[~EVIEWS 30 Van Laere, A.J. and Rivero, F. (1986) Arch. Microbiol.
136, 78-85
145, 290-294
37 Bergman, K., Eslava, A.P. and Cerd~i-Olmedo, E. (1973) Mol. Gen. Genet. 123, 1-16 38 Medina, J.R. and Cerdfi-Olmedo, E. (1977) Exp. Mycol. 1,
31 Rivero, F. and Cerd:i-Olmedo, E. (1987) Mol. Gen. Genet.
209, 149-153 32 Ruiz-Herrera, J. and Catvo-Mendez, C. (1987) Exp. Mycol. 33 34 35 36
286-292
11, 287-296 Martinez-Pacheco, M. et al. (1989) Arch. Microbiol. 151, 10-14 Galland, P. and Senger, H. (1988) Photochem. Photobiol. 48, 811-820 Gatland, P. and Senger, H. (1988)J. Photochem. Photobiol. B1,277-294 Weiss, J. and Weisenseel, M.H. (1990) J. Plant Physiol.
39 Lipson, E.D. and Terasaka, D.T. (1981) Exp. Mycol. 5,
101-111
L.M.
CORROCHANO AND E.
CERDlf-OLMEDO
ARE IN
THE
DEPA~TAMENTO DE GEN~TlCal, UmVERSlOAD DE SEVILL~ APARTADO 1095, E-41080 SEVILLa, SPAIN.
During development, the diversity of cell types found in the adult organism is generated through intensive cell divisions and differentiation. Differentiation can follow two major pathways: (1) Cytoplasmic determination: Determinative components are asymmetrically distributed within the cytoplasm; these components are inherited by only one of the two daughter cells after cleavage. Such asymmetries exist in higher organisms such as frogs, but are particularly apparent in the mosaic eggs of molluscs, ascidians and nematodes. For example, germ-line granules in the nematode Caenorhabditis elegans are redistributed in the zygote as a result of cytoplasmic movement, involving or driven by microfilaments, and after a series of divisions become restricted to the cells giving rise to the gametes 1. (2) Embryonic induction: The fate of inducible cells can be changed under the influence of signals emitted by other cells, often a long distance away. A well-studied example is mesoderm induction in Xenop u s laevis: cells from the animal pole of the blastula (which normally form the ectoderm) form mesodermal tissues when cultured with cells isolated from the vegetal pole 2. During early mouse development, a cytoplasmic segregation takes place that differs from the type described above, since asymmetries are set up in the cytoplasm by a more subtle reorganization of cytoplasmic and cortical networks, rather than by a redistribution of determinative molecules. This process is controlled by both cell interactions and intrinsic cellular mechanisms.
Cell polarity and cell diversification during early mouse embry0genesis CATHERINEGUETH-HALLONETAND BERNARDMARO At the eight-cell stage of mouse development, the organization of blastomeres changes from radially symmetrical to polarized This acquisition of cell polarity, followed by asymmetric divisions, leads to the formation of two phenotypically different cell types, which give rise to the first two cell lineages of the mouse blastocyst embryo, trophectoderm and the inner cell mass. Cellfate, controlled by positional information, is not irreversibly fixed during differentiation, providing the embryo with considerable developmental flexibility.
Early mouse development In mammals, the fertilized egg gives rise not only to the embryo but also to extraembryonic structures. These are elaborated during early development to allow the blastocyst-stage embryo to implant in the uterus. Thus, during the first four days of development, the blastocyst forms and the first two cell types are established: the inner cell mass (ICM), which will mainly give rise to the embryo proper, and the trophectoderm, which will give rise to many extraembryonic structures (Fig. 1). In ,the mouse, the embryonic genome is activated during the two-cell stage, much earlier than in most other organisms. The initial cleavage divisions are equal, asynchronous and without any particular orientation. The first signs of asymmetries
appear only at the eight-cell stage and they correspond to a surface and cytoskeletal reorganization referred to as polarization. The following cell divisions, between the fourth and the sixth cleavages, can be asymmetrical and give rise to two different cell populations3: the outer cells of the morula (16- and 32cell stages), which will give rise mainly to the trophectoderm, and the inner cells of the morula, which will mainly give rise to the ICM of the blastocyst (beginning at the 64-cell stage).
Totipotency and flexibility Tarkowski and Wroblewska showed 25 years ago that individual blastomeres at the eight-cell stage can still form both trophectoderm and the ICM4. This totipotency can be correlated with the fact that, at the beginning of the eight-cell stage, blastomeres are phenotypically (biochemically and morphologically) equivalent. But this totipotency persists at later stages, when the cells are phenotypically different. This provides the embryo with developmental flexibility within lineages during early development. One factor that could regulate the fate of a blastomere is the position of the cell within the embryo since, in experiments where two cleavage-stage embryos were aggregated, cells differentiated according to their new position, irrespective of their original location in the embryo: those positioned on the outside of the chimera
T16 AU6UST1992 rOE 8 NO. 8
m
]REVIEWS
FIGH Preimplantation mouse embryos. (A) Noncompacted early eight-cell embryo. The blastomeres are spherical and clearly visible. (B) Compacted late eight-cell embryo. The flattened blastomeres can no longer be distinguished. (C) Fully expanded blastocyst in phase contrast. Mouse embryo blastomeres have already given rise to two differentiated tissues: the trophectoderm (t) and the inner cell mass (icm). A voluminous cavity, the blastocoel (bl), has developed after cavitation of the embryo at the 32 cell stage. (D) Microtubule distribution in a compacted eight-cell embryo examined by confocal microscopy. Tubulin staining reveals the apical cytoplasmic poles• became trophectoderm cells, while those inside became ICM cells, as shown by the use of genetic markers 5. Such regulation allows aggregates of 16 outer or 16 inner cells to form apparently normal blastoc~sts 6. The influence of position on cell fate has also been demonstrated using single blastomeres: when moved to a new position in the morula, they differentiate according to this new position 7. More recently, flexibility has also been demonstrated in situ by following the fate of outside cells in morulae8: the ICM is derived from inside cells at the 16-cell stage, supplemented by occasional cells derived from outer cells during the fifth and sixth cleavages. It is only in fully expanded blastocysts (after the 64-cell stage) that ICM and trophectoderm cells cannot cross lineages. Flexibility is also evident in more quantitative aspects of development: for example, there is a great variability in the number of outer and inner cells at the 16-cell stage, but the relative proportion of these two cell populations is maintained within a narrow range after the fifth cleavage. Such variability at the 16-cell stage suggests that the number of asymmetrical divisions after polarization at the eight-cell stage does not determine the number of cells within the two different populations and that some cell lineages must therefore cross from one population to the other. All these observations underline the importance of understanding the cellular mechanisms (polarization and asymmetrical cell divisions) that precede the emergence of the two first cell populations of the embryo.
Setting up of cellular asymmetries during compaction Compaction The blastomeres of early eight-cell embryos are spherical, evenly covered with microvilli and can easily be dissociated, because cellular interactions are weak. During the eight-cell stage, cells start to flatten upon each other so that their boundaries can no longer be seen with an optical microscope, six or seven hours after the third cleavage (Fig. 1A, B). The major molecule involved in this cell flattening is the calciumdependent cell adhesion molecule uvomorulin (also called L-CAM or E-cadherin), as shown by experiments in which embryos cultured in the presence of antibodies against uvomorulin develop as grape-like structures 9. During flattening, uvomorulin is progressively
removed from the apical membrane domain of the blastomeres and redistributes predominantly in areas of cell--cell contact 1°,11. At the 16-cell stage, uw)morulin remains evenly distributed on the surface of inner blastomeres and is strictly basolateral on outer blastomeres. Simultaneously with this flattening process, cytoplasmic and surface components become asymmetrically distributed (or polarized), allowing the first distinction to be made between the basolateral and apical domains of the blastomeres. First, gap junctions assemble in the basal domain, and a cytoplasmic pole of endosomes, clathrin vesicles and cytoskeletal elements (microfilaments and microtubules) forms in the apical domain 12. In particular, the distribution of microtubules changes progressively during compaction: in early eight-cell blastomeres, microtubules are found mainly around the nucleus and in the cell cortex, whereas a few hours later many have disappeared from the area next to the zone of intercellular contact and have accumulated in the apical part of the cells (Fig. 1D) t3. In contrast, a subpopulation of more stable, acetylated microtubules becomes enriched in the basal part of the cell cortex during compaction 14. This cytoplasmic reorganization is associated with the apical redistribution of certain membrane proteins, such as the Na+-glucose cotransport system ~5. Second, the surrounding microvilli, consisting mainly of microfilaments, become restricted to the apical surface of the blastomere 16. Thus, at the end of the eight-cell stage, many surface and cytoplasmic components have changed their organization and become polarized rather than homogeneously distributed. Both processes - flattening and polarization - are referred to as compaction3.~e.lL Involvement of intercellular contacts Uvomorulin also seems to be involved in polarization, since an anti-uvomorulin antibody delays polarization and perturbs the axis of polarization, which is normally perpendicular to the plane of contact with adjacent cells (Fig. 2a) 1°. The molecular mechanism of cell adhesion through homophilic interactions of uw> morulin is beginning to be understood. However, the link between adhesion on the cell surface and the cytoplasmic triggering of compaction is not yet clear.
TIG AUGUST1992 VOL.8 NO. 8
m
~]~EVIEWS
S S'°J
No microtubules(~
No adhesion
(~
No microtubules No adhesion ( ~ time
4-
+
+
-I-
-
+
-
+
+
-
+ + ( if the nucleus is peripheral)
i,
P
FIG~ Alternative pathways for polarization. For clarity, these events are illustrated by an isolated pair of eight-cell stage blastomeres. Surface microvilli are shown as a black border, while cytoplasmic organelles are shown in light grey and the nucleus in dark grey. (a) General pathway mediated by intercellular adhesion: cytoplasmic and surface polarities develop opposite the area of cell contact. (b) With experimental disruption of microtubules: cytoplasmic polarity is inhibited. (c) Alternative pathway involving intrinsic mechanisms: cell polarity can develop in the absence of intercellular contacts in cells in which the nucleus is located near the cell surface. (d) With experimental disruption of microtubules and inhibition of adhesion: both cytoplasmic and surface polarity are inhibited. Many modifications in cell organization that occur during compaction seem to be regulated by changes in previously synthesized proteins: for example, uvomorulin is already present in eggs and cleaving embryos. In addition, some elements of compaction can occur or even be advanced when protein synthesis is inhibited as early as the late two-cell or early four-cell stage TM. An initiation signal for compaction acting at the post-translational level would allow compaction to proceed by removal of a prior inhibition. Some changes in protein phosphorylation associated specifically with passage through the eight-cell stage may have a role in compaction 19. Several levels of control of compaction by contact-directed mechanisms have been investigated. (1) Protein kinase C (PKC): Phorbol esters and diacylglycerides cause premature compaction of four-cell embryos, and ECCD-1, a monoclonal antibody directed against uvomorulin, completely blocks this induced compaction 2°,21. This suggests that PKC plays a role in the initiation of compaction through its effect on cell adhesion. Activation of PKC also affects the cytoskeleton, since it induces loss of microfilaments (both in microvilli and in the cortex) and cytoplasmic microtubules from compacted .eight-cell embryos. However, the mechanism underlying .PKC activation in the undisturbed eight-cell embryo is not clearly understood and a possible link between uvomorulin and activation of PKC remains to be determined.
(2) G proteins: Some of these proteins, normally involved in signal transduction, have been identified in the preimplantation mouse embryo; however, they may not be relevant to compaction, since interfering with their function with pertussis toxin has no effect on the development of a two-cell embryo to the blastocyst stage 22. (3) Others': Molecules possibly involved in direct transduction of the adhesion signal have n o w been characterized in other systems (Madin-Darby canine kidney cells, Xenopus laevis A6 cells and transfected mouse fibroblasts growing as monolayers): three proteins, catenins a, b and c, connect uvomorulin to actin filaments of the cytoskeleton, and this cytoplasmic anchorage is necessary for the adhesive function of uvomorulin 2-'-25. Uvomorulin can also be associated with membrane-cytoskeleton complexes containing ankyrin and fodrin in epithelial cells 26. Such complexes could help to set up basolateral membrane polarity. However, the link between these proteins and uvomorulin has not yet been investigated in the preimplantation mouse embryo.
Involvement of the cytoskeleton Microfilaments appear to be directly involved in compaction, since cytochalasin D (a drag that blocks microfilament polymerization) inhibits and reverses cell flattening. Cytochalasin D also affects the movement of the nucleus towards the domain of intercellular contact,
TIG AUGUST 1 9 9 2 VOL. 8 NO. 8
W
[~EVIEWS Spindle orientation
Division
Orthogonal
Symmetrical
@P°lafisati°na~(~)x~' ~
Phenotype Polar / Polar
Flatening Envelopment
8-cell stage
Mitosis
16-cell stage
FIGR] Polarization and asymmetric cell division. For clarity, these:events are illustrated by an isolated blastomere from the eight-cell stage. Surface microvilli are shown as a black border, while cytoplasmic organelles are shown in light grey. Cellular asymmetries are set up during compaction at the eight-cell stage. Cytoplasmic polarity is lost during mitosis but restored at the 16-cell stage, l)epending on the position of the spindle with respect to the axis of polarity (defined by the respective positions of the nucleus and of the surface and cytoplasmic poles), two different types of divisions can occur. (a) Symmetrical division: when the spindle is orthogonal to the axis of polarity, both 16-cell stage daughter cells inherit a polarized phenotype and the division is called conservative. After division. the two polarized 16-cell blastomeres flatten on each other. (b) Asymmetrical division: when the spindle is parallel to the axis of polarity, the two 16-cell stage daughter cells have a different phenotype and the division is called differentiative. After division, the polarized 16-cell blastomere tends to surround the nonpolarized blastomere. which normally occurs during polarization. When applied during compaction, cytochalasin D inhibits the formation of surface poles. However, once a surface pole of microvilli has formed, it persists even when the polar distribution of cytoplasmic microfilaments is 1ost27,28. The use of microtubule-inhibiting drugs, which also inhibit cytoplasmic polarity, confirms this possible dissociation of surface and cytoplasmic polarity 28. Since disruption of microtubules accelerates intercellular flattening and surface polarization, while stabilization of microtubules prevents and reverses both these processes, microtubules might exercise a constraining role during compaction 29 (Fig. 2a, b). The possible involvement of the microtubule network during compaction can be summarized in the following model: the reduction of the number of microtubules in the basal domain of the blastomeres facilitates formation of gap junctions and loss of microvilli in the basolateral domain, whereas it accelerates intercellular flattening. In contrast, the redistribution of microtubules in the apical domain would direct the movement of cytoplasmic components to this domain, and these apical microtubules would stabilize the newly formed poles of organelles and microvilli. Intracellular reorganization of the microtubule network can induce polarization of blastomeres at the eight-cell stage in the absence of intercellular contacts or when flattening is inhibited. Indeed, if the nucleus is near the cell surface (as it often is just after mitosis), a network of microtubules associated with cytoplasmic organelles forms between the cell surface and the nucleus, establishing a cytoplasmic pole 3°. These
microtubules are then necessary for the formation of a surface pole of microvilli, which is located just over the nucleus (Fig. 2a, c, d). So mechanisms intrinsic to the cell seem sufficient to set up asymmetries in the blastomeres in the absence of cell contact.
Asymmetric divisions at the 16-cell stage During the fourth cleavage, cytoplasmic polarity is lost, since the blastomeres entering mitosis locally decompact, gap junctions switch off" and their interphase network of microtubules disassembles to be replaced by the mitotic spindle (Fig. 3). In contrast, the polarized organization of the cell surface is retained during mitosis. However, when cells are blocked in mitosis for many hours by nocodazole, surface poles tend to spread over the whole blastomere surface, showing that surface polarization is also unstable, although stable enough to survive throughout mitosis (which normally lasts one hour) 31. Nonetheless, some elements of polarity established at the eight-cell stage are conserved during the fourth cleavage, since cytoplasmic polarity is restored in those blastomeres at the 16-cell stage that have inherited a sufficient part of the apical pole 32,33. In addition, gap junctions are switched on and blastomeres flatten on each other again 3
136, 78-85
145, 290-294
37 Bergman, K., Eslava, A.P. and Cerd~i-Olmedo, E. (1973) Mol. Gen. Genet. 123, 1-16 38 Medina, J.R. and Cerdfi-Olmedo, E. (1977) Exp. Mycol. 1,
31 Rivero, F. and Cerd:i-Olmedo, E. (1987) Mol. Gen. Genet.
209, 149-153 32 Ruiz-Herrera, J. and Catvo-Mendez, C. (1987) Exp. Mycol. 33 34 35 36
286-292
11, 287-296 Martinez-Pacheco, M. et al. (1989) Arch. Microbiol. 151, 10-14 Galland, P. and Senger, H. (1988) Photochem. Photobiol. 48, 811-820 Gatland, P. and Senger, H. (1988)J. Photochem. Photobiol. B1,277-294 Weiss, J. and Weisenseel, M.H. (1990) J. Plant Physiol.
39 Lipson, E.D. and Terasaka, D.T. (1981) Exp. Mycol. 5,
101-111
L.M.
CORROCHANO AND E.
CERDlf-OLMEDO
ARE IN
THE
DEPA~TAMENTO DE GEN~TlCal, UmVERSlOAD DE SEVILL~ APARTADO 1095, E-41080 SEVILLa, SPAIN.
During development, the diversity of cell types found in the adult organism is generated through intensive cell divisions and differentiation. Differentiation can follow two major pathways: (1) Cytoplasmic determination: Determinative components are asymmetrically distributed within the cytoplasm; these components are inherited by only one of the two daughter cells after cleavage. Such asymmetries exist in higher organisms such as frogs, but are particularly apparent in the mosaic eggs of molluscs, ascidians and nematodes. For example, germ-line granules in the nematode Caenorhabditis elegans are redistributed in the zygote as a result of cytoplasmic movement, involving or driven by microfilaments, and after a series of divisions become restricted to the cells giving rise to the gametes 1. (2) Embryonic induction: The fate of inducible cells can be changed under the influence of signals emitted by other cells, often a long distance away. A well-studied example is mesoderm induction in Xenop u s laevis: cells from the animal pole of the blastula (which normally form the ectoderm) form mesodermal tissues when cultured with cells isolated from the vegetal pole 2. During early mouse development, a cytoplasmic segregation takes place that differs from the type described above, since asymmetries are set up in the cytoplasm by a more subtle reorganization of cytoplasmic and cortical networks, rather than by a redistribution of determinative molecules. This process is controlled by both cell interactions and intrinsic cellular mechanisms.
Cell polarity and cell diversification during early mouse embry0genesis CATHERINEGUETH-HALLONETAND BERNARDMARO At the eight-cell stage of mouse development, the organization of blastomeres changes from radially symmetrical to polarized This acquisition of cell polarity, followed by asymmetric divisions, leads to the formation of two phenotypically different cell types, which give rise to the first two cell lineages of the mouse blastocyst embryo, trophectoderm and the inner cell mass. Cellfate, controlled by positional information, is not irreversibly fixed during differentiation, providing the embryo with considerable developmental flexibility.
Early mouse development In mammals, the fertilized egg gives rise not only to the embryo but also to extraembryonic structures. These are elaborated during early development to allow the blastocyst-stage embryo to implant in the uterus. Thus, during the first four days of development, the blastocyst forms and the first two cell types are established: the inner cell mass (ICM), which will mainly give rise to the embryo proper, and the trophectoderm, which will give rise to many extraembryonic structures (Fig. 1). In ,the mouse, the embryonic genome is activated during the two-cell stage, much earlier than in most other organisms. The initial cleavage divisions are equal, asynchronous and without any particular orientation. The first signs of asymmetries
appear only at the eight-cell stage and they correspond to a surface and cytoskeletal reorganization referred to as polarization. The following cell divisions, between the fourth and the sixth cleavages, can be asymmetrical and give rise to two different cell populations3: the outer cells of the morula (16- and 32cell stages), which will give rise mainly to the trophectoderm, and the inner cells of the morula, which will mainly give rise to the ICM of the blastocyst (beginning at the 64-cell stage).
Totipotency and flexibility Tarkowski and Wroblewska showed 25 years ago that individual blastomeres at the eight-cell stage can still form both trophectoderm and the ICM4. This totipotency can be correlated with the fact that, at the beginning of the eight-cell stage, blastomeres are phenotypically (biochemically and morphologically) equivalent. But this totipotency persists at later stages, when the cells are phenotypically different. This provides the embryo with developmental flexibility within lineages during early development. One factor that could regulate the fate of a blastomere is the position of the cell within the embryo since, in experiments where two cleavage-stage embryos were aggregated, cells differentiated according to their new position, irrespective of their original location in the embryo: those positioned on the outside of the chimera
T16 AU6UST1992 rOE 8 NO. 8
m
]REVIEWS
FIGH Preimplantation mouse embryos. (A) Noncompacted early eight-cell embryo. The blastomeres are spherical and clearly visible. (B) Compacted late eight-cell embryo. The flattened blastomeres can no longer be distinguished. (C) Fully expanded blastocyst in phase contrast. Mouse embryo blastomeres have already given rise to two differentiated tissues: the trophectoderm (t) and the inner cell mass (icm). A voluminous cavity, the blastocoel (bl), has developed after cavitation of the embryo at the 32 cell stage. (D) Microtubule distribution in a compacted eight-cell embryo examined by confocal microscopy. Tubulin staining reveals the apical cytoplasmic poles• became trophectoderm cells, while those inside became ICM cells, as shown by the use of genetic markers 5. Such regulation allows aggregates of 16 outer or 16 inner cells to form apparently normal blastoc~sts 6. The influence of position on cell fate has also been demonstrated using single blastomeres: when moved to a new position in the morula, they differentiate according to this new position 7. More recently, flexibility has also been demonstrated in situ by following the fate of outside cells in morulae8: the ICM is derived from inside cells at the 16-cell stage, supplemented by occasional cells derived from outer cells during the fifth and sixth cleavages. It is only in fully expanded blastocysts (after the 64-cell stage) that ICM and trophectoderm cells cannot cross lineages. Flexibility is also evident in more quantitative aspects of development: for example, there is a great variability in the number of outer and inner cells at the 16-cell stage, but the relative proportion of these two cell populations is maintained within a narrow range after the fifth cleavage. Such variability at the 16-cell stage suggests that the number of asymmetrical divisions after polarization at the eight-cell stage does not determine the number of cells within the two different populations and that some cell lineages must therefore cross from one population to the other. All these observations underline the importance of understanding the cellular mechanisms (polarization and asymmetrical cell divisions) that precede the emergence of the two first cell populations of the embryo.
Setting up of cellular asymmetries during compaction Compaction The blastomeres of early eight-cell embryos are spherical, evenly covered with microvilli and can easily be dissociated, because cellular interactions are weak. During the eight-cell stage, cells start to flatten upon each other so that their boundaries can no longer be seen with an optical microscope, six or seven hours after the third cleavage (Fig. 1A, B). The major molecule involved in this cell flattening is the calciumdependent cell adhesion molecule uvomorulin (also called L-CAM or E-cadherin), as shown by experiments in which embryos cultured in the presence of antibodies against uvomorulin develop as grape-like structures 9. During flattening, uvomorulin is progressively
removed from the apical membrane domain of the blastomeres and redistributes predominantly in areas of cell--cell contact 1°,11. At the 16-cell stage, uw)morulin remains evenly distributed on the surface of inner blastomeres and is strictly basolateral on outer blastomeres. Simultaneously with this flattening process, cytoplasmic and surface components become asymmetrically distributed (or polarized), allowing the first distinction to be made between the basolateral and apical domains of the blastomeres. First, gap junctions assemble in the basal domain, and a cytoplasmic pole of endosomes, clathrin vesicles and cytoskeletal elements (microfilaments and microtubules) forms in the apical domain 12. In particular, the distribution of microtubules changes progressively during compaction: in early eight-cell blastomeres, microtubules are found mainly around the nucleus and in the cell cortex, whereas a few hours later many have disappeared from the area next to the zone of intercellular contact and have accumulated in the apical part of the cells (Fig. 1D) t3. In contrast, a subpopulation of more stable, acetylated microtubules becomes enriched in the basal part of the cell cortex during compaction 14. This cytoplasmic reorganization is associated with the apical redistribution of certain membrane proteins, such as the Na+-glucose cotransport system ~5. Second, the surrounding microvilli, consisting mainly of microfilaments, become restricted to the apical surface of the blastomere 16. Thus, at the end of the eight-cell stage, many surface and cytoplasmic components have changed their organization and become polarized rather than homogeneously distributed. Both processes - flattening and polarization - are referred to as compaction3.~e.lL Involvement of intercellular contacts Uvomorulin also seems to be involved in polarization, since an anti-uvomorulin antibody delays polarization and perturbs the axis of polarization, which is normally perpendicular to the plane of contact with adjacent cells (Fig. 2a) 1°. The molecular mechanism of cell adhesion through homophilic interactions of uw> morulin is beginning to be understood. However, the link between adhesion on the cell surface and the cytoplasmic triggering of compaction is not yet clear.
TIG AUGUST1992 VOL.8 NO. 8
m
~]~EVIEWS
S S'°J
No microtubules(~
No adhesion
(~
No microtubules No adhesion ( ~ time
4-
+
+
-I-
-
+
-
+
+
-
+ + ( if the nucleus is peripheral)
i,
P
FIG~ Alternative pathways for polarization. For clarity, these events are illustrated by an isolated pair of eight-cell stage blastomeres. Surface microvilli are shown as a black border, while cytoplasmic organelles are shown in light grey and the nucleus in dark grey. (a) General pathway mediated by intercellular adhesion: cytoplasmic and surface polarities develop opposite the area of cell contact. (b) With experimental disruption of microtubules: cytoplasmic polarity is inhibited. (c) Alternative pathway involving intrinsic mechanisms: cell polarity can develop in the absence of intercellular contacts in cells in which the nucleus is located near the cell surface. (d) With experimental disruption of microtubules and inhibition of adhesion: both cytoplasmic and surface polarity are inhibited. Many modifications in cell organization that occur during compaction seem to be regulated by changes in previously synthesized proteins: for example, uvomorulin is already present in eggs and cleaving embryos. In addition, some elements of compaction can occur or even be advanced when protein synthesis is inhibited as early as the late two-cell or early four-cell stage TM. An initiation signal for compaction acting at the post-translational level would allow compaction to proceed by removal of a prior inhibition. Some changes in protein phosphorylation associated specifically with passage through the eight-cell stage may have a role in compaction 19. Several levels of control of compaction by contact-directed mechanisms have been investigated. (1) Protein kinase C (PKC): Phorbol esters and diacylglycerides cause premature compaction of four-cell embryos, and ECCD-1, a monoclonal antibody directed against uvomorulin, completely blocks this induced compaction 2°,21. This suggests that PKC plays a role in the initiation of compaction through its effect on cell adhesion. Activation of PKC also affects the cytoskeleton, since it induces loss of microfilaments (both in microvilli and in the cortex) and cytoplasmic microtubules from compacted .eight-cell embryos. However, the mechanism underlying .PKC activation in the undisturbed eight-cell embryo is not clearly understood and a possible link between uvomorulin and activation of PKC remains to be determined.
(2) G proteins: Some of these proteins, normally involved in signal transduction, have been identified in the preimplantation mouse embryo; however, they may not be relevant to compaction, since interfering with their function with pertussis toxin has no effect on the development of a two-cell embryo to the blastocyst stage 22. (3) Others': Molecules possibly involved in direct transduction of the adhesion signal have n o w been characterized in other systems (Madin-Darby canine kidney cells, Xenopus laevis A6 cells and transfected mouse fibroblasts growing as monolayers): three proteins, catenins a, b and c, connect uvomorulin to actin filaments of the cytoskeleton, and this cytoplasmic anchorage is necessary for the adhesive function of uvomorulin 2-'-25. Uvomorulin can also be associated with membrane-cytoskeleton complexes containing ankyrin and fodrin in epithelial cells 26. Such complexes could help to set up basolateral membrane polarity. However, the link between these proteins and uvomorulin has not yet been investigated in the preimplantation mouse embryo.
Involvement of the cytoskeleton Microfilaments appear to be directly involved in compaction, since cytochalasin D (a drag that blocks microfilament polymerization) inhibits and reverses cell flattening. Cytochalasin D also affects the movement of the nucleus towards the domain of intercellular contact,
TIG AUGUST 1 9 9 2 VOL. 8 NO. 8
W
[~EVIEWS Spindle orientation
Division
Orthogonal
Symmetrical
@P°lafisati°na~(~)x~' ~
Phenotype Polar / Polar
Flatening Envelopment
8-cell stage
Mitosis
16-cell stage
FIGR] Polarization and asymmetric cell division. For clarity, these:events are illustrated by an isolated blastomere from the eight-cell stage. Surface microvilli are shown as a black border, while cytoplasmic organelles are shown in light grey. Cellular asymmetries are set up during compaction at the eight-cell stage. Cytoplasmic polarity is lost during mitosis but restored at the 16-cell stage, l)epending on the position of the spindle with respect to the axis of polarity (defined by the respective positions of the nucleus and of the surface and cytoplasmic poles), two different types of divisions can occur. (a) Symmetrical division: when the spindle is orthogonal to the axis of polarity, both 16-cell stage daughter cells inherit a polarized phenotype and the division is called conservative. After division. the two polarized 16-cell blastomeres flatten on each other. (b) Asymmetrical division: when the spindle is parallel to the axis of polarity, the two 16-cell stage daughter cells have a different phenotype and the division is called differentiative. After division, the polarized 16-cell blastomere tends to surround the nonpolarized blastomere. which normally occurs during polarization. When applied during compaction, cytochalasin D inhibits the formation of surface poles. However, once a surface pole of microvilli has formed, it persists even when the polar distribution of cytoplasmic microfilaments is 1ost27,28. The use of microtubule-inhibiting drugs, which also inhibit cytoplasmic polarity, confirms this possible dissociation of surface and cytoplasmic polarity 28. Since disruption of microtubules accelerates intercellular flattening and surface polarization, while stabilization of microtubules prevents and reverses both these processes, microtubules might exercise a constraining role during compaction 29 (Fig. 2a, b). The possible involvement of the microtubule network during compaction can be summarized in the following model: the reduction of the number of microtubules in the basal domain of the blastomeres facilitates formation of gap junctions and loss of microvilli in the basolateral domain, whereas it accelerates intercellular flattening. In contrast, the redistribution of microtubules in the apical domain would direct the movement of cytoplasmic components to this domain, and these apical microtubules would stabilize the newly formed poles of organelles and microvilli. Intracellular reorganization of the microtubule network can induce polarization of blastomeres at the eight-cell stage in the absence of intercellular contacts or when flattening is inhibited. Indeed, if the nucleus is near the cell surface (as it often is just after mitosis), a network of microtubules associated with cytoplasmic organelles forms between the cell surface and the nucleus, establishing a cytoplasmic pole 3°. These
microtubules are then necessary for the formation of a surface pole of microvilli, which is located just over the nucleus (Fig. 2a, c, d). So mechanisms intrinsic to the cell seem sufficient to set up asymmetries in the blastomeres in the absence of cell contact.
Asymmetric divisions at the 16-cell stage During the fourth cleavage, cytoplasmic polarity is lost, since the blastomeres entering mitosis locally decompact, gap junctions switch off" and their interphase network of microtubules disassembles to be replaced by the mitotic spindle (Fig. 3). In contrast, the polarized organization of the cell surface is retained during mitosis. However, when cells are blocked in mitosis for many hours by nocodazole, surface poles tend to spread over the whole blastomere surface, showing that surface polarization is also unstable, although stable enough to survive throughout mitosis (which normally lasts one hour) 31. Nonetheless, some elements of polarity established at the eight-cell stage are conserved during the fourth cleavage, since cytoplasmic polarity is restored in those blastomeres at the 16-cell stage that have inherited a sufficient part of the apical pole 32,33. In addition, gap junctions are switched on and blastomeres flatten on each other again 3
