Differentiation requires continuous regulation

IFFERENTIATION has long been conceived of as a series of binary decisions. Wsddington's epigenetic landscape (1940) provides a particularly vivid visual image of this concept: differentiation is likened to the path taken by a ball as it rolls down a sloped surface grooved by valleys. Similarly, according to Stuart Kauffman's binary model (1973), early choices limit later probabilities for changes in cell fate that accompany determination and transdetermination. These theories have led to the still widely held view that progression along a developmental pathway entails changes that exclude future possibilities. This view is also embodied in commonly used terms such as "committed stem cell" and "terminally differentiated cell7 It suggests that cell states are locked in place by mechanisms that are not easily disrupted. Two molecular mechanisms that could silence genes that are inappropriate to a given differentiated cell type are "passive" and ~activeY It seems simplest to silence genes by a "passive control" mechanism: closing down unneeded genes so that they do not require active consideration in a given cell lineage for the life of the organism. Thus, commitment, like Lyonization of the X chromosome, would result in the permanent inactivation of many unnecessary genes. Indeed, what would be the advantage of keeping muscle genes accessible in the liver? Alternatively, differentiation could be governed by an ~active control" mechanism: the expression state of each gene being determined by the dynamic interaction of regulatory proteins present in the cell at any given time. This second possibility is often dismissed for the following reasons: (a) differentiation appears to be stable, (b) plasticity seems unnecessary, and (c) the number of regulators required appears cumbersome. In particular, the investment in negative regulators necessary to maintain the majority of genes in a silent state seems disproportionately large. In spite of the appeal of passive control, accumulating evidence suggests that differentiation is stably maintained by continuous regulation, both by positive (Britten and Davidson, 1969), and by negative regulators. We first present the evidence and then the implications of the continuous mode of regulation. Gurdon's (1962) nuclear transplantation experiments showed that genes were neither lost nor permanently inactivated during development. Upon transfer of an intestinal cell nucleus into an enucleated egg, entire swimming tadpoles developed. However, the frequency of this event was low, unless nuclei were first injected into oocytes (DiBerardino et al., 1986), a step that might allow reprogramming by stripping the DNA of mitotically heritable regulatory influences. Thus, although these experiments provided strong evidence that differentiation was reversible, they did not determine whether genes were silenced by active or passive mechanisms in the course of development. Cell fusion experiments showed that gene activation did not require ooplasm. Genes typical of other differentiated cell types, such as muscle genes in a liver cell, or adult globin genes in a fetal erythroid cell, were transcribed in heterokaryons (Blau et al., 1985; Baron and Maniatis, 1986). Moreover, the activation of silent genes occurred without DNA replication or a replay of the hierarchy of regulators characteristic of development from a fertilized egg to a differentiated tissue. Recently, the constitutive expression of a single cloned regulator, MyoD, was shown to activate the silent muscle genes, myosin hea W chain and desmin, in a range of nonmuscle cell types (Davis et al., 1987). Indeed, in fibroblasts, MyoD caused a complete phenotypic conversion: a muscle-specificdistribution of organelles and pattern ofgene expression was stably inherited. A muscle phenotype was also induced in more distantly related cell types when MyoD was presumably combined with additional regulators (Schaefer et al., 1990). These experimental manipulations demonstrate that silent genes in'committed" differentiated cells, are readily accessible suggesting that their repression is not passively, but actively controlled. Critical to the active control hypothesis is evidence that the continuous activity of positive and negative regulators is required to maintain differentiation in the course of normal development. A clear test would show that a disruption in expression of a nodal, or key, regulatory gene alters the fate of cells that are already differentiated. Two types of elegant experimental system have made this test possible: temperature-sensitive mutants and somatic mosaics. Both examine the temporal window during which a particular gene product is required by interfering with its expression, either through a shift in temperature or by x-ray-induced mitotic recombination or chromosome loss. In Drosophila and Caenorhabditis elegans such experiments have shown that unless regulatory gene expression is continuous throughout adult development, sexual characteristics, such as production of sperm and synthesis of egg yolk proteins, are lost (Kimble et al., 1984; Belote et al., 1985). Similarly, neural cell identity and pattern formation are altered even at late larval stages by disruption of the expression of critical genes, including members of the large polycomb family that encode negative regulators (Duncan and Lewis, 1982; Way and Chalfie, 1989). Perhaps the most striking example of plasticity is found in the adult Drosophila central nervous system: a female will engage in a complex male courtship behavior if exposed to a shift in temperature that disrupts the expression of the tra-2 gene (Belote