- Email: [email protected]
Plasticity of cell fate: Insights from heterokaryons
seminars in C E L L & D E V E L OP M E N T A L B I OL OG Y , Vol 10, 1999: pp. 267]272 Article No. scdb.1999.0311, available online at http:rrwww.idealibrary.com on
Plasticity of cell fate: Insights from heterokaryons Helen M. Blau and Bruce T. Blakely
Experiments with somatic cell hybrids and stable heterokaryons have demonstrated that differentiated cells exhibit a remarkable capacity to change. Heterokaryons have been particularly useful in determining the extent to which the differentiated state of a cell is plastic. Cell fate can be altered by a change in the balance of positive and negative trans-acting regulators. Although a single regulator may be sufficient in certain environments to trigger a change in cell fate, that regulator may be ineffective in other cell contexts where it encounters a different composition of regulators.
tained at a high frequency of 75% when the nuclei were initially injected into oocytes and conditioned by oocyte cytoplasm prior to transplantation into enucleated eggs;5 for review, see ref 6. That genetic material was not lost or permanently inactivated during vertebrate differentiation was apparent from these experiments. Moreover, they suggested that the specialized state of a cell is achieved by regulating the activity of its genes. However, the possibility remained that this degree of reactivation of dormant genes depended upon nuclei being exposed to a sequence of cues that accompanies the progression from undifferentiated zygote to specialized tissue. Experiments using somatic cell hybrids suggested that the differentiated state of a cell can be altered without recourse to the regulatory hierarchy characteristic of development. Moreover, cell fusion to form non-dividing heterokaryons showed that the differ entiated state of a cell can be changed in the absence of DNA replication or cell division. This plasticity of nuclear function is likely to result from a dynamic interaction of the combination of proteins the fused cells contain. Although, in muscle, the MyoD family of helix]loop]helix transcriptional regulators plays a major role, heterokaryons provide evidence that other critical regulators are also involved.
Key words: heterokaryon r myogenesis r differentiation r development r regulation Q1999 Academic Press
Introduction IN THE COURSE OF vertebrate development, totipotent cells in the early embryo give rise to cell types specialized for function in tissues, such as muscle cells. This process entails a series of stages during which the fate of a cell is influenced by extrinsic and intrinsic signals. Waddington’s epigenetic landscape suggested that differentiated cells are destined for a groove, or valley, and that a switch to a different valley is not easily achieved.1 Gurdon’s experiments,2 provided perhaps the first indication that the destiny of a differentiated cell could be altered; when nuclei from amphibian intestine were introduced into enucleated eggs, feeding tadpoles developed. Later experiments by Gurdon3 and DiBerardino and Hoffner 4 confirmed the hypothesis and clearly showed that, when transplanted, the nuclei of well-differentiated keratinocytes and non-cycling erythrocytes could display multipotentially. Indeed, feeding tadpoles were ob-
Changes in gene expression in somatic cell hybrids The combination of two different cell types through cell fusion to form somatic cell hybrids yielded early evidence that mammalian gene expression can be altered by diffusible trans-acting regulators. Following fusion with another cell type, the expression of differentiated functions frequently ceased;7 for review, see ref 8. This repression was reversed upon chromosome loss, which occurs as a result of nuclear fusion and continued cell division in hybrids. In some cases, repression was ascribed to specific genetic loci that act in trans.9,10 In a few cases, trans-activation of genes was also reported.11 ] 13 Studies with hybrids
From the Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, CA 94305, USA Q1999 Academic Press 1084-9521r 99 r 030267q 06 $30.00r 0
267
H. M. Blau and B. T. Blakely
also provided early evidence that the malignant state is, in some cases, recessive to the differentiated state;14,15 for review, see ref 16, a prediction confirmed at the molecular level for retinoblastoma.17 Taken together, these experiments suggested that the balance of positive and negative regulators might dictate the pattern of genes expressed in the differ entiated cell.
weeks, permitting an analysis of gene expression over much longer periods of time than was previously possible. Ž2. The choice of cell type}primary diploid cells, rather than aneuploid transformed cells were employed. Ž3. The differentiated state}the muscle cells used were multinucleated cells in which differ entiation was well underway. These findings were soon corroborated for muscle cells by Wright 26,27 and extended to other cell types, such as erythroid cells by Baron and Maniatis,28 pancreatic cells by Wu et al 29 and hepatic cells by Spear and Tilghman.30 Gene activation was detected in heterokaryons by assaying 10 different human muscle gene products using species-specific assays.31 These encompassed a range of muscle functions including structural proteins of the contractile apparatus, membrane components, and enzymes.31 The relative amounts produced and the time course of appearance of these gene products was typical of human myogenesis.32 In addition to muscle gene activation, repression of the production of proteins and transcripts typical of the non-muscle cell type was observed and the distribution of organelles within the cell rapidly changed to be like muscle.33 Within hours of cell fusion, the Golgi apparatus and the microtubule organizing center moved from a polar to a circumnuclear location and the centrioles were dispersed. This complexity of changes suggested that fusion with muscle cells in heterokaryons induced a fundamental alteration, or phenotypic change, in the differentiated function of non-muscle cells.
Changes in cell fate in heterokaryons A heterokaryon system is advantageous to analyze changes in gene activity in the context of the whole cell. The key feature of this system is that it is stable for the duration of the experiment. Since after fusion, there is no nuclear fusion or cell division, as occurs with hybrids, all genetic material remains intact within its own nucleus. This type of short-term, non-dividing fusion product makes possible an assessment of the influence of two or more sets of cytoplasmic and nuclear components on gene expression with minimal disruption. Moreover, since growth and genetic selection are not required to obtain the fusion product of interest, changes in gene expression can be monitored immediately following the fusion event and at well-defined time intervals thereafter. The use of non-dividing stable interspecific heterokaryons in the study of regulation of gene expression was pioneered by Harris and Ringertz. Following fusion with human cells, differentiated chick erythrocyte nuclei swelled, resumed RNA synthesis and contained human nuclear proteins;14,18,19 for review, see ref 20. Either coexpression or extinction of genes contributed by both cell types were frequent outcomes of heterokaryon experiments produced with cells of different species and differentiated states, including muscle.21 ] 24 However, in these early experiments gene activation was not detected. The first evidence that previously silent genes could be activated in heterokaryons was provided by Blau, Chiu and Webster.25 Following fusion with mouse muscle cells, muscle gene expression was induced in human amniotic fibroblasts. A combination of three features was probably responsible for the success of these experiments: Ž1. Culture media}the heterokaryons were maintained in media that was mitogen-poor and low in serum, thus promoting differ entiation, not proliferation. This was critical because conditions that stimulate proliferation are antagonistic to muscle differentiation. Moreover, this medium prevented heterokaryons from dividing for up to 2
The response of different cell types implicates additional regulators A particular advantage of heterokaryons in the analysis of differentiation is that changes in gene expression can be induced and studied in cells that would normally never express those genes. Thus, this type of analysis has advantages over the majority of studies of differentiation in tissue culture which typically utilize precursor cells, such as myoblasts, adipoblasts, or erythroleukemia cells which are already destined for the differentiated state they ultimately express. Studies using such precursors are likely to miss key regulatory steps, because the cells have already undergone a substantial number of the changes in protein composition and DNA conformation required by their particular cell type. By contrast, heterokaryons allow an analysis of a greater complexity of regulatory steps. These steps are necessary to in268
Plasticity of cell fate: insights from heterokaryons
duce a novel differentiated state in cells previously committed to a different fate. The effect of cell origin and its influence on the ability to express previously silent muscle genes was analyzed with heterokaryons produced with representatives of the three embryonic lineages: fibroblasts Žmesoderm., keratinocytes Žectoderm. and hepatocytes Žendoderm..31,33,34 Marked differences were detected among cell types in the time course and efficiency of muscle gene expression in heterokaryons. For heterokaryons containing fibroblasts, ker atinocytes and hepatocytes, the average frequency of expression of human muscle N-CAM on the cell surface was 95, 60, and 25%, respectively. In addition, the onset of expression of N-CAM differed by at least 2 days. To understand the regulatory mechanisms that control the differentiated state, knowledge of the molecular events that constitute the lag period prior to muscle gene expression is critical. The differences among cell types are likely to reflect a requirement for different numbers or concentrations of regulators necessary to achieve muscle gene activation.
activation of several muscle genes. No significant differences in the expression of the muscle gene encoding human N-CAM were observed in the presence or absence of DNA replication. These results indicate that conformational changes in chromatin either do not accompany gene activation in heterokaryons or do not require extensive DNA replication.
A network of positive and negative regulators mediates muscle differentiation In hybrids, gene dosage, or the relative genetic contribution of the two fused cell types, is critical to the suppression of malignancy Žfor review, see ref 16.. Similarly, in heterokaryons the relative contribution of muscle and non-muscle nuclei is critical to novel gene activation.31 Both the existence of negative and positive regulators is implicated in these cell fusion experiments. Their concentration and role differs among cell types. For example, irrespective of nuclear ratio, 95% of heterokaryons containing fibroblasts from three different sources Žfetal lung, fetal skin, and adult skin. ultimately expressed human muscle genes.34,37 These results suggest that if fi broblasts produce inhibitors of muscle differentiation, like the well-documented extinguishers of liver gene activation,9,10 they are readily overridden by the positive regulators contributed by differentiated muscle cells, due to differences in their nature or concentration. By contrast, in heterokaryons produced with hepatocytes the ultimate probability of activating muscle genes was dependent on the proportion of muscle and hepatocyte nuclei at all times.33 When hepatocyte nuclei outnumbered muscle cell nuclei, human muscle genes were not activated and the mouse muscle phenotype was extinguished38 presumably due to the relative levels of negative regulators that overwhelmed the positive regulators contributed by the muscle cell type.
Regulatory mechanisms for activating silent genes Muscle gene activation was generally observed in heterokaryons with primary diploid cells, but not in heterokaryons with transformed aneuploid cell types. In one case, the Hela cell, we showed that the lack of response could be reversed.35 If treated with 5azacytidine Ž5AC. prior to fusion, muscle gene expression was induced in HeLa cells in heterokaryons. Thus muscle gene activation in HeLa cells required changes induced by 5AC, presumably in the level of DNA methylation of structural genes or the genes encoding their regulators. Thus, unlike normal cell types, muscle genes are silenced by a mechanism that is not as easily reversed in this transformed cell type. It remains to be determined whether this mechanism is generally employed to repress tissue-specific genes in transformed cell types. We designed experiments to determine whether the lag period prior to muscle gene activitation reflected a requirement for DNA replication. Possibly, negative regulation was alleviated by changes in the chromatin structure during DNA synthesis. To address this possibility, the fibroblasts, keratinocytes and hepatocytes were each exposed to a DNA synthesis inhibitor, cytosine arabinoside, prior to and continuously after cell fusion31,33,36 and assayed for the novel
Heterokaryons reveal regulation of the helix–loop–helix family of myogenic regulators To examine the molecular basis for the musclespecific gene expression observed in multinucleated heterokaryons formed from the fusion of differentiated muscle cells to either hepatocytes or fibroblasts, we tested the role of the MyoD family of regulators. MyoD and its relatives are regulators of muscle269
H. M. Blau and B. T. Blakely
specific gene expression that have a helix]loop]helix motif.39 We tested whether these regulators alone could induce the phenotypic conversion observed in heterokaryons.40 MyoD or myogenin were stably or transiently introduced into fibroblasts or hepatocytes by microinjection, transfection or retroviral infection with complementary DNA in expression vectors. Fibroblasts expressed muscle-specific genes, whereas hepatocytes did not. However, following fusion of hepatocytes stably expressing MyoD to fibroblasts, activation in the heterokaryon of muscle-specific genes of both cell types was detected. These results imply that other regulators, present in fibroblasts but not in hepatocytes, are necessary for the activation of muscle-specific genes. They also show that the differentiated state of a cell is dictated by its history and a dynamic interaction among the proteins that it contains. A non-differentiating mouse muscle cell line, NFB, that represses the activity of the helix]loop]helix ŽHLH. family of myogenic regulators, yet expresses sarcomeric actins was analyzed.41 In NFB cells, the MyoD gene is silent, but can be activated upon transfection of a long terminal region-controlled chicken MyoD cDNA, resulting in myogenesis. When NFB cells are fused with H9c2 rat muscle cells in heterokaryons, the level of rat MyoD transcripts declines. Thus, the stoichiometry of MyoD and the putative repressor controls myogenesis. Although NFB cells express myogenin and Myf-5 transcripts Žother members of the MyoD family., the activity of these regulators is also repressed: myogenesis is not induced in 10T1r2 fibroblasts and is repressed in L6 muscle cells upon fusion with NFB cells. These findings led us to conclude that myogenesis is subject to control by dominant-negative regulators.
creatine kinase distribute over large portions of the heterokaryon, whereas other proteins, such as myosin heavy chain, are restricted to a smaller area near the nucleus that encodes the protein.42 Since heterokaryons have shown that regulatory proteins such as the myogenic HLH regulators can act on a nucleus other than the one that encodes them, one might expect that heterokaryons between muscle cells expressing different isoforms of muscle proteins, such as myosin, would undergo changes in expression. For example, in heterokaryons between mouse and rat muscle cells, myosin light chain isoform expression is reprogrammed in the rat nuclei to match that seen in the mouse nuclei.43 Heterokaryons of embryonic and fetal muscle cell lines, however, maintain expression of embryonic and fetal myosin heavy chain isoforms in distinct nuclear domains.44 Thus, myosin heavy chain isoform expression is not efficiently altered by diffusible transcriptional regulatory proteins in heterokaryons, possibly because such reprogramming is more dependent on extrinsic signals, as has been shown in vivo.45 Injection of normal myoblasts into diseased muscle to form heterokaryon fibers has been proposed to treat diseases in which a myopathy is caused by the lack of functional expression of a critical protein. Such therapy is more likely to succeed if the protein of interest can freely diffuse from the expressing nucleus throughout the muscle fiber. For example, normal myoblasts that express dystrophin have been injected into the muscle of Duchenne muscular dystrophy patients or mdx mice which lack functional dystrophin.46,47 If the dystrophin encoded by the normal nucleus was restricted to a small domain around the nucleus, such treatment would not be expected to succeed. However, dystrophin is not restricted to nuclear domains in heterokaryons in vitro 48 and can in fact spread several hundred micrometers away from the nucleus of origin in vivo.49 Treatment with normal myoblasts of several other myopathies, which are mitochondrial in origin, has also been proposed. The finding that normal and diseased mitochondria rapidly migrate and mix in heterokaryons 50 supports this approach.
Heterokaryons in fiber development and disease Once a muscle cell has made the decision to differ entiate, many gene expression decisions remain. Skeletal muscle proteins, such as myosin, have multiple isoforms which participate in determining the contractile properties of the muscle fiber. Questions about muscle fiber development can also be addressed using heterokaryons. Muscle cells from different species or individuals can spontaneously fuse in vitro or in vivo to form heterokaryons. This property was used to demonstrate that in heterokaryons of mouse and human muscle, some proteins, such as cell surface molecules and cytoplasmic
Conclusions The major conclusion from the heterokaryon studies described above is that the differentiated state is not fixed but can readily be altered. However, the differentiated state in vivo appears relatively stable. 270
Plasticity of cell fate: insights from heterokaryons
Thus, an understanding of the regulation of cell differentiation by mechanisms that allow for the type of plasticity observed in heterokaryons yet stably maintain a differentiated state is of particular interest. How is the change in differentiated state achieved? Heterokaryon experiments do not support molecular models which suggest that DNA replication is required for the activation of previously silent genes.51 ] 53 Changes in chromatin structure, such as the creation of DNase hypersensitive sites, removal of histones or changes in DNA methylation could still occur. However, if they occur, these changes are mediated by mechanisms that are independent of DNA replication. The elucidation of these mechanisms is now possible. How, then, is a heritable differentiated state achieved? Heterokaryon studies suggest that the differentiated state is largely governed by the dynamic interaction of the combination of proteins a cell contains. This is particularly clear from the striking effect on gene activation observed when the relative contribution of components, or dosage, of the two fused cell types is altered. How does this pertain to differentiation in vivo? The protein composition of a cell is part of its heritage, the product of a history of responses to cues in the course of development. The cell transmits these proteins to progeny through division. The response of cells to a single regulator, such as MyoD depends on the cell context, or set of proteins, it encounters. As a result, MyoD induces muscle gene expression in some cell types, but not in others. In cases where it fails to do so, such as liver cells, additional regulators, both positive and negative, are implicated. A threshold concentration of regulators may be critical to the expression and maintenance of the differentiated state. For some proteins this may be achieved by activating the expression of their own promoters, like c-jun.54 Positive autoregulation and feedback loops are levels of control that operate during the commitment of phage lambda to lysogeny Žfor review, see ref 55. and are likely to be characteristic of early acting regulators in mammalian cell differentiation. Protein]protein interactions provide another means of controlling regulator concentration. Transcriptional regulators with ‘helix]loop]helix’ motifs bind DNA sequences as heterodimers, 56 suggesting that a range of protein]protein combinations is possible. Indeed, the activity of the MyoD family of regulators, helix] loop]helix proteins, is determined, in part, by the concentration and nature Žinhibitory or facilitating. of their protein partners.57 In heterokaryons, differ -
ent partners lead to novel heterodimers that act either as positive or negative regulators. These heterodimers can act on the same or on different promoters from those recognized by the protein complexes originally present in each of the parental cells.
References 1. Waddington CH Ž1940. Organisers and Genes pp 91]93. Cambridge University Press, London 2. Gurdon JB Ž1962. The developmental capacity of nuclei taken from intestinal epithelium cells of feeding tadpoles. J Embryol Exp Morphol 10:622]640 3. Gurdon JB, Laskey RA, Reeves OR Ž1975. The developmental capacity of nuclei transplanted from keratinized skin cells of adult frogs. J Embryol Exp Morphol 34:93]112 4. DiBerardino MA, Hoffner NJ Ž1983. Gene reactivation in erythrocytes: nuclear transplantation in oocytes and eggs of Rana. Science 219:862]864 5. DiBerardino MA, Orr NH, McKinnell RG Ž1986. Feeding tadpoles cloned from Rana erythrocyte nuclei. Proc Natl Acad Sci USA 83:8231]8234 6. DiBerardino MA Ž1988. Genomic multipotentiality of differ entiated somatic cells. Cell Differ Dev 25 Suppl:129]136 7. Mevel-Ninio M, Weiss MC Ž1981. Immunofluorescence analysis of the time-course of extinction, reexpression, and activation of albumin production in rat hepatoma-mouse fibroblast heterokaryons and hybrids. J Cell Biol 90:339]350 8. Weiss MC, Cassio D, Sellem CH Ž1988. Cell differentiation: contributions of somatic cell genetics. Cancer Surv 7:295]302 9. Killary AM, Fournier RE Ž1984. A genetic analysis of extinction: trans-dominant loci regulate expression of liver-specific traits in hepatoma hybrid cells. Cell 38:523]534 10. Petit C, Levilliers J, Ott MO, Weiss MC Ž1986. Tissue-specific expression of the rat albumin gene: genetic control of its extinction in microcell hybrids. Proc Natl Acad Sci USA 83:2561]2565 11. Davidson RL Ž1972. Regulation of melanin synthesis in mammalian cells: effect of gene dosage on the expression of differentiation. Proc Natl Acad Sci USA 69:951]955 12. Peterson JA, Weiss MC Ž1972. Expression of differentiated functions in hepatoma cell hybrids: induction of mouse albumin production in rat hepatoma-mouse fibroblast hybrids. Proc Natl Acad Sci USA 69:571]575 13. Rankin JK, Darlington GJ Ž1979. Expression of human hepatic genes in mouse hepatoma]human amniocyte hybrids. Somatic Cell Genet 5:1]10 14. Harris H, Sidebottom E, Grace DM, Bramwell ME Ž1969. The expression of genetic information: a study with hybrid animal cells. J Cell Sci 4:499]525 15. Peehl DM, Stanbridge EJ Ž1982. The role of differentiation in the suppression of tumorigenicity in human cell hybrids. Int J Cancer 30:113]120 16. Harris H Ž1998. The analysis of malignancy by cell fusion: the position in 1988. Cancer Res 48:3302]3306 17. Friend SH, Bernards R, Rogelj S, Weinberg RA, Rapaport JM, Albert DM, Dryja TP Ž1986. A human DNA segment with properties of the gene that predisposes to retinoblastoma and osteosarcoma. Nature 323:643]646 18. Harris H, Watkins JF Ž1995. Hybrid cells derived from mouse and man: artificial heterokaryons of mammalian cells from different species. Nature 205:640]646 19. Ringertz NR, Carlsson SA, Ege T, Bolund L Ž1971. Detection of human and chick nuclear antigens in nuclei of chick
271
H. M. Blau and B. T. Blakely
20. 21.
22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.
33.
34. 35. 36. 37. 38. 39.
erythrocytes during reactivation in heterokaryons with HeLa cells. Proc Natl Acad Sci USA 68:3228]3232 Ringertz NR, Savage RE Ž1976. Cell Hybrids. Academic Press, New York Carlsson SA, Luger O, Ringertz NR, Savage RE Ž1974. Phenotypic expression in chick erythrocyte x rat myoblast hybrids and in chick myoblast x rat myoblast hybrids. Exp Cell Res 84:47]55 Lawrence JB, Coleman JR Ž1984. Extinction of muscle-specific properties in somatic cell heterokaryons. Dev Biol 101:463]476 Wright WE, Aronoff J Ž1983. The suppression of myogenic functions in heterokaryons formed by fusing chick myocytes to diploid rat fibroblasts. Cell Differ 12:299]306 Konieczny SF, Lawrence JB, Coleman JR Ž1983. Analysis of muscle protein expression in polyethylene glycol-induced chicken: rat myoblast heterokaryons. J Cell Biol 97:1348]1355 Blau HM, Chiu CP, Webster C Ž1983. Cytoplasmic activation of human nuclear genes in stable heterocaryons. Cell 32:1171]1180 Wright WE Ž1984. Expression of differentiated functions in heterokaryons between skeletal myocytes, adrenal cells, fi broblasts and glial cells. Exp Cell Res 151:55]59 Wright WE Ž1984. Induction of muscle genes in neural cells. J Cell Biol 98:427]435 Baron MH, Maniatis T Ž1986. Rapid reprogramming of globin gene expression in transient heterokaryons. Cell 46:591]602 Wu KJ, Samuelson LC, Howard G, Meisler MH, Darlington GJ Ž1991. Transactivation of pancreas-specific gene sequences in somatic cell hybrids. Mol Cell Biol 11:4423]4430 Spear BT, Tilghman SM Ž1990. Role of alpha-fetoprotein regulatory elements in transcriptional activation in transient heterokaryons. Mol Cell Biol 10:5047]5054 Blau HM, Pavlath GK, Hardeman EC, Chiu CP, Silberstein L, Webster SG, Miller SC, Webster C Ž1985. Plasticity of the differentiated state. Science 230:758]766. Hardeman EC, Chiu CP, Minty A, Blau HM Ž1986. The pattern of actin expression in human fibroblast x mouse muscle heterokaryons suggests that human muscle regulatory factors are produced. Cell 47:123]130 Miller SC, Pavlath GK, Blakely BT, Blau HM Ž1988. Muscle cell components dictate hepatocyte gene expression and the distribution of the Golgi apparatus in heterokaryons. Genes Dev 2:330]340 Pavlath GK, Chiu CP, Blau HM Ž1989. in vivo aging of human fibroblasts does not alter nuclear plasticity in heterokaryons. Somat Cell Mol Genet 15:191]202 Chiu CP, Blau HM Ž1985. 5-Azacytidine permits gene activation in a previously non-inducible cell type. Cell 40:417]424 Chiu CP, Blau HM Ž1984. Reprogramming cell differentiation in the absence of DNA synthesis. Cell 37:879]887 Pavlath GK, Blau HM Ž1986. Expression of muscle genes in heterokaryons depends on gene dosage. J Cell Biol 102:124]130 Blakely BT Ž1993. Negative and Positive Regulatory Mechanisms in Muscle and Liver Differentiation. Stanford University Dissertation, Stanford, CA Davis RL, Weintraub H, Lassar AB Ž1987. Expression of a single transfected cDNA converts fibroblasts to myoblasts. Cell 51:987]1000
40. Schafer BW, Blakely BT, Darlington GJ, Blau HM Ž1990. Effect of cell history on response to helix]loop]helix family of myogenic regulators. Nature 344:454]458 41. Peterson CA, Gordon H, Hall ZW, Paterson BM, Blau HM Ž1990. Negative control of helix]loop]helix family of myogenic regulators in the NFB mutant. Cell 62:493]502 42. Pavlath GK, Rich K, Webster SG, Blau HM Ž1989. Localization of muscle gene products in nuclear domains. Nature 337:570]573 43. Pajek L, Mariappan M, Wieczorek DF Ž1991. Reprogramming of myosin light chain 1r3 expression in muscle heterokaryons. Dev Biol 145:28]39 44. Pin CL, Merrifield PA Ž1997. Regionalized expression of myosin isoforms in heterotypic myotubes formed from embryonic and fetal rat myoblasts in vitro. Dev Dyn 208:420]431 45. Hughes SM, Taylor JM, Tapscott SJ, Gurley CM, Carter WJ, Peterson CA Ž1993. Selective accumulation of MyoD and myogenin mRNAs in fast and slow adult skeletal muscle is controlled by innervation and hormones. Development 118:1137]1147 46. Gussoni E, Pavlath GK, Lanctot AM, Sharma KR, Miller RG, Steinman L, Blau HM Ž1992. Normal dystrophin transcripts detected in Duchenne muscular dystrophy patients after myoblast transplantation. Nature 356:435]438 47. Karpati G, Pouliot Y, Zubrzycka-Gaarn E, Carpenter S, Ray PN, Worton RG, Holland P Ž1989. Dystrophin is expressed in mdx skeletal muscle fibers after normal myoblast implantation. Am J Pathol 135:27]32 48. Denetclaw WF Jr, Bi G, Pham DV, Steinhardt RA Ž1993. Heterokaryon myotubes with normal mouse and Duchenne nuclei exhibit sarcolemmal dystrophin staining and efficient intracellular free calcium control. Mol Biol Cell 4:963]972 49. Gussoni E, Blau HM, Kunkel LM Ž1997. The fate of individual myoblasts after transplantation into muscles of DMD patients. Nat Med 3:970]977 50. Walker UA, Walker WF, Miranda AF Ž1997. Mitochondrial mobility in differentiating muscle heterokaryons. J Neurol Sci 151:135]140 51. Holtzer H, Biehl J, Payette R, Sasse J, Holtzer S Ž1983. Cell diversification: differing roles of cell lineages and cell]cell interactions, in Limb Development and Regeneration pp. 272]280. Alan R. Liss, New York 52. Brown DD Ž1984. The role of stable complexes that repress and activate eucaryotic genes. Cell 37:359]365 53. Weintraub H Ž1985. Assembly and propagation of repressed and depressed chromosomal states. Cell 42:705]711 54. Angel P, Hattori K, Smeal T, Karin M Ž1988. The jun protooncogene is positively autoregulated by its product, JunrAP-1. Cell 55:875]885. 55. Ptashne M Ž1986. A Genetic Switch: Gene Control and Phage l. Cell Press and Blackwell Scientific Publications, Cambridge, MA. 56. Murre C, McCaw PS, Vaessin H, Caudy M, Jan LY, Jan YN, Cabrera CV, Buskin JN, Hauschka SD, Lassar AB et al Ž1989. Interactions between heterologous helix]loop]helix proteins generate complexes that bind specifically to a common DNA sequence. Cell 58:537]544 57. Lemercier C, To RQ, Carrasco RA, Konieczny SF Ž1998. The basic helix]loop]helix transcription factor Mist1 functions as a transcriptional of myoD. Embo J 17:1412]1422
272
Plasticity of cell fate: Insights from heterokaryons Helen M. Blau and Bruce T. Blakely
Experiments with somatic cell hybrids and stable heterokaryons have demonstrated that differentiated cells exhibit a remarkable capacity to change. Heterokaryons have been particularly useful in determining the extent to which the differentiated state of a cell is plastic. Cell fate can be altered by a change in the balance of positive and negative trans-acting regulators. Although a single regulator may be sufficient in certain environments to trigger a change in cell fate, that regulator may be ineffective in other cell contexts where it encounters a different composition of regulators.
tained at a high frequency of 75% when the nuclei were initially injected into oocytes and conditioned by oocyte cytoplasm prior to transplantation into enucleated eggs;5 for review, see ref 6. That genetic material was not lost or permanently inactivated during vertebrate differentiation was apparent from these experiments. Moreover, they suggested that the specialized state of a cell is achieved by regulating the activity of its genes. However, the possibility remained that this degree of reactivation of dormant genes depended upon nuclei being exposed to a sequence of cues that accompanies the progression from undifferentiated zygote to specialized tissue. Experiments using somatic cell hybrids suggested that the differentiated state of a cell can be altered without recourse to the regulatory hierarchy characteristic of development. Moreover, cell fusion to form non-dividing heterokaryons showed that the differ entiated state of a cell can be changed in the absence of DNA replication or cell division. This plasticity of nuclear function is likely to result from a dynamic interaction of the combination of proteins the fused cells contain. Although, in muscle, the MyoD family of helix]loop]helix transcriptional regulators plays a major role, heterokaryons provide evidence that other critical regulators are also involved.
Key words: heterokaryon r myogenesis r differentiation r development r regulation Q1999 Academic Press
Introduction IN THE COURSE OF vertebrate development, totipotent cells in the early embryo give rise to cell types specialized for function in tissues, such as muscle cells. This process entails a series of stages during which the fate of a cell is influenced by extrinsic and intrinsic signals. Waddington’s epigenetic landscape suggested that differentiated cells are destined for a groove, or valley, and that a switch to a different valley is not easily achieved.1 Gurdon’s experiments,2 provided perhaps the first indication that the destiny of a differentiated cell could be altered; when nuclei from amphibian intestine were introduced into enucleated eggs, feeding tadpoles developed. Later experiments by Gurdon3 and DiBerardino and Hoffner 4 confirmed the hypothesis and clearly showed that, when transplanted, the nuclei of well-differentiated keratinocytes and non-cycling erythrocytes could display multipotentially. Indeed, feeding tadpoles were ob-
Changes in gene expression in somatic cell hybrids The combination of two different cell types through cell fusion to form somatic cell hybrids yielded early evidence that mammalian gene expression can be altered by diffusible trans-acting regulators. Following fusion with another cell type, the expression of differentiated functions frequently ceased;7 for review, see ref 8. This repression was reversed upon chromosome loss, which occurs as a result of nuclear fusion and continued cell division in hybrids. In some cases, repression was ascribed to specific genetic loci that act in trans.9,10 In a few cases, trans-activation of genes was also reported.11 ] 13 Studies with hybrids
From the Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, CA 94305, USA Q1999 Academic Press 1084-9521r 99 r 030267q 06 $30.00r 0
267
H. M. Blau and B. T. Blakely
also provided early evidence that the malignant state is, in some cases, recessive to the differentiated state;14,15 for review, see ref 16, a prediction confirmed at the molecular level for retinoblastoma.17 Taken together, these experiments suggested that the balance of positive and negative regulators might dictate the pattern of genes expressed in the differ entiated cell.
weeks, permitting an analysis of gene expression over much longer periods of time than was previously possible. Ž2. The choice of cell type}primary diploid cells, rather than aneuploid transformed cells were employed. Ž3. The differentiated state}the muscle cells used were multinucleated cells in which differ entiation was well underway. These findings were soon corroborated for muscle cells by Wright 26,27 and extended to other cell types, such as erythroid cells by Baron and Maniatis,28 pancreatic cells by Wu et al 29 and hepatic cells by Spear and Tilghman.30 Gene activation was detected in heterokaryons by assaying 10 different human muscle gene products using species-specific assays.31 These encompassed a range of muscle functions including structural proteins of the contractile apparatus, membrane components, and enzymes.31 The relative amounts produced and the time course of appearance of these gene products was typical of human myogenesis.32 In addition to muscle gene activation, repression of the production of proteins and transcripts typical of the non-muscle cell type was observed and the distribution of organelles within the cell rapidly changed to be like muscle.33 Within hours of cell fusion, the Golgi apparatus and the microtubule organizing center moved from a polar to a circumnuclear location and the centrioles were dispersed. This complexity of changes suggested that fusion with muscle cells in heterokaryons induced a fundamental alteration, or phenotypic change, in the differentiated function of non-muscle cells.
Changes in cell fate in heterokaryons A heterokaryon system is advantageous to analyze changes in gene activity in the context of the whole cell. The key feature of this system is that it is stable for the duration of the experiment. Since after fusion, there is no nuclear fusion or cell division, as occurs with hybrids, all genetic material remains intact within its own nucleus. This type of short-term, non-dividing fusion product makes possible an assessment of the influence of two or more sets of cytoplasmic and nuclear components on gene expression with minimal disruption. Moreover, since growth and genetic selection are not required to obtain the fusion product of interest, changes in gene expression can be monitored immediately following the fusion event and at well-defined time intervals thereafter. The use of non-dividing stable interspecific heterokaryons in the study of regulation of gene expression was pioneered by Harris and Ringertz. Following fusion with human cells, differentiated chick erythrocyte nuclei swelled, resumed RNA synthesis and contained human nuclear proteins;14,18,19 for review, see ref 20. Either coexpression or extinction of genes contributed by both cell types were frequent outcomes of heterokaryon experiments produced with cells of different species and differentiated states, including muscle.21 ] 24 However, in these early experiments gene activation was not detected. The first evidence that previously silent genes could be activated in heterokaryons was provided by Blau, Chiu and Webster.25 Following fusion with mouse muscle cells, muscle gene expression was induced in human amniotic fibroblasts. A combination of three features was probably responsible for the success of these experiments: Ž1. Culture media}the heterokaryons were maintained in media that was mitogen-poor and low in serum, thus promoting differ entiation, not proliferation. This was critical because conditions that stimulate proliferation are antagonistic to muscle differentiation. Moreover, this medium prevented heterokaryons from dividing for up to 2
The response of different cell types implicates additional regulators A particular advantage of heterokaryons in the analysis of differentiation is that changes in gene expression can be induced and studied in cells that would normally never express those genes. Thus, this type of analysis has advantages over the majority of studies of differentiation in tissue culture which typically utilize precursor cells, such as myoblasts, adipoblasts, or erythroleukemia cells which are already destined for the differentiated state they ultimately express. Studies using such precursors are likely to miss key regulatory steps, because the cells have already undergone a substantial number of the changes in protein composition and DNA conformation required by their particular cell type. By contrast, heterokaryons allow an analysis of a greater complexity of regulatory steps. These steps are necessary to in268
Plasticity of cell fate: insights from heterokaryons
duce a novel differentiated state in cells previously committed to a different fate. The effect of cell origin and its influence on the ability to express previously silent muscle genes was analyzed with heterokaryons produced with representatives of the three embryonic lineages: fibroblasts Žmesoderm., keratinocytes Žectoderm. and hepatocytes Žendoderm..31,33,34 Marked differences were detected among cell types in the time course and efficiency of muscle gene expression in heterokaryons. For heterokaryons containing fibroblasts, ker atinocytes and hepatocytes, the average frequency of expression of human muscle N-CAM on the cell surface was 95, 60, and 25%, respectively. In addition, the onset of expression of N-CAM differed by at least 2 days. To understand the regulatory mechanisms that control the differentiated state, knowledge of the molecular events that constitute the lag period prior to muscle gene expression is critical. The differences among cell types are likely to reflect a requirement for different numbers or concentrations of regulators necessary to achieve muscle gene activation.
activation of several muscle genes. No significant differences in the expression of the muscle gene encoding human N-CAM were observed in the presence or absence of DNA replication. These results indicate that conformational changes in chromatin either do not accompany gene activation in heterokaryons or do not require extensive DNA replication.
A network of positive and negative regulators mediates muscle differentiation In hybrids, gene dosage, or the relative genetic contribution of the two fused cell types, is critical to the suppression of malignancy Žfor review, see ref 16.. Similarly, in heterokaryons the relative contribution of muscle and non-muscle nuclei is critical to novel gene activation.31 Both the existence of negative and positive regulators is implicated in these cell fusion experiments. Their concentration and role differs among cell types. For example, irrespective of nuclear ratio, 95% of heterokaryons containing fibroblasts from three different sources Žfetal lung, fetal skin, and adult skin. ultimately expressed human muscle genes.34,37 These results suggest that if fi broblasts produce inhibitors of muscle differentiation, like the well-documented extinguishers of liver gene activation,9,10 they are readily overridden by the positive regulators contributed by differentiated muscle cells, due to differences in their nature or concentration. By contrast, in heterokaryons produced with hepatocytes the ultimate probability of activating muscle genes was dependent on the proportion of muscle and hepatocyte nuclei at all times.33 When hepatocyte nuclei outnumbered muscle cell nuclei, human muscle genes were not activated and the mouse muscle phenotype was extinguished38 presumably due to the relative levels of negative regulators that overwhelmed the positive regulators contributed by the muscle cell type.
Regulatory mechanisms for activating silent genes Muscle gene activation was generally observed in heterokaryons with primary diploid cells, but not in heterokaryons with transformed aneuploid cell types. In one case, the Hela cell, we showed that the lack of response could be reversed.35 If treated with 5azacytidine Ž5AC. prior to fusion, muscle gene expression was induced in HeLa cells in heterokaryons. Thus muscle gene activation in HeLa cells required changes induced by 5AC, presumably in the level of DNA methylation of structural genes or the genes encoding their regulators. Thus, unlike normal cell types, muscle genes are silenced by a mechanism that is not as easily reversed in this transformed cell type. It remains to be determined whether this mechanism is generally employed to repress tissue-specific genes in transformed cell types. We designed experiments to determine whether the lag period prior to muscle gene activitation reflected a requirement for DNA replication. Possibly, negative regulation was alleviated by changes in the chromatin structure during DNA synthesis. To address this possibility, the fibroblasts, keratinocytes and hepatocytes were each exposed to a DNA synthesis inhibitor, cytosine arabinoside, prior to and continuously after cell fusion31,33,36 and assayed for the novel
Heterokaryons reveal regulation of the helix–loop–helix family of myogenic regulators To examine the molecular basis for the musclespecific gene expression observed in multinucleated heterokaryons formed from the fusion of differentiated muscle cells to either hepatocytes or fibroblasts, we tested the role of the MyoD family of regulators. MyoD and its relatives are regulators of muscle269
H. M. Blau and B. T. Blakely
specific gene expression that have a helix]loop]helix motif.39 We tested whether these regulators alone could induce the phenotypic conversion observed in heterokaryons.40 MyoD or myogenin were stably or transiently introduced into fibroblasts or hepatocytes by microinjection, transfection or retroviral infection with complementary DNA in expression vectors. Fibroblasts expressed muscle-specific genes, whereas hepatocytes did not. However, following fusion of hepatocytes stably expressing MyoD to fibroblasts, activation in the heterokaryon of muscle-specific genes of both cell types was detected. These results imply that other regulators, present in fibroblasts but not in hepatocytes, are necessary for the activation of muscle-specific genes. They also show that the differentiated state of a cell is dictated by its history and a dynamic interaction among the proteins that it contains. A non-differentiating mouse muscle cell line, NFB, that represses the activity of the helix]loop]helix ŽHLH. family of myogenic regulators, yet expresses sarcomeric actins was analyzed.41 In NFB cells, the MyoD gene is silent, but can be activated upon transfection of a long terminal region-controlled chicken MyoD cDNA, resulting in myogenesis. When NFB cells are fused with H9c2 rat muscle cells in heterokaryons, the level of rat MyoD transcripts declines. Thus, the stoichiometry of MyoD and the putative repressor controls myogenesis. Although NFB cells express myogenin and Myf-5 transcripts Žother members of the MyoD family., the activity of these regulators is also repressed: myogenesis is not induced in 10T1r2 fibroblasts and is repressed in L6 muscle cells upon fusion with NFB cells. These findings led us to conclude that myogenesis is subject to control by dominant-negative regulators.
creatine kinase distribute over large portions of the heterokaryon, whereas other proteins, such as myosin heavy chain, are restricted to a smaller area near the nucleus that encodes the protein.42 Since heterokaryons have shown that regulatory proteins such as the myogenic HLH regulators can act on a nucleus other than the one that encodes them, one might expect that heterokaryons between muscle cells expressing different isoforms of muscle proteins, such as myosin, would undergo changes in expression. For example, in heterokaryons between mouse and rat muscle cells, myosin light chain isoform expression is reprogrammed in the rat nuclei to match that seen in the mouse nuclei.43 Heterokaryons of embryonic and fetal muscle cell lines, however, maintain expression of embryonic and fetal myosin heavy chain isoforms in distinct nuclear domains.44 Thus, myosin heavy chain isoform expression is not efficiently altered by diffusible transcriptional regulatory proteins in heterokaryons, possibly because such reprogramming is more dependent on extrinsic signals, as has been shown in vivo.45 Injection of normal myoblasts into diseased muscle to form heterokaryon fibers has been proposed to treat diseases in which a myopathy is caused by the lack of functional expression of a critical protein. Such therapy is more likely to succeed if the protein of interest can freely diffuse from the expressing nucleus throughout the muscle fiber. For example, normal myoblasts that express dystrophin have been injected into the muscle of Duchenne muscular dystrophy patients or mdx mice which lack functional dystrophin.46,47 If the dystrophin encoded by the normal nucleus was restricted to a small domain around the nucleus, such treatment would not be expected to succeed. However, dystrophin is not restricted to nuclear domains in heterokaryons in vitro 48 and can in fact spread several hundred micrometers away from the nucleus of origin in vivo.49 Treatment with normal myoblasts of several other myopathies, which are mitochondrial in origin, has also been proposed. The finding that normal and diseased mitochondria rapidly migrate and mix in heterokaryons 50 supports this approach.
Heterokaryons in fiber development and disease Once a muscle cell has made the decision to differ entiate, many gene expression decisions remain. Skeletal muscle proteins, such as myosin, have multiple isoforms which participate in determining the contractile properties of the muscle fiber. Questions about muscle fiber development can also be addressed using heterokaryons. Muscle cells from different species or individuals can spontaneously fuse in vitro or in vivo to form heterokaryons. This property was used to demonstrate that in heterokaryons of mouse and human muscle, some proteins, such as cell surface molecules and cytoplasmic
Conclusions The major conclusion from the heterokaryon studies described above is that the differentiated state is not fixed but can readily be altered. However, the differentiated state in vivo appears relatively stable. 270
Plasticity of cell fate: insights from heterokaryons
Thus, an understanding of the regulation of cell differentiation by mechanisms that allow for the type of plasticity observed in heterokaryons yet stably maintain a differentiated state is of particular interest. How is the change in differentiated state achieved? Heterokaryon experiments do not support molecular models which suggest that DNA replication is required for the activation of previously silent genes.51 ] 53 Changes in chromatin structure, such as the creation of DNase hypersensitive sites, removal of histones or changes in DNA methylation could still occur. However, if they occur, these changes are mediated by mechanisms that are independent of DNA replication. The elucidation of these mechanisms is now possible. How, then, is a heritable differentiated state achieved? Heterokaryon studies suggest that the differentiated state is largely governed by the dynamic interaction of the combination of proteins a cell contains. This is particularly clear from the striking effect on gene activation observed when the relative contribution of components, or dosage, of the two fused cell types is altered. How does this pertain to differentiation in vivo? The protein composition of a cell is part of its heritage, the product of a history of responses to cues in the course of development. The cell transmits these proteins to progeny through division. The response of cells to a single regulator, such as MyoD depends on the cell context, or set of proteins, it encounters. As a result, MyoD induces muscle gene expression in some cell types, but not in others. In cases where it fails to do so, such as liver cells, additional regulators, both positive and negative, are implicated. A threshold concentration of regulators may be critical to the expression and maintenance of the differentiated state. For some proteins this may be achieved by activating the expression of their own promoters, like c-jun.54 Positive autoregulation and feedback loops are levels of control that operate during the commitment of phage lambda to lysogeny Žfor review, see ref 55. and are likely to be characteristic of early acting regulators in mammalian cell differentiation. Protein]protein interactions provide another means of controlling regulator concentration. Transcriptional regulators with ‘helix]loop]helix’ motifs bind DNA sequences as heterodimers, 56 suggesting that a range of protein]protein combinations is possible. Indeed, the activity of the MyoD family of regulators, helix] loop]helix proteins, is determined, in part, by the concentration and nature Žinhibitory or facilitating. of their protein partners.57 In heterokaryons, differ -
ent partners lead to novel heterodimers that act either as positive or negative regulators. These heterodimers can act on the same or on different promoters from those recognized by the protein complexes originally present in each of the parental cells.
References 1. Waddington CH Ž1940. Organisers and Genes pp 91]93. Cambridge University Press, London 2. Gurdon JB Ž1962. The developmental capacity of nuclei taken from intestinal epithelium cells of feeding tadpoles. J Embryol Exp Morphol 10:622]640 3. Gurdon JB, Laskey RA, Reeves OR Ž1975. The developmental capacity of nuclei transplanted from keratinized skin cells of adult frogs. J Embryol Exp Morphol 34:93]112 4. DiBerardino MA, Hoffner NJ Ž1983. Gene reactivation in erythrocytes: nuclear transplantation in oocytes and eggs of Rana. Science 219:862]864 5. DiBerardino MA, Orr NH, McKinnell RG Ž1986. Feeding tadpoles cloned from Rana erythrocyte nuclei. Proc Natl Acad Sci USA 83:8231]8234 6. DiBerardino MA Ž1988. Genomic multipotentiality of differ entiated somatic cells. Cell Differ Dev 25 Suppl:129]136 7. Mevel-Ninio M, Weiss MC Ž1981. Immunofluorescence analysis of the time-course of extinction, reexpression, and activation of albumin production in rat hepatoma-mouse fibroblast heterokaryons and hybrids. J Cell Biol 90:339]350 8. Weiss MC, Cassio D, Sellem CH Ž1988. Cell differentiation: contributions of somatic cell genetics. Cancer Surv 7:295]302 9. Killary AM, Fournier RE Ž1984. A genetic analysis of extinction: trans-dominant loci regulate expression of liver-specific traits in hepatoma hybrid cells. Cell 38:523]534 10. Petit C, Levilliers J, Ott MO, Weiss MC Ž1986. Tissue-specific expression of the rat albumin gene: genetic control of its extinction in microcell hybrids. Proc Natl Acad Sci USA 83:2561]2565 11. Davidson RL Ž1972. Regulation of melanin synthesis in mammalian cells: effect of gene dosage on the expression of differentiation. Proc Natl Acad Sci USA 69:951]955 12. Peterson JA, Weiss MC Ž1972. Expression of differentiated functions in hepatoma cell hybrids: induction of mouse albumin production in rat hepatoma-mouse fibroblast hybrids. Proc Natl Acad Sci USA 69:571]575 13. Rankin JK, Darlington GJ Ž1979. Expression of human hepatic genes in mouse hepatoma]human amniocyte hybrids. Somatic Cell Genet 5:1]10 14. Harris H, Sidebottom E, Grace DM, Bramwell ME Ž1969. The expression of genetic information: a study with hybrid animal cells. J Cell Sci 4:499]525 15. Peehl DM, Stanbridge EJ Ž1982. The role of differentiation in the suppression of tumorigenicity in human cell hybrids. Int J Cancer 30:113]120 16. Harris H Ž1998. The analysis of malignancy by cell fusion: the position in 1988. Cancer Res 48:3302]3306 17. Friend SH, Bernards R, Rogelj S, Weinberg RA, Rapaport JM, Albert DM, Dryja TP Ž1986. A human DNA segment with properties of the gene that predisposes to retinoblastoma and osteosarcoma. Nature 323:643]646 18. Harris H, Watkins JF Ž1995. Hybrid cells derived from mouse and man: artificial heterokaryons of mammalian cells from different species. Nature 205:640]646 19. Ringertz NR, Carlsson SA, Ege T, Bolund L Ž1971. Detection of human and chick nuclear antigens in nuclei of chick
271
H. M. Blau and B. T. Blakely
20. 21.
22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.
33.
34. 35. 36. 37. 38. 39.
erythrocytes during reactivation in heterokaryons with HeLa cells. Proc Natl Acad Sci USA 68:3228]3232 Ringertz NR, Savage RE Ž1976. Cell Hybrids. Academic Press, New York Carlsson SA, Luger O, Ringertz NR, Savage RE Ž1974. Phenotypic expression in chick erythrocyte x rat myoblast hybrids and in chick myoblast x rat myoblast hybrids. Exp Cell Res 84:47]55 Lawrence JB, Coleman JR Ž1984. Extinction of muscle-specific properties in somatic cell heterokaryons. Dev Biol 101:463]476 Wright WE, Aronoff J Ž1983. The suppression of myogenic functions in heterokaryons formed by fusing chick myocytes to diploid rat fibroblasts. Cell Differ 12:299]306 Konieczny SF, Lawrence JB, Coleman JR Ž1983. Analysis of muscle protein expression in polyethylene glycol-induced chicken: rat myoblast heterokaryons. J Cell Biol 97:1348]1355 Blau HM, Chiu CP, Webster C Ž1983. Cytoplasmic activation of human nuclear genes in stable heterocaryons. Cell 32:1171]1180 Wright WE Ž1984. Expression of differentiated functions in heterokaryons between skeletal myocytes, adrenal cells, fi broblasts and glial cells. Exp Cell Res 151:55]59 Wright WE Ž1984. Induction of muscle genes in neural cells. J Cell Biol 98:427]435 Baron MH, Maniatis T Ž1986. Rapid reprogramming of globin gene expression in transient heterokaryons. Cell 46:591]602 Wu KJ, Samuelson LC, Howard G, Meisler MH, Darlington GJ Ž1991. Transactivation of pancreas-specific gene sequences in somatic cell hybrids. Mol Cell Biol 11:4423]4430 Spear BT, Tilghman SM Ž1990. Role of alpha-fetoprotein regulatory elements in transcriptional activation in transient heterokaryons. Mol Cell Biol 10:5047]5054 Blau HM, Pavlath GK, Hardeman EC, Chiu CP, Silberstein L, Webster SG, Miller SC, Webster C Ž1985. Plasticity of the differentiated state. Science 230:758]766. Hardeman EC, Chiu CP, Minty A, Blau HM Ž1986. The pattern of actin expression in human fibroblast x mouse muscle heterokaryons suggests that human muscle regulatory factors are produced. Cell 47:123]130 Miller SC, Pavlath GK, Blakely BT, Blau HM Ž1988. Muscle cell components dictate hepatocyte gene expression and the distribution of the Golgi apparatus in heterokaryons. Genes Dev 2:330]340 Pavlath GK, Chiu CP, Blau HM Ž1989. in vivo aging of human fibroblasts does not alter nuclear plasticity in heterokaryons. Somat Cell Mol Genet 15:191]202 Chiu CP, Blau HM Ž1985. 5-Azacytidine permits gene activation in a previously non-inducible cell type. Cell 40:417]424 Chiu CP, Blau HM Ž1984. Reprogramming cell differentiation in the absence of DNA synthesis. Cell 37:879]887 Pavlath GK, Blau HM Ž1986. Expression of muscle genes in heterokaryons depends on gene dosage. J Cell Biol 102:124]130 Blakely BT Ž1993. Negative and Positive Regulatory Mechanisms in Muscle and Liver Differentiation. Stanford University Dissertation, Stanford, CA Davis RL, Weintraub H, Lassar AB Ž1987. Expression of a single transfected cDNA converts fibroblasts to myoblasts. Cell 51:987]1000
40. Schafer BW, Blakely BT, Darlington GJ, Blau HM Ž1990. Effect of cell history on response to helix]loop]helix family of myogenic regulators. Nature 344:454]458 41. Peterson CA, Gordon H, Hall ZW, Paterson BM, Blau HM Ž1990. Negative control of helix]loop]helix family of myogenic regulators in the NFB mutant. Cell 62:493]502 42. Pavlath GK, Rich K, Webster SG, Blau HM Ž1989. Localization of muscle gene products in nuclear domains. Nature 337:570]573 43. Pajek L, Mariappan M, Wieczorek DF Ž1991. Reprogramming of myosin light chain 1r3 expression in muscle heterokaryons. Dev Biol 145:28]39 44. Pin CL, Merrifield PA Ž1997. Regionalized expression of myosin isoforms in heterotypic myotubes formed from embryonic and fetal rat myoblasts in vitro. Dev Dyn 208:420]431 45. Hughes SM, Taylor JM, Tapscott SJ, Gurley CM, Carter WJ, Peterson CA Ž1993. Selective accumulation of MyoD and myogenin mRNAs in fast and slow adult skeletal muscle is controlled by innervation and hormones. Development 118:1137]1147 46. Gussoni E, Pavlath GK, Lanctot AM, Sharma KR, Miller RG, Steinman L, Blau HM Ž1992. Normal dystrophin transcripts detected in Duchenne muscular dystrophy patients after myoblast transplantation. Nature 356:435]438 47. Karpati G, Pouliot Y, Zubrzycka-Gaarn E, Carpenter S, Ray PN, Worton RG, Holland P Ž1989. Dystrophin is expressed in mdx skeletal muscle fibers after normal myoblast implantation. Am J Pathol 135:27]32 48. Denetclaw WF Jr, Bi G, Pham DV, Steinhardt RA Ž1993. Heterokaryon myotubes with normal mouse and Duchenne nuclei exhibit sarcolemmal dystrophin staining and efficient intracellular free calcium control. Mol Biol Cell 4:963]972 49. Gussoni E, Blau HM, Kunkel LM Ž1997. The fate of individual myoblasts after transplantation into muscles of DMD patients. Nat Med 3:970]977 50. Walker UA, Walker WF, Miranda AF Ž1997. Mitochondrial mobility in differentiating muscle heterokaryons. J Neurol Sci 151:135]140 51. Holtzer H, Biehl J, Payette R, Sasse J, Holtzer S Ž1983. Cell diversification: differing roles of cell lineages and cell]cell interactions, in Limb Development and Regeneration pp. 272]280. Alan R. Liss, New York 52. Brown DD Ž1984. The role of stable complexes that repress and activate eucaryotic genes. Cell 37:359]365 53. Weintraub H Ž1985. Assembly and propagation of repressed and depressed chromosomal states. Cell 42:705]711 54. Angel P, Hattori K, Smeal T, Karin M Ž1988. The jun protooncogene is positively autoregulated by its product, JunrAP-1. Cell 55:875]885. 55. Ptashne M Ž1986. A Genetic Switch: Gene Control and Phage l. Cell Press and Blackwell Scientific Publications, Cambridge, MA. 56. Murre C, McCaw PS, Vaessin H, Caudy M, Jan LY, Jan YN, Cabrera CV, Buskin JN, Hauschka SD, Lassar AB et al Ž1989. Interactions between heterologous helix]loop]helix proteins generate complexes that bind specifically to a common DNA sequence. Cell 58:537]544 57. Lemercier C, To RQ, Carrasco RA, Konieczny SF Ž1998. The basic helix]loop]helix transcription factor Mist1 functions as a transcriptional of myoD. Embo J 17:1412]1422
272