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First published online 14 March 2007
doi: 10.1242/dev.000703
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1 Shriners Hospitals for Children Portland, 3101 SW Sam Jackson Park Road,
Portland, OR 97239, USA.
2 Department of Cell Biology and Human Anatomy, Davis School of Medicine, 3301
Tupper Hall, University of California, Davis, CA 95616, USA.
3 Department of Cell and Developmental Biology, Oregon Health and Science
University, 3181 SW Sam Jackson Park Road, Portland, OR 97239, USA.
Author for correspondence (e-mail:
pjh{at}shcc.org)
Accepted 8 February 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Myc, N-Myc, Limb, Syndactyly, Apoptosis, Interdigital, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Niswander, 2003;
Tickle, 2003). Generating and
fixing the positional identity of limb bud cells to produce the complex
pattern of the developing cartilage templates requires the coordination of at
least three organizing centers. The first organizing center that forms is the
apical ectodermal ridge (AER), a morphologically distinct epithelium located
at the distal margin of the limb bud that produces fibroblast growth factors
(FGFs) required for sustained proximodistal (PD, shoulder to digits) growth.
The zone of polarizing activity (ZPA) forms soon after and directs
anteroposterior (AP, little finger to thumb) through the secretion of Sonic
hedgehog (Shh) from posterior mesenchyme. Finally, the dorsoventral axis of
the limb is patterned through Wnt signaling emanating from dorsal ectoderm. In
addition to controlling axis formation, elements of these pathways appear to
be integrated into the program that regulates cell death of interdigital
mesenchyme (IDM), which functions in the process of digit separation in
vertebrate species with free digits.
The Myc family proteins N-Myc (also known as Mycn - Mouse Genome
Informatics) and c-Myc (also known as Myc - Mouse Genome Informatics) play
essential roles during vertebrate development, as embryos lacking N-Myc or
c-Myc die by embryonic day (E) 11.5 and E10.5, respectively (reviewed by
Hurlin, 2005;
Murphy et al., 2005).
Lethality is associated with defective formation of many organs and is linked
to a general failure of different cell populations to sustain proliferation.
The characterization of mice following conditional, tissue-specific deletion
of N-Myc and c-Myc has further demonstrated their essential roles in
maintaining progenitor populations and controlling the balance between cell
proliferation and differentiation. For example, N-Myc is required in neuronal
and lung progenitor populations to promote their proliferation, prevent their
differentiation and maintain their plasticity
(Knoepfler et al., 2002;
Okubo et al., 2005).
The important activities of c-Myc and N-Myc in regulating cell
proliferation and differentiation during embryonic development are consistent
with their well-characterized roles in tumor formation. Forced expression of
Myc family proteins generally prevents cell differentiation, ultimately
leading to uncontrolled proliferation and tumorigenesis in a wide variety of
settings. However, in many cell types, such uncontrolled proliferation is
counteracted by a high rate of apoptosis
(Nilsson and Cleveland, 2003).
While it is well documented that forced expression of Myc family proteins can
trigger apoptosis, there is little or no direct evidence indicating that they
play normal physiological roles in programmed cell death. The possibility that
N-Myc might regulate programmed cell death of IDM was suggested by the finding
that haploinsufficiency of N-Myc causes Feingold syndrome
(van Bokhoven et al., 2005), a
pleomorphic disease syndrome often characterized by a mild form of syndactyly
(Celli et al., 2003).
Syndactyly is a relatively common human birth defect, and it is widely thought
that defects in molecular pathways controlling programmed cell death of
interdigital tissue are responsible. Thus, it is of considerable interest to
determine the possible role that N-Myc plays in interdigital cell death.
In addition to a potential role of N-Myc in regulating interdigital cell
death, early studies of N-Myc null mice suggest that N-Myc is important in
early limb development. Limb buds of N-Myc null embryos were small, and failed
to develop distal skeletal elements when cultured in vitro, leading to the
conclusion that N-Myc is required for proliferation of limb mesenchyme and
sustained PD limb growth (Sawai et al.,
1993). However, germline deletion of N-Myc causes lethality at an
early stage of limb development, raising the possibility that deficiencies in
other crucial supporting systems within the embryo regulated by N-Myc are
responsible for the small limb buds. Further, the limb bud culture
experiments, necessitated by the early lethality, probably do not accurately
recapitulate normal development. The lethality of N-Myc null mice also
precluded any investigation of the role N-Myc might play in later events of
limb morphogenesis, including interdigital programmed cell death.
To better define the role of N-Myc in limb development, we re-examined limb buds of N-Myc null mice and analyzed limb development following conditional deletion of N-Myc in the early limb bud. We found that N-Myc deficiency caused diminished cell proliferation, particularly in the early limb bud, and caused a relatively uniform reduction in the size of all skeletal elements. Most striking however, was a complete absence of programmed cell death of interdigital tissue and the development of profound soft-tissue syndactyly. Surprisingly, N-Myc expression is excluded from cells undergoing cell death and instead of regulating programs that control apoptosis, our results suggest that syndactyly is caused by a failure to produce N-Myc-dependent interdigital cells, of which the elimination by cell death mediates digit separation.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and
Prx1-Cre mice (Logan et al.,
2002) on a mixed background of C57BL/6 and 129 yielded
PC-NMycf/f mice. Mice and embryos were genotyped as
previously described (Knoepfler et al.,
2002; Logan et al.,
2002).
In situ hybridization
In situ hybridization of whole embryos and sections was performed using
digoxigenin-UTP-labeled riboprobes as previously described
(Brent et al., 2003).
BrdU incorporation
BrdU (Sigma) labeling (1.5 hours) was performed as described by Queva et
al. (Queva et al., 1998) using
BD Biosciences BrdU In Situ Detection Kit. Anti-BrdU stained sections were
counterstained with Hematoxylin.
Immunohistochemistry and electron microscopy
Embryos were fixed overnight in 4% paraformaldehyde and imbedded in OCT.
Frozen sections were blocked with 20% donkey or goat serum in PBS followed by
overnight incubation of primary antibodies [anti-E-cadherin (Santa Cruz),
anti-cyto pan keratin (Sigma), Pecam (Santa Cruz)] at 4°C. Cy3-conjugated
donkey anti-mouse F(ab)2 (Jackson ImmunoResearch Laboratories) and
goat anti-rabbit IgG (Alexa Fluor 488, Molecular Probes) were used as
secondary antibodies. For TEM, E13.5 forelimbs were fixed in 1.5%
glutaraldehyde/1.5% paraformaldehyde containing 0.05% tannic acid and
processed as previously described (Sakai
et al., 1986).
Skeletal preparation
De-skinned embryos and newborns were stained with 0.05% Alcian Blue and
0.015% Alizarin Red in 70% ethanol and 13% glacial acetic acid for 3 days,
cleared in 0.5% KOH overnight and stored in glycerin.
Cell death assays
Transverse sections of embryos were assayed by TUNEL using the Roche In
Situ Cell Death Detection Kit. Detection of cell death by LysoTracker
(Molecular Probes) staining was carried out according to the protocol of
Zucker et al. (Zucker et al.,
1999).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) were
mated with Prx1-Cre transgenic mice (Logan
et al., 2002). Logan et al.
(Logan et al., 2002) showed
that Prx1-Cre is active in the emerging forelimb bud mesenchyme at E9.5 and
hindlimb mesenchyme at E10.5. The region targeted by Prx1-Cre appears to
completely overlap with the regions of N-Myc expression in the early limb bud
(Sawai et al., 1990;
Kato et al., 1991;
Queva et al., 1998) (see
Fig. 2A). Using Cre-reporter
mice (Soriano, 1999), we found
Prx1-Cre to also be active in lateral mesenchyme at E8.5-9, which coincides
with the emergence of the forelimb bud (not shown). When compared with N-Myc
germline null and control Prx1-Cre (PC) embryos, the forelimb and hindlimb
buds of Prx1-Cre-N-Mycf/f (PC-NMycf/f)
embryos at E10.5 were intermediate in size
(Fig. 1A and not shown).
However, whereas the body size of PC-NMycf/f embryos was
otherwise close to that of control embryos, the small limb buds of
N-Myc-/- embryos corresponded to their overall small
embryonic size (Fig. 1A).
Furthermore, while N-Myc null embryos died by E11.5,
PC-NMycf/f embryos were viable and gave rise to fertile
adults. Thus, the effect of N-Myc deletion by Prx1-Cre on limb development can
be attributed to the intrinsic function of N-Myc in this process and not to
N-Myc-dependent regulation of extrinsic factors that might influence limb
development.
The small limb buds of N-Myc null embryos and
PC-NMycf/f embryos might be due to predicted effects of
N-Myc deficiency on cell proliferation (see more below), or defects in
establishing the early organizing centers that regulate cell proliferation.
However, expression of Fgf8, marking the formation of the AER, and
Sonic Hedgehog (Shh), marking formation of the ZPA, were
clearly present and appeared correctly positioned in limb buds of
N-Myc null and PC-NMycf/f embryos
(Fig. 1B). Gremlin,
which is a target of Shh signaling involved in feedback regulation between the
ZPA and AER (Zuniga et al.,
1999; Capdevila et al.,
1999), is also present in both N-Myc null and
PC-NMycf/f limb buds, but its expression is expanded
(Fig. 1B). Consistent with this
finding, expression of Fgf4 is elevated in the AER of N-Myc-deficient
limb buds (Fig. 1B). However,
Gremlin and Fgf4 expression is extinguished at later stages,
as in control limb buds (Fig.
6B and not shown). Bone morphogenetic protein 4 (Bmp4),
which is expressed in both the AER and the ZPA, and Hand2, which
marks cells competent to become the ZPA
(te Welscher et al., 2002),
were expressed in PC-NMycf/f and N-Myc null limb
buds at their proper locations (Fig.
1B). However, Hand2 appeared to be downregulated in
N-Myc-deficient limb buds. Finally, Gli3, which is restricted to
anterior mesenchyme at E10.5, at least in part by Hand2
(te Welscher et al., 2002),
had an anterior expression domain in N-Myc-deficient limb buds, but was
strongly downregulated (Fig.
1B). Although Hand2 and Gli3 were expressed at
low levels in N-Myc-deficient limb buds at E10.5, their levels and position
were comparable to control embryos at E11.5 (not shown). Taken together, these
results indicate that the AER and ZPA are formed in N-Myc-deficient limb buds,
but that at least some of their activities are transiently disrupted or
delayed.
N-Myc regulates cell proliferation in limb bud mesenchyme
The close association of Myc proteins with cell proliferation prompted an
examination of the relationship between cell proliferation and N-Myc
expression in the limb bud. Consistent with previous results
(Kato et al., 1991),
N-Myc was expressed in limb bud mesenchyme at E9.5, with highest
levels in the posterior bud and not expressed in limb bud ectoderm
(Fig. 2A). In contrast,
N-Myc expression was absent in most, if not all, mesenchymal cells of
PC-NMycf/f limb buds
(Fig. 2D) and was absent as
expected in germline null limb buds at E9.5
(Fig. 2G). The loss of
N-Myc expression in PC-NMycf/f limb buds is
consistent with robust Prx1-Cre activity at the earliest stages of limb bud
outgrowth (Logan et al., 2002)
(and data not shown). The absence of N-Myc in
PC-NMycf/f and N-Myc null limb buds at E9.5 was
associated with a decline in cell proliferation, as measured by the percentage
of cells that incorporated BrdU compared to control limb buds
(Fig. 2B,C,E,F,H,I and
Table 1). The decrease in BrdU
incorporation caused by loss of N-Myc corresponded to a decrease in cell
density (Table 1).
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Levels of cell death, as measured by TUNEL, in the mutant limb buds was very low or non-detectable between E9.5 and 11.5 and no different from control limb buds (not shown). Taken together, these results suggest that the reduced size and cell density of PC-NMycf/f and N-Myc limb buds is attributable to a decrease in proliferation of undifferentiated limb bud mesenchyme, particularly in the very early limb bud around E9.5. Further, the altered distribution of BrdU-positive cells was associated with a decrease in the size of the central mesenchymal condensation between E10.5 and 11.5, suggesting that N-Myc is an important regulator of the number or behavior of progenitor cells that give rise to the precartilaginous condensations.
Importantly, neither c-Myc nor L-Myc (Mycl1 - Mouse Genome Informatics) expression appeared to overlap with N-Myc expression in the developing limb, and no compensatory upregulation or changes in their expression pattern was seen in N-Myc-deficient limb buds (data not shown).
Loss of N-Myc leads to decreased skeleton size and defective digit formation
Staining with cartilage-specific Alcian Blue and bone-specific Alizarin Red
at E15.5 revealed that early limb skeletal elements of N-Myc-deficient limbs
were 20-40% smaller (length and diameter) than those of control littermates
(Fig. 3A-C and data not shown).
An exception is the most posterior digit (digit 5), the length (but not
diameter) of which was relatively unaffected. Notably, the reduced size of
limb elements was largely proportional to each other. Further, the most
proximal upper limb element, the scapula, which forms in lateral mesenchyme
and not the limb bud proper, was reduced in size
(Fig. 3A), consistent with
Prx1-Cre activity in presumptive limb regions of lateral mesenchyme. The small
limb skeletal elements were maintained during embryonic development
(Fig. 3B,C) and into young
mice, although the size differential with control mice was slightly decreased
in juveniles (not shown).
Little is known about the nature of mesenchymal cells that give rise to
chondrocytes, but their development and pool size appeared to be linked to
formation of the AER and production of AER-derived FGFs, particularly Fgf8
(Niswander, 2003;
Tickle, 2003). Although N-Myc
deficiency did not disrupt expression of Fgf8 and formation of the
AER or ZPA (Fig. 1B), it had a
marked effect on expression of Sox9. Sox9 is a marker of
precartilaginous condensations and is required for development of chondrocytes
(Bi et al., 1999).
Sox9 was very low in N-Myc-deficient limb buds at E10.5 and continued
to be low through E13.5 (Fig.
3D). The low Sox9 levels corresponded to a reduction in
the size of condensations observed at E10.5 and 11.5
(Fig. 2G,S). As illustrated in
sections through the developing digits at E13.5
(Fig. 3D), the low
Sox9 expression corresponded to small cartilaginous elements.
Similarly, expression of other chondrocyte markers, including Noggin
(Fig. 3E) and Collagen II (not
shown), was diminished in N-Myc-deficient limbs and probably reflects a
smaller domain of chondrogenic mesenchyme in the developing skeleton. Because
N-Myc expression appeared not to overlap with Sox9-positive
condensing mesenchyme in the early limb bud (compare
Fig. 2A,M with
Fig. 3D), these data, taken
together with the results of BrdU-incorporation assays, further suggest that
N-Myc regulates the size and/or behavior of undifferentiated limb bud
mesenchyme that gives rise to the Sox9-positive limb bud condensation. Thus,
the small skeletal elements cause by loss of N-Myc may reflect a significant
reduction in the initial pool of chondrogenic progenitor cells.
In addition to causing small skeletal elements, N-Myc deficiency disrupts
digit joint formation (Fig.
3B,C). Digits 2 to 5 had only one distinct joint distal to the
metacarpal joint, when they should have two, and all of other digit joints
that did form, including the metacarpal joints, were poorly formed. Expression
of Gdf5, a BMP family member that marks presumptive joints and is
required for proper joint formation (Archer
et al., 2003) was weak in PC-NMycf/f limbs and
lacked the prominent distal digit expression seen in control embryos at E12.5
(Fig. 3F). Gdf5 was
also very low at presumptive phalangeal joints at E13.5 and 14.5
(Fig. 3F). Gdf5 has strong
effects on chondrocyte proliferation
(Archer et al., 2003) and its
weak expression may contribute to reduced digit (and other skeletal element)
size and associated joint defects. Alternatively, its weak expression may
simply be a consequence of reduced numbers of chondrocytes or other cell types
in digit primordia that normally express Gdf5.
N-Myc-deficient forelimbs also exhibited fusions of digit 5 and digit 4 at their metacarpal bases (53/53) and between the metacarpal bases of digits 1 and 2 (26/53) (Fig. 3B,C and not shown). Fusion between the distal tips of the central three digits was also frequent (46/53), but whereas metacarpal fusions were apparent by E14.5, the distal fusions occurred at later stages (not shown).
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). There
was no evidence of syndactyly in mice heterozygous for N-Myc
(equivalent to Feingold syndrome in humans). To examine the relationship between N-Myc and cell death in the autopod, N-Myc expression was compared to whole-mount LysoTracker and TUNEL staining (marking apoptotic cells) of limb sections at E12.5, 13.5 and 14.5, stages when interdigital cell death is most active. At E12.5 N-Myc expression was highest in undifferentiated mesenchyme surrounding the anterior and posterior digit condensations (i.e. primordia of digits 1 and 5) (Fig. 5A). It was expressed at relatively low levels at the tips of digit primordia and in interdigital tissue of the central digits. At E12.5, cell death was detected primarily in the AER, but also weakly in IDM (Fig. 5B,C). N-Myc transcripts were not detected in PC-NMycf/f limbs at E12.5 (Fig. 5D), and apoptosis was detected only in the AER (Fig. 5E,F).
At E13.5 N-Myc expression was highest in peri-digital regions (Fig. 5G). In situ hybridization of distal limb sections at E13.5 confirmed peri-digital expression and absence of N-Myc from digit condensations (not shown). Strikingly, N-Myc expression was excluded from interdigital regions undergoing cell death at E13.5 (Fig. 5G-I). N-Myc was not detectable in PC-NMycf/f limbs at E13.5 (Fig. 5J), and these displayed a complete absence of interdigital cell death (Fig. 5K,L).
At E14.5, digit separation was nearly complete, and N-Myc continued to be expressed in peri-digital regions of the separated digits but was excluded from the remaining interdigital tissue undergoing cell death (Fig. 5M-O). It is notable that while N-Myc was not expressed in central interdigital regions, which undergo cell death linked to digit separation, N-Myc expression did appear to be coincident with some perichondrial and joint regions, where apoptosis was evident in the separated digits of E14.5 limbs (Fig. 5M-O). In PC-NMycf/f limbs at E14.5, N-Myc levels were very low or undetectable (Fig. 5P), and no interdigital or joint cell death was observed (Fig. 5Q,R).
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; Guha et al.,
2002; Wang et al.,
2004; Lu et al.,
2006). Therefore, one potential explanation for loss of
interdigital cell death and syndactyly in N-Myc-deficient autopods is
prolonged FGF signaling due to failure of AER regression. However, this
appears not to be the case, as Fgf8 expression was extinguished in
ectoderm overlaying interdigital regions in both PC-NMycf/f and
control limbs by E13.5 (Fig.
6A). Moreover, TEM indicates that the distinctive epithelium of
the AER became restricted to the distal digit tips in both PC and
PC-NMycf/f paws at E13.5 (not shown). The typically
transient Fgf4 expression in the posterior AER was not disrupted by
loss of N-Myc, with expression no longer detectable by E12.5
(Fig. 6B). Thus, the absence of
interdigital cell death and associated syndactyly is not due to failed AER
regression or persistent ectodermal expression of Fgf8 or Fgf4.
While the specific signals that trigger interdigital cell death remain to
be fully defined, BMP signaling is implicated in this process. Several BMP
family members are expressed in IDM, and ectopic BMP activity in IDM can
induce apoptosis (Zuzarte-Luis and Hurle,
2005). In N-Myc-deficient limbs, the interdigital and peri-digital
expression of Bmp2 and Bmp7 was very low relative to PC
limbs (Fig. 7A,B). Bmp4
expression was weakly expressed in the anterior autopod of both PC and
PC-NMycf/f limbs at E12.5 and 13.5 (not shown).
Msx2, a key downstream target of BMP signaling was expressed strongly
in the central IDM of control limbs but was very weakly expressed or absent in
N-Myc-deficient limbs (Fig.
7D). The Msx2 results suggest that either BMP signaling
is strongly downregulated in the IDM, or alternatively, that IDM tissue that
normally expresses Msx2 is absent in PC-NMycf/f
autopods. Consistent with the latter idea, expression of Fgfr2, which
is not known to be linked to BMP signaling in the limb, was largely lost from
its well-defined central IDM domain, but not from peri-digital locations
(Fig. 7E). In addition,
interdigital Twist expression was strongly downregulated
(Fig. 7F). Taken together,
these results raise the possibility that the absence of interdigital cell
death caused by N-Myc deficiency is due to the absence of a population of
interdigital cells, marked by expression of Fgfr2, Msx2 and to a
lesser extent Twist, that are normally destined to undergo programmed
cell death as a trigger for digit separation.
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As an additional readout for the spacing between digit primordia, we
examined blood vessels in the autopod, which form along tracts running
adjacent to the digit primordia (Ambler et
al., 2001) and delineate the interdigital space. In striking
contrast to control limbs, there was very little or no space between blood
vessel tracts of adjacent digits in PC-NMycf/f limbs, as
marked by Pecam staining (Fig.
8C). These data are consistent with the idea that the diminished
or altered expression of BMPs, Msx2, Fgfr2 and Twist in the
mutant autopod is not caused by their transcriptional downregulation in the
absence of N-Myc, but is related to the absence of a channel of interdigital
tissue in which they are normally expressed.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Boulet et al.,
2004). The reduced cell number and corresponding reduction in cell
density provide an explanation for the small Sox9-positive precartilaginous
condensation and the small skeletal elements that subsequently form in the
absence of N-Myc. How would this be linked to the syndactyly that subsequently
develops? We propose that the IDM cells targeted for elimination during digit
separation are derived from the pool of undifferentiated mesenchyme that
exists in the early limb bud. Consistent with this notion, it is well
documented that IDM cells, like the undifferentiated mesenchyme of the limb
bud, possess chondrogenic potential (Hurle
and Ganan, 1986; Ros et al.,
1997). We therefore speculate that the decreased number of
undifferentiated mesenchymal cells in the early limb bud of
PC-NMycf/f mice leads to their depletion as limb
development proceeds and is responsible for the absence of a central channel
of interdigital tissue, normally marked by the expression of Fgfr2
and Msx2 (Fig. 7D,E).
The absence or severe reduction in undifferentiated IDM cells normally
targeted for cell death as part of the program leading to digit separation
provides an explanation for the profound syndactyly that develops. Inherent in
this model is the idea that elimination of interdigital mesenchyme normally
serves both to initiate digit separation and to remove undifferentiated
mesenchymal cells that might otherwise form ectopic cartilage or other
structures after the skeletal template is established. Definitive proof of
this model will require fate mapping of early limb bud mesenchyme (e.g.
Vargesson et al., 1997),
performed in conjunction with markers specific for undifferentiated
mesenchyme, interdigital tissue and cartilage.
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). Moreover,
interdigital BMP expression can influence digit identity
(Drossopoulou et al., 2000;
Dahn and Fallon, 2000), and the
altered BMP, Msx2 and Twist expression in the interdigital
tissue of PC-NMycf/f autopods may contribute to the
defects in growth and segmentation of digit primordia. Further, interdigital
BMP expression appears to be under the control of Shh
(Drossopoulou et al., 2000),
raising the possibility that N-Myc functions downstream of Shh signaling in
either controlling the expression of molecules that determine AP patterning,
or in generating/maintaining the cells that express these factors. However,
the transient expression of Gremlin and Fgf4, which function
downstream of Shh in the limb bud, are robust in the absence of N-Myc. Indeed,
the expansion of Gremlin expression may be an underlying mechanism
responsible for the small precartilaginous condensations, as Gremlin is a
potent inhibitor of BMP-mediated chondrogenesis
(Merino et al., 1999). Thus,
unlike neuronal precursors, where Shh appears to control both N-Myc
gene expression and N-Myc protein function
(Kenney et al., 2003), N-Myc
expression and activity in the developing limb appears to be dissociated from
at least some important features of Shh signaling.
Implications for models of proximodistal patterning
Exactly how specification of limb cartilage elements takes place in space
and time is under debate, but two models are generally used to explain this
complicated process. The progressive specification or progress zone model
posits that positional value is specified in cells as a function of the length
of time spent in the distal mesenchyme or progress zone
(Summerbell et al., 1973).
Cells that spend the least time in the progress zone (and perhaps divide a
fewer number of times) give rise to more proximal structures, and as the limb
grows, more distal structures are produced from cells that have spent more
time in the progress zone. More recently, analyses of limb development
following AER removal in the chick (Dudley
et al., 2002) and following deletion of Fgf4 and
Fgf8 in the mouse limb (Sun et
al., 2002; Boulet et al.,
2004) have suggested an alternative model in which cells that give
rise to the different proximodistal parts of the limb are specified in the
early limb bud and expand during limb outgrowth (reviewed by
Mariani and Martin, 2003;
Niswander, 2003;
Tickle, 2003). Our data
indicate that reduced proliferation of undifferentiated limb bud mesenchyme
caused by N-Myc deficiency leads to a decrease in the size of the initial
condensation in the limb bud and leads to a relatively uniform decrease in the
size of all limb skeletal elements (depicted in
Fig. 9). If the pool of
chondrogenic cells is reduced in these limb buds as predicted, then according
to the progress zone model, distal truncations (and normal-sized proximal
elements) should occur due to depletion of the progenitor population over
time. Although loss of N-Myc causes defects in segmentation of distal
elements, their size is not significantly reduced relative to proximal
elements. Thus, our data appear not to support the progress zone model and
instead are more in line with a pre-specified model for proximodistal
patterning. However, the relatively severe defects in patterning of the digits
caused by N-Myc deficiency suggest that the mechanism responsible for
patterning the autopod (or digits) may be connected to, but distinct from, the
process that patterns more proximal elements (e.g. radius/ulnar and humerus).
Indeed, the notion that patterning of the autopod is performed, at least in
part, as a separate episode during limb development has been previously
suggested (Tickle, 2003).
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).
Indeed, we do not observe syndactyly following Prx1-Cre mediated
deletion of a single copy of N-Myc. Two of the most common
syndactyly-associated syndromes are Apert syndrome and Saethre-Chotzen
syndrome. Whereas Apert syndrome is caused by gain-of-function mutations in
Fgfr2 (reviewed by Wilkie et al.,
2002), Saethre-Chotzen syndrome is typically caused by
haploinsufficiency of Twist, but is also sometimes caused by
Fgfr2 and Fgfr3 mutations
(Paznekas et al., 1998). The
syndactyly associated with Apert syndrome is often severe
(Temtamy and McKusick, 1978)
and sometimes approaches the profound syndactyly caused by Prx1-Cre
mediated deletion of both copies of N-Myc described here.
Furthermore, there are other defects found in the hands and feet of Apert
syndrome patients that are remarkably similar to those caused by loss of
N-Myc. For example, both feature a very close proximity of the
digits, especially the central digits, and a corresponding decrease in the
space (tissue) between digits. Both also feature metacarpal fusions at the
base of digits 4 and 5, and the absence of distal phalangeal joints and
associated brachydactyly (Fig.
3B,C) (Wilkie et al.,
2002). Interestingly, Prx1-Cre is active in cranial
mesenchyme (Logan et al.,
2002) and PC-NMycf/f mice exhibit cranial
defects (Fig. 1A and not shown)
that closely resemble those associated with Apert, Saethre-Chotzen and related
syndromes (S.O., Z.Q.Z. and P.J.H., unpublished). Taken together, these
phenotypic similarities suggest that N-Myc, Fgfr2 and Twist function in the
same pathway(s) responsible for syndactyly and craniosynostosis in Apert and
Saethre-Chotzen syndromes (and perhaps other syndromes as well). Moreover,
while syndactyly is widely discussed in the context of defects in cell death
pathways, our results instead suggest that the underlying mechanism leading to
syndactyly in these and perhaps other syndromes may be related to a deficiency
of N-Myc-dependent interdigital cells that are targeted for cell death and
required for digit separation. |
ACKNOWLEDGMENTS |
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