Differentiation in Early Development

Chapter 14

Differentiation in Early Development Susana M. Chuva de Sousa Lopes and Christine L. Mummery Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, The Netherlands

Chapter Outline Preimplantation Development Cell Polarization Occurs during Compaction Blastocyst Formation (Cavitation) Axis Specification during Preimplantation in the Mouse Developmental Potency of the Early Mouse Embryo Genes Important during Preimplantation Mouse Development From Implantation to Gastrulation

139 139 140 141 142 143 147

PREIMPLANTATION DEVELOPMENT In mammals, fertilization occurs in the oviduct where sperm encounters and fuses with the oocyte. As a result, the oocyte nucleus, which had been arrested in metaphase II, completes meiosis, and the two parental pronuclei fuse to form the diploid zygotic nucleus (Figure 14.1A). Progressive DNA demethylation of first the paternal and then the maternal genomes (excluding the genomic imprints) begins after fertilization as part of the epigenetic reprogramming that takes place during preimplantation development (Reik et al, 2001). Transcription of the embryonic genome starts at the 2-cell stage in mice (Flach et al., 1982) and at the 4-cell to 8-cell stage in humans (Braude et al., 1988). Until then, the embryo relies solely on maternal mRNA but after activation of the embryonic genome, maternal transcripts are rapidly degraded, although maternally encoded proteins may still be present and functionally important. The embryo continues cleavage divisions without visible growth (Figure 14.1) as it travels inside a protective glycoprotein coat, the zona pellucida, through the oviduct into the uterus.

Cell Polarization Occurs during Compaction At the 8-cell stage in mice and the 8-cell to 16-cell stage in humans, the embryo undergoes a process known as

The Mouse Trophectoderm and Primitive Endoderm Cells Development of the Mouse Inner Cell Mass to the Epiblast The Human Embryo Implantation: Maternal versus Embryonic Factors The Role of Extraembryonic Tissues in Patterning the Mouse Embryo References

147 148 148 150 150 151

compaction to become a morula, a compact smooth spherical structure (Figure 14.1D). All blastomeres flatten, maximize their contacts, and become polarized. Their cytoplasm forms two distinct zones: the apical domain accumulates endosomes, microtubules, and microfilaments (Fleming and Pickering, 1985; Johnson and Maro, 1984; Reeve, 1981), whereas the nucleus moves to the basal domain (Reeve and Kelly, 1983). Furthermore, gap junctions form basally ensuring communication between blastomeres (Ducibella and Anderson, 1975; Lo and Gilula, 1979; Magnuson et al., 1977; Sheth et al., 1997) and numerous microvilli (Reeve and Ziomek, 1981) and tight junctions are formed apically (Ducibella and Anderson, 1975; Magnuson et al., 1977). The next cleavage plane of some of the blastomeres is perpendicular to their axis of polarity, resulting in two cells with different phenotypes (asymmetric division). One daughter cell is located inside the embryo (“inner” cell), is small and apolar, and contains only basolateral elements. The other daughter cell is located at the surface of the embryo (“outer” cell), is larger and polar, and contains the entire apical domain of the progenitor cell and some basolateral elements. These polar cells inherit the region containing the tight junctions, thereby creating a physical barrier between the inner apolar cells and the maternal environment (Magnuson et al., 1977; Ducibella et al., 1975).

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FIGURE 14.1 Mouse preimplantation development. After fertilization the two parental pronuclei fuse to form the zygote (A). The embryo cleaves, forming a 2-cell (B), 4-cell (C), and 8-cell embryo. The embryo then undergoes compaction to become a smooth spherical structure, the morula (D). Note that the second polar body remains attached to the embryo (*). The blastocoelic cavity then develops on one side of the embryo to form an early blastocyst (E). The cavity enlarges, occupying most of the expanded blastocyst (F, G). Around embryonic day (E) 4.5, the late blastocyst reaches the uterus, “hatches” from the zona pellucida, and is ready to implant (H). The late blastocyst consists of three cell subpopulations: the trophectoderm (green), the inner cell mass (orange), and the primitive endoderm (yellow). In the blastocyst three axes can be defined: the embryoniceabembryonic (abembeemb), the animalevegetal (aneveg), and a third axis on the same plane but perpendicular to the aneveg axis. (Photomicrographs courtesy of B. Roelen.)

Blastocyst Formation (Cavitation) After compaction, the presumptive trophectoderm (TE) cells form the outer layer of the embryo. Intercellular contacts strengthen between these cells, and a true epithelium is formed. This thin single cell layer develops a continuum of junctional complexes, including gap junctions, desmosomes, and tight junctions (Magnuson et al., 1977; Ducibella et al., 1975; Moriwaki et al., 2007). Furthermore, the composition of the basal and apical cell membranes of the TE cells becomes more distinct, with Na+/K+-ATPases accumulating in the basal membrane (Watson et al., 1990; Watson and Kidder, 1988). These ion pumps actively transport sodium ions into the embryo, which leads to accumulation of water molecules, possibly via aquaporins (Offenberg et al., 2000; Offenberg and Thomsen, 2005). A fluid-filled cavity, the blastocoelic cavity, is thus created on one side of the embryo (Wiley, 1984) in a process known as cavitation (Figure 14.1E). The presumptive inner cell mass (ICM) cells stay closely associated during this process, not only because of gap junctions, tight junctions, and interdigitating microvilli between the cells but also because processes from TE cells fix the ICM to one pole of the embryo and partially isolate it from the blastocoel (Ducibella et al., 1975). The intercellular permeability seal of the TE cells prevents fluid loss, and as a consequence the blastocoelic cavity gradually

expands to occupy most of the blastocyst between the 64-cell and 128-cell stages (Figure 14.1E to G). At this point, the embryo is not radially symmetric around the embryoniceabembryonic axis but is bilaterally symmetric because the zona pellucida is slightly oval. The outer TE layer and the ICM are composed of descendants of the “outer” and “inner” cell population of the morula, respectively. The TE in turn consists of two subpopulations: the polar TE that contacts the ICM and the mural TE that surrounds the blastocoelic cavity. The TE descendants give rise to extraembryonic structures such as the placenta and do not contribute to the embryo proper. The cells of the ICM also consist of two subpopulations mixed initially distributed in a salt-and-pepper fashion. However, due to adherence differences, one of the subpopulations (the GATA6-positive cells) segregates to the surface of the ICM where it contacts the blastocoelic cavity and differentiates into primitive endoderm, also an extraembryonic tissue (Chazaud et al., 2006; Plusa et al., 2008). The ICM gives rise not only to the embryo proper but also to extraembryonic mesoderm that contributes cells to the visceral yolk sac, amnion, chorion and the allantois, a structure that will later develop into the umbilical cord. An overview of cell lineage relationships in the early mouse embryo is shown in Figure 14.2. During preimplantation development (3 to 4 days in mice, 5 to 7 days in humans), the embryo has remained inside the zona

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Differentiation in Early Development

141

FIGURE 14.2 Cell lineages in mouse development. Trophectoderm-derived tissues are depicted in green, endoderm-derived tissues in yellow, ectoderm-derived tissues in orange, and mesoderm-derived in blue. The cell/tissues represented in gray are regarded as pluripotent. All extraembryonic tissues are enclosed by hatched lines, whereas embryonic tissues are enclosed by solid lines.

pellucida which prevents its premature implantation while still in the oviduct. Reaching the uterus, the blastocyst “hatches” from the zona pellucida using the enzyme strypsin (Perona and Wassarman, 1986) and is then ready to implant in the uterine wall (Figure 14.1H). Stages of mouse and human preimplantation development are summarized in Table 14.1.

Axis Specification during Preimplantation in the Mouse In lower vertebrates, the body axes are already specified (determined) in the undivided egg or very soon thereafter, whereas in mammalian embryos, axis specification was thought to be completed only at gastrulation, with the appearance of the primitive streak. However, the first morphological sign of the axis determination (anterioreposterior) is now considered to be the migration of the slightly cuboidal visceral endoderm at the distal tip of the embryo towards the more anterior part, forming the anterior visceral endoderm (AVE) at embryonic day (E) 5.5e6.0 (Thomas and Beddington, 1996). Data suggest that the anterioreposterior axis of the embryo is molecularly determined even earlier at E4.0e4.5 by asymmetric

expression of Lefty1 on one side of the primitive endoderm that will eventually correspond to the “tilt” (see the section on implantation) (Takaoka et al., 2006). This view of relatively late axis determination is supported by the observation that the mammalian embryo is extremely plastic and able to ignore disturbances such as the removal or reaggregation of blastomeres. The prevailing concept, therefore, became one of no embryonic prepatterning before implantation. However, several studies challenge this view suggesting that the mammalian zygote may in fact be polarized and that the body axes could somehow be specified at the time of fertilization in a fashion similar to that in lower vertebrates. In the mouse zygote, the position of the animal pole, marked by the second polar body (Plusa et al., 2002), or the sperm entry point, which triggers Ca2+ waves (Deguchi et al., 2000) or the plane defined the two pronuclei in the zygote (Hiiragi and Solter, 2004), have all been discussed as defining the plane of first cleavage. However, it is still unclear whether the position of these cues is directly responsible for the zygote polarity and the subsequent position of the first cleavage plane. Alternatively, zygote polarity, the position of the second polar body and the sperm entry point (and of the paternal pronucleus) might be determined by an intrinsic asymmetry already present in

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TABLE 14.1 Summary of Mouse and Human Preimplantation Development Stage (M)

Time (M)

Stage (H)

Time (H)

Developmental processes

Zygote

0e20 h

Zygote-2-cell

0e60 h

Axis determination?

2-cell

20e38 h

4e8-cell

60e72 h

Activation of embryo genome

4-cell

38e50 h

8-cell

50e62 h

16-cell

62e74 h

32-cell

~3.0 d

64-cell

~3.5 d

128e256-cell

~4.5 d

Lineage determination? 8e16-cell

32-cell

166e286-cell

~3.5 d

Compaction

~4.0 d

Two phenotypically different cells emerge

~4.5 d

Blastocoelic cavity forms (cavitation)

~5.5 d

Blastocyst consists of two cell populations (ICM and TE)

~6.0 d

Part of ICM differentiates to PrE; hatching, followed by implantation

During mouse and human development, the timing of each cleavage division is dependent on environmental factors (in vitro versus in vivo), individual variation, and mouse strain. The cleavage times presented here are ranges from several published sources. Adapted from Nagy, A., Gertsenstein, M., Vintersten, K., Behringer, R. (2003) Manipulating the Mouse Embryo. New York: Cold Spring Harbor Laboratory Press and from Larsen, W.J. (1997) Human Embryology. New York: Churchill Livingstone. d, days; h, hours; H, human; ICM, inner cell mass; M, mouse; PrE, primitive endoderm; TE, trophectoderm.

the oocyte. The oocyte has been shown, for example, to have an asymmetric distribution of mitochondria (Calarco, 1995; Van Blerkom and Runner, 1984) and other factors including Leptin and Stat3 (Antczak and Van Blerkom, 1997). The first cleavage plane coincides with the embryoniceabembryonic boundary of the blastocyst. However, it is still unsettled whether the fates of the two blastomeres are distinguishable and can be anticipated. The blastomere containing the sperm entry site generally divides first and contributes preferentially to the embryonic region of the blastocyst, whereas its sister cell preferentially forms the abembryonic region (Gardner, 2001; Piotrowska and Zernicka-Goetz, 2001). Parthenogenetic eggs that do not contain a sperm entry point are able to divide and develop into blastocysts, although there is no tendency for the two blastomeres to follow different fates (Piotrowska and Zernicka-Goetz, 2002). This indicates that, although during normal development the site of sperm penetration correlates with the later spatial arrangement of the blastocyst, it is not essential for patterning the embryo. Moreover, several studies have claimed that the two blastomeres are similar but it is the ellipsoidal shape of the zona pellucida that defines the embryoniceabembryonic axis (Alarcon and Marikawa, 2008; Kurotaki et al., 2007; Motosugi et al., 2005). The topographic relationship between the zygote and the blastocyst axes and between the blastocyst and body axes of the future fetus remains a matter of debate, although the prevalent view is that of existing cues, but those could be easily overruled (Rossant and Tam, 2009).

Developmental Potency of the Early Mouse Embryo In the mouse, both blastomeres of a 2-cell stage embryo transplanted separately into foster mothers develop into identical mice (Tsunoda and McLaren, 1983). To assess the developmental potential of each blastomere of 4-cell and 8-cell mouse embryos, Kelly (1977) combined isolated blastomeres with genetically distinguishable blastomeres of the same age, creating chimeric composites. Each blastomere was shown to contribute extensively to both embryonic and extraembryonic tissues (TE and visceral yolk sac) and to generate viable and fertile mice. This indicated that at these developmental stages all blastomeres are still totipotent. However, single isolated 4-cell and 8-cell stage blastomeres were able to develop only to blastocysts and implant but they were incapable of generating viable concepti (Rossant, 1976; Tarkowski and Wro´blewska, 1967). This may be explained by the fact that a defined number of cell divisions (five) occurs before blastocyst formation. Thus, in contrast to the normal 32-cell blastocyst, isolated blastomeres from 4-cell and 8-cell embryos resulted in 16-cell and 8-cell blastocysts, respectively. Blastocysts generated from isolated 4-cell and 8-cell blastomeres contain progressively fewer cells in the ICM, making it likely that a minimum number of ICM cells is necessary for survival beyond the blastocyst stage. According to Tarkowski and Wro´bleswska (1967) it is the position of a cell in the blastocyst that determines its fate: cells at the surface of the embryo become TE, whereas cells enclosed in the embryo become ICM. Recent studies

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Differentiation in Early Development

have revealed that molecular heterogeneity already detectable at the 4-cell stage could direct a developmental bias towards TE or ICM (Plachta et al., 2011; TorresPadilla et al., 2007), perhaps by dictating the preferential axis of division (giving rise to two “outer” cells by symmetric division or one “inner” and one “outer” cell by asymmetric division). Although different phenotypically, the 2-cell subpopulations in the 16-cell morula are still plastic and able to produce cells of the other lineage provided they are at the correct position in the embryo, that is, inside or at its surface. Cells of the ICM of 32-cell and 64-cell embryos are also still capable of contributing to all tissues of the conceptus (embryonic and extraembryonic) and are thus still totipotent (Pedersen, 1986). The potency of TE cells has been difficult to determine because TE cells are not easy to isolate (tightly connected with each other) and because they are not readily integrated inside the embryo (low adhesiveness). After the 64-cell stage, the ICM loses totipotency (Gardner and Rossant, 1979). Once the mouse embryo has implanted (up to E7.0), the embryonic cells (including the primordial germ cells formed slightly later in development) lose their ability to contribute to the embryo when introduced directly into a host blastocyst to give rise to a chimeric embryo or chimera (Donovan, 1994; Gardner et al., 1985). In agreement, stem cells isolated from E5.5 and E6.5 embryonic (or epiblast) cells, the so-called EpiSCs, are also unable to form chimeras (Brons et al., 2007; Han et al., 2010). Remarkably, when introduced directly into genetically identical adult mice, epiblast cells are able to generate teratocarcinomas, tumors containing a spectrum of differentiated tissues derived from the three germ layers (endoderm, mesoderm, and ectoderm) and a stem cell population called embryonal carcinoma (EC) cells. These epiblastderived EC cells are also able to form mouse chimeras (but unable to go germ line) when introduced into blastocysts (Brinster, 1974; Mintz and Illmensee, 1975; Papaioannou et al., 1975), suggesting that, although pluripotency is lost in the epiblast, it can be regained to a certain extent. Similarly, primordial germ cells isolated from E8.5 mouse embryos cultured to become embryonic germ (EG) cells and also adult hematopoietic and neural stem cells are able to regain pluripotency and can contribute to the embryo when introduced into blastocysts (Clarke et al., 2000; Geiger et al., 1998; Stewart et al., 1994).

Genes Important during Preimplantation Mouse Development Before implantation, the embryo is relatively self-sufficient and can, for example, develop in vitro in simple culture media without growth factors supplements (Hardy and

143

Spanos, 2002). Of particular importance during preimplantation development are genes that regulate activation of the embryonic genome, genome DNA demethylation and chromatin remodeling, the cell cycle, compaction, cavitation, and hatching (Shi and Wu, 2009; Warner, and Brenner, 2001). However, only relatively few mutations (specific gene deletions, insertions, and more extensive genetic abnormalities) have been reported to result in preimplantation lethality (Table 14.2). The reasons for this are not clear but one may be that the initial presence of maternal transcripts in the zygote effectively results in maternal rescue. Ablation of specific maternal transcripts in the zygote is not always feasible using conventional knockout techniques because deficiency in candidate genes often results in lethality before adulthood. However, a growing number of maternal-effect genes involved in preimplantation development are being identified (Li et al., 2010) (see Table 14.3). Interestingly, most genes transcribed during preimplantation development are detected immediately after embryonic genome activation and continue to be transcribed, resulting in mRNA accumulation (Kidder, 1992). Therefore, to trigger the different specific developmental events during preimplantation development, posttranscriptional regulation may play an important role. ES cells are derived from the ICM; therefore, it is not surprising that ES and ICM cells express common genes. Some of these genes have been described as being necessary for maintaining the undifferentiated phenotype of ES cells and could be expected to play important roles in the segregation of the pluripotent ICM from the differentiated TE cell population. However, when deleted in the mouse, most of those genes appear to be crucial during implantation or gastrulation but not during the preimplantation period when both ICM and TE are formed. Most pertinent in this respect are the genes for leukemia inhibitory factor (LIF) and LIF receptors. Although mouse ES cells are highly dependent on LIF for maintenance of pluripotency in culture, deletion of neither receptor nor ligand genes appears to affect the pluripotency of the ICM at the blastocyst stage (Stewart et al., 1992; Ware et al., 1995; Yoshida et al., 1996). Interestingly, in vivo LIF signaling appears important for regulation of implantation (see the section on implantation). The POU transcription factor Oct4 has the best-characterized involvement in regulating potency in mammals. Oct4 is initially expressed by all blastomeres but expression becomes restricted to the ICM as the blastocyst forms (Figure 14.3). Thereafter, a transient upregulation of Oct4 occurs in the ICM cells that differentiate to primitive endoderm (Scholer, 1991). Interestingly, expression levels of Oct4 in mouse ES cells also regulate early differentiation choices, mimicking events in the blastocyst: mouse ES cells lacking Oct4 differentiate to TE, whereas a twofold increase in Oct4 expression leads to endoderm and

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TABLE 14.2 Lethal Mouse Mutations Affecting Differentiation during Early Development Gene/Locus

Mutant phenotype

References

Wt1 (Wilms’ tumor 1)

Zygotes fail to undergo mitosis

Kreidberg et al. (1999) Mol. Reprod. Dev (background dependent) 52, 366

Faf1

2-cell stage arrest

Adham et al. (2008) Mol. Hum. Reprod. 14, 207

Tgfb1

2- to 4-cell arrest

Kallapur et al. (1999) Mol. Reprod. Dev (background dependent) 52, 341

C 25H (pid)

2- to 6-cell stage embryos fail to undergo mitosis

Lewis (1978) Dev. Biol. 65, 553

L2dtl

4- to 8-cell stage arrest

Liu et al. (2007) J. Biol. Chem. 282, 1109

Geminin

4- to 8-cell stage arrest

Hara et al. (2006) Genes Cells 11, 1281

E-cadherin (uvomorulin, Cdh1)

Defects in compaction

Larue et al. (1994) PNAS 91, 8263; Riethmacher et al. (1995) PNAS 92, 855

Trb (Traube)

Defects in compaction

Thomas et al. (2000) Dev. Biol. 227, 324

Cdk8

Defects in compaction

Westerling et al. (2007) Mol. Cell Biol. 27, 6177

Mdn (morula decompaction)

Defects in compaction

Cheng and Costantini (1993) Dev. Biol. 156, 265

Om (ovum mutant)

Failure to form blastocysts

Wakasugi et al. (1967) J. Reprod. Fert. (background dependent) Fertil. 13, 41; Baldacci et al. (1992) Mamm. Genome 2, 100

SRp20

Failure to form blastocysts

Jumaa et al. (1999) Curr. Biol. 9, 899

Rbm19

Failure to form blastocysts

Zhang et al. (2008) BMC Dev. Biol. 8, 115

Wdr36

Failure to form blastocysts

Gallenberger et al. (2011) Hum. Mol. Genet. 20, 422

w32

Failure to form blastocysts

Bennett (1975) Cell 6, 441; Smith (1956) J. Exp. Zool. 132, 51

T hp (hairpin)

Failure to form blastocysts

Babiarz (1983) Dev. Biol. 95, 342

Ts (Tail short)

Failure to form blastocysts

Paterson (1980) J. Exp. Zool. 211, 247

a-E-catenin

Failure to form blastocysts (TE defect)

Torres et al. (1997) PNAS 94, 901

SNEV (Prp19, Pso4, NMP200)

Failure to form blastocysts

Fortschegger et al. (2007) Mol. Cell Biol. 27, 3123

Emi1

Failure to form blastocysts

Lee et al. (2006) Mol. Cell Biol. 26, 5373

Vav

Blastocysts fail to hatch

Zmuidzinas et al. (1995) EMBO J. 14, 1

Os (oligosyndactyly)

Metaphase arrest at the early blastocyst

van Valen (1966) J. Embryol. Exp. stage Morphol. 15, 119; Magnuson and Epstein (1984) Cell 38, 823

Brg1

Abnormal blastocyst development

Bultman et al. (2000) Mol. Cell 6, 1287

Ax (lethal nonagouti)

Abnormal blastocyst development

Papaioannou and Mardon (1983) Dev. Genet. 4, 21

l(5)-1

Abnormal blastocyst development

Papaioannou (1987) Dev. Genet. 8, 27

t wPa-1

Abnormal blastocyst development

Guenet et al. (1980) Genet. Res. 36, 211

PL16

Abnormal blastocyst development

Sun-Wada et al. (2000) Dev. Biol. 228, 315

CpG binding protein (CGBP)

Abnormal blastocyst development

Carlone and Skalnik (2001) Mol. Cell Biol. 21, 7601

Mbd3

Abnormal blastocyst development

Kaji et al. (2007) Development 134, 112

Thioredoxin (Txn)

Abnormal blastocyst development

Matsui et al. (1996) Dev. Biol. 178, 179

Gpt

Abnormal blastocyst development

Marek et al. (1999) Glycobiology 9, 1263

12

t ,t

(Continued)

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Differentiation in Early Development

145

TABLE 14.2 Lethal Mouse Mutations Affecting Differentiation during Early Developmentdcont’d Gene/Locus

Mutant phenotype

References

Ltbp2

Abnormal blastocyst development

Shipley et al. (2000) Mol. Cell Biol. 20, 4879

Wdr74

Abnormal blastocyst development

Maserati et al. (2011) PLoS One 6, e22516

Hbath-J

Decreased TE cell number

Hendrey et al. (1995) Dev. Biol. 172, 253

Ay (lethal yellow)

Defects in TE formation

Papaioannou and Gardner (1979) J. Embryol. Exp. Morphol. 52, 153

Evx1

Defects in TE formation

Spyropoulos and Capecchi (1994) Genes Dev. 8, 1949

Eomes (Eomesodermin)

Defects in TE formation

Russ et al. (2000) Nature 404, 95

Cdx2

Defects in TE formation

Chawengsaksophak et al. (1997) Nature 386, 84

Tead4

Defects in TE formation

Yagi et al. (2007) Development 134, 3827; Nishioka et al. (2008) Mech. Dev. 125, 270

Arp3

Defects in TE formation

Vauti et al. (2007) FEBS Lett. 581, 5691

Egfr

Defects in ICM formation

Threadgill et al. (1995) Science 269, (background dependent) 230

b1 integrin

Defects in ICM formation

Stephens et al. (1995) Genes Dev. 9, 1883; Fa¨ssler and Meyer (1995) Genes Dev. 9, 1896

Lamc1

Defects in ICM formation

Smyth et al. (1999) J. Cell Biol. 144, 151

B-myb

Defects in ICM formation

Tanaka et al. (1999) J. Biol. Chem. 274, 28067

Fgf4

Defects in ICM formation

Feldman et al. (1995) Science 267, 246

Fgfr2

Defects in ICM formation

Arman et al. (1998) PNAS 95, 5082

Taube Nuss (Tbn)

Defects in ICM formation

Voss et al. (2000) Development 127,5449

Oct4 (Oct3, Pou5f1)

Defects in ICM formation

Nichols et al. (1998) Cell 95, 379

Nanog

Defects in ICM formation

Mitsui et al. (2003) Cell. 113, 631; Chambers et al. (2003) Cell 113, 643

Eset

Defects in ICM formation

Dodge et al. (2004) Mol. Cell Biol. 24, 2478

Ronin

Defects in ICM formation

Dejosez et al. (2008) Cell 133, 1162

Sall4

Defects in ICM formation

Elling et al. (2006) PNAS 103, 16319

Grb2

Defects in ICM formation

Chazaud et al. (2006) Dev. Cell 10, 615

The table is divided in two sections. The top includes genes and loci that, when deleted, cause embryonic lethality before implantation. The lower section includes genes and loci that, when deleted, cause embryonic lethality during implantation but before the formation of the egg cylinder. Embryos deficient in most of these genes develop to normal blastocysts and are able to hatch and implant, but the whole embryo or selectively the TE or ICM (ICM- or primitive endoderm-derived cells) degenerates soon thereafter, leading to resorption. ICM, inner cell mass; TE, trophectoderm.

mesoderm formation (Niwa et al., 2000). Mouse embryos deficient in Oct4 are unable to form mature ICM and die around the time of implantation (Nichols et al., 1998). Other genes described as being involved in cell fate determination during preimplantation development include Taube nuss, B-myb, Nanog, Cdx2, and Eomes (see Table 14.2). Both Taube nuss and B-myb homozygous-deficient mice develop to normal blastocysts. At the time of implantation, however, Taube nuss / ICM cells undergo massive apoptosis and the embryo becomes a ball of

trophoblast cells (Voss et al., 2000); in B-myb knockout mice, the ICM also degenerates although the reason for this is unclear (Tanaka et al., 1999). Taube nuss and B-myb seem to be necessary for ICM survival, whereas Oct4 is required for establishment and maintenance of the ICM identity but not cell survival. Nanog is expressed exclusively in the ICM and, whereas Oct4 prevents TE differentiation, Nanog prevents differentiation of ICM to primitive endoderm. In agreement, Nanog / blastocysts are formed but derivative ICM in culture differentiates into

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TABLE 14.3 Maternal Effect Mutations Affecting Preimplantation Embryonic Development Gene/Locus

Mutant phenotype

References

Zar1

Zygote arrest

Wu et al. (2003) Nat. Genet. 33, 187

Dicer

Zygote arrest

Tang et al. (2007) Genes Dev. 21, 644

Brwd1

Zygote arrest

Philipps et al. (2008) Dev. Biol. 317, 72

Hsf1

Zygote to 2-cell stage arrest

Christians et al. (2000) Nature 407, 693

Ago2

Zygote to 2-cell stage arrest

Kaneda et al. (2009) Epigen. Chrom. 2, 9

Npm2

Zygote to 2-cell stage arrest

Burns et al. (2003) Science 300, 633

Mater (Nalp5)

2-cell stage arrest

Tong et al. (2000) Nat. Genet. 26, 267

Hr6a (Ube2a)

2-cell stage arrest

Roest et al. (2004) Mol. Cell Biol. 24, 5485

E-cadherin (uvomorulin, Cdh1)

2-cell stage arrest

Kanzler et al. (2003) Mech. Dev. 120, 1423

Padi6

2-cell stage arrest

Yurttas et al. (2008) Development 135, 2627

Floped (Ooep)

2-cell stage arrest

Li et al. (2008) Dev. Cell 15, 416

Basonuclin (Bnc1)

2-cell stage arrest

Ma et al. (2006) Development 133, 2053

Pdk1 (Pdpk1, Pkb kinase)

2-cell stage arrest

Zheng et al. (2010) EMBO Rep. 11, 890

Zfp36l2

2-cell stage arrest

Ramos et al. (2004) Development 131, 4883

Importin a7

2-cell stage arrest

Rother et al. (2011) PLoS One 6, e18310

Brg1

2- to 4-cell stage arrest

Bultman et al. (2006) Genes Dev. 20, 1744

Tcl1

4- to 8-cell stage arrest

Narducci et al. (2002) PNAS 99, 11712

Atg5

4- to 8-cell stage arrest

Tsukamoto et al. (2008) Science 321, 117

Dppa3 (Stella, PGC7)

Failure to form blastocysts

Payer et al. (2003) Curr. Biol. 13, 2110; Bortvin et al. (2004) BMC Dev. Biol. 4, 2

Uchl1

Defects in compaction

Mtango et al. (2012) J. Cell Physiol. 227, 2022

CTCF

Failure to form blastocysts

Wan et al. (2008) Development 135, 2729

The table lists a growing number of maternal-effect genes. Embryos lacking these genes develop normally at least until implantation/gastrulation in heterozygous, but not in homozygous, mothers. If the gene of interest is homozygous lethal, the deletion of the maternal pool of transcripts is achieved by the conditional deletion of the gene by crossing mice carrying a “floxed” allel (fl/fl or fl/e) with transgenic mice expressing a zona pellucida 3 (ZP3) promotermediated CRE recombinase [de Vries et al. (2000) Genesis 26, 110], which deletes the gene specifically in growing oocytes.

FIGURE 14.3 Oct4 expression at morula and blastocyst stages. GFP expression driven by distal elements of the Oct4 promoter was used here to mimic endogenous Oct4 expression. In the morula, all blastomeres express high levels of Oct4 (A). In the early blastocyst, the inner cell mass expresses high levels of Oct4, whereas weaker expression is observed in trophectoderm cells (B).

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endoderm (Chambers et al., 2003; Mitsui et al., 2003). In contrast, Cdx2 and Eomes are involved in trophoblast development; embryos lacking these genes die soon after implantation because of defects in the trophoblast lineage (Chawengsaksophak et al., 1992; Russ et al., 2000).

FROM IMPLANTATION TO GASTRULATION The mechanisms used by the mammalian embryo to implant are species dependent, contrasting with the general developmental steps during the preimplantation period. In addition, an intimate and highly regulated cross-talk between mother and embryo makes implantation in mammals a complex process. Upon reaching the uterus, the blastocyst hatches from the zona pellucida and the TE cells become adhesive, expressing integrins that enable the embryo to bind the extracellular matrix (ECM) of the uterine wall (Wang and Armant, 2002). The mouse embryo adheres to the uterine wall via the mural TE cells of the abembryonic region and is slightly tilted (Smith, 1980). In contrast, human embryos bind through the embryonic region. Once attached to the uterus, trophoblast cells secrete enzymes that digest the ECM (Brenner et al., 1989; Strickland et al., 1976), allowing them to infiltrate and start uterine invasion. At the same time, the uterine tissues surrounding the embryo undergo a series of changes collectively known as the decidual response. These include the formation of a spongy structure known as deciduum in mice (decidua in humans), vascular changes leading to the recruitment of inflammatory and endothelial cells to the implantation site, and apoptosis of the uterine epithelium (Cross et al., 1994).

(A)

(B)

(C)

The Mouse Trophectoderm and Primitive Endoderm Cells Apoptosis occurring in the uterine wall gives TE cells the opportunity to invade the deciduum by phagocytosing dead epithelial cells. At about E5.0, the mural TE cells cease division but continue endoreduplicating their DNA to become primary trophoblastic giant cells (Varmuza et al., 1988). This cell population is joined by polar TE cells that migrate around the embryo and similarly become polytene (secondary trophoblastic giant cells). However, other polar TE cells continue dividing and remain diploid, giving rise to the ectoplacental cone and the extraembryonic ectoderm that pushes the ICM into the blastocoelic cavity (Figure 14.4). These proliferative TE cells when cultured in the presence of fibroblast growth factor 4 (FGF4) and heparin give rise to the so-called trophoblast stem (TS) cells, which are able to either self-renew or differentiate into trophoblastic giant cells (Tanaka et al., 1998). During implantation, the primitive endoderm layer forms two subpopulations: the visceral endoderm (VE) and the parietal endoderm (PE), both of which are extraembryonic tissues (Gardner, 1982). The VE is a polarized epithelium closely associated with the extraembryonic ectoderm and the ICM/epiblast (Figures 14.4, 14.5) which is heterogeneous in character (with prominent vacuolization in the extraembryonic VE). When in culture, the primitive/visceral endoderm is also able to give rise to a self-renewing stem cell population of extraembryonic endoderm (XEN) cells (Kunath et al., 2005). Later in development, the VE contributes to the formation of the visceral yolk sac but some VE cells may end up intercalated in the definitive gut (Kwon et al., 2008). PE cells migrate largely as individual cells over the TE (Figures 14.4, 14.5) and secrete large amounts of ECM to form a thick

FIGURE 14.4 Tissue formation and movements during and shortly after implantation of the mouse embryo (E5.0eE5.5). During implantation, cell division rate in the embryo increases, leading to rapid growth (AeC). The primitive endoderm cells segregate into visceral endoderm (VE) and parietal endoderm (PE). The polar trophectoderm cells (pTE) form the ectoplacental cone (ec) and the extraembryonic ectoderm (ex). pTE cells together with mural trophectoderm cells (mTE) contribute to form the trophoblastic giant cells (TGC). The inner cell mass (ICM) cavitates and organizes into an epithelium known as the epiblast (e).

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FIGURE 14.5 Tissue formation and movements in the pregastrulation mouse embryo (E5.5eE6.0). During this period, the extraembryonic ectoderm organizes into an epithelium. The proamniotic cavity initially restricted to the epiblast now expands into the extraembryonic ectoderm, forming the proamniotic canal. At E5.5, the most distal visceral endoderm cells (red) express a different set of markers then the surrounding visceral endoderm (VE). These (or other) distal VE cells move from the distal tip to surround the prospective anterior part of the epiblast and form the anterior visceral endoderm (AVE). The VE surrounding the extraembryonic ectoderm consists of a columnar epithelium, whereas the VE cells surrounding the epiblast are more flattened.

basement membrane known as Reichert’s membrane. The PE cells together with the trophoblastic giant cells and Reichert’s membrane form the parietal yolk sac.

Development of the Mouse Inner Cell Mass to the Epiblast The ICM located between the TE-derived extraembryonic ectoderm and the primitive endoderm-derived VE gives rise to all cells of the embryo proper. During implantation, the ICM organizes into a pseudostratified columnar epithelium (also referred to as primitive or embryonic ectoderm, epiblast, or egg cylinder) surrounding a central cavity, the proamniotic cavity (Figure 14.4). Signals from the VE (and perhaps also from the extraembryonic ectoderm), including bone morphogenetic proteins (BMPs), are responsible for apoptosis in the core of the epiblast leading to its cavitation (Coucouvanis and Martin, 1995, 1999). Between E5.5 and E6.0, the proamniotic cavity expands to the extraembryonic ectoderm, forming the proamniotic canal (Figure 14.5). After implantation, a wave of de novo DNA methylation occurs, leading to epigenetic reprogramming (finished by E6.5). This affects the entire genome to a different extent in embryonic and extraembryonic lineages (Reik et al., 2001) and may be responsible for the observed loss of the ability to contribute to chimeras. After implantation, the rate of cell division increases, followed by rapid growth. At E4.5, the ICM consists of approximately 20 to 25 cells, at E5.5 the epiblast has about 120 cells, and at E6.5 it consists of 660 cells (Snow, 1977). At E6.5, the embryo is not cylindrical but has already a long and a short axis. Gastrulation starts with the

formation of a morphologically visible structure (the primitive streak) that marks the future posterior side of the embryo. Surprisingly, the primitive streak forms at one side of the short axis. However, the embryo undergoes an apparent shift in orientation inside the deciduum and the primitive streak ends up at one side of the long axis (Mesnard et al., 2004; Perea-Gomez et al., 2004). In addition, during gastrulation the three definitive germ layers are formed, the germ line is set aside, and the extraembryonic mesoderm that contributes to the visceral yolk sac, placenta, and umbilical cord is generated. An overview of tissue formation and movement during mouse gastrulation is shown in Figure 14.6.

The Human Embryo Human development during implantation and gastrulation is significantly different from that of the mouse. Briefly, the human trophoblast cells invade the uterine tissue and form the syncytiotrophoblast, a syncytial tissue. The trophoblast cells that contact the ICM and the blastocoelic cavity stay as single cells, remain diploid, and are known as cytotrophoblasts. These cells proliferate and can fuse with the syncytiotrophoblast or develop into the column cytotrophoblast or the (to some extent polyploid) extravillous cytotrophoblast. In humans, a structure equivalent to the mouse extraembryonic ectoderm is not thought to form. Human primitive endoderm cells, also known as hypoblast cells, segregate on the surface of the ICM and proliferate. Some of these cells migrate to line the blastocoelic cavity leading to the formation of the exocoelomic membrane (or Heuser’s membrane). Analogous to the formation of the mouse Reichert’s membrane, a spongy layer of acellular material known as the extraembryonic reticulum is formed

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FIGURE 14.6 Tissue formation and movements during the gastrulation of the mouse embryo (E6.5eE7.5). Gastrulation begins with the formation of the primitive streak (ps) in the posterior side of the E6.5 embryo at the junction of the extraembryonic ectoderm (ex) and epiblast (e) (A). As more cells ingress through the streak, it elongates toward the distal tip of the embryo, between epiblast and visceral endoderm (VE) (B). While the newly formed embryonic mesoderm (m) moves distally and laterally to surround the whole epiblast, the extraembryonic mesoderm (xm) pushes the extraembryonic ectoderm upwards and to the center (C, D). The extraembryonic mesoderm develops lacunae, creating a mesoderm-lined cavity known as exocoelom (exo). The exocoelom enlarges and as a consequence, the tissue at the border of extraembryonic and embryonic ectoderm fuses, dividing the pro-amniotic cavity (ac) in two and forming the amnion (am) and the chorion (ch) (E). The layer of extraembryonic mesoderm and the visceral endoderm together form the visceral yolk sac (vys). At the posterior side of the embryo the allantois (al) and the primordial germ cells are formed (E, F). The tissues colored green (the extraembryonic ectoderm and ectoplacental cone (ec)) are derived from the trophectoderm. The tissues in yellow are derived from the primitive endoderm and epiblast cells that passed through the streak, generating the definitive endoderm. The definitive endoderm cells intercalate with the visceral endoderm in the embryonic part of the embryo. The tissues colored orange are derived from the inner cell mass and remain ectoderm. The tissues colored blue are formed during gastrulation and represent primitive streak and mesoderm-derived tissues (excluding the primordial germ cells present at the basis of the allantois). For the lineages of early mouse development see Figure 14.2.

between the cytotrophoblast and the exocoelomic membrane. Thereafter, the extraembryonic reticulum is invaded by extraembryonic mesoderm. The origin of this tissue in humans is still uncertain (epiblast derived or hypoblast derived). The extraembryonic mesoderm proliferates to line both Heuser’s membrane (forming the primary yolk sac) and cytotrophoblast (forming the chorion). The extraembryonic reticulum then breaks down and is replaced by a fluid-filled cavity, the chorionic cavity. The human primary yolk sac is thus not equivalent to the mouse parietal yolk sac, although both are transient structures. Moreover, it is still unclear whether human embryos develop a PE-like cell type. A new wave of hypoblast proliferation generates cells that contribute to the formation of the definitive yolk sac. This new structure displaces the primary yolk sac, which buds off and breaks up into small vesicles that remain present in the abembryonic pole. The definitive yolk sac in humans is equivalent to the visceral yolk sac in the mouse. The human ICM organizes into a pseudostratified columnar epithelium and cavitates, producing the amniotic cavity. The ICM cells that lie on the hypoblast are known as the epiblast and will give rise to the embryo proper. The ICM cells that contact the trophoblast form the amnion. The human embryo forms a bilaminar embryonic disc, similar the chick embryo and the patterns of cell movement

during gastrulation are relatively conserved between chick and human. With such diversity in extraembryonic structures supporting the development of the ICM in mice and humans, it is not surprising that ES cells derived from mice and humans are not equivalent. They differ in developmental potency, for example, in their ability to differentiate to TElike cells. Human ES cells can form TE in culture (Xu et al., 2002) but under normal circumstances mouse ES cells do not (Beddington and Robertson, 1989). Furthermore, mouse ES cells in culture have been shown to develop into cells with some properties of mature germ cells (sperm- and oocyte-like cells) (Chuva de Sousa Lopes and Roelen, 2010). The potential of these cells to fertilize or to be fertilized and generate viable mice is still unclear. It is not yet known whether human ES cells have the potential to form mature gamete-like cells in culture. Mouse and human ES cells also express different cell surface markers, have different requirements in culture for self-renewal and respond differently to growth and differentiation cues (Kuijk et al., 2011; Pera et al., 2000) although their expression profile of core pluripotency genes is similar. Recent literature has shown that mouse ES cells are a heterogeneous population in terms of expression of markers, containing cells that resemble either ICM or

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epiblast cells (Hayashi et al., 2008). Mouse ES cells that are most related to the epiblast cells resemble mouse EpiSCs. In contrast to ICM-like mouse ES cells, EpiSCs and human ES cells have similar characteristics (Brons et al., 2007; Tesar et al., 2007). Current thought suggests that the unusual characteristics of mouse ES cells arise from the fact that the mouse uses a reproductive strategy known as facultative embryonic diapause. This means that the mouse blastocyst has the capacity to wait (temporarily arrested in development) in the uterus until the conditions for implantation become favorable (for example, the embryos wait until the mother stops lactating). The mouse ES cells would reflect this “arrested” stage in culture. In humans and most other mammals, the blastocyst is not able to arrest its development and when in the uterus it either implants and develops or degenerates.

Implantation: Maternal versus Embryonic Factors In mice, the presence of the blastocyst in the uterus is sufficient to trigger ovarian production of progesterone and estrogen. These two hormones are absolutely required for embryo survival because they prime the uterus for implantation and decidualization. The uterus starts producing LIF and members of the epidermal growth factor (EGF) family, including EGF, heparin-binding EGF, transforming growth factor-alpha (TGFalpha), and amphiregulin. Those molecules, together with HoxA10, induce the production of cyclo-oxygenase (COX) enzymes, the rate-limiting enzymes in the production of prostaglandins. These factors and corresponding receptors play crucial roles during the “window of implantation” and when genetically deleted or mutated lead to female infertility due to a defective uterine response and embryonic lethality during or soon after implantation (Paria et al., 2001, 2002; Rashid et al., 2011). The embryo, on the other hand, also produces important molecules including interleukin-1b, TGFa, and insulin growth factor (IGF) that act in autocrine and paracrine ways to stimulate embryoe uterine cross-talk leading to implantation (Hardy and Spanos, 2002). The suppression of the maternal immune response is also essential during implantation but is still incompletely understood. TE cells, the only cell population of the conceptus that physically contacts maternal cells, have developed several mechanisms to avoid rejection (Cross et al., 1994; Mor et al., 2011). Examples are the production of numerous factors and enzymes, including indoleamine 2,3-dioxygenase (IDO) (Munn et al., 1998) by the TE cells that suppress the maternal immune system and the lack of polymorphic class I and II major histocompatibility complex (MHC) antigens in TE cells.

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The Role of Extraembryonic Tissues in Patterning the Mouse Embryo Extraembryonic tissues not only are necessary for nutrition and regulating implantation during development, but also play crucial roles in patterning the embryo before and during gastrulation (Tam and Loebel, 2007). Unequivocal evidence for this comes from the analysis of chimeric embryos generated from blastocysts colonized with ES cells (Beddington and Robertson, 1989). In chimeras, ES cells preferentially colonize epiblast-derived tissues. It is, therefore, possible to generate embryos with extraembryonic tissues of one genotype and epiblast-derived tissues of another genotype. For example, Nodal is expressed embryonically and extraembryonically (depending on the developmental stage). Furthermore, Nodal-deficient embryos fail to gastrulate (Conlon et al., 1991, 1994). It was, thus, initially difficult to distinguish embryonic from extraembryonic functions. However, when Nodal / ES cells were introduced into wild-type blastocysts, the extraembryonic tissues were wild type, whereas epiblastderived tissue lacked Nodal. The developing chimera was essentially normal until mid-gestation, suggesting that the presence of Nodal (exclusively) in the extraembryonic tissues was sufficient to rescue embryonic patterning (Varlet et al., 1997). In contrast to the extensive mixing of epiblast cells, labeled primitive endoderm cells develop as more coherent clones, consistent with the function of the VE in embryo patterning. The primitive endoderm cells in the vicinity of the second polar body preferentially form VE cells surrounding the epiblast, whereas cells away from the second polar body preferentially form VE cells surrounding the extraembryonic ectoderm (Weber et al., 1999). At E5.5, the most distal VE (DVE) cells are characterized by the expression of the genes Hex and Lefty1 (Thomas et al., 1998; Yamamoto et al., 2004). Until recently it was thought that this cell population migrated towards the prospective anterior side of the embryo during the next day of development, producing an endodermal stripe known as the anterior visceral endoderm (AVE) (Figure 14.5). However, recent data proposes that the migratory AVE may not directly descend from DVE cells, but constitute a newly formed population (Takaoka et al., 2011). The AVE is responsible for the production of innumerous secreted signaling molecules. Of particular interest is the production of antagonists of the Nodal (Lefty1 and Cer1) and the Wnt (Dkk1) signaling pathways, which play important roles in the specification of anterior fate in the embryo (Tam et al., 2006). There is less known about specific gene products produced by the posterior part of the VE (PVE), but expression of Wnt3, Wnt2b, and BMP2 by the PVE is important for posterior embryonic

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patterning and development (Kemp et al., 2005; RiveraPerez and Magnuson, 2005; Ying and Zhao, 2001). Before gastrulation, the extraembryonic ectoderm also signals to the proximal epiblast, inducing expression of several genes important for posterior proximal identity, in particular via BMP4 and BMP8b (Lawson et al., 1999; Ying et al., 2000). By controlling the levels of activity of the Wnt and Nodal/ BMP signaling pathways, the extraembryonic tissues VE and extraembryonic ectoderm determine both anterior and posterior fate in the embryo. The same two signaling pathways will also play further roles in dorsaleventral patterning and organogenesis. Both VE and VE-like cell lines secrete signals that are able to induce differentiation of mouse and human ES cells at least towards cardiomyocytes (Mummery et al., 2003; Nijmeijer et al., 2009). Making use of the tissues or sequences of signal transduction pathways used by the embryo for its own patterning and differentiation seems to be the most efficient way to direct ES cell differentiation and therefore it is paramount to understand the events that take place early during embryonic development to define differentiation signals more precisely.

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Differentiation in Early Development

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