Proc. Natl. Acad. Sci. USA Vol. 92, pp. 2254-2258, March 1995 Developmental Biology

Myoblast differentiation during mammalian somitogenesis is dependent upon a community effect G. Cossu*t1, R. KELLY*, S. DI DONNAt, E. VIVARELLIt, AND M. BUCKINGHAM* *Department of Molecular Biology, Pasteur Institute, Centre National de la Recherche Scientifique URS67, 25 rue du Dr. Roux, 75724 Paris Cedex 15, France; and tIstituto Pasteur-Cenci Bolognetti, Institute of Histology and General Embryology, University of Rome "La Sapienza," Via A. Scarpa 14, 00161 Rome, Italy Communicated by J. B. Gurdon, Wellcome/CRC Institute, Cambridge, United Kingdom, November 22, 1994 (received for review July 12, 1994)

ABSTRACT The differentiation potential of early mam- neural tube) in inducing myogenesis in paraxial mesodermal malian myogenic cells was tested under clonal culture condi- cells (5-10). It is also unknown whether and when a commu- tions. Cells were isolated from paraxial mesoderm and limb nity effect is necessary for mammalian mesodermal cells to buds oftransgenic mouse embryos at 9.5 days after conception undergo skeletal muscle differentiation. We addressed this and grown in culture at clonal density either on collagen- question by using transgenic mice carrying the bacterial gene coated dishes or on various feeder cell layers. The transgene encoding 13-galactosidase with a nuclear localization signal used contained a reporter gene encoding j3-galactosidase with (nlacZ) under the control of muscle specific regulatory se- a nuclear localization signal under the control of regulatory quences (11). In these mice, cells which have initiated muscle sequences from the gene for fast myosin light chain 3, so that differentiation are 13-galactosidase-positive (f3-gal+), allowing 13-galactosidase staining indicated the presence of differenti- reconstitution experiments with ortho- or heterotopic and ated muscle cells. After 5 days in culture, the number and size ortho- or heterochronic nontransgenic tissues from the same of 13-galactosidase-positive (.8-gal+) clones were recorded. species. Cells isolated from somites I-V (the last five somites to have In this paper we report a community effect for mammalian formed) or from unsegmented paraxial mesoderm did not give myogenesis, which occurs during the initial stages of somito- rise to any j8-gal+ clones. Cells isolated from somites VI-X or genesis, and we discuss these results in relation to the require- from the forelimb bud gave rise to .8-gal+ clones, but only on ment for inductive signals from the neural tube. feeder cells. Cells from somites XI or older gave rise to f8-gal+ clones independently of the substrate. However, when cells MATERIALS AND METHODS isolated from unsegmented paraxial mesoderm or somites I-V were cultured with nontransgenic cells from the trunk (in- Transgenic Mouse Lines. The MLC3F-nlacZ construct cluding neural tube and notochord), differentiation occurred contains 2 kb of the mouse fast myosin light chain 3 (MLC3F) on condition that the cells were in a three-dimensional ag- upstream sequence, including the promoter, with 1 kb of gregate, even though their specific position in the somite had sequence downstream of the transcription initiation site and a been lost. By culturing explants ranging in size from 1 to < 100 3' muscle-specific enhancer from this gene, fused to an nlacZ- cells in the presence of an inhibitor of cell division, we simian virus 40 polyadenylylation sequence. In two independent determined that a minimal number of 30-40 cells is required transgenic lines the nlacZ reporter gene is strongly expressed in for mesodermal cells to differentiate. skeletal muscle from 9 days of development (11). Heterozygous transgenic males were crossed with CD1 outbred female mice. The mechanisms by which mesoderm is induced and muscle Embryos were dated by taking 0.5 day postcoitus (dpc) as the day differentiation is initiated are still poorly understood. Work of the vaginal plug. Mice with nlacZ targeted by homologous mainly carried out with Xenopus embryos has implicated recombination to the vimentin locus were generated and kindly members of the fibroblast growth factor and transforming made available by E. Colucci-Guyon and C. Babinet (12). growth factor f families as inductive molecules released by the Cell Cultures. MLC3F-nlacZ transgenic mouse embryos, endoderm and capable of inducing mesodermal gene expres- ranging in age from 20 to 25 somites (9.5 dpc) were isolated in sion in competent ectodermal cells (1). phosphate-buffered saline. The hearts, where the transgene is Furthermore, in order to respond to inductive signals, also expressed (11), were stained for 13-galactosidase activity competent cells must be surrounded by similar cells (2). This and 13-gall and 3-gal- embryos were pooled separately. The phenomenon, the "community effect," has been described in trunk, initially comprising axial structures, including noto- the amphibian embryo as a prerequisite for mesodermal cells chord and neural tube as well as paraxial mesoderm, was to undergo differentiation into skeletal muscle (3) and into dissected into separate blocks of 5 somites (namely I-V, VI-X, other mesodermal derivatives such as notochord (4). During and XI-XV, somite I being the most recently formed caudal gastrulation, single cells isolated from areas fated to give rise somite). The unsegmented paraxial mesoderm (UPM) and the to muscle and transplanted to a different environment will fail forelimb bud (FLB) were also dissected. The tissues were then to differentiate into muscle, whereas groups of cells similarly digested with 0.1% collagenase/0.1% dispase for 5 min at transplanted will do so. At the end of gastrulation mesodermal 30°C, washed in complete medium (see below), and then gently cells no longer require such a community effect and will pipetted through a siliconized capillary to obtain a single-cell terminally differentiate independently oftheir location and the suspension. Where indicated, the neural tube-notochord com- type of neighboring cell. At this point the cells are assumed to plex was separated from somites by treatment with 0.25% be irreversibly committed to differentiation. pancreatin/0.1% trypsin for 5 min at 4°C. In this case, gentle In the case of higher vertebrates, virtually nothing is known pipetting of the digested structures also resulted in a single-cell about the induction of mesoderm. Several studies, however, suspension. About 80% of isolated cells were viable at the time have suggested a role for axial structures (i.e., notochord and Abbreviations: UPM, unsegmented paraxial mesoderm; MLC3F, fast myosin light chain 3; FLB, forelimb bud; dpc, day(s) postcoitus; f-gal+, The publication costs of this article were defrayed in part by page charge ,B-galactosidase-positive; nlacZ, gene encoding ,B-galactosidase with a payment. This article must therefore be hereby marked "advertisement" in nuclear localization signal. accordance with 18 U.S.C. §1734 solely to indicate this fact. 4To whom reprint requests should be addressed. 2254 Downloaded by guest on September 26, 2021 Developmental Biology: Cossu et aL Proc. Natl. Acad ScL USA 92 (1995) 2255 of plating, as judged by trypan blue exclusion. Approximately (paraxial mesoderm and limb buds) of 9.5-dpc mouse embryos 10 cells from MLC3F-nlacZ transgenic mouse embryos were carrying the nlacZ reporter gene under the control of regu- plated in 5 ml of medium in collagen-coated 60-mm dishes, latory regions of the MLC3F gene. After 3 days in standard either without a feeder layer or with growing or confluent high-density culture, staining for j3-galactosidase revealed 10T½/2 fibroblasts, growing or differentiated C2C7 myogenic many 13-gall nuclei in mononucleated cells and oligonucleated cells, differentiated BC3H myocytes, or confluent embryonic myotubes which were also stained by MF20, a monoclonal cells from the trunk of nontransgenic siblings. antibody that recognizes sarcomeric myosin heavy chain. Thus In experiments to analyze the capacity for differentiation in this histochemical stain reliably identifies differentiated mus- three-dimensional structures, 10 transgenic cells isolated cle cells from these mice. from UPM or somites I-V were mixed with 105 nontransgenic cells (either similar cells from nontransgenic siblings, or 10T1/2 To establish when a single somitic cell acquires the ability to or C2C7 cells), pelleted in a 15-ml Falcon tube, and cultured undergo terminal differentiation independently from signals for 3 days as an aggregate (13). In some of the experiments, derived from surrounding cells, we dissected blocks of 5 axial structures were retained with UPM or somites in the somites (I-V, VI-X, and XI-XV), UPM, and FLB from trunk, as specified in the text. 9.5-dpc MLC3F-nlacZ embryos (at a stage of 20-25 somites) In experiments to determine the minimum number of cells and dissociated these tissues into a single-cell suspension. Cells required for the community effect, tissues were mechanically (104) were plated on collagen-coated 60-mm dishes or on a dissociated by gently pipetting through the yellow tip of a feeder layer prepared from cell lines (10T/2 embryonic fibro- Gilson pipette to produce a suspension containing single cells blasts, C2C7 muscle cells, or BC3H myocytes) or from primary and clusters ranging up to 100-200 cells. This suspension was cells derived from the trunk region of nontransgenic embryos plated in complete medium on collagen-coated 60-mm dishes. (which includes UPM, somites, neural tube, and notochord). It was difficult to count cell number at the time of plating; After 5 days of culture, the cells were stained for 13-galacto- hence each suspension was plated at 1:3 successive dilutions sidase activity and the number and size of positive clones were and only dishes containing about 50 adherent cells or clusters recorded (Fig. 1 and Table 1). Cells isolated from UPM or 2 hr after plating were chosen for further analysis. Half of these somites I-V did not give rise to differentiated cells under any cultures were treated for 2 hr with mitomycin C (5 jg/ml), of the conditions tested, even when plated on primary embry- washed five times, and incubated in complete medium. onic cells, where some single mesodermal cells must come into All cultures were grown in RPMI 1640 medium (GIBCO) close contact with neuronal or notochordal cells. This suggests supplemented with 10% fetal bovine serum (Flow Laborato- that at low density, single mesodermal cells fail to respond to ries), 300 ,uM 2-mercaptoethanol, and gentamicin (50 ,ug/ml). inductive from axial structures (5-10). In contrast to At the times indicated, cultures and pellets were fixed and signals stained for f3-galactosidase activity (14) and/or incubated with these results, cells isolated from somites VI-X and from FLB various antibodies. After staining, pellets were embedded in differentiated at very low frequency on collagen but gave rise 7% gelatin/15% sucrose for sectioning. to many muscle clones on feeder layers. Cells isolated from Immunocytochemistry. Immunocytochemistry on tissue somites XI-XV differentiated as well on collagen as on feeder sections and cultured cells was carried out with the MF20 layers, thus indicating that, by this stage, a cell-autonomous anti-myosin heavy chain and anti-,3III-tubulin monoclonal capacity for terminal differentiation has been acquired. antibodies and a rabbit anti-sarcomeric myosin polyclonal That single cells from somites I-V or from UPM failed to antibody (15). give rise to myogenic clones may be due to the fact that cells die once dissociated from tissue. To address this point, we isolated and cloned, under identical conditions, cells from a RESULTS transgenic mouse line with nlacZ targeted to the vimentin Myogenic Differentiation of Mesodermal Cells Under locus. In this case, most mesodermal cell types express the Clonal Conditions. Cells were isolated from myogenic areas reporter gene in vitro, allowing clones to be identified on

D FIG. 1. Examples of clones, grown on feeder layers for 5 days, of cells isolated from somites VI-X of 9.5-dpc MLC3F-nlacZ mouse embryos (A-C) or from UPM of vimentin-nlacZ embryos (D) stained for ,3-galactosidase activity. Downloaded by guest on September 26, 2021 2256 Developmental Biology: Cossu et al. Proc. NatL Acad ScL USA 92 (1995) Table 1. Number and size of myogenic clones (f3-gal+) from from axial structures is required, in addition to a community paraxial mesoderm and limb buds of 9.5-dpc mouse embryos effect, we performed a second set of experiments. This time VI-X XI-XV FLB aggregation was carried out with a 10-fold excess of cells isolated from the trunk (UPM or somites together with neural Feederlayer A B C A B C A B C tube and notochord) of nontransgenic embryos, from an None 2 1 1.0 69 8 1.7 3 1 1.0 equivalent rostrocaudal level. Many positive cells were de- 10T½/2 sparse 49 10 1.9 98 10 2.2 72 5 1.8 tected in pellets obtained by reaggregation of cells from UPM 10T/2 confluent 48 8 2.4 ND 143 10 3.2 with embryonic cells from the trunk (Fig. 2B). Axial structures C2C7 myoblasts 28 12 2.5 ND 19 5 2.5 also influenced differentiation of cells from somites I-V: C2C7 myotubes 22 9 2.9 74 11 3.2 11 6 3.0 pellets aggregated with cells from the trunk gave about twice BC3H myocytes 60 18 3.7 101 16 3.8 45 8 3.2 as many I3-gall cells as pellets aggregated with cells from Embryonic cells 15 8 3.2 ND 18 4 2.0 somites alone (data not shown). UPM and the last five somites formed (I-V) gave no clones; VI-X The fact that we did not observe any differentiation when and XI-XV: more mature somites, counting from the last formed isolated cells from UPM or early somites were cultivated with somite (16). A, total number of clones recorded in three separate cells from the trunk, which includes axial structures, might experiments; B, maximal number of 3-gal+ cells per clone; C, average have been due to loss of inductive capacity of the latter when number of cells per clone; ND, not done. Embryonic cells were present as dissociated cells. To test this possibility, neural tube obtained from the trunk (paraxial and axial structures) of similarly and notochord cells from nontransgenic embryos, since they aged (18-25 somites) nontransgenic embryos. do not survive without a cellular support, were plated at low feeder layers independently of their differentiation into mus- density on 10T/2 fibroblasts. Isolated cells or aggregates of cle. In contrast to our observations with MLC3F transgenic UPM from MLC3F-nlsZ transgenic mouse embryos were embryos, many }3-gall clones from UPM and from somites I-V plated on these cultures. Under these conditions, the isolated of these embryos were observed on feeder layers (Fig. 1D); the neural cells induced differentiation after 5 days in aggregated number and size of clones increased in cultures established UPM but not in isolated UPM cells (data not shown). from more mature somites. Thus, mesodermal cells do not die From these experiments it appears that cells from early upon dissociation into a single-cell suspension and can even- somites or UPM need to be surrounded by similar cells in a tually give rise to clonal progeny in vitro. three-dimensional structure in order to differentiate but that Myogenic Differentiation of Mesodermal Cells in Reconsti- cells do not need to maintain a precise position within such a tuted Three-Dimensional Structures. The experiments re- structure, since the aggregates are disorganized; moreover, it ported above do not rule out the possibility that premyogenic cells are preferentially killed by enzymatic dissociation. We therefore carried out experiments to test this possibility and also to establish whether reconstitution of a three-dimensional organization would be sufficient to trigger differentiation. Somites I-V and UPM were dissociated into a single-cell suspension as described above. In a first set of experiments, 104 cells from MLC3F-nlacZ embryos were mixed with 105 10T½/2 or C2C7 cells or with 105 cells isolated from similarly ages somites or UPM or nontransgenic embryos. The suspensions were centrifuged in 15-ml conical tubes and grown as pellets for 3 days. Unlike organ cultures, this culture system does not retain precise three-dimensional organization but nevertheless allows more mature mesodermal cells to develop into well- differentiated cartilage and muscle (13). After 3 days, the pellets were analyzed for f3-galactosidase activity. No f3-gal+ cells were detected in pellets aggregated with 10T/2 or C2C7 cells. When isolated cells from UPM were aggregated with similar cells from nontransgenic UPM, no cells expressed ,3-galactosidase in the resultant pellets. However f-gall cells did appear in pellets formed by cells isolated from somites I-V which had been aggregated with cells from somites I-V derived from nontransgenic embryos (Fig. 2A). Thus, cells from newly formed somites can undergo terminal differenti- ation, although to a moderate extent, in a three-dimensional structure where they are surrounded by similar cells. In contrast, cells from UPM cannot differentiate under these conditions. A Role for Axial Structures in Regulating Myogenic Dif- ferentiation of Mesodermal Cells in Reconstituted Three- Dimensional Structures. Absence of differentiation in cells from UPM even when cultured as aggregates (and the modest ...e;. .. differentiation of cells from newly formed somites) may be due to their requirement for signals coming from axial structures FIG. 2. (A) Cryostat section of pellets of 104 cells isolated from tube and Such structures were not present somites I-V of 9.5-dpc MLC3F-nlacZ mouse embryos and reaggre- (neural notochord). gated with 105 cells from somites I-V of similarly aged nontransgenic in the pellet cultures described above. We had, however, embryos. (B) Similar section of pellets of 104 cells isolated from UPM established from the cloning experiments that inductive signals of 9.5-dpc MLC3F-nlacZ mouse embryos and reaggregated with 105 from axial structures are not sufficient to trigger differentia- cells from the trunk (UPM plus axial structures) of similarly aged tion of single mesodermal cells from UPM or somites I-V. nontransgenic embryos. All pellets were cultured for 3 days and then Therefore, in order to understand whether an inductive signal stained for ,B-galactosidase activity. Downloaded by guest on September 26, 2021 Developmental Biology: Cossu et aL Proc. Natl Acad. Sci USA 92 (1995) 2257

Table 2. Size and composition of a community of embryonic cells p - A necessary for myogenic differentiation CELL No. of Minimal COMUITY EFFECT ONDIFFERENIATIONFEEDER LAYER DIFFERENTIATIONSNGLE Sample Mit C X3-gal+ cells P3-gal+ cluster UPM + NT, NC - 3 >100 UPM only - 0 UPM + NT, NC + 0 UPM only + 0 I-V + NT, NC - 37 45 I-V only - 11 52 I-V + NT, NC + 18 38 AXIAL AIAL INDUCTION AAL INDUCTION I-V only + 2 63 INDUCTION PARTIALLY NO LONGER REQaIRED VI-X + NT, NC - 220 32 REQUIRED REQUIRED VI-X only - 195 36 FIG. 3. Scheme showing the developmental changes in mesoder- VI-X + NT, NC + 107 38 mal cell requirements for a community effect and axial induction VI-X only + 115 46 during mouse somitogenesis. A, anterior; P, posterior; NT, neural XI-XV + NT, NC - 198 1 tube. XI-XV only - 177 1 XI-XV + NT, NC + 101 1 differentiation to occur in newly formed somites is 30-40 cells. XI-XV only + 94 1 Furthermore, at least a fraction of cells from somites I-V Cultures were established from either isolated groups of somites undergo terminal differentiation without further cell division. (and UPM) or from the corresponding trunk segments which also Fig. 3 shows a scheme summarizing the developmental depen- contained the axial structures [neural tube and notochord (+ NT, dence of mouse paraxial mesoderm upon neural induction and NC)]. Tissues were mechanically fragmented to give a suspension of a community effect. single cells and clusters ranging in size up to about 100 cells. Half of the cultures were treated with mitomycin C (Mit C, 5 ,ug/ml) for 2 hr after plating. Cultures were stained for ,3-galactosidase activity after DISCUSSION 5 days. The average size of aggregates was smaller in mitomycin C-treated cultures, but large aggregates (up to >100 cells) were The data reported here show the existence of a community present also in cultures that had no ,B-gal+ cells. effect in mammalian myogenesis. They also define the tem- poral limits of this phenomenon and the minimal size of such appears that differentiation is also dependent upon inductive a community necessary to permit muscle differentiation. signals. This dependence is total before somitogenesis but only Presumptive myogenic cells from UPM and newly formed partial in newly formed somites. somites need to be surrounded by sister cells in order to Size and Cellular Composition of Aggregates Which Are differentiate. At this stage the presence of axial structures is Permissive for Myogenic Differentiation. We next investigated also necessary. Cells found in more mature somites (i.e., the minimal size of an explant which will allow terminal somites VI-X) can undergo autonomous differentiation pro- myogenic differentiation. For this purpose we isolated blocks vided that they are grown on a feeder layer of other cells. of 5 somites (I-V, VI-X, XI-XV) and UPM from 9.5-dpc Although differences exist among different feeder cells, the MLC3F-nlacZ embryos. In some experiments we included fact that at this stage similar embryonic cells did not promote axial structures, in order to discriminate between a community more myogenic differentiation than established cell lines sug- effect and axial induction. In cultures derived from UPM gests that the contribution of the feeder layer may well be which were treated with mitomycin C, no ,B-gal+ cells were metabolic. Cells which are located in mature somites (cranial observed (Table 2), despite the fact that many cells as well as to somite X) can differentiate in isolation and therefore can be small and large aggregates survived the treatment. This was considered no longer dependent upon any community effect. also the case when axial structures were included. In untreated Successful cloning of UPM cells from mouse embryos where cultures established from UPM and including axial structures, the nlacZ gene was under the control of the vimentin promoter rare f3-gal+ cells were observed, always in large clusters (which marks most mesodermal cells) proved that these cells containing > 100 cells. As expected, when axial structures were did not die upon dissociation. Furthermore, experiments absent, no X3-gal+ cells were observed in UPM-derived clusters. where single cells from newly formed somites and UPM In cultures derived from somites I-V, several ,B-gal+ cells were differentiated when mixed in an aggregate with a 10-fold observed in both control and mitomycin C-treated cultures: excess of similarly aged nontransgenic cells, proved that a the minimal size of a cluster containing ,B-gall cells was 38 specific position within the somite was not a prerequisite for cells. These clusters usually contained both myosin-positive, differentiation, in agreement with the observed plasticity of ,B-gal+ myocytes and 13III-tubulin-positive neurons (data not cells in newly formed avian somites (16, 17). shown). In cultures derived from somites I-V in the absence of An uncommitted cell (from somites I-V) needs to be axial structures, ,B-gal+ cells were observed in clusters of surrounded by at least 30-40 similar cells in order to acquire similar size (30-40 cells), although at a lower frequency. The competence for differentiation, as shown by growing explants same minimal size was observed in cultures from somites of various sizes in the presence of mitomycin C to prevent cell VI-X, although the total number of positive nuclei was now division. In comparison with controls, treated cultures con- much higher, and control cultures contained approximately tained approximately half the number of differentiated cells, twice the number of positive nuclei present in treated cultures. suggesting that, at least under these conditions, explanted cells Similar results were observed in control and mitomycin C- in the absence of mitomycin C undergo one or, at most, two treated cultures from somites XI-XV, the major difference more cell divisions before differentiating. Cells from UPM being the presence of positive nuclei in isolated cells, in survived but did not differentiate in the presence of mitomycin agreement with our observations in clonal cultures. No effect C. of axial structures was seen on somites older than VI, thus Comparison of these data with the results of similar exper- indicating that any inductive effect is no longer needed after iments in Xenopus (3) reveals that the size of a community is the fifth last-formed somite. From this experiment it appears smaller in mammals than in amphibia and that the phenom- that the minimal size of a cluster which will permit terminal enon occurs later in mammals. This is not surprising, since Downloaded by guest on September 26, 2021 2258 Developmental Biology: Cossu et al. Proc. Natl. Acad ScL USA 92 (1995) myotomes form very early in frogs and also constitute the critical reading of the manuscript. This work was supported by grants quasi-totality of somites, whereas in mammals they form later from Telethon A.13, Cenci Bolognetti, and the Ministero Universita and represent a minor proportion of somites. e Ricerca Scientifica e Tecnologica (to G.C.); by grants from the The existence of a community effect in mammals requires Association Frangaise Contre les Myopathies, Association pour la discussion in the context of the demonstrated induction effect Recherche contre le Cancer, and Institute National de la Sante et de on somites of the axial structures in la Recherche Medical (to M.B.); and by a European Community grant (Fig. 3). Experiments from the Human Capital and Mobility Program (to M.B. and G.C.). chicken and in mammals have demonstrated (with the excep- R.K. was supported initially by a Wellcome traveling fellowship and tion of ref. 18) that UPM and newly formed somites require then by a European Community fellowship under the Human Capital contact with, or close proximity to, axial structures in order to and Mobility Program grant. undergo muscle differentiation (5-10). The timing of this phenomenon is roughly comparable to the timing of the 1. Smith, J. (1993) EMBO J. 12, 4463-4470. community effect; i.e. only UPM and newly formed somites 2. Gurdon, J. B. (1988) Nature (London) 336, 772-774. require axial induction to undergo myogenesis, whereas more 3. Kato, K. & Gurdon, J. B. (1993) Proc. Natl. Acad. Sci. USA 90, mature somites differentiate independently of this influence 1310-1314. (9, 10). However, since at present it is not possible to identify 4. Weston, M. J. D., Kato, K. & Gurdon, J. B. (1994) Rouxs Arch. with certainty possible axial inducers (candidates include Dev. Biol. 203, 250-253. neural crest, neurons, and notochord) in reaggregated pellets 5. Vivarelli, E. & Cossu, G. (1986) Dev. Biol. 117, 319-325. and undissociated clusters, we repeated the experiments using 6. Kenny-Mobbs, T. & Thorogood, P. (1987) Development (Cam- either isolated somites and UPM or trunk segments, which are bridge, U.K) 100, 449-462. likely to contain these potential inducers. 7. Rong, P. M., Teillet, M. A., Ziller, C. & Le Douarin, N. M. (1992) Differentiation of Development (Cambridge, U.K) 115, 657-672. UPM cells appears to be dependent upon axial structures, 8. Pourquie, O., Coltey, M., Teillet, M. A., Ordhal, C. & Le since these cells cannot differentiate in either clusters or pellets Douarin, N. M. (1983) Proc. Natl. Acad. Sci. USA 90,5242-5246. containing sister cells but not axial cells. In contrast, cells from 9. Borman, W. H. & Yorde, D. E. (1994) Dev. Dyn. 200, 68-78. somites I-V are only in part dependent on axial cells and can 10. Buffinger, N. & Stockdale, F. E. (1994) Development (Cambridge, therefore undergo terminal differentiation in a community U.K) 120, 1443-1452. composed only of sister cells, although at a reduced frequency. 11. Kelly, R., Alonso, S., Tajbakhsh, S., Cossu, G. & Buckingham, M. In conclusion, the data reported here show that a commu- (1995) J. Cell Biol., in press. nity effect exists in mammalian myogenesis for cells located in 12. Colucci-Guyon, E., Portier, M., Dunia, I., Paulin, D., Pournin, S. the UPM and in the first five formed somites. Cells in somites & Babinet, C. (1994) Cell 79, 679-694. VI-X require support which can be provided by any cell type, 13. Cossu, G., Warren, L., Boettiger, D., Holtzer, H. & Pacifici, M. and cells from somites older than X differentiate in the (1982) J. Biol. Chem. 257, 4463-4468. absence of a cellular community. Response to axial inducers 14. Sanes, J. R., Rubenstein, J. L. R. & Nicolas, J. F. (1986) EMBO in J. 5, 3133-3142. occurs an overlapping but temporally shorter period, such 15. Tajbakhsh, S., Vivarelli, E., Cusella De Angelis, G., Rocancourt, that cells in somites I-V are still dependent on a cellular D., Buckingham, M. & Cossu, G. (1994) Neuron 13, 813-821. community but only partially on induction. More detailed 16. Christ, B., Brand-Saberi, B., Grim, M. & Wilting, J. (1992) Anat. understanding of the relationship between these two phenom- Embryol. 186, 505-510. ena awaits the cellular and molecular identification of myo- 17. Ordhal, C. P. & Le Douarin, N. (1992) Development (Cambridge, genic inducer(s). U.K) 114, 339-353. 18. George-Weinstein, M., Gerhart, J. V., Foti, G. J. & Lash, J. W. We thank S. Tajbakhsh and M. Primig for helpful discussions and (1994) Exp. Cell Res. 211, 263-274. Downloaded by guest on September 26, 2021