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Cell Growth & Differentiation Vol. 13, 185-193, April 2002
© 2002 American Association for Cancer Research

c-Myc Overexpression Increases Cell Size and Impairs Cartilage Differentiation during Chick Limb Development1

M. Elisa Piedra2, M. Dolores Delgado, Maria A. Ros3 and Javier León

Departamento de Anatomía y Biología Celular (M. E. P., M. A. R.) and Grupo de Biología Molecular del Cáncer, Departamento de Biología Molecular y Unidad Asociada al Centro de Investigaciones Biológicas (CSIC) (M. D. D., J. L.). Universidad de Cantabria, 39011 Santander, Spain.


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
c-Myc is a transcription factor involved in the control ofcell proliferation, differentiation, and apoptosis, all basicprocesses 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 extra-phalangeal 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
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
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 demonstrated that c-Myc is required for proliferation of many cell types, and that promotes cell cycle progression by up-regulating 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, 2, 3, 4, 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 <=9.5–10.5 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, 10, 11, 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 {alpha}; Refs. 3 and 18, 19, 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 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
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
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. 1ACitation ). Endogenous c-myc mRNA was also detected in the described areas of expression, such as somites (Fig. 1ACitation ; 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).



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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).

 
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)Citation . 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. 1ACitation ), 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 CCitation and 4Citation ). 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 CCitation ; Fig. 4Citation ). 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.



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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.

 
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.

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)Citation . 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)Citation . 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)Citation . 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)Citation . Therefore, we concluded that overexpression of c-myc resulted in cells of larger size than normal.



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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.

 
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)Citation . 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 BCitation (day 10, stage 36). The cartilage elements of c-myc-infected wings were shorter and thicker than normal (the control left wing of Fig. 3ACitation is shown in Fig. 3CCitation for comparison). Interestingly, infected wings showed minuscule extra cartilage elements at the end of each digital ray (arrowsin Fig. 3, A and BCitation ). Very frequently, the first 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 BCitation ). 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. 3BCitation ). 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–CCitation ). 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)Citation 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 Fig. 3ECitation ). The arrowheadsin Fig. 3, D and ECitation point to the collar bone that has formed both in infected and control limbs.



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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 (*). 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 (* 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.

 
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-c-myc-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. 3FCitation ). The TUNEL4 assay demonstrated comparable patterning and extension of interdigital apoptosis in infected and control limbs (Fig. 3, G and H)Citation , with the only difference that the time course was delayed in the infected limb (Fig. 3F)Citation . We concluded that overexpression of c-myc delayed but did not 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. 3ICitation for the autopod/foot and Fig. 3JCitation 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 (arrowin Fig. 3ICitation ), 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. 3JCitation ) 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 replaced 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)Citation . However, its expression was undetectable in the equivalent infected cartilages of the contralateral control limb (Fig. 4A)Citation . 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)Citation . 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)Citation . 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)Citation , whereas it continued as a single central domain in the infected cartilage (Fig. 4D)Citation . 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)Citation . 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 up-regulate 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-myc-infected 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)Citation , 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.

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 chondrocytes of infected limb was similar to normal (Fig. 4, H and I)Citation , indicating that c-Myc does not act through the Wnt5a pathway.

Thus, we can conclude that the forced expression of c-myc 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)Citation , but expression decreases when these cells differentiate (35) . Previous studies have indicated that forced c-myc expression could inhibit myogenic differentiation (36, 37, 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)Citation and 27 (Fig. 5BCitation ; 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)Citation . Note the positive phenotype in the infected limb, which appears thicker than the contralateral control (Fig. 5C)Citation . 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)Citation . Our results thus indicate that enforced c-myc expression in vivo does not interfere with myogenic differentiation, which appears to progress normally.



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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. 4FCitation .

 

    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
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, 14, 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 ß-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-myc-overexpressing 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 apoptosis (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-Myc-infected 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, {Delta}/Notch, and Ihh/PTHrP signaling pathways have been shown to participate (Refs. 31 , 33 , 42 , and 43 ; Fig. 6Citation ). Here, we demonstrate that c-Myc negatively regulates the progression from prehypertrophic to hypertrophic chondrocyte maturation (Fig. 6)Citation , 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.



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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.

 
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, 48, 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, 52, 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
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
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 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-µm 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 µl 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',6-diamidino-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.


    Acknowledgments
 
We thank Stephen Hughes for RCAS-myc viral construct and Intervet Laboratories S. A., especially Q. C. Department, for the pathogen-free eggs. We thank Françoise Dieterlen-Lievre, Andrew MacMahon, Clifford J. Tabin, and Andrea Vortkam for the probes used in this work and Andrei Tikhonenko for useful comments. We also thank Marisa Junco for her excellent technical assistance.


    Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by Grants PM98-0151 and FIS 01/1219 (to M. A. R.), PM98-109 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. Back

2 M. E. P. and M. D. D. contributed equally to this work. Back

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: 34-942-201903; E-mail: rosm{at}unican.es Back

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 hormone-related protein receptor; FGFR, fibroblast growth factor receptor. Back

Received for publication 12/ 6/01. Revision received 3/ 4/02. Accepted for publication 3/ 4/02.


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