Genomic Imprinting, Action, and Interaction of Maternal and Fetal Genomes

Genomic imprinting, action, and interaction of maternal and fetal genomes

Eric B. Keverne1

Sub-Department of Animal Behaviour, University of Cambridge, Cambridge CB23 8AA, United Kingdom

Edited by Donald W. Pfaff, The Rockefeller University, New York, NY, and approved October 16, 2014 (received for review July 9, 2014) Mammalian viviparity (intrauterine development of the fetus) and striatum (4). Moreover, the growth of the brain in parthe- introduced a new dimension to brain development, with the fetal nogenetic chimeras was enhanced by this increased gene dosage hypothalamus and fetal placenta developing at a time when the from maternally expressed alleles, whereas the brains of an- fetal placenta engages hypothalamic structures of the maternal drogenetic chimeras were smaller, especially relative to body generation. Such transgenerational interactions provide a basis for weight (3). These spatially distinct anatomical outcomes matched ensuring optimal maternalism in the next generation. This success to those brain regions found to be affected in human clinical has depended on genomic imprinting and a biased role of the studies on brain dysfunction seen in Prader–Willi and Angelman’s matriline. Maternal methylation imprints determine parent of origin syndromes (5, 6). The uniparental disomies of restricted chromo- expression of genes fundamental to both placental and hypotha- somal regions found in these clinical disorders were congruent lamic development. The matriline takes a further leading role for with the neural distribution of the chimeric cells. transgenerational reprogramming of these imprints. Developmen- Since these early findings, ∼100 genes have been identified tal errors are minimized by the tight control that imprinted genes to be “imprinted,” the majority of which have been shown to be have on regulation of downstream evolutionary expanded gene expressed in the placenta (7). Interestingly, a number of these families important for placental and hypothalamic development. placental genes have also been shown to be expressed in the de- Imprinted genes themselves have undergone purifying selection, veloping hypothalamus, most of which are maternally imprinted providing a framework of stability for in utero development with and paternally expressed and many of which influence cell pro- most growth variance occurring postnatally. Mothers, not fathers, liferation, cell cycle arrest, and apoptosis (Table 1). It is these take the lead in the endocrinological and behavior adaptations that placental/hypothalamic coexpressed imprinted genes and the nurture, feed, and protect the infant. In utero coadaptive develop- networks they regulate that are of special interest to a consideration ment of the placenta and hypothalamus has thus required a con- of mammalian brain development and its evolution. comitant development to ensure male masculinization. Only placental male mammals evolved the sex determining SRY, which activates Genomic Imprinting Sox9 for testes formation. SRY is a hybrid gene of Dgcr8 expressed There has been a strong bias for the matriline in determining the in the developing placenta and Sox3 expressed in hypothalamic evolutionary reproductive success of mammals, primarily through development. This hybridization of genes that take their origin the disproportionate investment shown by females in pregnancy, from the placenta and hypothalamus has enabled critical in utero in postnatal nurturing, and in maternal care for offspring. Geno- timing for the development of fetal Leydig cells, and hence tes- mic imprinting has not been found in egg-laying monotreme tosterone production for hypothalamic masculinization. mammals, and there are relatively few genes recruited to the imprint control regions (ICRs) of those marsupials with a genomic imprinting | genomic reprogramming | hypothalamus | short-lived chorio-vitelline placenta (8). Thus, fewer genes with placenta | development imprinted expression are observed in marsupials (six to date), and all are expressed in the placenta, whereas a number of genes enomic imprinting is an unusual epigenetic process in that imprinted in the eutherian placenta are expressed but not Git is heritable and results in autosomal gene expression imprinted in the marsupial placenta. The evolutionary trend has according to parent of origin. The recognition of genomic im- been to increase the number of heritable ICRs or differentially printing as a prominent feature of mammalian development came methylated region (DMRs) and at the same time increase the about through the generation of diploid mouse embryos with two number of genes recruited to these ICRs/DMRs. The ICRs are copies of maternal (parthenogenetic) or two copies of paternal particularly important for monoallelic regulation of gene dosage (androgenetic) genomes (1, 2). Although the developmental out- as revealed by the disrupted phenotypes when perturbations are comes of these maternal and paternal disomies produced differ- seen in the ICR but without any changes to the genes that are ences in placental size (1, 2), both procedures were lethal before imprinted. Such ICR perturbations are seen in a subset of the brain development. A significant experimental approach to in- human syndromes for Prader–Will and Angelman’s syndrome (9). vestigating genomic imprinting in the brain was first achieved by ICRs act on clusters of genes (3–12 in mouse) and typically in- the construction of chimeras (3, 4). Mouse embryos constructed clude a long noncoding RNA (lncRNA). The lncRNA is usually from a mixture of cells that were either androgenetic/normal or expressed from the opposite parental chromosome to the pro- parthenogenetic/normal survived, provided that these disomic tein coding RNA, but a common regulatory theme with respect cells were not greater than 40% of total cells. The precise loca- tions in the brain to which these chimeric cells participate was made possible to observe by the insertion of a LacZ or multiple β This paper results from the Arthur M. Sackler Colloquium of the National Academy of copies of the -globin gene marker. At birth, the neural cells that Sciences, “Epigenetic Changes in the Developing Brain: Effects on Behavior,” held March were disomic for paternal alleles contributed substantially to those 28–29, 2014, at the National Academy of Sciences in Washington, DC. The complete pro- regions of the brain engaged with primary motivated behavior gram and video recordings of most presentations are available on the NAS website at (hypothalamus, medial preoptic area (MPOA), bed nucleus of www.nasonline.org/Epigenetic_changes. stria terminalis and amygdala (BNST), and amygdala) and were Author contributions: E.B.K. designed research, performed research, and wrote the paper. excluded from the developing cortex and striatum. In contrast, The author declares no conflict of interest. parthenogenetic cells were excluded from these hypothalamic This article is a PNAS Direct Submission. regions of the brain but selectively accumulated in the neocortex 1Email: [email protected].

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Table 1. Imprinted gene expression in placenta and developmental integrity, forming the development foundations COLLOQUIUM hypothalamus on which the nonimprinted genome builds. Imprint control re- Gene Molecular actions Cellular actions gions have a sexual dimorphism in that they are predominantly matrilineal and have significant effects for embryo survival early Grb10 (P) (M) Maternal expression: Apoptosis in development, primarily by pathways concerned with mother/ placenta; paternal fetus interactions (16). The reproductive success of mammals expression; hypothalamus also places a considerable burden on matrilineal time and en- Phlda2 (M) Placental Apoptosis ergy, with some 95% of mammalian female adult life committed growth/neuroendocrine to pregnancy, lactation, and maternal care. Because stability and tumors purifying selection are features of imprinted genes, and matri- P57 kip2 (M) Tumor suppressor Cell cycle regulation; lineal ICRs result primarily in paternal expression, the question cdk inhibitor arises as to how gene stability survived the mutagenic environ- Dlk (P) Interacts with Igf1 binding Cell cycle ment of spermatogenesis. Point mutations, expanded tandem protein arrest/apoptosis repeats, and structural chromosomal mutations usually pre- Gnas (P) YY1 binding/activates Energy homeostasis dominate through patrilineal transmission (17). However, the adenylyl cyclase sperm genome is tightly packaged with protamines and not ini- Magel2 (P) MAGE family of proteins Apoptosis tially available for transcription following fertilization. Post- Necdin (P) Regulates Hif1α/Arnt Apoptosis fertilization male chromatin is remodeled from the nucleoprotamine 2+ Nnat (P) Phosphorylates CREB Increase intracell Ca state to the nucleohistone state, and at the nuclear replication β Peg1 (P) Hydrolase fold family Growth regulation phase (E2–3), sperm DNA undergoes mismatch repair (18). The Peg3 (P) YY1 binding/regulates Apoptosis mammalian zygote has several unique features influencing DNA α Hif1 /Arnt repair depending on proteins and mRNAs of maternal origin in Zac1 (P) Bind to Igf2 enhancer Cell cycle the oocyte. Maternal and paternal chromatin at this zygotic arrest/apoptosis stage is epigenetically very dissimilar, with male chromatin un- dergoing widespread demethylation linked to DNA repair mechanisms (19). This active demethylation of the patrilineal to lncRNAs and imprinted clusters has yet to be determined genome, through Tet3 and UG mismatch repair and base exci- (10). One imprinted cluster in particular, that of Dlk-Dio3,has sion repair, ensures greater genomic stability of imprinted pa- three protein coding genes that are expressed from the paternal ternally expressed genes. Hence, DNA repair at the zygotic phase allele (Dlk, Rtl1,andDio3), whereas several noncoding RNAs of development can be regarded as a maternal trait for repairing (Meg3,anti-Peg11, Meg8, Meg9, AK50713, and AK053394) are paternally expressed genetic errors. Indeed, many maternal zygotic characterized by expression from the maternal allele. The ma- factors are important for paternal allele demethylation (20), jority of these noncoding RNAs contain microRNAs that are epigenetic stability, and protection and maintenance of maternal predicted to negatively regulate this cluster (11). imprints at this zygotic phase of patrilineal methylation reprog- Rtl1 is a member of the Suchi-ichi long terminal repeat (LTR) ramming (21) (Fig. 1). retrotransposons that has lost its LTRs and codes for a protein Reprogramming of the genome differs for the male and fe- essential for placental development and fetal growth (12). Rtl1 is male germ lines in that the male genome is reprogrammed after thought to have retrotransposed into this locus before the di- fertilization (i.e., current in utero generation), whereas female vergence of the placental eutherian mammals from the marsu- reprogramming occurs in the developing fetal ovary, providing pials and is thought to have played a significant role in driving reprogrammed oocytes for the next generation. In this way, the imprinting at the locus (8). Once imprinted through the gain of female germ line expresses forward transgenerational planning an ICR/DMR, gene expression was fixed at this locus and un- by providing protection for developing zygotic development of derwent purifying selection against short interspersed nuclear the next generation. Maternal zygotic factors aid demethylation elements (SINE) and long interspersed nuclear elements (LINE) of paternal alleles (Gse) (20), protect maternal imprints from insertions. The large number of anti-Rtl1 mRNAs is likely to demethylation (Pgc7 and stella) (22), maintain these imprints have arisen by duplication, possibly as a host defense mechanism (Zfp57) (21), and regulate epigenetic stability and initiate spe- associated with the retrotransposon insertion (13). The selection cific differentiation (Cdx) of the trophectoderm (23) (Fig. 1). against SINE repeats is an evolutionary feature of imprinted In addition, removal of the compact protamine packaging of domains, as is the elevated GC content and short introns. sperm paternal DNA and replacement with maternal histones is Imprinted genes are under purifying selection with constraints on domain size such that cis-acting elements can function correctly (13). An important feature of the genes that are imprinted is their stability achieved through purifying selection. Purifying selection, sometimes called negative selection, selects against extreme variance, leading to strong stabilization of DNA in these genomic regions. Much of the theory behind genomic imprinting has tended to focus on the genes themselves, which are either ma- GENETICS ternally or paternally expressed, rather than on the heritable epigenetic process that brings this about. Imprinted genes are remarkably stable, and the majority have not undergone subsequent gene duplication, nor is there evidence for positive selection on their protein coding sequences in placental mammals (14, 15). Many of these same genes have, however, been subject Fig. 1. Zygotic patriline methylation reprogramming. Reprogramming of to positive selection in species where they are not imprinted. The paternal methylation marks engages Tet3 UG mismatch base excision repair evolutionary stability of the genes that are subject to imprinting (BER) and is dependent on maternal zygotic factors that determine epige- can be viewed as an essential contribution to the early stages of netic stability and ensure the maternal methylated imprints are sustained.

Keverne PNAS | June 2, 2015 | vol. 112 | no. 22 | 6835 Downloaded by guest on September 28, 2021 necessary for subsequent male genome expression (24). Some genes escape demethylation during oocyte reprogramming (25), but a second wave of demethylation occurs later in development. This second wave of demethylation (around E13.5 in mouse) is Tet1 dependent and is particularly important for the few paternal imprints regulating the genes that are maternally expressed (Dlk/ Meg3, Mirg, and Rasgrf1) (26). Tet1 KO males have been shown to exhibit aberrant hypermethylation in the differentially methylated regions of certain imprinted genes in the next generation. This hypermethylation can be traced back to their primordial germ cells. Analysis of DNA methylation dynamics in reprogramming primordial germ cells indicates that Tet1 functions at this late developmental phase to erase any remaining methylation, including the few paternal imprint control regions (27). – Genetic Stability and Variance Fig. 2. Peg3 target genes (E11 12) coexpressed in the hypothalamus/placenta. Evolutionary expanded gene families (Prls, Psgs, Ceacams) of mammalian de- Stability is a common feature of the genes that are imprinted, velopmental genes that are coexpressed in placenta and hypothalamus providing monoallelic expression always from the same allele. (E11–12)andshowchangedexpressioninthePeg3 mutant mouse. Many of these imprinted genes are members of a network of coregulated genes, comprising a hub for other imprinted genes (H19, Dlk, Peg3, and Cdkn 1c) and being directly regulated by ternal hypothalamus, become reorganized during pregnancy by the H19/Ifg2 locus through binding to a shared enhancer (28). the influence of hormones from the fetal placenta, as does the The stability and robustness of functional imprinted gene net- regulation of postpartum delivery of milk, warmth, and maternal works, especially those involved with complex phenotypes (growth, behavior (36). Progesterone in particular suppresses fertility and metabolism, brain and placental development), is provided by increases maternal food intake in anticipation of subsequent compensatory actions through downstream genes (29) common fetal demands in the later stages of pregnancy. The same feto- to the interacting hubs. Thus, errors may be absorbed or the cells placental hormone promotes synthesis of maternal hypothalamic in which they occur are eliminated at early stages of development, oxytocin in anticipation of its requirements for parturition, ma- thereby sustaining the robustness of complex biological systems ternal behavior, and milk ejection (37) (Fig. 3). like the brain and placenta. Network-based studies of imprinted Placental progesterone is also a precursor for the production genes have revealed insights into complex disorders. The imprinted of the remarkably high concentration of allopregnenalone in the gene products network (IGPN) (29) as part of the human inter- mother’s brain and in the fetal brain and shows a rapid decline actome reveals ∼214 genes of the IGPN to be significantly up- with placental loss at birth (38). Progesterone and allopregnenalone regulated in psychotic individuals. Interestingly, the IGPN genes act at the GABA receptor to provide neuroprotection against show a contrasting pattern of dysregulation in psychosis and au- ischemia and lack of oxygen in the brain (39). These placental tism, up-regulated in psychosis and down-regulated in autism, steroids are also alleviators of maternal stress. Allopregnenalone which is congruent with the contrasting behavioral symptoms of restrains the responses of the maternal hypothalamo-pituitary- these disorders (29). adrenal axis to stress by activation of neural opioids, which also Genetic variance is also an important component of evolu- prevents any premature secretion of oxytocin (40). In contrast, tionary development, but in the context of genomic imprinting, glucocorticoids that are produced by chronic prenatal stress of this is provided by the expansion of gene families that are the mother are associated with the programming of diseases downstream from those imprinted genes that provide the robust in the fetus (metabolic, cardiovascular, and psychiatric) (41). In framework for development. Three large gene families (pro- short, the fetal genome determines its own destiny via the pla- lactins, ceacams, and pregnancy-specific glycoproteins) have in- centa, hormonally regulating the maternal hypothalamus to serve creased in number across placental mammalian evolution and the interests of the fetus that, at the same time, is developing its are expressed in the placenta and hypothalamus. The pregnancy- own hypothalamus. The question arises as to how the developing specific glycoproteins (Psgs) are members of the Ig superfamily fetal placental genome knows the optimal way of regulating the and induce the release of anti-inflammatory cytokines (30). adult maternal hypothalamus of the previous generation, and These glycoproteins are thought to be important for modulating how has the adult mature hypothalamus developed to respond to the innate and adaptive immune response to assure a successful future demands of this next generation? These transgenerational pregnancy (31). Psgs activate TGF-β and are also important in coadaptive events also require coadaptations across the maternal brain development, neural stem cell fate decisions, and neuro- and fetal genomes, the success of which is epigenetically carried protection (32). In the placenta, prolactins stimulate cell mi- forward to the next generation through genomic imprinting. The gration and uterine invasion by trophoblast (33). They also have same imprinted genes that develop both the fetal hypothalamus a strong anti-inflammatory effect at the placenta–uterine in- and fetal placenta do so at a time when the placenta is instructing terface (34). In the brain, prolactin induces neurogenesis in the the maternal hypothalamus for provisioning the fetus. In this subventricular zone, and low prolactin levels in pregnancy are way, the developing hypothalamus and placenta serve as a tem- associated with increased maternal anxiety (35). The mammalian plate on which positive selection pressures for good mothering imprinted Peg3 gene impacts on these three large families and operate, driven by the effectiveness and success of placental brings about changes in coexpression in both the brain and interactions with the adult maternal hypothalamus of the pre- placenta (Fig. 2). vious generation (Fig. 4). Molecular genetic studies have identified several of the imprinted Coadaptive Development of Brain and Placenta genes that are coexpressed in the developing hypothalamus and In utero placental development introduces a new dimension to placenta and have been shown to be concerned with the regu- understanding how the environment may influence brain de- lation of maternal behavior and physiology (Table 1) (42). De- velopment of the next generation through regulatory influences tailed studies with mice carrying a mutation in the maternally on maternal behavior of the current generation. The primary imprinted gene (Peg3) have provided some insights into how the motivated behaviors of sex and feeding, regulated by the ma- functioning of the hypothalamus, which develops in utero, is adapted

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Genetic linkages between the hypothalamus and placenta are COLLOQUIUM also illustrated by the finding that placental stem cells, even from the human placenta, can be induced to differentiate into neurons (46). Moreover, the placenta itself has been shown to be a source of the neurotransmitter serotonin (47). Whether or not this serotonin can affect the fetal or maternal brain is not known, but serotonin has been shown to increase placental aromatase ac- tivity, a key enzyme controlling placental steroids that have important actions on the maternal hypothalamus during pregnancy. Coadaptation of Hypothalamus and Placenta: Consequences for Masculinization of the Brain The coadaptive development of the placenta and maternal hypothalamus has required a concomitant evolutionary develop- Fig. 3. Placental regulation of maternal neuroendocrine and neural func- ment to ensure masculinization of the male hypothalamus (48). tion. Placentally produced steroid hormones determine forward maternal Males do not, of course, undergo pregnancy and parturition, but planning by directing/orchestrating maternal physiology and postnatal maternalism to synchronize with the development of the fetus. they nevertheless benefit from viviparity and have been subject to the same placental hormones and selection pressures from viviparous in utero development. Indeed, the male brain is ca- to respond to the placental hormones that determine maternal- pable of maternalism in monogamous mammals, whereas cas- ism. When Peg3 is mutated in the maternal hypothalamus, but not tration at birth enables the paternal care for offspring in many in the placenta or fetus, many aspects of the mutant phenotype mammalian species (49). Mammalian brain masculinization has are similar to the functional consequences of this same gene being long been established to be a consequence of testosterone aro- mutated in the placenta but not in the mother’s hypothalamus matization to estrogen, which epigenetically regulates sex dif- (43) (Fig. 5). The functional consequences of hypothalamic gene ferences in the neuronal structure of certain hypothalamic nuclei deletion are manifest through reduced cell numbers in the hy- (50), many of which are concerned with regulating maternalism. pothalamus paraventricular nucleus (PVN), supra-optic nucleus Thus, an important evolutionary step in masculinization of the (SON), and medial preoptic area (MPOA) (44), which results in hypothalamus in placental mammals has been the evolution of impaired maternal care and impaired suckling, which in turn, the male Sry sex-determining gene. Sry is not present in the delay growth and onset of puberty and adult reproduction. This earliest mammals, the egg-laying monotremes, and is thought to same Peg3 gene deleted in the placenta also impairs placental have evolved in parallel with placentation. Interestingly, Sry is a growth, which in turn impairs hormonal priming of the hypo- hybrid gene of Dgcr8 and Sox3 (51) and serves as the master thalamus for maternal care and milk letdown. Thus, offspring that switch for testes development (52). It provides the trigger for sex receive optimal nourishment and improved maternal care will be determination by activating Sox9, which not only brings about predisposed to develop a hypothalamus that is both genetically and testes formation but blocks the genetic pathway to the differ- epigenetically predisposed to this same type of good mothering. entiation of ovarian cells (53). Mechanistically, imprinted genes have played an important Sox3, an X-linked maternally expressed gene from which Sry role in the mother–infant coadaptive process. Certain genes are evolved, is also required for development of the hypothala- coexpressed at critical times in the developing placenta and de- mus and pituitary (54), whereas Dgcr8, which became inserted veloping hypothalamus (Peg1 and Peg3). Mutations to these genes in the placenta produce remarkably similar functional deficits when the same gene is selectively mutated in the developing hypothalamus. Indeed, these convergent phenotypic outcomes are congruent with and support the notion of the hypothalamus and placenta having coadaptively evolved. Because these outcomes are developmentally synchronized, the placenta is in a pivotal position for providing resources for fetal hypothalamic development at the same time as commandeering the adult maternal hypothalamus to provide these resources. Chal- lenging this developmental lineage of hypothalamus and placenta with 24-h food deprivation in animal studies at the late stages of pregnancy results in disruption to coexpression of imprinted genes, primarily by affecting placental gene expression (45). Food deprivation produces a significant decrease in Peg3 expression in the placenta, with consequences similar to many of the gene changes induced by Peg3 mutation. Such genomic dysregulation does not occur in the developing fetal hypothalamus where Peg3 expression increases with maternal food deprivation (45). GENETICS Such changes in gene expression brought about by food deprivation are consistent with the fetal genome maintaining hypothalamic development at a cost to its placenta. This biased change Fig. 4. Coadaptive evolution. Imprinted genes are coexpressed in the fetal to gene dysregulation in the placenta is linked to autophagy and placenta and fetal hypothalamus (Table 1) during the development period ’ ribosomal turnover that sustains, in the short term, nutrient supply when the fetal-placental instructs the mother s hypothalamus for maternal behavior. This fetal-placental transgenerational engagement of the mother’s for the developing hypothalamus. Thus, the fetus controls its own hypothalamus is essential for the successful destiny of its own generation. destiny in times of acute starvation, especially in the last trimester Thus, a transgenerational template is provided for selection pressures to of pregnancy, by short-term sacrifice of its placenta to preserve evolve successful development of fetal placental-maternal hypothalamic resources critical for brain development (45). transgenerational interactions.

Keverne PNAS | June 2, 2015 | vol. 112 | no. 22 | 6837 Downloaded by guest on September 28, 2021 amygdala, BNST, MPOA, and nucleus accumbens) involved with reproductive behavior, pheromonal processing, and reward (44). Discussion The reproductive success of eutherian mammals has been achieved through in vivo placentation, postnatal lactation, and maternal care. Hence, the greater investment of time, energy, and physi- ological resources for mammalian offspring is primarily a function of the matriline. The male fetus also benefits from such provisioning and care, but their adult reproductive success is achieved by mating with many females. The success of such a strategy in males depends on the finding of reproductively Fig. 5. Coadaptive functioning of the Peg3 gene The similarity of functional available estrous females and competing with other males for phenotypes with Peg3 mutation in the developing placenta or independently priority over sexual access. Olfaction and pheromonal cues play in the developing hypothalamus illustrates its coadaptive functions across a very significant role in this process, both for male sexual and generations. aggressive behavior and for the male’s pheromonal priming of female reproductive status (64). These reproductive strategies are primarily determined by basal forebrain structures to which upstream of the ancestral Sox3 in Sry evolution, is important the hypothalamus is integral. Such neural and endocrine mech- for regulating microRNAs of the Dgcr8–Drosha complex – anisms were in place some millions of years before the arrival of expressed in the placenta (55). The Dgcr8 Drosha complex, primates. Although the basic principles of basal forebrain de- also known as a microprocessor, recognizes the RNA substrate velopment also apply to primates, including humans, major de- and the Dgcr8 protein recognizes and binds to pri-miRNAs to velopmental changes have also occurred in their neocortical trigger their cleavage by Drosha (56). The largest microRNA cluster forebrain brain development. In particular, substantial postnatal (C19 MC) discovered thus far is regulated by genomic imprinting, development of the neocortex has enabled emancipation of be- is expressed in the placenta, and displays altered expression pat- havior from deterministic hormonal mechanisms and the de- terns in placentas from complicated pregnancies (57). It has a pendence on olfactory/pheromonal cues. Complex social learning maternal-specific methylation imprint acquired in oocytes and and hierarchically determined interactions are integral to pri- generates a population of large compartmentalized noncoding mate behavior and are served by the evolutionary enlargement of RNA species (58). Conditional KO studies of Dgcr8 have also the neocortex. Albeit complex, there is nevertheless an impor- demonstrated a crucial role for miRNAs in development of the tant role for genomic imprinting in postnatal neocortical de- brain (59), especially in the context of olfactory neuron morpho- velopment through neurogenesis and preprogrammed cell death genesis (60). MeCP2 binds directly to Dgcr8, regulating gene ex- (42). Nevertheless, the role which genomic imprinting provides pression posttranscriptionally by suppressing nuclear miRNA for in utero and early postnatal development has been funda- processing, and gain of function of MeCP2 strongly inhibits den- mental to development of the eutherian basal forebrain. dritic spine growth (61). Another member of the Sox gene family Mammalian genomic imprinting evolved with placentation (Sox15) has 75% sequencing in common with Sox3 and is also and, indeed, most of the imprinted genes that are engaged with expressed in the placenta. Maternally expressed Sox3 is important development are expressed in the placenta. The epigenetic mark, for the development of the placental giant cells that produce those which regulates the monoallelic paternal or maternal allele ex- hormones that regulate the maternal hypothalamus. This hybrid- pression, is heritable and reprogrammed primarily in the matri- ization of placental and hypothalamic genes in to the mammalian line. Successful placentation has engaged a complex evolutionary Sry sex-determining gene has ensured critical in utero timing for process, for although the placenta is of fetal origin, at the ge- the development of fetal Leydig cells. Unlike the postnatal con- nomic level, there is a strong biased role for matrilineal en- dition, fetal Leydig cells produced testosterone without the need gagement. Not only is there a strong bias for epigenetic inheritance for gonadotrophin releasing hormone (GnRH). Fetal Leydig cell of ICRs through the matriline, but from the very earliest stages of production of testosterone counteracts the maternalizing influence germ cell production, germ cell reprogramming, decidualization, of placental hormones and masculinizes the male hypothalamus, transfer of mitochondrial DNA, DNA repair mechanisms, and an effect that continues into the early postpartum period when postfertilization placentation, the selection pressures introduced by viviparity have further endowed the matriline with a biased fetal Leydig cells subsequently undergo apoptosis (42). contribution, ensuring reproductive success and fetal survival. Few studies have examined the significance of genomic im- This in utero development has not only provided a safe environ- printing on male sexual behavior, although the imprinted Dlk1 ment for fetal development, but also one in which the maternal affects postnatal neurogenesis, and mutants are depleted of genome plays a dominant role. mature neurons in the olfactory bulb (62). The determinant role Mammalian viviparity requires the action and interaction of of testosterone in regulation of mammalian brain development two genomes in the mother, thereby providing a template for and male sexual behavior is, however, closely linked to structures intergenomic coadaptive functions. In short, the fetal genome that engage chemoreception. Male sexual behavior is severely determines its own destiny via the placenta, hormonally regu- impaired by genetic ablation of chemosensory/pheromonal sig- lating the maternal hypothalamus to serve the interests of the naling (63, 64) or by neural ablation of hypothalamic regions that fetus that, at the same time, is developing its own hypothalamus. receive this chemosensory information (65). The imprinted gene Thus, the success of this placental–maternal interaction in de- (Peg3) is expressed in basal forebrain nuclei, particularly in regions termining good mothering will shape the success of imprinted concerned with processing chemoreception and determining gene expression in developing a hypothalamus for good mothering male sexual behavior. Mutant Peg3 males are poor at sexual in the next generation. Each of these coexpressed imprinted genes discrimination of estrous urine and, unlike nonmutant males, are part of a network, where interacting alleles have evolved to they fail to improve this behavior through sexual experience (66). become coinherited (e.g., Psgs, Prls, and Ceacams). Thus, genomic Further studies have shown that the neuronal and behavioral imprinting has become a coordinator of coadapted gene expression, deficits seen in mice lacking a functional Peg3 gene are mediated a view point that has recently been given strong theoretical sup- by changes to apoptosis in anatomical regions (cortico-medial port (67).

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The complexity of mammalian in utero development not only the male germ line initially occurs after fertilization in the zygote COLLOQUIUM reveals coadapted interaction across different organs (placenta by the action of Tet3 (68). Tet1, a gene that is also engaged with and brain) but also genomic coadaptation across generations. oxidation of 5-methylcytosine, is important for the demethyla- The coexistence of three matrilineal generations in the pregnant tion erasure of imprints at a later developmental reprogramming female (maternal genome, fetal genome, and matrilineal genome phase (25). This second, later wave of demethylation in germ of the developing ovarian oocytes) enables transgenerational cells is important for removing any remaining methylation from forward planning (fetal placenta instructing maternal brain ICRs and DMRs inherited from males but of maternal origin contingent with fetal hypothalamic development). Moreover, from the previous generation. A failure of this late phase deme- reprogramming of the matrilineal genome is maturationally one thylation in Tet1 KO mice has resulted in paternally expressed generation ahead of the patrilineal genome. Development of the genes (notably Peg3 and Peg10) being down-regulated (27). This oocyte and reprogramming of the matrilineal genome occurs in later phase of ICR reprogramming is essential for demethylation the fetal developing ovary, whereas reprogramming of the male germ cells occurs after fertilization in the zygote primarily under of the maternal ICRs inherited from the father, which, if in- the regulatory control of maternal zygotic factors (20, 22, 23). complete, could potentially induce biallelic silencing of paternal Thus, the oocyte, which is fertilized by the male sperm, arose in expressed genes. the epiblast of the fetus in the grandmother’s uterus and un- ACKNOWLEDGMENTS. My thanks to Kevin Broad, James Curley, Frances derwent demethylation reprogramming at this very early stage. Champagne, and William Swaney for their contributions to these studies. Spermatogenesis in male offspring occurs primarily after birth, These studies were supported by grants from the Biotechnology and and after puberty and reprogramming of this next generation, Biological Sciences Research Council.

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