molecular mechanisms underlying abnormal placentation …171093/fulltext01.pdf · classification...

ACTA

UNIVERSITATIS

UPSALIENSIS

UPPSALA

2007

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology 371

Molecular Mechanisms UnderlyingAbnormal Placentation in theMouse

YANG YU

ISSN 1651-6214ISBN 978-91-554-7037-1urn:nbn:se:uu:diva-8331

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List of Papers

This thesis is based on the following papers, which will be referred to in the text by their Roman numerals:

I. Singh U, Yang Y, Kalinina E, Konno T, Sun T, Ohta H, Wakayama T, Soares MJ, Hemberger M, Fundele R. Carboxypeptidase E in the mouse placenta. Differentiation 2006 Dec;74(9-10):648-60.

II. Geng T*, Singh U*, Yang Y*, Elsworth B, Hemberger M, Geyer R,

Stewart MD, Behringer RR, Fundele R. Expression and function of the LIM-homeobox containing genes Lhx3 and Lhx4 in the mouse placenta. Manuscript.

III. Yang Y, Singh U, Wei S, Konno T, Soares MJ, Geyer R, Fundele R.

Influence of murine maternal diabetes on placental morphology, gene expression, and function. Submitted.

IV. Yang Y, Wei S, Bullwinkel J, Billur M, Geyer R, Singh PB, Fun-

dele R. Loss of the heterochromatin protein-1� encoding gene Cbx1 leads to defective placental development. Manuscript.

*Equal contribution Reprints were reproduced with the permission of the publishers

Contents

Introduction.....................................................................................................7 Species concepts.........................................................................................7 Interspecies hybridization and hybrid dysgenesis effects ..........................8 The placenta and placental functions .........................................................9

Classification Based on Placental Shape and Contact Points ..............10 Classification Based on Layers Between Fetal and Maternal Blood...11

Mouse placental development ..................................................................11 Genetic regulation of mouse placental development ...........................13 Marker genes for specific cell types ....................................................17 Epigenetic regulation of mammalian development .............................18 Epigenetic regulation and genomic imprinting in mouse placental development.........................................................................................22

Placental dysplasia models.......................................................................24 Abnormal placentation in mouse interspecies hybrids ........................25 Placental over growth in diabetic pregnancy.......................................29

Aims of the present studies ...........................................................................32 Paper I ......................................................................................................32 Paper II .....................................................................................................32 Paper III....................................................................................................33 Paper IV ...................................................................................................33

Results and discussion ..................................................................................35 Paper I ......................................................................................................35 Paper II .....................................................................................................36 Paper III....................................................................................................36 Paper IV ...................................................................................................37

Conclusions...................................................................................................39

Summary in Swedish ....................................................................................41

Acknowledgements.......................................................................................43

References.....................................................................................................44

Abbreviations

ASMA Alpha smooth muscle actin BrdU Bromodeoxyuridine cDNA Complementary DNA DXMit DNA segment, X chromosome, Massachusetts Institute

of Technology E Embryonic day post coitum F1 First filial generation IHPD Interspecies hybridization placental dysplasia IUGR Intrauterine growth restriction DNMTs DNA methyl transferases HDACs Histone deacetylases HMTs Histone methyl transferases H3K9me Methylated Histone 3 lysine 9 MMA Mus macedonicus MMU Mus musculus MSP Mus spretus MS MMU x MSP (female always shown first in all crosses) MSM MS x MMU backcross 1 MSS MS x MSP MX MMU x MMA MXM MX x MMU MXX MX x MMA PcG Polycomb group qRT-PCR Quantitative reverse transcription polymerase chain reaction SM MSP x MMU SMS SMx MSP STZ Streptozotocin TrxG Trithorax group XM MMA x MMU

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Introduction

Species concepts

What are species? The question of how best to define "species" is one that has occupied natural philosophers and then biologists for centuries, and the debate itself has become renowned as the species problem. In the 18th cen-tury, Linnaeus classified organisms according to differences in the form of reproductive apparatus. Although his system of classification sorts organ-isms according to degrees of similarity, it made no claims about the relation-ship between similar species. At that time, it was still a dogma that there was no kinship or lineage between species, no matter how similar they appeared, as this would have been in stark contrast to the Biblical creation myth. In the 1940’s, the behavioral biologist Ernst Mayr developed the widely used bio-logical species definition, which states that a species is a group of actually or potentially interbreeding populations that are reproductively isolated from other such groups. In consequence, gene flow between different species should be difficult or impossible (Dobzhansky, 1951; Mayr, 1963). Since then, several other species concepts have been formulated. The morphologi-cal species concept defines a species as a group of individuals with funda-mental resemblance in morphology and anatomy; the recognition species concept is based on reproductive isolation mechanisms, assuming that members of one species will have the ability to recognize potential mates from the same species but will ignore potential mates from similar but dif-ferent species; the evolutionary species concept interprets species as a sin-gle lineage of ancestor-descendant population which maintains its identity from other such lineages and which has its own evolutionary tendencies and historical fate; and the ecological species concept, which states that all or-ganisms of a species share same ecological resources and compete for them, that they tend to be on the same trophic level, at the same nodes in the food web and compete for same habitats (Mayr, 1942; Dobzhansky, 1951; Schlu-ter and Rambaut, 1996; Schluter, 2000).

In this thesis, the biological species concept has been followed.

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Interspecies hybridization and hybrid dysgenesis effects

As the ‘biological species concept’ would be nonsensical if individuals of different species were to produce offspring on a regular basis, interspecies hybridization is not a common event under natural conditions. This is due to reproductive barriers, which may act either pre- or post-zygotically (Hal-dane, 1922; Wu and Palopoli, 1994). Still, interspecies hybridization occurs between closely related species, and the best-known examples of hybrids in the animal kingdom are the mule and the hinny. They are produced inten-tionally by humans in matings between female horse (Equus caballus) and male donkey (Equus asinus) and between male horse and female donkey, respectively. However, hybridizations between other closely related species of other mammalian groups have been described For example Zeedonk (Equus grevy x Equus asinus), Liger (Panthera leo x Panthera tigris) (Fig. 1).

Such reports have been systematically collected in ‘Mammalian Hybrids’

by A.P. Gray (Gray, 1971). Hybridization between the two rodent species Mus musculus (MMU), the common house mouse, and M. spretus (MSP) was described approximately 30 years ago (Bonhomme et al., 1978) and F1 hybrids and backcrosses derived from them have been used extensively in scientific research. Although interspecies hybridization can rarely occur naturally, various abnormalities, named hybrid dysgenesis effects, are com-monly observed in the hybrids. The most prominent hybrid dysgenesis ef-fects are sterility (mostly in male mammalian hybrids) and inviability. Other effects are altered growth and abnormal placental development (Rogers and Dawson, 1970; Gray, 1971; Zechner et al., 1996). Both growth and placental hybrid dysgenesis effects exhibit parent-dependent effects, as reciprocal

Fig. 1: Zeedonk (hybrid between a Zebra and a Donkey) is shown to the left. Liger (hybrid between Lion and Tiger) is shown to the right. Pic-tures adopted from comons.wikimedia.org/wiki/Image:Zeedonk_800.jpg and www.gangwar.com/blog/

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phenotypes are observed in reciprocal matings. At present, the best-studied example of interspecies hybridization is between MMU and MSP. F1 hy-brids from MMU x MSP matings (female always shown first) tend to be smaller than the F1 hybrids from MSP x MMU mating. This is also the case for placental dysplasia effects. Depending on the direction of the crosses and backcrosses, the placental phenotype can be manifested as increased or de-creased placental size (Zechner et al., 1996).

The work presented in this thesis focuses on molecular and biological as-pects of altered placental development in different mouse models. In the following sections, I will describe the placenta, it’s development and func-tion.

The placenta and placental functions As the embryo grows and develops a vascular system, it must establish a

‘bridge’ to obtain nutrients and eliminate waste products. This ‘bridge’ is the placenta. The word placenta is derived from the Latin term meaning ‘flat cake’. It is a unique organ that forms both the interface for selective delivery of nutrients from the mother to the fetus and also re-directs maternal meta-bolic, endocrine, cardiovascular and immune functions to promote fetal sur-vival and growth (Cross, 2005).

A true placenta refers to mammals only, however, in some non-mammalian species, the similarly functioning organ has also been described. For example, some sharks, snakes and lizards (Enders and Carter, 2006). Generally, placentas consist of four extraembryonic membranes; yolk sac, allantois, chorion and amnion. In the reptiles, birds and monotreme mam-mals which lay shelled eggs, these extraembryonic membranes have special-ized functions. The yolk sac contains mainly lipoproteins, which serve as nutritional reserve. The allantois can collect nitrogenous wastes, and the amnion, filled with amniotic fluid, provides a cushion to the growing em-bryo. The chorion forms the outermost extraembryonic membrane, which is in close proximity with the porous eggshell and is involved in respiratory gas exchange. In the marsupials, the egg is not shelled and becomes embedded in the uterine wall. The yolk sac then establishes a close connection with the maternal blood supply through which nutrient exchange takes place. How-ever, this association between the fetal and maternal tissues does not develop further proximity and complexity and is often called a yolk sac placenta (Enders and Carter, 2006). In the eutherian mammals, the allantois and the chorion fuse with each other and form a ramified meshwork of highly vascu-larized fetal tissue, which is in close proximity with maternal blood. Such a combination of the maternal and fetal tissues called a chorioallantoic pla-centa enables very efficient biochemical exchange across the two circula-tions (Georgiades et al., 2002; Enders and Carter, 2004).

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Unless were pointed out specifically, the term placenta will only be used to imply a chorioallantoic placenta hereafter. In addition to its primary goal of facilitating transport between mother and fetus, the placenta is also a ma-jor endocrine organ. In almost all mammals the placenta synthesizes and secretes steroid hormones - progestins and estrogens. The placenta also pro-duces a number of protein hormones. Depending on the species, it is the source of chorionic gonadotropins, relaxin, and placental lactogens. Placen-tal hormones have profound effects on both fetal and maternal physiology. Despite the fact that all placentas carry out the same basic processes of transport and hormone secretion, there are important differences in structure and function between different families of mammals. The placentas of hu-mans, cattle, horses and dogs are all very different from one another at both gross and histological levels. They also differ in certain functions that are clinically important - for example, in the ability to transport maternal immu-noglobulins to the fetus (Georgiades et al., 2002; Enders and Carter, 2004).

Based on different parameters, for instance, area of contact, proximity between the fetal and maternal circulations, and the direction of blood flow of the two circulations, the placenta can be classified into different types (Georgiades et al., 2002; Enders and Carter, 2004).

Classification Based on Placental Shape and Contact Points

As we had mentioned above, there are striking differences among species

in gross and microscopic structure of the placenta. Two characteristics are particularly divergent and form bases for classification of placental types: the gross shape of the placenta and the distribution of contact sites between fetal membranes and endometrium, and, the number of layers of tissue be-tween maternal and fetal vascular systems.

Depending the shape of placenta and the area of contact between fetal and maternal tissue, placentas can be categorized into different types. Dif-fuse placenta: almost the entire surface of the allantochorion is involved in formation of the placenta; seen in horses and pigs. Cotyledonary placenta: multiple, discrete areas of attachment called cotyledons are formed by inter-action of patches of allantochorion with endometrium. The fetal portions of this type of placenta are called cotyledons, the maternal contact sites (carun-cles), and the cotyledon-caruncle complex a placentome; seen in ruminants. Zonary placenta: the placenta takes the form of a complete or incomplete band of tissue surrounding the fetus; seen in carnivores like dogs and cats, seals, bears, and elephants. Discoid placenta: a single placenta is formed and is discoid in shape; seen in primates and rodents (Balinsky B, 1975).

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Classification Based on Layers Between Fetal and Maternal Blood

There are a total of six layers of tissue separating maternal and fetal

blood. Among them, three layers are fetal extraembryonic membranes: endo-thelium lining allantoic capillaries, connective tissue in the form of chorioal-lantoic mesoderm and chorionic epithelium, the outermost layer of fetal membranes derived from trophoblast. All of these layers are components of the mature placenta.

There are also three layers on the maternal side, but the number of these layers, which are retained - that is, not destroyed in the process of placenta-tion - varies greatly among species. The three potential maternal layers in a placenta are endothelium lining endometrial blood vessels, connective tissue of the endometrium and endometrial epithelial cells. Depends on the number of these three layers, placenta can be classified into the following types:

� Epithelicochorial placentas: all these three layers, which are en-dometrial epithelium, connective tissue and uterine endothelium, are retained. This type of placenta can be seen in horses, swines and ruminants.

� Endotheliochorial placentas: only the uterine endothelium is re-tained. Seen in dogs and cats.

� Hemochorial placentas: fetal chorionic epithelium is bathed in maternal blood directly. Seen in humans and rodents (Balinsky B, 1975; Enders and Carter, 2004).

Mouse placental development

Although the gross architecture of the human and mouse placentas differ somewhat in their details, their overall structures and the molecular mecha-nisms underlying placental development are thought to be quite similar (Rossant and Cross, 2001).

In mice, placental development begins in the blastocyst at embryonic day (E) 3.5 when the trophectoderm layer is separated from the inner cell mass (Fig. 2). At this stage, two distinct cell lineages are formed: the outer layer, specialized trophectodermal epithelium, which pumps fluid internally to form the blastocoelic cavity, and the inner cell mass (ICM), which lies to one side of the cavity (Balinsky B, 1975; Kaufmann, 1982; Georgiades et al., 2002; Enders and Carter, 2004; Watson and Cross, 2005).

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At the time of implantation (E4.5), the inner cell mass divides rapidly.

The monolayer of cells surrounding the blastocoel is called mural trophecto-derm, and the trophectoderm overlying the immer cell mass is called polar trophectoder. The mural trophectoderm cells stop dividing, yet they continue to replicate DNA (endoreduplication) to become polyploid, and differentiate into primary trophoblast giant cells that are analogous to human extravillous cytotrophoblast cells. In contrast, two diploid cell types emerge from the polar trophectoderm, which are those cells immediately adjacent to the inner cell mass: the proximal extraembryonic ectoderm and the distal ectoplacental cone (Fig. 2). The ectoplacental cone consists of secondary giant cells whe-reas the extraembryonic ectoderm retains the trophoblast stem cells (Ba-linsky B, 1975; Kaufmann, 1982; Georgiades et al., 2002; Enders and Carter, 2004; Watson and Cross, 2005).

By E6, implantation sites measuring about 4-6 mm2 and are visible in the uterine horns. The secondary giant cells extensively invade the uterine tissue

Fig. 2: Different stages of mouse extraembryonic development. See text for details. Figure adopted from Watson and Cross, 2005.

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and the rapidly growing ectoplacental cone starts to come in contact with the maternal blood vessels. The inner cell mass further differentiates into ex-traembryonic and embryonic regions. The primary endoderm completely lines the mural trophectoderm and the parietal and visceral yolk sacs become clearly distinguished at this stage (Fig. 2) (Balinsky B, 1975; Kaufmann, 1982; Georgiades et al., 2002; Enders and Carter, 2004; Watson and Cross, 2005).

Subsequently, the extraembryonic ectoderm will grow proximity with the trophoblast cells of the chorion layer and, later, forms the labyrinth. While developing, the labyrinth is supported structurally by an ectoplacental cone-derived layer called the spongiotrophoblast. It forms a compact layer of cells sandwiched between the labyrinth and the outer giant cell layer and corre-sponds to the column cytotrophoblast of the human placenta. During later gestation, glycogen trophoblast cells begin to differentiate within the spon-giotrophoblast layer, and subsequently they diffusely invade the uterine wall. The vascular portion of the placenta is derived from extraembryonic meso-derm (allantois) that extends from the posterior end of the embryo at E8.0 (Rossant and Cross, 2001; Adamson et al., 2002; Watson and Cross, 2005).

At E8.5, the mesodermal part of the allantoic bud continues to grow in the form of multiple fingers-like projections until it obliterates the extraem-bryonic coelm and further continues to grow and push against the mesoder-mal side of the chorion. This process of the allantois and the chorion join together called chorioallantoic fusion. Soon thereafter, the chorion begins to fold to form the villi, creating a space into which the fetal blood vessels grow from the allantois. At this time, the chorionic trophoblast cells begin to differentiate into two labyrinth cell types. Multinucleated syncytiotro-phoblast cells, formed by the fusion of trophoblast cells, surround the fetal endothelium of the capillaries. A mononuclear trophoblast cell type lines the maternal blood sinuses. Together the trophoblast and fetal vasculature gen-erate extensively branched villi of the labyrinth (comparable with human chorionic villi). By E11.5, all these structures are clearly organized and a functional placenta is formed as a distinct organ. Branched villi of the laby-rinth become larger and more extensively branched until birth (E18.5–19.5) (Adamson et al., 2002). The maternal and the fetal bloods flow in a counter-current manner within the labyrinth to maximize nutrient transport. If the labyrinth is not appropriately vascularized with suitable patterning, branch-ing, and dilation, placental perfusion is impaired, resulting in poor oxygen and nutrient diffusion (Rossant and Cross, 2001; Adamson et al., 2002; Wat-son and Cross, 2005).

Genetic regulation of mouse placental development

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The precise molecular control of placental development is still not fully understood. However, in the past few decades, there have been significant advances in our understanding of the genetic control of placental develop-ment, which have been achieved mainly through the transgenic mice and targeted mutations in the mouse (Rossant and Cross, 2001; Watson and Cross, 2005).

In the early mouse embryonic development, the trophoblast depends on signals from the ICM and its later derivatives for its proliferation and differ-entiation (Copp, 1979). Fibroblast growth factor (FGF) signalling pathway is the key player involved in the interaction between the trophoblast and ICM. The FGF family consists of at least 23 members in vertebrates, but the most important factor in promoting trophoblast development is FGF4. FGF4 is widely expressed during early preimplantation development, becomes re-stricted to the ICM at the blastocyst stage, and is expressed throughout the early postimplantation epiblast (Niswander and Martin, 1992; Rappolee et al., 1994). Four FGF4 receptors that can receive the embryo-derived FGF4 signal have been identified, and the main trophoblast stem cell-specific re-ceptor is FGFR2 (Arman et al., 1998). Few downstream targets of FGF sig-naling had been also identified. They are specifically expressed in the re-gions of the trophoblast that are adjacent to the source of embryonic FGF4 signal and are necessary for trophoblast lineage development. Cdx2 (caudal-type homeobox 2) and Eomes (eomesodermin homologue) are particularly notable because they belong to transcription factor gene families and have important function in embryonic patterning (Beck et al., 1999; Russ et al., 2000). Another signaling pathway, which is oestrogen-related receptor (Errb) pathway, is also very important in early trophoblast stem cell mainte-nance and differentiation (Luo et al., 1997). Few other factors are also im-portant at early embryonic stage, such as Hand1 and Stra3 can promote giant cell proliferation (Cross et al., 1995; Hughes et al., 2004), Socs3 and Lif1 are responsible for restricting giant cell growth and counteract the effects of Hand1 and Stra3 (Roberts et al., 2001; Takahashi et al., 2003).

At mid-gestational stage, the most important event is chorioallantoic fu-sion. There are many genes involved in regulation of this process (reviewed by (Hemberger and Cross, 2001; Rossant and Cross, 2001; Watson and Cross, 2005). Several core factors are required during this stage, such as Brachyury, Bmp5 and Bmp7 are important for embryonic patterning; Vcam1 and Itga4 are important for chorioallantoic attachment (Gurtner et al., 1995; Kwee et al., 1995; Yang et al., 1995); Gcm1 is responsible for initiation of chorioallantoic branching (Schreiber et al., 2000).

At mid-late gestational stage, the matured mouse placental structure will be formed gradually. Genes involved in different stages of this process have been identified. Lifr, Egfr and Met are required for chorioallantoic branching morphogenesis (Gharib-Hamrouche et al., 1995; Hemberger and Cross, 2001). The mutation of downstream genes of these receptors, such as Grb2,

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Gab1, Sos1 and Mek1, also show labyrinth specific phenotype (Cheng et al., 1998; Giroux et al., 1999; Itoh et al., 2000; Qian et al., 2000). Tcf1/Lef1, Wnt2 and Fzd5, which are the factors in Wnt signaling pathway, are also important for labyrinth development (Monkley et al., 1996; Galceran et al., 1999; Ishikawa et al., 2001). Ascl2 is responsible for differentiation of spon-giotrophoblast out of the cells of the ectoplacental cone, which are otherwise destined to differentiate into secondary giant cells only (Guillemot et al., 1995; Tanaka et al., 1997). Esx1, Arnt and Tcfeb are crucial for vasculariz-tion (Kozak et al., 1997; Li and Behringer, 1998; Adelman et al., 2000). The genes governing mouse embryonic development had been extensively re-viewed in many literatures (Cross et al., 1994; Hemberger and Cross, 2001; Rossant and Cross, 2001; Simmons and Cross, 2005; Watson and Cross, 2005) and are summarized in Table 1.

Table 1. Genes involved in trophoblast and placental development

Gene name Gene product Trophoblast defect

Trophoblast lineage development

Cdx2 Homeobox transcription factor yes

Eomes Homeobox transcription factor yes

Fgf4 Fibroblast growth factor 4 no

Fgfr2 Fibroblast growth factor receptor ?

Trophoblast stem cell maintenance

Estrb (Err2) Nuclear hormone receptor yes

Trophoblast giant cell proliferation

Hand1 bHLH transcription factor yes

Mdfi (I-mfa) Inhibitor of bHLH factors yes

Ectoplacental cone function

Ets2 Ets domain transcription factor yes

Spongiotrophoblast maintenance

Ascl2 (Mash2) bHLH transcription factor yes

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Egfr Epidermal growth factor receptor yes

Hsf1 Heat-shock factor1 ?

Chorioallantoic fusion

Bmp5/Bmp7 Bone morphogenetic proteins ?

Dnmt1 DNA methyltransferase ?

Itga4 (Integrin �4) Integrin that binds Vcam1 no

Lhx1 (Lim1) Lim-domain transcription factor no

Mrj Co-chaperone no

Tcf/Lef1 double Transcription factor downstream of Wnt ?

T (brachyury) T-box transcription factor no

Vcam1 Cell adhesion molecule that binds integrin �4

Chorioallantoic branching and labyrinthine development

Dlx3 Homeobox transcription factor yes

Fgfr2 null Fibroblast growth factor receptor yes

Fra1 AP1 transcription factor yes

Fzd5 Frizzled5, Wnt receptor yes

Gab1 Signaling adaptor molecule yes

Gcm1 Transcription factor yes

Grb2 hypomorph Signaling adaptor molecule ?

Gja7 (Cx45) Connexin, gap junction protein ?

Hgf Hepatocyte growth factor yes

Hsp84-1 (Hsp90b) Heat-shock protein no

Itgav(Integrin �v) Adhesion molecule ?

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Junb AP1 transcription factor yes

Lifr Leukemia inhibitory factor receptor ?

Map2k1 (Mek1) MAP kinase cascade yes

Map2k2 (p38alpha)

MAP kinase cascade yes

Map3k3 (Mekk3) MAP kinase cascade ?

Met Hepatocyte growth factor receptor yes

Pdgfra Platelet-derived growth factor receptor ?

Pdgfb Platelet-derived growth factor chain � ?

Pparg Nuclear hormone receptor yes

Rxra/b double Nuclear hormone receptor yes

Sos1 GDP/GTP exchange factor yes

Vhlh Tumour suppressor ?

Wnt2 Wingless homologue, secreted glycoprotein no

Vascularization of labyrinth

Esx1 Homeobox transcription factor yes

Arnt bHLH/PAS transcription factor yes

Tcfeb (Tfeb) bHLH-Zip transcription factor yes

Nutrient transport across labyrinth

Gjb2 (Cx26) Connexin, gap junction protein yes

Marker genes for specific cell types Several genes are expressed in a certain cell type during placental devel-

opment and for some of them, precise functions and mechanisms of action are unknown till now. These genes are frequently used as markers for spe-cific cell types.

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Giant cells: Prolactin family 2, subfamily c, member 2 (Prl2c2) and Pro-lactin family 3, subfamily b, member 1 (Prl3d1) are expressed only by giant cells (Lee et al., 1988). Prl2c2 and Prl3d1 are widely used as early and late giant cell markers (Hall and Talamantes, 1984; Faria et al., 1990).

� Spongiotrophoblast: Tpbpa and Flt1 are expressed only in the ec-toplacental cone and later in the spongiotrophoblast exclusively (Lescisin et al., 1988; He et al., 1999). Tpbpa is one of the most commonly used markers.

� Glycogen cells: Cdkn1c is strongly expressed by glycogen cell in a mature placenta and serves to identify glycogen cells(Takahashi et al., 2000; Georgiades et al., 2002).

� Labyrinth: Eomes is expressed only in small clusters of trphoblast stem cells located in the labyrinth and has been used as tro-phoblast stem cell marker (Russ et al., 2000). Peg1 is exclusively expressed by the fetal capillaries in the labyrinth (Mayer et al., 2000) and �-smooth muscle actin is specifically expressed by the pericytes (Ohlsson et al., 1999).

Overall, it becomes clear that the mouse placental development is a proc-

ess regulated by concerted expression and silencing of different genes in a stage and cell lineage-specific manner. The differential regulation of gene expression in different genetically identical cell types in a developing pla-centa, just like in the other parts of a developing embryo, is taken care of by several different mechanisms, which can override the genetic information. Such mechanisms, collectively covered under the commonly used term “epi-genetics”, are important in placental development.

Epigenetic regulation of mammalian development

The primary DNA sequence is only a foundation for understanding how the genetic information is read. Without changing the DNA base sequence, how is gene expression differently regulated in different parts of a develop-ing embryo, eventually guiding the development of different organs and tissues? The answer comes from the analysis of different modifications of the DNA. Although the term ‘epigenetics’ literally means ‘above the genet-ics’, it is now generally used to mean those heritable modifications in DNA and associated proteins, which can change gene expression without changing the DNA base sequence. Such modifications are reversible and yet trans-mitotically heritable. Epigenetic modifications affect either DNA or DNA-binding proteins, which in turn affect the local structure and composition of chromatin, thus affecting the factors-accessibility and transcriptional compe-tence of the nucleosomal DNA template. Several types of epigenetic modifi-

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cations have been identified: DNA methylation, histone modification, poly-comb/trithorax mediated gene regulation and chromatin remodeling.

DNA methylation

The best-studied epigenetic modification in mammals is DNA methyla-tion. DNA methylation is a covalent modification that triggers heritable gene silencing(Levenson and Sweatt, 2005). This reaction is catalyzed by DNA methyltransferases (DNMTs), which can transfer a methyl group to the car-bon-5 position of cytosine in CpG dinucleotides. Gene silencing mediated by DNA methylation can be achieved in two ways. First, methylation can di-rectly interfere with transcription factor binding to recognition sites on DNA. Second, methyl-CpG binding domain proteins (MBPs) can reinforce silenc-ing by recruiting corepressor complexes that can be used as a harbor to ac-commodate histone deacetylases (HDACs) or histone methyltransferases (HMTs). MBP-associated HMTs form a positive feedback circle between DNA methylation and another epigenetic silencing mark, methylated lysine 9 on histone 3 (H3K9)(Fujita et al., 2003; Fuks, 2003).

Mammalian DNA methyltransferases can be classified into two groups based on their preferred DNA substrate. The de novo methyltransferases DNMT3a and DNMT3b are mainly responsible for initiation of cytosine methylation at unmethylated CpG sites. Targeted disruption of DNMT3a and/or DNMT3b in mice resulted in embryonic lethality (Okano et al., 1998; Jaenisch and Bird, 2003). DNMT1 copies pre-existing methylation patterns onto the new DNA strands during DNA replication and thus functions as the maintenance methyltransferase (Bestor and Ingram, 1983; Bestor, 1988). A fourth DNA methyltransferase, DNMT2, shows weak DNA methyltrans-ferase activity in vitro (Hermann et al., 2003), but targeted deletion of DNMT2 gene in embryonic stem cells causes no detectable effect on global DNA methylation, suggesting that this enzyme has no important function in setting DNA methylation patterns (Okano et al., 1998; Liu et al., 2003). DNMT3L is a DNMT-related protein that does not contain DNA methyl-transferase activity, but physically associates with DNMT3a and DNMT3b and modulates their catalytic activity (Okano et al., 1998; Hata et al., 2002). Briefly, different combinations of these de novo and maintenance methyl-transferases seem to constitute the core enzymatic components of the DNA methylation system in mammals.

Histone modification

The nucleosome, basic repeating unit of chromatin, consists of 146 bp of DNA wrapped around an octameric histone core containing two copies each of histones H2A, H2B, H3, and H4 (Margueron et al., 2005). Nucleosomal

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DNA can be further compacted by association with the linker histone H1 and additional nonhistone proteins, as well as by higher order looping and fold-ing of the chromatin fiber. The organization of chromatin not only restricts physical access of nuclear factors to the underlying DNA, but it is now clear that posttranslational modifications of histone proteins can alter chromatin conformation and play direct regulatory roles in gene expression. Histones have a central globular domain and relatively unstructured N- and C- termi-nal tails. The N-terminal tail is primarily subjected to post-translational modifications, such as acetylation, phosphorylation, ubiquitination, and me-thylation, which can result in significant changes in local chromatin structure (Berger, 2002). Different combinations of histone modifications can form a specific ‘language’ that instructs the alteration of structural state of chroma-tin: on or off. This ‘language’ is called histone code (Strahl and Allis, 2000).

Histone methylation is an imortant modification that is linked to epige-netic activation and silencing mechanisms. It has long been known that his-tones, particularly H3 and H4, are methylated at a number of lysine (Lys) and arginine (Arg) residues. So far, the major sites of Lys-methylation on histones identified are: Lys4, Lys9, Lys27, Lys36, Lys79 on H3 and Lys20 on H4 (Li, 2002; Sims et al., 2003). In addition, the Lys residue can be me-thylated in the forms of mono-, di-, or trimethylation. These varied methyla-tion patterns provide further functional diversity to each site of Lys methyla-tion. The methylation of Lys9 and Lys27 on histone H3 (H3K9, H3K27) is associated with gene silencing, whereas the methylation of Lys4 on histone H3 (H3K4) is associated with gene activation (Khorasanizadeh, 2004). A major breakthrough in the understanding of H3 Lys-methylation function was the discovery that one of the well studied Su(var) genes which encodes a histone methyltransferase (HMT). The Su(var)3–9 gene is the first gene that showed repression activity by methylation of Lys9 on Histone H3 (H3K9) (Rea et al., 2000), and this modification targets a silencing protein, called heterochromatin protein 1 (encoded by Cbx gene), to heterochromatin (Ban-nister et al., 2001; Lachner et al., 2001).

Histone acetylation normally occurs at the N-terminal on H3 and H4 re-sidues. In general, acetylation of histones loosens chromatin packaging and correlates with transcriptional activation, whereas deacetylation of histones is associated with transcriptional repression and silencing (Grunstein, 1997a; Grunstein, 1997b; Turner, 2000). Histone acetylation and deacetylation are mediated by histone acetyltransferases (HATs) and histone deacetylases (HDACs) respectively.

Histone phosphorylation, involving Serines 10 of histone H3, has also emerged as an important modification, both in transcriptional activation and in chromosome condensation during mitosis (Cheung et al., 2000).

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Polycomb/trithorax mediated gene regulation

Studies on Drosophila genetics provide additional link between epigenet-ics and histone modifications. Two evolutionarily conserved proteins fami-lies, the Polycomb Group (PcG) and the Trithorax Group (TrxG), had been found to be involved in modification of histones. The PcG proteins function in multimeric complexes and they are believed to maintain long-term gene silencing during development by altering local chromatin structure and in-volving post-translational modification of core histones (Czermin et al., 2002). Further analysis revealed that PcG proteins act in two forms to facili-tate chromatin condensation. TrxG proteins, which are antagonist of PcG in regulation of homeotic genes during devleopment, promote methylation of H3K4 and mediate transcriptionally active chromatin (Ringrose and Paro, 2004; Ruthenburg et al., 2007).

Chromatin-remodeling In eukaryotic cells, genomic DNA is folded with histone and non-histone

proteins to form chromatin. On the basis of different compaction manner, heterochromatin can be distinguished from euchromatin. Euchromatin is less condensed, more accessible and generally more easily transcribed, whereas heterochromatin is typically highly condensed, inaccessible and highly or-dered in nucleosomal arrays (Huisinga et al., 2006). The chromosomal re-gions that contain a high density of repetitive DNA elements and transpos-able elements are the main targets of heterochromatin formation (Birchler et al., 2000; Hall et al., 2003; Martens et al., 2005). These regions remain con-densed throughout the cell cycle.

A key feature of heterochromatin is its ability to propagate, and thereby influence gene expression in a region-specific, sequence-independent man-ner. When heterochromatin spreads across domains, it generally causes epi-genetic repression of nearby sequences and inhibits potentially deleterious recombination between homologous repetitive sequences, and it can partici-pate in the developmental regulation of specific genes. Although hetero-chromatin formation is widely involved in epigenetic gene silencing, the activation of gene expression associated with heterochromatin formation was also reported in several cases (Weiler and Wakimoto, 1995; Lu et al., 2000; Yasuhara and Wakimoto, 2006).

Many factors are required for the formation of heterochromatin structure. Histone modification and heterochromatin protein HP1 have a crucial role in this process. HP1 belongs to a highly conserved chromatin proteins family, their homologues are found from fission yeast (Swi6, Chp2 and Chp1) to humans (HP1�, HP1� and HP1�) (Huisinga et al., 2006). Each HP1 protein interacts with diverse factors that are involved in different aspects of hetero-chromatin structure and function.

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Based on unique histone modification patterns heterochromatin and eu-chromatin can be distinguished. In heterochromatin formation, histone modi-fications are characterized by hypoacetylation and H3K9 methylation. In contrast, histone H4 acetylation and H3K4 methylation is the hallmark for euchromatin (Grunstein, 1998; Litt et al., 2001; Nakayama et al., 2001; Noma et al., 2001). The general strategy of heterochromatin formation had been described in many literatures (Singh and Georgatos, 2002; Grewal and Jia, 2007). The formation of heterochromatin structure can be initiated by two mechanisms, either by silencer elements or by the RNAi machinery that targets repetitive DNA sequences (reviewed in (Grewal and Jia, 2007). Sub-sequently, histone methyltransferase (HMTs) and histone deacetylase (HDACs) can be recruited and cause methylation of H3K9. H3K9me pro-vides binding site for heterochromatin proteins such as Swi6/HP1, which can bind to the modified histone tails. Chromatin-bound Swi6/HP1 in turn serve as an assembly platform to recruit a number of factors that are involved in maintenance and spreading of heterochromatin. For example, Swi6/HP1 can recruit Su(var)3-9, which is a HMTs and the main producer of H3K9me, and promote the formation of H3K9me on adjacent histones. This heterochro-matin assembly process can be repeated continually on neighboring histones and spread to nearby sequences (Fig. 3) (Grewal and Jia, 2007).

Epigenetic regulation and genomic imprinting in mouse placental development

The epigenetic mechanisms are involved in the establishment and mainte-

nance of allele-specific gene expression in different tissues, especially the placenta. Majority of genes in our genome are expressed from both maternal

Fig. 3: Heterochromatin formation can be initiated by two different factors: (1) Silencer elements and (2) localization of non-coding RNA to repetitive DNA. Figure adopted from Grewal and Jia, (2007).

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and paternal alleles. However, there is a subset of genes, which are normally expressed only monoallelically. In the studies on mouse embronic develop-ment using parthenogenetic (PG) cells, which contain an exclusively mater-nally derived genome, and androgenetic (AG) cells, which contain only pa-ternally derived genome, it had been shown that paternally expressed genes are necessary for the growth of extra-embryonic tissues, whereas maternally expressed genes are important in embryonic development (McGrath and Solter, 1984; Surani et al., 1984; Barton et al., 1991). Their functional non-equivalence is due to genomic imprinting, a process by which one allele becomes epigenetically modified and inactivated based on parental origin (McGrath and Solter, 1984; Surani et al., 1984; Barton et al., 1991). Those genes, which are expressed only from the maternal allele, are frequently called paternally imprinted genes. Conversely, genes expressed only from the paternal allele are called maternally imprinted genes. It has been hy-pothesized that in the placental mammals, there is a competition between the maternal and the paternal genomes in the developing embryo, for the avail-able nutritional resources during gestation. The paternally expressed genes extract nutrition and promote growth of the placenta and hence the fetus. The maternally expressed genes on the other hand conserve nutritional resources for other offspring and hence antagonize the growth promoting effects of the paternally expressed genes (Moore and Haig, 1991).

The epigenetic modifications play a crucial role in the mouse embryonic and placental development through the maintenance/loss of imprinting. The clearest demonstration for imprinted genes is the allelic-specific DNA me-thylation patterns. Most imprinted genes contain at least one differentially methylated region (DMR) located in the promoter region or in the gene it self. When Dnmt1 or Dnmt1o (an oocyte specific DNA methyltransferase) was mutated, the imprinted genes showed either biallelic expression or bial-lelic repression except Ascl2 (Lewis et al., 2004). However, DNA metylation is not the only requirement for the maintenance of imprinting in mouse pla-centa, and histone modification is another important mechanism involved in imprinting control. It had been found that imprinting on mouse distal chro-mosome 7 was controlled by repressive histone methylation potentially trough a noncoding antisense RNA (Lewis et al., 2004). For several pater-nally imprinted genes, which are located on the imprinting center 2 (IC2) on mouse distal chromosome 7 lack parent-specific DNA methylation and de-methylation of DNA does not lead to loss of imprinting. Instead, disruption of dimethylation of H3K9 and trimethylation of H3K27 in IC2 can lead to reactivation of paternal allele (Lewis et al., 2004). Intriguingly, the Eed-Ezh2 Polycomb complexes are also required for paternal repression along this domain (Umlauf et al., 2004). Since noncoding RNA and H3K9me are involved in the formation of heterochromatin by recruitment of HP1, there-fore, different epigenetic regulations are linked in placental development.

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Allelic-specific acetylation and methylation of specific histone residues have also been reported for Igf2, H19, Igf2r, Snrpn and U2af1-rs1 (Grand-jean et al., 2001; Gregory et al., 2001). Several proteins, including DNMTs, MBPs and others with chromatin insulator functions, have been identified to be involved in epigenetic regulation of these loci.

Approximately 80 imprinted genes had been identified so far. Most of them are expressed in the placenta. Notably, Gatm, Obph1, Ascl2, Dcn, Slc22a2, Slc22a3 and Esx1 are expressed in an exclusively placenta-specific imprinted manner (Reik et al., 2003). The imprinted genes from different parental origin often have opposite effects on placental development in agreement with parent conflict hypothesis. Thus, paternally expressed genes enhance growth and maternally expressed genes suppress growth. For in-stance, mice with mutation of paternally expressed genes Igf2, Peg1 and Peg3 have decreased placental size, in contrast, mutation of maternally ex-pressed genes Igf2r, Cdkn1c, Ipl, Grb10 and Esx1 have increased placental size (Reik et al., 2003). However, parent conflict hypothesis is not univer-sally valid for all imprinted genes in placental development. One example is the maternally expressed gene Ascl2. The mutation of Ascl2 can cause lack of spongiotrophoblast and underdeveloped labyrinthine trophoblast (Guillemot et al., 1995). Imprinted genes can also affect placental transport function. This was proved by the placental phenotype observed in Slc22a2, Slc22a3 and Slc38a4/Ata3 mutation mice (Reik et al., 2003). Another exam-ple is Igf2. The mutation of Igf2 P0 promoter (labyrinthine trophoblast spe-cific promoter) caused reduced passive permeability and nutrient transfer across the placenta, leading to intrauterine growth retardation (IUGR), thus, small fetal size (Constancia et al., 2002).

Placental dysplasia models As mention above, many factors can affect placental development. Stud-

ies on the mouse models during the last few decades have added to our knowledge about the importance of various genes in placental growth. Based on the etiology, placental growth defects of mouse models can be classified into three groups:

� Entirely genetic model: a defect in placental growth is manifested as a result of heritable mutation of any specific gene or a chromo-somal segment.

� Entirely non-genetic model: placental growth defect cannot be de-termined by any genotype and is not genetically inherited by the next generation. For example, gestational diabetes and reproduc-tive cloning.

� Model with mixed etiology: the placental phenotype is deter-mined by a combination of genetic and non-genetic mechanisms.

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The best-known example is interspecies hybrid placental dyspla-sia (IHPD) mouse model (Zechner et al., 1996).

Abnormal placentation in mouse interspecies hybrids Abnormal placentation had been observed in several mammalian inter-

species hybrids. Interspecies hybrid placental dysplasia in the mouse (IHPD) was reported by our laboratory in 1996 (Zechner et al., 1996). This study showed two main features of IHPD: strong linkage of IHPD phenotype to the alleles on proximal X chromosome, and, reciprocal placental phenotypes in reciprocal hybrids. Also, different ranges of placental phenotypes were observed even in the littermates.

Reciprocal placental dysplasia derived from reciprocal hybrids Depending on the direction of crosses and backcrosses, the placentas can

be either hyperplastic or hypoplastic, determined by parent-species combina-tion used to generate the hybrids (Fig. 4). With the female always mentioned first in each cross, the placentas derived from crosses MMU x MSP (MS) and MMU x MMA (MX) are hypoplastic (Zechner et al., 1996), whereas the re-

ciprocal crosses MSP x MMU (SM) and MMA x MMU (XM) cause hyper-plastic placentas (Zechner et al., 1996). Notably, it is very difficult to gener-ate offspring from SM cross and only one conceptus with an 848mg placenta has been obtained so far (Zechner et al., 1996). In the backcrosses, MSM (MS x MMU) and MXM (MX x MMU) fetuses have hyperplastic placentas whereas the reverse backcrosses MSS (MS x MSP) and MXX (MX x MMA)

Normal weight MXM placenta, with MMU alleles on proximal X chromo-some. Hyperplastic MXM placenta with MMA alleles on proximal X chromo-some Hypoplastic MSS placenta with MSP alleles on the proximal X chromosome

Fig. 4: Reciprocal placental phenotypes in different mouse interspecies hybrids. Figure adopted from Zechner et al., 1996.

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give hypoplastic placentas (Zechner et al., 1996). The reciprocation is mani-fested as not only placental dysplasia, but also as reversal of histo-morphological features in reciprocal crosses. These reciprocal placental phe-notypes cannot be attributed to a difference in the intra-uterine environment, since the contrasting phenotypes are produced by using the mother of same genotype (MS F1 female in the case of MSM and MSS back crosses).

Placental phenotypes with different intensities IHPD is the only model in which strongly contrasting ranges of placental

phenotypes are observed, even among the littermates (Zechner et al., 1996). In the genetic models of placental dysplasia and in the cloned placentas, the hyperplasia or hypoplasia occurs according to an all-or-none law. In such models, there is a clear distinction between normal and abnormal and a very narrow range of abnormal phenotypes exists. Depending on the cross, the IHPD placental phenotypes may vary from as low as 12 mg to as high as 800 mg (Zechner et al., 1996). The hyperplastic phenotype is more pronounced in the males compared to female littermates (Zechner et al., 1996). The se-vere hypoplasia in MSS backcross is frequently associated with fetal death. The hybrids between MMU and MMA produce more severe placental phe-notypes compared to the hybrids between MMU and MSP. In addition to the weight, histo-morphological features also present as a gradient in such pla-centas (Zechner et al., 1996).

IHPD is X-linked It had been found that the MSP alleles on the X chromosome are associ-

ated with hyperplasia in MSM backcross and hypoplasia in MSS backcross and the same holds true for MMA alleles in MXM and MXX backcrosses, this indicates that IHPD segregates with alleles on the X chromosome, in MSM, MXM, MSS and MXX backcrosses and further backcrosses (Zechner et al., 1996; Zechner et al., 1997). This linkage is spread among a large por-tion of the X chromosome from DXMit57 to DXMit116, the highest score is the region from DXMit8 and proximal to it on the X chromosome. This lo-cus is also called IHPD locus (Zechner et al., 1996; Zechner et al., 1997). Several loci on the X chromosome, synergistically with other autosomal loci give rise to placental hyperplasia in the MSM backcross as MMU mice con-genic for MSP alleles on different parts of the X chromosome present with significantly increased placental weight. The homozygous congenic strain AT24, having MSP alleles from 12 cM to 31 cM on X chromosome in MMU background gives rise to moderately hyperplastic placentas which are histo-morphologically comparable to hyperplastic IHPD placentas (Hemberger et al., 1999). Nevertheless, the occurrence of X linked placental hyperplasia could not be attributed to any single gene locus on the X chromosome as MMU mice, homozygous congenic for regions smaller than 20 cM (between 12 cM and 31 cM) did not produce any hyperplasia. This indicates that

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though multiple regions on X chromosome can determine placental weight independently, there is a threshold for the minimum amount of introgression needed to initiate placental dysplasia. However, this has been tested only for the AT24 region on the X chromosome and not for the other regions of which only heterozygous congenic mice could be made owing to male steril-ity (Hemberger et al., 1999). The AT24 mice provide us with a very useful tool to study placental hyperplasia caused by MSP alleles on X chromosome in an otherwise homogenous genetic background.

One very interesting aspect of the linkage of IHPD to X chromosome is that the genetic linkage is very strong to a locus of paramount epigenetic interest as the X chromosome is paternally imprinted in placenta normally. It has been shown that under the experimentally induced conditions of absence of a maternally inherited X chromosome, MSP or MMA alleles inherited through paternal X chromosome can cause IHPD phenotypes similar to that of MSM or MXM placentas. However, whether abnormalities in preferential paternal X inactivation also underlie IHPD, has not been tested. Neverthe-less, through genetic studies, it is indicated that complex genetic interactions in-trans and associated epigenetic changes on the X chromosome underlie IHPD (Zechner et al., 1997).

Histo-morphological features of IHPD placentas Abnormal spongiotrophoblast growth is the main feature in many dys-

plastic placentas. In the hypoplastic placentas, the spongiotrophoblast is poorly grown and is very rudimentary; in the hyperplastic placentas, spongi-otrophoblast overgrows into the labyrinth and the boundary between the basal layer and the labyrinthine layer is lost (Zechner et al., 1996). Glycogen trophoblasts almost do not exist in the hypoplastic placentas, whereas this cell type is abundant in hyperplastic placentas. The differentiation of spongi-otrophoblast into glycogen trophoblasts is also increased in hyperplastic placentas (Zechner et al., 1996). Although the function of glycogen tro-phoblasts in placenta is not fully understood, it has been shown that there is a correlation between fetal weight and increased differentiation of glycogen cells in IHPD placentas (Kurz et al., 1999). The number of giant cells is also significantly increased in the hyperplastic placentas. The features of ede-matic cysts in the cloned placentas are also seen in extremely hyperplastic IHPD placentas (Zechner et al., 1996). The labyrinthine structure and func-tioning is apparently normal in the hyperplastic IHPD placentas and there are no signs of IUGR. Despite the striking overgrowth of IHPD placentas, the size of the litters from MSM or MXM crosses is comparably normal (6-10 conceptuses per litter) (Zechner et al., 1996).

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Comparative gene profiling of hyperplastic IHPD, Esx1 mutant and cloned placentas

Placental hyperplasia was also observed in other mouse models, including a genetic model, Esx1 mutant (Li and Behringer, 1998) and an epigenetic model, cloned mice (Tanaka et al., 2001). Comparative gene profiling be-tween hyperplastic IHPD, Esx1 mutant and cloned placentas was described recently (Singh et al., 2004). It was found that a set of genes were deregu-lated in hyperplastic IHPD irrespective of the extent of hyperplasia and the species used to generate hybrids (Singh et al., 2004). Only one gene with known function, Ramp2, was identified commonly deregulated in all three models. Another EST, belonging to an unknown UniGene cluster was also commonly deregulated in these placentas. The genes, Ppp2r4 and Ncam1, were commonly deregulated in cloned and Esx1 mutants, and, IHPD and Esx1 mutants, respectively (Singh et al., 2004). Some deregulated genes were deregulated in opposite directions between different models. For in-stance, Ncam1 expression was increased in IHPD and decreased in Esx1 mutant placentas (Singh et al., 2004). Poor similarity of gene expression between IHPD, cloned and Esx1 mutant placentas revealed that different mechanisms underlie placental hyperplasia in these different models (Singh et al., 2004).

Till today, the precise mechanism that regulates placental overgrowth in IHPD is still not fully understood. Genes that showed up-regulation in hy-perplastic IHPD placentas, probably are not determinants of IHPD pheno-type and also do not play a crucial role in normal placental growth. This was revealed as the mutation of some of these genes, for instance, Car2, Ncam1, Fbln1 and Cacnb3, did not induce placental dysplasia (Singh et al., 2004; Singh et al., 2005; Singh et al., 2006; Singh et al., 2007). Furthermore, there was no rescue of placental phenotype when heterozygous mutations of these genes were bred into the congenic mice AT24 (Singh et al., 2005; Singh et al., 2006; Singh et al., 2007). Interestingly, many of these genes were been found co-expressed in placenta with other genes of the same family, indicat-ing a possibility of functional redundancy. One example is Fbln1. Not only the other splice variant of Fbln1, Fbln1C, but also the other members of this gene family, Fbln2 and Fbln5 were found to be expressed in the placenta. This observation raised the possibility that perhaps there is a functional re-dundancy for the Fbln1 gene in placentas (Singh et al., 2006). Therefore, other genes can take over Fbln1 function when it is deleted. Similar observa-tion was fond for different carbonic anhydrases and neural cell adhesion molecules in the mouse placentas (Singh et al., 2005; Singh et al., 2006; Singh et al., 2007).

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Placental over growth in diabetic pregnancy Pregnancy in diabetic women is associated with placental overgrowth and

is known since decades (Neiger, 1992). The gross morphological changes in human diabetic placentas are increased placental weight, central thickness and diameter (Ashfaq and Campbell, 1980). Histological analysis showed that increased glycogen content is a dominant characteristic, observed in diabetic placentas and might attribute to increased placental size (Shafrir and Barash, 1991; Desoye et al., 1992). Due to maternal insulin resistance and fetal hyperinsulinemia, the majority of glycogen deposits are distributed in a manner surrounding the fetoplacental vasculature, i.e. endothelial cells, smooth muscle cells and pericytes (Robb and Hytten, 1976).

Impaired placental transport functions that subsequently lead to abnormal fetal development had been identified. However, the assessment of placental transport function is complicated to some extent, by the controversial results from different experiments. Measurements of maternal placental blood flow and volume in the umbilical circulation clearly showed that blood flow is reduced on both sides of the placental exchange barrier, and results in IUGR. However, the similar observations have also been made in diabetic preg-nancy associated with macrosomia. The increased level of glucose trans-porter-1 (GLUT1) in the basal membrane of human diabetic placenta sug-gests that glucose transport across the placenta is elevated in diabetic preg-nancy (Gaither et al., 1999). The fact that the activity of System A, a Na+- dependent transporter mediating the uptake of non-essential neutral amino acids, is markedly increased in the microvillous membrane of diabetic pla-centas indicates enhanced amino acid transport through diabetic placentas (Jansson et al., 2002). However, opposite results had been observed in type I diabetic pregnancy (Kuruvilla et al., 1994). Whereas, the commonly ac-cepted concept is that, pregnancy in diabetic women is associated with a high incidence of fetal macrosomia (Neiger, 1992; Garcia-Patterson et al., 1998) combined with enhanced placental transport. However, it is also known that poorly controlled, long-standing maternal diabetes may lead to vascular disease involving uterine and decidual vessels resulting in de-creased placental perfusion and IUGR (Neiger, 1992).

Gene expression profiling revealed that stress-associated and inflamma-tory response genes represent the largest cluster of deregulated genes in hu-man diabetic placenta (Radaelli et al., 2003). Upregulation of interleukins, leptin, and tumor necrosis factor-� receptors and their downstream molecu-lar adaptors indicated an activation of pathways recruiting stress-activated protein/c-Jun NH2-terminal kinases. Transcriptional activation of extracellu-lar matrix components and angiogenic activators pointed to a major struc-tural reorganization (over growth) of the placenta (Radaelli et al., 2003). These results provided the molecular basis of abnormal placental develop-ment in human diabetic pregnancy.

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There are several animal models, which had been set up for studying dia-betic placentopathy. The best-studied model is the diabetic rat model, in whichdiabetes is induced by chemicals or diet. Similar to the placental phe-notype observed in human diabetic pregnancy, these rat models also exhib-ited increased placental size and glycogen content (Padmanabhan and al-Zuhair, 1987). In contrast to enhanced placental transport in human diabetes, reduced placental transport function and subsequent IUGR had been reported (Padmanabhan and al-Zuhair, 1987). However, the molecular basis underly-ing placental over growth in diabetic rat model is still not described in detail. Diabetic mouse model have also been used (Ogura et al., 1999). High glyco-gen content and reduced placental transport function are common features of diabetic mouse placentas. Although there are some similarities shared be-tween these gestational diabetic models, few distinct differences also exist. One example is the varied expression of glucose transporter in diabetic rat and mouse placentas. It has been demonstrated that glucose transporter-1 (GLUT1) mRNA and protein levels in diabetic rat placentas remain un-changed, whereas GLUT3 mRNA and protein expression are increased in diabetic rat placentas (Boileau et al., 1995). In contrast to this finding, the expression of GLUT1 is decreased in diabetic mouse placentas (Ogura et al., 1999).

Notably, unlike diabetic pregnancy in human that usually can lead to ma-crosomia, all experimental diabetic models can exclusively lead to IUGR to different extents depending on the level of maternal diabetes and when the diabetes was induced. This suggests that the potential mechanisms underly-ing abnormal placentation in diabetic animal models are different from hu-man diabetic pregnancy.

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Aims of the present studies

Paper I In our previous study, we had reported a set of genes, which were differ-

entially expressed in IHPD, cloned and Esx1 mutant placentas. The deregu-lated genes identified in these three different hyperplastic placental models are potentially important in murine placental development. Some of these genes had never been reported to play a role in placental development. Thus, it was a major challenge to identify the functions of these genes in murine placentation. In this study, we focused on two genes, first on carboxypepti-dase E (Cpe) and subsequently also on carboxypeptidase D (Cpd). Cpe was downregulated in IHPD as well as cloned placentas. The principal aim of this study was to determine if Cpe has a functional role in placental devel-opment. To attain this aim, we analyzed the spatio-temporal expression pat-tern of Cpe and the placental phenotypes of the Cpe null mutation. We then identified by microarray hybridization the target genes of CPE in order to elucidate how it interacts with other factors in placentation. Expression of Cpd, which encodes another carboxypeptidase that has identical functions as CPE, was also examined to determine if CPD can rescue the Cpe mutation in the placenta.

Paper II To further determine the functions of different genes that had been shown

deregulated in IHPD placentas, we focused on Lhx3 in this study. Lhx3 was over expressed in hyperplastic IHPD placentas. It was already known that Lhx3, which is a member of the LIM-3 homeobox gene group, is critical for normal development. It is involved in formation of the pituitary and the mo-toneuron system and loss of the gene is a perinatal lethal. This important role in developmental processes made Lhx3 an attractive candidate for a placen-tation gene and it was decided to analyze the function of LHX3 in the mouse placenta. For this, Lhx3 spatio-temporal expression and phenotype of Lhx3-/-

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placentas were assessed. When it was found that Lhx3-/- placentas develop normally, double mutants for Lhx3 and Lhx4 were produced. Lhx4 is the second member of the LIM-3 group and can in part take over the functions of Lhx3.

Paper III Increased placental weight is a common feature observed in human dia-

betic pregnancy. Clinical research showed that enhanced placental nutrient transport might attribute to macrosomia in the pregnancy of type I diabetic women. The transcriptional activation of extracellular matrix components and angiogenic activators is the potential mechanism that triggers placental overgrowth. Placentas derived from rat females with experimentally strepto-zotocin-induced diabetes also exhibited abnormally increased placental weight and histological analysis had shown that morphology of these rat placentas is very similar to that of IHPD or cloned mouse placentas. This opened the possibility that similar mechanisms could underlie these similar placental phenotypes. Furthermore, there are at present no reports that de-scribe placental development in diabetic mouse females, a species that is much better suited for genetic and embryological manipulation than the rat. Therefore, this study was initiated to obtain a first impression of the conse-quences of maternal hyperglycaemia on the mouse placenta. For this pur-pose, we combined histological techniques, gene expression analysis and placental transport studies to characterize the ‘diabetic’ mouse placenta.

Paper IV This work was initiated independent from our IHPD work and is derived

from another project whose focus was the developmental and cell biological characterization of the targeted mutation of the Cbx1 gene. This gene en-codes a heterochromatin protein-1 isoform, CBX1 or HP1� and thus plays a crucial role in long range gene silencing. The primary objective of this study was to determine if the Cbx1 mutation results in a placental phenotype at all. When this was found to be the case, a variety of methodologies were applied to provide a comprehensive first characterization of the phenotype. These included placental transport assays, large-scale and in situ expression analy-sis, and proliferation and apoptosis.

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Results and discussion

Paper I By mRNA in situ hybridization, Cpe transcripts were detected in the E8

and E10. This expression persisted until the end of gestation. Between E12 and E16 high transcripts levels were located in the decidua but weak expres-sion was also seen in the labyrinth. Towards the end of gestation expression in decidua and labyrinth was reduced, however, strong expression was still found in the chorionic plate by E18. No expression could be detected in the junctional zone, i.e., the spongiotrophoblasts and the glycogen cells. In E18 IHPD placentas, Cpe expression was similar to that observed in E10 wild-type placentas. A total of 12 litters, containing 94 conceptuses were obtained from matings between heterozygous Cpe mutant mice. Of the 94 concep-tuses, 22 were homozygous mutants. There was no consistent and significant correlation between loss of Cpe, which in adults causes obesity and hyper-pro-insulinemia, and placental or fetal weight, even though in two E18 litters Cpe mutation was associated with mild IUGR. Morphological analysis showed that some Cpe mutant placentas had increased giant cell number with enlarged nuclei. The glycogen cell population was increased in all mu-tant placentas. Gene expression profiling showed that only 7 genes with known functions were deregulated in Cpe mutant placentas. Dtprp, which is a decidua and trophoblast-specific protein coding gene, was found to over expressed in Cpe mutant placentas consistently.

It is believed that expression of Cpd, which codes for an enzyme with the same substrate-specificity as CPE, can compensate for the loss of CPE activ-ity in Cpe mutants. Therefore, expression of this gene was also analyzed. Overlapping expression pattern was confirmed by both in situ hybridization and immunohistochemisty. Interestingly, in E12 IHPD placentas, a stage at which hyperplasia is not yet apparent, the expression of Cpe and Cpd was found consistently to be correlated with fetal genotypes indicative of hyper-plastic placentas by E18. This indicates that changed expression of Cpe and Cpd occurs in early stages of IHPD and hence is likely to be involved in regulation of IHPD.

Finally, DNA methylation assay was performed. We assayed CpG me-thylation in a 139bp region which is located upstream of the Cpe transcrip-tion start site. Our results showed that this region was unmethylated in wild type as well as IHPD placentas of different weights.

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Paper II

As no previous reports were available that described the expression of Lhx3 in the mouse placenta, this was investigated in detail by in situ hybridi-zation analysis. This showed that no expression of Lhx3 can be detected in E8 placentas. At E10, the expression was in the ectoplacental cone and the developing spongiotrophoblast. From E12 onwards, Lhx3 was expressed in parts of the spongiotrophoblast and in many small foci in the labyrinth. Ex-pression declined after E16 and extremely weak expression was detected in the spongiotrophoblast of E18 placentas. Lhx3 is also expressed in decidual glycogen cells at E16 and E18. No significant effects on placental and fetal weights were found in association with the Lhx3 mutation. Furthermore, morphological analysis did not detect any significant alteration in Lhx3-/- placentas. Thus loss of LHX3 does not cause any obvious placental pheno-types.

To determine if Lhx4 can substitute for Lhx3 in placental development and rescue the Lhx3 mutation, , we set up Lhx3+/-;Lhx4+/- double het x het matings. A total of 17 E18 litters with 176 conceptuses were obtained. 7 Lhx3-/-;Lhx4-/- conceptuses were identified among them. No significant differences in placental weight were observed between the different geno-types. However, decreased fetal weights were observed in three E18 double mutant fetuses compared to their littermates. Comparative histological anal-ysis showed that three double-mutant placentas exhibited reduced density and loose organization of labyrinthine architecture, and immuhistochemistry revealed disorganization of pericytes in three three abnormal Lhx3-/-;Lhx4-/- placentas. The placental phenotype did not correlate with reduced fetal size. Furthermore, lipid transport analysis provided no indication for aberrant placental transport in double mutant placentas. qRT-PCR found that Gnrhr, a known target gene of Lhx3, was inconsistently up regulated in Lhx3-/-;Lhx4-/- placentas as compared to littermate placentas. However, no signifi-cant placental morphological defects can be detected in Gnrhr mutant mice. From AT24/AT24 x Lhx3+/-;Lhx4+/- matings, we found that reduced Lhx3 and Lhx4 activity can not rescue AT24 phenotypes. Hence, it was confirmed that Lhx3 and Lhx4 are not causally involved in IHPD.

Paper III Following standard procedures using STZ induced ablation of pancreatic

island cells, we generated a mouse model placental development in diabetic mothers. 182 placentas and were collected, 80 from controls and 102 from diabetic females. According to maternal blood glucose level, these concep-tuses were divided into two groups: moderate diabetes (derived from females

37

with blood glucose levels between 11.1 and 16.7 mM), or severe diabetes (derived from females with blood glucose levels � 16.7 mM). Our data show that severe diabetes was associated with strong IUGR.. Morphological anal-ysis revealed that spongiotrophoblast expansion and increased glycogen cell numbers were major placental phenotype in the severe diabetic group. How-ever, this placental phenotype was not correlated with placental transport function, since lipid composition was not different between diabetic and control conceptuses. Gene expression profiling identified a set genes, whichwere deregulated in severely diabetic placentas. Some of these genes have known function in normal placental development. In addition, some imprinted genes including Igf2 and Gatm, were identified in the expression screen. For Igf2 and Gatm the microarray results were further confirmed by in situ hybridization analysis, which showed decreased expression of Igf2 and increased expression of Gatm. For some genes that were identified in the expression screen, a direct link to hyperglycemia has been reported. Thus, the up-regulated gene Txnip codes for thioredoxin interacting protein (TXNIP), a protein primarily involved in response to oxidative stress. TXNIP was shown to be a central regulator of insulin secretion. Sh2bpsm, which was downregulated in diabetic placentas, is believed to be a physio-logical enhancer of insulin receptor activation and to be essential for main-taining insulin sensitivity and glucose homeostasis. These findings provide a starting point for future research that may potentially provide insight into the molecular mechanisms underlying abnormal placentation in diabetic mouse model.

Paper IV In this study, we first examined the expression of other epigenetic marks

and CBX isoforms in Cbx1 mutant mice by western blot. These experiments showed no measurable changes in histone modification marks such as tri-methylated H3K9, di-methylated H3K9 and trimethylated H4K20. However, levels of CBX3 and CBX5 protein were altered, with decreased CBX3 and increased CBX5. Reduced expression of CBX3 was however not visible by immunofluorescence staining on sections. From Cbx1+/- x Cbx1+/- het x het matings, we collected 13 E18 litters containing 94 conceptuses and 4 E12 litters containing 25 conceptuses. At both gestational stages the frequency of genotypes roughly followed Mendelian distruibution, indicatingthat the dele-tion of Cbx1 is compatible with prenatal development. E18 placental weight showed no significant difference dependent on genotype. However, Cbx1-/- fetal weights were significantly reduced as compared to Cbx1+/+ and Cbx1+/- fetuses. Several phenotypes in Cbx1-/- placentas were identified by morphological analysis. Enlargement of the spongiotrophoblast was ob-served in all E18 Cbx1-/- placentas. An additional placental phenotype was

38

characterized by obvious alterations in labyrinthine structure that were con-sistent reduced maternal and fetal exchange area In E12 Cbx1-/- placentas, enlargement of fetal blood vessels was observed. Unexpectedly, these pla-cental phenotypes have no effect on lipid transport system since no differ-ence of fetal EFAs content could be detected between Cbx1-/- and Cbx1+/+ conceptuses. Gene expression profiling identified a panel of genes that have earlier been shown to be involved in placental development. Examples are Igf2, Hand1, Cited1, Cited2, Birc6, Jup and Pdgfrb. Several imprinted genes, Gatm, Sgce, H19, Igf2, Slc38a4 also altered expression in Cbx1-/- mice, in addition to Igf2. Interestingly, Igf2 had an ectopic expression in Cbx1-/- placentas. In the E18 Cbx1+/+ placentas Igf2 was expressed specifi-cally in the labyrinth and the glycogen cells, whereas in the Cbx1-/- placen-tas Igf2 transcripts were detected mainly in the spongiotrophoblast and weakly in the labyrinth.

39

Conclusions

My studies looked at mouse placental development from three different angles, by addressing: 1) single gene deletions (genetic regulation); 2) al-tered epigenetic ‘steady-state’ brought about by deletion of a heterochro-matin protein-1 (epigenetic regulation); and disturbed maternal physiology (environmental condition).

The first gene that was sudied, Cpe, provided a number of interesting re-sults. Thus loss of Cpe leads to a consistent phenotype of varying severity that recapitulates some features of the placental hyperplasia caused by inter-species hybridization or cloning. Variability of the phenotype is possibly caused by expression of Cpd, which encodes an enzyme of equivalent func-tion to CPE. Our finding that both Cpe and Cpd are differentially expressed in IHPD placentas even before these develop a significant phenotype sug-gests that these genes are causally involved in IHPD and thus possibly func-tion as speciation genes in the genus Mus. These findings open the way for future studies directed towards the identification of the genes that control Cpe and Cpd expression in the placenta.

We had earlier observed that deletion of genes whose deletion caused se-vere fetal phenotypes was not associated with any placental abnormalities. In all of these cases we found that these genes belonged to gene families and that other family members were also expressed in the placenta. This sug-gested that functional redundancy was underlying the lack of placental phe-notypes. When I found that loss of Lhx3, which is a perinatal lethal, does not interfere with placental development, the obvious explanation was that Lhx4, the second member of the LIM-3 group, was able to substitute for the ab-sence of Lhx3. The finding that loss of both Lhx3 and Lhx4 did not produce a consistent phenotype is difficult to explain as it seems to be certain that no other LIM homeodomain containing transcription factors can substitute for LHX3 and LHX4, at least in pituitary and motoneuron differentiation. As a candidate gene analysis for the placental target genes of LHX3 and LHX4 was inconclusive, conclusions regarding their role in placental development are not possible.

Maternal diabetes causes a physiological environment which can induce placental over growth in human and rat. Strangely, the effect of maternal diabetes on placental development in the mouse has not been investigated. As diabetic rat placentas and mouse cloned and IHPD placentas exhibit nearly identical phenotypes, a project was initiated whose aim it was to pro-

40

vide a first characterization of the mouse diabetic placenta. Application of various experimental approaches resulted in several novel results that pro-vide a strong basis for further work.

In the last project, which was initiated independent of our IHPD work, the role of a gene, Cbx1, encoding a heterochromatin protein-1 was ana-lyzed. CBX1 has a central role in epigenetic gene silencing, which makes the analysis of this gene of especial interest. We found that Cbx1 is indeed re-quired for normal placentation. We also identified a panel of genes with known function in normal placental development that exhibited altered ex-pression in the Cbx1-/- placentas. This finding is of especial interest as it opens the way for future research, which will address the possibility that CBX1 directly regulates expression of these genes.

41

Summary in Swedish

Sammanfattning

Placentans utveckling kan störas av olika faktorer, såsom mutationer i specifika gener eller maternell diabetes. Abnormal placentabildning sker även hos klonade djur och interspecies-hybrider. Våra tidigare studier av interspecies hybrid placental dysplasi (IHPD) i släktet Mus visade att ab-normala placentala fenotyper kan sammankopplas med regioner på X-kromosomen. Genom att jämföra genexpressionsprofiler identifierades ett flertal gener som var deregulerade i IHPD-placenta och i ytterligare två mo-deller av placental hyperplasia, klonade möss och Esx1-mutanter. Dessa kandidatgener är potentiella placenteringsgener. Två av dessa gener behand-las i denna avhandling, Cpe och Lhx3. I de två studier som behandlar dessa gener, utförde vi in situ hybridiseringar för att bestämma deras spatio-temporala expressionsmönster i placenta. Därefter undersöktes placental fenotyp och genexpression i muterade möss. Våra resultat visar att den pla-centala fenotypen i Cpe mutant-möss efterliknar vissa IHPD fenotyper. De-regulerad expression av Cpe och Cpd, en funktionellt ekvivalent gen, före-gick manifestering av IHPD-fenotyp vilket indikerade att Cpe och Cpd är gener av potentiell betydelse för uppkomsten av IHPD. I motsats till detta orsakade deletion av Lhx3 ingen tydlig placental fenotyp. Dock kunde en inkonsekvent fenotyp observeras när båda medlemmarna av LIM3-gruppen av homeoboxgener, Lhx3 och Lhx4, raderades. Denna fenotyp hade dock ingen effekt på placental transport. En analys av kandidatgener gav ingen information om vilka gener som är mål för LHX3/LHX4 i placenta. En orsa-kande roll för Lhx3 och Lhx4 i IHPD kunde uteslutas eftersom nedreglering av Lhx3/Lhx4 inte räddade den placentala fenotypen hos AT24-möss. Det tredje projektet, analys av placentans utveckling i diabetiska mödrar, initie-rades eftersom diabetiska råttplacentor uppvisar fenotyper som kan observe-ras I IHPD hos möss. Våra resultat visar att svår maternell diabetes resulte-rade i fetal intrauterin tillväxthämning (IUGR) men placentans vikt påverka-des ej. Emellertid uppvisade diabetisk placenta onormal morfologi. Placental transportanalys visade att IUGR ej orsakades av placental funktionsoduglig-het. Analys av genexpressionsprofiler identifierade ett antal gener som bidrar till patologin vid human diabetes. I det sista projektet undersökte jag funk-tionen av ett heterokromatinprotein, CBX-1, I placentabildningen hos mus.

42

Denna analys visade att CBX-1 är nödvändig för normal placentabildning, eftersom deletion av genen konsekvent orsakar spongiotrofoblast- och laby-rint-fenotyper. Genexpressionsanalys och spatio-temporal expressionsanalys visade att ett flertal gener med känd funktion i placental utveckling var dere-gulerade i placenta I hos mutanter som saknar Cbx1.

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Acknowledgements

This is my doctoral thesis, but it belongs not only to me. It belongs to all the people who have supported me during my PhD study. Thanks to Prof. Reinald Fundele, my PhD supervisor. I can imagine how hard it is to incubate a student from a very beginner stage to the final stage of a senior PhD student. Thanks for his knowledge, kindness and patience. The most important thing I learned from him is the attitude towards the sci-ence: strict, strict and stricter. Thanks to Prof. Rolf Ohlsson. I always sensed your creative sparks in the seminar. Thanks to all the people surrounding me: Chandrasekhar, Elena Jazin, He-lena, Anita Mattson, Rose-Marie, Tong, Tian, Tiger, Anita, Piero, Sha, Rosita, Mikeal, Sylvain, Carole, Kuljeet, Gholamreza, Claes, Pandey, Fi-zaan, Julia, Björn, Eva, Samer, Noriyuki, Marta, Lin, and Chengxi; you all have helped me a lot. I especially thank Noopur and Umashankar, you are my best friends. I will miss the time we have spent together. Finally, I thank Wei Shi, not only because she is my wife, but also because of her guidance in science.

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