Download c-Myc Overexpression Increases Cell Size and Impairs Cartilage

Vol. 13, 185–193, April 2002
Cell Growth & Differentiation
c-Myc Overexpression Increases Cell Size and Impairs
Cartilage Differentiation during Chick
Limb Development1
M. Elisa Piedra,2 M. Dolores Delgado, Maria A. Ros,3
and Javier León
Departamento de Anatomı́a y Biologı́a Celular (M. E. P., M. A. R.) and
Grupo de Biologı́a Molecular del Cáncer, Departamento de Biologı́a
Molecular y Unidad Asociada al Centro de Investigaciones Biológicas
(CSIC) (M. D. D., J. L.). Universidad de Cantabria, 39011 Santander,
Spain.
Abstract
c-Myc is a transcription factor involved in the control
of cell proliferation, differentiation, and apoptosis, all
basic processes for embryogenesis. To analyze c-Myc
roles in limb development, we overexpressed c-myc in
chick embryos using a retroviral vector. Forced c-myc
expression resulted in enlarged limbs, because of an
increase in cell size not accompanied by modifications
in cell proliferation. However, at later stages, limbs
overexpressing c-myc showed a marked shortening of
their skeletal elements, because of the inhibition of
chondrocyte maturation. c-Myc interfered with
chondrogenesis, independently of the Indian
hedgehog/parathyroid hormone-related protein and
Wnt5a/Wnt5b regulatory loops. c-myc-infected limbs
also exhibited patterning defects, such as extraphalangeal elements and delayed interdigital apoptosis
that occasionally led to interdigital chondrogenesis. In
contrast, c-myc overexpression did not interfere with
other processes, such as muscle differentiation.
Although based on overexpression experiments, our
results suggest that endogenous c-Myc may be
implicated in the control of cell size and skeletal
differentiation during normal limb development.
Introduction
c-Myc is an oncogenic transcription factor of the HLH-LZ
family involved in the control of cell proliferation, differentiation, and apoptosis. All these processes are crucial events
highly regulated during development. In vitro studies have
Received 12/6/01; revised 3/4/02; accepted 3/4/02.
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
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1
Supported by Grants PM98-0151 and FIS 01/1219 (to M. A. R.), PM98109 and FD97-241 (to J. L.), and FIS 01/1129 (to M. D. D.). M. E. P. was
the recipient of a postdoctoral fellowship from the Fundacion Marques de
Valdecilla.
2
M. E. P. and M. D. D. contributed equally to this work.
3
To whom requests for reprints should be addressed, at Departamento
de Anatomia y Biologia Celular, Facultad de Medicina, Universidad de
Cantabria, 39011 Santander, Spain. Phone: 34-942-201933; Fax: 34-942201903; E-mail: [email protected].
demonstrated that c-Myc is required for proliferation of many
cell types, and that promotes cell cycle progression by upregulating the activity of proteins controlling key events in G1
phase, such as cyclin D2, E2F, cyclin E, or cyclin-dependent
kinase 4, while down-regulating the levels or activity of
p21Waf1 and p27Kip1 (reviewed in Refs. 1–5). Consistently,
c-myc-null cells show delayed cell cycle times as compared
with parental cells (6, 7). However, during embryogenesis,
c-Myc appears to be dispensable for cell division because
c-myc-null mice develop ⱕ9.5–10.5 days of gestation (8),
and, therefore, a great deal of cell proliferation occurs in the
absence of c-Myc before that developmental stage. Moreover, during vertebrate development, c-myc shows a dynamic and precise temporo-spatial pattern of expression
that does not parallel the distribution of highly proliferative
areas (9 –12).
Recently, the identification of dMyc, the Drosophila ortholog of c-myc, and the study of its mutations yielded the
discovery of a new role for c-Myc in the control of cell size.
Thus, the size of Drosophila imaginal disc cells is reduced in
the absence of dMyc, whereas it is increased by dMyc overproduction (13). This effect is not specific of the developing
wing and has been reported in other systems, including
human lymphoid cell lines (14), murine B cells (15), human
keratinocytes (16), and mouse hepatocytes (17). The mechanism by which c-Myc increases cell size may be mediated
by its stimulatory effect on protein synthesis (18). Consistently, many of the reported c-Myc target genes are involved
in protein synthesis both at the levels of ribosome biogenesis
(e.g., MrDb rRNA helicase, nucleolin, and ⬃30 ribosomal
proteins) and protein synthesis (e.g., translation initiation factors eIF4E and eIF2 ␣; Refs. 3 and 18 –20).
Another recognized function of c-Myc is the control of cell
death. Overexpression of c-Myc promotes apoptosis when
cells are in suboptimal growth conditions (21, 22). However,
during embryonic development, the pattern of c-myc expression does not correlate with known areas of apoptosis, e.g.,
vertebrate limb development courses with well-defined areas
of programmed cell death (23), but expression of c-myc does
not overlap with any of these areas (24). In addition, experimentally induced cell death, such as that after deprival of the
apical ridge (25), an important source of survival factors
during vertebrate limb development, is not accompanied by
c-myc expression (24).
In this study, we have further investigated the function that
c-Myc plays during vertebrate development. We selected the
avian developing limb as a valuable model in which to overexpress normal chicken c-myc by retroviral vectors. To our
knowledge, this is the first study of c-Myc gain-of-function
during vertebrate limb development. We performed such
studies and found that excess of c-Myc resulted in increased
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c-Myc and Chick Limb Development
cell size and delay in endochondral ossification. Our results
suggest that endogenous c-Myc may be involved in the
control of cell size and skeletal development.
Results
Overexpression of c-myc Resulted in Limb Buds of
Larger Size than Normal. To obtain forced expression of
c-myc, we used a retroviral vector (RCASBP-A) containing
the c-myc coding region (26). Concentrated viral stocks were
injected into the prospective right-wing field of stage 12–14
embryos or directly into the wing or leg buds of stage 17–18
embryos. The infection’s success was assessed by hybridization with an antiviral probe (data not shown) or for c-myc
(inset in Fig. 1A). Endogenous c-myc mRNA was also detected in the described areas of expression, such as somites
(Fig. 1A; Ref. 24). Although early injections resulted frequently in partial infections, with only ⬃40% of the injected
embryos exhibiting adequate targeting of the limb, later injections resulted in a high level of c-myc expression all
across the limb bud of most (95%) infected embryos. The
higher technical difficulty in injecting the prospective limb
field versus the limb bud may explain the lower infection
efficiency of earlier injections. Because the phenotype observed was similar with both types of injections, especially
for the analysis of the skeleton (see below), we mainly performed injections in the limb bud (stages 17–18).
The development of the limb was examined sequentially
after infection with RCAS-c-myc. We found that infected
limbs were enlarged in size than the contralateral noninfected limb (Fig. 1A). This effect was specific for c-myc,
because limbs infected with the same retroviral vector carrying the green fluorescent protein did not show this phenotype (data not shown). Furthermore, the limb is a widely used
system for experiments with retroviral vectors, and this phenotype has not been reported previously. The increase in size
of the c-myc-infected limbs was consistent and clearly discernible but moderate. When the transversal thickness of the
infected and contralateral control wing of the same embryo
was measured and compared (red lines in Fig. 1A), we found
that RCAS-c-myc-infected limbs were ⬃17% thicker than
the control limbs on the average (n ⫽ 5). The paired t test for
the mean difference on thickness between the control and
infected wings led to the rejection of the null hypothesis that
they were equal (P ⫽ 0.022). Infected limbs showed normal
histology, with no signs of edema or other gross alterations
of morphology (data not shown; Figs. 1, B and C and 4). In
situ mRNA hybridization of limb sections revealed that c-myc
overexpression was not confined preferentially to specific
cell types but was rather distributed uniformly through the
infected limb (Fig. 1, B and C; Fig. 4). Specific areas of limb
cells are normally fated to die by apoptosis during limb
development (23, 27). These are well-defined areas positioned mainly to the anterior and posterior margin and in the
interdigits. It is worth noticing that these areas were not
modified by c-myc overexpression (data not shown). In particular, we concentrated our study in the interdigital areas of
cell death that are implicated in separating the individual
digits. As will be explained later, we only found some delay
in the course of the apoptosis process in the wing interdigits.
Fig. 1. Overexpression of c-myc increases limb size but does not modify
cell proliferation. A, cranial view of left (control) and right (infected) wings
hybridized for c-myc showing high levels of c-myc mRNA in the right wing
bud 4 days after infection. The increase in size of the infected limb is
⬃20% when their thickness (red bars) is compared at equivalent transversal levels (indicated by the yellow longitudinal line). Inset, the c-myc
expression of the infected embryo. In B and C, in situ c-myc hybridization
in tissue sections from control (B) and infected (C) limbs shows generalized expression of c-myc in the infected limb. D–G, BrdUrd incorporation
in limbs. Embryos were injected with BrdUrd in vivo as described in
“Materials and Methods,” and immunofluorescence was performed with
anti-BrdUrd-fluorescein antibody in paraffin sections of control (D) and
infected limbs (E). Immunofluorescence was also performed on cytospin
preparations of dissociated cells from control (F) and infected limbs (G).
During subsequent developmental stages, the enlarged
limb phenotype evolved into a thickening and shortening of
the limb, a consequence of cartilage differentiation impairment, described below.
Overexpression of c-myc Increases Cell Size. Limbs of
increased size, the observed phenotype after c-myc infection, could result from an increase in proliferation or an
increase in cell size, both putative c-myc-controlled processes. Thus, we examined the status of proliferation and
cell size in the infected limbs.
Cell Growth & Differentiation
To analyze proliferation, we determined BrdUrd incorporation. The right (infected) and left (control) limbs of infected
embryos injected with BrdUrd in the amniotic sac were processed in parallel for BrdUrd immunofluorescence. The percentage of BrdUrd-positive nuclei in sections of infected and
noninfected limbs appeared similar (Fig. 1, D and E). Furthermore, the percentage of BrdUrd-positive nuclei was also
analyzed in cytospin preparations performed with dissociated cells from infected and noninfected limbs (Fig. 1, F and
G). At least 1000 4⬘,6-diamidino-2-phenylindole-stained nuclei were counted, and the percentage of BrdUrd-positive
nuclei was 10.6 ⫾ 0.71 (mean ⫾ SE; data from eight limbs)
for noninfected limbs and 12 ⫾ 1.18 (mean ⫾ SE; data from
nine limbs) for c-myc-infected limbs. Statistical analysis
showed no significant differences between these data. Thus,
our results indicate that c-Myc overexpression did not significantly modify the rate of proliferation in limb buds.
To analyze cell size, we subjected dissociated cells of
infected and control limbs to flow cytometry and compared
the size of cells in both populations by forward scattering.
For each experiment, the infected limb bud was hemisected
in two longitudinal halves. Half of the infected limb was
dissociated to single-cell level, and cell size was measured
by forward scattering. The remaining of the limb was hybridized for c-myc to confirm the infection (Fig. 2, A–C). In every
case analyzed (eight out of eight), the cells from strongly
infected limbs appeared larger than the cells from comparable normal, noninfected limbs, as assessed by forward scattering (Fig. 2, D and E). Therefore, we concluded that overexpression of c-myc resulted in cells of larger size than
normal.
Because cell size increases during cell cycle progression,
we evaluated the possibility that an accumulation of cells in
the G2 phase attributable to c-Myc overproduction could
also contribute to the observed increase in cell size, as
described (16, 28). To test this, we performed flow-cytometry
analysis of the DNA content of cell populations from infected
and noninfected limbs. We found no differences in cell cycle
distribution between control and c-myc-overexpressing
limbs (Fig. 2F). In human keratinocytes (16) and in mouse
fibroblasts (29), enforced c-Myc expression induces endoreplication, which results in the appearance of multinuclear larger cells. However, we did not detect a higher fraction of polyploid cells in infected limbs (data not shown). As
a whole, our results indicate that the increase in cell size
observed in the c-myc-infected limbs is caused by an increase in the size of the infected cells.
c-myc Misexpression Resulted in Shortening of the
Skeletal Elements. At later stages, the development of the
c-myc-infected limbs was marked by a significant shortening
of the skeletal elements. This phenotype was observed in all
c-myc-infected limbs but with variable degrees of severity.
Representative skeleton patterns of c-myc-infected wings
are shown in Fig. 3, A and B (day 10, stage 36). The cartilage
elements of c-myc-infected wings were shorter and thicker
than normal (the control left wing of Fig. 3A is shown in Fig.
3C for comparison). Interestingly, infected wings showed
minuscule extra cartilage elements at the end of each digital
ray (arrows in Fig. 3, A and B). Very frequently, the first
Fig. 2. Overexpression of c-myc increases cell size. c-myc mRNA expression in embryos infected with RCAS-c-myc (A and B) and in noninfected control embryo (C). Longitudinal half of the wing was dissected out
(arrowheads), and the cells were dissociated as described in “Materials
and Methods.” D, forward scattering of cells of the half wing dissected
from the c-myc-infected embryo shown in A, compared with control
embryo shown in C. E, forward scattering of cells from the half wing
dissected in the c-myc-infected embryo shown in B, compared with
control embryo shown in C. F, analysis of cell cycle distribution. Data
show the fraction of cells in G0-G1, S, and G2-M stages for control
noninfected limbs and c-Myc-overexpressing limbs as determined by flow
cytometry analysis after propidium iodide staining. Bars, SD from 8 (Control) and 13 (c-Myc) independent experiments.
phalange of digit 2 appeared split into two elements, resulting in a digit 2 with three phalanges instead of the normal two
elements (open arrowheads in Fig. 3, A and B). In ⬃20% of
the cases (6 out of 33), an extra cartilage element developed
in the first interdigital space bridging digits 2 and 3 (asterisk
in Fig. 3B). This occurred in the limbs with the stronger
shortening of the cartilage elements that we interpreted as
the limbs with the heavier c-myc infection. Finally, ossification was retarded, and signs of bone formation were not
observed in the cartilage elements of infected wings at a time
in which they were clearly discernible in the control wing
(black arrowheads in Fig. 3, A–C). The sequential analysis of
H&E-stained sections of infected and control wings demonstrated a clear delay in the process of cartilage resorption
and invasion by osteoclast in infected cartilages. A magnified
longitudinal section through the diaphysis of the infected
radius (Fig. 3D) confirmed the total absence of endochondral
bone invasion in clear contrast with the control radius in
which the signs of bone invasion were prominent (asterisks in
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c-Myc and Chick Limb Development
Fig. 3. Skeleton abnormalities induced by c-myc overexpression. A and B, two c-myc-infected wings with shortening and thickening of the skeletal
elements. C, cartilage pattern of the noninfected control limb of the embryo shown in A for comparison. To facilitate comparison, the picture of the control
left wing has been reversed. The extra-distal phalange elements and the appearance of extra joints are indicated in the infected wings by the arrows and
open arrowheads, respectively. The phenotype in B is stronger, with the appearance of an interdigital cartilage (ⴱ). Black arrowheads, the delay in ossification
of the infected wing (A and B) compared with the control wing (C). D and E, magnified H&E-stained sections through the radius dyaphysis (area indicated
by the rectangle in B and C) of infected (D) and control (E) wings. Although the collar bone has formed in both infected and control radius (arrowheads),
the process of cartilage resorption is absent in the infected dyaphysis, whereas it has considerably progressed in the control radius (ⴱ in E). F, scanning
electron micrographs of a control wing and an infected wing to show the delay in the regression of interdigital tissues (arrowhead). In G and H, the pattern
of programmed cell death detected by TUNEL in the third interdigital space of a control wing (G) and an infected (H) wing is similar, except for the temporal
delay. I, skeletal pattern of the leg autopod of a control limb and a c-myc-infected limb. Note the shortening and broadening of the cartilage elements. Arrow,
an extra joint in the second digit of the infected leg. J, skeletal pattern of the leg zeugopod of a control leg and a c-myc-infected leg, showing dramatic
tibia shortening. Arrow, a frequently observed diaphysis bend. Note the longer of the infected leg versus the control (arrowheads). The staining of the skeletal
elements was performed with Victoria blue.
Fig. 3E). The arrowheads in Fig. 3, D and E point to the collar
bone that has formed both in infected and control limbs.
Interdigital cells can form cartilage when diverted from
their normal apoptotic pathway (30). Because c-Myc has
been implicated in apoptosis control, the formation of a
cartilage element in the first interdigital space of RCAS-cmyc-infected wings made us consider the possible interference between c-myc overexpression and cell death. When
the regression of the interdigital tissue was analyzed sequentially with scanning electron microscopy, we found that this
process was retarded in the infected wings compared with
their contralateral control limb (five out of five; arrowheads in
Fig. 3F). The TUNEL4 assay demonstrated comparable patterning and extension of interdigital apoptosis in infected and
control limbs (Fig. 3, G and H), with the only difference that
the time course was delayed in the infected limb (Fig. 3F). We
concluded that overexpression of c-myc delayed but did not
4
The abbreviations used are: TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling; ColX, Collagen X; Ihh, Indian Hedgehog; PTH, parathyroid hormone; PTHrP, parathyroid hormone-related
protein; FGF, fibroblast growth factor; PTHrP-R, parathyroid hormonerelated protein receptor; FGFR, fibroblast growth factor receptor.
impede the apoptosis process and that interdigital cartilages
probably developed only in the cases of extreme delay.
When c-myc was overexpressed in leg buds, the resulting
phenotype was comparable with that described for the
wings, but the shortening of the cartilage elements was even
more dramatic (Fig. 3I for the autopod/foot and Fig. 3J for the
zeugopod/tibia and fibula). The minuscule distal phalanges
typical of the infected wings were never observed in infected
legs. However, the second phalange of leg digit 2 appeared
frequently split into two elements (arrow in Fig. 3I), thus
resulting in a digit 2 with an extra phalangeal element. Finally,
a striking feature of c-myc-infected legs was that the length
of the fibula was comparable with that of the tibia (compare
control with infected leg in Fig. 3J) that frequently exhibited
a diaphysis bend.
c-Myc Misexpression Resulted in Delayed Chondrocyte Differentiation. The most prominent characteristic of
the c-myc-infected wings and legs was the shortening and
broadening of the cartilage elements. Both delay and acceleration of chondrocyte differentiation may lead to this phenotype (31). The skeleton of the limbs forms by endochondral
ossification (32). Each bone develops from a cartilage element in which terminally differentiated chondrocytes are re-
Cell Growth & Differentiation
placed by bone. The differentiation of the chondrocytes is a
process tightly regulated both temporary and spatially.
Chondrocyte differentiation advances centrifugally from the
center to the ends of the cartilage element, with the chondrocytes passing sequentially through the proliferative, prehypertrophic, and then hypertrophic state. Several molecular
markers have been identified, e.g., hypertrophic chondrocytes are marked by their expression of ColX, whereas prehypertrophic cells specifically express Ihh and PTH/PTHrP
receptor (33).
We first verified c-myc overexpression by in situ hybridization, and only the cases with extensive c-myc expression
were considered. To examine chondrogenic differentiation in
the c-myc-infected limbs, we analyzed the expression patterns of genes considered markers for different phases of
chondrocyte differentiation. By day 7 (stage 30 –31), the expression of ColX detected the presence of terminally differentiated chondrocytes in the central region of the zeugopod
cartilages of the control limb (Fig. 4A). However, its expression was undetectable in the equivalent infected cartilages of
the contralateral control limb (Fig. 4A). One day later (stage
33), expression of ColX had normally progressed in the control cartilage. However, in the equivalent cartilage element,
only very few cells expressed ColX in the infected limb (Fig.
4B). This result indicates a clear delay in the process of
cartilage differentiation and prompted us to examine the
prehypertrophic chondrocytes characterized normally by
their expression of Ihh. The analysis of Ihh expression in
alternate sections of the same infected cartilages analyzed
for ColX showed that the domain of expression of Ihh was
comparable in infected and noninfected limbs at stage
30 –31 (Fig. 4C). The Ihh area of expression was reduced
slightly in infected limbs, as corresponding to the shortening
of the infected cartilage element. At stage 33, Ihh domain of
expression had progressed to two separate areas of expression in the control cartilage (Fig. 4D), whereas it continued as
a single central domain in the infected cartilage (Fig. 4D). This
result is consistent with the strong reduction observed in
ColX expression in infected limbs. Contiguous sections hybridized for c-myc show the elevated c-myc misexpression
(Fig. 4, E and F). Taken together, our results indicate a
negative effect of c-Myc in the progression from prehypertrophic to hypertrophic chondrocyte differentiation.
Ihh is a member of the conserved Hedgehog family of
transcription factors that has important functions in cartilage
development (33, 34). Its expression is restricted to the prehypertrophic chondrocytes from where it signals to upregulate PTHrP expression in the periarticular region. Signaling of PTHrP through its receptor PTH/PTHrP-R expressed in
the prehypertrophic chondrocytes down-regulates Ihh expression, establishing a negative feedback loop that regulates the onset to the hypertrophic transition. In our c-mycinfected limbs, we found that the expression of PTH/
PTHrP-R in the prehypertrophic chondrocytes, as well as the
PTHrP in the periarticular cartilage (Fig. 4G), was normal.
This indicates that the Ihh/PTHrP regulatory loop was not
affected by c-Myc overexpression and reinforced the notion
that the c-Myc-dependent block in the chondrogenic maturation was subsequent to the prehypertrophic stage.
Fig. 4. Cartilage differentiation is delayed in c-myc-infected limbs. A–F,
in situ hybridization with the indicated probes. The infected limb is always
shown on the right, and the stage is indicated at the top of the figure. A,
C, and E, consecutive serial sections from the same pair of stage 30 –31
infected limb and left control limb. B, D, and F, consecutive serial sections.
A, undetectable ColX expression in the infected limb, which is clearly
detectable in the control limb. B, a very reduced domain of ColX in the
infected limb, compared with the control at stage 33. In C, the domain of
expression of Ihh is reduced in the infected limb, compared with the
normal one. In D, at stage 33, the domain of expression of Ihh in the
infected limb is still continuous, indicating a marked delay compared with
the control. E and F, contiguous sections hybridized for c-myc to confirm
the elevated misexpression. G, three contiguous longitudinal sections
from an infected limb, showing that elevated levels of c-myc expression
(left) does not affect the pattern of expression of the PTHrP mRNA (middle)
or PTH/PTHrP-R (right). H and I, contiguous sections through the control
limb (left) and the infected (right) limb, showing normal patterns of Wnt5a
and Wnt5b expression.
An additional level of regulation depending on Wnt5a signaling has been recently identified (31). It has been proposed
that Wnt5a signaling from the perichondrium, probably mediated by Wnt5b, negatively controls the transition from the
prehypertrophic to the hypertrophic state. Interestingly,
overexpression of Wnt5a gives a similar phenotype to that
observed here after c-myc overexpression (31). Thus, we
wanted to analyze if the Wnt5a pathway was modified in our
c-myc-infected limbs. However, the expression of Wnt5a in
the perichondrium and Wnt5b in the prehypertrofic chondro-
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c-Myc and Chick Limb Development
Discussion
Fig. 5. c-Myc does not interfere with normal myogenic differentiation. A,
dorsal view of a stage 20 control embryo hybridized for c-myc, showing
the normal pattern of expression in the dorsal premuscular mass of the
developing wings. B, dorsal view of a stage 27 control wing hybridized for
c-myc, showing the normal pattern of expression in the limb’s developing
muscular components. C, control (left) and c-myc-infected (right) wings
hybridized for MyoD. Note the thickened phenotype of the infected wing
and that the forced c-myc expression does not modify the normal pattern
of MyoD expression. In situ hybridization of contiguous sections of control
(left) and infected (right) limbs for MyoD (D) and Myogenin (E), showing that
the infection does not alter the normal pattern of expression. The expression of c-myc is shown in Fig. 4F.
cytes of infected limb was similar to normal (Fig. 4, H and I),
indicating that c-Myc does not act through the Wnt5a pathway.
Thus, we can conclude that the forced expression of cmyc results in a marked delay of chondrocyte maturation,
without interfering with the Ihh/PTHrP regulatory loop, and
independent of the Wnt5a pathway.
Muscular Differentiation in the Infected Limbs
Progresses Normally. During myogenesis, c-myc is expressed in proliferating myoblasts (Fig. 5, A and B), but
expression decreases when these cells differentiate (35).
Previous studies have indicated that forced c-myc expression could inhibit myogenic differentiation (36 –38). Our
model makes it possible to analyze myogenic differentiation
in vivo under conditions of forced c-myc expression. As
indicated above, c-myc is highly expressed in the muscular
component of the developing limb, as shown for stages 20
(Fig. 5A) and 27 (Fig. 5B; Ref. 24). However, overexpression
of c-myc did not alter the pattern of expression of MyoD,
which remained similar in control and infected limbs (Fig. 5,
C and D). Note the positive phenotype in the infected limb,
which appears thicker than the contralateral control (Fig. 5C).
The expression of myogenin, a member of the MyoD family
with important functions controlling muscular differentiation,
also occurred in a normal pattern in the infected limb (Fig.
5E). Our results thus indicate that enforced c-myc expression
in vivo does not interfere with myogenic differentiation, which
appears to progress normally.
Here we report on the consequences of the overexpression
of the proto-oncogene c-myc during the development of the
chick limb bud. Our results show that c-Myc: (a) increases
the size of the embryonic limb; and (b) interferes with skeletal
differentiation, resulting in elements shorter and thicker than
normal. These effects occur without affecting the general
structure of the growing limb and, particularly, myogenic
differentiation. Interestingly, RCAS-c-myc-infected limbs, albeit enlarged, showed no neoplastic growth in any infected
embryo, despite the high level of ectopic c-Myc expression.
This was an expected result since c-myc overexpression has
only been demonstrated to be oncogenic in lymphoid cells.
It also supports the idea that c-Myc overexpression per se is
insufficient for tumorogenesis and is consistent with findings
from c-myc transgenic mice, in which tumors appear after a
long latency period and are monoclonal (1). Finally, it must be
noted that we expressed the chicken wild-type c-myc gene,
which is a less potent inducer of proliferation of chicken cells
than v-myc (26).
Our analysis of cell proliferation and cell size indicated that
the enlarged size of c-myc-infected limbs is a result, at least
in part, of increased cell size. A number of reports have
established that c-Myc is able to increase cell size in different
cell culture systems and in vivo models (13–15, 17). We have
now shown that c-Myc increases cell size during vertebrate
limb development. c-Myc-mediated increase in limb size is
consistent with the smaller size of c-myc⫺/⫺ embryos, compared with wild-type or heterozygous embryos (8). The results are also consistent with the phenotype of mad1 transgenic mice, where the transgene is expressed in most
tissues under the control of the ␤-actin promoter. In this
model, the ectopic expression of Mad1, a biological antagonist of c-Myc, results in perinatal lethality and dwarfism (39).
Recently, it has been reported that the reduction of c-myc
expression in mice carrying hypomorphic c-myc alleles results in overall decrease in body size (40). Although the cell
size was not specifically analyzed in developing limbs, the
effect in lymphoid organs was attributed to decreased cell
number rather than reduced cell size (40). Thus, it is conceivable that c-Myc-mediated increase of cell size in vertebrates operates in particular cell types and developmental
stages.
In other models, c-Myc induces endoreplication, accompanied by the appearance of multinuclear larger cells (16,
29), or accumulation of cells in G2 phase, which are larger
than G1 cells (16, 28). However, we did not find a higher
fraction of multinucleated cells in c-myc-expressing limbs,
compared with noninfected limbs, as determined by DNA
flow cytometry. In addition, we did not detect an increased
fraction of cells in the G2 phase of cell cycle in the c-mycoverexpressing cells, indicating that changes in cell cycle
were not contributing to the cell size increase observed in
infected limbs.
In addition, our observation that c-myc misexpression did
not significantly modify proliferation in chicken growing limb
buds concurs with previous reports showing that c-Myc
overexpression in chicken lymphocyte precursors affects differentiation and migration, rather than proliferation and ap-
Cell Growth & Differentiation
Fig. 6. c-Myc function in chondrocyte differentiation. Schematic representation of a cartilage element showing the progression of chondrocyte
differentiation through the steps indicated on the left of the scheme. The
corresponding molecular markers for each zone of differentiation are
indicated on the right side of the skeletal element. Our results indicate that
c-Myc negatively regulates the progression from the prehypertrophic to
hypertrophic state. Several factors known to participate in the maturation
of the chondrocytes are indicated on the left of the figure (see text for
details and references), but we do not intend to be exhaustive.
optosis (41). Similarly, the normal areas of apoptosis in developing limbs were not affected in RCAS-c-myc-infected
limbs. Neither syndactyly, caused by reduced apoptosis of
the interdigital tissue, nor signs of increased cell death were
observed in heavily infected legs. In wings, we observed a
delay in the normal process of apoptosis, although it was
patterned normally. A conclusion of our study is that overexpression of c-Myc during limb development does not significantly interfere with the normal processes of cell death.
At later developmental stages, the enlarged size of c-Mycinfected limbs was substituted by a clear limb shortening,
attributable to impaired chondrocyte maturation. Recent
work has shown that chondrocyte maturation is a highly
regulated process, in which the FGF, bone morphogenetic
protein, Wnt, ⌬/Notch, and Ihh/PTHrP signaling pathways
have been shown to participate (Refs. 31, 33, 42, and 43; Fig.
6). Here, we demonstrate that c-Myc negatively regulates the
progression from prehypertrophic to hypertrophic chondrocyte maturation (Fig. 6), a step that was strongly delayed in
infected limbs, yielding short and thick skeletal elements.
c-Myc overexpression does not appear to interfere with progression of chondrocytes from the proliferating to the prehypertrophic state, a process regulated by the Ihh/PTHrP
regulatory loop (33), because the expression of Ihh, PTHrP,
and PTH/PTHrP-R is normal. Wnt5a has been shown recently to regulate chondrocyte maturation at the same level
as c-Myc (31). However, since misexpression of c-myc does
not influence the normal pattern of expression of Wnt5a and
Wnt5b, it is likely that c-Myc action is also independent of
this regulatory pathway.
Deregulated FGF signaling caused by overexpression of
FGF or mutations of FGFRs results in impairment of bone
development and shortening of the appendicular skeleton,
the basis of Apert syndrome and other dwarfisms (reviewed
in Ref. 42). These phenotypes are similar to the c-myc overexpression phenotype described here and raise the question
of a possible interaction between FGF and c-Myc pathways.
However, several observations do not support this hypothesis: (a) proliferation is inhibited by FGF signaling, whereas it
is not modified appreciably in RCAS-c-myc-infected limbs;
(b) the pattern of FGFR3 expression in c-myc-overexpressing limbs was normal (data not shown); and (c) although
overexpression of FGF results in a failure of joint formation
(44), our c-myc-overexpressing limbs showed extra joint formation. It is tempting to speculate that both the formation of
minuscule distal cartilages and the extra joints are the result
of the block of normal cartilage differentiation we have found
after c-myc overexpression.
Our results are consistent with previous work showing that
Myc prevents differentiation of cultured chondrocytes into
hypertrophic and calcifying cartilage (45, 46). We have extended this observation to an in vivo model. Because c-myc
is expressed by proliferating chicken and mouse chondrocytes (11, 47– 49), we propose a role for endogenous c-Myc
in the modulation of chondrogenic maturation. If endogenous c-Myc expression prevents passage of chondrocytes
to the hypertrophic state during normal development, the
expression of c-myc should be switched off before the chondrocytes reach this step. Interestingly down-regulation of
c-myc expression has been shown to occur at this check
point (48). Thus, c-myc down-regulation of expression may
be a prerequisite for terminal differentiation of chondrocytes,
adding a different cell-autonomous level of regulation to this
process. The inhibition of differentiation induced by c-Myc in
other culture models has been explained by the proliferative
stimulation mediated by c-Myc (reviewed in Refs. 1 and 50).
Although we did not detect increased DNA synthesis in the
chondrocytes of infected limbs, we cannot discard that a
small increase in BrdUrd-positive cells, under our detection
level, could have a significant effect on the development of
the limb skeleton, as described in a Mad1 transgenic model
(39). Given the high nonphysiologic levels of c-myc expression achieved by retroviral infection and the large number of
c-Myc target genes (20, 51–53), there are multiple putative
mechanisms by which c-Myc could cause the phenotypes
described in this work.
In the present study, we have identified the level at which
c-Myc interferes with endochondral differentiation, but its
precise mechanisms of action merit future investigation. Our
work clearly indicates that c-Myc plays different roles during
vertebrate avian development, including control of cell size
and skeletal development.
Materials and Methods
Embryos and Viral Infections. Pathogen-free eggs (Intervet, Spain and Lohmann, Germany) were routinely incubated, opened, and staged according to (54). The replicant
competent RCASBP(A)-c-myc retroviral vector (26) was
kindly provided by Stephen Hughes (National Cancer Institute, Bethesda, MD). Transfection of retroviral constructs
and production of virus concentrated stocks was carried out
as described previously (55). Injections were performed at
two different stages: (a) early injections in the prospective
191
192
c-Myc and Chick Limb Development
wing field of embryos stages 12–14; and (b) late injections
into the limb buds (wing and leg) of stage 17–18 embryos.
Flow Cytometry of Limb Cells. Four days after infection,
the embryos were removed from the eggs and placed in
PBS, where a longitudinal half of the infected right wing bud
was dissected out. The whole embryo with half of the right
wing was fixed in 4% paraformaldehyde and processed for
whole mount in situ hybridization with the c-myc probe to
confirm infection. The isolated longitudinal half of the right
wing was dissociated to single-cell level as described (56).
Then the cellular suspension was filtered through a 100-␮m
membrane, and cell suspension was subjected to flow cytometry analysis in a Becton Dickinson FacsCalibur cytometer. Forward scattering was measured and analyzed with
Cell Quest software. For the analysis of cell cycle stages,
dissociated cells were fixed in ethanol and stained with propidium iodide as described (57). Cell cycle distributions were
estimated using ModFit software.
BrdUrd Incorporation into Cellular DNA. Four days after
infection, 250 ␮l of 5-bromo-2⬘-deoxyuridine (Roche; 3
mg/ml in PBS) were injected into the amniotic cavity. After
1 h, the embryos were fixed in 4% paraformaldehyde, and
paraffin sections were prepared. Alternatively, the wing buds
were dissected out, the cells were dissociated as above, and
cytospins were prepared and fixed in 4% paraformaldehyde.
Immunofluorescence was performed by standard procedures using anti-BrdUrd-fluorescein mouse monoclonal antibody (Roche). Cell nuclei were counterstained with 4⬘,6diamidino-2-phenylindole.
In Situ Hybridization and Cartilage Staining. Hybridization with digoxigenin-labeled riboprobes was performed on
whole mount and tissue sections as described (58). Hybridization with 35S-labeled riboprobes was performed (59). The
probes used were: c-myc (24), MyoD (60), Myogenin (61), Ihh,
Col X, PTHrP and PTH/PTHrP-R (33), and Wnt5a and Wnt5b
(31).
To analyze the skeleton pattern, the embryos were allowed
to develop to the desired stage, fixed in 10% formalin,
stained with Victoria blue, and cleared in methyl salicylate.
When necessary, the specimens were embedded in paraffin,
sectioned, and routinely stained with H&E.
TUNEL Assay. For the analysis of apoptosis, in situ detection of DNA fragmentation was performed using terminal
transferase to incorporate fluorescein-dUTP (In Situ Cell
Death Detection Kit, Fluorescein; Roche), following the manufacturer’s instructions.
Scanning Electron Microscopy. The specimens were
fixed in 2.5% glutaraldehyde in cacodylate buffer at pH 7.2,
dehydrated in acetone, dried by the critical-point method,
and sputtered with gold. Observations were made using a
Jeol T-100 microscope.
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