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Biological Signifi cance and Evolution

Cytogenet Genome Res 113:12–16 (2006) DOI: 10.1159/000090809

Imprinting today: end of the beginning or beginning of the end? D. Solter Max Planck Institute of Immunobiology, Freiburg (Germany)

second polar body extrusion following fertilization and then removed the male pronucleus. These embryos failed to de-velop in the way parthenogenones usually fail despite being fertilized and thus possessing a putative sperm contribution. The authors suggested that excessive homozygosity was the cause. It was apparent that these two sets of data were con-tradictory and it was unlikely that both were correct. The question remained open and sharply outlined.

The ability to transplant pronuclei from one zygote to an-other seemed essential to address this question and we devel-oped the necessary technique (McGrath and Solter, 1983) which then provided the other element necessary for the dis-covery of imprinting. Once it was clear that the exchange of pronuclei between two zygotes is a biologically neutral process (McGrath and Solter, 1983), experiments in which the male pronucleus was replaced by a female pronucleus and vice ver-sa were obviously the next step. Since zygotes could easily be constructed in which two male or two female pronuclei were derived from two different mouse strains, excessive homozy-gosity would no longer be the problem and the use of fertilized zygotes as recipients would also eliminate the issue of non-chromosomal sperm contribution. Thus we fully expected that androgenones and gynogenones constructed in such a manner would develop normally and we were rather surprised and took a long time to convince ourselves when they did not (McGrath and Solter, 1984a). Androgenones and gynoge-nones failed in their development soon after implantation: androgenones due to the poor development of the embryo proper and gynogenones (similar to parthenogenones) due to the poor development of extraembryonic membranes (most-ly trophectoderm derivatives). These results were simultane-ously and independently confi rmed by Surani’s group (Barton et al., 1984; Surani et al., 1984). We proposed that maternal and paternal genomes are functionally different and that both are necessary for normal development. We also suggested that specifi c genes are inherited in a functional form from one par-ent and in a non-functional form from the other and that this

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The beginning – a brief history

In the early eighties, just before the discovery of imprint-ing, two elements crucial for this discovery coincided. One element was the recurring question in mammalian develop-mental biology, namely why diploid parthenogenetic embry-os do not develop to term. Parthenogenetic embryos had pre-viously been produced by activating the ovulated oocyte and suppressing either the second meiotic or fi rst mitotic division in order to restore diploidy. Such embryos did have a normal set of chromosomes but despite this they failed mainly be-tween implantation and mid-gestation. Two hypotheses were advanced to explain this failure: one suggesting that excessive homozygosity due to chromosome duplication reveals hidden lethal genes and the other suggesting that the sperm makes an essential physical (non-genetic) or physiological contribution without which development cannot be completed. Experi-mental evidence seemed to support and contradict both of these hypotheses. Hoppe and Illmensee (1977) removed ei-ther the male or female pronucleus from the zygote and then prevented fi rst division in order to duplicate the remaining chromosomal complement. They reported that several mice were born from embryos containing either only paternal (an-drogenones) or only maternal (gynogenones) chromosomes and concluded that non-chromosomal sperm contribution is essential for development and that its absence explains the failure of parthenogenesis. These results were never repro-duced and, in view of what emerged later and what we now know, they are extremely unlikely. A few years later Surani and Barton (1983) produced triploid embryos by suppressing

Manuscript received 5 August 2005; accepted in revised form for publication by F. Ishino, 26 September 2005.

Cytogenet Genome Res 113:12–16 (2006) 13

is due to a specifi c conditioning during gametogenesis (Mc-Grath and Solter, 1984a). Surani et al. (1984) suggested the term ‘imprinting’ for such conditioning, and this term is used today for the process which renders two parental alleles func-tionally different depending on the sex of the contributing parent.

Although the above-presented interpretation for our fi nd-ings seemed plausible, we immediately realized that other explanations were equally possible. If one pronucleus is re-moved and replaced with a pronucleus derived from the oth-er sex parent, this does eliminate the haploid genome but it also eliminates unknown nucleus-associated components (chromatin proteins, nuclear membrane, etc.). It was thus es-sential to demonstrate the genetic basis of the imprinting phe-nomenon. A fi rst tentative indication also came from nuclear transfer experiments. For this purpose we used embryos car-rying a T hp deletion inherited maternally or paternally (Mc-Grath and Solter, 1984b). T hp deletion had been known for some time and it possessed a unique phenotype (Johnson, 1974, 1975), namely paternal inheritance results in viable heterozygous mice with short tail but maternal inheritance of T hp results in late fetal lethality, the rare newborns are edem-atous and die immediately after birth. Phenotypic differ-ences observed after reciprocal crossing in presumably ge-netically identical individuals are always diffi cult to interpret, since the phenotype following maternal transmission could be alternatively caused by a change in the oocyte cytoplasm or by gestation in the uterus of a heterozygous female. Nucle-ar transplantation is essential to resolve such issues and we were thus able to show that a maternal pronucleus carrying T hp reproduces the phenotype of T hp maternal inheritance even when it is transplanted into a normal zygote (after re-moval of the endogenous female pronucleus) which is then transferred into a normal pseudopregnant female (McGrath and Solter, 1984b); the same is true for the paternal T hp pro-nucleus. These results strongly suggested that the T hp pheno-type is caused by gene(s) which function differently when in-herited maternally than when inherited paternally, as previ-ously hypothesized by McLaren (1979).

Further evidence for a genetic basis of the imprinting phe-nomenon came from work using chromosomal translocation. By mating mice with Robertsonian and other chromosomal translocations, progeny can be produced in which the entire chromosome pair or part of it is derived from one parent only (uniparental disomies). Using this model, Cattanach and Kirk (1985) demonstrated that mice with disomy of chromo-some 11 have unique phenotypes which differed depending on whether the disomy was maternal or paternal. They sub-sequently refi ned these results by using partial translocation to demonstrate that only the proximal part of chromosome 11 is responsible for the observed phenotypes. These and sim-ilar results strongly suggested the existence of genes which function differently depending on the parent of origin (im-printed genes) and also demonstrated that these genes are only found in restricted chromosomal areas while the major-ity of the genome contains only non-imprinted genes.

Though all these data argued for the genetic nature of im-printing, the fi nal and decisive proof came with the identifi ca-

tion of imprinted genes. It took six years since the initial de-scription of imprinting but in 1991 within a fi ve-month pe-riod the fi rst three imprinted genes were described (Barlow et al., 1991; Bartolomei et al., 1991; DeChiara et al., 1991). In-terestingly, the fi rst to be described was discovered based on a logical extension of information about the already men-tioned T hp mutation. Knowing that the T hp deletion must contain a differentially functioning gene (presumably differ-entially expressed) depending on the parent of origin, Barlow et al. (1991) scanned the appropriate region and found that the insulin-like growth factor-2 receptor (Igf2r) is only ex-pressed from the maternal chromosome while all other genes in the region were expressed biallelically. Maternal-only ex-pression of Igf2r did fi t in with the fact that the lethal pheno-type was only observed in fetuses with maternal T hp deletion, though the exact mechanism was not clear.

The second imprinted gene was identifi ed following anal-ysis of the phenotype in reciprocal crosses of mice heterozy-gous for the deletion of the insulin-like growth factor II gene (Igf2) produced by homologous recombination (DeChiara et al., 1991). When heterozygous females were crossed with nor-mal males, all progeny was normal but in the reciprocal cross half of the progeny was signifi cantly smaller. This result is consistent with the concept that only the paternal copy of Igf2 is expressed, which was confi rmed by molecular analysis (DeChiara et al., 1991).

The third imprinted gene, H19 , was identifi ed because it was known that it maps to the imprinted region of chromo-some 7, actually quite close to Igf2 (Bartolomei et al., 1991). H19 is abundantly expressed only from the maternal chromo-some, it codes for non-coding RNA and even today its func-tion is not known.

These results established beyond doubt the existence of imprinted genes and provided us with initial information about their nature. As expected, only one allele was tran-scribed and parental origin was the determining factor. The allele not expressed in one individual, for example the mater-nal allele of Igf2 in a male mouse, will nevertheless be ex-pressed in his progeny. This generational switching on and off depending on the sex of the transmitting individual is the hallmark of imprinted genes and demonstrates that, while the imprint is quite stable and can last for a lifetime of an indi-vidual, it can be and is easily reversed during gametogenesis. The fi nding of two reciprocally imprinted genes (Igf2 and H19) in close proximity suggested the possibility of imprint-ed gene clusters and of common controlling elements. Analy-sis of these two genes in the subsequent decade provided a wealth of information about the molecular control of expres-sion of imprinted genes.

Thus by the beginning of the nineties we had established that imprinting is a genetic phenomenon, imprinted genes are expressed monoallelically depending on the parental sex and that the presence of both maternal and paternal genome is essential for normal development, suggesting the crucial role of imprinted genes in embryonic development. In the follow-ing one-and-a-half decades a substantial number of imprinted genes (now more than 50) have been identifi ed. As we learned more about the mechanisms which control the expression of

Cytogenet Genome Res 113:12–16 (2006) 14

the active allele and the silence of the inactive allele, we began to realize that these mechanisms can be exceedingly complex and that they vary signifi cantly from one imprinted gene to another. One could argue that, with the possible exception of DNA methylation, there is not a single molecular mechanism common to all imprinted genes. My purpose here is not to elaborate on the control of expression of imprinted genes on which most of the work has concentrated and which has been extensively reviewed (Tilghman, 1999; Ferguson-Smith and Surani, 2001; Reik and Walter, 2001; Reik et al., 2001; Kaneko-Ishino et al., 2003). I will try instead to assess very briefl y our current understanding of imprinting and which aspects require further clarifi cation. In order to do this, I will attempt to answer the basic questions used to approach any unsolved problem, i.e. the what, who, where, when, why and (w)how of imprinting.

When and where

The major problem in answering these and other questions is that we are not completely sure how to recognize an im-printed gene at the moment of imprint setting. Parent-of-ori-gin-specifi c, monoallelic expression is certainly an indication of imprinting but biallelic expression does not necessarily ex-clude imprinting, since it could be caused by the absence of imprint-reading mechanisms. A parent-of-origin-specifi c dif-ference in DNA methylation is also a likely indication of im-printing but we are not sure if DNA methylation is indeed the mark nor if it precedes or follows the establishment of im-printing. For these reasons we have to resort to functional tests in an attempt to determine at which point imprinting takes place and when it is completed.

The mammalian organism starts as a fertilized egg with one haploid maternally imprinted and one paternally im-printed genome. Since it will produce germ cells whose ge-nome is imprinted according to its sex, it is obvious that the sex-inappropriate imprint must be lost in the germ line at some point during gametogenesis. At the beginning we did not know whether the imprint is retained in somatic cells but in the meantime the success of cloning by nuclear transfer indicates that the correct (or at least suffi cient) imprint must be retained in at least some somatic cells. At the same time the low success rate of cloning and the haphazard expression of imprinted genes in clones indicate that there is a substantial loss of imprinting in somatic cells. This is especially true when ES cells are used as nuclear donors, possibly refl ecting the la-bility of correct imprinting of several imprinted genes in ES cell clones (Dean et al., 1998; Humpherys et al., 2001; Ogawa et al., 2003). The role of imprinting in the failure of clones derived from adult cells is less clear. While imprinted genes were expressed correctly in 9.5-day-old fetuses and placentas and in 12.5-day-old fetuses, they were disregulated in 12.5-day-old placentas (Inoue et al., 2002). It is possible that the observed state of imprinting in these surviving clones is some-what misleading since analysis of cloned preimplantation em-bryos demonstrated a signifi cant misexpression of imprinted genes (Mann et al., 2003). To return to germ cells, it is not

clear whether both imprints are fi rst erased and then the sex-appropriate imprint is established or whether the sex-appro-priate imprint is retained and only the sex-inappropriate erased, though the fi rst possibility is more likely. Erasure of imprinting starts in primordial germ cells (PGC) upon enter-ing the genital ridge. It is obviously diffi cult to judge how quickly and effi ciently the erasure is completed and this may differ for individual genes. Accepting the biallelic expression or absence of expression, and absence of DNA methylation as evidence of imprint erasure, the majority of imprinted genes in PGC lose the imprint between day 10.5 and 12.5 of fetal development (Hajkova et al., 2002; Lee et al., 2002). However, for some genes or in some germ cells the erasure of imprinting may be a slow and gradual process which may run concomitantly with the establishment of a new sex-appropri-ate imprint (Yamazaki et al., 2003). Completion of imprint-ing may occur at different points in oogenesis and spermato-genesis. It seems that the male imprint is established before the beginning of meiosis, since the genome of the primary spermatocyte is capable of functioning as an imprinted male genome (Ogura et al., 1998), as is obviously also true for the genome of the secondary spermatocyte (Kimura and Yanagi-machi, 1995a) and the round spermatid (Kimura and Yana-gimachi, 1995b). However, the very low success rate reported (Ogura et al., 1998) may be an indication that only some of the primary spermatocytes have completed the process of im-printing (see later for similar experiments using oocyte ge-nomes). Even the nuclei of round spermatids injected into the oocyte were substantially inferior in supporting normal de-velopment when compared to the nuclei of testicular sperma-tozoa. Though this gradual increase in capacity to support development from primary spermatocyte to testicular sperm may be due to technical problems, it is entirely possible that acquisition of imprints is a gradual process and that comple-tion of imprinting does not take place until spermiogenesis.

It has recently been shown that the genome of non-grow-ing oocytes isolated from newborn females can partially sub-stitute for the male genome and, if genetically manipulated, can in very rare cases support normal development in com-bination with the haploid genome of an ovulated mature oo-cyte (Kono et al., 2004). These results would indicate that erasure of the male imprint during oogenesis is a very slow process and that it is not completed before the beginning of oocyte growth. Indeed certain imprinted genes show clear pa-ternal imprint-related expression in fetuses derived from eggs containing both haploid genomes derived from female ga-metes (Kono et al., 2004). It is not clear whether the extreme-ly low success rate in these experiments indicates that the speed of erasure of the male imprint varies between oocytes, thus only very few retain a suffi cient male imprint at birth to support normal development. Neither is it clear whether this process of erasure continues into the phase of oocyte growth or whether that phase is entirely devoted to the establishment of new imprints which are also established gradually, the tim-ing of imprinting being gene-specifi c (Obata and Kono, 2002). At present it is unknown whether imprinting is completed by the time the egg is fully grown or if it continues during oocyte maturation.

Cytogenet Genome Res 113:12–16 (2006) 15

Who and what

Who is doing the imprinting and which genes are imprint-ed should be relatively easy questions to answer. The number of imprinted genes identifi ed is steadily growing and it is like-ly that new methods for scanning the entire genome will even-tually identify them all (Nikaido et al., 2003). A quick visit to http://www.mgu.har.mrc.ac.uk/research/imprinting/index.html will provide the reader with the current list of imprinted genes and what is known about them. As to who is doing the imprinting, we know that, without the action of certain DNA methyltransferases, imprinting would be lost (Li et al., 1993; Howell et al., 2001), but this does not necessarily mean that these enzymes are involved in the establishment of the im-print, though they are certainly essential for its maintenance.

Why and how

These are certainly the most diffi cult questions to answer and at present there is much more speculation than facts. The fi rst two imprinted genes to be identifi ed were (serendipitous-ly) a ligand-receptor pair (Igf2-Igf2r) , one expressed from the paternal and one from the maternal allele, the fi rst stimulat-ing and the second inhibiting fetal growth. This observation immediately prompted the formulation of a confl ict hypoth-esis to explain the evolution of imprinting (Haig and Graham, 1991; Moore and Haig, 1991). Briefl y stated, this hypothesis claims that in any situation of direct codependence between fetus and mother (best exemplifi ed in placental mammals) there will be competition for resources; the paternal genome will favor growth of each individual fetus at the expense of all other fetuses and the mother, while the maternal genome will favor an equal distribution of resources among all fetuses and preservation of itself for future pregnancies. Many imprinted genes fi t into this confl ict hypothesis so that paternally ex-pressed genes favor the growth of fetus and placenta while maternally expressed genes tend to reduce the growth of fetus and placenta. The confl ict hypothesis, though plausible and intellectually pleasing, cannot explain all imprinting phenom-ena (Hurst and McVean, 1997; Iwasa, 1998) and it is likely that further acquisition of data will result in its modifi cation

(Wilkins, 2005). It is tempting to consider that imprinting, conceivably unique for mammals, was a necessary evolution-ary gimmick which enabled the emergence of placental mam-mals (Reik and Lewis, 2005).

How imprinting is accomplished in molecular terms is al-most entirely unclear. Research in the last decade has pro-vided a vast amount of information addressing the mecha-nisms which secure monoallelic expression of imprinted genes (Reik and Walter, 2001; Kaneko-Ishino et al., 2003). It is now clear that these mechanisms are complex and vary from one imprinted gene to another. However, before mono-allelic expression can be maintained, there must be one or more mechanisms which can identify unique DNA se-quences during gametogenesis and mark them in a specifi c way. This marking could be done by methylation but we do not yet know what it is that exactly guides the methylases to the specifi c sequence. If the initial mark is in fact something else and not methylation, our ignorance of how the imprint is established is indeed complete. Very imaginative and in-sightful experiments will be required to crack the ‘whys’ and ‘hows’ of imprinting.

The future

Imprinting as an underlying mechanism may be crucial to explain mammalian evolution but it is also becoming relevant in many areas concerning clinical medicine from oncogenesis (Robertson, 2005) to the study of behavior (Allen et al., 1995; Davies et al., 2005; Raefski and O’Neill, 2005) and pre-eclampsia (van Dijk et al., 2005), to name but a few. As the genetic mechanisms of diseases are becoming known and more imprinted genes are being identifi ed, it is likely that the role of imprinting and its failures in pathogenesis will become even more prominent. The progress we have achieved in un-derstanding imprinting merely illustrates how much remains unknown and, considering the importance of this phenome-non, a substantial effort needs to be and probably will be ex-pended to ameliorate this situation. Indeed despite the tre-mendous amount of knowledge we have amassed since the discovery of imprinting just over twenty years ago, we are barely at the end of the beginning.

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