Epigenetic Control of Early Development

Chapter 4

Epigenetic Control of Early Development Unlike unicellulars, metazoans do not produce copies of themselves. They simply provide gametes with information for building the Bauplan and the CNS at the phylotypic stage, when the embryo becomes competent of generating the information for individual development up to adulthood.

Early embryonic development in metazoans is regulated by epigenetic information that is parentally provided to gametes (egg and sperm cells) in the form of parental cytoplasmic factors. Initially by their own activities, and later on by determining the temporal order and the spatial patterning of expression of zygotic genes, these factors determine cell divisions, establishment of body axes, formation of the embryonic layers, and gastrulation, culminating at the phylotypic stage with formation of the operational central nervous system (CNS) and the resulting integrated control system (ICS). Thus, the epigenetic program put in the zygote is an interim developmental program for early embryonic development, until the phylotypic stage, when the embryonic CNS is operational and takes over the individual development. It bridges the physical gap between the parental and embryonic CNSs. Earlier I have argued and presented supporting evidence that the synthesis of maternal cytoplasmic factors and their placement in the egg cell by the follicle and/or nurse cells via the active uptake by the oocyte from the intercellular environment takes place under the control of the integrated control system (ICS). Now I will attempt to prove that the epigenetic information (parental cytoplasmic factors) determines the early individual development up to the phylotypic stage.

EPIGENETIC CONTROL OF FORMATION OF PRIMORDIAL GERM CELLS All forms of animal life start from germ cells that are specified in the earliest stages of the development of their parents. These cells are endowed with the unique ability to start the development of a new animal organism of the parental Epigenetic Principles of Evolution. https://doi.org/10.1016/B978-0-12-814067-3.00004-1 © 2019 Elsevier Inc. All rights reserved.

119

120 PART

I Epigenetic Basis of Metazoan Inheritance

species. The fact that germ cells possess qualitatively the same genes and DNA with other somatic cells of the body, clearly indicates that their singular ability to develop into a multicellular organism of its own species, depends not on the genetic material alone, but on something else, on the presence in the germ cells of another type of information that is provided them by the parent(s) in the form of parental (maternal and paternal) cytoplasmic factors. Segregation of the first primordial germ cells (PGCs), precursors of gametes, takes place during the early development, that is, at a time when the genomic transcription has not begun yet and the development is determined by the epigenetic information (Weidinger et al., 2003) provided to gametes in the form of parental factors (e.g., mRNAs, sncRNAs, hormones, and neuromodulators), placed orderly in their cytoplasm as shown in Chapter 3, subsection Neural Control of Deposition of Maternal Factors in Insect Oocytes). Two are the modes of the specification of the PGCs during the individual development: the preformation mode and the epigenesis mode. In the preformation mode (Fig. 4.1), epitomized by Drosophila melanogaster, the formation of the PGCs is determined by maternal factors, a number of mRNAs such as vasa, and so on, by nurse cells that are transported to the posterior tip of the egg (Tomancak et al., 1998), where the information for PGC differentiation and specification of the abdomen is stored (Vanzo and Preformation

FIG. 4.1 During oogenesis in D. melanogaster, RNAs and proteins are synthesized by the nurse cells. These products (far right) are transported through cytoplasmic bridges (arrows) to the oocyte. They become localized to the posterior of the ooplasm both by molecular anchoring at the posterior of the oocyte and by posterior-specific translational and transcriptional regulation. This posterior ooplasm is the germ plasm or germ line determinant. During early embryogenesis, cells which inherit the germ plasm become the primordial germ cells (PGCs). (From Extavour, C.G., Akam M., 2003. Mechanisms of germ cell specification across the metazoans: epigenesis and preformation. Development 130, 5869–5884.)

Epigenetic Control of Early Development Chapter

4

121

Epigenesis

FIG. 4.2 No maternally deposited germ plasm has been observed in the oocytes of the mouse Mus musculus. Instead, PGC determination takes place after the segregation of embryonic and extraembryonic tissues. A subpopulation of the pluripotent epiblast cells express “germline competence genes” (striped). These cells are able to interpret the inductive signals that arrive from neighboring tissues and differentiate into PGCs. The inductive signals come from the extraembryonic ectoderm (downward arrow) and endoderm (oblique arrow). (From Extavour, C.G., Akam M., 2003. Mechanisms of germ cell specification across the metazoans: epigenesis and preformation. Development 130, 5869–5884.)

Ephrussi, 2002). They form the so-called germ plasm, the zone of the cytoplasm where mRNA and protein determinants of the germ cells are concentrated. The role of this pole plasm as determinant of the germ cell line (precursors of the gametes) has been verified in experiments where its transplantation to the anterior pole leads to formation in the somatic region of ectopic germ cells Illmensee and Mahowald (1974). These transplants reach gonads and contribute to germ line (Illmensee and Mahowald, 1976). In species employing preformation, PGCs appear as early as the first divisions after fertilization. In species employing the epigenesis mode (Fig. 4.2), formation of PGCs results from inductive signals, such as the bone morphogenetic proteins (BMP) (Lawson et al., 1999), transforming growth factor β (TGFβ), and other ribonucleoproteins (Eno and Pelegri, 2016) originating in the neighboring cells. Mouse is the organism where the epigenesis mode of germ cell specification is studied extensively. PGCs in mouse embryos are observed first at the early gastrulation stage in the 6 dpc (day post coitus) (Lawson and Hage, 1994).

EPIGENETIC CONTROL OF MIGRATION OF PGCs As pointed out earlier, the fate of primordial germ cells (PGCs) is determined by deposition of the maternal vasa mRNA in the vegetal pole and its asymmetric allocation among the early blastula cells. In order to fully differentiate into germ cells, PGCs have to migrate to the genital ridge, the Anlage of the future gonads. The directed migration of PGCs from the vegetal pole to their target sites, traveling distances exceeding thousand times their diameter, is made possible

122 PART

I Epigenetic Basis of Metazoan Inheritance

by the presence on these cells of specific membrane receptors, CHCRs (chemokine receptors). PGCs find their way to the target site by binding their receptor, the specific ligand, chemokine stromal cell-derived factor-1 (SDF-1). This almost universal ligand of cell migration is a downstream element of signal cascades originating in the CNS. At the level of hormonal control, recently it has been demonstrated that E2 (17-β-estradiol), via its nuclear receptor, induces expression of the gene coding for SDF-1 (Coser et al., 2003; Hall and Korach, 2003; Ouyang et al., 2016), and the latter is a direct target of that hormone (Hall and Korach, 2003). A downregulator of the SDF-1 is TGF-β1 (transforming growth factorβ1), which inhibits transcription of the sdf-1 gene in the bone marrow stromal cells, thus affecting their migration and adhesion ability (Wright et al., 2003). Chick and mouse PGCs use a different way of reaching their target sites; they use the blood flow as a transport vehicle and leave the blood vessels at specific sites where they sense the presence of the chemoattractant SDF-1α and, passing through the blood vessels’ walls, they follow the chemokine to find their way to the genital ridge (Stebler et al., 2004). By experimentally inhibiting the normal pattern of SDF-1 expression and by supplying SDF-1 to sites where it is normally absent, changes in the direction of migration are induced so that PGCs reach ectopic sites of the SDF-1 sources (Doitsidou et al., 2002; Stebler et al., 2004). Upon arrival in the genital ridge, the PGC population proliferates under the influence of FGFs (fibroblast growth factors) (Kawase et al., 2004) and TGF-β1 (transforming factor-β1), but these processes are regulated by a balanced proliferation/programmed cell death or apoptosis (Tres et al., 2004).

EPIGENETIC CONTROL OF EARLY DEVELOPMENT IN INSECTS Cell division during the cleavage is regulated by maternal cyclins and the maternal String proteins, whose transcripts are present in the zygote and are among the earliest mRNAs to be translated. Because of the uniform distribution of these factors, the early cycles of nuclear divisions are uniformly executed but no cell divisions occur. The resulting naked nuclei move to the outer edge of the embryo, thus leading to formation of a syncytial blastoderm. This lasts until the 14th cell cycle, when normal cell divisions begin to occur and the syncytial blastoderm is transformed into a cellularized blastoderm. This change is related to the fact that, at this point in time, the maternal reserve of the String protein is exhausted and differential expression of the gene for the String protein in various parts of the embryo begins. After the 17th or 18th cycle, cells in the epidermis and mesoderm stop dividing, and differentiate. This cessation of proliferation is caused by the exhaustion of maternal cyclin E, originally laid down in the egg, which is required for progression through the cell cycle. (Wolpert et al., 1998)

Epigenetic Control of Early Development Chapter

4

123

In insects, the anterior-posterior axis is established by the combined action of three systems of maternal cytoplasmic factors placed in the egg by nurse cells: the bicoid mRNA in the anterior part of the egg cell, nanos mRNA and caudal mRNA in the posterior, and the hunchback mRNA initiate a transcription sequence of genes gap-pair-rule-engrailed homeotic. In Drosophila these genes are expressed in phase with the gene for the neurotransmitter serotonin and serotonin receptor in characteristic stripes pattern (Colas et al., 1995) suggesting a role of the neurotransmitter in the process. Maternal cytoplasmic factors also induce the synthesis of neurotransmitters which are among the first products of zygotic gene activity in the early stages of embryonic development (Shmukler et al., 1999). Application of serotonin antagonists causes a transient regression of the first cleavage furrow in sea urchin embryos, suggesting a morphogenic role of the neurotransmitter in the early embryonic development (Shmukler et al., 1999). Maternal signals determine a peak of expression of the neurotransmitter serotonin and its receptors in Drosophila, which is coincident with the onset of the germ band extension (corresponding to the phylotypic stage). How important that peak of serotonin is for the normal gastrulation is shown by the fact that mutant Drosophila embryos that fail to reach that peak do not develop proper germ band extension and die with a cuticular organization that is characteristic of embryos that do not express the serotonin receptor (Colas et al., 1999). Early during the phylotypic stage, the incipient CNS is involved in patterning of neighboring embryonic mesoderm and underlying ectoderm and in determining their cell fates. Among the inductive effects of the CNS midline cells is the formation of somatic muscles from mesodermal progenitors (L€uer et al., 1997).

EPIGENETIC CONTROL OF EARLY DEVELOPMENT IN VERTEBRATES The dorsal side of the embryo is established opposite of where the sperm cell enters the egg cell. β-catenin, a maternal transcription factor, and other maternal cytoplasmic factors such as mRNAs for Vg1, Xwnt11, Noggin, and Activin, are accumulated in the dorsal side of the developing embryo, as a result of the cortical reaction. The first divisions of the zygote are stimulated by the synthesis of the cyclin protein Cdc6 from the maternal Cdc6 mRNA, which makes possible the replication of zygotic chromosomes by activating the minichromosome maintenance helicase complex (Lemaitre et al., 2002). Cyclins combine with a cyclin-dependent kinase to form the maturation promoting factor (MPF), a phosphoprotein that is responsible for cell division since the early stages of the embryonic development. In this context, one must remember that MPF is an active form of the pre-MPF, which in turn, is induced by progesterone (Murray and Kirshner, 1989).

124 PART

I Epigenetic Basis of Metazoan Inheritance

By the 14th cycle of cell divisions, the reserve of maternal cyclin mRNA and the String, a protein phosphatase, are exhausted. The zygotically coded String protein (cdc 25 phosphatase) is rhythmically translated to phosphorylate the pre-MPF (maturation promoting factor) and transform it into active MPF, just before the mitotic cell division. The zygotic string gene will be differentially expressed, only in the cells that have inherited transcription factors of the gap, pair ruled, and other early patterning genes, which are expressed in phase with the gene for the neurotransmitter serotonin and serotonin receptor in characteristic stripes pattern. Expression of various types of RA receptors in the inner cell mass and trophoectoderm of blastocysts suggests that maternal RA is likely to directly regulate gene expression during preimplantation development (Mohan et al., 2001) of bovine embryos. In some species RA (retinoic acid) is present as maternal factor in the egg cytoplasm. Later in the mouse embryogenesis (E11), RA is produced in an endocrine way in the incipient adrenal gland. RA may cooperate with growth factors to provide positional information (Cho and De Robertis, 1990; Langston et al., 1997). Regulatory functions of RA are chiefly related to its ability to regulate expression of Hox genes, which have retinoic acid response elements (RARE) in their enhancers (Langston et al., 1997). RA is a common immediate regulator of the activity of almost all of the known homeotic genes, since the earliest stages of the embryonic development and during the postnatal development in metazoans (Clagett-Dame and Plum, 1997; Conlon, 1995; Cupp et al., 1999; De Luca and Ross, 1996; Malpel et al., 2000; Marshall et al., 1996). By regulating expression of the Hox genes, RA is crucially involved in establishing the anterior-posterior axis during gastrulation in vertebrates. Vitamin A (RA precursor) deficiency causes early death of quail embryos but administration of retinoids to those bird embryos up to the 5-somite stage (not later) rescues embryonic development, suggesting the existence of a narrow developmental window in early development during which presence of retinoids is necessary for the embryonic development (Kostetskii et al., 1998; Knezevic and Mackem, 2001). Based on the facts that the hormone RA synergistically induces expression of Hox genes in the blastoderm cell culture and that exogenous RA mimics the effects of hypoblast rotation on primitive streak extension, it is suggested that RA (maternal and/or embryonic-N.C.) plays some role in the development of the pregastrula embryo (Knezevic and Mackem, 2001).

EPIGENETIC CONTROL OF EARLY DEVELOPMENT IN MAMMALS Like other metazoan groups, the early embryonic development in mammals is under control of parental epigenetic information (parental cytoplasmic factors, imprinted genes, and probably other factors). The close contact of the

Epigenetic Control of Early Development Chapter

4

125

developing embryo with the mother and the maternal dependence of the embryonic development in these animals led to one of the most visible features of the maternal control in mammals, which is the comparably early termination of the role of maternal cytoplasmic factors provided with the egg cell in mammalian embryos (Gilbert, 2000). In mice, the embryonic genome is active from the 2-cell stage (Piko and Clegg, 1982) and in rabbits transcription of zygotic genes starts from the one-cell stage (Brunet-Simon et al., 2001), whereas in a nonmammal species, the clawed frog, Xenopus laevis, expression of the zygotic genes first starts in the
5000 cell embryo. In other mammals, however, the transition to zygotic gene expression takes place after several cell cycles (Henrion et al., 2000). The physical continuity of the mother and the developing embryo makes it possible for mammals to depend less on the deposition of maternal factors and rely more on direct maternal control, via the neuroendocrine system, a “real time” control and regulation of the embryonic development. Maternal hormones, growth factors, and other secreted proteins that reach the embryo transplacental are essentially involved in mammal embryogenesis from its beginning. Implantation involves interactions between the endometrium and blastocyst. Endometrial secretions are considered to be regulators of implantation and placentation. Among the early secreted maternal proteins that are involved in the implantation of the blastocyst, during the “implantation window” in mammals, are growth factors, cytokines, and Hox genes. Numerous growth factors are specifically expressed in the maternal reproductive tract (Hardy and Spanos, 2002). In preparation for implantation of the blastocyst, at the site of blastocyst attachment to the endometrium, the latter expresses 22 genes for growth factors (Paria et al., 2001). EGF induces expression of its receptor, EGFR, in the mouse 8-cell stage blastocyst (Kim et al., 1999). EGFR is expressed in oviducal and endometrial membranes of pregnant pigs during the preimplantation period. This, and the fact that its receptor, EGFR, is also present in the zygote, suggests that maternal EGF acts on the blastocyst at this early stage. Expression of EGF in the pig oviduct is stimulated by estradiol (Wollenhaupt et al., 1999), a downstream element of a signal cascade that starts with an epigenetic brain signal that is communicated to the ovary via the hypothalamic-pituitary axis. The same is true for the expression of the egf-R gene that is induced by the pituitary GH. Progesterone alone regulates several specific factors such as TGF-β, interleukin-1, insulin-like growth factor binding protein-1, tissue inhibitors of metalloproteinases (TIMPs), and fibronectin (Johansson et al., 1989). Estradiol and progesterone regulate synthesis of the transmembrane receptor for all the interleukin-6 type cytokines, which are necessary for blastocyst implantation (Classen-Linke et al., 2004). Pituitary prolactin, estrogen, and progesterone increase expression of estrogen receptors (ERs), but this does not explain why this expression is spatially restricted mainly to the antimesometrial site of the uterus where the decidua

126 PART

I Epigenetic Basis of Metazoan Inheritance

capsularis forms in rats (Tessier et al., 2000). It is possible that, as it is observed in many other cases, the limited site-specific expression of ER may be function of the neural arm of the binary neural control of gene expression, performed by local innervation. Maternal progesterone, via its receptor (PR), induces expression of a number of genes, playing a crucial role in the process of implantation in mammals, by inducing endometrial epithelium to secrete adhesion-promoting proteins such as galectin and osteopontin (Spencer et al., 2004). Note that not only the pituitary prolactin but the “primary effectors,” estrogen and progesterone, are under ultimate CNS control via the hypothalamus-pituitary-ovarian axis and the local ovarian innervation. A number of maternal Hox genes encode transcription factors that are essential for the uterus receptivity and implantation. Such a function has been demonstrated for Hox10 (Cermik et al., 2001; Daftary and Taylor, 2000) and Hox11 (Daftary and Taylor, 2001). In the preimplantation endometrium and myometrium, Hox10 is downregulated by maternal progesterone (Cermik et al., 2001). Maternal hormones insulin and IGF-1 (insulin-like growth factor-1) are present in secretions of oviduct and uterus and bind to morula cells that express their respective receptors, thus stimulating the blastocyst formation (Herrler et al., 1998). Expression in the mesometrium and uterine epithelium of maternal activin and its receptor in pigs shows that “both embryonic and uterine activin are involved in intrauterine processes, such as attachment and early embryonic development” (van de Pavert et al., 2001). Let us remember that the synthesis of activin itself may be neurally regulated via the hypothalamus-pituitary axis by gonadotropins, FSH and LH (Demura et al., 1993). Under the control of ovarian hormones, a group of angiogenic factors (VEGF, FGF-2) and their specific receptors, as well as EGFR, are expressed in and around the human endometrial blood vessels, a fact that is considered to be related to their contribution to the “regulation of angiogenesis” (Moeller et al., 2001). The neurohormonally regulated pattern of expression of genes of the EGF family during the menstrual cycle and early pregnancy may be involved in monkey embryo implantation (Yue et al., 2000). TGF β-1 is downregulated by neuroactive opioids and their specific receptors (Chatzaki et al., 2000). The deciduation or transformation of the mammal endometrium into a receptive state for implantation of the blastocyst requires expression of a number of cytokines. LIF (leukemia inhibitory factor) is hormonally induced by estradiol and cytokines (Sawai et al., 1997) and IL-11 (interleukin-11), as well as by genes for protein D9K, as it may be concluded from the fact that null mutant mice for those factors fail to implant embryos in the endometrium (Salamonsen, et al., 2001). The optimal levels of the neurotransmitter nitric oxide are crucial for endometrial function and embryo implantation (Ota et al., 1999).

Epigenetic Control of Early Development Chapter

4

127

Blastocyst Ovarian estrogen

Uterus

Diffusible factor(s)

Wnts Initiation of implantation Expression of Wnt target genes at implantation site

FIG. 4.3 Uterine-embryonic communication at implantation. Estradiol is proposed to act on uterine cells to induce synthesis of a secreted molecule that acts on the blastocyst to trigger expression of estradiol-regulated genes encoding factors such as Wnt11. These may induce expression of target genes in the uterus or in the blastocyst that facilitate implantation. (From Mohamed, O.A., Dufort, D., Clarke, H.J., 2004. Expression and estradiol regulation of Wnt genes in the mouse blastocyst identify a candidate pathway for embryo-maternal signaling at implantation. Biol. Reprod. 71, 417–424.)

In mice, the transplacentally provided maternal estradiol and progesterone upregulates expression of the zygotic wnt genes which are crucial for implantation and deciduation (Chen et al. 2009; Hayashi et al., 2009; Mohamed et al., 2004; Fig. 4.3). Placenta is believed to be regulated by imprinted genes in a process in which paternally expressed genes promote and maternally expressed genes restrain its growth (Coan et al., 2004). It has been shown that decrease of LH and prolactin during the second half of pregnancy in mares is centrally regulated by deactivation of the opioidergic system (Aurich et al., 2001). In mice embryos, the neurotransmitter serotonin starts to be produced by E11. Before this stage, it is the maternal serotonin that circulates in the embryo and, due to the incomplete formation of the blood-brain barrier, the maternal serotonin enters the brain and participates in the development of particular embryonic brain structures (C^ ote et al., 2007). Maternal thyroid hormones (T3 and T4) are detected in the human fetus since the first gestation trimester, that is, before the fetus starts producing thyroid hormones. At this stage, maternal T3 is detected only in the fetal brain. Even after activation of the fetal thyroid gland in the second trimester, maternal thyroid hormones are still detected in the human fetal brain. There is evidence that the maternal hypothalamic neurohormone TRH (thyroid-releasing hormone) transplacental regulates the function of the fetal thyroid gland before the maturation of the fetal hypothalamic-pituitary-thyroid axis (Chan and Kilby, 2000). Maternal thyroid hormone (T4) crosses the placenta and enters the embryonic brain, where it regulates neuronal proliferation, neuronal migration, process outgrowth, and myelin formation (Porterfield and Hendry, 1998). It may also induce several genes, including RC3/neurogranin gene, in the brain cortex of rat fetus (Dowling and Zoeller, 2000). The fact that GHR (growth

128 PART

I Epigenetic Basis of Metazoan Inheritance

hormone receptor) mRNA is expressed in preimplantation bovine embryos and that supplementation of the GH (growth hormone) to those embryos in vitro favorably affects their cleavage, blastocyst formation, and hatchability (Izadyar et al., 2000), suggests that the maternal pituitary GH (growth hormone), which is regulated by secretion of the neurohormone GHRH (growth hormone-releasing hormone), plays an essential role in regulation of the preimplantation development of bovine embryos. Embryo transfer experiments have shown a strong maternal influence on the skeleton development, especially of the embryonic craniofacial skeleton and teeth (Nonaka et al. 1993; Sasaki et al., 1995). Under experimental conditions, various stressors, via the neuroendocrine system, induce hormonal imbalances in pregnant laboratory mammals, causing nonadaptive behavioral and morphological changes in their offspring (Hopper and Hart, 1985). The representative facts presented herein on the regulation of the activity of fertilized egg and early embryonic development in mammals show that besides the control of the embryonic development via parental cytoplasmic factors deposited in gametes, this group of animals transplacentally exerts a maternal epigenetic control on the early development of the embryo.

EPIGENETIC CONTROL OF FORMATION OF EMBRYONIC GERM LAYERS Maternal Control of Endoderm Formation It is generally admitted that formation of endoderm is induced by Nodal signaling (Poulain et al., 2006; Schier, 2003). But Nodal subfamily is not where the signal cascade and regulatory network for endoderm formation begins: three main groups of maternal factors are involved in formation of endoderm: VegT, β-catenin, and Otx (Grapin-Botton and Constan, 2004). Maternal vegT mRNA is concentrated in the vegetal pole of the Xenopus egg. This transcript codes for VegT, which is a transcription factor (Zhang and King, 1996) that induces expression of numerous endodermal genes (Bix1, Bix2, Bix3, Bix4, Mix1, Mix2, Mixer, Xsox17 alpha, Gata4, Gata5, Gata6), including the anterior endodermal genes, Xhex and cerberus) in the vicinity, leading to the formation of endoderm (Xanthos et al., 2001; Fig. 4.3). Another maternal factor involved in endoderm induction in zebrafish and amphioxus is eomesodermin (Grapin-Botton and Constan, 2004). A selflimiting mechanism enables VegT to inhibit induction of endodermal genes, thus determining the endoderm boundary in Xenopus (Clements and Woodland, 2003). The previous evidence proves that it is the epigenetic information deposited in strictly determined sites of the egg cell in the form of maternal VegT, β-catenin, and Otx mRNAs, rather than the genetic information (respective

Epigenetic Control of Early Development Chapter

4

129

genes are not involved in the process of development of endoderm) that determines formation of endoderm.

Maternal Control of Mesoderm Formation Maternal cytoplasmic factors (transcripts for growth factors of the TGF-β superfamily of the secreted proteins, Vg1, and activin) of the dorsal region are responsible for mesoderm induction (Kofron et al., 1999). The activin type II receptor is expressed in all blastula cells and a number of maternal TGF-β growth factors, including Vg-1, may bind it. FGF and bone morphogenetic protein4 (BMP4) are considered to be mesoderm-inducing secreted proteins present in the Nieuwkoop center (Stennard et al., 1996; Zhang and King, 1996). Maternal vegt transcripts are translated into VegT, which induces formation of mesoderm by activating expression of zygotic growth factors of the TGF-β superfamily (Nodal subfamily) Xnr1, Xnr2, Xnr4, and derriere. Elimination or lack of maternal VegT prevents formation of mesoderm (Clements et al., 1999; Kimelman and Bjornson, 2004; Kofron et al., 1999). Ectopic expression of VegT converts prospective ectoderm into ventral mesoderm (Zhang and King, 1996) and elimination of vegT mRNA prevents formation of mesoderm (Zhang et al., 1998). Maternal β-catenin is also involved in expression of Xnr genes and formation of the organizer, which induces mesoderm formation along the dorsoventral axis (Kimelman and Bjornson, 2004).

Epigenetic Control of Neural Induction and Formation of the CNS The development of the nervous system is “a largely epigenetic phenomenon” (Tierney, 1996), which is determined by maternal cytoplasmic factors deposited in gametes under control of the parental nervous system(s). Neural induction and specification of the neural cells starts as early as the blastula stage (Wilson and Edlund, 2001) if not earlier: The cascade of inductive interactions leading to the formation of the central nervous system starts in the uncleaved egg. (M€ uller, 1996)

The role of the CNS in the metazoan individual development is of paramount importance but here I will only briefly outline the epigenetic control of the neural induction by maternal factors. The development of the central nervous system starts before any other organ or system of organs in metazoans. β-Catenin, a maternal transcription factor, accumulates in the dorsal part of the egg and falls to the vegetal cells of the Xenopus embryo (Schneider et al., 1996). Ectodermal explants from the animal cap in the absence of BMP differentiate into neural cells whereas in the presence of BMP are transformed into epidermal cells (Rogers et al., 2009; Fig. 4.4). As early as the 32-cell blastula these cells represent the Nieuwkoop center where

130 PART

I Epigenetic Basis of Metazoan Inheritance

dnActR or Noggin Neural cells

Epidermal cells

Animal cap AC ectodermal explant

BMP Epidermal cells

Neural cells FIG. 4.4 The default model of neural induction in Xenopus. An ectodermal explant dissected from the animal cap of a blastula stage embryo forms epidermis (gray). If BMP signaling is inhibited in those cells by expressing a dominant-negative Activin receptor (dnActR) or the extracellular antagonist Noggin, then neural tissue forms. Dissociation of the explant into single cells without the addition of exogenous factors also leads to the formation of neural tissues. However, if exogenous BMP is added to the dispersed cells, they adopt an epidermal fate (gray). (From Rogers, C., Moody, S.A., Casey, E., 2009. Neural induction and factors that stabilize a neural fate. Birth Defects Res. C Embryo Today 87, 249–262.)

maternal FGF and BMP4 are also present (Stennard et al., 1996). There, the maternal β-catenin forms a complex with the Tcf3, thus enabling the transcription of several genes (Yang et al., 2002). The product of one of those genes, Siamois, then induces expression of goosecoid, producing a protein that triggers expression of a number of genes in the cells dorsal to Nieuwkoop center, thus determining the formation of the Spemann’s organizer (Laurent et al., 1997; Schneider et al., 1996). Neural induction implies suppression of the BMP (bone morphogenetic protein) signaling pathway. Development of the neural plate results from expression of maternal Fgf mRNA which blocks expression of maternal Bmp mRNA in the medial epiblast cells (prospective neural plate) but not in lateral epiblast cells. At early blastula stage a blastula Chordin- and Noggin-expressing (BCNE) group of cells in the dorsal animal cap represents both the prospective neuroectoderm and Spemann organizer. Expression of the BMP antagonists, Chd and Nog, in these cells is determined by accumulation of maternal β-catenin. The BCNE center contains much of the presumptive anterior CNS and is required for brain formation in Xenopus embryos (Kuroda et al., 2004). Neural induction ends by the end of the gastrula stage in different vertebrate classes (Stern, 2004). In X. laevis, for example, it ends before the beginning of large-scale expression of zygotic genes (after 12 cell divisions, i.e., at 4096-cell embryonic stage), implying that neural induction in this amphibian is

Epigenetic Control of Early Development Chapter

4

131

determined by epigenetic information provided parentally to the zygote in the form of cytoplasmic factors. In humans, neurulation starts with the formation of a flat sheet of about 125,000 cells in the dorsal side of the embryo, known as neural plate “from which all the neurons and glial cells derive” (Dowling, 2004). Soon, the neural plate rises on both sides to form the neural tube/ CNS, along almost the whole length of the embryo in the process of the primary neurulation. At the phylotypic stage, the nervous system is operational. In mouse the first neurons are differentiated between the E9 and E10 (Martynoga et al., 2012), implying that at this point in time neurons not only secrete neurotransmitters to communicate with other neurons, but based on their unique self-assembling ability, form specific circuits, which receive and process sensory information by E11 (Momose-Sato and Sato, 2016). Even this glimpse of the processes of neural induction and development of the central nervous system reveals some thought-provoking facts and coincidences: First, formation of the CNS coincides with the exhaustion of the parental epigenetic information (parental cytoplasmic factors) at the phylotypic stage, right before the beginning of organogenesis, when the demand of the developing embryo for morphogenetic information is greater than ever. Second, the nervous system is the first organ system (with neurons being the first differentiated cells) to develop, although conventional wisdom says that systems of blood circulation and excretory system would be necessary to develop before all else. The over early embryonic development of the CNS suggests that it might serve something other than the communication with the external world, during early embryogenesis when that communication is next to absent. Third, initially the CNS is excessively large (in some cases representing almost a quarter of the overall embryonic mass), a fact that in view of the insignificant low level of communication of the embryo with the external environment suggests some other important role. Fourth, formation of the neural ectoderm marks the establishment of the primary embryonic axis as supreme source of inductive radiations in metazoans. The incipient CNS immediately engenders a network of inductions that give rise to the different cells, tissues, and organs of embryos and adults (Hall, 1998a). Its further structures arise in relation to this central axis. This is especially evident in the development of paired elements such as the somites that presage the vertebrae, and paired organ rudiments such as left and right limb buds and the primordia of the gonads, kidney, lung and heart. (Hall, 1998b)

If all the previous coincidences are not determined by chance, as it seems to be the case, they may point to a central role of the nervous system in the individual development.

132 PART

I Epigenetic Basis of Metazoan Inheritance

REFERENCES Aurich, C., Aurich, J.E., Parvizi, N., 2001. Opioidergic inhibition of luteinising hormone and prolactin release changes during pregnancy in pony mares. J. Endocrinol. 169, 511–518. Brunet-Simon, A., et al., 2001. Onset of zygotic transcription and maternal transcript legacy in the rat embryo. Mol. Reprod. Dev. 58, 127–136. Cermik, D., Karaca, M., Taylor, H.S., 2001. HOXA10 expression is repressed by progesterone in the myometrium: differential tissue-specific regulation of HOX gene expression in the reproductive tract. J. Clin. Endocrinol. Metab. 86, 3387–3392. Chan, S., Kilby, M.D., 2000. Thyroid hormone and central nervous system development. J. Endocrinol. 165, 1–8. Chatzaki, E., et al., 2000. Kappa opioids and TGFbeta1 interact in human endometrial cells. Mol. Hum. Reprod. 6, 602–609. Chen, Q., Zhang, Y., Lu1, J., Wang, Q., Wang, S., Cao, Y., Wang, H., Duan, E., 2009. Embryo– uterine cross-talk during implantation: the role of Wnt signaling. Mol. Hum. Reprod. 15, 215–221. Cho, K.W., De Robertis, E.M., 1990. Differential activation of Xenopus homeobox genes by mesoderm-inducing growth factors and retinoic acid. Genes Dev. 4, 1910–1916. Clagett-Dame, M., Plum, L.A., 1997. Retinoid regulated gene expression. Crit. Rev. Eukaryot. Gene Expr. 7, 299–342. Classen-Linke, I., Mueller-Newen, G., Heinrich, P.C., Beier, H.M., von Rango, U., 2004. The cytokine receptor gp 130 and its soluble form are under hormonal control in human endometrium and deciduas. Mol. Hum. Reprod. 10, 495–504. Clements, D., Woodland, H.R., 2003. VegT induces endoderm by a self-limiting mechanism and by changing the competence of cells to respond to TGF-beta signals. Dev. Biol. 258, 454–463. Clements, D., Friday, R.V., Woodland, H.R., 1999. Mode of action of VegT in mesoderm and endoderm formation. Development 126, 4903–4911. Coan, P.M., Burton, G.J., Ferguson-Smith, A.C., 2004. Imprinted genes in the placenta—a review. Placenta 26, S10–S20. Colas, J.F., Launay, J.M., Kellermann, O., Rosay, P., Maroteaux, L., 1995. Drosophila 5-HT2 serotonin receptor: coexpression with fushi-tarazu during segmentation. Proc. Natl. Acad. Sci. USA 92, 5441–5445. Colas, J.F., Launay, J.M., Maroteaux, L., 1999. Maternal and zygotic control of serotonin biosynthesis are both necessary for Drosophila germband extension. Mech. Dev. 87, 67–76. Conlon, R.A., 1995. Retinoic acid and pattern formation in vertebrates. Trends Genet. 11, 314–319. Coser, K.R., et al., 2003. Global analysis of ligand sensitivity of estrogen inducible and suppressible genes in MCF7/BUS breast cancer cells by DNA microarray. Proc. Natl. Acad. Sci. USA 100, 13994–13999. C^ ote, F., et al., 2007. Maternal serotonin is crucial for murine embryonic development. Proc. Natl. Acad. Sci. USA 104, 329–334. Cupp, A.S., Dufour, J.M., Kim, G., Skinner, M.K., Kim, K.H., 1999. Action of retinoids on embryonic and early postnatal testis development. Endocrinology 140, 2343–2352. Daftary, G.S., Taylor, H.S., 2000. Implantation in the human: the role of Hox genes. Semin. Reprod. Med. 18, 311–320. (Abstract). Daftary, G.S., Taylor, H.S., 2001. Molecular markers of implantation: clinical implications. Curr. Opin. Obstet. Gynecol. 13, 269–274. De Luca, L.M., Ross, S.A., 1996. Retinoic acid response elements as positive and negative regulator of the expression of the homeobox 1 gene. Nutr. Rev. 54, 61–63.

Epigenetic Control of Early Development Chapter

4

133

Demura, R., et al., 1993. Activin and inhibin secretion by cultured porcine granulosa cells is stimulated by FSH and LH. Endocr. J. 40, 447–451. Doitsidou, M., et al., 2002. Guidance of primordial cell migration by the chemokine SDF-1. Cell 111, 647–659. Dowling, J.E., 2004. The Great Brain Debate-Nature or Nurture? Joseph Henry Press, Washington, DC, p. 8. Dowling, A.L., Zoeller, R.T., 2000. Thyroid hormone of maternal origin regulates expression of RC3/neurogranin mRNA in the fetal rat brain. Mol. Brain Res. 82, 126–132. Eno, C., Pelegri, F., 2016. Germ cell determinant transmission, segregation, and function in the zebrafish embryo. In: Carreira, R.P. (Ed.), Insights from Animal Reproduction. IntechOpen. https://doi.org/10.5772/62207. Available from: https://www.intechopen.com/books/insightsfrom-animal-reproduction/germ-cell-determinant-transmission-segregation-and-function-inthe-zebrafish-embryo. Gilbert, S.F., 2000. Developmental Biology, sixth ed. Sinauer Associates Inc. Publishers, Sunderland, MA, p. 223. Grapin-Botton, A., Constan, D., 2004. Endoderm development. In: Stern, C.D. (Ed.), Gastrulation: From Cells to Embryo. Cold Spring Harbor Laboratory Press, New York, pp. 433–448. Hall, B.K., 1998a. Evolutionary Developmental Biology, second ed. Chapman & Hall, London, p. 134. Hall, B.K., 1998b. Evolutionary Developmental Biology, second ed. Chapman & Hall, London, p. 163. Hall, J.M., Korach, K.S., 2003. Stromal cell-derived factor 1, a novel target of estrogen receptor action, mediates the mitogenic effects of estradiol in ovarian and breast cancer cells. Mol. Endocrinol. 17, 792–803. Hardy, K., Spanos, S., 2002. Growth factor expression and function in the human and mouse preimplantation embryo. J. Endocrinol. 172, 221–236. Hayashi, K., Erikson, D.W., Tilford, S.A., Bany, B.M., Maclean II, J.A., Rucker III, E.B., Johnson, G.A., Spencer, T.E., 2009. Wnt genes in the mouse uterus: potential regulation of implantation. Biol. Reprod. 80, 989–1000. Henrion, G., et al., 2000. Differential regulation of the translation and the stability of two maternal transcripts in preimplantation rabbit embryos. Mol. Reprod. Dev. 56, 12–25. Herrler, A., Krusche, C.A., Henning, M.B., 1998. Insulin and insulin-like growth factor-I promote rabbit blastocyst development and prevent apoptosis. Biol. Reprod. 59, 1302–1310. Hopper, A.F., Hart, N.H., 1985. Foundations of Animal Development. Oxford University Press, New York, p. 286. Illmensee, K., Mahowald, A.P., 1974. Transplantation of posterior polar plasm in Drosophila. Induction of germ cells at the anterior pole of the egg. Proc. Natl. Acad. Sci. USA 71, 1016–1020. Illmensee, K., Mahowald, A.P., 1976. The autonomous function of germ plasm in a somatic region of the Drosophila egg. Exp. Cell Res. 97, 127–140. Izadyar, F., et al., 2000. Preimplantation bovine embryos express mRNA of growth hormone receptor and respond to growth hormone addition during in vitro development. Mol. Reprod. Dev. 57, 247–255. Johansson, S., Husman, B., Norstedt, G., Andersson, G., 1989. Growth hormone regulates the rodent hepatic epidermal growth factor at a pretranslational level. J. Mol. Endocrinol. 3, 113–120. Kawase, E., Hashimoto, K., Pedersen, R.A., 2004. Autocrine and paracrine mechanisms regulating primordial germ cell proliferation. Mol. Reprod. Dev. 68, 5–16. Kim, C.H., et al., 1999. The effect of epidermal growth factor on the preimplantation development, implantation and its receptor expression in mouse embryos. J. Obstet. Gynaecol. Res. 25, 87–93.

134 PART

I Epigenetic Basis of Metazoan Inheritance

Kimelman, D., Bjornson, C., 2004. Vertebrate mesoderm induction. In: Stern, C.D. (Ed.), Gastrulation: From Cells to Embryo. Cold Spring Harbor Laboratory Press, New York, pp. 363–372. Knezevic, V., Mackem, S., 2001. Activation of epiblast gene expression by the hypoblast layer in the prestreak chick embryo. Genesis 30, 264–273. Kofron, M., et al., 1999. Mesoderm induction in Xenopus is a zygotic event regulated by maternal VegT via TGFbeta growth factors. Development 126, 5759–5770. Kostetskii, I., et al., 1998. Initial retinoid requirement for early avian development coincides with retinoid receptor coexpression in the precardiac fields and induction of normal cardiovascular development. Dev. Dyn. 213, 188–198. Kuroda, H., Wesely, O., De Robertis, E.M., 2004. Neural induction in Xenopus: requirement for ectodermal and endoesodermal signals via chordin, noggin, beta-catenin, and cerberus. PloS Biol. 2, e92. Langston, A.W., et al., 1997. Retinoic acid-responsive enhancers located 30 of the Hox A and Hox B homeobox gene clusters. J. Biol. Chem. 272, 2167–2175. Laurent, M.N., Blitz, I.L., Hashimoto, C., Rothbacher, U., Cho, K.W., 1997. The Xenopus homeobox gene twin mediates Wnt induction of goosecoid in establishment of Spemann’s organizer. Development 124, 4905–4919. Lawson, K., Hage, W.J., 1994. Clonal analysis of the origin of primordial germ cells in the mouse. CIBA Found. Symp. 182, 68–91. Lawson, K.A., Dunn, N.R., Roelen, B.A.J., Zeinstra, L.M., David, A.M., Wright, C.V., Korving, J.P.W.F.M., Hogan, B.L.M., 1999. Bmp4 is required for the generation of primordial germ cells in the mouse embryo. Genes Dev. 13, 424–436. Lemaitre, J.-M., Bocquet, S., Mechall, M., 2002. Competence to replicate in the unfertilized egg is conferred by Cdc6 during meiotic maturation. Nature 419, 718–722. L€ uer, K., Urban, J., Kl€ambt, C., Technau, G.M., 1997. Induction of identified mesodermal cells by CNS medline progenitors in Drosophila. Development 124, 2681–2690. Malpel, S., Mendelsohn, C., Cardoso, W.V., 2000. Regulation of retinoic acid signaling morphogenesis. Development 127, 3057–3067. Marshall, H., Morrison, A., Studer, M., Popperl, H., Krumlauf, R., 1996. Retinoic acid and Hox genes. FASEB J. 10, 969–978. Martynoga, B., Drechsel, D., Guillemot, F., 2012. Molecular control of neurogenesis: a view from the mammalian cerebral cortex. Cold Spring Harb. Perspect. Biol. 4, a008359. Moeller, B., et al., 2001. Expression of angiogenic growth factors VEGF, FGF-2, EGF and their receptors in normal human endometrium during the menstrual cycle. Mol. Hum. Reprod. 7, 65–72. Mohamed, O.A., Dufort, D., Clarke, H.J., 2004. Expression and estradiol regulation of Wnt genes in the mouse blastocyst identify a candidate pathway for embryo-maternal signaling at implantation. Biol. Reprod. 71, 417–424. Mohan, M., et al., 2001. Expression of retinol binding protein messenger RNA and retinoic acid receptors in preattachment bovine embryos. Mol. Reprod. Dev. 60, 289–296. Momose-Sato, Y., Sato, K., 2016. Development of synaptic networks in the mouse vagal pathway revealed by optical mapping with a voltage-sensitive dye. Eur. J. Neurosci. 44, 1906–1918. M€ uller, W.A., 1996. Developmental Biology. Springer, New York, p. 90. Murray, A.W., Kirshner, M.W., 1989. Cyclin synthesis drives the early embryonic cell cycle. Nature 339, 275–280. Nonaka, K., et al., 1993. Intrauterine effect of dam on parental development of craniofacial complex of mouse embryo. J. Craniofac. Genet. Dev. Biol. 13, 206–212(Abstract).

Epigenetic Control of Early Development Chapter

4

135

Ota, H., et al., 1999. Optimal levels of nitric oxide are crucial for implantation in mice. Reprod. Fertil. Dev. 11, 183–188. Ouyang, L., Chang, W., Fang, B., Qin, J., Qu, X., Cheng, F., 2016. Estrogen-induced SDF-1α production promotes the progression of ER-negative breast cancer via the accumulation of MDSCs in the tumor microenvironment. Sci. Rep. 6, 39541. Paria, B.C., et al., 2001. Cellular and molecular responses of the uterus to embryo implantation can be elicited by locally applied growth factors. Proc. Natl. Acad. Sci. USA 98, 1047–1052. Piko, I., Clegg, K.B., 1982. Quantitative changes in total RNA, total poly(A), and ribosomes in early mouse embryos. Dev. Biol. 89, 362–378. Porterfield, S.P., Hendry, L.B., 1998. Impact of PCBs (polychlorinated biphenyls) onthyroid hormone directed brain development. Toxicol. Ind. Health 14, 103–120. Poulain, M., F€ urthauer, M., Thisse, B., Thisse, C., Lepage, T., 2006. Nodal, FGF and BMP signaling. Development 133, 2189–2200. Rogers, C., Moody, S.A., Casey, E., 2009. Neural induction and factors that stabilize a neural fate. Birth Defects Res. C Embryo Today 87, 249–262. Salamonsen, L.A., et al., 2001. Genes involved in implantation. Reprod. Fertil. Dev. 13, 41–49. Sasaki, Y., et al., 1995. The strain effect of dam on intrauterine incisal growth in mouse fetuses. J. Craniofac. Genet. Dev. Biol. 15, 140–145(Abstract). Sawai, K., Matsuzaki, N., Okada, T., Shimoya, K., Koyama, M., Azuma, C., et al., 1997. Human decidual cell biosynthesis of leukemia inhibitory factor: regulation by decidual cytokines and steroid hormones. Biol. Reprod. 56, 1274–1280. Schier, A.F., 2003. Nodal signaling in vertebrate development. Annu. Rev. Cell Dev. Biol. 19, 589–621. Schneider, S., Steinbeisser, H., Warga, R.M., Hausen, P., 1996. Beta-catenin translocation into nuclei demarcates the dorsalizing centers in frog and fish embryos. Mech. Dev. 57, 191–198. Shmukler, Y.B., Buznikov, G.A., Whitaker, M.J., 1999. Action of serotonin antagonists on cytoplasmic calcium levels in early embryos of sea urchin Lytechinus pictus. Int. J. Dev. Biol. 43, 179–182. Spencer, T.E., Johnson, G.A., Bazer, F.W., Burghardt, R.C., 2004. Implantation mechanisms: insights from the sheep. Reproduction 128, 657–668. Stebler, J., et al., 2004. Primordial germ cell migration in the chick and mouse embryo: the role of the chemokine SDF-1/CHCL12. Dev. Biol. 272, 351–361. Stennard, F., Carnac, G., Gurdon, J.B., 1996. The Xenopus T-box gene, antipodean, encodes a vegetally localised maternal mRNA and can trigger mesoderm formation. Development 122, 4179–4188. Stern, C.D., 2004. Neural induction. In: Stern, C.D. (Ed.), Gastrulation. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, p. 427. Tessier, C., et al., 2000. Estrogen receptors α and ß in rat decidua cells: cell-specific expression and differential regulation by steroid hormones and prolactin. Endocrinology 141, 3842–3851. Tierney, A.J., 1996. Evolutionary implications of neural circuit structure and function. Behav. Process. 35, 173–182. Tomancak, P., Guichet, A., Zavorsky, P., Ephrussi, A., 1998. Oocyte polarity depends on regulation of gurken by vasa. Development 125, 1723–1733. Tres, L.L., Rosselot, C., Kierszenbaum, A.L., 2004. Primordial germ cells: what does it take to be alive? Mol. Reprod. Dev. 68, 1–4. van de Pavert, S.A., et al., 2001. Uterine-embryonic interaction in pig: activin, follistatin, and activin receptor II in uterus and embryo during early gestation. Mol. Reprod. Dev. 59, 390–399.

136 PART

I Epigenetic Basis of Metazoan Inheritance

Vanzo, N.F., Ephrussi, A., 2002. Oskar anchoring restricts pole plasm formation to the posterior of the Drosophila oocyte. Development 129, 3705–3714. Weidinger, G., et al., 2003. Dead end, a novel vertebrate germ plasm component, is required for zebrafish primordial germ cell migration and survival. Curr. Biol. 13, 1429–1434. Wilson, S.I., Edlund, T., 2001. Neural induction: toward a unifying mechanism. Nat. Neurosci. 4, 1161–1168. Wollenhaupt, K., Einspanier, R., Gabler, C., Schneider, F., Kanitz, W., Br€ussow, K.P., 1999. Identification of the EGF/EGF-R system in the oviduct and endometrium of pigs in early stages of pregnancy and early conceptus. Exp. Clin. Endocrinol. Diabetes 107, 530–538. Wolpert, L., Beddington, R., Lawrence, P., Jessell, T.M., 1998. Principles of Development. Current Biology Ltd. and Oxford University Press, Oxford; New York; Tokyo, p. 259. Wright, N., et al., 2003. Transforming growth factor-beta 1 down-regulates expression of chemokine stromal cell-derived factor-1: functional consequences in cell migration and adhesion. Blood 102, 1978–1984. Xanthos, J.B., Kofron, M., Heasman, J., 2001. Maternal VegT is the iniator of a molecular network specifying endoderm in Xenopus laevis. Development 128, 167–180. Yang, J., Tan, C., Darken, R.S., Wilson, P.A., Klein, P.S., 2002. Beta-catenin/Tcf-regulated transcription prior to midblastula transition. Development 129, 5743–5752. Yue, Z.-P., Yang, Z.-M., Li, S.-J., Wang, H.-B., Harper, M.J.K., 2000. Epidermal growth factor family in rhesus monkey uterus during the menstrual cycle and early pregnancy. Mol. Reprod. Dev. 55, 164–174. Zhang, J., King, M.I., 1996. Xenopus VegT RNA is localized to the vegetal cortex during oogenesis and encodes a novel T-box transcription factor involved in mesoderm patterning. Development 122, 4119–4129. Zhang, J., et al., 1998. The role of maternal VegT in establishing the primary germ cell-layers in Xenopus embryos. Cell 94, 515–524.

FURTHER READING Di, W.L., et al., 2001. Oestriol and oestradiol increase cell-to-cell communication and connexin43 protein expression in human myometrium. Mol. Hum. Reprod. 671–679. Dimitriadis, E., et al., 2000. Expression of interleukin-11 during the human menstrual cycle: coincidence with stromal cell decidualization and relationship to leukemia inhibitory factor. Mol. Hum. Reprod. 6, 907–914. Eddy, E.M., 1975. Germ plasm and the differentiation of the germ cell line. Int. Rev. Cytol. 42, 229–280. Weaver, C., Kimelman, D., 2004. Move it or lose it: axis specification in Xenopus. Development 131, 3491–3499.