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.