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REVIEWS Genomic imprinting effects on brain development and function Lawrence S. Wilkinson, William Davies and Anthony R. Isles Abstract | In a small fraction of mammalian genes — at present estimated at less than 1% of the total — one of the two alleles that is inherited by the offspring is partially or completely switched off. The decision as to which one is silenced depends on which allele was inherited from the mother and which from the father. These idiosyncratic loci are known as imprinted genes, and their existence is an evolutionary enigma, as they effectively nullify the advantages of diploidy. Although they are small in number, these genes have important effects on physiology and behaviour, and many are expressed in the brain. There is increasing evidence that imprinted genes influence brain function and behaviour by affecting neurodevelopmental processes. Parthenogenetic embryos Embryos that develop in the absence of fertilization by a male. Parthenogenetic cells contain two copies of the maternal genome, but have no paternal contribution. Androgenetic embryos Embryos that develop in the absence of a contribution from a female. Androgenetic cells contain two copies of the paternal genome. Behavioural Genetics Group, School of Psychology and Department of Psychological Medicine, School of Medicine, Cardiff University, Tower Building, Park Place, Cardiff, CF10 3AT, UK. Correspondence to L.S.W. e-mail: [email protected] doi:10.1038/nrn2235 Published online 10 October 2007 At the moment of conception, each parent contributes one copy of each autosomal gene to their offspring. In most cases, the expression of these genes is indifferent to parental origin. However, imprinted genes are special in that they are subject to a form of epigenetic control that is mediated by chemical marking of the DNA and associated proteins. This leads to the selective expression of one of the two alleles, depending on whether they pass through the egg or the sperm1 (FIG. 1). The upshot of this epigenetic control is a ‘parent-of-origin’ pattern of imprinted gene expression in the somatic tissues of the offspring. Many of the genes that are known to be subjected to imprinting are expressed in the placenta and the brain2,3. The existence of genomic imprinting raises many questions, including why it exists at all: where silencing is complete, imprinted genes are effectively haploid, which negates the considerable benefits of diploidy. The most resilient (but not exclusive; see BOX 1 for alternative views) hypothesis that has been put forward to explain this evolutionary conundrum is that imprinted genes represent the consequences of a form of ‘intragenomic conflict’ that arises from the differing interests of maternal and paternal genomes in aspects of the physiology and behaviour of the offspring. These different interests arise, in turn, from asymmetries of relatedness between the offspring and the parents4 (BOX 1). In this Review, we focus on genomic imprinting in the brain and discuss the emerging evidence that brainexpressed imprinted genes can influence brain function and behaviour through effects on neurodevelopment. In particular, we emphasise recent data that indicate how 832 | November 2007 | volume 8 imprinted genes affect cell survival and differentiation and associated cell-signalling pathways. Our Review brings together a number of diverse areas, including brain development, mother–infant interactions, long-term programming of behaviour and the risk of neuropsychiatric disorders. It also addresses the extent to which the pattern of genomic-imprinting effects on neurodevelopment can be explained by the way in which imprinted genes have evolved. Effects on brain size and organization Imprinted genes and growth. Growth and development are key processes for the action of imprinted genes. Some of the first experimental evidence for the existence of genomic imprinting arose from the failure of parthenogenetic (PG) and androgenetic (AG) mouse embryos to develop normally and survive to term5,6. Subsequently, the first imprinted genes to be identified encode growth factors, namely paternally expressed insulin-like growth factor 2 (Igf2) 7 and maternally expressed insulin-like growth factor 2 receptor (Igf2r)8. The discovery of these genes and the fact that they were reciprocally imprinted caused great excitement, as their existence confirmed earlier theoretical predictions, and their antagonistic actions on growth provided strong initial evidence in favour of the general notion of intragenomic conflict 9. These seminal data also encouraged the idea that the allocation of resources from the mother to the offspring is an important arena for conflict between the male and female genomes10. According to this idea, the paternal interest seeks to maximise resource allocation to the fetus in the current www.nature.com/reviews/neuro © 2007 Nature Publishing Group REVIEWS Zygote Adult animal Gene 1 Gene 2 Gene 1 Gene 2 Somatic lineages Gene 1 Gene 2 Fertilization Gene 1 Gene 2 Germline lineage Sperm Gene 1 Gene 2 Gene 1 Gene 2 Gene 1 Gene 2 Germline establishment Egg Gene 1 Gene 2 Gene 1 Gene 2 Gene 1 Gene 2 Figure 1 | The cycle of genomic imprinting. The parental-allele expression that is displayed by imprinted genes is achieved by parent-of-origin-specific epigenetic Nature Reviews | Neuroscience modifications (represented by orange lollipops) — chiefly, DNA methylation at key control regions and/or histone modifications1. This epigenetic marking results in differential reading by the transcriptional machinery and, as a consequence, the gene is expressed predominantly from one parental allele. The epigenetic marks are erased and reset in the nascent germline of each developing embryo. Thus, depending on whether an individual is destined to become a mother or a father, the marking that controls imprinted-gene expression is reset in the developing eggs and sperm, respectively. This inherited set of marks is then maintained in the somatic cell lineages of the offspring, but in their germ cells the whole cycle is repeated once again. As a result of this cycle of epigenetic marking, an allele of a maternally expressed gene (represented as pink boxes) in a male individual will not be expressed in that male’s offspring, as it will now be inherited as a paternal allele (represented as blue boxes). As a consequence, the inheritance of imprinted traits is non-Mendelian, and is often characterized by passage down a parental line and/or skipping of generations. Figure modified, with permission, from REF. 114 (2004) Elsevier Science. pregnancy (IGF2 is growth-enhancing), whereas it is in the interest of the maternal genome to share resources equally across all of the mother’s offspring (IGF2R is growth-inhibiting) (BOX 1). In terms of growth phenotypes, this view is supported by most, but not all, of the findings that have been obtained to date. Early work in mouse chimaeras. A powerful early demonstration that, in addition to their influence on growth in general, imprinted genes can have specific effects on brain size and organization, came from elegant work using mouse chimaeras. These chimaeras were created by adding normal cells (N) to PG and AG embryos at an early stage of development, which produced PG–N and AG–N animals, respectively. Both types of low contribution (<40% PG or AG) chimaera mice survived to term, but they had different brain phenotypes11,12: the brains of PG–N chimaeras, especially the forebrain, were relatively large in comparison with those of wildtype controls, despite the animals’ smaller overall body nature reviews | neuroscience size. By contrast, AG–N chimaeras had relatively small brains but larger bodies. In an additional experimental refinement, the PG and AG cells were engineered to express the reporter gene lacZ, which made it possible to observe their location in the brain in detail. This analysis showed that the distribution of PG and AG cells was not random — rather, it had distinct patterns. Although both AG and PG cells were distributed throughout the brain early in development, they each established persistent localizations in different brain regions later in embryogenesis (BOX 2). AG cells were preferentially localized to the hypothalamic, septal and pre-optic areas of the bed nucleus of the stria terminalis, but were excluded from the overlying cortex. Conversely, PG cells contributed substantially to neocortical areas, to the striatum and to the hippocampus, but were not present in the brain regions that were colonized by the AG cells. The chimaera data were important because they strongly implicated imprinted genes in processes of neurodevelopment and indicated that the maternal and paternal genomes could have dissociable effects on functionally distinct brain systems. This observation made it tempting to place the chimaera data in the context of ‘genomic conflict’ as applied to brain functioning. Did the parent-of-origin effects on neurodevelopment in the chimaeras reflect differing interests of the parental genomes in the brain function and behaviour of the offspring12? If so, one interpretation of the data might be that the maternal interests are, in broad terms, mediated by neurodevelopmental effects on ‘higher’ cognitive systems, whereas the paternal interests are mediated by effects on brain systems that sub-serve ‘emotional’ or ‘autonomic’ functions13. However, two caveats indicate that the situation might be more complex than was first envisaged. First, there is no reason to assume that the final locations of the AG and PG cells are a result of the net effects of all paternally (in AG cells) and maternally (in PG cells) expressed imprinted genes. Instead, the localization of AG and PG cells in the brain might arise from the large effects of a few imprinted genes. Indeed, this possibility has been put forward as an explanation for the failure of pure (as opposed to chimeric) parthenogenetic embryos to survive14. Second, as data on the spatial expression patterns of individual imprinted genes have accumulated, it has become apparent that, although for many genes the pattern of imprinting corresponds to that seen in the chimaeras, there are several exceptions2. This discrepancy might arise from the ‘few genes, large effects’ caveat, but it could also be the result of a contribution to conflict at the molecular level (in addition to functional brain systems), with maternally and paternally expressed genes counteracting one another’s expression, or acting in an antagonistic manner on biochemical pathways2. Imprinted-gene expression in neurodevelopment. Any consideration of the specific mechanisms by which genomic imprinting might influence neurodevelopment has to take account of the complex spatial and temporal expression patterns that are exhibited by imprinted genes in the brain2. A large proportion of the volume 8 | November 2007 | 833 © 2007 Nature Publishing Group REVIEWS Box 1 | The evolution of genomic imprinting The unique epigenetic status of imprinted genes has led to several theories as to why they exist. These include ideas that they occur to prevent parthenogenesis99, as a means to give rise to rapid evolution at certain loci100, and to promote the coadaptation of the physiology and behaviour of the mother and her offspring67,101. Although all these scenarios are valid, the theory that best explains most current data, at least in terms of growth phenotypes, is the idea that genomic imprinting evolved as a consequence of intragenomic conflict, which occurs when the maternal and paternal genomes have different ‘interests’. In this respect, imprinted genes represent classic ‘selfish’ genes102, in that paternal and maternal genes ‘act’ in their own interests, despite the fact that these interests might not be optimal for the individual in which the genes find themselves. One scenario in which intragenomic conflict might arise is when asymmetries of relatedness occur. This is best summarized by David Haig, who said that “my mother’s kin are not my father’s kin”103. The ‘maternal half’ of an individual’s genome is closely related to that of relatives in the maternal line, and it is generally not at all related to the genome of relatives in the paternal line (and vice versa): what is good for maternally related genes might not be good for paternally related genes. In mammals, this ‘battle of the sexes’ is felt most acutely with regard to the allocation of resources between mother and offspring9. Here, paternal genes are expected to maximize resources to an individual offspring, whereas maternal genes limit them so that they are equally distributed among all the mother’s offspring — an idea that is supported by the number of imprinted genes that affect placental function, in utero growth and suckling behaviour10. The classic examples are the Igf2 and Igf2r genes in the mouse7,8,9. IGF2, the product of the paternally expressed Igf2 gene, promotes growth in the offspring when it binds to its main receptor, IGF1R. However, IGF2 also binds to the IGF2 receptor (IGF2R, the product of Igf2r), which is maternally expressed, and here it is targeted for destruction, thus reducing any effects on growth. Such asymmetries of relatedness also occur in matrilineal and patrilineal social groups, in which maternal and paternal genes, respectively, are more common and are shared with other group members. Here, the differential interests between parental genomes are predicted to affect social and affiliative behaviours81,103. Yeast two-hybrid screen A molecular method for determining whether proteins interact. The binding and activation domains of the transcription factor GAL4 are split and fused to the proteins in the assay. If the two proteins interact, the reconstituted GAL4 initiates the transcription of a reporter gene. BAX A well characterized regulator of apoptosis that exerts its effects through heterodimerization with BCL2. 100 or so imprinted genes that have been discovered so far are expressed in brain tissue (although not necessarily exclusively). These genes can show substantial variability in terms of where (including in what cell type) and when they are expressed and also, in some cases, in their imprinting status. For example, imprinting of Ube3a, which encodes the ubiquitin ligase protein UBE3A (formerly known as E6-associated protein, E6AP) and is maternally expressed, is confined to discrete neuronal populations of the olfactory bulb, the hippocampus and the cerebellum; elsewhere in the brain and in other body tissues, UBE3A is biallelically expressed15,16. Imprinting can also be short-lasting, as in the case of copper metabolism (Murr1)-domaincontaining 1 (Commd1), which is preferentially maternally expressed in adulthood but exhibits biallelic expression in neonates17. These examples emphasize the dynamic nature of imprinted-gene expression in the brain, which can add a further subtle level of control to imprinted-gene function. We do not yet have a full picture of the developmental profiles of brain-expressed imprinted genes and, on the basis of these limited data, no organizational patterns have yet emerged with regard to the direction of imprinting (that is, whether a gene is maternally or paternally expressed), apart from a bias in terms of spatial location, which might be argued to reflect the dichotomy that is seen in the mouse chimaeras. Nonetheless, it is clear that 834 | November 2007 | volume 8 imprinted genes are active at key developmental time points (TABLE 1). Note, however, that the expression of these genes is not confined to the prenatal period: expression and imprinting can persist into the postnatal period and beyond, sometimes into the adult brain15,18. It would seem that, although many imprinted genes are in a position to influence early brain development and differentiation in utero, they might also contribute to processes of brain development and sculpting that continue after birth. The persistence of imprinting in the adult brain might also, of course, indicate that imprinted genes have functions that are independent of neurodevelopment altogether. Effects on cell survival and differentiation. Our understanding of the cellular and molecular mechanisms by which imprinted genes can modify brain development has recently been enhanced by in vitro molecular screening methods (commonly yeast two-hybrid screens) that have been used to examine the binding and interactions of imprinted-gene products with other molecules. One of the most extensively characterized imprinted genes in this regard is paternally expressed gene 3 (Peg3), which encodes a large multidomain protein that might act as a transcription factor owing to its putative DNA-binding activity19. Peg3 is highly expressed in the brain20. The main phenotypic feature of female mice that lack paternally inherited Peg3 is a reduction in the number of oxytocin-producing neurons in the hypothalamus, consistent with the functional deficits in maternal behaviour and milk let-down that have been observed in these mice20. The precise events that lead to a reduction in oxytocin neurons are unknown, but there is indirect evidence that they might involve interactions of PEG3 protein with p53-mediated cell-death pathways (FIG. 2a). In normal transfected mouse fibroblasts, Peg3 expression is upregulated in cells that co-express p53 and the proto-oncogene Myc; these cells are poised to enter apoptosis. Elegant follow-up studies indicated that PEG3 and the p53-inducible gene product, seven in absentia 1A (SIAH1A), cooperate as downstream mediators of p53-induced cell death, with PEG3 being the critical ‘switch’ protein that enables cells to undergo apoptosis as opposed to growth arrest21. Importantly, the findings obtained in non-neural tissue and from molecular screens regarding the role of PEG3 in apoptosis seem to be generalizable to the brain in the context of both an ischaemia/hypoxia model22 and work in neuronal cell cultures. Indeed, data from neuronal cultures indicate that PEG3 enables apoptosis by encouraging the translocation of BCL2-associated X protein (BAX) from the cytosol to the mitochondria23. PEG3 has been suggested to interact with TNF receptor-associated factor 2 (TRAF2), which potentiates nuclear factor κB (NF‑κB) activation in response to tumour necrosis factor (TNF)24; as NF‑κB activation confers resistance to apoptosis25, PEG3 might therefore, in some instances, promote cell survival. However, the sensitivity of primary embryonic fibroblasts derived from Peg3-knockout mice to the cytotoxic action of TNF does not differ from that of cells derived from control mice26. www.nature.com/reviews/neuro © 2007 Nature Publishing Group REVIEWS Box 2 | The distribution of parthenogenetic and androgenetic cells in the brain of chimeras Results from mouse chimaera experiments11,12 have Striatum Hippocampus indicated a reciprocal distribution of parthenogenetic Frontal cortex (PG; shown in pink) and androgenetic (AG; shown in blue) cells in the brain. Although early in brain development both cell types are found throughout the brain, PG cells are selectively excluded from hypothalamic and pre-optic areas, and instead accumulate in the cortex and the striatum. The opposite pattern is seen for AG cells, which are excluded from the cortex and the striatum, and are enriched in hypothalamic and pre-optic areas. AG and PG cells are also found in other brain regions, but not to any great extent and not in numbers that exceed the numbers of normal cells in N–N control chimaeras11,12. PG cells have Pre-optic area Hypothalamus also been found to contribute to other neural areas, including the retina, the main olfactory mucosa and the vomeronasal organ (VNO). The main olfactory mucosa and Nature Reviews | Neuroscience the VNO are of particular interest, because in the main olfactory system, the distribution of PG cells is bilaterally symmetrical, possibly reflecting a contribution of imprinted genes to the patterning of specific olfactory neurons11. In addition to the reciprocal distribution pattern of cells, the neurodevelopmental abnormalities in AG–N or PG–N chimaeras are striking. AG–N chimaeras, despite having increased body weight, have reduced brain weight and a reduced brain/body weight ratio, relative to controls12. Conversely, PG–N chimaeras have reduced body weight and, when normalized for body weight, an increased brain/body weight ratio, possibly due in its entirety to the proliferation of PG cells in the forebrain area11. Furthermore, in PG–N chimaeras, this increased brain/body weight ratio correlates with the relative contribution of PG cells. This paradoxical effect (brain size normally positively co-varies with body weight104) suggests that maternal and paternal genomes have differential interests with regard to the allocation of resources to brain and body development13. Given the difficulty of generating these chimaeras, behavioural studies of adult low contribution chimaeras are limited. However, some analysis of sexual and aggressive behaviours has been performed for PG–N chimaeras. Remarkably, in a similar manner to the brain-weight data, the level of aggressive behaviour positively correlated with the contribution of PG cells11. Angelman syndrome A neurodevelopmental disorder that is characterized by mental retardation, ataxia and a ‘happy’ disposition. It results from a lack of maternally expressed genes on chromosome 15, at positions q11–q13. Ube3a is another well studied imprinted gene that is expressed in the brain. The paternal allele of this gene is silenced and the maternal allele is expressed. Like PEG3, UBE3A protein is present in both the cytoplasm and the nucleus27. A priori, UBE3A seems to have the potential to affect neurodevelopment in several ways (FIG. 2b). UBE3A was originally identified as a factor that associates with the papillomavirus E6 oncoprotein to bring about the ubiquitin-dependent degradation of p53 (hence its original name, E6-associated protein)28. In vivo studies have shown that UBE3A can also promote the degradation of p53, even in the absence of viral E6 (Ref. 29). In addition, UBE3A can ubiquitylate, and thereby target for destruction, the DNA-repair and cell-cycle-progression proteins HHR23 and O-6-methylguanine-DNA methyltransferase (MGMT)30,31 and the putative neuronal outgrowth Rho guanine nucleotide exchange factor ECT2 (Ref. 32). In addition to its ubiquitin ligase activity, UBE3A functions as a transcriptional co-activator, directly interacting with the progesterone and androgen receptors in a hormone-dependent manner. Recent experiments in mice have indicated that a fundamental role of UBE3A (in the prostate gland at least) might be to promote growth and development through effects on androgenreceptor function and by limiting p53-mediated apoptosis29,33,34. UBE3A is also thought to mediate degradation of the oestrogen receptor, with this interaction being modulated by a Ca2+–calmodulin complex35. Although their interpretation is speculative, these data indicate that UBE3A might modulate the effects of gonadal hormones on brain development and function. nature reviews | neuroscience At the gross level, Ube3a-knockout mice in which the maternal allele is deleted and the paternal allele is not expressed have a normal neuroanatomy. However, more subtle anatomical abnormalities might be present, as these animals show deficits in context-dependent learning, long-term potentiation, hippocampal electroencephalographic (EEG) recordings and cerebellar activity36–40. Furthermore, consistent with an in vivo role for UBE3A in degrading p53, cytoplasmic p53 levels are elevated in Ube3a-knockout mice37. In humans, specific mutations of UBE3A are associated with the neurodevelopmental disorder Angelman syndrome (see below). Individuals with Angelman syndrome have anatomical abnormalities, including cortical atrophy, cerebellar dysmyelination and Purkinje cell loss, and ventricular enlargement2. In most cases of Angelman syndrome, it appears that it is the ubiquitin ligase function of UBE3A that is lost, rather than the co-activator function41, implying that the neurodevelopmental phenotype of Angelman syndrome results primarily from aberrant ubiquitylation. The three other brain-expressed imprinted genes that have been characterized to some extent (in terms of the molecular interactions of their products) are paternally expressed necdin (Ndn), growth-factor-receptor-bound protein 10 (Grb10) and delta-like 1 (Dlk1). Ndn, the most extensively characterized of the three, is a member of the melanoma-associated antigen gene (MAGE) superfamily. Work in cell-culture systems indicates that its gene product, necdin, can influence multiple pathways that are involved in neuronal volume 8 | November 2007 | 835 © 2007 Nature Publishing Group REVIEWS Table 1 | Imprinted gene expression in the brain is a spatiotemporally dynamic process Features of the gene Examples Direction of imprinting Function of the gene product Expression dynamics Chromosomal location Highly expressed during embryogenesis and in the neonatal period Ndn Paternally expressed Regulator of neuronal differentiation and axonal outgrowth In the mouse brain, Ndn is expressed during early periods of neuronal generation and differentiation; peak levels of expression occur between postnatal days 1–4 (Ref. 115) Central chromosome 7 (mouse); 15q11–15q13 (human) Gtl2 Maternally expressed Untranslated RNA with In the mouse brain, the highest expression of Gtl2 Chromosome 12 possible regulatory is between E12.5 and P0 (Ref. 116) (mouse); 14q32 function (human) H19 Maternally expressed Untranslated RNA with In the fetal brain (6–12 weeks of gestation), H19 possible regulatory is highly expressed; in the adult brain, H19 has function low expression that is limited to the pons and the globus pallidus117 Chromosome 7 (mouse); 11p15.5 (human) Nesp Maternally expressed Neuroendocrine secretory protein, involved in both constitutive and regulated secretion Chromosome 2 (mouse); 20q13.2 (human) SnrpnsnoRNA Paternally expressed The gene is a long In the brain, Snrpn-snoRNA is imprinted into transcript that encodes adulthood118 various products Chromosome 7 (mouse); 15q11– 15q13 (human) Biallelic Commd1 expression during embryogenesis; imprinted Igf2 expression in adulthood Maternally expressed Involved in copper metabolism; the encoded protein interacts with NF‑κB Commd1 is biallelically expressed in the embryonic and neonatal mouse brain, but maternally expressed in the adult mouse brain17 Chromosome 11 (mouse); 2p15 (human) Paternally expressed Promotes growth Igf2 undergoes biallelic expression in the fetal brain (6–12 weeks of gestation), with monoallelic expression in other fetal tissues; in the adult brain, there is biallelic expression in the pons and monoallelic expression in the globus pallidus, the Raphe nucleus and the hypothalamus117 Chromosome 7 (mouse); 11p15.5 (human) Brain (region)specific imprinting Ube3a Maternally expressed Ubiquitin ligase and transcriptional coactivator activities In the mouse brain, Ube3a is maternally expressed in Purkinje neurons, the hippocampus and the mitral cells of the olfactory bulb, and exhibits biallelic expression elsewhere15; in humans, UBE3A imprinting is restricted to the brain16. Ube3a shows cell-type-specific expression119 Central chromosome 7 (mouse); 15q11–15q13 (human) Imprinted in all tissues apart from the brain Zim1 Maternally expressed Transcriptional regulator In the mouse, Zim1 is biallelically expressed in the Chromosome 7 neonatal and adult brain120 (mouse); no human orthologue Speciesspecific imprinting Commd1/ COMMD1, U2af1-rs1 Maternally expressed, paternally expressed COMMD1 protein is involved in copper metabolism. U2af1-rs1 encodes a zinc finger protein In the mouse, Commd1 is maternally expressed, Chromosome 11 and contains the paternally expressed (mouse); 2p15 (human) U2af1-rs1 in the first intron. In humans, COMMD1 is biallelically expressed, and does not contain a homologue of U2af1-rs1 (Ref. 121) Polymorphic imprinting Dlx5 Maternally expressed DNA binding; downstream effects on axonogenesis Dlx5 is polymorphically imprinted in the human brain122 but biallelically expressed in the mouse brain50 Chromosome 6 (mouse); 7q22 (human) Htr2a/ HTR2A Maternally expressed? Serotonin receptor subunit HTR2A is monoallelically expressed in 4 out of 18 adult human brains123 Chromosome 14 (mouse); 13q14 (human) Imprinted into adulthood Nesp is imprinted throughout development and in adulthood79 Additional information is available on the author’s homepage (see Further information). Commd1, copper metabolism (Murr1)-domain-containing 1; Dlx5, distal-less homeobox 5; E12.5, embryonal day 12.5; Gtl2, gene trap locus 2; Htr2a, 5-hydroxytryptamine receptor 2A; Igf2, insulin-like growth factor 2; Ndn, necdin; Nesp, neuroendocrine secretory protein; P0, postnatal day 0; snoRNA, small nucleolar RNA; Snrpn, small nuclear ribonucleoprotein N; U2af1-rs1, U2 small nuclear ribonucleoprotein auxiliary factor, small subunit 1; Ube3a, ubiquitin protein ligase E3A; Zim1, zinc finger, imprinted 1. survival, differentiation and outgrowth (FIG. 2c). The main cellular targets for necdin are proteins that are involved in nerve growth factor (NGF)-mediated cell survival and apoptosis. In the cytoplasm, necdin facilitates the association of the NGF receptors p75 and TRKA with each other42,43; following endocytosis, the intracellular 836 | November 2007 | volume 8 domains of these receptors are cleaved by γ-secretase44 and translocated to the nucleus, possibly with necdin attached. In the nucleus, necdin binds to the transcription factors E2F1 (Ref. 45) and p53 (Ref. 46), and also to its homologous MAGE protein, MAGED1 (also known as DLXIN1 and NRAGE)47, which is a specific facilitator www.nature.com/reviews/neuro © 2007 Nature Publishing Group REVIEWS a p53 PEG3 MYC p53 MYC p53 Peg 3 Siah1a p53 BAX SIAH1A Apoptosis Nucleus Mitochondrion b Ubiquitin Ubiquitin ligase activity Transcriptional co-activator activity Androgen receptor Androgen-receptordependent gene transcription UBE3A protein Target protein Ubiquitin Target protein Ubiquitin c Necdin MAGED1 DLX2 DLX5 p53 Necdin E2F1 Wnt1 Cdc2 Nucleus Figure 2 | Molecular interactions of imprinted-gene products. In vitro data from molecular screens (including yeast two-hybrid screens), followed in vivo |confirmation NaturebyReviews Neuroscience studies, are beginning to further our knowledge of the cellular processes and pathways that are affected by imprinted-gene products. Three of the best-characterized imprinted-gene products (in terms of their molecular interactions) are paternally expressed gene 3 (PEG3), the ubiquitin ligase protein UBE3A and necdin. a | Increased expression of the tumour-suppressor protein p53 and of the oncoprotein MYC in response to cellular signals results in the induction of the Peg3 and seven in absentia 1A (Siah1a) genes21. The PEG3 and SIAH1A proteins might cooperate to induce neuronal apoptosis, possibly by mediating the transport of BCL2-associated X protein (BAX) to the mitochondria23. b | UBE3A has two distinct functions: it is a ubiquitin ligase that facilitates ubiquitin addition to specific proteins (for example, p53), thereby targeting them for destruction, and a transcriptional co-activator for gonadal hormone receptors29. In Angelman syndrome, it appears to be the ligase function of the enzyme that is lost41. c | In the nucleus, necdin associates with melanoma antigen, family D, 1 (MAGED1), distal-less homeobox 2 (DLX2) and DLX5 to promote cellular differentiation through WNT1 signalling51. Necdin might also bind p53 and E2F1 to inhibit apoptosis by repression of cell division cycle 2 (CDC2) transcription49. nature reviews | neuroscience of NGF-dependent apoptosis48. E2F1 is a cell-cycleprogression factor that transcriptionally activates many pro-apoptotic genes, including that which encodes the cyclin-dependent protein kinase cell division cycle 2 (CDC2). Recent work indicates that the interaction between necdin and E2F1 has some functional relevance, insofar as necdin can repress E2F1-dependent CDC2 gene transcription in neuronal cultures and thereby attenuate the apoptosis of postmitotic neurons49. The functional relevance of necdin binding to p53 in neural tissues is less clear, although this binding inhibits p53-mediated apoptosis in osteosarcoma cells46. In addition to its functions as an anti-apoptotic factor, necdin can also modify neuronal differentiation. It binds through MAGED1 to the homeodomain proteins distal-less homeobox 2 (DLX2) and DLX5 (the genes for which are imprinted in a species-specific manner50) to upregulate activation of the Wnt1 promoter and hence enhance the differentiation of GABA (g-aminobutyric acid)-releasing neurons in the embryonic mouse forebrain51. Furthermore, necdin modulates intracellular processes that are essential for neurite outgrowth, by binding to the axonal outgrowth protein FEZ1 and to Bardet–Biedl syndrome protein 4 (BBS4) in the vicinity of the centrosomes52. The findings about necdin in cell-culture systems are recapitulated, to an extent, in vivo. Consistent with predicted roles for necdin in neuronal survival and differentiation, mice that lack Ndn show a reduced number of neurons in the hypothalamus that produce oxytocin and luteinizing hormone-releasing hormone53, as well as a reduced number of forebrain GABA-releasing neurons 51, dorsal root ganglion neurons and substance-P‑producing neurons42. Moreover, they show morphological abnormalities in axonal outgrowth and fasciculation in several neuronal cell types54. In humans, expression of NDN is lost in Prader–Willi syndrome (PWS), a neurodevelopmental disorder in which dysregulation of GABA-mediated signalling is thought to be a key contributor to some aspects of the phenotype55. Grb10 is paternally expressed in the mouse brain, owing to the presence of brain-specific promoter regions that are active on the paternal allele56 (elsewhere, Grb10 is maternally expressed), and it encodes a SRC homology domain 2 (SH2)-containing adaptor protein. There is some indirect evidence linking the GRB10 protein to the growth and development of the brain, through its ability to bind many plasma membrane receptors (including tyrosine kinases and growth receptors) that are involved in the development of the nervous system57,58. It has been postulated that GRB10 regulates signalling between these receptors and the apoptosis-inducing machinery on the mitochondrial outer membrane by modulating the apoptotic activity of the mitochondrial protein RAF1 (Ref. 59). Paternally expressed Dlk1 encodes a membranespanning and secreted protein in the epidermal growth factor-like homeotic family (with homology to members of the Notch–Delta developmental signalling pathway)60. It is expressed in the monoaminergic nuclei in the rodent and human brains61 and has been implicated in the differentiation of midbrain dopaminergic neurons62. volume 8 | November 2007 | 837 © 2007 Nature Publishing Group REVIEWS Currently, little is known about the specific molecular mechanisms by which DLK1 protein influences neuronal survival and differentiation. In adipocytes, it seems to inhibit differentiation, and might be co-regulated with necdin63. DLK1 is also more highly expressed in gliomas than in normal brain tissue, which indicates that it might interact with p53 (Ref. 64). Wnt1 promoter Wnt1 encodes a secreted signalling protein that is involved in developmental processes, including the induction of the mesencephalon and the cerebellum. Prader–Willi syndrome A neurodevelopmental disorder that is characterized by early hypotonia, followed by compulsive eating, mild mental retardation, several behavioural abnormalities and hypogonadism. It results from a lack of paternally expressed genes on chromosome 15, at positions q11–q13. SH2-containing adaptor protein A protein that is an accessory to the main proteins in a signal transduction pathway and that contains an SH‑2 domain (which binds phosphorylated tyrosine residues). Epidermal growth factor-like homeotic family A group of proteins that are involved in cell signalling and subsequent neurodevelopment or neural patterning and which have homology with epidermal growth factor. In vivo significance. Although some degree of caution is advisable in the interpretation of all molecular screens (it is in their nature to produce significant numbers of false positives65), the products of imprinted genes seem to affect several signalling cascades that are involved in cell survival and differentiation, notably acting through the tumour suppressor and key neurodevelopmental protein p53. However, because much of this screening work has been carried out in cell culture, often in the context of investigating the molecular processes that are involved in cancer, the extent to which these findings can be extrapolated to in vivo events that occur during normal mammalian neurodevelopment remains to be determined. In some cases, findings from humans and whole-animal models are consistent with the in vitro molecular findings, for example, regarding the role of Ube3a/UBE3A and Ndn/NDN in brain development and neurodevelopmental disorders. However, there is a relative lack of systematic data linking the effects of particular brain-expressed imprinted genes on cell survival and differentiation pathways with specific neurodevelopmental outcomes. Certainly, on the basis of the current data, we do not have clear evidence of any pattern of effects with respect to the direction of imprinting that might throw light on the curious behaviour of AG and PG cells during brain development. Functional consequences Mother–infant interactions. Whatever the eventual usefulness of the molecular findings that implicate imprinted genes in the cellular processes of brain growth and differentiation, there is substantial evidence for the influence of genomic imprinting, evident early in development, on brain function and behaviour. An important time during which imprinting influences the brain is the pre-weaning period, when mammalian infants are still highly dependent on the mother for resources. In animals, genomic imprinting has important effects on suckling behaviour by the offspring. For example, Gnasxl is paternally expressed and encodes an isoform of the stimulatory G‑protein subunit Gsα (XLαs). Mouse pups bearing a targeted deletion of Gnasxl fail to thrive, and many die before weaning. Their core deficit is an inability to suckle, which is consistent with the expression of Gnasxl in the motor areas of the brain that control the muscles of the jaw and tongue66. Advocates of the genomic conflict hypothesis in the context of maternal resource distribution might argue that these data are predictable, insofar as they reflect the paternal interest in increasing the extraction of food resources from the mother — here, imprinted genes in the brain are proposed to continue in postnatal life the effects that were mediated by the placenta in utero. 838 | November 2007 | volume 8 Consistent with this idea, knocking out Peg3, another imprinted gene that is paternally expressed in the brain, also leads to problems in suckling behaviour67. The intragenomic conflict hypothesis cannot, however, explain all imprinted-gene effects on mother– infant interactions. For example, Peg3-null mothers show deficits in aspects of maternal care, such as milk let-down, nest building and pup retrieval. These effects are independent of those that arise in the pups67, but are due (in all probability) to the reduction in hypothalamic oxytocin-expressing neurons in Peg3-null adult females20. These effects are difficult to reconcile with the genomic conflict hypothesis because of the erasure and re-setting of the imprinting mark in the germ line (FIG. 1) and the fact that relatedness asymmetries are not carried over generations. Therefore, because grandoffspring are just as related to their maternal grandmother as to their grandfather, there is no selective advantage (under intragenomic conflict) of a paternally derived gene influencing maternal behaviour and survival of the grand-offspring68. Rather, it has been suggested that the effects of Peg3 on mother and infant behaviours are an example of co-adaptation of imprinted-gene traits that, through different evolutionary routes and presumably different neurodevelopmental mechanisms, ensure adequate provisioning of the pre-weaning infant67. Studies in humans also emphasize mother–infant interactions as a key area for imprinted-gene function. Angelman syndrome and PWS are complex neurodevelopmental conditions that are associated with the disruption of a cluster of imprinted genes on human chromosome 15, specifically at 15q11–15q13. Angelman syndrome arises from disruptions that lead to a lack of maternally expressed gene product(s); PWS results from disruptions that lead to a lack of paternally expressed gene product(s)69. Angelman syndrome is characterized by severe learning difficulties, ataxia, seizures and EEG abnormalities70, whereas PWS is characterized by a failure to thrive in infancy, mild learning difficulties and, on emerging from infancy, a grossly abnormal satiety response to food intake71. Both conditions are associated with various changes in the structure 72,73 and function 74,75 of the brain. GABAA-receptor function is particularly affected 76, consistent with the major contribution of the lack of paternally expressed NDN to the PWS phenotype and the possibility of cytogenetic abnormalities disrupting GABAA-receptor-subunit gene expression in Angelman syndrome and PWS (BOX 3). Again, these human imprinted syndromes give rise, apparently, to effects on the mother–infant relationship that can be accommodated (tentatively, given the complex nature of the conditions) in the predictions of the genomic conflict hypothesis in terms of drawing on maternal resources. First, there is the well-established observation that infants with PWS have difficulty suckling71. More recently, work has focused on the unusually sociable disposition of individuals with Angelman syndrome — these individuals laugh and smile frequently and show much-reduced displays of negative-affect signals, such as crying and tantrums70,77. Previously, www.nature.com/reviews/neuro © 2007 Nature Publishing Group REVIEWS Disomies The presence of two sets of chromosomes — the normal situation in diploid organisms such as humans and mice. In uniparental disomies, both sets of chromosomes originate from one parent. Box 3 | Angelman syndrome and Prader–Willi syndrome (PWS): the GABAA connection There are several mechanisms that can cause the lack of maternally expressed gene products that results in Angelman syndrome. Most common are deletions in maternally inherited chromosomes, followed by paternal uniparental disomies and then mutations of the imprinting centre, which are the rarest variants. In PWS, which is often termed the sister disorder to Angelman syndrome, similar but reciprocal disruptions lead to a lack of paternally expressed gene product(s)69. Disruption of the GABA (γ-aminobutyric acid) type A (GABAA) receptor system is thought to be an important contributor to some of the behavioural and neurological abnormalities that are seen in individuals with Angelman syndrome or PWS. This is evidenced by their phenotypes (epilepsy in Angelman syndrome; psychosis in PWS) and their response to GABA-related treatments. One candidate imprinted gene that has been shown to have a direct action on GABA-mediated neuron differentiation is the paternally expressed necdin (see main text), but there are now indications that additional mechanisms lead to GABA-related problems in Angelman syndrome and PWS. Adjacent to the imprinted-gene cluster that is disrupted in Angelman syndrome and PWS are three genes that encode the GABAA-receptor subunits β3, α5 and γ3. These are not thought to be part of the imprinting cluster per se, but recent data indicate that they are sensitive to the changes in epigenetic status that occur as a result of the mutations that lead to Angelman syndrome and PWS92. Of particular importance is the β3 subunit which, along with ubiquitin protein ligase E3A (UBE3A), is regulated by the methyl CpG binding protein MECP2. This provides a molecular link between Angelman syndrome, PWS and other neurodevelopmental disorders with similar behavioural abnormalities, such as autism and Rett syndrome94. Therefore, loss of expression of these GABAA subunits, either directly (by physical deletion) or through epigenetic mechanisms, might underlie some aspects of the neurological and behavioural deficits that are seen in Angelman syndrome and PWS. This idea is supported by positron emission tomography studies of the benzodiazepine binding site in individuals with Angelman syndrome. These studies showed a dissociation in the binding deficits between individuals with a maternal 15q11–15q13 deletion encompassing the β3 subunit and individuals with Angelman syndrome resulting from a mutation in UBE3A105 — findings that were supported by similar results in two different mouse models of Angelman syndrome76. A similar pattern of decreased binding has been seen in individuals with PWS caused by the 15q11–15q13 deletion that encompasses the β3 subunit106. However, the conclusive study (comparing the phenotype that results from the 15q11–15q13 deletion with those that result from chromosome 15 maternal uniparental disomy and/or imprinting centre genotypes (where no direct disruption of the GABAA cluster has occurred)) has yet to be performed. these positive-affect behaviours were thought to occur indiscriminately. However, more detailed analysis has provided evidence that they occur specifically in a social context, namely in response to adult speech, eye contact and touch78. These findings, and the increased negativeaffect signals shown by children with PWS71, have led to the idea that, in addition to influencing resource allocation from the mother to the offspring through effects on suckling behaviour, brain-expressed imprinted genes can also influence the social resources that the offspring receives, through effects on behaviours that elicit care from the mother (BOX 4). The direction of the effects seen in Angelman syndrome and, to some extent, those seen in PWS would, according to the conflict hypothesis, suggest that the interest of the paternal genome is to maximize the amount of care and attention received from the mother by the current offspring and that the interest of the maternal genome is to divide these resources more equally across all her maternally related kin. It is unknown what neurobiological mechanisms, including effects on neurodevelopment, might mediate these influences on the early social interplay between mother and infant, and how these mechanisms relate to the complex genomic aetiology of Angelman syndrome and PWS. Adult behaviour. The persistence, in many cases, of genomic imprinting in the brain into adulthood begs the question of whether all effects of brain-expressed imprinted genes have a developmental basis. The gene Commd1, for example, is only fully imprinted in the adult brain17. A steadily increasing number of examples show that manipulating brain-expressed imprinted genes in animal models gives rise to effects on a range of adult nature reviews | neuroscience behaviours2,20,37,53. In a limited number of situations, for example, in mice that lack the maternally imprinted gene product NESP55, this occurs in the absence of any obvious gross developmental effects79. However, in the absence of ‘conditional’ genetic models, which allow the temporal control of gene activity, subtle effects on brain development and function cannot be excluded. Nonetheless, it seems that genomic imprinting has the potential to influence adult behaviour through several mechanisms. First, it can have effects on brain development. This can occur either through the influence of imprinted genes on nutrient supply by the placenta 3 or through more specific effects of brain-expressed imprinted genes on brain structure, connectivity and differentiation. Second, there is ongoing, persistent imprinted-gene function in the adult brain. Third, it is possible that brain-expressed imprinted genes have enduring effects on adult behaviour indirectly, through their influence on mother–infant interactions, variations in which have been consistently shown to have significant long-term effects on offspring behaviour80. The existence of imprinted-gene effects on adult behaviour has stimulated the debate about whether these effects could be adaptive in evolutionary terms. Here, in another facet of genomic conflict, it has been argued that the effects of imprinted genes on adult behaviour might be a consequence of the asymmetries of relatedness that arise in social groups (BOXes 1,4), and that the behaviours that are affected are likely to be those that underlie the effective manipulation of social interactions in a group. This would extend the functional consequences of imprinted-gene action in the brain into complex areas such as language, empathy and cooperative behaviours81. volume 8 | November 2007 | 839 © 2007 Nature Publishing Group REVIEWS Linkage A technique for identifying candidate chromosomal regions that underlie a particular trait, based on the extent to which that trait is co-inherited with certain genetic markers. Autism spectrum disorders (ASD). A number of phenotypically overlapping neurodevelopmental conditions, including autism, Asperger’s disorder, childhood disintegrative disorder and pervasive developmental disorder — not otherwise specified. Linkage peaks Chromosomal regions, identified by linkage analysis, that contain closely linked markers that are often co-inherited in probands. Epigenetic hotspot A region of the genome that is especially sensitive to perturbation of gene expression through effects on DNA methylation and/or histone modification. Risk of neuropsychiatric disorders. Alongside the widening repertoire of behavioural and cognitive functions that are influenced by genomic imprinting, there is increasing evidence that suggests there is an important role for imprinted genes in a number of common mental and neurological disorders, many of which have suspected neurodevelopmental antecedents 82. The evidence ranges from parent-of-origin effects on the symptomatology or inheritance patterns of certain disorders (for example, Tourette’s syndrome82 and multiple sclerosis83) to more convincing parent-of-origin effects in specific linkage studies (for example, studies of bipolar disorder84, of handedness and susceptibility to schizophrenia85, of the maternal origin of the X chromosome in Turners syndrome, and of autism86). In all cases, it is important to differentiate between parent-of-origin effects that are due to imprinting and those that arise as a result of other mechanisms, such as the preferential expansion of trinucleotide repeats or mutations in mitochondrial DNA (which is always maternally inherited)82. Some of the clearest evidence linking genomic imprinting to neuropsychiatric disease has come from genome-wide scans that examined linkage to autism spectrum disorders (ASD). Here, several of the linkage peaks overlap with, or are in close proximity to, imprinted regions, especially the imprinted-gene clusters located at 7q21–7q31.31, 7q32.3–7q36.3 and 15q11–15q13 (Ref. 87) . The linkage peak at 15q11–15q13, which encompasses the region that is disrupted in Angelman syndrome and PWS, has been termed an epigenetic Box 4 | Conflict in the mind? An important arena for the genomic ‘battle of the sexes’ might be the allocation of maternal resources. There is strong evidence supporting this idea from imprinted genes that are known to influence placental function and suckling behaviour10. Although the idea is speculative at this stage, it is thought that some genes in the q11–q13 region of chromosome 15 (the interval that is disrupted in Angelman syndrome and Prader–Willi syndrome (PWS)) might also act antagonistically on the way in which infants elicit care from their mother. A key characteristic of Angelman syndrome is an unusually sociable disposition and reduced display of negative-affect signals70,77. These positive-affect behaviours were originally considered to occur inappropriately107, but now they are thought to occur specifically in a social context108–110. This indicates that one or both of the two known maternally expressed genes in the critical region (ubiquitin protein ligase E3A (UBE3A) and ATP10A) normally act as brakes that limit positive-affect signals. In Angelman syndrome, in which these gene products are absent, this ‘braking’ function is lost. Conversely, individuals with PWS show increased negative-affect signals (stubbornness and temper tantrums) and are prone to mood instability and non-psychotic depression111. In maternal chromosome-15 uniparental disomy, and in PWS imprinting-centre genetic sub-types in particular, there is a ‘double dose’ of the maternally expressed UBE3A and ATP10A gene products (owing to the presence of two maternal copies and to a relaxation of the silencing of the paternal copy, respectively). This might lead to a greater-than-normal application of the brake that limits positive-affect signals, resulting in an increased propensity to develop affective psychosis112,113. This idea is predicted by the kinship theory (see BOX 1)81, the suggestion being that these positive-affect signals manipulate the sensory systems of receivers with respect to the allocation of social ‘resources’. In the same manner in which it manipulates nutrient resources through its effects on placental function and suckling, it is in the ‘interest’ of the paternal genome to maximize the amount of social resources received from the mother, whereas it is in the ‘interest’ of the maternal genome to equalize the amounts of these resources across all maternally related offspring. 840 | November 2007 | volume 8 hotspot for ASD gene candidates87. Cytogenetic abnor- malities that disrupt gene expression in this region are a common feature in patients with ASD (they are found in up to 5% of ASD patients), particularly abnormalities that give rise to duplications of the maternally inherited copy of chromosome 15 (Refs 87,88). This observation has focused attention on the two known maternally expressed imprinted genes in the 15q11–15q13 interval, UBE3A89 and ATP10A90,91. However, there are several other candidate genes that might convey an ASD risk in this complex genomic region, including the genes that encode the GABAA-receptor subunits GABRB3, GABRA5 and GABRG3. These genes are probably not imprinted themselves, but they lie close to the imprinted cluster and could become aberrantly expressed as a result of disrupted epigenetic regulation92. The linkage of 15q11–15q13 to the risk of ASD has generated numerous hypotheses, one of the most attractive of which suggests that abnormalities in the methylation of UBE3A lead to reduced UBE3A protein levels in the brain. Considering that reduced expression of UBE3A is also found in post-mortem brain samples taken from individuals with Angelman syndrome and Rett syndrome, it is possible that misexpression of UBE3A constitutes a common molecular lesion that underlies these phenotypically related disorders87,93. In addition to UBE3A, several of the imprinted genes in the vicinity of 15q11–15q13 might have a role in ASD, owing to their position on the chromosome and to their known neurobiological function; however, exactly how specific molecular abnormalities in these genes translate into abnormal brain development and function remains to be established. An important challenge with respect to ASD and other neurodevelopmental conditions in which imprinted mechanisms have been implicated, such as Angelman and Rett syndromes, is how to explain the delay in behavioural symptoms: individuals with these disorders often have normal development initially and only begin to show deficits later in infancy. This pattern suggests an important role for postnatal brain maturation, including experience-dependent plasticity94. One provocative hypothesis on which to end this Review is the suggestion that brain-expressed imprinted genes have the potential to contribute to the long-term risk of psychopathology by modifying the postnatal experience of infants, and the idea that they could do this by controlling key components of the early-life environment, notably mother–infant interactions. Conclusions and future directions Since the discovery and characterization of the first imprinted genes approximately sixteen years ago, it has become clear that growth and development, and in particular, brain development, are prime areas for imprintedgene function. However, it is equally clear that, in terms of neurodevelopment, there is much more to learn. At the molecular level, data are accumulating that show possible interactions of brain-expressed imprinted genes with several of the signalling pathways that coordinate the initial patterning, pruning and differentiation of brain cells. But www.nature.com/reviews/neuro © 2007 Nature Publishing Group REVIEWS Rett syndrome A neurodevelopmental disorder that occurs mainly in females and is characterized by cognitive impairments and autism-like behaviours. In most cases, the disorder is caused by a mutation in the X‑linked methyl CpG binding protein 2 gene. In males, this mutation is nearly always lethal. we still do not know, in many cases, how these molecular interactions ultimately lead to variations in brain structure and function. We are also unsure of the significance of the persistence of genomic imprinting in the postmitotic brain throughout adulthood. Genetic animal models will allow the dissociation of imprinted-gene effects that occur early in life (in utero and postnatally) from those that influence adult brain function and behaviour. Another important issue is the extent to which genomic-imprinting effects on neurodevelopment reflect the selective pressures under which imprinted genes have evolved. This is a controversial area, but the concepts of intragenomic conflict and the ultimate selfish gene account for many of the data that have been obtained from functional studies in which imprintedgene functioning is perturbed, such as in certain human conditions or as a result of genetic engineering in animal models. The issue is, are these functional effects subserved by the effects of differential maternal and paternal interests on neurodevelopment? The short answer is that, with the notable exception of the findings in mouse Reik, W. & Walter, J. Genomic imprinting: parental influence on the genome. Nature Rev. Genet. 2, 21–32 (2001). 2. Davies, W., Isles, A. R. & Wilkinson, L. S. Imprinted gene expression in the brain. Neurosci. Biobehav. Rev. 29, 421–430 (2005). 3. Fowden, A. L., Sibley, C., Reik, W. & Constancia, M. Imprinted genes, placental development and fetal growth. Horm. Res. 65 (Suppl. 3), 50–58 (2006). 4. Wilkins, J. F. & Haig, D. 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L.S.W. is a member of the MRC Co-operative on Imprinting in Health and Disease. DATABASES Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query. fcgi?db=gene ATP10A | BBS4 | CDC2 | Commd1 | Dlk1 | DLX2 | DLX5 | E2F1 | ECT2 | FEZ1 | GABRA5 | GABRB3 | GABRG3 | Grb10 | HHR23 | Igf2 | Igf2r | MAGED1 | MGMT | Ndn | p53 | p75 | Peg3 | RAF1 | SIAH1A | TNF | TRAF2 | TRKA | Ube3a | OMIM: http://www.ncbi.nlm.nih.gov/entrez/query. fcgi?db=OMIM Angelman syndrome | Bardet–Biedl syndrome | PWS | Rett syndrome FURTHER INFORMATION Lawrence S. Wilkinson’s homepage: http://www.BGG. cardiff.ac.uk ALL LINKS ARE ACTIVE IN THE ONLINE PDF volume 8 | November 2007 | 843 © 2007 Nature Publishing Group