZPA (Tanaka et al.,
2000). There is evidence of a role for retinoids in Shh
induction and maintenance from studies using retinoid inhibitors and retinoid
deficiency models (e.g. Lu et al.,
1997
; Power et al.,
1999
; Stratford et al.,
1997
; Stratford et al.,
1999
), and from Shh induction after treatment with
retinoids (reviewed by Tickle and Eichele,
1994
). Misexpression of Hoxb8 in anterior limb mesoderm
results in ectopic Shh expression but only in the proximity of the
AER (Charité et al.,
1994
). Interestingly, Hoxb8 expression precedes
retinoid-induced Shh expression in the anterior limb bud mesoderm
(Lu et al., 1997
;
Stratford et al., 1999
).
Similarly, ectopic expression of the transcription factor dHAND results in
ectopic Shh expression
(Charité et al., 2000
;
Fernandez-Teran et al., 2000
;
McFadden et al., 2002
) and
dHAND-/- mice fail to express Shh in the limb
(Charité et al., 2000
).
At present, it is not clear how these observations can be integrated to
explain how the ZPA is spatially delineated or how Shh expression is
maintained.
While it would appear that a cohort of cells in the emerging limb bud has
the competence to express Shh when exposed to FGFs
(Ros et al., 1996), analyses
of mouse mutants with anterior polydactyly, and other studies, indicate the
existence of negative regulators that restrict Shh expression to the
posterior bud. The transcription factors Alx4 and Gli3 have domains of
expression in the limb bud complementary to that of Shh and have been
proposed to repress Shh expression in the anterior limb mesoderm
(Büscher et al., 1997
;
Marigo et al., 1996b
;
Masuya et al., 1997
;
Masuya et al., 1995
;
Qu et al., 1997
;
Qu et al., 1998
;
Takahashi et al., 1998
). A
gradient of the repressor form of Gli3 has been described in the limbs of mice
and chickens with the highest concentration in the anterior portion of the
limb (Litingtung et al., 2002
;
Wang et al., 2000
). It has
also been proposed recently that Gli3 is an obligate component of ZPA
function, required in responding cells for Shh mediated polarizing activity
(Litingtung et al., 2002
).
The mechanisms involved in skeletal patterning downstream of Shh are being
actively investigated. Bmp2 has been considered a candidate Shh
effector gene because it is expressed in a domain that overlaps Shh
expression and because it is induced in the anterior limb mesoderm by ectopic
Shh expression (Duprez et al.,
1996; Yang et al.,
1997
). Recently, it was proposed that Shh acts to specify digit
formation, while concurrently setting up a gradient of Bmp2 that subsequently
specifies digit identity in a dose-dependent manner
(Drossopoulou et al., 2000
).
It has been demonstrated that BMP activity in the interdigital mesoderm at
autopod stages is required for the interdigits to specify digit identity
(Dahn and Fallon, 2000
).
We have analyzed a new limb mutant in the chicken first described by Smyth
et al. (Smyth et al., 2000)
and previously named Ametapodia 2. These chickens develop limbs that
lack ulna and fibula and all digits except digit 1 (d1) of the foot. Digit
identity was proposed on the basis of genetic evidence. Here we rename this
mutation as oligozeugodactyly (ozd) meaning reduced zeugopod
and digits and report data consistent with the complete absence of
Shh expression and activity specifically in the developing limb buds.
We report that the stylopod is normal in ozd limbs and the zeugopod
develops with only radius or tibia. While the wing lacks digits, the leg
develops a clearly identifiable d1. Consistent with the absence of Shh
signaling, neither Ptc1 nor Gli1 are detectable in mutant
limb buds, and we observe that the expression of Bmp2, dHAND and
5' Hoxd genes in posterior wing and leg bud mesoderm is
comparable to that observed in the limbs of Shh-/- mice.
We conclude that Shh becomes necessary for limb skeletal patterning distal to
the elbow and knee joints, similar to Shh-/- mice
(Chiang et al., 2001
;
Kraus et al., 2001
). The data
presented are consistent with a developmental model proposing the PD axis is
specified in the limb field, and that the radius/tibia and d1 are Shh
independent, while the ulna/fibula and other digits are Shh dependent.
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MATERIALS AND METHODS |
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To analyze gene expression in ozd embryos by whole-mount in situ
hybridization, we used two methods: the batch method and the hemisection
method. By the batch method we analyzed groups of appropriately staged embryos
from the ozd flock, of which approximately one quarter should be
homozygous for the ozd mutation. For each gene expression analyzed we
used a minimum of 16 embryos of the ozd flock, giving a probability
of 0.99 that at least one of them is homozygous. The hemisection method was
based on the fact that limb buds of ozd homozygous embryos do not
express Shh at any stage. Embryos were hemisected along their midline
and one half was hybridized for the gene of interest and the other half for
Shh. Embryos in which Shh was not detected were confirmed
ozd mutants. In order to analyze gene expression before the
ozd mutant phenotype was discernible, we surgically removed the right
wing buds from embryos in ovo and allowed the embryo to develop to show the
phenotype (Carrington and Fallon,
1988). Similar results were obtained by all three methods.
Recombinant limb experiments
Right wing buds of stage (st.) 19-21 embryos from the ozd flock
were removed in ovo, and embryos allowed to develop to confirm the phenotype.
The isolated buds were incubated in 0.5% trypsin for 1 hour at 4°C to
separate the ectoderm from the mesoderm. The isolated ectoderm was recombined
with wild-type mesoderm. Using the same approach, isolated limb bud mesoderm
from the ozd mutant flock was recombined with ectoderm from wild-type
embryos. The recombinant limbs were allowed to heal for 1 hour and then
grafted to the flank level somites of host embryos as described previously
(Fernandez-Teran et al.,
1999).
Grafts of ZPA and applications of Shh or RA
The ZPA was removed in ovo from st. 19-20 embryos of the ozd
flock, and donor embryos were allowed to develop to confirm the phenotype. ZPA
grafts were performed as described previously
(Tickle, 1981). Heparin
acrylic beads (Sigma, H5263) were soaked in recombinant mouse Shh (4 mg/ml).
The beads were implanted into the posterior wing bud mesoderm of st. 20
embryos from the ozd flock.
For application of retinoic acid (RA; all-trans-retinoic acid,
Sigma), beads (AG1X2, Bio-Rad) were soaked for 20 minutes at room temperature
in 0.1 mg/ml, 0.6 mg/ml or 1 mg/ml RA suspended in DMSO and rinsed several
times in saline before use. RA-soaked beads were implanted under the AER at
the anterior or posterior border of the developing wing and leg buds
(Tickle et al., 1985).
In situ hybridization in whole mounts and to tissue sections
Digoxigenin-labeled antisense riboprobes were prepared, and wholemount in
situ hybridization analysis performed according to standard procedures
(Nieto et al., 1996).
S35-labeled riboprobes were prepared and hybridized to tissue
sections as described previously
(Wilkinson and Nieto, 1993
).
The probes used were Shh, Fgf4, Fgf8, Bmp2, Hoxd11, Hoxd12, Hoxd13, Gli1,
Gli3, Ptc1, Hoxb8 and dHAND (kindly provided by C. Tabin, T.
Jessel, J-C Izpisua-Belmonte, P. Beachy and D. Srivastava).
Cell death analysis
In situ detection of DNA fragmentation was performed using terminal
deoxynucleotidyl transferase (TdT) mediated deoxyuridine-triphosphate (dUTP)
nick end-labeling (TUNEL) with the In Situ Cell Death Detection Kit,
Fluorescein (Boehringer-Mannheim).
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RESULTS |
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Anatomy of ozd limbs
Limb development in ozd embryos proceeds normally until st. 23/24
when the limb buds become abnormally narrow across the AP axis. The narrowing
becomes more evident during subsequent stages of development; by st. 26 the
mutant limb buds acquire a pointed and hooked shape; eventually, the mutant
limbs adopt a spiked shape (Fig.
1).
|
Skeletal preparations at 10 days of incubation
(Fig. 1A,B) showed ozd
mutant wings composed of humerus, radius and a hypoplastic carpal element
while the ulna, metacarpals and digits were absent
(Fig. 1B). ozd mutant
legs displayed femur, tibia, tibiale and first toe with a total absence of
fibula, and digits 2, 3 and 4 (Fig.
1B). It is important to emphasize that the skeletal elements
present in the mutant limb were of normal morphology and easily recognizable
except for the rudimentary carpal. The single element present in the leg
autopod showed the characteristic morphologies of the first metatarsal and
proximal phalanx of d1, making the identification unequivocal
(Fig. 1C). According to the
current classification of limb mutations, ozd can be considered a
longitudinal postaxial defect (Stoll et
al., 1998).
Alcian Green staining of st. 25 and 27 mutant and wild-type limbs failed to detect evidence of cartilage condensations corresponding to the absent skeletal elements in the day-10 mutant limb, indicating the development of these elements was never initiated (Fig. 1D).
Unexpected patterns of apoptosis in ozd limb buds
To determine whether ozd limb bud narrowing resulted from abnormal
cell death we performed TUNEL analysis in wild-type and ozd limbs at
st. 24, when the mutant phenotype became discernible
(Fig. 2). Wild-type wing buds
showed two areas of well-defined mesodermal apoptosis, one in the center of
the wing bud, known as the opaque patch (OP), and another along the posterior
border called the posterior necrotic zone (PNZ;
Fig. 2A)
(Fell and Canti, 1934;
Hinchliffe, 1982
;
Hurlé et al., 1995
;
Saunders and Fallon, 1967
). In
contrast, comparably staged ozd wing buds showed extensive abnormal
apoptosis in the anterior border mesoderm that extended into the distal
mesoderm (Fig. 2B) as well as
increased apoptosis in the OP (Fig.
2A,B). However, cell death was not detected in the posterior
border mesoderm in mutant wing buds (arrow in
Fig. 2B, compared with the
control in Fig. 2A). TUNEL
analysis of leg buds gave similar results. st. 24 wild-type leg buds show
apoptosis in a fairly extensive anterior zone, called the anterior necrotic
zone (ANZ) as well as in the OP and a small PNZ
(Fig. 2C). The ozd leg
buds showed massive apoptosis along the anterior and distal borders of the
limb and increased central cell death (Fig.
2D). No evidence of cell death in the posterior mesoderm was found
(arrow in Fig. 2D). During
subsequent development of the mutant limb, the anterior-distal area of cell
death persisted, but posterior apoptosis was not observed (not shown). The
absence of posterior cell death was a surprising result since the shape of the
mutant buds gives the appearance of a less substantial posterior border that
eventually formed a concavity. Our results indicate that the increased
apoptosis in the mutant contributes to the progressive narrowing of the bud to
a pointed shape over the course of development. However, the predominantly
anterior pattern of apoptosis in the mutant cannot account for the loss of
posterior structures characteristic of ozd wings and legs.
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The mesoderm is the defective tissue layer in ozd
To investigate which tissue layer is affected by the ozd mutation
we performed recombination experiments interchanging mesoderm and ectoderm
between mutant and normal donors
(Fernandez-Teran et al.,
1999). Control experiments exchanging mesoderm and ectoderm from
normal limb buds resulted in completely normal skeletal patterns
(Fig. 2E). Recombinant limbs
constructed with mutant ectoderm and wild-type mesoderm also developed into
limbs with a normal skeletal pattern (Fig.
2F), indicating that the ozd ectoderm is capable of
supporting normal development. However, mutant mesoderm recombined with normal
ectoderm resulted in limbs exhibiting the mutant phenotype
(Fig. 2G). These experiments
demonstrate that the mesoderm is defective in the mutant while the ectoderm is
capable of normal function.
Shh expression is undetectable in ozd limb
buds
The lack of posterior elements in both the zeugopod and autopod of the
ozd mutants indicated a defect along the AP axis, so we began our
molecular analysis by looking at Shh expression.
Batch analysis of st. 19 and older embryos revealed that approximately one quarter (14/50) lacked normal posterior Shh expression (Fig. 3E-H). This correlated with the expected percentage of homozygous embryos, suggesting ozd mutants did not express detectable levels of Shh in the limb.
|
We detected Shh transcripts in st. 17/18 wild-type embryos (cf.
Riddle et al., 1993) by
whole-mount in situ hybridization (n=10;
Fig. 3A). But, since there is
some variability in the developmental time at which Shh expression is
initiated in the limb bud (cf. Riddle et
al., 1993
), the batch method was not completely satisfactory for
the study of these stages. In order to determine if mutant embryos expressed
transient levels of detectable Shh prior to st. 19, we analyzed
Shh expression in st. 17/18 wing buds of confirmed ozd
embryos. For this specific experiment, we removed the right limb buds in ovo
and allowed the embryo to develop to determine the phenotype. Confirmed
ozd limbs were embedded, sectioned and hybridized with
35S-labeled Shh riboprobe. We found that Shh
expression was undetectable in all confirmed ozd buds (n=4,
Fig. 3D) while control buds,
acquired in the same way, expressed Shh (n=11,
Fig. 3C). Thus, ozd
embryos do not express detectable levels of Shh in the posterior limb
bud at any stage. We stress at this point that the defect in Shh
expression is specific for the limb bud, since expression at other embryonic
sites, e.g. the floor plate of the neural tube, appeared normal and these
structures had no morphological defects
(Fig. 1A-B and
Fig. 3).
We also analyzed the posterior ozd mesoderm for polarizing
activity. ZPA grafts from confirmed ozd limbs gave no duplications
(n=3, not shown) while ZPA tissue from non-ozd siblings gave
the expected digital duplications (n=8, not shown); polarizing
activity of 71.8%, calculated according to the method of Drossopoulou et al.
(Drossopoulou et al.,
2000).
The Shh pathway is not activated in ozd posterior limb bud
mesoderm
To confirm that Shh was not expressed in ozd limbs, we analyzed
the expression of Patched1 (Ptc1) and Gli1, genes
directly regulated by Shh and considered to be highly sensitive indicators of
Shh signaling (Ingham and McMahon,
2001).
During normal limb development Ptc1, the receptor for Shh, and Gli1, a target of Shh signaling, are expressed in domains overlapping the expression domain of Shh but extend more anteriorly (Fig. 4A). Using the batch method, we found that roughly 25% of embryos from the ozd flock did not express detectable levels of Ptc1 in the wing bud (5/22; Fig. 4A). Utilizing the hemisection technique, expression of Ptc1 was never detected in st. 18/19 ozd mutant limb buds (st. 18-19, 36-38 somites: n=5; Fig. 5B). Similar to Ptc1, we found that roughly 25% of hybridized embryos (3/18) did not express detectable Gli1 in the limb (compare Fig. 4C with 4D). These data confirm that detectable Shh activity is not present in ozd limb buds.
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|
Gli3 and Shh have mutually exclusive expression domains
in the developing limb and are believed to repress one another's expression
(Büscher et al., 1997;
Marigo et al., 1996a
;
Masuya et al., 1997
;
Schweitzer et al., 2000
). By
the batch method, at st. 18/19, no differences in Gli3 expression
were detected among embryos of the ozd flock
(Fig. 4E). This was confirmed
in mutant limb buds (n=4) as early as late st. 18/19 (37-40 somites)
by hemisection analysis. However, at st. 21, ozd embryos failed to
down-regulate Gli3 expression at the posterior border of the limb
(25% of batch, Fig. 4F).
We next compared expression patterns of the bHLH transcription factor dHAND
which has been proposed to act upstream of Shh and establish a
positive feedback loop with Shh later in development
(Charité et al., 2000;
Fernandez-Teran et al., 2000
).
Expression of dHAND in the ozd limbs started normally (batch
method), but then was reduced to a weak domain of expression restricted to the
posterior border of the limb, in a very similar pattern to that observed in
the limbs of the Shh-/- mice
(Fig. 4G,H)
(Charité et al., 2000
;
Fernandez-Teran et al.,
2000
).
We also analyzed the expression patterns of other genes considered to be major downstream targets of Shh. Bmp2, previously thought to be a downstream target of Shh, was expressed in the mesoderm and AER of both mutant wing and leg buds as early as st. 18/19 (Fig. 4I,J). It was expressed in a reduced area and at a slightly lower level than normal as determined by both batch (n=2/4, st. 20-23) and hemisection methods (st. 19, 37-39 somites; n=3).
Expression of Hoxd11-13 was also analyzed in ozd limbs. Using the batch method, it was determined that Hoxd11-13 expression was initiated in a temporally and spatially normal pattern (not shown), but progressively declined with time (Fig. 5). Hoxd11 pattern of expression was virtually normal in ozd wings up to st. 25, although its level of expression was slightly reduced compared to wild-type (Fig. 5A-B). During subsequent stages Hoxd11 expression in the mutant wing was restricted to the posterior border (Fig. C,D). In the ozd leg bud Hoxd11 expression was very reduced compared to wild type at st. 21/22 (Fig. 5A), becoming undetectable at st. 24/25 (Fig. 5B-D). Hoxd12 expression was reduced in the ozd wing buds as early as st. 21/22 (Fig. 5E) and its expression continued restricted to the posterior border (Fig. 5F-H). Expression of Hoxd12 was much more affected in the mutant leg where it became undetectable at st. 24 (Fig. 5E-H). In the mutant wing and leg, Hoxd13 expression was very reduced and became undetectable by st. 23/24 (Fig. 5I-L). Interestingly, Hoxd13 was re-expressed in the distal mutant leg mesoderm at st. 27 (Fig. 5K) and persisted in the distal leg mesoderm (Fig. 5L). Re-expression of Hoxd11 or 12 was never observed.
Genes involved in PD and DV patterning were normally expressed in ozd limbs. For PD specification we analyzed the expression of Meis1 and 2 and Hoxa11 and Hoxa13 genes. We found that expression of Meis1 and 2 was not modified in ozd limbs (not shown). While the expression of Hoxa11 was normal in ozd wing buds, the expression of Hoxa13, considered a marker for the autopod, was dramatically diminished to a thin low-level stripe of distal expression in the mutant wing mesoderm. In the mutant leg Hoxa13 expression was similar to normal (not shown). For DV specification we analyzed the expression of Wnt7a and Lmx1; both showed a normal pattern of expression in ozd limbs (not shown).
Gene expression in the ozd AER
Although our molecular characterization and experimental study of the
ozd mutant limb indicates that the defect is in the mesoderm,
reciprocal interactions between the mesoderm and the AER are well documented
(Deng et al., 1997;
Ohuchi et al., 1997
).
Therefore, we analyzed the expression of Fgf8 and Fgf4 in
the AER of ozd limbs. The mutant AER always expressed high levels of
Fgf8 throughout development of both the wing and leg
(Fig. 6A-F). Coincident with
the progressive narrowing of the mutant limb, the posterior extent of the AER
was reduced, showing an abrupt end at the posterior border at the point of the
posterior concavity in the mutant limb shape
(Fig. 6B). Fgf8
expression persisted in the mutant AER up to st. 27 in the wing and st. 28 in
the leg. At later stages, Fgf8 was dramatically reduced throughout
the anterior AER (Fig. 6C). The
anterior loss of Fgf8 together with its reduced posterior extension
resulted in a discrete point of Fgf8 expression at the very tip of
the mutant limbs at st. 28 (not shown). The expression of Fgf4
appeared reduced except in the most posterior of the mutant AER
(Fig. 6D), where a spot of
elevated expression became apparent by st. 22/23
(Fig. 6E). Fgf4
expression was not maintained in the mutant AER and declined with time, so
that by st. 25 it was undetectable except for residual levels of expression in
the posterior spot of high-level expression seen at st. 22/23
(Fig. 6F, compare with
Fig. 6E).
|
Recently, it was proposed that Fgf4 upregulation by Shh in the
posterior AER is mediated by the BMP antagonist Gre and expression of
Gre in the limb mesoderm is considered necessary for AER maintenance
(Capdevila et al., 1999;
Zúñiga et al.,
1999
). During development of the ozd limb buds
Gre expression appeared reduced and restricted to the posterior
border (Fig. 6G-I) as confirmed
by the hemisection technique (st. 20/21, 40-44 somites; n=5). This
pattern of Gre expression is similar to that reported in the
Shh mutant mice
(Zúñiga et al.,
1999
) and is consistent with the reduced Fgf4 expression
observed in ozd limb buds.
ZPA or SHH application rescues the ozd phenotype
Since Shh expression and signaling is undetectable in mutant
limbs, we tried to rescue the mutant phenotype by grafting a normal ZPA or
applying exogenous SHH-N to the posterior border of st. 20 mutant limb buds.
ZPA fragments from st. 20 wild-type limb buds were grafted under the posterior
AER of either the wing or leg of embryos from the mutant flock. For wings,
pieces of leg ZPA were used and for legs, pieces of wing ZPA were used. When
the ZPA was grafted to an ozd limb, the mutant phenotype was restored
to normal (n=2; Fig.
7B). In the specimen showed in
Fig. 7B, the piece of ZPA of
leg origin has also formed a digit characteristic of the leg (asterisk in
Fig. 7B). The appearance of a
digit of graft (leg) origin may occur if the grafted ZPA is large. ZPA grafts
into the ozd leg buds gave equivalent results (not shown).
|
Next, heparin acrylic beads loaded with SHH-N protein (4 mg/ml) were applied to the posterior border, attempting to mimic a normal ZPA. In these cases, a total pattern restoration of the AP axis was observed at the zeugopod level with formation of a normal ulna, and improved development of carpals, although the limbs were truncated at wrist level (Fig. 7C). The sequential application of a second SHH-N loaded bead 24 hours after the first restored wing patterning at both zeugopod and autopod levels (Fig. 7D).
Retinoic acid is unable to induce Shh expression in the
ozd mutant limb mesoderm
Retinoic acid (RA) induces Shh expression when applied to the
anterior wing bud mesoderm (Helms et al.,
1994; Riddle et al.,
1993
) and is implicated in the normal induction of the ZPA
(Lu et al., 1997
;
Stratford et al., 1997
). We
applied RA to either the anterior or posterior mesoderm of st. 20/21
ozd limb buds to determine if Shh could be induced and the
mutant phenotype rescued. We first applied beads soaked in RA (0.1 and 1
mg/ml) under the posterior AER. In wild-type limb buds (n=10), the
level of normal Shh expression was reduced when analyzed 24 hours
after the operation (Fig. 7E)
and resulted in a range of skeletal alterations varying from a loss of digits
(n=9) to the complete inhibition of outgrowth (n=1). These
data are consistent with previous reports
(Tickle et al., 1985
).
Application of an RA bead to the posterior mesoderm of ozd wings did
not induce Shh expression after 24 hours (n=3;
Fig. 7F) and resulted in the
total absence of the right wing (n=2; not shown).
RA-soaked beads (0.1 and 1 mg/ml) applied to anterior ozd limb
mesoderm did not induce Shh at 24 hours (n=2; compare
Fig. 7H with
Fig. 7G) or 48 hours
(n=1; not shown) after the operation and the mutant phenotype was not
modified (n=2). It has been shown in the wing that the induction of
Shh by RA may be mediated by the early, transient activation of
Hoxb8 (Lu et al.,
1997; Stratford et al.,
1997
), and that it is also preceded by the activation of
dHAND expression (Fernandez-Teran
et al., 2000
). Thus, we analyzed at what point RA induction of
Shh failed in the mutant. RA-soaked beads (1 mg/ml and 0.6 mg/ml)
were placed in the anterior border of wing buds, and embryos were fixed after
5 hours to analyze Hoxb8 expression, and after 12 or 20 hours to
analyze dHAND expression. Hoxb8 was normally expressed by
ozd limb mesoderm in response to RA signaling (n=5;
confirmed by hemisection technique; not shown). RA applications also induced
dHAND expression in the anterior mutant limb mesoderm, similar to the
normal limb (n=5; Fig.
7I-L). These observations indicate that the ozd mutation
lies downstream of Hoxb8 and dHAND activation by RA.
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DISCUSSION |
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AP molecular asymmetry in the absence of Shh function
Bmp2 and 5' Hoxd genes are considered to be
downstream effectors of Shh signaling since Shh application to the anterior
border induces their ectopic expression
(Laufer et al., 1994;
Riddle et al., 1993
;
Yang et al., 1997
). We report
that in ozd limbs these genes are activated in a pattern similar to
that in normal limbs. The 5' Hoxd genes were also shown to be
asymmetrically expressed in the limbless mutant limb bud in the
absence of detectable Shh expression
(Grieshammer et al., 1996
;
Noramly et al., 1996
;
Ros et al., 1996
) and in the
Shh-/- mouse (Chiang
et al., 2001
; Kraus et al.,
2001
). Phase II of 5' Hoxd genes expression,
proposed to be Shh dependent (Nelson et
al., 1996
), starts normally in ozd but is not fully
developed and expression declines with time. Phase III of expression, which
corresponds to the autopod (Nelson et al.,
1996
), is dramatically affected. The more 5' the
Hoxd gene, the earlier and more severely its pattern of expression is
affected. For example, in the st. 25 ozd wing bud, Hoxd11 is
expressed in a pattern similar to normal, while Hoxd12 and
Hoxd13 expression is progressively diminished. This may indicate a
progressive differential requirement for Shh among 5' Hoxd
genes. However, it is of interest that the distal tip of the ozd leg
bud re-expresses Hoxd13 at later stages correlating with the
formation of d1 and, interestingly, precedes activation of Indian
hedgehog (Ihh) in the digital cartilage (data not shown). This
late Hoxd13 expression was also reported to occur in the
Shh-/- hindlimb
(Chiang et al., 2001
;
Kraus et al., 2001
). Also,
dHAND is expressed in a reduced but posteriorly polarized domain of
expression in ozd limb buds. Thus, activation and polarization of
Bmp2, the 5' Hoxd and dHAND expression in the
posterior limb bud does not require Shh and reflects AP patterning asymmetries
in the early limb bud that are independent of Shh. However, Shh inputs are
required to stabilize and augment initial gene expressions so that the AP
polarization of the limb bud is realized.
Shh-dependent and -independent limb skeletal elements
Because Shh signaling is absent in the limbs of ozd embryos, it is
useful to compare the limb phenotype of ozd mutants and
Shh-/- mice (Chiang et
al., 2001; Kraus et al.,
2001
). Interestingly, both types of limbs show a very similar
phenotype forming a complete PD axis with a normal stylopod. One digit,
identified as d1, forms in the Shh-/- hindlimb
(Chiang et al., 2001
;
Kraus et al., 2001
;
Lewis et al., 2001
) and also
d1 forms in the ozd leg. The main differences between ozd
and Shh-/- limbs occur at the zeugopod. The skeletal
elements in the zeugopod of the Shh-/- mice (one in
forelimb and two in hindlimbs) are abnormal while the morphology of the single
fore and hindlimb zeugopod element in ozd mutants are virtually
normal. Despite the differences, both genotypes demonstrate the necessity for
Shh distal to the elbow/knee region, since either loss of AP identity and/or
posterior deficits are observed without it. Thus, it is possible to classify
the skeletal elements of the limb according to their requirement for Shh
signaling. The ozd mutation indicates that in the chick the
humerus/femur, radius/tibia and d1 are Shh independent, while the ulna/fibula
and rest of the digits require Shh inputs for normal development
(Fig. 8). However, the
Shh-independent potential of the limb varies between chick and mouse at the
zeugopod level since the element that forms in chick is well shaped while it
is unidentifiable in mouse.
|
Experimental removal of the posterior wing mesoderm in chick, including the
whole ZPA leads to limbs with a phenotype very similar to ozd limbs
(Pagan et al., 1996;
Todt and Fallon, 1987
). The
operated wings form a normal radius with or without d2 and since the surgery
is performed at st. 20, before the determination of the zeugopod
(Summerbell, 1974
), it can be
concluded, on the basis of various approaches to this issue, that a completely
normal radius can develop in the chick in the absence of Shh input.
Morphological differences between the wing and the leg reflect differences
in the response to common molecular signals that pattern them. Moreover, wing
buds and leg buds may respond differently to experimental manipulation (e.g.
Todt and Fallon, 1987;
Wada and Nohno, 2001
). The
formation of a properly patterned digit in the leg but not the wing indicates
that Shh is required for the most anterior digit to form in the wing. The
identity of the three avian wing digits remains controversial
(Burke and Feduccia, 1997
) (see
also Kundrát et al.,
2002
; Larsson et al., 2002). However, if we assume the
conventional nomenclature of d2, d3, d4, our hypothesis that d1 is Shh
independent predicts no wing digits will develop in the absence of Shh
function. Admittedly, the loss of d1 in the Shh-/- mouse
forelimb is difficult to explain. It is possible that global loss of Shh
function has more deleterious effects on limb development than limb-specific
loss of Shh function alone. A conditional null of Shh in the mouse
limb will permit a direct comparison of the mouse with the ozd
limb.
The role of Shh in mesoderm cell survival and proliferation
Removal of posterior mesoderm was shown to cause cell death similar to our
findings for ozd and was attributed to the loss of ZPA function
(Todt and Fallon, 1987). It is
notable that grafting a bead loaded with Shh protein prevents normal anterior
cell death in the chick wing
(Sanz-Ezquerro and Tickle,
2001
), suggesting a role for Shh in regulating cell death in the
limb.
Abnormal cell death correlates with the progressive narrowing of
ozd limbs. Interestingly, while the anterior mesoderm undergoes
increased apoptosis, neither the PNZ nor abnormal cell death are detected in
the posterior border. However, there is a significant change in the shape of
the posterior border, most notably in the leg, where a concavity forms that
contributes to the spike shape of the ozd phenotype. Determination of
the mechanism of posterior limb bud shape change is made more challenging by
the observation that there are no gross differences in BrdU incorporation in
posterior cells as compared to wild type at the stages examined (st. 19, 23
and 25; not shown). It is possible that those cells that will later contribute
to posterior structures failed to proliferate and were left behind, beginning
slightly before the phenotype becomes obvious, around st. 23/24. A slight
change in proliferation at st. 17 and 18, or even at the stages analysed with
BrdU, but below a detectable level could still account for the loss of
posterior structures. Clarification of this point will require further
investigation. Also, it is worth mentioning that a mitogenic effect for Shh
has been reported in several developing systems
(Bellusci et al., 1997;
Duprez et al., 1998
;
Jensen and Wallace, 1997
) and
that Hh signaling can induce proliferation during development by promoting
expression of cyclin D and cyclin E
(Duman-Scheel et al., 2002
).
Thus, in the absence of Shh, stimulus from the AER would not be sufficient to
support enough mesoderm to permit the specification of the whole
anterior-posterior axis.
The ozd mutation potentially affects a Shh
regulatory element
Disruptions in AP limb pattern are among the most common human birth
defects (Castilla et al., 1996;
Castilla et al., 1998
), and
understanding the affected developmental mechanisms is of significant clinical
importance. Interestingly, studies in human and mouse have mapped several
mutations and transgene insertions causing limb-specific AP patterning defects
to a syntenic locus near or within the Limb region 1 (Lmbr1)
gene, located less than 1 Mbp from the Shh coding region
[(Clark et al., 2001
;
Lettice et al., 2002
), and
references therein]. Recent genetic analyses demonstrate the Lmbr1
gene is incidental to the limb phenotypes; rather, evidence suggests these
mutations affect long-range cis regulatory elements, embedded within
the Lmbr1 locus, that control Shh expression in the limb.
While the majority of these mutations cause dominant pre-axial polydactyly,
the small deletion responsible for the autosomal recessive human disorder
Acheiropodia maps within the Lmbr1 locus
(Ianakiev et al., 2001
), and
causes longitudinal postaxial deficiencies closely resembling the limb
phenotypes of ozd chicks and Shh-/- mice. Here we
have shown that ozd limb mesoderm is incapable of expressing
Shh, clearly indicating that the mutation affects a limb-specific
regulatory element of Shh expression. Although the data presented
here are compatible with the mutation affecting either a cis- or
trans-acting element, we hypothesize that the ozd mutation
disrupts a cis-acting regulatory element directing Shh
expression in the limb, which lies within the Lmbr1 locus such as in
Acheiropodia individuals
(Ianakiev et al., 2001
); this
hypothesis is currently being investigated.
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ACKNOWLEDGMENTS |
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