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Quantitative genetic variation and developmental clocks
£ theor. Biol. (1991) 151, 351-358
Quantitative Genetic Variation and Developmental Clocks ROBIN HOLLIDAY
CSIRO Laboratory for Molecular Biology, Division of Biomolecular Engineering, P.O. Box 184, North Ryde, N S W 2113, Australia (Received on 17 July 1990, Accepted on 18 December 1990) It is well-known that most genetic variation affects quantitative traits, and natural or artificial selection can act to change quantitative features of organisms more rapidly than qualitative ones. Surprisingly, variability is not confined to outbred species, but also occurs in inbred mice at a much higher rate than expected from known mutation rates. The size and shape of organisms and their constituent parts are, at least in part, controlled by the number of cell divisions, and there is published evidence for the existence of developmental clocks, which may count cell divisions. A molecular model for a developmental clock was previously proposed. It depends on the D N A methylation of repeated sequences of DNA, where the methylation of each additional sequence is tied to D N A synthesis and therefore celt division. The number of repeats specifies the number of divisions which will occur before a signal is produced which can activate or inactivate one or more genes. It is known that crossing over occurs between sister chromatids, and where tandemly repeated sequences occur unequal exchange can generate a larger or smaller number of repeats. An example of this is seen in the well-known variability of "minisatellite" sequences in human DNA. Unequal sister chromatid exchange can occur in mitotic and meiotic cells in the germ line, and in the case of developmental clock sequences could generate variation in clock length which in turn would directly affect quantitative traits. These events can be regarded as a special case of molecular drive during evolution.
Introduction It is a w e l l - k n o w n t a x o n o m i c p r i n c i p l e t h a t q u a n t i t a t i v e traits are m u c h m o r e v a r i a b l e t h a n q u a l i t a t i v e ones. T h e m a j o r t a x o n o m i c g r o u p s a r e c h a r a c t e r i z e d b y c o n s t a n t s t r u c t u r a l features, r e f e r r e d to as h o m o l o g o u s s t r u c t u r e s , w h i c h v a r y in size, s h a p e , length in the species, g e n e r a , o r d e r s a n d so on, w i t h i n e a c h m a j o r g r o u p . It c a n also b e s t a t e d with c e r t a i n t y that n o r m a l g e n e t i c v a r i a t i o n affects q u a n t i t a t i v e f e a t u r e s o f an o r g a n i s m to a far g r e a t e r extent t h a n it c h a n g e s the q u a l i t a t i v e o r h o m o l o g o u s s t r u c t u r e s o f the t a x o n o m i c g r o u p in q u e s t i o n . D a r w i n (1868) was w e l l - a w a r e o f this a n d he d o c u m e n t e d t h e e x t r e m e v a r i a b i l i t y o f d o m e s t i c a t e d p l a n t s a n d a n i m a l s . H e s h o w e d t h a t artificial s e l e c t i o n c o u l d r a p i d l y p r o d u c e these differences, the g r e a t m a j o r i t y o f w h i c h are q u a n t i t a t i v e . F o r e x a m p l e , the e x t r e m e v a r i a t i o n in leg length, b o d y a n d facial f e a t u r e s o f different b r e e d s o f d o g s has b e e n a c h i e v e d o v e r a t i m e scale w h i c h is trivial in c o m p a r i s o n to t h a t r e q u i r e d for c o m p a r a b l e e v o l u t i o n a r y c h a n g e s b y n a t u r a l selection. In n a t u r a l e v o l u t i o n t h e s a m e v a r i a t i o n o n a t h e m e is seen. I n m a m m a l s t h e n u m b e r o f l i m b s a n d t h e i r u n d e r l y i n g s k e l e t a l s t r u c t u r e s r e m a i n c o n s t a n t , b u t t h e v a r i a t i o n in t h e d i m e n s i o n s o f s u c h l i m b s is e n o r m o u s . 351
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Similarly basic physiological features o f a taxonomic group remain remarkably constant. Whales retain the need to breathe air, maintain a constant body temperature, reproduce and feed newborn offspring in the same way as land based mammals, or bats. It is often the case that homologous structures retain their basic morphology, but vary primarily in size. For example, vertebrae in rodents are anatomically very similar to those in whales, but the difference in bone weight can be up to 107-fold. Morphometric analysis o f plants and animals was carried out by D'Arcy Thompson (1942) and he showed that the changes in shape or form o f a particular part of a plant or animal could be due to spatial determinants in two or three dimensions. Such determinants could be easily altered by natural selection. In outbreeding species, the response to artificial selection shows that the genetic control of many quantitative traits is polygenic, since the changes can occur over many generations before becoming stabilized (reviewed by Falconer, 1981). According to basic genetic principles, highly inbred mice should be homozygous at all loci, and therefore show no variability in constant laboratory environments, apart from occasional mutations. These animals show unexpected variability in skeletal structures (Deol et al., 1957; Grewal, 1962; Gruneberg, 1963; Hoi-Sen, 1972; Festing, 1973). Gruneberg (1970) pointed out that this variability was far higher than that expected from known mutation frequencies, and demanded special explanation. He went on to suggest that the skeletal variability seen in inbred strains might be accounted for by the presence of one or more latent viruses transmitted vertically from generation to generation. Here, a different mechanism for quantitative variability is proposed, which could operate in inbred or outbred plants and animals. It addresses particularly the question of variation in size, but if we bear in mind D'Arcy Thompson's analysis, we can conclude that an increase or decrease of a particular morphometric determinant can also affect the shape o f structures in two or three dimensions.
Developmental Clocks The size of an organism or its constituent parts in a uniform environment is genetically controlled. In many cases adults cease growth at a particular size, in other cases, such as perennial plants, growth may continue, but the size and shape o f individual components, such as leaves or flowers, are largely invariant. Any limit to growth means that the actual number o f cell divisions is controlled. It follows that mechanisms must exist which are capable o f measuring or counting cell divisions, and these are referred to as developmental clocks. Evidence o f the existence of developmental clocks is both general and specific. For example, the growth o f the chick retina has been analysed in detail both experimentally and theoretically (Morris & Cowan, 1984). There is proliferation until a given number of cells is produced, and then mitotic activity ceases. The interpretation o f the results is certainly consistent with the existence o f an intrinsic clock or cell division counting mechanism. In studies o f the development of the chick wing bud it was shown by grafting experiments that a terminal progress zone
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exists which consists o f dividing cells and is responsible for the ordered formation of the bones of the wing (Summerbell et al., 1973). When the progress zone from a later stage of development was substituted for one at an earlier stage, it gave rise to a truncated limb with absence of distal bone structures. The reciprocal grafts resulted in a wing consisting of two sets of bones, tandemly duplicated. These observations are also consistent with the existence of a developmental clock in which cells in the progress zone form the appropriate structures in sequence, and the specification depends on the number o f times these cells have divided. More recently studies on cell lineages in the developing rat optic nerve show that type 2 astrocyte progenitor cells undergo a specific number of mitotic divisions, in the presence of type 1 astrocytes, before differentiation to oligodendrocytes occurs (Temple & Raft, 1986). More generally, the existence of invariant cell pedigrees during development (reviewed by Davidson, 1986) implies strict control over cell division and the subsequent fate of individual cells. A developmental clock need not count several or many cell divisions, but also one or two divisions, which is the requirement in pedigrees. Moreover, there is no reason, in principle, why there should not be two or more overlapping clocks in a pedigree, or any other developmental situation. Imagine a clock at zero time set to run five divisions, but which also triggers a second clock after three. For the first three divisions one clock is operating, for the next, two clocks, and thereafter only one again for whatever time is specified. In developmental biology there has been much emphasis placed on pattern formation and the organization of cells and tissues in three-dimensional space. In this type of analysis, whether experimental or theoretical, the significance of temporal controls is not usually emphasized. It is obvious that cell divisions are essential for normal development, but Holtzer has long argued more specifically that quantal cell cycles are an essential component of the differentiation process (reviewed by Holtzer et al., 1975). Further evidence for the importance o f cell divisions tied to gene expression and developmental clocks has also been reviewed by Satoh (1982) and Stubblefield (1986). The significance of strict temporal controls has recently come to light in detailed studies of the development of the embryos of Drosophila (Foe, 1989) and Xenopus (Cooke & Smith, 1990). However, these particular controlling events are not tied to cell division counting mechanisms.
A Molecular Mechanism for Counting Cell Divisions It was previously proposed that cell division counting mechanisms might be based on the processive modification of short tandemly repeated D N A sequences (Holliday & Pugh, 1975). Since that time considerable evidence has accumulated for the functional significance o f 5-methylcytosine in a variety of biological contexts. These include the inactivation and reactivation of the X chromosome and autosomal genes, genomic imprinting, the expression of genes with specialized functions, the activity o f viruses and transposable elements, and the induction of specific differentiation pathways in cultured mammalian cells. For recent reviews see Cedar, 1988; Volpe
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& Nicolini, 1989; Doerfler, 1989; Holliday et al., 1990; Holliday, 1990a). The clock mechanism based on D N A methylation invokes two enzymes. First, a switch or setting enzyme which starts the clock; and secondly a "clock" enzyme which methylates both strands o f one repeated sequence after each round of D N A synthesis, as shown in Fig. 1. Once the clock has been set in a given cell, the counting of cell divisions will be synchronized in all progeny. After a given number of divisions, specified by the tandemly repeated D N A substrates, it is assumed that a methylation signal activates or represses one or more genes downstream from the clock sequences. Alternatively, it is possible that there is progressive demethylation tied to cell division. A D N A binding protein might recognize adjacent non methylated and a hemimethyINITIAL SIGNAL
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FIG. 1. A molecular model for a cell division counting mechanism or clock, essentially the same as that proposed by Holtiday & Pugh (1975). There are three components to the model. First, a switch or setting DNA methylase recognizes and methylates (closed circle) one strand of a DNA sequence to the left of the series of repeated elements which constitute the clock itself. This initial signal provides the substrate for the second component, the clock methylating enzyme. This has the property methylating both strands of the first of the repeated sequences, but no further repeats (arrows and closed circles). It specifically requires a hemimethylated substrate, which arises after DNA replication (dashed lines), and then the second repeat is methylated. There is thus a "'growing point" of methylation tied to cell division. The sequences in newly replicated DNA behind the growing point may or may not be methylated (open circles). In the example shown there are five repeats, and after four cell divisions, the last one, on the right, will become methylated. This triggers the third component o f t h e clock, namely, the elicitation o f a signal, such as the activation or repression of a gene to the right of the clock sequences.
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lated sequence at the replication fork and prevents the maintenance methylase acting on that particular hemimethylated substrate. The clock principle is the same as in Fig. 1, but the active process is the progressive demethylation of tandemly repeated sequences. A third mechanism was also proposed by Holliday & Pugh (1975), which depends on the specific modification of bases and leads to A - T ~ G-C transitions at individual sites in tandemly repeated sequences. It had earlier been suggested by Scarano (1971) that such transitions in DNA might be important in the control of developmental processes. So far evidence for this proposal has not been forthcoming, although such base change modifications are known to occur in RNA (reviewed by Benne, 1990).
Unequal Crossing Over Between Sister Chromatids Differential staining of sister chromatids containing bromodeoxyuridine in one or both polynucleotide chains in DNA makes it possible to demonstrate crossing-over between these chromatids (reviewed by Wolff, 1977). Such crossing-over appears to be randomly distributed and is relatively common, for example, in a human diploid cell there are five to six events per mitosis. DNA damaging agents greatly increase the frequency of these sister chromatid exchanges (SCEs), which suggests that they may have a role in DNA repair. The exact reciprocity of SCEs shows that homologous DNA sequences are required, and also indicates that the exchanges are accurate, without the loss of gain of genetic material. However, in the case of tandemly repeated sequences, the possibility of unequal exchange arises. Starting with n repeats, an unequal exchange in the participating chromatids could generate n + l a n d n - l , or n + 2 a n d n - 2 repeats, and so on. There has been much discussion of the evolution of multigene families, particularly by Dover (reviewed by Dover, 1986). In the case of tandemly repeated DNA sequences, such as those coding for ribosomal DNA or histones, unequal sister chromatid exchange can generate greater or smaller numbers of repeats. In conjunction with other genetic mechanisms, mutations in one repeat can spread to others to bring about the end result of "homogenization". Jeffreys and his co-workers (Jeffreys et al., 1985a, b; Jeffreys, 1987) have demonstrated the existence of much smaller tandemly repeated sequences in the DNA of humans and other species. The remarkable features of this "minisatellite" DNA is that the number of repeats varies from individual-to-individual, as judged by the pattern of bands in genomic DNA hybridized in Southern blots to probes containing these repeated sequences. The restriction enzyme(s) used cut the genomic DNA on each side of the repeats, so the variation in fragment size is due to the variation in the copy number of the repeated sequence. The polymorphism seen follows normal Mendelian inheritance, since offspring almost invariably contain individual bands derived from one or the other parent, but in several pedigrees a new variant occurred in one case, which could have arisen by unequal crossing over. Originally it was thought that the minisatellite sequences comprised recombination hot spots, which promoted recombination between homologous chromosomes. More recent data indicate that
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unequal crossing over between sister chromatids, either during mitosis of germ line cells or at meiosis, is the more likely mechanism for generating changes in repeat copy number (Jeffreys et al., 1990). The same process could also produce changes in the counting specification of developmental clocks at frequencies much higher than normal mutations, and this event would be expected to occur as readily in inbred organisms as outbred ones. The variability generated, on which natural selection could act, could be regarded as a special case of "molecular drive" acting on repeated sequences of DNA during evolution (Dover, 1986). It was mentioned that quantitative genetic traits are commonly under polygenic control, so it is important to emphasize that a change in clock length would be only one of several components in the regulation of size or shape which is subject to natural selection. Variation in the number of tandem repeats could occur in both homologous chromosomes. Such "heterozygosity" in the specification of clock length could have abnormal effects on development. The same problem arises if important developmental switches in gene activity are operating independently in homologues. For this reason it was suggested that important regulatory genes, or loci, would be subject to allelic exclusion so that they are effectively haploid (Holliday & Pugh, 1975; Holliday, 1990b). The same argument can be made for developmental clocks. Conclusions
Several arguments have been brought together in formulating a general hypothesis. The first is the well-known variability in quantitative traits in plants and animals and the rapid response to the selection of any particular trait. The second is the continuing variability which is seen to occur in inbred mouse populations, which occurs at a rate much higher than expected from single gene mutations. The third is concerned with the specifications of size and shape in higher organisms. It is obvious that there are controls on the extent of cell division, but the more specific proposal is that developmental clocks exist which count cell divisions and therefore provide temporal control of development. The existence of strict cell pedigrees argues that the segregation o f different cell types is tied to cell division, and other evidence o f developmental clocks also exists. Fourth, the D N A methylation model for the control o f gene activities in the development of higher organisms can be extended to a cell division counting mechanism (Fig. 1), based on the processive methylation of short tandemly repeated sequences of DNA. After each round of D N A synthesis one additional repeat is methylated. Fifth, it is well-known that unequal crossing over can occur between tandemly repeated homologous DNA sequences, and this can generate greater or smaller numbers of repeats. Such unequal crossing over can occur between sister chromatids in mitotic and meiotic cells, and probably also between homologous chromosomes at meiosis. The frequency of these events in germ line cells are likely to be far higher than normal mutation frequencies. They also provide a source o f genetic variability in homozygous inbred organisms. The unifying proposal is that developmental clocks are important in controlling major quantitative features of organisms, that such clocks are based on the processive modification of short tandemly repeated sequences of DNA, and unequal crossing
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over can change the number of repeats and therefore generate quantitative variation. I t h a n k Alec Jeffreys, Gabriel Dover, Richard C o w a n a n d J o h n Pugh for their interest a n d helpful c o m m e n t s . REFERENCES BENNE, R. (1990). RNA editing in trypanosomes: is there a message? Trends Genet. 6, 177-181 CEDAR, H. (1988). DNA methylation and gene activity. Cell 53, 3-4. COOKE, J. & SMITH, J. C. (1990). Measurement of developmental time by cells of early embryos. Cell 60, 891-894. DARWIN, C. R. (1868). The Variation o f Animals and Plants under Domestication. London: Murray. DAV1DSON, E. H. (t986). Gene Activity in Early Development 3rd edn. New York: Academic Press. DEOL, M. S., GRUNEaERG, H., SEARLE, A. G. & TRUSLOVE,G. M. (1957). Genetical differentiation involving morphological characters in an inbred strain of mice. I. A British branch of the C57BL strain. Jr. Morphol. 100, 345-376. DOERFLER, W. (1989). Complexities in gene regulation by promoter methylation. In: Nucleic Acids and Molecular Biology (Eckstein, F. & Lilley, D. M. J., eds) 3, 92-119. Berlin: Springer Verlag. DOVER, G. A. (1986). Molecular drive in multigene families: how biological novelties arise, spread and are assimilated. Trends Genet. 2, 159-165. FALCONER, D. S. (1981). Introduction to Quantitative Genetics 2nd edn. New York: Longman. FESTING, M. (1973). A multivariate analysis of subline divergence in the shape of the mandible in C-57BL-GR mice. Genet. Res. 21, 121-132. FOE, V. E. (1989). Mitotic domains reveal commitment of cells in Drosophila embryos. Development 107, 1-22. GREWAL, M. S. (1962). The rate of genetic divergence of sublines in the C57BL strain of mice. Genet. Res. 3, 226-237. GRUNEBERG, H. (1963). The Pathology of Development: A Study of Inherited Skeletal Disorders in Animals. Oxford: Blackwell. G RUN EaERG, H. (1970). Is there a viral component in the genetic background ? Nature, Lond. 225, 39-41. HoI-SEN, Y. (1972). Is subline differentiation a continuing process in inbred strains of mice? Genet. Res. 19, 53-59. HOLLIDAY, R. (1990a). Genetic mechanisms for the control of gene activity during development. Biol. Rev. 65, 431-47 I. HOLLIDAY, R. (1990b). Genomic imprinting and allelic exclusion. Development (Suppl.), 125-129. HOLLIDAY, R., MONK, M. & PUGH, J. E. (eds) (1990). DNA Methylation and Gene Regulation. London: Royal Society. HOLLIDAY, R. & PUGH, J. E. (1975). DNA modification mechanisms and gene activity during development. Science 187, 226-232. HOLTZER, H., RUBINSTEIN, N., FELLINI, S., YEOH, G., CHI, J., BtRNBAUM, J. & OKAYAMA, M. (1975). Lineages, quantal cell cycles and the generation of cell diversity. Quant. Reo. Biophys. 8, 523-557. JEFFREYS, A. J. (1987). Highly variable minlsatellites and DNA fingerprints. Biochem. Soc. Trans. 15, 309-317. JEFFREYS, m. J., NEUMANN, R. & WILSON, V. (1990). Repeat unit sequence variation in minisatellites: a novel source of DNA polymorphism for studying variation and mutation by single molecule analysis. Cell 60, 473-485. JEFFREYS, A. J., WILSON, V. & THEI N, S. L. (1985a). Hypervariable "minisatellite" regions in human DNA. Nature, Lond. 314, 67-73. JEFFREYS, A. J., WILSON, V. & THEI N, S. L. (1985b). Individual-specific "'fingerprints" of human DNA. Nature, Lond. 316, 76-79. MORRIS, V. B. & COWAN, R. (1984). A growth curve of cell numbers in the neural retina of embryonic chicks. Cell Tissue Kinet. 17, 199-208. SATOH, N. (1982). Timing mechanisms in early embryonic development. Differentiation 22, 156-163. SCARANO, E. (1971). The control of gene function in cell differentiation and in embryogenesis. Adv. Cytopharmacol. 1, 13-24. STUBBLEFIELD, E, (1986). A theory for developmental control by a programme encoded in the genome. J. theor. Biol. 118, 129-143.
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SUMMERBELL,D., LEwls, J. H. & WOLPERT,L. (1973). Positional information in chick limb morphogenesis. Nature, Lond. 244, 492-496. TEMPLE, S. & RAFF, M. C. (1986). Clonal analysis of oligodendrocyte development in culture: evidence for a developmental clock that counts cell divisions. Cell 44, 773-779. THOMPSON, D'ARCY, W. (1942). On Growth and Form 2nd edn. Cambridge University Press. VOLPE, E. & NICOLINI, C. (eds) (1989). Forum on eukaryotic DNA methylation, Cell Biophys. 15, 1-155. WOLFF, S. (1977). Sister chromatid exchange. Annu. Rev. Genet. 11, 183-201.
Quantitative Genetic Variation and Developmental Clocks ROBIN HOLLIDAY
CSIRO Laboratory for Molecular Biology, Division of Biomolecular Engineering, P.O. Box 184, North Ryde, N S W 2113, Australia (Received on 17 July 1990, Accepted on 18 December 1990) It is well-known that most genetic variation affects quantitative traits, and natural or artificial selection can act to change quantitative features of organisms more rapidly than qualitative ones. Surprisingly, variability is not confined to outbred species, but also occurs in inbred mice at a much higher rate than expected from known mutation rates. The size and shape of organisms and their constituent parts are, at least in part, controlled by the number of cell divisions, and there is published evidence for the existence of developmental clocks, which may count cell divisions. A molecular model for a developmental clock was previously proposed. It depends on the D N A methylation of repeated sequences of DNA, where the methylation of each additional sequence is tied to D N A synthesis and therefore celt division. The number of repeats specifies the number of divisions which will occur before a signal is produced which can activate or inactivate one or more genes. It is known that crossing over occurs between sister chromatids, and where tandemly repeated sequences occur unequal exchange can generate a larger or smaller number of repeats. An example of this is seen in the well-known variability of "minisatellite" sequences in human DNA. Unequal sister chromatid exchange can occur in mitotic and meiotic cells in the germ line, and in the case of developmental clock sequences could generate variation in clock length which in turn would directly affect quantitative traits. These events can be regarded as a special case of molecular drive during evolution.
Introduction It is a w e l l - k n o w n t a x o n o m i c p r i n c i p l e t h a t q u a n t i t a t i v e traits are m u c h m o r e v a r i a b l e t h a n q u a l i t a t i v e ones. T h e m a j o r t a x o n o m i c g r o u p s a r e c h a r a c t e r i z e d b y c o n s t a n t s t r u c t u r a l features, r e f e r r e d to as h o m o l o g o u s s t r u c t u r e s , w h i c h v a r y in size, s h a p e , length in the species, g e n e r a , o r d e r s a n d so on, w i t h i n e a c h m a j o r g r o u p . It c a n also b e s t a t e d with c e r t a i n t y that n o r m a l g e n e t i c v a r i a t i o n affects q u a n t i t a t i v e f e a t u r e s o f an o r g a n i s m to a far g r e a t e r extent t h a n it c h a n g e s the q u a l i t a t i v e o r h o m o l o g o u s s t r u c t u r e s o f the t a x o n o m i c g r o u p in q u e s t i o n . D a r w i n (1868) was w e l l - a w a r e o f this a n d he d o c u m e n t e d t h e e x t r e m e v a r i a b i l i t y o f d o m e s t i c a t e d p l a n t s a n d a n i m a l s . H e s h o w e d t h a t artificial s e l e c t i o n c o u l d r a p i d l y p r o d u c e these differences, the g r e a t m a j o r i t y o f w h i c h are q u a n t i t a t i v e . F o r e x a m p l e , the e x t r e m e v a r i a t i o n in leg length, b o d y a n d facial f e a t u r e s o f different b r e e d s o f d o g s has b e e n a c h i e v e d o v e r a t i m e scale w h i c h is trivial in c o m p a r i s o n to t h a t r e q u i r e d for c o m p a r a b l e e v o l u t i o n a r y c h a n g e s b y n a t u r a l selection. In n a t u r a l e v o l u t i o n t h e s a m e v a r i a t i o n o n a t h e m e is seen. I n m a m m a l s t h e n u m b e r o f l i m b s a n d t h e i r u n d e r l y i n g s k e l e t a l s t r u c t u r e s r e m a i n c o n s t a n t , b u t t h e v a r i a t i o n in t h e d i m e n s i o n s o f s u c h l i m b s is e n o r m o u s . 351
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Similarly basic physiological features o f a taxonomic group remain remarkably constant. Whales retain the need to breathe air, maintain a constant body temperature, reproduce and feed newborn offspring in the same way as land based mammals, or bats. It is often the case that homologous structures retain their basic morphology, but vary primarily in size. For example, vertebrae in rodents are anatomically very similar to those in whales, but the difference in bone weight can be up to 107-fold. Morphometric analysis o f plants and animals was carried out by D'Arcy Thompson (1942) and he showed that the changes in shape or form o f a particular part of a plant or animal could be due to spatial determinants in two or three dimensions. Such determinants could be easily altered by natural selection. In outbreeding species, the response to artificial selection shows that the genetic control of many quantitative traits is polygenic, since the changes can occur over many generations before becoming stabilized (reviewed by Falconer, 1981). According to basic genetic principles, highly inbred mice should be homozygous at all loci, and therefore show no variability in constant laboratory environments, apart from occasional mutations. These animals show unexpected variability in skeletal structures (Deol et al., 1957; Grewal, 1962; Gruneberg, 1963; Hoi-Sen, 1972; Festing, 1973). Gruneberg (1970) pointed out that this variability was far higher than that expected from known mutation frequencies, and demanded special explanation. He went on to suggest that the skeletal variability seen in inbred strains might be accounted for by the presence of one or more latent viruses transmitted vertically from generation to generation. Here, a different mechanism for quantitative variability is proposed, which could operate in inbred or outbred plants and animals. It addresses particularly the question of variation in size, but if we bear in mind D'Arcy Thompson's analysis, we can conclude that an increase or decrease of a particular morphometric determinant can also affect the shape o f structures in two or three dimensions.
Developmental Clocks The size of an organism or its constituent parts in a uniform environment is genetically controlled. In many cases adults cease growth at a particular size, in other cases, such as perennial plants, growth may continue, but the size and shape o f individual components, such as leaves or flowers, are largely invariant. Any limit to growth means that the actual number o f cell divisions is controlled. It follows that mechanisms must exist which are capable o f measuring or counting cell divisions, and these are referred to as developmental clocks. Evidence o f the existence of developmental clocks is both general and specific. For example, the growth o f the chick retina has been analysed in detail both experimentally and theoretically (Morris & Cowan, 1984). There is proliferation until a given number of cells is produced, and then mitotic activity ceases. The interpretation o f the results is certainly consistent with the existence o f an intrinsic clock or cell division counting mechanism. In studies o f the development of the chick wing bud it was shown by grafting experiments that a terminal progress zone
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exists which consists o f dividing cells and is responsible for the ordered formation of the bones of the wing (Summerbell et al., 1973). When the progress zone from a later stage of development was substituted for one at an earlier stage, it gave rise to a truncated limb with absence of distal bone structures. The reciprocal grafts resulted in a wing consisting of two sets of bones, tandemly duplicated. These observations are also consistent with the existence of a developmental clock in which cells in the progress zone form the appropriate structures in sequence, and the specification depends on the number o f times these cells have divided. More recently studies on cell lineages in the developing rat optic nerve show that type 2 astrocyte progenitor cells undergo a specific number of mitotic divisions, in the presence of type 1 astrocytes, before differentiation to oligodendrocytes occurs (Temple & Raft, 1986). More generally, the existence of invariant cell pedigrees during development (reviewed by Davidson, 1986) implies strict control over cell division and the subsequent fate of individual cells. A developmental clock need not count several or many cell divisions, but also one or two divisions, which is the requirement in pedigrees. Moreover, there is no reason, in principle, why there should not be two or more overlapping clocks in a pedigree, or any other developmental situation. Imagine a clock at zero time set to run five divisions, but which also triggers a second clock after three. For the first three divisions one clock is operating, for the next, two clocks, and thereafter only one again for whatever time is specified. In developmental biology there has been much emphasis placed on pattern formation and the organization of cells and tissues in three-dimensional space. In this type of analysis, whether experimental or theoretical, the significance of temporal controls is not usually emphasized. It is obvious that cell divisions are essential for normal development, but Holtzer has long argued more specifically that quantal cell cycles are an essential component of the differentiation process (reviewed by Holtzer et al., 1975). Further evidence for the importance o f cell divisions tied to gene expression and developmental clocks has also been reviewed by Satoh (1982) and Stubblefield (1986). The significance of strict temporal controls has recently come to light in detailed studies of the development of the embryos of Drosophila (Foe, 1989) and Xenopus (Cooke & Smith, 1990). However, these particular controlling events are not tied to cell division counting mechanisms.
A Molecular Mechanism for Counting Cell Divisions It was previously proposed that cell division counting mechanisms might be based on the processive modification of short tandemly repeated D N A sequences (Holliday & Pugh, 1975). Since that time considerable evidence has accumulated for the functional significance o f 5-methylcytosine in a variety of biological contexts. These include the inactivation and reactivation of the X chromosome and autosomal genes, genomic imprinting, the expression of genes with specialized functions, the activity o f viruses and transposable elements, and the induction of specific differentiation pathways in cultured mammalian cells. For recent reviews see Cedar, 1988; Volpe
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& Nicolini, 1989; Doerfler, 1989; Holliday et al., 1990; Holliday, 1990a). The clock mechanism based on D N A methylation invokes two enzymes. First, a switch or setting enzyme which starts the clock; and secondly a "clock" enzyme which methylates both strands o f one repeated sequence after each round of D N A synthesis, as shown in Fig. 1. Once the clock has been set in a given cell, the counting of cell divisions will be synchronized in all progeny. After a given number of divisions, specified by the tandemly repeated D N A substrates, it is assumed that a methylation signal activates or represses one or more genes downstream from the clock sequences. Alternatively, it is possible that there is progressive demethylation tied to cell division. A D N A binding protein might recognize adjacent non methylated and a hemimethyINITIAL SIGNAL
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METHYLATION
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FIG. 1. A molecular model for a cell division counting mechanism or clock, essentially the same as that proposed by Holtiday & Pugh (1975). There are three components to the model. First, a switch or setting DNA methylase recognizes and methylates (closed circle) one strand of a DNA sequence to the left of the series of repeated elements which constitute the clock itself. This initial signal provides the substrate for the second component, the clock methylating enzyme. This has the property methylating both strands of the first of the repeated sequences, but no further repeats (arrows and closed circles). It specifically requires a hemimethylated substrate, which arises after DNA replication (dashed lines), and then the second repeat is methylated. There is thus a "'growing point" of methylation tied to cell division. The sequences in newly replicated DNA behind the growing point may or may not be methylated (open circles). In the example shown there are five repeats, and after four cell divisions, the last one, on the right, will become methylated. This triggers the third component o f t h e clock, namely, the elicitation o f a signal, such as the activation or repression of a gene to the right of the clock sequences.
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lated sequence at the replication fork and prevents the maintenance methylase acting on that particular hemimethylated substrate. The clock principle is the same as in Fig. 1, but the active process is the progressive demethylation of tandemly repeated sequences. A third mechanism was also proposed by Holliday & Pugh (1975), which depends on the specific modification of bases and leads to A - T ~ G-C transitions at individual sites in tandemly repeated sequences. It had earlier been suggested by Scarano (1971) that such transitions in DNA might be important in the control of developmental processes. So far evidence for this proposal has not been forthcoming, although such base change modifications are known to occur in RNA (reviewed by Benne, 1990).
Unequal Crossing Over Between Sister Chromatids Differential staining of sister chromatids containing bromodeoxyuridine in one or both polynucleotide chains in DNA makes it possible to demonstrate crossing-over between these chromatids (reviewed by Wolff, 1977). Such crossing-over appears to be randomly distributed and is relatively common, for example, in a human diploid cell there are five to six events per mitosis. DNA damaging agents greatly increase the frequency of these sister chromatid exchanges (SCEs), which suggests that they may have a role in DNA repair. The exact reciprocity of SCEs shows that homologous DNA sequences are required, and also indicates that the exchanges are accurate, without the loss of gain of genetic material. However, in the case of tandemly repeated sequences, the possibility of unequal exchange arises. Starting with n repeats, an unequal exchange in the participating chromatids could generate n + l a n d n - l , or n + 2 a n d n - 2 repeats, and so on. There has been much discussion of the evolution of multigene families, particularly by Dover (reviewed by Dover, 1986). In the case of tandemly repeated DNA sequences, such as those coding for ribosomal DNA or histones, unequal sister chromatid exchange can generate greater or smaller numbers of repeats. In conjunction with other genetic mechanisms, mutations in one repeat can spread to others to bring about the end result of "homogenization". Jeffreys and his co-workers (Jeffreys et al., 1985a, b; Jeffreys, 1987) have demonstrated the existence of much smaller tandemly repeated sequences in the DNA of humans and other species. The remarkable features of this "minisatellite" DNA is that the number of repeats varies from individual-to-individual, as judged by the pattern of bands in genomic DNA hybridized in Southern blots to probes containing these repeated sequences. The restriction enzyme(s) used cut the genomic DNA on each side of the repeats, so the variation in fragment size is due to the variation in the copy number of the repeated sequence. The polymorphism seen follows normal Mendelian inheritance, since offspring almost invariably contain individual bands derived from one or the other parent, but in several pedigrees a new variant occurred in one case, which could have arisen by unequal crossing over. Originally it was thought that the minisatellite sequences comprised recombination hot spots, which promoted recombination between homologous chromosomes. More recent data indicate that
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unequal crossing over between sister chromatids, either during mitosis of germ line cells or at meiosis, is the more likely mechanism for generating changes in repeat copy number (Jeffreys et al., 1990). The same process could also produce changes in the counting specification of developmental clocks at frequencies much higher than normal mutations, and this event would be expected to occur as readily in inbred organisms as outbred ones. The variability generated, on which natural selection could act, could be regarded as a special case of "molecular drive" acting on repeated sequences of DNA during evolution (Dover, 1986). It was mentioned that quantitative genetic traits are commonly under polygenic control, so it is important to emphasize that a change in clock length would be only one of several components in the regulation of size or shape which is subject to natural selection. Variation in the number of tandem repeats could occur in both homologous chromosomes. Such "heterozygosity" in the specification of clock length could have abnormal effects on development. The same problem arises if important developmental switches in gene activity are operating independently in homologues. For this reason it was suggested that important regulatory genes, or loci, would be subject to allelic exclusion so that they are effectively haploid (Holliday & Pugh, 1975; Holliday, 1990b). The same argument can be made for developmental clocks. Conclusions
Several arguments have been brought together in formulating a general hypothesis. The first is the well-known variability in quantitative traits in plants and animals and the rapid response to the selection of any particular trait. The second is the continuing variability which is seen to occur in inbred mouse populations, which occurs at a rate much higher than expected from single gene mutations. The third is concerned with the specifications of size and shape in higher organisms. It is obvious that there are controls on the extent of cell division, but the more specific proposal is that developmental clocks exist which count cell divisions and therefore provide temporal control of development. The existence of strict cell pedigrees argues that the segregation o f different cell types is tied to cell division, and other evidence o f developmental clocks also exists. Fourth, the D N A methylation model for the control o f gene activities in the development of higher organisms can be extended to a cell division counting mechanism (Fig. 1), based on the processive methylation of short tandemly repeated sequences of DNA. After each round of D N A synthesis one additional repeat is methylated. Fifth, it is well-known that unequal crossing over can occur between tandemly repeated homologous DNA sequences, and this can generate greater or smaller numbers of repeats. Such unequal crossing over can occur between sister chromatids in mitotic and meiotic cells, and probably also between homologous chromosomes at meiosis. The frequency of these events in germ line cells are likely to be far higher than normal mutation frequencies. They also provide a source o f genetic variability in homozygous inbred organisms. The unifying proposal is that developmental clocks are important in controlling major quantitative features of organisms, that such clocks are based on the processive modification of short tandemly repeated sequences of DNA, and unequal crossing
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over can change the number of repeats and therefore generate quantitative variation. I t h a n k Alec Jeffreys, Gabriel Dover, Richard C o w a n a n d J o h n Pugh for their interest a n d helpful c o m m e n t s . REFERENCES BENNE, R. (1990). RNA editing in trypanosomes: is there a message? Trends Genet. 6, 177-181 CEDAR, H. (1988). DNA methylation and gene activity. Cell 53, 3-4. COOKE, J. & SMITH, J. C. (1990). Measurement of developmental time by cells of early embryos. Cell 60, 891-894. DARWIN, C. R. (1868). The Variation o f Animals and Plants under Domestication. London: Murray. DAV1DSON, E. H. (t986). Gene Activity in Early Development 3rd edn. New York: Academic Press. DEOL, M. S., GRUNEaERG, H., SEARLE, A. G. & TRUSLOVE,G. M. (1957). Genetical differentiation involving morphological characters in an inbred strain of mice. I. A British branch of the C57BL strain. Jr. Morphol. 100, 345-376. DOERFLER, W. (1989). Complexities in gene regulation by promoter methylation. In: Nucleic Acids and Molecular Biology (Eckstein, F. & Lilley, D. M. J., eds) 3, 92-119. Berlin: Springer Verlag. DOVER, G. A. (1986). Molecular drive in multigene families: how biological novelties arise, spread and are assimilated. Trends Genet. 2, 159-165. FALCONER, D. S. (1981). Introduction to Quantitative Genetics 2nd edn. New York: Longman. FESTING, M. (1973). A multivariate analysis of subline divergence in the shape of the mandible in C-57BL-GR mice. Genet. Res. 21, 121-132. FOE, V. E. (1989). Mitotic domains reveal commitment of cells in Drosophila embryos. Development 107, 1-22. GREWAL, M. S. (1962). The rate of genetic divergence of sublines in the C57BL strain of mice. Genet. Res. 3, 226-237. GRUNEBERG, H. (1963). The Pathology of Development: A Study of Inherited Skeletal Disorders in Animals. Oxford: Blackwell. G RUN EaERG, H. (1970). Is there a viral component in the genetic background ? Nature, Lond. 225, 39-41. HoI-SEN, Y. (1972). Is subline differentiation a continuing process in inbred strains of mice? Genet. Res. 19, 53-59. HOLLIDAY, R. (1990a). Genetic mechanisms for the control of gene activity during development. Biol. Rev. 65, 431-47 I. HOLLIDAY, R. (1990b). Genomic imprinting and allelic exclusion. Development (Suppl.), 125-129. HOLLIDAY, R., MONK, M. & PUGH, J. E. (eds) (1990). DNA Methylation and Gene Regulation. London: Royal Society. HOLLIDAY, R. & PUGH, J. E. (1975). DNA modification mechanisms and gene activity during development. Science 187, 226-232. HOLTZER, H., RUBINSTEIN, N., FELLINI, S., YEOH, G., CHI, J., BtRNBAUM, J. & OKAYAMA, M. (1975). Lineages, quantal cell cycles and the generation of cell diversity. Quant. Reo. Biophys. 8, 523-557. JEFFREYS, A. J. (1987). Highly variable minlsatellites and DNA fingerprints. Biochem. Soc. Trans. 15, 309-317. JEFFREYS, m. J., NEUMANN, R. & WILSON, V. (1990). Repeat unit sequence variation in minisatellites: a novel source of DNA polymorphism for studying variation and mutation by single molecule analysis. Cell 60, 473-485. JEFFREYS, A. J., WILSON, V. & THEI N, S. L. (1985a). Hypervariable "minisatellite" regions in human DNA. Nature, Lond. 314, 67-73. JEFFREYS, A. J., WILSON, V. & THEI N, S. L. (1985b). Individual-specific "'fingerprints" of human DNA. Nature, Lond. 316, 76-79. MORRIS, V. B. & COWAN, R. (1984). A growth curve of cell numbers in the neural retina of embryonic chicks. Cell Tissue Kinet. 17, 199-208. SATOH, N. (1982). Timing mechanisms in early embryonic development. Differentiation 22, 156-163. SCARANO, E. (1971). The control of gene function in cell differentiation and in embryogenesis. Adv. Cytopharmacol. 1, 13-24. STUBBLEFIELD, E, (1986). A theory for developmental control by a programme encoded in the genome. J. theor. Biol. 118, 129-143.
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SUMMERBELL,D., LEwls, J. H. & WOLPERT,L. (1973). Positional information in chick limb morphogenesis. Nature, Lond. 244, 492-496. TEMPLE, S. & RAFF, M. C. (1986). Clonal analysis of oligodendrocyte development in culture: evidence for a developmental clock that counts cell divisions. Cell 44, 773-779. THOMPSON, D'ARCY, W. (1942). On Growth and Form 2nd edn. Cambridge University Press. VOLPE, E. & NICOLINI, C. (eds) (1989). Forum on eukaryotic DNA methylation, Cell Biophys. 15, 1-155. WOLFF, S. (1977). Sister chromatid exchange. Annu. Rev. Genet. 11, 183-201.